Abstract
Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) lack nanoscale structures essential for efficient excitation–contraction coupling. Such nanostructures, known as dyads, are frequently disrupted in heart failure. Here we show that the reduced expression of cardiomyopathy-associated 5 (CMYA5), a master protein that establishes dyads, contributes to dyad disorganization in heart failure and to impaired dyad assembly in hiPSC-CMs, and that a miniaturized form of CMYA5 suitable for delivery via an adeno-associated virus substantially improved dyad architecture and normalized cardiac function under pressure overload. In hiPSC-CMs, the miniaturized form of CMYA5 increased contractile forces, improved Ca2+ handling and enhanced the alignment of sarcomere Z-lines with ryanodine receptor 2, a protein that mediates the sarcoplasmic release of stored Ca2+. Our findings clarify the mechanisms responsible for impaired dyad structure in diseased cardiomyocytes, and suggest strategies for promoting dyad assembly and stability in heart disease and during the derivation of hiPSC-CMs.
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Data availability
The data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.
Code availability
Custom code is provided as Supplementary Information and is also available in Zenodo at https://doi.org/10.5281/zenodo.13366870 (ref. 48).
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Acknowledgements
We thank the Gift of Life Donor Program of Philadelphia for enabling the procurement of human hearts from deceased organ donors; W. Chen, University of Calgary, for providing us with the Ryr2-gfp mouse line; J. Milosh for coverslip preparation; and M. Rosnach for cell-patterning artwork. F.L. discloses support for the research described in this study from Fudan University (JIH1340085Y) and the American Heart Association (20POST35200233); W.T.P. discloses support for the research described in this study from the National Institutes of Health (R01HL163937, R01HL146634 and UH3TR003279). K.K.P. discloses support for the research described in this study from the National Institutes of Health (UH3TR003279). K.B.M. discloses support for the research described in this study from the National Institutes of Health (R01HL149891 and R01HL105993).
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F.L. and W.T.P. conceptualized the project. F.L., W.X., M.A.T., K.S., K.K.P., D.Z. and W.T.P. developed the methodology. F.L., C.L., Q.M., Z.W., R.H.B., M.P., P.B., B.X., Y.X., S.X., X.Z. and A.P. conducted investigations. F.J.N., K.C.B. and K.B.M. produced materials and reagents. W.T.P., K.K.P. and K.B.M. acquired funding. W.T.P., K.K.P. and K.B.M. administered the project. F.L. wrote the original draft. F.L. and W.T.P. reviewed and edited the manuscript.
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K.B.M. holds research grants from Amgen and serves as a scientific consultant/advisory board member for Bristol Myers Squibb. W.T.P. and F.L. have filed for intellectual property rights on miniCMYA5. All other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Echocardiographic analysis of AAV-miniCMYA5 treatment in a murine pressure-overload model.
a–d, Cmya5KO (a and b) or WT (c and d) mice were treated with AAV-Ctrl or AAV-miniCMYA5 and then Sham or TAC surgery. Left ventricular internal diameter at end-diastole (LVID;d, a and c) or fractional shortening (FS, b and d) were measured at the indicated time point. Mann Whitney test with Bonferroni correction for 2 comparisons. Data are shown as mean ± SD.
Extended Data Fig. 2 Assessment of contractility, Ca2+ transient, and SR load in dissociated murine cardiomyocytes.
Cmya5KO or WT mice were treated with AAV-GFP or AAV-miniCMYA5 following the timeline shown in Fig. 3a. Cardiomyocytes were dissociated from mouse hearts, loaded with Ca2+-sensitive dye Rhod2, and analyzed by confocal line scan imaging during 1 Hz pacing. a-f, Cmya5KO. g-i, WT. Representative confocal line scan images are shown in a, c, and e. SR load was measured as the peak fluorescent intensity after cells were treated with caffeine (SR load F/F0, b and g). Ca2+ transient amplitude was determined from the peak fluorescent intensity divided by the baseline intensity (Ca2+ transient F/F0, d and h). Contractility was measured as the fractional shortening of the cardiomyocyte (Fractional shortening: f and i). Mann-Whitney with Bonferonni correction for two tests. Data are shown as mean ± SD.
Extended Data Fig. 3 Expression and localization of CMYA5 in hiPSC-CMs and murine cardiomyocytes.
a-c. Expression of Cmya5, Fsd2, and Ryr2 mRNAs during murine heart development. Gene expression was measured in purified ventricular cardiomyocytes at the indicated stage by RNA-seq. Data are from GSE195902. n = 3 per time point. d. Co-localization of miniCMYA5 with ACTN2 in patterned hiPSC-CMs. e. Increased organization of RYR2/jSR induced by miniCMYA5 in hiPSC-CMs was not sufficient to stimulate detectable formation of T-tubules, marked by CAV3. Bar = 5 µm. Data are shown as mean ± SD.
Extended Data Fig. 4 MiniCMYA5 enhanced excitation-contraction coupling and force generation by hiPSC-CMs.
a. Representative images of engineered heart tissues (EHTs) generated from hiPSC-CMs treated with Ad:GFP (Ctrl) or Ad:miniCMYA5. EHTs were electrically paced at 2 Hz. b. Active force produced by EHTs as a function of passive stretch above the resting length. c. Contraction duration of EHTs as a function of passive stretch. The contraction duration was measured at 80% maximal force. d. Representative traces of EHT active force over time as a function of initial stretch. Data are shown as mean ± SD.
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Lu, F., Liou, C., Ma, Q. et al. Virally delivered CMYA5 enhances the assembly of cardiac dyads. Nat. Biomed. Eng 9, 730–741 (2025). https://doi.org/10.1038/s41551-024-01253-z
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DOI: https://doi.org/10.1038/s41551-024-01253-z
