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

Nature Biomedical Engineering (2018) | Download Citation

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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References

  1. 1.

    Carrier, L., Mearini, G., Stathopoulou, K. & Cuello, F. Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology. Gene 573, 188–197 (2015).

  2. 2.

    Strande, J. L. Haploinsufficiency MYBPC3 mutations: another stress induced cardiomyopathy? Let’s take a look! J. Mol. Cell. Cardiol. 79, 284–286 (2015).

  3. 3.

    Feric, N. T. & Radisic, M. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv. Drug Deliv. Rev. 96, 110–134 (2016).

  4. 4.

    Mathur, A. et al. In vitro cardiac tissue models: current status and future prospects. Adv. Drug Deliv. Rev. 96, 203–213 (2016).

  5. 5.

    Eng, G. et al. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes. Nat. Commun. 7, 10312 (2016).

  6. 6.

    Turnbull, I. C. et al. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. FASEB J. 28, 644–654 (2014).

  7. 7.

    Conradi, L. et al. Immunobiology of fibrin-based engineered heart tissue. Stem Cells Transl. Med. 4, 625–631 (2015).

  8. 8.

    Legant, W. R. et al. Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues. Proc. Natl Acad. Sci. USA 106, 10097–10102 (2009).

  9. 9.

    Mathur, A. et al. Human iPSC-based cardiac microphysiological system for drug screening applications. Sci. Rep. 5, 8883 (2015).

  10. 10.

    Abramson, S. V. et al. Pulmonary hypertension predicts mortality and morbidity in patients with dilated cardiomyopathy. Ann. Intern. Med. 116, 888–895 (1992).

  11. 11.

    Engler, A. J. et al. Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating. J. Cell Sci. 121, 3794–3802 (2008).

  12. 12.

    Kijlstra, J. D. et al. Integrated analysis of contractile kinetics, force generation, and electrical activity in single human stem cell-derived cardiomyocytes. Stem Cell Rep. 5, 1226–1238 (2015).

  13. 13.

    Liau, B., Christoforou, N., Leong, K. W. & Bursac, N. Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function. Biomaterials 32, 9180–9187 (2011).

  14. 14.

    Schaaf, S. et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS ONE 6, e26397 (2011).

  15. 15.

    Ribeiro, A. J. et al. Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness. Proc. Natl Acad. Sci. USA 112, 12705–12710 (2015).

  16. 16.

    Hirt, M. N. et al. Increased afterload induces pathological cardiac hypertrophy: a new in vitro model. Basic Res. Cardiol. 107, 307 (2012).

  17. 17.

    Hinson, J. T. et al. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science 349, 982–986 (2015).

  18. 18.

    Stoppel, W. L., Kaplan, D. L. & Black, L. D.III Electrical and mechanical stimulation of cardiac cells and tissue constructs. Adv. Drug Deliv. Rev. 96, 135–155 (2016).

  19. 19.

    Kawata, S., Sun, H. B., Tanaka, T. & Takada, K. Finer features for functional microdevices. Nature 412, 697–698 (2001).

  20. 20.

    Klein, F. et al. Elastic fully three-dimensional microstructure scaffolds for cell force measurements. Adv. Mater. 22, 868–871 (2010).

  21. 21.

    Jeon, H., Hidai, H., Hwang, D. J. & Grigoropoulos, C. P. Fabrication of arbitrary polymer patterns for cell study by two-photon polymerization process. J. Biomed. Mater. Res. A 93, 56–66 (2010).

  22. 22.

    Ma, Z. et al. Three-dimensional filamentous human diseased cardiac tissue model. Biomaterials 35, 1367–1377 (2014).

  23. 23.

    Burridge, P. W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014).

  24. 24.

    Tohyama, S. et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127–137 (2013).

  25. 25.

    Thavandiran, N. et al. Design and formulation of functional pluripotent stem cell-derived cardiac microtissues. Proc. Natl Acad. Sci. USA 110, E4698–E4707 (2013).

  26. 26.

    Huebsch, N. et al. Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. Sci. Rep. 6, 24726 (2016).

  27. 27.

    Gautel, M., Furst, D. O., Cocco, A. & Schiaffino, S. Isoform transitions of the myosin binding protein C family in developing human and mouse muscles: lack of isoform transcomplementation in cardiac muscle. Circ. Res. 82, 124–129 (1998).

  28. 28.

    Bennett, P. M., Furst, D. O. & Gautel, M. The C-protein (myosin binding protein C) family: regulators of contraction and sarcomere formation? Rev. Physiol. Biochem. Pharmacol. 138, 203–234 (1999).

  29. 29.

    Moss, R. L., Fitzsimons, D. P. & Ralphe, J. C. Cardiac MyBP-C regulates the rate and force of contraction in mammalian myocardium. Circ. Res. 116, 183–192 (2015).

  30. 30.

    Previs, M. J. et al. Molecular mechanics of cardiac myosin-binding protein C in native thick filaments. Science 337, 1215–1218 (2012).

  31. 31.

    Lan, F. et al. Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. Cell Stem Cell 12, 101–113 (2013).

  32. 32.

    Argenziano, M. et al. Electrophysiologic characterization of calcium handling in human induced pluripotent stem cell-derived atrial cardiomyocytes. Stem Cell Rep. 10, 1867–1878 (2018).

  33. 33.

    Hidai, H., Jeon, H., Hwang, D. J. & Grigoropoulos, C. P. Self-standing aligned fiber scaffold fabrication by two photon photopolymerization. Biomed. Microdevices 11, 643–652 (2009).

  34. 34.

    Mandegar, M. A. et al. CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell 18, 541–553 (2016).

  35. 35.

    Musunuru, K. Genome editing of human pluripotent stem cells to generate human cellular disease models. Dis. Models Mech. 6, 896–904 (2013).

