Methods and compositions for detecting and promoting cardiolipin remodeling and cardiomyocyte maturation and related methods for treating mitochondrial dysfunction
阅读说明：本技术 检测和促进心磷脂重塑和心肌细胞成熟的方法和组合物以及治疗线粒体功能障碍的相关方法 (Methods and compositions for detecting and promoting cardiolipin remodeling and cardiomyocyte maturation and related methods for treating mitochondrial dysfunction ) 是由 J·W·米克拉斯 H·劳霍拉-贝克 Y·王 于 2018-12-07 设计创作，主要内容包括：本公开的实施方案涉及用于诱导心肌细胞成熟的方法和组合物。在一些实施方案中,心肌细胞体外衍生自干细胞。在一些实施方案中,该组合物和方法通过在心肌细胞中诱导Let7i microRNA(miRNA)的过表达、miR-452的过表达、miR-122的降低表达和/或miR-200a的降低表达来诱导成熟。在其他实施方案中,本公开涉及用于治疗诸如脂肪酸氧化紊乱等以线粒体功能障碍为特征的病症的方法。在其他实施方案中,本公开涉及筛选影响心脏肌肉功能的化合物的方法。在其他实施方案中,本公开涉及通过检测或监测细胞的心磷脂概况来检测或监测细胞中的线粒体功能障碍的方法。(Embodiments of the present disclosure relate to methods and compositions for inducing cardiomyocyte maturation. In some embodiments, the cardiomyocytes are derived in vitro from stem cells. In some embodiments, the compositions and methods induce maturation by inducing overexpression of Let7i microrna (mirna), overexpression of miR-452, decreased expression of miR-122, and/or decreased expression of miR-200a in cardiomyocytes. In other embodiments, the disclosure relates to methods for treating conditions characterized by mitochondrial dysfunction, such as fatty acid oxidation disorders. In other embodiments, the disclosure relates to methods of screening for compounds that affect cardiac muscle function. In other embodiments, the disclosure relates to methods of detecting or monitoring mitochondrial dysfunction in a cell by detecting or monitoring a cardiolipin profile of the cell.)
1. A method of inducing cardiomyocyte maturation comprising inducing two or more of the following in immature cardiomyocytes: overexpression of Let7i microRNA (miRNA), overexpression of miR-452, decreased expression of miR-122 and decreased expression of miR-200 a.
2. The method of claim 1, comprising inducing three or more of the following in immature cardiomyocytes: overexpression of Let7imiRNA, overexpression of miR-452, decreased expression of miR-122 and decreased expression of miR-200 a.
3. The method of claim 2, comprising inducing overexpression of Let7i miRNA, overexpression of miR-452, decreased expression of miR-122, and decreased expression of miR-200a in immature cardiomyocytes.
4. The method of any one of claims 1-3, wherein inducing overexpression comprises contacting immature cardiomyocytes with a vector comprising a nucleic acid encoding a miRNA to be overexpressed.
5. The method of claim 4, wherein the vector is configured to facilitate transient expression of a nucleic acid encoding the miRNA to be overexpressed.
6. The method of claim 4, wherein the vector is a viral vector configured to integrate a nucleic acid encoding the miRNA to be overexpressed into the genome of the immature cardiomyocytes.
7. The method of claim 6, wherein the viral vector is a lentiviral vector or an adeno-associated viral vector.
8. The method of any one of claims 1 to 3, wherein inducing decreased expression of the miRNA comprises contacting the immature cardiomyocytes with a nucleic acid fragment that hybridizes to the miRNA targeted for decreased expression, or with a vector comprising a nucleic acid encoding a transcript that hybridizes to the miRNA targeted for decreased expression.
9. The method of claim 1, wherein inducing decreased expression comprises effecting knock-out of a gene encoding a miRNA.
10. The method of any one of claims 1-3, wherein inducing reduced expression comprises providing immature cardiomyocytes with a nuclease and a guide nucleic acid having a sequence that facilitates specific cleavage by the nuclease of a nucleic acid encoding the miRNA targeted for reduced expression.
11. The method of claim 10, wherein providing a nuclease to an immature cardiomyocyte comprises contacting the immature cardiomyocyte with the nuclease or a vector encoding the nuclease, wherein the vector is configured to promote expression of the enzyme in the cardiomyocyte.
12. The method of claim 10, wherein providing the immature cardiomyocyte with the guide nucleic acid comprises contacting the immature cardiomyocyte with the guide nucleic acid or a vector encoding the guide nucleic acid, wherein the vector is configured to facilitate expression of the guide nucleic acid in the cardiomyocyte.
13. The method of claim 10, wherein the nuclease is an endonuclease, such as Cas9 or TALENS.
14. The method of claim 8, 11 or 12, wherein the vector is a viral vector.
15. The method of claim 14, wherein the viral vector is a lentiviral vector or an adeno-associated viral vector.
16. The method of claim 1, wherein the immature cardiomyocytes are derived from stem cells.
17. The method of claim 14, wherein the immature cardiomyocytes are derived in vitro from stem cells.
18. The method of claim 16 or claim 17, wherein the stem cell is an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
19. The method of any one of claims 1-18, further comprising contacting the immature cardiomyocytes with two or more long chain fatty acids selected from the group consisting of palmitic acid, oleic acid, and linoleic acid.
20. The method of claim 19, wherein the one or more long chain fatty acids comprise palmitate, oleic acid and linoleic acid.
21. The method of any one of claims 1-20, wherein the cardiomyocytes comprise a genetic aberration.
22. The method of claim 21, wherein the genetic aberration is associated with a metabolic or pathological disease state in the heart.
23. The method of claim 22, wherein the genetic aberration is associated with a Fatty Acid Oxidation (FAO) disorder.
24. The method of claim 22, wherein the cardiomyocyte comprises a mutation in a gene encoding one of: HADHA, FATP1, FACS1, OCTN2, L-CPTI, M-CPTI, CAT, CPT II, VLCAD, LCAD, MCAD, SCAD, LCHAD, SHYD, M/SCHAD, SKAT, MKAT, HS, HL, ETF, and ETF QO.
25. A cardiomyocyte produced by any of the methods described in any one of claims 1-24.
26. The cardiomyocyte of claim 25, wherein said cardiomyocyte comprises a genetic aberration.
27. The cardiomyocyte of claim 26, wherein the genetic aberration is associated with a Fatty Acid Oxidation (FAO) disorder.
28. The cardiomyocyte of claim 27, wherein the genetic aberration is a mutation in a gene encoding hadoa.
29. A method of treating a subject having a disorder treatable by administration of cardiomyocytes having a mature cardiolipin profile, comprising administering to the subject an effective amount of the cardiomyocytes of claim 25.
30. The method of claim 29, wherein the subject has damaged cardiac tissue or cells.
31. The method of claim 29, wherein the subject has diabetes, congenital heart disease, ischemia, myopathy, mitochondrial disease, and/or has an infarction.
32. The method of claim 29, wherein the mitochondrial disease is a Fatty Acid Oxidation (FAO) disorder.
33. The method of claim 29, wherein the subject has a mutation in the gene encoding HADHA.
34. The method of claim 29, wherein the subject is experiencing arrhythmia.
35. The method of claim 29, wherein the subject is at elevated risk of Sudden Infant Death Syndrome (SIDS).
36. A method of screening for a compound for modulating cardiac function comprising:
contacting one or more cardiomyocytes according to any one of claims 25-28 with a candidate agent; and
measuring a cardiac functional parameter of the one or more cardiomyocytes;
wherein a change in the cardiac function parameter indicates that the candidate agent modulates cardiac function.
37. The method of claim 36, wherein the mature cardiomyocytes comprise a genetic aberration.
38. The method of claim 37, wherein the genetic aberration is associated with a Fatty Acid Oxidation (FAO) disorder.
39. The method of claim 38, wherein the genetic aberration is a mutation in a gene encoding HADHA.
40. The method of claim 36, wherein the cardiac functional parameters comprise lipid profile, cardiolipin profile, metabolic profile, oxygen consumption rate, mitochondrial proton gradient, contractility, calcium transport, conduction rate, glucose stress, and cell death.
41. A method of treating a mitochondrial Fatty Acid Oxidation (FAO) disorder in a subject, the method comprising administering an effective amount of a composition that stabilizes a cardiolipin profile in the subject or promotes mature cardiolipin remodeling in the mitochondria of the subject.
42. The method of claim 41, wherein said FAO disorder is associated with conditions of diabetes, heart failure, neurodegeneration, advanced age, congenital heart disease, ischemia, myopathy, and/or infarction.
43. The method of claim 41, wherein the FAO disorder is a Fatty Acid (FA) β -oxidation disorder.
44. The method of claim 41, wherein the mitochondrial dysfunction phenotype is associated with an increased risk of sudden infant death syndrome.
45. The method of claim 41, wherein stabilizing the cardiolipin profile comprises preventing cardiolipin oxidation.
46. The method of claims 41-45, wherein said composition is or comprises elaiprentide.
47. A method of detecting a pathological state of a cultured cardiomyocyte, comprising:
determining a profile of cardiolipin in the cardiomyocytes, wherein a relative increase in cardiolipin with acyl chains greater than 18 carbons indicates and a relative decrease in cardiolipin with acyl chains less than 18 carbons indicates a decrease in the pathological state of the cardiomyocytes.
48. The method of claim 47, wherein the increase or decrease in cardiolipin is relative to wild-type immature cardiomyocytes.
49. The method of claim 47, wherein the cultured cardiomyocytes are derived in vitro from stem cells.
50. The method of claim 49, wherein said stem cell is an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
51. The method of claim 47, wherein said pathological condition is associated with mitochondrial dysfunction.
52. The method of claim 51, wherein the mitochondrial dysfunction is a mitochondrial trifunctional protein deficiency.
53. The method of claim 47, further comprising contacting the cultured cardiomyocytes with a candidate agent to reduce the pathological state of the cultured cardiomyocytes.
54. The method of claim 53, comprising determining a cardiolipin profile in the cultured cardiomyocytes a plurality of times before, during, and/or after the step of contacting the cultured cardiomyocytes with the candidate agent to determine the effect of the candidate agent on the pathological state of the cultured cardiomyocytes.
55. A composition for inducing maturation of cultured cardiomyocytes, comprising two or more of: a nucleic acid construct encoding Let7imicroRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of the sequence encoding miR-122, and a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of the sequence encoding miR-200 a.
56. The composition of claim 55, comprising three or more of: a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of a sequence encoding miR-122, and a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of a sequence encoding miR-200 a.
57. The composition of claim 55, comprising a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of a sequence encoding miR-122, and a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of a sequence encoding miR-200 a.
58. The composition of any one of claims 55 to 57, wherein the nucleic acid constructs encoding microRNAs and/or encoding oligomers are each operably linked to one or more promoter sequences.
59. The composition of any one of claims 55-57, wherein one or more of said constructs is incorporated into one or more vectors configured for delivery to a cell.
60. The composition of claim 59, wherein the one or more vectors are viral vectors.
61. The composition of claim 60, wherein at least one viral vector is a lentiviral vector or an AAV vector.
62. The composition of any one of claims 55-61, wherein the oligomer that hybridizes to a portion of the sequence encoding miR-122 and the oligomer that hybridizes to a portion of the sequence encoding miR-200a are guide RNA molecules configured to induce gene editing enzymes to cleave miR-122 and miR-200a, respectively.
63. The composition of claim 62, wherein said gene-editing enzyme is a nuclease.
64. The composition of any one of claims 55-63, further comprising a nuclease.
65. The composition of claim 63 or claim 64, wherein the nuclease is Cas 9.
66. The composition of any of claims 55-65, further comprising one or more long chain fatty acids.
67. The composition of claim 66, wherein the one or more long chain fatty acids comprise two or more of palmitate, oleic acid and linoleic acid.
68. The composition of claim 67, wherein the one or more long chain fatty acids comprise palmitate, oleic acid and linoleic acid.
Drawings
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
FIGS. 1A-1F illustrate the generation of HADHA mutant (Mut) and Knockout (KO) stem cell-derived cardiomyocytes FIG. 1A) is a schematic representation of fatty acid β -oxidation detailing four enzymatic steps FIG. 1B) is a schematic representation of HADHA KO DNA and protein sequences from WTC iPSC lines showing a22 bp deletion leading to an early stop codonWTThe DNA fragment has a sequence shown in SEQ ID NO: 1, corresponding HADHAWTThe sequence of the protein fragment is shown as SEQ ID NO: 2, respectively. Schematic HADHAKOThe DNA fragment has a sequence shown in SEQ ID NO: 3, corresponding HADHAKOThe sequence of the protein fragment is shown as SEQ ID NO: 4, respectively. Exons, introns and In/Del domains are indicated. Figure 1C) schematic representation of the hadaa Mut DNA and protein sequences from WTC iPSC lines showing a 2bp deletion and 9bp insertion on the first allele and a 2bp deletion on the second allele. Schematic HADHAWTThe DNA fragment has a sequence shown in SEQ ID NO: 5, respectively. Schematic HADHAMutDNA fragment sequences are shown in
FIGS. 2A-2F illustrate aspects of cardiomyocyte maturation microRNA screening. FIG. 2A) is a schematic workflow diagram for determining candidate microRNAs for screening cardiomyocyte maturation. Fig. 2B) is a schematic of a workflow performed to generate stem cell-derived cardiomyocytes transduced with microRNA. FIG. 2C) cell area analysis of microRNA treated hipSC-CM. MicroRNA-208b OE resulted in a significant increase in cell area, while miR-205KO resulted in a significant decrease. Cells were stained for alpha actinin, phalloidin and with DAPI and imaged with a confocal microscope. Fig. 2D) microelectrode array analysis of microRNA treated hiPSC-CM corrected field potential duration (cppd). MiR-452OE resulted in longer cpds. FIG. 2E) Single cell twitch force (twitch force) analysis using a micro-post assay. MiR-200aKO resulted in a significant increase in the twitch force of the hiPSC-CM. FIG. 2F) hippocampal analysis of the maximal change in Oxygen Consumption Rate (OCR) due to FCCP after oligomycin treatment of hipSC-CM treated with micro RNA. MiR-122KO resulted in a significant increase in maximum OCR, while miR-208b OE, -378e OE and-200 a KO resulted in a significant decrease in maximum OCR.
FIGS. 3A-3O illustrate that MiMaC accelerates the maturation of hipSC-CM. Fig. 3A) a schematic of the combination of four mirrorrnas to produce miMaC. Figure 3B) single cell contractility assay on microcolumns showed that treatment of hiPSC-CM with MiMaC resulted in a significant increase in twitch force. Fig. 3C) representative tracking of EV (control) and MiMaC treated hiPSC-CM. Fig. 3D) single cell twitch force analysis on the microcolumn showed that hiPSC-CMs treated with MiMaC resulted in a significant increase in power. Fig. 3E) cell size analysis showed that the MiMaC-treated hiPSC-CM resulted in a significant increase in area. Fig. 3F) representative confocal microscope images of EV and MiMaC treated hiPSC-CM. Alpha actinin (green), phalloidin (red) and DAPI are shown. Fig. 3G) hippocampal analysis of fatty acid oxidation capacity showed that the mipsc-treated hiPSC-CM matured to the point where palmitate could be oxidized to generate ATP, whereas control cells failed to utilize palmitate. The OCR of mimochipc-CMs was significantly increased due to the addition of palmitate. Figure 3H) Venn plot of KOmicroRNA predicted targets, and identification of HOPX as a common predicted target among all KO mirs screened for cardiomyocyte maturation. Figure 3I) HOPX expression profile from RNA sequence data during cardiomyocyte maturation. HOPX expression was significantly higher in D30 and in 1-year-old hESC-CM compared to D30 hESC-CM, whereas 1-year-old hESC-CM had statistically significantly higher HOPX. Indicates significance relative to D20. # denotes the relationship with respect to D30. Fig. 3J) HOPX expression in adult human ventricular tissue was significantly higher than in human fetal ventricular tissue. RNA sequencing data was used for mapping. Fig. 3K) RT-qPCR for HOPX expression of HOPX showed that mipc treated hiPSC-CM had statistically significantly higher HOPX levels at D30 compared to EV control D30 hiPSC-CM. FIG. 3L) Single cell RNA-Seq tSNE map of unbiased clustering of microRNA-treated hPSC-CM. Fig. 3M) cluster map detailing which treatment groups were enriched in each cluster. Fig. 3N) heatmap based on maturity category of MiMaC cluster. Figure 3O) heat map (yellow) of in vivo human maturation markers upregulated with maturation.
FIGS. 4A-4L depict that fatty acid-challenged HADHA Mut CM exhibited elevated cytosolic calcium levels, resulting in increased beat rate irregularity. Figure 4A) hippocampal mitochondrial pressure (mitostress) assay that analyzed maximum oxygen consumption after addition of oligomycin and FCCP. The MiMaC-treated CM showed a significant increase in maximum OCR compared to the control EV CM. Fig. 4B) representative trace of mitochondrial stress measurements. Fig. 4C) hippocampal analysis of fatty acid oxidation capacity showed that the mipsc-treated hiPSC-CM matured to the point where palmitate could be oxidized to generate ATP, whereas control cells failed to utilize palmitate. The OCR of mimochipc-CMs was significantly increased due to the addition of palmitate. Both MiMaC treated Mut and KOhipSC-CM were unable to oxidize palmitate. FIG. 4D) representative trace of fluorescence change during calcium transient analysis. FIG. 4E) quantification of the maximum change in fluorescence during calcium transients. Calcium changes were statistically significantly lower in Mut CM compared to WT CM after 12 days of Glc + FA medium treatment. FIG. 4F) quantification of tau-decay constant. Mut CM has a higher tau decay constant than WT CM after 12 days of Glc + FA medium treatment. Fig. 4G) representative trace of fluorescence change during fluovert, action potential, assay. Fig. 4H) the maximum change in fluorescence during the action potential was quantified. FIG. 4I) the wave duration of Mut CM was significantly longer than WT CM by 50% after 12 days of Glc + FA medium treatment. FIG. 4J) representative pulse rate tracking of Mut CM in Glc or Glc + FA media. Fig. 4K) quantification of beat interval change (Δ BI). Mut CM in Glc + FA medium has a statistically significantly higher Δ BI than Mut CM in Glc medium. Fig. 4L) Poincare plot shows ellipses with 95% confidence intervals for each group. The more rounded ovals under Mut Glc + FA conditions show greater beat-to-beat instability of these cells compared to Mut Glc CM.
