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Contractile deficits in engineered cardiac microtissues as a result of MYBPC3 deficiency and mechanical overload

Abstract

The integration of in vitro cardiac tissue models, human induced pluripotent stem cells (hiPSCs) and genome-editing tools allows for the enhanced interrogation of physiological phenotypes and recapitulation of disease pathologies. Here, using a cardiac tissue model consisting of filamentous three-dimensional matrices populated with cardiomyocytes derived from healthy wild-type (WT) hiPSCs (WT hiPSC-CMs) or isogenic hiPSCs deficient in the sarcomere protein cardiac myosin-binding protein C (MYBPC3–/– hiPSC-CMs), we show that the WT microtissues adapted to the mechanical environment with increased contraction force commensurate to matrix stiffness, whereas the MYBPC3–/– microtissues exhibited impaired force development kinetics regardless of matrix stiffness and deficient contraction force only when grown on matrices with high fibre stiffness. Under mechanical overload, the MYBPC3–/– microtissues had a higher degree of calcium transient abnormalities, and exhibited an accelerated decay of calcium dynamics as well as calcium desensitization, which accelerated when contracting against stiffer fibres. Our findings suggest that MYBPC3 deficiency and the presence of environmental stresses synergistically lead to contractile deficits in cardiac tissues.

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Fig. 1: Cardiac microtissue remodelling on filamentous matrices.
Fig. 2: Force measurement based on fibre deflection.
Fig. 3: Contractile deficits of MYBPC3–/– cardiac microtissues.
Fig. 4: No structural disarray on MYBPC3–/– cardiac microtissues.
Fig. 5: Mechanical load altered contractile force dynamics.
Fig. 6: Calcium transient abnormalities on MYBPC3–/– cardiac microtissues.
Fig. 7: EP300- and GATA4-directed contractile deficits due to mechanical stress.

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Acknowledgements

This work was supported in part by the NIH NHLBI (R01HL096525, R01HL108677, U01HL100406 and U01HL098179), NIH NIBIB (R21EB021003) and NIH NCATS (UH3TR000487). Z.M. acknowledges support from NSF (1804875), American Heart Association postdoctoral fellowship (16POST27750031) and the Nappi Family Foundation Research Scholar Project. N.H. acknowledges support from NIH T32 (HL007544). M.A.M. acknowledges support from the Canadian Institute of Health Research Postdoctoral Fellowship (129844). B.S. acknowledges support from the California Institute for Regenerative Medicine (TBI-01197). We acknowledge assistance from the Berkeley CIRM/QB3 Shared Stem Cell Facility for flow cytometry, Biological Imaging Facility for confocal microscopy (supported by NIH S10 programme 1S10RR026866-01) and Biomolecular Nanotechnology Center for scanning electron microscopy. We thank E. Nora and P. Devine (Gladstone Institute of Cardiovascular Disease) for helpful discussions and advice regarding p300 expression analysis. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the California Institute for Regenerative Medicine and/or other agencies of the State of California.

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Contributions

Z.M., S.K., N.H. and K.E.H. conceived and designed the sample fabrication and experiments. Z.M. performed the biological experiments and analysed the data. N.H. performed the gene expression, GCaMP6f analysis and quantitative fluorescence microscopy studies to identify the molecular basis. S.K. fabricated the filamentous matrices, performed the COMSOL analysis and wrote the MATLAB script for force measurement. N.H. developed the software for quantitative sarcomere analysis, as well as the motion-tracking software for contractility analysis. M.A.M. developed the MYBPC3 hiPSC lines using the TALEN-mediated genome-editing method. B.S. helped with hiPSC culturing, and hiPSC-CM differentiation and characterization. Z.M., N.H., S.K. and K.E.H. wrote the manuscript with discussions and improvements from all authors. K.E.H., B.R.C. and C.P.G. funded the study. C.P.G., B.R.C. and K.E.H. supervised the project development and management.

Corresponding author

Correspondence to Kevin E. Healy.

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Competing interests

B.R.C. is a founder of Tenaya Therapeutics, a company focused on finding treatments for heart failure, including the use of CRISPR interference to interrogate genetic cardiomyopathies. B.R.C. holds equity in Tenaya Therapeutics, and Tenaya Therapeutics provides research support for heart failure-related research to B.R.C. K.E.H. and N.H. have a financial relationship with Organos, and both themselves and the company may benefit from commercialization of the results of this research. The other authors declare no competing interests.

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Supplementary methods, figures, tables, references and video captions

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Supplementary Video 1

The fabrication process of filamentous matrices. One fibre was fabricated with one single shot of laser irradiation

Supplementary Video 2

Five days after seeding WT hiPSC-CMs into a 5 μm filamentous matrix, the cardiac microtissue formed and bent the fibres during the contraction

Supplementary Video 3

Time-dependent transient stress presented to the fibres was simulated using COMSOL during the cardiac microtissue contraction

Supplementary Video 4

Twenty days after seeding MYBPC3/ hiPSC-CMs into a 10 μm filamentous matrix, the cardiac microtissue formed and bent the fibres during the contraction

Supplementary Video 5

Twenty days after seeding GCaMP6f-WT hiPS-CMs into a 10 μm filamentous matrix, the calcium dynamics was recorded under a fluorescent microscope

Supplementary Video 6

Twenty days post-differentiation, WT hiPSC-CMs beat as a sheet and were analysed by motion-tracking software to show the contraction vectors

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Ma, Z., Huebsch, N., Koo, S. et al. Contractile deficits in engineered cardiac microtissues as a result of MYBPC3 deficiency and mechanical overload. Nat Biomed Eng 2, 955–967 (2018). https://doi.org/10.1038/s41551-018-0280-4

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