Skip to main content

Advertisement

SpringerLink
Log in

Attenuation of Cardiomyocyte Hypertrophy via Depletion Myh7 using CASAAV

  • Published:
Cardiovascular Toxicology Aims and scope Submit manuscript

Abstract

Myh7 is a classic biomarker for cardiac remodeling and a potential target to attenuate cardiomyocyte (CM) hypertrophy. This study aimed to identify the dominant function of Myh7 after birth and determine whether its removal would affect CM maturation or contribute to reversal of pathological hypertrophy phenotypes. The CASAAV (CRISPR/Cas9-AAV9-based somatic mutagenesis) technique was used to deplete Myh6 and Myh7, and an AAV dosage of 5 × 109 vg/g was used to generate a mosaic CM depletion model to explore the function of Myh7 in adulthood. CM hypertrophy was induced by transverse aortic constriction (TAC) in Rosa26Cas9-P2A-GFP mice at postnatal day 28 (PND28). Heart function was measured by echocardiography. Isolated CMs and in situ imaging were used to analyze the structure and morphology of CM. We discovered that CASAAV successfully silenced Myh6 and Myh7 in CMs, and early depletion of Myh7 led to mild adulthood lethality. However, the Myh7 PND28-knockout mice had normal heart phenotype and function, with normal cellular size and normal organization of sarcomeres and T-tubules. The TAC mice also received AAV-Myh7-Cre to produce Myh7-knockout CMs, which were also of normal size, and echocardiography demonstrated a reversal of cardiac hypertrophy. In conclusion, Myh7 has a role during the maturation period but rarely functions in adulthood. Thus, the therapeutic time should exceed the period of maturation. These results confirm Myh7 as a potential therapeutic target and indicate that its inhibition could help reverse CM hypertrophy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Cunningham, K. S., Spears, D. A., & Care, M. (2019). Evaluation of cardiac hypertrophy in the setting of sudden cardiac death. Forensic Science Research, 4(3), 223–240.

    Article  Google Scholar 

  2. Wu, Q. Q., et al. (2017). Mechanisms contributing to cardiac remodelling. Clinical Science (Lond), 131(18), 2319–2345.

    Article  CAS  Google Scholar 

  3. Sucharov, C., Bristow, M. R., & Port, J. D. (2008). miRNA expression in the failing human heart: functional correlates. Journal of Molecular and Cellular Cardiology, 45(2), 185–192.

    Article  CAS  Google Scholar 

  4. Liu, J., et al. (2007). Progressive troponin I loss impairs cardiac relaxation and causes heart failure in mice. American Journal of Physiology-Heart and Circulatory Physiology, 293(2), H1273–H1281.

    Article  CAS  Google Scholar 

  5. James, J., et al. (2010). Effects of myosin heavy chain manipulation in experimental heart failure. Journal of Molecular and Cellular Cardiology, 48(5), 999–1006.

    Article  CAS  Google Scholar 

  6. Dirkx, E., da CostaMartins, P. A., & De Windt, L. J. (2013). Regulation of fetal gene expression in heart failure. Biochimica et Biophysica Acta, 1832(12), 2414–2424.

    Article  CAS  Google Scholar 

  7. Shih, Y. H., et al. (2015). Cardiac transcriptome and dilated cardiomyopathy genes in zebrafish. Circulation: Cardiovascular Genetics, 8(2), 261–269.

    CAS  Google Scholar 

  8. Lowes, B. D., et al. (1997). Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium. Journal of Clinical Investigation, 100(9), 2315–2324.

    Article  CAS  Google Scholar 

  9. Marian, A. J., & Braunwald, E. (2017). Hypertrophic cardiomyopathy: genetics, pathogenesis, clinical manifestations, diagnosis, and therapy. Circulation Research, 121(7), 749–770.

    Article  CAS  Google Scholar 

  10. Krenz, M., et al. (2003). Analysis of myosin heavy chain functionality in the heart. Journal of Biological Chemistry, 278(19), 17466–17474.