FIGS. 5A-5J depict scRNA-Seq revealing various disease states of fatty acid-challenged HADHA Mut CM. FIG. 5A) Single cell RNA sequencing tSNE plot of WT compared to HADHA Mut CM shows a clear difference between these two groups. Four conditions of D30 CM: the FA treatment of MiMaC WT CM was 6 days, the FA and SS-31MiMaC WT CM was 6 days, the FA treatment of MiMaCHADHA Mut CM was 6 days, and the FA and SS-31 treatment of MiMaC HADHA Mut CM was 6 days. Fig. 5B) unbiased clustering analysis revealed 6 unique groups. Figure 5C) detail the heat map of enrichment of conditions in each cluster. Fig. 5D) heatmap based on maturity category of MiMaC cluster. Figure 5E) heat map of in vivo mouse maturation markers upregulated with maturation. FIG. 5F) confocal microscope showed that HADHA Mut CM has more nuclei than WT CM. blue-DAPI, green-ATP synthase β subunit, pink-Titin (Titin). The inset is the kernel displayed in grayscale. Fig. 5G) histograms of cell frequencies with 1, 2, 3 or 4 or more nuclei. The HADHA mutant CM possesses a large number of cells with 3 or more nuclei. Figure 5H) downregulated metabolic pathways in cluster 0 (non-replicating hadaa CM) compared to cluster 3(WT CM). Figure 5I) down-regulated metabolic pathways in cluster 2 (internal replication type hadoa CM) compared to cluster 3(WT CM). Figure 5J) upregulated metabolic pathways in cluster 2 (internal replication type hadaa CM) compared to cluster 3(WT CM). The circle size of the metabolic bubble map is proportional to the statistical significance. The smaller the p-value, the larger the circle. The adjusted p-value of 0.01 was used as a critical value.
FIGS. 6A-6H depict HADHA Mut CMs challenged with fatty acids displaying swollen mitochondria with severe mitochondrial dysfunction. FIG. 6A) representative confocal images of WT and Mut CM in Glc + FA medium for 12 days. Figure 6B) quantitates co-localization and intensity of mitochondrial tracer and ATP synthase beta. FIG. 6C) TEM image of WT and Mut CM after 12 days in Glc + FA medium, showing sarcomere and mitochondrial structure. FIG. 6D) mitochondrial circularity index histograms of WT and HADHAMut CM after 12 days in Glc + FA medium show that the mitochondria of HADHA Mut CM are more rounded. FIG. 6E) mitochondrial area histograms of WT and HADHA Mut CM after 12 days in Glc + FA medium showed that the mitochondria of HADHA Mut CM were smaller. Fig. 6F) quantification of maximum OCR from mitochondrial stress assay (mitostress assay). Mut and KO CM had significantly lower max OCR compared to WT CM after 12 days of Glc + FA medium. Figure 6G) quantification of ATP production from mitochondrial stress assay, calculated as the difference between baseline OCR and post-oligomycin OCR. Mut and KO CMs have significantly lower ATP production than WT CMs after 12 days of Glc + FA medium. Figure 6H) quantification of proton leakage from mitochondrial stress assay, calculated as the difference between OCR after oligomycin and OCR after antimycin and rotenone. After 12 days of Glc + FA medium, Mut and KO CMs had significantly higher proton leakage compared to WT CMs. After 12 days of Glc + FA, SS-31 treated Mut CM had significantly reduced proton leakage compared to untreated Mut CM.
FIGS. 7A-7K are graphical representations of fatty acid-challenged HADHA KO and Mut CM with elevated fatty acids and abnormal cardiolipin profiles. Fig. 7A) model of long-chain FA intermediate accumulation after the first step of long-chain FAO due to hadoa loss. FIG. 7B) the sum of all long-chain acylcarnitines in WT, Mut and KO FA treated hPSC-CM. FIG. 7C) amount of sperm whale acid in free fatty acid state in WT, Mut and KO FA treated hPSC-CM. FIG. 7D) amount of palmitoleic acid in the free fatty acid state in WT, Mut and KO FA treated hPSC-CM. FIG. 7E) amount of oleic acid in the free fatty acid state in WT, Mut and KO FA treated hPSC-CM. Fig. 7F) tetra [ 18: 2-CL. Figure 7G) cardiolipin profiles generated from targeted lipidomics against Glc or Glc + FA treated WT and HADHA KO CM. Figure 7H) cardiolipin profiles of WT CM12D Glc + FA,
FIG. 8 graphically depicts the maturation of phospholipids in the center of CM. The WTiPSC-derived CM has a reduced selectivity compared to the immature iPSC-derived CM by having [ 14: 0],[14: 1][16: 1] or [ 16: 0] and increasing CL with acyl chains of greater than 18 carbons (including the intermediate [ 18: 1] [ 18: 2] [ 18: 2] [ 20: 2] alter their CL profile during maturation.
Detailed description of the invention
The present disclosure is based on the inventors' analysis of mitochondrial trifunctional protein deficiencies. As described in more detail below, the inventors addressed the major deficiencies in current cell models by generating new stem cell-derived cardiomyocytes from HADHA-deficient human-induced pluripotent stem cells (hipscs). The inventors have developed methods for accelerating cardiomyocyte maturation using engineered microRNA maturation mixtures that are capable of up-regulating the epigenetic regulator homeobox protein (HOPX). Fatty Acid (FA) -challenged hadaa mutant cardiomyocytes showed abnormal calcium regulation, delayed repolarization, and irregular beating, suggesting a proarrhythmic state. These pathological cardiac manifestations are the result of underlying mitochondrial pathology, which is manifested as mitochondrial dysfunction due to loss of proton gradient and mitochondrial depletion of normal mitochondrial cristae (cristae). The underlying mechanism of this pathological mitochondrial state is thought to be the deregulation of cardiolipin homeostasis and tetra [ 18: 2] the consequences of a decrease in cardiolipin species. These data reveal a fundamental dual role of hadoa as an acyltransferase in fatty acid beta-oxidation and in cardiolipin remodeling of homeostasis in the heart.
These studies provide new methods of promoting cardiomyocyte maturation for therapeutic, experimental modeling, or drug screening applications. In addition, the basic observations of CL modeling provide a means to detect and monitor CL remodeling states to infer cell health or maturation.
In light of the foregoing, in one aspect, the present disclosure provides a method of inducing cardiomyocyte maturation. The method comprises inducing two, three or all of the following in immature cardiomyocytes: overexpression of Let7 microRNA (miRNA), overexpression of miR-208b, overexpression of miR-452, decreased expression of miR-1222 and decreased expression of miR-200 a. Agents that induce regulated miRNA expression are collectively referred to herein as microRNA maturation mixtures (mimacs).
In some embodiments, the method comprises inducing overexpression of at least Let 7miRNA and miR-452 in immature cardiomyocytes. In another embodiment, the method comprises inducing at least overexpression of a Let 7miRNA and decreased expression of miR-122 in immature cardiomyocytes. In another embodiment, the method comprises inducing at least overexpression of a Let 7miRNA and decreased expression of miR-200a in immature cardiomyocytes. In another embodiment, the method comprises inducing at least overexpression of miR-452 and decreased expression of miR-122 in immature cardiomyocytes. In another embodiment, the method comprises inducing at least overexpression of miR-452 and decreased expression of miR-200a in immature cardiomyocytes. In another embodiment, the method comprises inducing at least decreased expression of miR-122 and decreased expression of miR-200a in immature cardiomyocytes.
In some embodiments, the method comprises inducing at least overexpression of Let 7miRNA, overexpression of miR-452, and decreased expression of miR-122 in immature cardiomyocytes. In another embodiment, the method comprises inducing at least overexpression of Let 7miRNA, overexpression of miR-452 and decreased expression of miR-200a in immature cardiomyocytes. In another embodiment, the method comprises inducing at least overexpression of a Let 7miRNA, decreased expression of miR-122 and decreased expression of miR-200a in immature cardiomyocytes. In another embodiment, the method comprises inducing at least overexpression of miR-452, decreased expression of miR-122, and decreased expression of miR-200a in immature cardiomyocytes.
And, () Let7, miR-452, miR-208b, miR-122 and miR-200a are all microRNAs in Cardiomyocytes (CM) that are shown herein to have an effect on various aspects of SM maturation (see experimental discussion below). As described, manipulation of these mirnas has been shown to affect signaling pathways leading to higher maturation and cardiolipin remodeling in CM. When performed in cultured (e.g., stem cell-derived) CM, these miRNA manipulations result in CM that is more similar to Adult Cardiomyocytes (ACM).
Let7 is a family of miRNAs described in more detail in Kuppusamy, K.T. et al, Let-7family of microRNA amplified for formation and adult-like metabolism in step cell-derived cardio cells, Proc Natl Acad Sci U S A,2015, and US 9,624,471, the entire contents of which are incorporated herein by reference. The Let-7miRNA may be selected from Let7a-1, Let7a-2, Let7b, Let7c, Let7e, Let7f-1, Let7f-2, Let7g and Let7 i. In some embodiments, the Let 7miRNA is Let7 i. Representative DNA sequences encoding Let7imiRNA include those set forth in SEQ ID NO: 11, which is the sequence of an amplicon generated from a human genomic template, inserted into an expression vector to facilitate expression of the Let7i miRNA. The nucleic acid molecule encoding the indicated Let 7miRNA can be obtained by any conventional method. In some embodiments, the nucleic acid may be obtained by amplifying the sequence from the coding genome using specific primers. For example, as described in more detail below, this amplification process is used to amplify and obtain the coding sequence of Let7i (plus additional sequences upstream and downstream) so that it can be incorporated into an expression vector for overexpression in a cell. Exemplary forward and reverse primers for amplifying such a region including Let7i are set forth in SEQ id nos: 12 and 13.
Representative sequences encoding miR-452 are included in SEQ ID NO: 14, which is the sequence of an amplicon generated from a human genomic template that is inserted into an expression vector to facilitate expression of the mi-452 miRNA. Exemplary forward and reverse primers for amplifying a region comprising human miR-452 are set forth in SEQ ID NO: 41 and 42.
miR-208b is described, for example, in Callis, T.E. et al, Callis, T.E., et al, MicroRNA-208a isa regulator of cardiac hyperthermia and control in micro.J. Clin Invest,2009.119(9): p.2772-86, which is incorporated herein by reference in its entirety. Exemplary forward and reverse primers for amplifying a region comprising human miR-452 are set forth in SEQ ID NO: 39 and 40.
Representative sequences encoding miR-122 are included in SEQ ID NO: 46, in the sequence shown in figure 46. Representative sequences encoding miR-200a are included in SEQ ID NO: 45, or a sequence shown in seq id no. Each of these sequences represents an amplicon of the human genome sequence, including the indicated miRNA coding regions in addition to additional sequences upstream and downstream. The sequence of the entire amplicon can be used to transgenically express the entire miRNA. One of ordinary skill in the art can readily use this sequence to generate a guide RNA that hybridizes to the coding sequence, or to generate a single-stranded nucleic acid fragment that hybridizes to a portion of a miRNA, to reduce functional expression of the target miRNA (discussed in more detail below).
With respect to Let7i, miR-452, and miR-208b, the term "induce overexpression" and grammatical variants thereof encompasses any additional level of miRNA within the cell. In some embodiments, the expression level of the target miRNA is increased by at least about 1%, 5%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more. Overexpression may be induced by enhancing the endogenous expression activity of the cell itself from its coding region, or by providing the cell with additional foreign coding regions for additional transcriptional activity. In some embodiments, "inducing overexpression" entails simply providing additional copies of the miRNA itself to the cell.
In some embodiments, induction of miRNA (e.g., one or more of Let7i, miR-452, and miR-208b) overexpression can include the step of contacting immature CM with a vector comprising a nucleic acid encoding a miRNA to be overexpressed. The vector may be configured to promote transient or constitutive expression of the miRNA in the cell. In this regard, the nucleic acid is operably linked to a promoter sequence that can drive transcription of the miRNA coding region within the cell. Promoter regions may be selected by one of ordinary skill in the art to accommodate the type of expression desired.
In some embodiments, the vector is configured to facilitate integration of a nucleic acid encoding a miRNA to be overexpressed (e.g., one or more of Let7i, miR-452, and miR-208b in the same or separate vectors). For example, the vector may be a viral vector comprising a nucleic acid encoding a miRNA to be overexpressed. Any suitable viral vector for such genomic integration of the encoding nucleic acid is contemplated herein. Non-limiting exemplary viral vectors for this purpose are lentiviral vectors and adeno-associated viral vectors (AAV). The use of lentiviral embodiments is described in more detail below for illustration.
With respect to miR-122 and miR-200a, the term "reduced expression" encompasses any reduction in the expression level of a functional miRNA within the cell. In some embodiments, reduction of the expression level of a target miRNA encompasses "sponge" methods, wherein a single-stranded nucleic acid hybridized to at least a portion of the miRNA (i.e., miR-122 or miR-200a) interferes with the ability of the miRNA to affect transcription of its genomic target. As indicated above, the sequences encoding miR-122 and mi-200a are included in SEQ ID NO: 46 and 35, respectively. In this sense, the added nucleic acid "absorbs" the target miRNA from the immature cardiomyocytes and removes it from the environment of the intracellular transcriptional modulator. In some embodiments, hybridization results in degradation of the miRNA, for example, in RNA interference. The single-stranded nucleic acid may be administered directly. In other embodiments, immature cardiomyocytes can be transformed with sequences encoding single-stranded nucleic acids using vectors configured to facilitate transient or constitutive expression of the single-stranded nucleic acids. Non-limiting examples of vector platforms that can be used for this purpose include lentivirus and AAV vectors. See the discussion above regarding suitable vectors, which also applies in this context.
In other methods, mirnas targeted to reduce expression (i.e., miR-122 and/or miR-200a) target genomic alterations in immature cardiomyocytes that permanently reduce or knock out expression of functional target mirnas in the cells. In one embodiment, immature cardiomyocytes are provided having a guide RNA and a nuclease. The guide RNA has a sequence that allows it to hybridize to a region of the genomic sequence encoding the target miRNA. After hybridization, the guide RNA promotes specific cleavage of the genomic region by nucleases. Immature cardiomyocytes have endogenous DNA repair enzymes that periodically introduce repair errors that occur as substitutions, insertions or deletions within the coding sequence, resulting in functional knock-outs of mirnas. Even if the repair process is precise in the first few rounds, eventually the guide RNA/nuclease/repair combination will lead to a wrong repair, leading to a functional knockout. Exemplary guide RNAs for
In some embodiments, the nuclease has endonuclease activity. Exemplary non-limiting nucleases include Cas9 and TALENS. Other such nucleases that can specifically edit or cleave DNA based on guide RNA are known and encompassed by the present disclosure.
The guide RNA may be provided to immature cardiomyocytes directly or by transgenically expressing the guide RNA in the immature cardiomyocytes. Independently of the guide RNA, the nuclease may be provided to the immature cardiomyocytes directly or by transgenically expressing the nuclease in the immature cardiomyocytes.
In one embodiment, guide RNAs that hybridize to a region of the genomic sequence encoding a target miRNA are administered directly to immature cardiomyocytes to promote nuclease specific cleavage of the genomic region. In other embodiments, the guide RNA is transiently or constitutively transgene expressed in immature cardiomyocytes by transforming the cells with a nucleic acid construct encoding the guide RNA. Suitable vectors can be selected for the desired expression. For example, non-limiting examples of vector platforms that can be used to integrate guide RNA coding sequences into the genome of immature cardiomyocytes include lentiviral and AAV vectors. See the discussion above regarding suitable vectors, which also applies in this context.
As indicated above, the nuclease may be provided directly to the immature cardiomyocytes, or may be transiently or constitutively transgenic expressed within the immature cardiomyocytes. To induce nuclease expression in immature cardiomyocytes, the cells can be transformed with a nucleic acid construct encoding a nuclease using a vector configured to promote transient or constitutive expression of a single-stranded nucleic acid. For example, non-limiting examples of vector platforms that can be used to integrate a nuclease coding sequence in the genome of immature cardiomyocytes include lentiviruses and AAV vectors. See the discussion above regarding suitable vectors, which also applies in this context.
In view of the foregoing, illustrative examples of specific embodiments for inducing cardiomyocyte maturation are described.
In one embodiment, the method comprises inducing a decrease in expression of one or both of miR-122 and miR-200a in immature cardiomyocytes. The corresponding guide RNA and nuclease (e.g., Cas9) transduce expression in immature cardiomyocytes. The DNA encoding the guide RNA (guide RNA if both mirnas are targeted) and the DNA encoding the nuclease may be integrated into the same or separate vectors. Each coding DNA region is operably linked to its own promoter sequence configured to drive transcription in immature cardiomyocytes. The sequence encoding the guide RNA is typically operably linked to an RNA promoter to ensure that the transcribed guide RNA remains an RNA construct. In some embodiments, the DNA encoding the guide RNAs for miR-122 and miR-200a are integrated into the same vector. In other embodiments, the DNA encoding the guide RNAs for miR-122 and miR-200a are integrated into separate vectors. In some embodiments, the DNA encoding the nuclease and the DNA encoding one or both guide RNAs are integrated into the same vector. In other embodiments, the DNA encoding the nuclease and the DNA encoding one or both guide RNAs are integrated into different vectors.