  36. 36.

    Harris, S. P. et al. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ. Res. 90, 594–601 (2002).

  37. 37.

    Korte, F. S., McDonald, K. S., Harris, S. P. & Moss, R. L. Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C. Circ. Res. 93, 752–758 (2003).

  38. 38.

    De Lange, W. J. et al. Neonatal mouse-derived engineered cardiac tissue: a novel model system for studying genetic heart disease. Circ. Res. 109, 8–19 (2011).

  39. 39.

    Fraysse, B. et al. Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice. J. Mol. Cell. Cardiol. 52, 1299–1307 (2012).

  40. 40.

    Chen, P. P. et al. Dissociation of structural and functional phenotypes in cardiac myosin-binding protein C conditional knockout mice. Circulation 126, 1194–1205 (2012).

  41. 41.

    Baumgarten, G. et al. Myocardial injury modulates the innate immune system and changes myocardial sensitivity. Basic Res. Cardiol. 101, 427–435 (2006).

  42. 42.

    Birket, M. J. et al. Contractile defect caused by mutation in MYBPC3 revealed under conditions optimized for human PSC-cardiomyocyte function. Cell Rep. 13, 733–745 (2015).

  43. 43.

    Ribeiro, A. J. S. et al. Multi-imaging method to assay the contractile mechanical output of micropatterned human iPSC-derived cardiac myocytes. Circ. Res. 120, 1572–1583 (2017).

  44. 44.

    McCain, M. L. et al. Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip. Proc. Natl Acad. Sci. USA 110, 9770–9775 (2013).

  45. 45.

    Yang, H. et al. Dynamic myofibrillar remodeling in live cardiomyocytes under static stretch. Sci. Rep. 6, 20674 (2016).

  46. 46.

    Hirt, M. N. et al. Deciphering the microRNA signature of pathological cardiac hypertrophy by engineered heart tissue- and sequencing-technology. J. Mol. Cell. Cardiol. 81, 1–9 (2015).

  47. 47.

    Stohr, A. et al. Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice. J. Mol. Cell. Cardiol. 63, 189–198 (2013).

  48. 48.

    Tanaka, A. et al. Endothelin-1 induces myofibrillar disarray and contractile vector variability in hypertrophic cardiomyopathy-induced pluripotent stem cell-derived cardiomyocytes. J. Am. Heart Assoc. 3, e001263 (2014).

  49. 49.

    Bostrom, P. et al. C/EBPβ controls exercise-induced cardiac growth and protects against pathological cardiac remodeling. Cell 143, 1072–1083 (2010).

  50. 50.

    Wei, J. Q. et al. Quantitative control of adaptive cardiac hypertrophy by acetyltransferase p300. Circulation 118, 934–946 (2008).

  51. 51.

    He, A., Kong, S. W., Ma, Q. & Pu, W. T. Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart. Proc. Natl Acad. Sci. USA 108, 5632–5637 (2011).

  52. 52.

    He, A. et al. Dynamic GATA4 enhancers shape the chromatin landscape central to heart development and disease. Nat. Commun. 5, 4907 (2014).

  53. 53.

    Dai, Y. S., Cserjesi, P., Markham, B. E. & Molkentin, J. D. The transcription factors GATA4 and dHAND physically interact to synergistically activate cardiac gene expression through a p300-dependent mechanism. J. Biol. Chem. 277, 24390–24398 (2002).

  54. 54.

    Sunagawa, Y. et al. Cyclin-dependent kinase-9 is a component of the p300/GATA4 complex required for phenylephrine-induced hypertrophy in cardiomyocytes. J. Biol. Chem. 285, 9556–9568 (2010).

  55. 55.

    Hutter, J. L. & Bechhoefer, J. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64, 1868–1873 (1993).

  56. 56.

    Laughner, J. I. et al. Processing and analysis of cardiac optical mapping data obtained with potentiometric dyes. Am. J. Physiol. Heart C 303, H753–H765 (2012).

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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.

Author information

Author notes

    • Zhen Ma

    Present address: Department of Biomedical and Chemical Engineering, Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, USA

    • Nathaniel Huebsch

    Present address: Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA

    • Sangmo Koo

    Present address: Department of Mechanical Engineering, Incheon National University, Incheon, Republic of Korea

  1. These authors contributed equally to this work: Zhen Ma, Nathaniel Huebsch, Sangmo Koo.

Affiliations

  1. Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA

    • Zhen Ma
    • , Nathaniel Huebsch
    • , Brian Siemons
    •  & Kevin E. Healy
  2. California Institute for Quantitative Biosciences, Berkeley, CA, USA

    • Zhen Ma
    • , Nathaniel Huebsch
    •  & Kevin E. Healy
  3. Department of Mechanical Engineering, University of California, Berkeley, Berkeley, CA, USA

    • Sangmo Koo
    •  & Costas P. Grigoropoulos
  4. Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA

    • Mohammad A. Mandegar
    •  & Bruce R. Conklin
  5. Department of Medicine, University of California, San Francisco, CA, USA

    • Mohammad A. Mandegar
    •  & Bruce R. Conklin
  6. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA

    • Mohammad A. Mandegar
    •  & Bruce R. Conklin
  7. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    • Steven Boggess
  8. Department of Material Science and Engineering, University of California, Berkeley, Berkeley, CA, USA

    • Kevin E. Healy

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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.

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.

Corresponding author

Correspondence to Kevin E. Healy.

Supplementary information

  1. Supplementary Information

    Supplementary methods, figures, tables, references and video captions

  2. Reporting Summary

  3. Supplementary Video 1

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

  4. 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

  5. Supplementary Video 3

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

  6. 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

  7. 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

  8. 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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DOI

https://doi.org/10.1038/s41551-018-0280-4