In some embodiments, the cell line of the immature cardiomyocytes is genetically modified to integrate a gene encoding a nuclease into the cell genome. In this embodiment, the modification may be performed on immature cardiomyocytes or stem cell progenitors thereof. For example, the cell is contacted with a vector (e.g., a lentiviral vector) comprising a DNA encoding a nuclease (e.g., Cas9) operably linked to a promoter, wherein the lentiviral vector permanently integrates the expression cassette with the nuclease gene and promoter into the genome of the cell. The promoter may be configured to facilitate conditional or constitutive expression of the nuclease. With such a genetic background, immature cardiomyocytes can be contacted with one or more guide RNAs, as described above, that promote specific cleavage of DNA encoding a target miRNA (e.g., miR-122 or miR-200 a). The guide RNA may be produced in advance using a typical method (e.g., recombinant expression in bacteria, etc.). This allows for efficient production of cell cultures of cardiomyocytes with knock-out of the desired target miRNA (e.g., miR-122 and/or miR-200 a). Alternatively, immature cardiomyocytes can be contacted with one or more plasmids or other vectors that incorporate guide RNA-encoding sequences as described above that can promote specific cleavage of DNA encoding a target miRNA (e.g., miR-122 or miR-200 a). The DNA encoding the different guide RNAs may be incorporated into the same or different plasmids or other vectors. Integration into the genome is not necessary, and transient expression of the guide RNA is sufficient to cause knock-out of the cell.
In yet another embodiment, a nuclease (e.g., Cas9) and a guide RNA (e.g., against DNA encoding miR-122 or miR-200a) can be exogenously produced and administered directly to immature cardiomyocytes, independent of transgene expression in the immature cardiomyocytes themselves.
In other embodiments where both overexpression of a target miRNA and reduced expression of other target mirnas are sought, the nucleic acid constructs driving overexpression or reduced expression of each respective target miRNA may be integrated into the same vector construct. In exemplary embodiments, the cell is contacted with a vector comprising an expression cassette having a plurality of nucleic acid expression constructs. In this embodiment, the cells may be immature cardiomyocytes or stem cell progenitors thereof. The expression cassette may facilitate inducible expression of the transcript encoded therein using an inducer specific for the vector. The expression cassette may include any combination of the encoding constructs described above. To illustrate, in one embodiment, an expression cassette comprises a DNA sequence encoding one or more mirnas targeted for overexpression (e.g., one or more of Let7, miR-452, and miR-208b) and a DNA sequence encoding a single-stranded nucleic acid that hybridizes to one or more mirnas targeted for decreased expression (e.g., one or both of miR-122 and miR-200 a). An exemplary vector suitable for use in this embodiment is pAC150-PBLHL-4xHS-EF1a-DEST (Addgene, #48234) with insulator sequences flanking the expression cassette to ensure that the expression construct in the cassette is not silenced. Such inducible vectors can be induced by, for example, doxycycline to facilitate expression of the MiMaC members contained therein.
As described above, immature cardiomyocytes can be derived from stem cells. In some embodiments, the cells are derived in vitro from stem cells by promoting differentiation of the stem cells into immature stem cells, as described in more detail in the examples. This process is also described in more detail in, for example, Palpant, N.J., et al, Generation high-throughput cardiac and synthetic derivatives from patterned media using human pluripotent cells. Nat. Protoc,2017.12(1): p.15-31; burridge, P.W., et al, chemical refining generation of human cardio cells. nat Methods,2014.11(8): p.855-60; and Tohyama, S.et al, Distingt methyl flow enabled large-scale discovery of mouse and mouse pluripotential Stem-derived cardiac cells, 2013.12(1): p.127-37, each of which is incorporated herein by reference in its entirety. The stem cell may be an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
In some embodiments, the method further comprises contacting immature cardiomyocytes with an effective amount of a long chain fatty acid. As used herein, the term "effective amount" refers to an amount sufficient to promote cardiomyocyte maturation and/or the remodeling of cardiolipin in the cell to a more mature state. In some embodiments, the method further comprises contacting immature cardiomyocytes with at least two long chain fatty acid species. In some embodiments, the method further comprises contacting immature cardiomyocytes with at least three long chain fatty acid species. The long chain fatty acid species may be selected from palmitic acid, oleic acid and linoleic acid. Typically, palmitic acid is used with oleic or linoleic acid, as it may be cytotoxic to cells itself.
The long chain fatty acid may be contacted with immature cardiomyocytes in a form in which it is conjugated to a carrier such as BSA which may aid in its uptake and stability. Exemplary oleic acid/BSA conjugate concentrations or ranges in cell culture media include: about 10-14. mu.g/mL, about 11-13. mu.g/mL, about 12-13. mu.g/mL, e.g., about 11. mu.g/mL, about 11.5. mu.g/mL, about 12. mu.g/mL, about 12.5. mu.g/mL, about 12.7. mu.g/mL, about 13. mu.g/mL, and about 13.25. mu.g/mL. Exemplary linoleic acid/BSA conjugate concentrations or ranges in cell culture media include: about 5.5-8.5. mu.g/mL, about 6.5-8. mu.g/mL, about 6.75-8.0. mu.g/mL, e.g., about 6. mu.g/mL, about 6.5. mu.g/mL, about 7. mu.g/mL, about 7.05. mu.g/mL, about 7.5. mu.g/mL, about 8. mu.g/mL, and about 8.25. mu.g/mL. Exemplary monopalmitic acid (sodium palmitate form)/BSA conjugate concentrations or ranges in cell culture media include: about 40-60. mu.M, about 45-55. mu.M, about 50-55. mu.M, e.g., about 45. mu.g/mL, about 48. mu.M, about 50. mu.M, about 52.5. mu.M, about 55. mu.M, about 58. mu.M and about 60. mu.M. In an exemplary embodiment, as described in the examples, the fatty acid medium utilizes the concentration of oleic acid (SigmaO3008) conjugated to BSA: 12.7 μ g/mL, concentration of linoleic acid conjugated to BSA (Sigma L9530): 7.05 μ g/mL, concentration of sodium palmitate (Sigma P9767) conjugated to BSA (Sigma a 8806): 52.5. mu.M.
In further embodiments, the method further comprises contacting immature cardiomyocytes with carnitine at a concentration of about 100-. Exemplary concentrations include about 100. mu.g/mL, 110. mu.g/mL, about 120. mu.M, about 125. mu.M, about 130. mu.M, about 135. mu.M, about 140. mu.M, and about 150. mu.M. Carnitine helps transport the long chain fatty acids administered into the mitochondria.
In some embodiments, the immature cardiomyocytes comprise a genetic aberration. The genetic aberration may be associated with a metabolic or pathological disease state of the heart. For example, the genetic aberration is associated with a disorder of Fatty Acid Oxidation (FAO). In some embodiments, the cardiomyocyte comprises a mutation in a gene encoding one of: HADHA, FATP1, FACS1, OCTN2, L-CPTI, M-CPT I, CAT, CPT II, VLCAD, LCAD, MCAD, SCAD, LCHAD, SHYD, M/SCHAD, SKAT, MKAT, HS, HL, ETF and ETF QO, which cause fatty acid disorders. By implementing genetic aberrations in the induced mature cardiomyocytes, the resulting cardiomyocytes provide a disease model for ACM with the indicated aberrations.
In another aspect, the present disclosure provides a cardiomyocyte produced by the above method. As noted, the cardiomyocytes can be derived from stem cells, such as embryonic stem cells, pluripotent stem cells or induced pluripotent stem cells. In some embodiments, the stem cell is from a human.
Furthermore, as described in more detail, the cells may contain genetic aberrations, such as aberrations associated with Fatty Acid Oxidation (FAO) disorders. Target genes containing exemplary genetic aberrations are listed above. In one embodiment, the genetic aberration is a mutation in the gene encoding hadoa.
Considering a method of inducing cardiomyocyte maturation using a MiMaC, the cells may comprise an exogenous nucleic acid that is or encodes a miRNA to be overexpressed (i.e., Let7, miR-452 and/or miR-208 b). Alternatively or additionally, the cell may comprise an exogenous nucleic acid that is or encodes a single-stranded nucleic acid that can hybridize to a target miRNA targeted for reduced expression (i.e., miR-122 and/or miR-200 a). Alternatively or additionally, the cell may comprise an exogenous nucleic acid that is or encodes a guide RNA that can hybridize to genomic sequences encoding mirnas targeted for reduced expression (i.e., miR-122 and/or miR-200 a). In some embodiments involving a guide RNA for reducing expression of a target miRNA, the cell further comprises a nuclease (e.g., Cas9 or TALENS) or a nucleic acid construct encoding the nuclease. In one embodiment, the cell comprises an expression cassette having a first nucleic acid encoding a Let 7miRNA, a second nucleic acid encoding miR-452, a third nucleic acid encoding a single-stranded nucleic acid that hybridizes to at least a portion of miR-122, and a fourth nucleic acid encoding a single-stranded nucleic acid that hybridizes to at least a portion of miR-200 a. The nucleic acid sequence is operably linked to one or more promoters. In a further embodiment, the expression of said nucleic acid sequence may be induced by the use of doxycycline.
In another aspect, the present disclosure provides a method of treating a subject having a disorder treatable by administration of cardiomyocytes having a mature cardiolipin profile. The method comprises administering to the subject an effective amount of cardiomyocytes produced by the methods described herein to promote maturation in culture. For example, the method may comprise culturing induced stem cells obtained from the subject to differentiate into immature cardiomyocytes, administering the MiMaC to the cells in any of the forms described herein, and allowing the cells to develop into adult cardiomyocytes during their maturation. The cells can then be administered to a subject in need thereof. In other embodiments, the stem cells may be from different subjects of the same species. As described above, the stem cell may be an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
The subject may be any mammal. In some embodiments, the subject is a rodent or primate. In some embodiments, the subject is a human, dog, cat, mouse, rat, rabbit, or the like.
In some embodiments, the cardiac myocytes in the cardiac tissue of the subject are damaged. This may include a subject suffering from diabetes, congenital heart disease, ischemia, myopathy, mitochondrial disease, and/or suffering from an infarct event. In some embodiments, the mitochondrial disease is a Fatty Acid Oxidation (FAO) disorder. In some embodiments, the subject has a mitochondrial trifunctional protein (MTP/TFP) deficiency. In some embodiments, the subject has a mutation in the gene encoding hadoa. In other embodiments, the subject has a mutation in a gene encoding at least one of FATP1, FACS1, OCTN2, L-CPTI, M-CPT I, CAT, CPT II, VLCAD, LCAD, MCAD, SCAD, LCHAD, SHYD, M/SCHAD, SKAT, MKAT, HS, HL, ETF, and ETF QO.
In some embodiments, the condition or disorder may be manifested as experiencing an arrhythmia. The subject may be a neonate or infant with a high risk of Sudden Infant Death Syndrome (SIDS), such as for example in the case of a deficiency in mitochondrial triple function protein (MTP/TFP).
The cells can be readily formulated for administration to damaged cardiac tissue according to techniques understood in the art.
In another aspect, the present disclosure provides a method of treating a mitochondrial Fatty Acid Oxidation (FAO) disorder in a subject. The method comprises administering an effective amount of a composition that stabilizes a cardiolipin profile in mitochondria in the subject or promotes remodeling of mature cardiolipin.
The subject may be any mammal, such as a human.
Mitochondrial dysfunction may be associated with diabetes, heart failure, neurodegenerative diseases, advanced age, congenital heart disease, ischemia, myopathy, and/or infarction. In some embodiments, the FAO disorder is a fatty acid beta-oxidation disorder. The FAO disorder may be associated with a mutation in any of the genes described above. In some embodiments, mitochondrial dysfunction is associated with increased risk of arrhythmia and/or sudden infant death syndrome.
In some embodiments, stabilizing the cardiolipin profile comprises preventing cardiolipin oxidation. In some embodiments, the composition is or comprises elalopeptide (also known as SS-31) (steath BioTherapeutics Inc, newton, MA), a small tetrapeptide that targets mitochondria, which is known to reduce the production of toxic reactive oxygen species and stabilize cardiolipin. In one embodiment, an effective amount of an elapsin is administered to a subject suffering from a mitochondrial trifunctional protein deficiency.
In another aspect, the present disclosure provides a method of screening for candidate compounds for potential cardiac function modulation. In this regard, the methods and compositions described herein enable the production of cultured cardiomyocytes to be performed during their maturation to more accurately reflect adult cardiomyocytes. Thus, such cells can be readily produced in vitro to provide a screening process for candidate agents/compounds.
The method comprises contacting one or more cardiomyocytes produced by the methods described herein with a candidate agent; and measuring a cardiac functional parameter in the one or more cardiomyocytes. A change in a cardiac function parameter indicates that the candidate agent modulates cardiac function. Candidates that promote favorable functional parameters and/or reduce negative functional parameters may be selected as strong candidates or compounds for treatment or continued research.
The cardiac function parameter may include any relevant, measurable parameter that has an effect on cardiac tissue function. Non-limiting exemplary cardiac functional parameters include lipid profile, cardiolipin profile, metabolic profile, oxygen consumption rate, mitochondrial proton gradient, contractility, calcium transport, conduction velocity, glucose stress, and cell death in a particular situation. In addition, potential toxicity and dosing concentrations can be tested in the disclosed cells.
In addition to screening for the effect of a candidate agent on a model of healthy adult cardiomyocytes, the method further includes embodiments of screening for the effect of a candidate compound on a model of disease with a more mature cardiomyocyte status. Thus, in some embodiments, mature cardiomyocytes used in the screening can comprise a genetic aberration, such as described elsewhere herein. Aberrations can be associated with, for example, Fatty Acid Oxidation (FAO) disorders. To illustrate, the following experimental description is directed to cells having a genetic mutation in the HADHA protein. The cells were induced to develop to a more mature state to provide an adult cardiomyocyte model with a mitochondrial triple function protein (MTP/TFP) deficiency. This allows testing compounds to counteract dysfunction.
In another aspect, the present disclosure provides a method of detecting a pathological state of cultured cardiomyocytes. The method includes determining a cardiolipin profile in a cardiomyocyte. A relative increase in cardiolipin with acyl chains greater than 18 carbons indicates and/or a relative decrease in cardiolipin with acyl chains less than 18 carbons indicates a decrease in the pathological state of the cardiomyocytes.
Exemplary lipidomics methods for determining the profile of cardiolipin are described in more detail in the examples below.
The relative increase or decrease in cardiolipin can be compared to a reference standard for cardiomyocytes, such as derived from wild-type adult cardiomyocytes or cultured cardiomyocytes that have been established to exhibit normal or acceptable mitochondrial function or have an established normal or mature cardiolipin profile. In other embodiments, the relative increase or decrease in cardiolipin is compared to wild-type immature cardiomyocytes or cultured cardiomyocytes that have been established to exhibit normal or acceptable mitochondrial function or to have an established normal or mature cardiolipin profile. The following experimental disclosure describes the profile of phospholipids in human cardiomyocytes cultured during the maturation process.
As described above, the cultured cardiomyocytes may be derived from autologous stem cells, such as embryonic stem cells, pluripotent stem cells or induced pluripotent stem cells.
The pathological state may be a state associated with mitochondrial dysfunction, as detailed above. In some embodiments, the mitochondrial dysfunction is a mitochondrial trifunctional protein deficiency.
The method may be performed to determine whether the cultured cells are sufficiently mature, i.e., have sufficient cardiolipin remodeling to serve their intended purpose. In addition, the method may be performed one or more times during in vitro screening of candidate agent compounds to determine their effect on cardiac homeostasis or other mitochondrial function. Thus, the method may comprise further contacting the cultured cardiomyocytes with a candidate agent to reduce the pathological state of the cultured cardiomyocytes. The timing of the detection step can be suitably designed for a particular screening or treatment. The determining step may be performed, for example, multiple times before, during and/or after the step of contacting the cultured cardiomyocytes with the candidate agent to determine the effect of the candidate agent on the pathological state of the cultured cardiomyocytes.
It will be appreciated that the assay method may also be extended to be performed on cells obtained from a subject to diagnose a pathological state of cardiomyocytes.
In this regard, mitochondrial trifunctional protein deficiency is often manifested synergistically in cells from the heart (cardiomyocytes), liver (hepatocytes) and retina. Thus, one or more cells can be obtained from any of these tissues (typically the liver) and the profile of cardiolipin can be determined. As described herein, a relatively low level of cardiolipin with 18 (or more) carbon chains indicates that the cell is unable to fully reconstitute cardiolipin from an immature state to a mature state. Failure of cardiolipin remodeling indicates that cells are unable to efficiently utilize fatty acids as a primary energy source. This failure may be experienced in parallel with the subject's cardiomyocytes, resulting in an increased risk of pathologies such as, for example, arrhythmia and SIDS.
In another aspect, the present disclosure provides a composition or kit of compositions for inducing maturation of cultured cardiomyocytes. The composition can be used in the above method to promote maturation of immature cardiomyocytes. The composition relates to a MiMaC formulation and comprises two or more of: a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes a nucleic acid fragment that hybridizes to a portion of a sequence encoding miR-122, and a nucleic acid construct that is or encodes a nucleic acid fragment that hybridizes to a portion of a sequence encoding miR-200 a.
In one embodiment, the composition comprises three or more of: a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes a nucleic acid fragment that hybridizes to a portion of a sequence encoding miR-122, and a nucleic acid construct that is or encodes a nucleic acid fragment that hybridizes to a portion of a sequence encoding miR-200 a. In a further embodiment, the composition comprises a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes a nucleic acid fragment that hybridizes to a portion of a sequence encoding miR-122, and a nucleic acid construct that is or encodes a nucleic acid fragment that hybridizes to a portion of a sequence encoding miR-200 a.
Target mirnas are described in more detail above. Also described are nucleic acid fragments that hybridize to a portion of the sequence encoding the target miRNA to prevent functionality and result in reduced functionality of expression.
The nucleic acid constructs encoding micrornas and/or encoding nucleic acid fragments are each operably linked to one or more promoter sequences.
One or more constructs may be incorporated into one or more vectors configured for delivery to a cell. The one or more vectors may be viral vectors, such as lentiviral or AAV vectors.
In some embodiments, each nucleic acid construct is incorporated into a separate vector, and thus, the composition comprises multiple vectors (with different incorporated expression constructs) and mixed. In another embodiment, each nucleic acid construct is incorporated into the same vector. For example, multiple nucleic acid constructs can be incorporated into the same expression cassette incorporated into a single vector. The vector may be configured to facilitate expression of all of the constructs in the cassette, either constitutively or transiently (e.g., by induction). The vector may provide an insulator sequence to prevent inactivation following delivery to the cell. An exemplary vector suitable for use in this embodiment is pAC150-PBLHL-4xHS-EF1a-DEST (Addgene, # 48234).
The nucleic acid fragment that hybridizes to a portion of the sequence encoding miR-122 and the nucleic acid fragment that hybridizes to a portion of the sequence encoding miR-200a are guide RNA molecules configured to induce gene editing enzymes to cleave miR-122 and miR-200a, respectively. As described above, the gene-editing enzyme may be a nuclease and/or have an endonuclease function. Examples are Cas9 and TALENS, although other examples are known and encompassed by the present disclosure.
In some embodiments, the kits or compositions disclosed herein further comprise a nuclease. In other embodiments, the kit or composition further comprises a nucleic acid construct encoding a nuclease. The nucleic acid construct encoding the nuclease is operably linked to a promoter sequence that facilitates expression of the nuclease in a target cell.
In some embodiments, the kit or composition further comprises one or more long chain fatty acids, which are described in more detail above. The one or more long chain fatty acids include palmitic acid, oleic acid and/or linoleic acid. In some embodiments, the kit or composition comprises a combination of palmitic acid, oleic acid, and linoleic acid.
In some embodiments, the kits disclosed herein may further comprise cell culture media and instructions to facilitate the preparation of mature cultured cardiomyocytes from stem cell-derived cardiomyocytes.
In another embodiment, the kits disclosed herein may further comprise stem cell-derived cardiomyocytes, which may be metabolically active or frozen. In another embodiment, the kit and/or any of its components may be transported and/or stored at ambient or room temperature or, for example, 4 ℃. The stem cell-derived cardiomyocytes can ultimately be derived from a subject having a disease or disorder (e.g., mitochondrial dysfunction as described herein) or genetically modified to mimic a disease or disorder, including, for example, a heart disease or disorder.
Unless specifically defined herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention belongs. With regard to definitions and terminology in the technical field, practitioners make specific reference to: sambrook J.et al (eds.), Molecular Cloning, A Laboratory Manual, 3 rd edition, Cold Spring Harbor Press, Plainview, New York (2001); ausubel, f.m., et al (eds.), Current Protocols in molecular biology, John Wiley & Sons, New York (2010); coligan, J.E., et al (eds.), Current protocols in Immunology, John Wiley & Sons, New York (2010); mirzaei, H. and Carrasco, M. (eds.), Modern Proteomics-Sample Preparation, Analysis and practical application sites in Experimental Medicine and Biology, Springer International Publishing, 2016; and Commai, L, et al (eds.), genomic: Methods and protocols in Molecular Biology, Springer International Publishing, 2017.
For convenience, certain terms employed herein in the specification, examples, and appended claims are provided herein. These definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims.
The term "or" is used in the claims to mean "and/or" unless explicitly indicated to refer only to alternatives or alternatives are mutually exclusive, although the disclosure supports definitions that refer only to alternatives and "and/or".
Following long-standing patent law, the terms "a" and "an" when used in conjunction with the term "comprising" in the claims or the specification mean one or more unless specifically indicated.
Throughout the specification and claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, unless the context clearly requires otherwise. Meaning "including but not limited to". Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above" and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application. The word "about" denotes a number within a slight variation above or below the reference number. For example, "about" may refer to a number within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the range above or below the indicated reference number.
Materials, compositions, and components useful in the disclosed methods and compositions products are disclosed that can be used in conjunction with, can be used in the preparation of, or can be the product. It is to be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each individual combination and permutation of these compounds may not be explicitly disclosed, each and every combination, both individual and collective, is specifically contemplated. This concept applies to all aspects of this disclosure including, but not limited to, steps in the methods described. Thus, particular elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. For example, if multiple additional steps can be performed, it is understood that each of these additional steps can be performed with any specific method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Additionally, it should be understood that embodiments described herein may be performed using any suitable material, such as those described elsewhere herein or known in the art.
The publications cited herein and the subject matter to which they are cited are hereby expressly incorporated by reference in their entirety.
The following describes the study of the etiology of mitochondrial triple function protein (MTP) deficiency, a disease that can lead to Sudden Infant Death Syndrome (SIDS). This study involves the development of a novel method that promotes the maturation of Cardiomyocytes (CMs) from induced pluripotent stem cells, thereby generating a model of CMs that reflects the relevant disease state.
Abstract
Mitochondrial trifunctional protein deficiency is caused by a hydratase subunit a (hadha) mutation. To reveal the etiology of the disease, stem cell-derived cardiomyocytes were generated from HADHA-deficient hipscs and their maturation was accelerated by up-regulating a novel engineered MicroRNA maturation cocktail of epigenetic regulator HOPX ("MiMaC"). Fatty acid challenged MiMaC treated HADHA mutant cardiomyocytes exhibited a disease phenotype: defective calcium kinetics and repolarization kinetics that lead to a pre-arrhythmic state. Based on metabolic gene expression, single-cell RNA-seq discloses a novel intermediate for cardiomyocyte development. This intermediate produces mature-like cardiomyocytes in control cells, but the mutant cells are transformed to a pathological state with reduced fatty acid beta-oxidation (FAO), reduced mitochondrial proton gradient, disrupted mitochondrial crest structure and defective cardiolipin remodeling. This study shows that TFPa/HADHA, an MLCL-AT-like enzyme, is essential for FAO and cardiolipin remodeling, which is critical for functional mitochondria in human cardiomyocytes.
Results
Production of mitochondrial trifunctional protein-deficient cardiomyocytes
To recapitulate the heart pathology of mitochondrial trifunctional protein deficiency at the cellular level in vitro, mutations were generated in the gene HADHA of human ipscs using the CRISPR/Cas9 system. From the wild-type (WT) hiPSC line used as our isogenic control, multiple HADHA mutant hiPSC lines were generated using two different guides targeting the
Examination of HADHAKOThe DNA sequence of the line shows a homozygous 22bp deletion, which results in the early stop codon of exon 1 (FIG. 1B). HADHAMutThe lines had a 2bp deletion and 9bp insertion on the first allele and a 2bp insertion on the second allele (FIG. 1C). Both lines showed no off-target mutations at the first three predicted sites (not shown). In HADHAMutMutations found in the lines result in predicted early stop codons on both alleles. (representative HADHA corresponding to the wild-type sequence shown in FIG. 1CMutThe protein fragment sequence is shown in SEQ ID NO: and 6, listing. Corresponding to the two HADHA shown in FIG. 1CMutMutant allele of HADHAMutThe protein fragment sequences are respectively shown as SEQ ID NO: listed as 8 and 10). However, when examining proteins in each line, expression of hados in the WT hiPSC line was observed, in which hados were expressedKOIs not expressed in the line and remains in HADHAMutExpressed to a lesser extent in the lines (FIG. 1D). Then examined in WT and HADHAMutTranscripts of HADHA expressed in lines. The WT line was found to express full-length HADHA transcripts from exons 1-20, whereas HADHAMutThe line skipped exons 1-3 and expressed HADHA exons 4-20 (FIG. 1C). Because of the lack of the known transcript of the HADHA of exons 4-20, mutations made at the intron-exon junction may induce alternative splicing events and new transcripts. The observed decrease in molecular weight of the hadoa mutant (fig. 1D) supports this hypothesis. Expressed HADHAMutThe protein skips exons 1-3, and expression of 60 amino acids produces a truncated C1 pP/crotonase domain, which may impair mitochondrial localization and protein folding of the enzyme pocket, resulting in the inability to stabilize enolate anion intermediates during FAO.
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) were generated from these three lines using a monolayer directed differentiation protocol [ palpot, n.j., et al, Generating high-purity and endothial derivatives from a patternednodesusing human pluripotent stem cells, nat protocol, 2017.12(1): p.15-31 ]. It was found that the reduction or loss of hadoa did not hinder the ability to generate cardiomyocytes (see image in fig. 1E). To assess the functional phenotype of MTP-deficient cardiomyocytes, a hippocampal assay was performed to measure the increase in oxygen consumption due to the presence of long-chain FA (palmitate). Compared to WT CM, MTP-deficient CM is expected to exhibit a hindered ability to utilize long-chain FAs. However, it was found that all CMs, even control CMs, were unable to utilize long chain FAs (fig. 1F). hiPSC-CM are immature cells representing FCM but not ACM, which is why they cannot use FA as a substrate for ATP production. Therefore, there is a need for a strategy to mature hipscs-CMs so that they can utilize FA to better assess the functional phenotype of MTP-deficient CMs.
Screening of microRNA (miR) for hPSC-CM maturation
To better understand the biological changes that occur during human cardiac maturation, the inventors previously conducted a miR screen in which many significantly regulated mirs were observed during the in vitro transition between day 20 (D20) hPSC-CM and 1 year mature hPSC-CM [ Kuppusamy, k.t. et al, Let-7family of microRNA is required for formation and adut-like metabolism in stem cell-derived cardiac cytograms, proc natl Acad Sci u.s.a.,2015 ]. This list is cross-referenced to human fetal ventricular to adult ventricular myocardial in vivo miR sequencing data [ Akat, K.M. et al, Comparative RNA-sequencing analysis of myocardial and circulating RNAs in human heart failure and the human clinical importance activators Proc Natl Acad Sci U S A,2014.111(30): p.11151-6; yang, K.C. et al, DeepRNA sequencing measurements dynamic regulation of biochemical coding RNAs influencing human heart and modifying with a mechanical circulation system circulation,2014.129(9): p.1009-21 ]. The inventors previously found that the highest up-regulated family of mirs, Let-7, can be overexpressed in hESC-CM to drive a robust (though incomplete) maturation response [ Kuppusamy, k.t. et al Proc Natl Acad Sci u.s.a.,2015 ]. Here, additional miRs are combined with Let-7 to rapidly mature hPSC-CM by promoting a more complete adult-like transcriptome. The top 15 up-and down-regulated mirs were selected from the screen and the top 200 predicted targets (TargetScanHuman) were identified for each miR. For the predicted target of each miR, pathway analysis was performed using GeneAnalytics software to determine which mirs affect pathways associated with cardiomyocyte maturation. These include glucose and/or fatty acid metabolism, cell growth and hypertrophy, and the cell cycle. Many of the down-regulated mirs are involved in maintaining a pluripotent state and therefore have not been selected for screening cardiomyocytes for their maturation state. Finally, based on pathway analysis, six mirs were selected for further analysis to assess their CM maturation potency: three up-regulated miRs (miR-452, -208b and-378 e) and three down-regulated miRs (miR-122, -200a and-205) (FIG. 2A).
Specifically, the three candidate highly upregulated miRs selected are miR-378e [ Nagalingam, R.S. et al, cardiac-rich microRNA miR-378e [ Nagalingam, R.S. et al, A cardiac-derived microRNA, miR-378, blocks cardiac hyperthyroidism by targeting Ras signaling, 2013.288(16): p.11216-32], -208b [ Callis, T.E. et al, MicroRNA-208a regulator of cardiac hyperthermia and control in mice.J. ClinInvest,2009.119(9): p.2772-86] and-452. The family of 378 mirs was chosen because of their high expression levels in mature CM and involvement in cardiac hypertrophy. MiR-378e and-378 f share the same seed region, and miR-378e was selected as a representative miR of family 378. Mir-208b was chosen because it is predicted to be involved in both metabolic and cardiac hypertrophy pathways. Moreover, miR-208b is an intron miR in the myosin beta heavy chain gene (MYH7) and has been reported to function in specifying slow muscle fibers while inhibiting the rapid myofiber gene program in the mouse heart [ van Rooij, E.et al, A family of microRNAs encoded by muscle genes in a dev Cell,2009.17(5): p.662-73 ]. MiR-452 is the second highest up-regulated MiR after Let-7, found to have a predicted target associated with metabolism.
The three highly down-regulated candidate miRs selected were miR-200a, -122 and-205. MiR-141 and-200 a share the same seed region and are involved in both hypertrophy and metabolic pathways. MiR-200a was selected as a representative miR for study. The other two highly down-regulated miRs, miR-205 and miR-122, showed the greatest degree of down-regulation.
Functional analysis of candidate microRNA (miR)
The six mirs described above were evaluated using four functional assays to determine hPSC-CM maturation: cell area, contractility, metabolic capacity and electrophysiology. WT D15 hiPSC-CM was transduced with lentivirus using CRISPR/Cas9 to OEmiR or KO miR. Cells were then lactate-selected to enrich for cardiomyocyte populations and puromycin-selected to enrich for cell populations containing viral vectors. Functional assessment was performed two weeks after D30miR perturbation (fig. 2B).
An important feature of cardiomyocyte maturation is an increase in cell size. Among the tested miRs, only miR-208b OE was found to induce a significant increase in cell area (EV: 2891 μm)2，208b：5802μm2，P<0.05) (fig. 2C). The immature hPSC-CM spontaneously beats at a high rate and the field potential duration is short when studied by extracellular microelectrodes. OE miRs were evaluated using microelectrode arrays to determine whether they increased the field potential duration to physiologically relevant lengths. Of the tested miRs, only miR-452OE increased the corrected field potential duration (cFPD) to a more adult-like duration (cFPD, EV: 296ms, 452: 380ms) (FIG. 2D). One of the hallmarks of cardiomyocyte maturation is an increase in the contractile force produced by the cells. Single cell contractility analysis was performed using a micro-column platform [ Kuppusamy, k.t. et al, Proc Natl Acad Sci us a, 2015; rodriguez, M.L. et al, measuring the concentration of human induced plodditive stem cells with areas of microposts.J. biomechEng,2014.136(5): p.051005; methods of treatment of microbial cells-derived cardiac cells, 2016.94: p.43-50]. Among the tested miRs, only KO of miR-200a caused a significant increase in contractility (EV: 30.8nN, miR-200 a: 51.7nN, P)<0.05) (fig. 2E). Finally, the metabolic capacity of miR-treated hPSC-CM was assessed. Cardiomyocytes are the type of cell required for metabolism, requiring mitochondria, which have a high ATP synthesis capacity. Among the tested mirs, only miR-22 KO resulted in a significant increase in maximal Oxygen Consumption (OCR), indicating more active mitochondria (miR-122 KO: 1.35 fold change compared to EV, P<0.001) (fig. 2F).
Bioinformatic analysis of candidate microRNAs (miRs)
RNA sequencing was performed after alteration of certain miR (miR-378e OE, -208b OE, -452OE, -122KO or-205 KO) to assess its overall transcriptional impact in hPSC-CM. To determine whether each miR is capable of producing a differential effect at the global transcription level, samples were analyzed using Principal Component Analysis (PCA). In each sample, approximately 11,000 protein-encoding genes were expressed, and aggregate expression of at least three FPKMs across all samples was used for PCA. PCA showed that each miR was able to bring significant changes from its respective control (not shown). MiR-452OE showed the greatest separation on PC1, while miR-122KO showed the greatest separation on
The function of each miR was then analyzed in a more targeted manner by specifically examining the pathways essential for heart maturation. Pathway enrichment heatmaps were generated showing how each miR affected 7 different pathways selected as markers of cardiomyocyte maturation (characterized by cardiac hypertrophy, cardiac identity, cell cycle, electrophysiology, fatty acid metabolism, glucose metabolism and cytoskeleton; not shown). MiR-122KO has up-regulated cell cycle and fatty acid metabolism genes. MiR-452OE showed cardiac hypertrophy, electrophysiology and upregulation of the cytoskeleton. MiR-208b OE showed cardiac identity along with strong upregulation of cell cycle and electrophysiological genes. Finally, miR-378e OE showed up-regulation of electrophysiological genes, whereas miR-205KO showed poor up-regulation of pathways associated with cardiac maturation. This heatmap potentiates the unique effect of each miR on cardiomyocyte maturation, as each miR brings a different set of pathway enrichments. Furthermore, based on the heat map data, miR-205KO has a weak capacity to cause cardiomyocyte maturation, while miRs-122KO, -452OE and-208 b OE all show a strong capacity to influence the marker pathways of myocardial maturation.
From these data, a MicroRNA maturation cocktail called MiMaC was generated that included the following constructs: let7i OE, miR-452OE, miR-122KO and miR-200a KO. Let7i was chosen as a result of preliminary studies by the inventors that showed the potential of this miR to induce cardiomyocyte maturation [ Kuppusamy, KT et al, Proc natl acad Sci El SA, 2015 ]. From each functional assay, mirs that give significantly improved maturity were selected to produce a mixture consisting of the fewest number of mirs.
Functional assessment of MiMaC
To assess the maturity of MiMaC-treated hPSC-CM, we performed contractility, cell area and metabolic assays (fig. 3A). The hiPSC-CMs treated with MiMaC had a statistically significant increase in twitch force (average force: 36nN, P ═ 0.002) compared to control cells (average force: 24nN) (fig. 3B). There was also a statistically significant increase in the power produced by the MiMaC-treated hiPSC-CM (mean power: 38fW, P ═ 0.016) compared to the control cells (mean power: 22fW) (fig. 3D).
The mmac treated hPSC-CM had a statistically significant increase in cell area. Using hiPSC-CM, with an average area of 2389 μm2Average area of MiMaC-treated CM compared to control cells of 3022 μm2，P<0.001 (fig. 3E and 3F). In addition to the effect of MiMaC on hiPSC-CM, MiMaC significantly increased the cellular area of treated hESC-CM (not shown).
One of the hallmarks of cardiomyocyte maturation is the ability to gain ATP production from FA. Immature hPSC-CM cannot utilize long-chain FA to produce ATP via beta-oxidation. To assess whether hmsc-CM treated with MiMaC can oxidize long-chain FA, cells were acutely challenged with palmitate and measured for increased OCR. Both the MiMaC-treated hESC-CM and the hipSC-CM were able to utilize palmitate significantly more than the control CM (see FIG. 3G for hipSC-CM; similar results for hESC-CM not shown).
Transcriptional evaluation of MiMaC
To better understand how MiMaC affects the transcriptome of hiPSC-CM, RNA sequencing was performed comparing D30EV control CM with D30 MiMaC-treated CM. Pathway enrichment analysis using a marker gene set indicates that many cellular maturation and muscle processes are upregulated, for example: myogenesis and epithelial mesenchymal transition [34 ]. The apical down-regulation pathway is associated with the cell cycle, a key feature of cardiomyocyte maturation. Using STRING analysis, we identified a network of genes that are significantly up-regulated and correlated in relation to two pathways: myogenesis and epithelial-mesenchymal transition. The genes that have also been shown to be significantly down-regulated and correlated with cell cycle inhibition using STRING analysis are the mitotic spindle and the G2M checkpoint. These findings indicate that the MiMaC tool can promote a more mature transcriptome in hiPSC-CM.
HOPX is a novel modulator of CM maturation
To better understand the molecular mechanisms crucial for cardiac maturation, overlapping predicted targets of the six mirs selected were determined. One of the predicted targets, HOPX (FIG. 3H), is important for the specification of cardiomyocytes [ Jain, R. et al, HeartDEVELOPMENT. integration of Bmp and Wnt signaling by Hopx ligands of cardiovascular communities of cardiovascular diseases.science, 2015.348(6242): p.aa6071 ], but work on this transcriptional regulator has not addressed the latter process, i.e., the maturation of human cardiomyocytes. Here, it was determined that HOPX expression was upregulated in vitro (fig. 3I), in vivo (fig. 3J) and in MiMaC-treated hiPSC-CM (fig. 3K). To analyze how selected MiMaCmiR might individually modulate HOPX expression during maturation, HOPX levels in miR-122KO and Let7i OE hiPSC-CM were analyzed. HOPX was found to be up-regulated 6.8-fold in D30 miR-122KO hipSC-CM, whereas Let7i OE mature hipSC-CM had no effect on HOPX expression (not shown). These data indicate that Let7i OE maturation does not dominate the HOPX cardiac maturation pathway. This highlights the necessity of combining multiple mirs together to produce a strong maturation effect in hPSC-CM, and HOPX appears to be a strong candidate for post-directed cardiomyocyte maturation.
scRNA sequencing analysis of miR-treated CM maturation
By using single cell RN a sequencing (scra-Seq), the MiMaC tool was used to further gain insight into the underlying mechanisms of cardiomyocyte maturation and to better understand the behavior of each miR that makes up MiMaC in CM maturation. Five groups of miR-treated CM were subjected to scRNA-Seq: EV, Let7i and miR-452OE, miR-1222 and-200 a KO, MiMaC and MiMaC + FA. Unbiased clustering was performed to determine how miR perturbation changes CM; five subgroups were found (fig. 3L) and whether miR perturbation resulted in enrichment of these five clusters was assessed using the chi-square test (fig. 3M). The EV group was enriched in
To rank which clusters have a higher degree of myocardial maturation, the scRNA-Seq clusters were evaluated in two different ways. First, highly up-and down-regulated genes in the MiMaC-enriched cluster (cluster 2) were evaluated along with cardiac markers and oxidative phosphorylation genes (fig. 3N). Next, the in vivo human cardiac maturation markers in the identified clusters were examined (fig. 3O).
Finally, the addition of fatty acids to the MiMaC formula was evaluated to increase cardiomyocyte maturation. Three long chain fatty acids, palmitate, linoleic acid and oleic acid, were added to the basal heart medium used. We found that MiMaC + FA cells were enriched in
scRNA-Seq revealed an intermediate cardiomyocyte maturation phase
After unbiased analysis of miR-treated CM, it is clear that each miR combination results in an enrichment of different states of CM maturation. Interestingly, Let7i and miR-452 OE-enriched
MTP/HADHA deficient CM displaying reduced mitochondrial function
The generation of the MiMaC tool allows the study of the cause of hadaa CM disease. Since immature hPSC-CM cannot oxidize fatty acids, the HADHA Mut and KO CM must be matured with MiMaC, which results in fatty acid oxidation in WT CM. First, the maximum OCR of WT, hadaa Mut and KO CM was evaluated. The maximal OCR (2.2-fold change) of the MiMaC-treated WT CM was statistically significantly increased compared to control cells (fig. 4A and 4B). Interestingly, the control and MiMaC-treated hadham amut CM had similar maximum OCR as the control WT-CM, while the hadoa KO CM had reduced maximum OCR. These data indicate that the mitochondrial activity of HADHA is deficient in Mut and KO CM.
Next, it was evaluated whether the hadaa Mut and KO CM treated with MiMaC could produce ATP using fatty acid palmitate. There was a statistically significant increase in oxygen consumption for the mmac-treated WT CM alone due to the addition of palmitate (fig. 4C). WT control CM along with control and MiMaC treated HADHA Mut and KO CM were unable to utilize FA. These data show that MiMaC-treated CM has the ability to utilize long-chain FA. However, the hadaa Mut and KO CM treated with MiMaC failed to do this. MiMaC is crucial for assessing the FAO limits of HADHA Mut and KO CM.
Abnormal calcium management of HADHA Mut CM
Infants with MTP deficiency may experience sudden, initially unexplained death after birth. It has been suggested that the stress of lipids, the major substrate for ATP production found in mother's breast milk, is responsible for early infant death due to MTP deficiency. To address this hypothesis, a combination of three long chain fatty acids was supplemented into basal heart medium with glucose (Glc + FA medium): palmitate, oleic and linoleic acids, since these FAs are most abundant in the serum of breast-fed human infants. Palmitate, as a substrate for fatty acids, is one of the most abundant fatty acids circulating during the newborn, representing 36% of all long chain free fatty acids. Although challenge of CM with FA may lead to lipotoxicity, care was taken to develop concentrations and combinations of three fatty acids that do not lead to lipotoxicity (fig. 3L).
To better understand the manner in which MTP-deficient CMs may cause arrhythmic states leading to SIDS, calcium transients were measured in WT and HADHA Mut CMs (see, e.g., fig. 4D). Fold change in circulating calcium in WT CM was significantly higher (fig. 4E) than had amaut CM (WT CM: 2.03, Mut CM: 1.55, P <0.001) (fig. 4E) with unchanged calcium ramp rate (not shown). This indicates that calcium is circulating out of the cytoplasm and is stored in an abnormal manner in the HADHA MutCM. When tau-decay constants were examined, the HADHA Mut CM was found to have higher mean values (WT CM: 0.63s, Mut CM: 0.76s) (FIG. 4F). This indicates that the rate of calcium pumping back to the muscular/endoplasmic reticulum is slower in HADHA Mut CM.
Delayed repolarization and beat rate abnormalities in HADHA Mut CM
Since the HADHA Mut CMs cultured in Glc + FA medium exhibit abnormal calcium cycling, it was evaluated whether these CMs also exhibit abnormal electrophysiology. Changes in membrane potential were determined using the voltage sensitive fluorescent dye fluovert. It was found that although the HADHA Mut CM had no change in the maximum change in voltage amplitude, time to maximum depolarization or depolarization rate (fig. 4G and 4H), significant differences were observed when examining the repolarization rate. The time to reach 50% (WD50) and 90% (WD90) of the wave time limit (WD) in HADHA Mut CM was found to be significantly longer than that of WT CM (WD50 is shown in FIG. 4I; similar results were observed for WD90 and not shown). These data indicate that repolarization of the HAHDA Mut CM is impaired. This phenotype may be due to observed calcium kinetic abnormalities, resulting from impaired calcium circulation back to the sarcoplasmic reticulum.
Since the HADHA Mut CMs exhibit calcium handling and electrophysiological defects, whether these CMs exhibit abnormal beat rates was evaluated. Spontaneous beats of HADHA Mut CM were followed in the presence of FA to quantify beat rate abnormalities. The hadfamut CM exhibits abnormal beat rate variation due to temporal non-uniformity between beats (e.g., see fig. 4J). Upon quantifying these findings, the beat interval for the HADHA Mut CM was found to be significantly higher (not shown), with significantly greater changes in beat interval (Δ BI) (fig. 4K). These data indicate that the average beats of the hadoa Mut CM are slower and the time variation between beats is greater. Furthermore, the Δ BI percentage of greater than 250ms was quantified, and the HADHA Mut CM after 12D in Glc + FA medium had on average a higher percentage of potential arrhythmic Δ BI (-30%) compared to Mut CM in Glc medium (10%). This quantification of the number of cells with a Δ BI greater than 250ms suggests cells that are unstable beating. Finally, a Poincare map (fig. 4L) is generated with fitted ellipses (95% confidence intervals) around the heartbeat interval data of each group. The narrower and elongated ellipses indicate uniform beat intervals, while the rounder ellipses indicate abnormal beat rates. Taking the ratio of the major axis to the minor axis of each ellipse, we found that the HADHA MutGlcGlc condition had a ratio of 4.36, while the HADHA mutglc + FA condition had a ratio of 3.12, indicating that the HADHA mutglc + FA condition had more rounded ellipses, meaning that the beat-to-beat differences in these CMs were greater.
Single cell RNA sequencing identification of HADHA Mut CM subgroup
Single cell RNA sequencing was performed to better understand the behavior of the HADHA Mut CM population when challenged with FA. Detailed description tSNE plots of each sequenced cell group showed a clear distinction between WT and HADHA Mut CM with little but significant overlap (fig. 5A). When unbiased clustering was performed, 6 clusters were found: 0 hadaa Mut CM was not replicated, 1 intermediate maturation population of WT and Mut CM, 2 hadaa Mut CM replicates, 3 healthy CMs, 4 fibroblast-like populations, 5 epicardial-like populations (fig. 5B and 5C).
To assess maturity and disease status, each cluster was classified based on the key categories described above (fig. 3N). The up-regulated genes in
Examining the hallmark pathway for significant changes between the hadaa Mut CM cluster and the WT CM cluster, it was found that oxpos, cardiac processes and myogenesis were inhibited in mutant cells. Furthermore, although WT CM showed strong expression of the cell cycle repressor CDKN1A, both HADHA Mut CM populations lost this expression.
To ensure that the cell cycle is not a fundamental difference between all clusters, cell cycle genes were examined in each cluster. Unlike previous studies that found that the bias for cluster differences was determined by which cell cycle state the cells were in, only cluster 2 (fig. 5B) was found to show up-regulated cell cycle genes. Clustering data was also reprocessed by removing cell cycle genes, all but the original cluster 2 (high cell cycle hadoa Mut CM) was retained. These findings suggest that the cell cycle is the root cause of cluster 2and not the rest of the cell population (fig. 5A and 5B).
Based on this data, three different pathological states were assumed in FA challenged hadaa Mut CM: an intermediate state: : non-duplicated CM state: : the CM state is copied.
Unbiased metabolic pathway analysis was performed and 68 metabolic pathways were screened, and it was found that
Myoganglion degradation and abnormal mitochondrial Activity of HADHA Mut and KO CM
When HADHA Mut and KO CM were cultured only in glucose medium, no significant defect was observed in HADHA Mut and KO compared to WT CM (not shown; confocal images were of D24 and D30 WT; HADHA Mut and HADHA KO hipSC-CM cultured in glucose medium, myofibril staining of alpha actinin and actin (phaloidin) did not show abnormality; mitochondrial staining of ATP synthase beta subunit and mitochondrial potential gradient shown via mitochondrial tracing staining did not show mitochondrial abnormality; however, when cultured for 6-12 days in FA medium, sarcomere and mitochondrial defects were shown in HADHA Mut and KO CM, while WT CM appeared normal (FIG. 6A; not shown; HADHAMut and KO HIPSC-CM exhibited signs of sarcomere actin destruction, as seen by less defined alpha actinin, and onset of loss of mitochondrial proton gradient as seen by mitochondrial tracing staining in the mutant; ATP synthase β subunit shows normal mitochondrial network in both WT and hadnauthi psc-CM and onset of mitochondrial network loss in hadoa KO hiPSC-CM). After 12 days of Glc + FA medium treatment, WT CM had healthy myofibrils, whereas hadaa Mut CM showed sarcomere lysis due to punctate alpha-actinin staining and difficult actin filament detection (fig. 6A). Mitochondrial health was next assessed because the HADHA Mut and KO CM were unable to process long-chain FA. Mitochondria were stained using ATP synthase β subunit to assess the presence of mitochondrial network. Both WT and HADHA Mut CM have many connected mitochondria, while KO CM has lost the mitochondrial network to smaller, more rounded mitochondria at 6D FA. To assess the functionality of these mitochondria, mitochondrial proton gradients were analyzed via mitochondrial tracing orange staining. After 12 days in Glc + FA rich medium, the HADHA MutCM had a highly reduced mitochondrial membrane proton gradient (fig. 6A and 6B).
To better assess the phenotype of sarcomere and mitochondrial disease, Transmission Electron Microscopy (TEM) was performed on WT and hadfamut CM12 days after Glc + FA exposure (fig. 6C). WT CM showed abundant myofibrils, clear Z-bands, but unclear a-and I-bands, with no M-lines, indicating that CM myofibril formation is in the middle normal stage. In addition, WT CM showed healthy mitochondria with good inner ridge formation. In contrast, the HADHA Mut CM showed poor myofibrils with Z-disc structural discontinuities replaced by punctate Z-bodies and myofilaments scattered in the cytoplasm. Interestingly, the hadaa Mut CM mitochondria were small and swollen with a very basic mitochondrial ridge morphology (fig. 6C). Quantification of WT and hadaa Mut CM mitochondria showed that hadaa Mut mitochondria were smaller in area and more rounded compared to WT mitochondria (fig. 6D and 6E). Finally, western blot analysis examining complex I-V proteins showed that complex I-IV protein expression of HADHA Mut CM was reduced under Glc + FA conditions (not shown). These data show that hadoa CM loses sarcomeric structure, mitochondrial membrane potential and morphology when exposed to FA.
SS-31 rescues abnormal proton leakage in HADHA Mut CM of long-term exposure to FA
To better understand the pathological state of the chronic FA-exposed hadaa Mut and KO CM, their mitochondria were functionally evaluated. Maximum OCR was significantly reduced for the Glc + FA treated HADHA Mut and KO CM compared to WT cells (Mut CM: 190pmoles/min/cell, KO CM: 125pmoles/min/cell, WT CM: 359pmoles/min/cell, P <0.05) (FIG. 6F). Furthermore, HADHA Mut CM showed reduced oxygen-dependent ATP production (Mut CM: 51pmoles/min/cell, KO CM: 43pmoles/min/cell, WT CM: 93pmoles/min/cell, P <0.05) (FIG. 6G) and HADHA Mut CM showed reduced glycolytic capacity (Mut CM: 14mpH/min/cell, KO CM: 18mpH/min/cell, WT CM: 23mpH/min/cell, Mut Vs WT P < 005) (not shown: observed via mitochondrial stress assay and calculated as the difference between extracellular acidification rates after oligomycin and 2-deoxy-D-glucose. mitochondrial membrane potential reduction and ATP production reduction due to FA exposure, it is speculated that this may be due in part to increased proton leakage. inhibition of ATP synthase (oligomycin treatment) and inhibition of the electronic chain (antimycin, rotenone), demonstrating that the proton leakage of HADHA Mut and KO CM is significantly higher than that of WT CM (Mut CM: 7.66pmoles)/min/cell, KO CM: 10.52pmoles/min/cell, WT CM: 3.64pmoles/min/cell, P < 0.05). Previous studies have shown that the mitochondrial targeting peptide elaiprentide (SS-31) can prevent mitochondrial depolarization and proton leakage. Interestingly, 1nM treatment of HADHA Mut cardiomyocytes with elaiprentide (SS-31) rescued the increase in proton leakage in the Glc + FA challenged Mut CM (FIG. 6H). These data suggest that HADHA Mut and KO CM exposure to FA causes a decrease in mitochondrial capacity due in part to an increase in proton leakage.
Loss of the function of HADHA leads to accumulation of long chain fatty acids
In the first step of fatty acid beta-oxidation, acyl-coa dehydrogenase generates a double bond between the alpha and beta carbons. Thus, after the first step, disruption of hadoa should lead to accumulation of FA intermediates (fig. 7A). To assess the disruption of long chain fatty acid oxidation in hadaa Mut and KO CM, non-targeted lipidomic analysis was performed to characterize overall lipidomic changes.
The long-chain acylcarnitines in HADHA Mut and KO CM were increased compared to WT CM, while the medium-chain acylcarnitine levels were not significantly changed (FIG. 7B; results for medium-chain acylcarnitine levels are not shown). These data indicate that mutations in hadas result in accumulation of long chain fatty acids in mitochondria in the absence of hadas. In the first step of long chain FAO, saturated fatty acids are processed into fatty acids with a single double bond, such as: 14:0 → 14:1,16:0 → 16:1 and 18:0 → 18:1, while the unsaturated fatty acid at the carboxy terminus undergoes the first step of FAO and acquires another double bond, for example: 18:1 → 18:2and 18:2 → 18: 3. Thus, it was found that saturated fatty acids in hadaa Mut and KO CM: 14: 0. 16:0 and 18: the level of 0 changes minimally (not shown), but 14: 1. 16: 1. 18:1 was greatly increased, along with 18 in the HADHA KO CM: 2and 18:3 increased slightly (FIGS. 7C-7E; results for 18:2and 18:3 in HADHA KO CM not shown). These data indicate that disruption of hadoa and KO lead to accumulation of specific long-chain FA intermediates. However, one of the striking phenotypes observed is mitochondrial rounding and collapse, rather than mitochondria that have broken due to potential fatty acid overload. Therefore, the next step is to examine another class of phospholipids that regulate mitochondrial structure, namely cardiolipin.
The HADHA and TAZ series of actions leads to a mature cardiolipin remodeling
Cardiolipin (CL) is the phospholipid necessary for optimal mitochondrial function and homeostasis because it maintains electron transport chain function along with other mitochondrial functions. CL is the major phospholipid of the inner mitochondrial membrane, synthesized in mitochondria, and dynamically remodelled during postpartum development and disease [ see, e.g., Kiebish, M.A., et al, molecular regulation of lipid flux by cardiac lipophilic synthase: setting the bed for bioenergetic efficiency.J Biol Chem,2012.287(30): p.25086-97; and He, Q. and X.Han, Cardiolipine modeling in diabatic heart. chem Phys Lipids,2014.179: p.75-81 ]. The most abundant class of CL in the human heart is tetraoleoyl-CL (tetra [ 18: 2] -CL). In cardiac diseases such as diabetes, ischemia/reperfusion and heart failure, or due to a specific mutation in the cardiolipin remodeling enzyme tazapine (TAZ; leading to Barth syndrome), tetra [ 18: 2-CL levels are abnormal. Specific cardiolipin maturation was observed in the early postnatal mouse heart. The inventors have used the maturation paradigm to reach a maturation step of iPSC-derived Cardiomyocytes (CMs) that allows the use of fatty acids as an energy source. Various maturation paradigms were identified that could mimic not only the steps of FAO, but also the process of Cardiolipin (CL) remodeling early after birth (fig. 8). Maturation in Wild Type (WT) CM showed tetra [ 18: a significant increase in 2-CL is similar to that previously observed during postnatal maturation of cardiomyocytes in vivo. These data indicate that CL maturation in cardiomyocytes can be induced in vitro. Furthermore, as shown by early postpartum in vivo development, WT CM is differentiated by differentiation into a protein with [ 14: 0],[14: 1],[16: 1] and [ 16: 0] reducing most CL and increasing CL with acyl chains having more than 18 carbon atoms includes intermediates [ 18: 1][18: 2][18: 2][20: 2] (fig. 8) to change its CL profile. Although this CL maturation has not yet reached the adult CL remodeling stage, the observed postpartum maturation can be used as a useful assay for interrogation with the aim of ultimately understanding and manipulating the first step of CL maturation in CM in the case of normal and pathological mutants 12-2 l. WT CM supplemented and without FA was analyzed using targeted lipidomics. FA-treated WT CMs result in tetra [ 18: 2] -CL was significantly increased (FIG. 7F), similar to previous findings observed during postnatal maturation of cardiomyocytes in vivo [ see Kiebish, M.A. et al, J Biol Chem,2012.287(30): p.25086-97; and He, Q, and X.Han, Chem Physlipids,2014.179: p.75-81, supra. These data indicate that maturation of CL in cardiomyocytes can be induced in vitro. However, ha KO CM after FA treatment did not increase tetra [ 18: 2-CL. Furthermore, as shown by postpartum in vivo development, WT CM shifted its CL profile to a more mature CL profile, showing [ 16: 1], while CL of greater than 18 carbons increased, including the intermediate [ 18: 1][18: 2][18: 2][20: 2] [ see, Kiebish, MA et al, J Biol Chem,2012.287(30): p.25086-97 ]. However, the HADHA KO CM was not able to reshape its CL profile as efficiently as the WT CM (fig. 7G). These data surprisingly show that besides its role in long-chain FAO, HADHA is also essential for the cardiomyocyte CL remodeling process.
Since the hados KO CM appeared to be deficient in CL remodeling, the cardiolipin species were subsequently analyzed in more detail in WT, hados Mut and KO CM using complete lipidomics. By boosting our targeted lipidomics results, we found that FA challenged hadaa Mut and KO CM showed increased abundance of the lighter chain CL and depletion of the heavier chain CL (fig. 7H). Three CL species, tetra [ 18: 1],[18: 1][18: 1][18: 1][18: 2] and [ 18: 1][18: 1][18: 2][18: 2] significantly enriched in HADHA Mut and KO CM (FIG. 7H). Interestingly, the [ 18: 1] [ l 8: 1] [ l 8: 2] [ l 8: 2] CL is particularly depleted in Barth syndrome patients with TAZ mutations.
It has previously been demonstrated that the HADHA protein has an enzymatic function similar to that of monosaccharide-based cardiolipin acyltransferase (MLCL AT). MLCL AT primarily transfers unsaturated fatty acyl chains to lysocl. Therefore, it seems reasonable that hados reconstitutes cardiolipin to produce mature tetra [ 18: 2-CL has a direct effect in the class. Both TAZ and hadoa should equally consume MLCL pools if they act in parallel to produce a reconstructed CL. When TAZ was knocked out, MLCL increased dramatically, indicating that TAZ directly generated mature CL using MLCL. However, it was observed here that there was no change in the MLCL pool when the haddha was knocked out (not shown). This suggests that hadaa does not remodel MLCL but rather CL. If TAZ and HADHA act in parallel, each KO should not result in an inverse cumulative relationship with a particular CL intermediate. For example, TAZ KO results in [ 18: 1][18: 1][18: 2][18: 2] CL decreases. However, in the current HADHA KO, the same kind of accumulation is observed. Thus, it is speculated that TAZ first remodels MLCL into an intermediate of CL, such as [ 18: 1][18: 1][18: 2][18: 2], then HADHA proceeds to remodel the CL species to tetra [ 18: 2-CL.
Loss of the function of the HADHA does not increase the function of ALCAT1
To better understand how the cardiolipin profile changes due to lack of HADHA, it was investigated which new CL species were enriched in HADHA Mut and KO CM. CL species with fatty acid acyl chains of saturated fatty acids (such as 14:0 and 16: 0) were enriched in HADHA Mut and KO CM (fig. 7I, J). No identification was made of a peptide having 18:0 acyl chain. Typically, nascent CL (CL) with multiple saturated fatty acid acyl chains has been synthesized from cardiolipin synthase (CLS)Sat) (see, e.g., FIG. 7K). At CLsatThe saturated fatty acid acyl chain is replaced by an unsaturated fatty acid acyl chain during remodeling. These data suggest that nascent CL is present in the HADHA mutantsatAccumulation of (2).
Next, we examined ALCAT1 as a method for hadaa Mut and KO CM for CL remodeling. Since ALCAT1 has no preference for fatty acyl substrates, it should take advantage of any fatty acyl CoA substrate present. An indication of ALCAT1 activity is an increase in polyunsaturated fatty acid acyl chains incorporated with CL. However, when CL species having acyl chains of fatty acids with carbon lengths of 20 or more were examined, most of the hadna Mut and KO CMs actually have fewer species than WT CMs (fig. 7H). In addition, there is no increase in CL species having a plurality of acyl chains of fatty acids having a carbon length of 20 or more in any group. Thus, these data suggest that ALCAT1 does not participate in the hadoa Mut and KO CM to compensate for the loss of hadoa.
Discussion of the related Art
This study involved the development of the first human MTP-deficient heart model in vitro using the mipac mature hiPSC-CM and led to the discovery that TFP α/HADHA deficiency in long-chain FAO and CL remodeling could cause unstable pulsatile disease similar to what suggests arrhythmic states. In addition, a mechanism of action was demonstrated; due to the acyl-coa transferase activity of hadoa, the mutation results in an abnormal component of the prominent phospholipid cardiolipin. Abnormalities in CL composition lead to defects in the mitochondrial inner cristae and a greatly reduced mitochondrial proton gradient. These mitochondrial defects are manifested by sarcolysis, calcium handling and electrophysiological defects. Calcium storage and repolarization delays cause an uneven beating pattern of cardiomyocytes, which in turn can exacerbate tissue-level arrhythmias seen in MTP-deficient SIDS newborns.
The use of pluripotent stem cell-derived cardiomyocytes for the study of MTP deficiency requires the generation of a tool that rapidly and efficiently matures cardiomyocytes in vitro to a stage exhibiting postnatal cardiac FAO disease. A number of tools have been developed to mature hPSC-CM, including: electrical and/or mechanical stimulation, cell microenvironment and culture time. However, none of these methods directly affects the maturation aspects that allow analysis of FA metabolism. Based on the preliminary work of the inventors investigating the role of Let-7 in hPSC-CM maturation, a microRNA maturation cocktail (MiMaC) was developed that was able to mature hPSC-CM size, contractility and metabolism. MiMaC facilitates the study of MTP deficiency in hPSC-CMs and is a useful tool for the maturation of hPSC-CM for the study of FAO disorders. In addition, the MiMaC system is used to better understand the late development, maturation process. Importantly, the common microRNA target HOPX was discovered as a new key regulator of cardiomyocyte maturation.
Previous studies have shown that metabolic gene expression increases as myocardial cells are metabolically remodeled from fetal development to adult stages. An increase in OXPHOS gene expression may indicate an increase in mitochondrial copy number or biosynthesis by more mature mitochondria, or both. These scRNA-seq studies found a novel, intermediate subset of cardiomyocytes with hypermetabolic gene expression for the OXHPA and Myc targets. These data suggest a possible intermediate stage from fetal-like CM to more mature CM, which requires transient upregulation of the OXPHOS gene. Since Parkin (Parkin) is also up-regulated at this stage, these data support the following assumptions: quality control of mitochondria during fetal phase mitochondrial autophagy and biosynthesis of mature mitochondria occurred in mmac-induced myocardial maturation. This is similar to the previously shown mitophagy-mediated response via parkin during perinatal mouse cardiac development. Importantly, an intermediate stage of maturation was also observed in MTP/HADHA mutant cardiomyocytes prior to the development of the pathological state. Further dissection of this stage will allow a positive understanding of the regulation of the process in both normal and diseased states.
We used pluripotent stem cell-derived cardiomyocytes to search for the cause of arrhythmia observed in patients with a mutation in hadaa (causing MTP deficiency). Importantly, the hiPSC-derived hadaa mutant cardiomyocytes recapitulate the arrhythmic phenotype observed in patients, underscoring the utility of hiPSC-CM in modeling human disease. To better understand the cause of arrhythmia, fatty acid challenged hada cardiomyocytes were used to assess phenotype and identify potentially clue cardiolipin for disease progression. One novel therapeutic intervention that rescues a portion of the HADHA mutant phenotype is SS-31. SS-31 is a mitochondrial targeting peptide that has been shown to bind to cardiolipin and prevent conformational changes in cardiolipin under stress (e.g., peroxidation) [ Birk, A.V., et al, The mitochondled compound SS-31 re-energys immunochemistry. J. Am Soc Nephrol,2013.24(8): p.1250-61 ]. SS-31 has been shown to inhibit mitochondrial depolarization and swelling of cardiomyocytes and islets and rescue cardiolipin defects in cardiomyocytes. Since SS-31 was found in this study to rescue one aspect of mitochondrial pathology, i.e. increased proton leakage, and since abnormal CL species were observed in HADHA Mut CM, it was suggested that cardiolipin defects would lead to the observed mitochondrial dysfunction.
Cardiolipin is an important component of the inner mitochondrial membrane. CL is an atypical phospholipid consisting of four (rather than two) acyl chains linked to a glycerol moiety. This atypical structure of cardiolipin results in a cone shape, which is believed to be critical to the structure and function of the internal mitochondrial membrane. In particular, cardiolipin has been shown to play a role in the Electron Transport Chain (ETC) high-order structure of tissues important for ETC activity and to act as a proton trap on the outer leaflet of the mitochondrial inner membrane. Thus, a decrease in the mature form of CL leads to mitochondrial abnormalities such as a loss of proton gradient, a decrease in ETC, leading to decreased ATP production and abnormalities in mitochondrial structure.
Pathological remodeling of CL involves mitochondrial dysfunction observed in diabetes, heart failure, neurodegeneration, and aging. However, in the case of hadaa Mut and KO CM, the pattern and composition of aberrant CL species is more specific than previously seen in heart failure or diabetes, suggesting that hadaa may be directly involved in CL processing. Interestingly, previous studies using HeLa cells suggested that hadoa exhibited acyl-coa transferase activity on MLCL as it remodels into cardiolipin. Thus, these data suggest that defects in hadoa directly result in impaired cardiolipin remodeling leading to failure to produce and possibly maintain the acyl chain composition of mature cardiolipin. However, the exact contribution of this acyltransferase to physiological CL remodeling is not known. It is now reported that in the FA challenged human HADHA Mut and KO cardiomyocytes, mature tetra [ 18: 2-CL decreases and mitochondrial activity is impaired. This is similar to the previous findings seen in TAZ mutants leading to Barth syndrome, an X-linked cardiac and skeletal muscle mitochondrial myopathy. These data establish for the first time the exact contribution of hadoa acyltransferase to physiological CL remodeling in human cardiomyocytes.
TAZ is a transacylase that is essential for remodeling MLCL to mature cardiolipin. Both HADHA and TAZ play a key role in the production of mature cardiolipin and both diseases have similar pathological phenotypes, including unexplained sudden death due to ventricular arrhythmia. Cardiolipin species with reduced specific abundance in TAZ mutants [ 18: 1][18: 1][18: 2][18: 2] show increased abundance in the HAHDA Mut and KO CM. Furthermore, no accumulation of MLCL was observed in the HADHA Mut and KO CM, which usually occurs when there is a mutation in the TAZ. These data indicate that CL remodeling is first treated by TAZ and then by HADHA to generate tetra [ 18: 2-CL (FIG. 7K).
Mutations in HADHA apparently result in CM being unable to produce large amounts of tetra [ 18: 2-CL. It is also clear from the literature that once tetra [ 18: the 2-CL species begin to deplete and CM can be trapped in the pathological state of mitochondrial disorders. Interesting about this finding is that HAADHA Mut and KO CM do not suffer from FA challenges even though they have less tetra [ 18: 2-CL, nor do they enter the disease state. It was also demonstrated here that the addition of FA to the hadaa Mut and KO CM did result in long-chain FA accumulation. However, this accumulation of FA did not result in mitochondrial swelling and eventual rupture. Instead, it was found that mitochondrial collapse in the HADHA mutant had rounded. These data indicate that the FAO phenotype alone may not account for the defects observed in hadaa Mut and KOCM, and that CL remodeling is particularly important during CM maturation.
When all CL species were examined in CM, it was evident that the side chain was correctly remodeled to [ 18: 2], HADHA Mut and KO CM failed to acquire a mature CL profile. Furthermore, long carbon groups of 20 carbons or greater were not forcibly incorporated multiple times in CL, indicating that ALCAT1 did not compensate. It is evident that there is a saturated FA side chain 14 in CL: 0 and 16: 0. it is likely that accumulation of CL species in the HADHA Mut and KO with saturated side chains leads to a breakdown of the mitochondrial structure and subsequent heart pathology. Thus, these data suggest that mutations in the hadoa enzyme during CM maturation lead to an excessive accumulation of immature CL-saturated species, which may be responsible for the mitochondrial defects and pathology observed in hadoa CM (fig. 7K).
It is demonstrated here that long chain fatty acids, the normal substrates used to produce energy and phospholipids in postpartum and adult CM, contribute to the pathology of MTP deficiency in CM, leading to abnormal cardiolipin patterns, resulting in severe mitochondrial defects and calcium abnormalities, inducing CM instability in HADHA Mut CM. Identification of SS-31 as a new therapy can rescue the FA challenged proton leak phenotype of hadaa Mut CM. This suggests that SS-31 or other cardiolipin affecting compounds can be used as potential therapies for alleviating aspects of mitochondrial dysfunction in MTP deficiency.
Examples
The following methods are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the disclosed invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.
Method of producing a composite material
hESC and hipSC with cardiac differentiation
The hESC line RUES2(NIHhESC-09-0013) and the hipSC line WTC #11, previously derived in the Conklin laboratory [ Kreitzer, F.R., et al, A robust method to derivative functional neural cell from human pluripotent Cells. am J Stem Cells,2013.2(2): p.119-31], were cultured on Matrigel growth factor-reduced basement membrane matrix (Corning) in mTeSR medium (Stemell Technologies). Monolayer-based directed differentiation protocols were followed to generate hESC-CM and hipSC-CM as previously performed [ Palpant, N.J., et al, Generation high-purity card and endothial derivatives from patterned media using human pluripotent cells. Nat Protoc,2017.12(1): pages 15-31 ]. The hiPSC-CM cardiolipin assay is performed using a directed differentiation protocol based on small molecule monolayers, as previously done [ [ palpot, n.j. et al, Generation high-purity card and endothial derivatives from patterned media using human pluripotent cells. natProtoc,2017.12(1): p.15-31 ]. 15 days after differentiation, hPSC-CM was enriched for cardiomyocyte populations using the lactate selection process [ Palpant, N.J., et al, Generation high-purity cardiac and endothiel derivatives from patterned media using human pluripotent cells. Nat. Protoc,2017.12(1): p.15-31 ]. The resulting cardiomyocyte population was 40-60%, then enriched to 75-80% cardiomyocytes after 4 days of lactic acid enrichment.
Establishment of HADHA line
Two different
Table 1: gRNA of LentiCRISPRV2 (SEQ ID NOS in parentheses)
CRISPR off-targets
Potential off-targets of HADHA gRNA were identified using the Cas-OFFinder tool from Crispr-RGEN [ Bae, S., J.park and J.S.Kim, Cas-OFFinder: a fast and versatic oligonucleotide primers for potential off-target sites of Cas9 RNA-defined end effectors, 2014.30(10): p.1473-5 ]. The highest predicted off-target was then amplified by GoTaq PCR and sequenced. Off-target assay primers can be found in table 2.
Table 2: primers for off-target analysis (SEQ ID NOS in parentheses)
RNA extraction and qPCR analysis
RNA was extracted from cells using Trizol and analyzed by SYBR green qPCR using a 7300 real-time PCR system (Applied Biosystems). The primers used are listed in table 3. The delta-delta Ct method was used to calculate linear expression values for all qPCR experiments.
Table 3: quantitative RT-qPCR primers for human genes (SEQ ID NOS in parentheses)
Protein extraction and western blot analysis
Cells were lysed directly on the plate with lysis buffer containing 20mM Tris-HCl pH 7.5, 150mM NaCl, 15% glycerol, 1% Triton X-100, 1M β -glycerophosphate, 0.5M NaF, 0.1M sodium pyrophosphate, orthovanadate, PMSF and 2% SDS [ Moody, J.D., et al, First clinical prescription H3K27me3 marks in organizing cells identified using labeled protein inhibitor. Proc Natl Acad Sci U SA,2017 ]. Prior to use, 25U Benzonase nuclease (EMD Chemicals, Gibbstown, NJ) was added to the lysis buffer. Protein quantification was performed by Bradford assay (Bio-Rad) using BSA (bovine serum albumin) as standard and EnWallac Vision. Protein samples were combined with 4x Laemmli sample buffer, heated (95 ℃, 5 min), and run on SDS-PAGE (
MicroRNA overexpression and knockout
The LentiCrisprV2 plasmid (Addgene 52961) was used to knock-out (KO) microRNA-141, -200a, -205 and-122. The grnas for each miR were selected and tested for their pre-spacer adjacent motif (PAM) NGG cleavage sites adjacent to or in the seed region of the mature microRNA, respectively. grnas can be found in table 1. The overall reduction of each miR was assessed by TaqMan RT-qPCR with probes specific for the mature form of each representative miR.
The pLKO.1TRC vector (pLKO.1-TRC cloning vector (Addge plasmid #10878) was used to Overexpress (OE) microRNAs [ Moffat, J. et al, Alentiviral RNAi library for human and mouse genetic applied to an arrayed viral high-content search. cell,2006.124(6): p.1283-98 ]. genomic sequences 200bp upstream and downstream of the mature microRNAs were amplified and purified.primers for each microRNA are listed in Table 4.
Table 4: primers for genomic amplification of microRNA regions (SEQ ID NOS in brackets)
Virus production
HEK 293FT cells were plated the day before transfection. On the day of transfection, the selected OE or KO plasmid was combined with the packaging vectors psPAX2(psPAX2 as gift from the dier troaddge, plasmid #12260) and pmd2.g (pmd2.g as gift from the dier troaddge, plasmid #12259) in the presence of 1 μ g/μ L Polyethyleneimine (PEI) per 1 μ g of dna. After 24 hours the medium was changed and lentiviruses were harvested 48 and 72 hours after transfection. The viral particles were concentrated using PEG-it (System Biosciences, Inc).
hiPSC-CM transduction and selection
On day 14 post-induction, hiPSC-CM was transduced in the presence of hexadimethrine bromide (mebendammonium bromide, 6 μ g/ml). Lentivirus was applied for 17-24 hours and then removed. Cells were cultured for two more weeks. An enriched population of cardiomyocytes was obtained using lactic acid selection [ Tohyama, S. et al, Distingt metabolic flow enabled large-scale purification of mice and human pluratent step Cell-derived myocardial cells. Cell step Cell,2013.12(1): p.127-37 ]. Puromycin selection was used to select cells that had been positively incorporated into the vector. After two weeks of culture, cells were harvested for endpoint analysis. For the MiMaC group, hiPSC-CM was simultaneously transduced with lower doses of four different lentiviruses, while controls were with two control vectors: pLKO.1 and LentiCRISPRV2 empty vectors.
Immunocytochemistry and morphological analysis
Cells were fixed in 4% (vol/vol) paraformaldehyde, blocked with 5% (vol/vol) Normal Goat Serum (NGS) for 1 hour and incubated overnight with primary antibody in 1% NGS, followed by secondary antibody staining in NGS. The CM area was measured using Image J software. Quantification of mitochondrial tracer intensity was performed using Image J software and following previously published methods for co-localization quantification [ Li, Q, et al, A
Microelectrode array
Electrophysiological recordings of spontaneously beating cardiomyocytes were collected for 2 minutes using AxIS software (AxIS Biosystems). After raw data was collected, the signal was filtered using a Butterworth bandpass filter and a 90 μ V spike detection threshold. A polynomial fit T-wave detection algorithm is used to automatically determine the field potential duration.
Microcolumn (contraction force and pulsation rate)
Polydimethylsiloxane (PDMS) micropillar arrays [ Beussman, K.M. et al, Micropost arrays for measuring cell-derived cardiac cytometric interactions. methods,2016.94: p.43-50. RTM.]. The tips of the microcolumns were coated with mouse laminin (Life Technologies) and incubated in RPMI medium containing B27 supplement and 10% fetal bovine serum at about 75,000 per cm2Inoculating cells to
Hippocampus assay
Mitochondrial function was assessed using a Seahorse XF96 extracellular flux analyzer [ Kuppusamy, K.T., et al, Let-7family of microRNA is required for formation and adut-lipolytics in step cell-derived cardiac cells, Proc Natl Acad Sci U.S.A.,2015], as previously described. Plates were pretreated with Matrigel reduced growth factor (Corning) diluted 1: 60. Approximately 28 days after differentiation, cardiomyocytes were seeded onto the plates at a density of 50,000 cells per XF96 well. Hippocampal analysis was performed 3 days after plating onto XF96 well plates. One hour prior to assay, the medium was changed to basal medium (unbuffered DMEM; Seahorse XF assay medium) supplemented with sodium pyruvate (Gibco/Invitrogen, 1mM) and 25mM glucose (for the MitoStress assay), 25mM glucose and 0.5mM carnitine (for the palmitate assay). Injections of substrate and inhibitor were used to achieve a final concentration of 1 μ M of 4- (trifluoromethoxy) phenylhydrazone (FCCP; Seahorse Biosciences), oligomycin (2.5 μ M), antimycin (2.5 μ M) and rotenone (2.5 μ M) for MitoStress analysis during the measurement; 200mM palmitate or 33. mu.M BSA, and 50. mu.M Etomoxide (ETO) were used for palmitate determination. The OCR values were further normalized to the number of cells present in each well and quantified by Hoechst staining (Hoechst 33342; Sigma-Aldrich) using fluorescence measurements at 355nm excitation and 460nm emission. Maximum OCR is defined as the change in OCR in response to FCCP compared to OCR after oligomycin addition. ATP production was calculated as the difference between basal respiration and post-oligomycin respiration. Proton leak was calculated as the difference between respiration after oligomycin and after antimycin and rotenone. The ability of the cells to utilize palmitate as an energy source was calculated as the difference between the average OCR after the second palmitate addition and the final respiration value before the second palmitate addition. Unless otherwise stated, these reagents were from Sigma.
RNA sequencing
Day 30hiPSC-CM was harvested for RNA preparation and whole genome RNA-seq (reads over 2000 ten thousand). RNA-seq samples were aligned to hg19[ Trapnell, C., L.Pathter, and S.L.Salzberg, TopHat: converting spot junctions with RNA-seq.Bioinformatics,2009.25(9): p.1105-11] using Tophat alignment version 2.0.13. Ensembl GRCh37 gene annotation was used to quantify gene level read counts using HTSeq-counts [ Anders, S., P.T.Pyl and W.Huber, HTseq- -a Python frame to work with high-through put sequence data. Bioinformatics,2015.31(2): p.166-9 ]. Differential analysis was performed using DESeq [ Anders, S. and W.Huber, Differential expression analysis for sequence count data. genome biol,2010.11(10): p.R106] to retain genes that collectively expressed a normalized read count of greater than 1 in the RNA-seq samples in each binary comparison. The Princomp function from R was used for principal component analysis. The TopGO R software packages [ Alexa, A., J.Rahnenfuhr, and T.Lengauer, Improved sequencing of functional groups from gene expression data by biochemical engineering GO graphics, 2006.22(13): p.1600-7] were used for gene ontology enrichment analysis. To assess the effect of miR perturbation on the cardiac maturation pathway, each case was compared to its Empty Vector (EV) and up-regulated genes (>1.5 fold change) and down-regulated genes (< 1.5 fold change) were identified. The up-and down-regulated genes were separately tested in high geometry to enrich for a panel of selected pathways beneficial for heart maturation, resulting in an m by n matrix, where m is the pathway number (m-7) and n is the condition number (n-6, including EV). The negative log10 of the ratio of the enriched p-values of the up-and down-regulated genes was calculated to represent the overall net "benefit" of treatment: a positive value (>0) means that the treatment results in more upregulation of genes in the cardiac maturation pathway than down-regulation of these genes, and a more negative value means that the treatment results in more down-regulation of genes in the cardiac maturation pathway.
Single cell RNA sequencing
Raw single cell RNA-seq data were processed through the CellRanger pipeline of 10 Xgenomics. The output of the CellRanger pipeline was further analyzed using the Seurat R software package [ Satija, R. et al, spatial correlation of single-cell gene expression data. Nat Biotechnol,2015.33(5): p.495-502 ]. Cells with more than 40% of reads mapped to mitochondrial genes, less than 200 genes detected or less than 2000 Unique Molecular Identifiers (UMIs) were removed. The remaining cells were scaled by the number of UMIs and the percentage mapped to mitochondrial genes. The parameters for tSNE analysis of mature single cell RNA-seq data were the first 2905 variable genes, the first 10 major components, and a resolution of 0.5. The parameters for tSNE analysis of single cell RNA-seq data for HADHA mutants were the first 3375 variable genes, the first 10 major components and a resolution of 0.4. Cell cycle effects on clustering were assessed using cell cycle genes from Kowalczyk et al [ Kowalczyk, M.S. et al, Single-cell RNA-seq derived samples in cell cycles and differential considerations of nucleic acids cells genome Res,2015.25(12): p.1860-72] and cell cycle score functions in the Seurat software package. Genes detected in at least 25% of the cells in any cluster and with a false discovery rate <0.1 were defined as differentially expressed. The expression values for each gene in all cells plotted in the heatmap were normalized (i.e., Z score). The in vivo maturation markers in humans are based on genes that are up-regulated in the adult heart compared to the fetal heart in the "Roadmap Epigenomics program" [ Roadmap Epigenomics, c. et al, integrated analysis of 111reference humanepidemics. nature,2015.518(7539): p.317-30 ]. The in vivo maturation markers in mice are based on genes upregulated by Single Cell RNA-seq data from cardiomyocytes in vivo from Delaughter et al [ Delaughter, D.M., et al, Single-Cell resolution of Temporal Gene Expression vector development. Dev. Cell,2016.39(4): p.480-490 ]. Genes significantly higher than fetal in adult hearts (2-fold higher in adults, FDR <0.05) were selected using DESeq. We then intersected these genes with the first 30 most highly expressed genes in each scra-seq cluster to obtain the final gene list for the heatmap in fig. 3O. Enrichment of gene entities was performed using the TopGO software package [ Alexa, A., J.Rahnenfuhr, and T.Lengauer, Improved sequencing of functional groups from genetic expression data by systematic transformation GO graphics structure. Bioinformatics,2006.22(13): p.1600-7 ].
Calcium transient analysis method
Cardiomyocytes were plated on Matrigel-coated round glass coverslips. Cardiomyocytes were incubated with 1mM Fluo-4AM (Life Technologies, F14201) in Tyrode buffer (1.8mM CaCl)2，1mM MgCl2、5.4mM KCl，140mM NaCl，0.33mM NaH2P0410mM HEPES, 5mM glucose, pH 7.4) at 37 ℃ for 25 minutes. The substrate was then transferred to a60 mm Petri dish (Petri dish) containing fresh pre-warmed Tyrode buffer for imaging. The samples were imaged using a Hamamatsu ORCA-flash2.8 Scientific CMOS camera mounted on a Nikon Eclipse Ti vertical microscope. Video was captured using a 40-fold water immersion objective at a frame rate of at least 20 frames per second. The fluorescence intensity was adjusted to ensure that the fluorescence change was captured sufficiently during depolarization without whitening, and the same fluorescence intensity was used for all experiments. Cardiomyocytes were stimulated bi-phasic with carbon electrodes (Ladd Research, 30250) at a frequency of 0.5Hz or 1Hz at 5V/cm, and at least 5 beats were captured in each video for analysis.
The video is analyzed by using a self-defined MATLAB code; calcium transients were acquired, cell boundaries were found and fluorescence within each video frame boundary was averaged. Background fluorescence was automatically determined for each video frame and subtracted from the calcium transient. Calcium transients were then analyzed to find peak fluorescence (F), baseline fluorescence (F)0) Time to peak (T)peak) And time to 50% and 90% relaxation (T)50R，T90R). The rates of peak, 50% and 90% relaxation (R) were calculated by dividing the respective fluorescence changes by the respective timespeak，R50R，R90R). Exponentially decaying function (e)-t/τ) Fit to the slack between 10% and 90% slack to determine the slack coefficient τ. All these measurements were taken at least 4 beats per video and averaged for comparison.
Transmission Electron Microscope (TEM)
Cells were immobilized in 4% glutaraldehyde in sodium cacodylate buffer, columns were immobilized in osmium tetroxide, bulk stained in 1% uranyl acetate, dehydrated through a series of ethanol, and embedded in Epon Araldite. 70nm sections were cut on a LeicaEM ETC7 microtome and viewed on a JEOL 1230 TEM.
Glucose and fatty acid medium
The minimal medium (we call glucose medium) was RPMI supplemented with B27 and insulin. The fatty acid medium was a glucose medium with oleic acid conjugated to BSA (Sigma O3008): 12.7 μ g/mL, linoleic acid conjugated to BSA (Sigma L9530): 7.05 μ g/mL, sodium palmitate conjugated to BSA (Sigma P9767) (Sigma a 8806): 52.5 μ M, L-carnitine (carnitine): 125 μ M.
Elamipretide(SS-31)
SS-31 was obtained from Stealth Biotherapeutics, dissolved in PBS. The final concentration used in the experiment was 1 nM.
Box diagram
The "x" in each box plot represents the mean, the horizontal bar represents the median, and outliers are not shown. Denotes P < 0.05.
Bar graph
Bar graphs show mean ± SEM. Bar graphs not showing SEM were generated from RNA sequencing data from sequencing one or two samples.
STRING analysis
Protein correlation plots were generated using STRING version 10.5. In each figure, genes linked to each other are associated with each other. There are three effects: arrow- > positive, - - | -negative, line with a circle at the end-unspecified. Line colors also represent eight different action types: green-activation, blue-binding, cyan-phenotype, black-response, red-inhibition, violet-catalysis, pink-post-translational modification and yellow-transcriptional regulation. Kmeans clustering was used to identify significantly altered genes due to MiMaC, with respect to: muscle structure development and extracellular matrix organization. Markov clustering algorithm (MCL) was used to identify genes that mmac has been down-regulated to control cell division.
Statistical analysis
Statistical analysis was performed with experiments where N was equal to or greater than 3. P values were calculated using student's t-test or one-way analysis of variance. For student's t-test, the Shapiro-Wilk normality test was performed. For one-way anova, a Kolmogorov-Smirnov normality test was performed. For multiple comparisons, the Holm-Sidak method was used. For one-way anova that did not pass the normality test, Kruskal-Wallis one-way anova of rank difference was performed. For multiple comparisons, the Dunn method was used. All statistical tests used α ═ 0.05.
Targeted cardiolipin analysis using LC-MS/MS
12D Wild Type (WT) hipSC-CM was treated with Glc + FA medium and 6D and 12D HADHA Mut hipSC-CM was treated with Glc + FA medium. Just prior to extraction, each cell pellet was dissolved in 40 μ L DMSO and cell membranes were disrupted by sonication. Cells were sonicated using 3 cycles consisting of 20 seconds on, 10 seconds off. Care was taken to keep the cells on ice during sonication. After shaking, the suspension was transferred to a 2mL glass LC vial.
For cardiolipin extraction, an extraction mixture consisting of 20mL of chloroform/methanol mixture (2: 1v/v) and 30 μ L of internal standard solution (5mg PC (18: 0/18: 1(9Z)) (Avanti Polar Lipids, Inc., Alabaster, AL) was prepared next, 600 μ L of the extraction mixture was added to the sample, then vortexed and incubated at-20 ℃ for 20 minutes, then the sample was sonicated in an ice bath for 15 minutes pure water (100 μ L) was added, and the sample was shaken at room temperature for 30 minutes after centrifugation at 12,000Xg for 10 minutes at 4 ℃, the bottom phase was transferred to a new glass LC vial and dried under vacuum, then 150 μ L of acetonitrile/isopropanol/H was added2The residue was reconstituted with O (65: 30: 5, v/v/v) and centrifuged at 20,000g for 10 min at 4 ℃. The supernatant was transferred to individual glass vials for MS analysis. All samples were n-3.
For targeted cardiolipin measurements, 2 μ L of each prepared sample was injected into a 6410Agilenttriple Quad LC-MS/MS system to use electrospray ionizationThe source and negative ionization modes were analyzed. Chromatographic separation was done on an Agilent300SB-C8 RRHD chromatography column (1.8 μm, 2.1X50 mm). The mobile phase A is in acetonitrile/H210mM ammonium acetate in O (6: 4, v/v), mobile phase B in isopropanol/acetonitrile/H210mM ammonium acetate in O (90: 10: 1, v/v/v). During the 12 minute separation, the composition of the mobile phase changed from 60% a to 1% a, then increased rapidly to 60% a and equilibrated to prepare for the next injection. The total experimental time for each injection was 20 minutes. The flow rate was 0.26mL/min, the autosampler temperature was 4 ℃ and the column oven temperature was set to 55 ℃. Targeted MS/MS data was acquired using a Multiple Reaction Monitoring (MRM) mode. The MassHunter workstation software of QQQ b.07.00(Agilent) quantitatively analyzed the MRM peaks for integral extraction.
Non-targeted lipidomic analysis
One million cells were extracted with 225. mu.l methanol at-20 ℃ and contained RE (17: 0/17: 0), PG (17: 0/17: 0), PC (17: 0/0: 0), C17 sphingosine, ceramide (d 18: 1/17: 0), SM (d 18: 0/17: 0), palmitic acid-d3PC (12: 0/13: 0), cholesterol-d7，TG(17：0/17：1/17：0)-d5DG (12: 0/12: 0/0: 0), DG (18: 1/2: 0/0: 0), MG (17: 0/0: 0/0: 0), PE (17: 1/0: 0), LPC (17: 0), LPE (17: 1) and a mixture of cholesterol esters 22: 750 μ L of MTBE (methyl tert-butyl ether) (Sigma Aldrich) from 1. Cells were vortexed for 20 seconds, sonicated for 5 minutes, and shaken with an orbital mixing cooling/heating plate (Torrey pipes Scientific Instruments) for 6 minutes at 4 ℃. Then 188. mu.l of LC-MS grade water (Fisher) was added. Samples were vortexed and centrifuged at 14,000rcf (Eppendorf 5415D). The upper layer (non-polar organic layer) was collected in two 350 μ L aliquots and evaporated to dryness. An aliquot of the organic phase was resuspended in a solution containing 50ng/mL of CUDA ((12- [ [ ((cyclohexylamino) carbonyl)]Amino group]100 μ L methanol of dodecanoic acid) (Cayman Chemical): toluene (9: 1, v/v) mixture. The samples were then vortexed, sonicated for 5 minutes, and centrifuged at 16,000rcf in preparation for lipidomic analysis. Method blanks and pooled human plasma (bioremodelionivt) were included as quality control samples. WT FA CM,
Chromatographic and mass spectrometric conditions for lipidomic RPLC-QTOF analysis
Resuspended samples were injected at 3. mu.L and 5. mu.L for ESI positive and negative modes, respectively, onto a Waters AcquisyUPLC CSH C18(100mm length x 2.1mm inner diameter; particle size 1.7 μm) and a Waters AcquisyVanGuard CSH C18 pre-column (inner diameter 5mm x 2.1 mm; particle size 1.7 μm) maintained at 65 ℃ was coupled to a Vanqish UHPLC system. To improve lipid coverage, different mobile phase modifiers were used for positive and negative pattern analysis [ Cajka, T. and O.Fiehn, incorporated lipid coverage by selective optical-phase modulators in LC-MS of blood plasma, Meta-molomics, 2016.12(2): p.34]. For the positive mode, 10mM ammonium formate and 0.1% formic acid were used, and for the negative mode, 10mM ammonium acetate (Sigma-Aldrich) was used. Both positive and negative modes use the same mobile phase composition: (A)60:40v/v acetonitrile: water (LC-MS grade) and (B)90:10v/v isopropanol: and (3) acetonitrile. The gradient started at 15% (B) in 0 min, 30% (B) in 0-2 min, 48% (B) in 2-2.5 min, 82% (B) in 2.5-11 min, 99% (B) in 11-11.5 min, 99% (B) in 11.5-12 min, 15% (B) in 12-12.1 min and 15% (B) in 12.1-15 min. A flow rate of 0.6mL/min was used. For data acquisition, a Q-exact HFhybrid Quadrupole-Orbitrap mass spectrometer was used with the following parameters: mass range m/z 100-1200; MS (Mass Spectrometry)1Resolution was 60,000: data dependent MS2Resolution was 15,000;
LC-MS data processing Using MS-DIAL and statistics
Non-targeted lipidomic data processing for deconvolution, peak picking, alignment and identification was performed using MS-DIAL [ Tsugawa, H. et al, MS-DIAL: data-independent MS/MS controlled volume for comprehensive metabolism Methods,2015.12(6): p.523-6 ]. In addition to MS/MS spectral databases in msp format, internal m/z and retention time libraries [ Kind, T. et al, Lipidplast in silica tandem mass spectrometry data base for labeled identification. Nat Methods,2013.10(8): p.755-8] were used. The signature is reported when at least 50% of the samples in each group are present. The statistical analysis is first performed by normalizing the data or mTIC using known sums to scale each sample. The normalized peak heights were then submitted to R for statistical analysis. Analysis of variance was performed by FDR correction and post hoc detection.
Exemplary embodiments
For purposes of illustration only, a non-limiting list of exemplary embodiments encompassed by the present disclosure includes:
A1. a method of inducing cardiomyocyte maturation comprising inducing two or more of the following in immature cardiomyocytes: overexpression of Let7i microRNA (miRNA), overexpression of miR-452, decreased expression of miR-122 and decreased expression of miR-200 a.
A2. The method of embodiment a1, comprising inducing in immature cardiomyocytes three or more of: overexpression of Let7i miRNA, overexpression of miR-452, decreased expression of miR-1222 and decreased expression of miR-200 a.
A3. The method of embodiment a1 or a2, comprising inducing overexpression of Let7i miRNA, overexpression of miR-452, decreased expression of miR-122, and decreased expression of miR-200a in immature cardiomyocytes.
A4. The method of one of embodiments a1-A3, wherein inducing overexpression comprises contacting immature cardiomyocytes with a vector comprising a nucleic acid encoding a miRNA to be overexpressed.
A5. The method of embodiment a4, wherein the vector is configured to facilitate transient expression of a nucleic acid encoding a miRNA to be overexpressed.
A6. The method of embodiment a4 or a5, wherein the vector is a viral vector configured to integrate a nucleic acid encoding a miRNA to be overexpressed into the genome of an immature cardiomyocyte.
A7. The method of any one of embodiments a4-a6, wherein the viral vector is a lentiviral vector or an adeno-associated viral vector.
A8. The method of any one of embodiments a1-a7, wherein inducing decreased expression of a miRNA comprises contacting immature cardiomyocytes with a nucleic acid fragment that hybridizes to a miRNA targeted for decreased expression, or with a vector comprising a nucleic acid encoding a transcript that hybridizes to a miRNA targeted for decreased expression.
A9. The method of any one of embodiments a1-A8, wherein inducing decreased expression comprises effecting knock-out of a gene encoding the miRNA.
A10. The method of one of embodiments a1-a9, wherein inducing reduced expression comprises providing immature cardiomyocytes with a nuclease and a guide nucleic acid having a sequence that facilitates specific cleavage by the nuclease of a nucleic acid encoding the miRNA targeted for reduced expression.
A11. The method of one of embodiments a1-a10, wherein providing the immature cardiomyocyte with a nuclease comprises contacting the immature cardiomyocyte with the nuclease or a vector encoding the nuclease, wherein the vector is configured to promote expression of the enzyme in the cardiomyocyte.
A12. The method of embodiment a10, wherein providing a guide nucleic acid to an immature cardiomyocyte comprises contacting the immature cardiomyocyte with the guide nucleic acid or a vector encoding the guide nucleic acid, wherein the vector is configured to facilitate expression of the guide nucleic acid in the cardiomyocyte.
A13. The method of embodiment a10 or a11, wherein the nuclease is an endonuclease, such as Cas9 or TALENS.
A14. The method of any one of embodiments A8-a12, wherein the vector is a viral vector.
A15. The method of embodiment a14, wherein the viral vector is a lentiviral vector or an adeno-associated viral vector.
A16. The method of any one of embodiments a1-a15, wherein the immature cardiomyocytes are derived from stem cells.
A17. The method of one of embodiments a1-a16, wherein the immature cardiomyocytes are derived in vitro from stem cells.
A18. The method of embodiment a16 or a17, wherein the stem cell is an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
A19. The method of one of embodiments a1-a18, further comprising contacting the immature cardiomyocytes with two or more long chain fatty acids selected from palmitic acid, oleic acid, and linoleic acid.
A20. The method of embodiment a19, wherein the one or more long chain fatty acids include palmitate, oleic acid and linoleic acid.
A21. The method of any one of embodiments a1-a20, wherein the cardiomyocyte comprises a genetic aberration.
A22. The method of embodiment a21, wherein the genetic aberration is associated with a metabolic or pathological disease state in the heart.
A23. The method of embodiment a22, wherein the genetic aberration is associated with a Fatty Acid Oxidation (FAO) disorder.
A24. The method of embodiment a22 or a23, wherein the cardiomyocyte comprises a mutation in a gene encoding one of: HADHA, FATP1, FACS1, OCTN2, L-CPTI, M-CPT I, CAT, CPT II, VLCAD, LCAD, MCAD, SCAD, LCHAD, SHYD, M/SCHAD, SKAT, MKAT, HS, HL, ETF, and ETF QO.
B1. A cardiomyocyte produced by any of the methods described in one of embodiments a1-a 24.
B2. The cardiomyocyte of embodiment B1, wherein said cardiomyocyte comprises a genetic aberration.
B3. The cardiomyocyte of embodiment B2, wherein the genetic aberration is associated with a disorder of Fatty Acid Oxidation (FAO).
B4. The cardiomyocyte of embodiment B3, wherein the genetic aberration is a mutation in the gene encoding HADHA.
C1. A method of treating a subject having a disorder treatable by administration of cardiomyocytes having a mature cardiolipin profile, comprising administering to the subject an effective amount of the cardiomyocytes described in embodiment B1.
C2. The method of embodiment C1, wherein the subject has damaged cardiac tissue or cells.
C2. The method of embodiment C1 or C2, wherein the subject has diabetes, congenital heart disease, ischemia, myopathy, mitochondrial disease and/or has an infarction.
C3. The method of one of embodiments C1-C3, wherein the mitochondrial disease is a Fatty Acid Oxidation (FAO) disorder.
C4. The method of one of embodiments C1-C4, wherein the subject has a mutation in the gene encoding HADHA.
C5. The method of one of embodiments C1-C5, wherein the subject experiences an arrhythmia.
C6. The method of one of embodiments C1-C6, wherein the subject is at elevated risk of Sudden Infant Death Syndrome (SIDS).
D1. A method of screening for a compound for modulating cardiac function comprising:
contacting one or more cardiomyocytes described in one of embodiments B1-B4 with a candidate agent; and
measuring a cardiac functional parameter of the one or more cardiomyocytes;
wherein a change in the cardiac function parameter indicates that the candidate agent modulates cardiac function.
D2. The method of embodiment D1, wherein the mature cardiomyocytes comprise a genetic aberration.
D3. The method of embodiment D1 or D2, wherein the genetic aberration is associated with a disorder of Fatty Acid Oxidation (FAO).
D4. The method according to any one of embodiments D1-D3, wherein the genetic aberration is a mutation in the gene encoding hadoa.
D5. The method according to any one of embodiments D1-D4, wherein the cardiac function parameters comprise lipid profile, cardiolipin profile, metabolic profile, oxygen consumption rate, mitochondrial proton gradient, contractility, calcium transport, conduction velocity, glucose stress, and cell death.
E1. A method of treating a mitochondrial Fatty Acid Oxidation (FAO) disorder in a subject, the method comprising administering an effective amount of a composition that stabilizes a cardiolipin profile in the subject or promotes mature cardiolipin remodeling in the mitochondria of the subject.
E2. The method of embodiment E1, wherein the FAO disorder is associated with a condition of diabetes, heart failure, neurodegeneration, advanced age, congenital heart disease, ischemia, myopathy, and/or infarction.
E3. The method of embodiment E1 or E2, wherein the FAO disorder is a Fatty Acid (FA) β -oxidation disorder.
E4. The method of one of embodiments E1-E3, wherein the phenotype of mitochondrial dysfunction is associated with an increased risk of sudden infant death syndrome.
E5. The method of one of embodiments E1-E4, wherein stabilizing the cardiolipin profile comprises preventing cardiolipin oxidation.
E6. The method according to any one of embodiments E1 to E4, wherein the composition is or comprises elamipeptide.
F1. A method of detecting a pathological state of a cultured cardiomyocyte, comprising:
determining a profile of cardiolipin in the cardiomyocytes, wherein a relative increase in cardiolipin with acyl chains greater than 18 carbons indicates and a relative decrease in cardiolipin with acyl chains less than 18 carbons indicates a decrease in the pathological state of the cardiomyocytes.
F2. The method of embodiment F1, wherein the increase or decrease in cardiolipin is relative to a wild-type immature cardiomyocyte.
F3. The method of embodiment F1 or F2, wherein the cultured cardiomyocytes are derived in vitro from stem cells.
F4. The method of embodiment F3, wherein the stem cell is an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
F5. The method according to one of embodiments F1-F4, wherein the pathological state is associated with mitochondrial dysfunction.
F6. The method of embodiment F5, wherein the mitochondrial dysfunction is a mitochondrial trifunctional protein deficiency.
F7. The method of one of embodiments F1-F6, further comprising contacting the cultured cardiomyocyte with a candidate agent to reduce the pathological state of the cultured cardiomyocyte.
F8. The method of embodiment F7, comprising determining a cardiolipin profile in the cultured cardiomyocytes a plurality of times before, during and/or after the step of contacting the cultured cardiomyocytes with the candidate agent to determine the effect of the candidate agent on the pathological state of the cultured cardiomyocytes.
G1. A composition for inducing maturation of cultured cardiomyocytes, comprising two or more of: a nucleic acid construct encoding Let7imicroRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of the sequence encoding miR-122, and a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of the sequence encoding miR-200 a.
G2. The composition of embodiment G1 comprising three or more of the following: a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of a sequence encoding miR-122, and a nucleic acid construct that is or encodes an oligomer that hybridizes to a portion of a sequence encoding miR-200 a.
G3. The composition of embodiment G1 or G2, comprising a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct being or encoding an oligomer hybridizing to a portion of the sequence encoding miR-122, and a nucleic acid construct being or encoding an oligomer hybridizing to a portion of the sequence encoding miR-200 a.
G4. The composition according to one of embodiments G1 to G3, wherein the nucleic acid constructs encoding micrornas and/or encoding oligomers are each operably linked to one or more promoter sequences.
G5. The composition of one of embodiments G1-G4, wherein one or more of the constructs is incorporated into one or more vectors configured for delivery to a cell.
G6. The composition of embodiment G5, wherein the one or more vectors are viral vectors.
G7. The composition of embodiment G5 or G660, wherein at least one viral vector is a lentiviral vector or an AAV vector.
G8. The composition of one of embodiments G1-G7, wherein the oligomer that hybridizes to a portion of the sequence encoding miR-122 and the oligomer that hybridizes to a portion of the sequence encoding miR-200a are guide RNA molecules configured to induce gene editing enzymes to cleave miR-122 and miR-200a, respectively.
G9. The composition of embodiment G8, wherein the gene-editing enzyme is a nuclease.
G10. The composition of one of embodiments G1-G9, further comprising a nuclease.
G11. The composition of embodiments G9 or G10, wherein the nuclease is Cas 9.
G12. The composition of one of embodiments G1-G11, further comprising one or more long chain fatty acids.
G13. The composition of embodiment G12 wherein the one or more long chain fatty acids comprise two or more of palmitate, oleate and linoleate.
G14. The composition of embodiments G12 or G13, wherein the one or more long chain fatty acids comprise palmitate, oleic acid and linoleic acid.
H1. A kit comprising one or more compositions of embodiments G1-G14.
H2. The kit of embodiment H1, further comprising cell culture medium and/or one or more immature cardiomyocytes.
While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110>University of Washington
Miklas, W.
Ruohola-Baker, H.
Wang, Y.
<120> methods and compositions for detecting and promoting cardiolipin remodeling and cardiomyocyte maturation
Compositions and related methods for treating mitochondrial dysfunction
<130>UWOTL167910
<150>US 62/596438
<151>2017-12-08
<150>US 62/674978
<151>2018-05-22
<160>46
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<400>44
acccaaagtg tacaatcatt gact 24
<210>45
<211>90
<212>DNA
<213> human
<400>45
ccgggcccct gtgagcatct taccggacag tgctggattt cccagcttga ctctaacact 60
gtctggtaac gatgttcaaa ggtgacccgc 90
<210>46
<211>85
<212>DNA
<213> human
<400>46
ccttagcaga gctgtggagt gtgacaatgg tgtttgtgtc taaactatca aacgccatta 60
tcacactaaa tagctactgc taggc 85
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