    Article  CAS  Google Scholar 

  11. Tardiff, J. C., et al. (2000). Expression of the beta (slow)-isoform of MHC in the adult mouse heart causes dominant-negative functional effects. American Journal of Physiology-Heart and Circulatory Physiology, 278(2), H412–H419.

    Article  CAS  Google Scholar 

  12. Krenz, M., & Robbins, J. (2004). Impact of beta-myosin heavy chain expression on cardiac function during stress. Journal of the American College of Cardiology, 44(12), 2390–2397.

    Article  CAS  Google Scholar 

  13. Guo, Y., et al. (2018). Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor. Nature Communications, 9(1), 3837.

    Article  Google Scholar 

  14. Guo, Y., et al. (2017). Analysis of cardiac myocyte maturation using CASAAV, a platform for rapid dissection of cardiac myocyte gene function in vivo. Circulation Research, 120(12), 1874–1888.

    Article  CAS  Google Scholar 

  15. VanDusen, N. J., et al. (2018). CASAAV: A CRISPR-based platform for rapid dissection of gene function in vivo. Current Protocols in Molecular Biology. https://doi.org/10.1002/cpmb.46

    Article  Google Scholar 

  16. Grieger, J. C., Choi, V. W., & Samulski, R. J. (2006). Production and characterization of adeno-associated viral vectors. Nature Protocols, 1(3), 1412–1428.

    Article  CAS  Google Scholar 

  17. O’Connell, T. D., Rodrigo, M. C., & Simpson, P. C. (2007). Isolation and culture of adult mouse cardiac myocytes. In F. Vivanco (Ed.), Cardiovascular proteomics: methods and protocols (pp. 271–296). Totowa, NJ: Humana Press.

    Google Scholar 

  18. Guo, Y., et al. (2014). Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins. Molecular Biology of the Cell, 25(8), 1287–1297.

    Article  Google Scholar 

  19. Guo, Y., & Zheng, Y. (2015). Lamins position the nuclear pores and centrosomes by modulating dynein. Molecular Biology of the Cell, 26(19), 3379–3389.

    Article  CAS  Google Scholar 

  20. Chen, B., et al. (2015). In situ single photon confocal imaging of cardiomyocyte T-tubule system from Langendorff-perfused hearts. Frontiers in physiology, 6, 134. https://doi.org/10.3389/fphys.2015.00134

    Article  PubMed  PubMed Central  Google Scholar 

  21. Spitzer, M., et al. (2014). BoxPlotR: a web tool for generation of box plots. Nature Methods, 11(2), 121–122.

    Article  CAS  Google Scholar 

  22. Porrello, E., et al. (2015). Novel roles of GATA4/6 in the postnatal heart identified through temporally controlled, cardiomyocyte-specific gene inactivation by adeno-associated virus delivery of Cre recombinase. PLoS ONE, 10(5), e0128105.

    Article  Google Scholar 

  23. Zhang, D., et al. (2018). Mitochondrial cardiomyopathy caused by elevated reactive oxygen species and impaired cardiomyocyte proliferation. Circulation Research, 122(1), 74–87.

    Article  CAS  Google Scholar 

  24. VanDusen, N. J., et al. (2017). CASAAV: A CRISPR-based platform for rapid dissection of gene function in vivo. Current Protocols in Molecular Biology. https://doi.org/10.1002/cpmb.46

    Article  PubMed  PubMed Central  Google Scholar 

  25. Guo, Y., & Pu, W. T. (2020). Cardiomyocyte maturation: new phase in development. Circulation Research, 126(8), 1086–1106.

    Article  CAS  Google Scholar 

  26. Akerberg, B. N., et al. (2019). A reference map of murine cardiac transcription factor chromatin occupancy identifies dynamic and conserved enhancers. Nature Communications, 10(1), 4907.

    Article  Google Scholar 

  27. Ho, C. Y., et al. (2018). Genotype and lifetime Burden of Disease in Hypertrophic Cardiomyopathy: Insights from the Sarcomeric Human Cardiomyopathy Registry (SHaRe). Circulation, 138(14), 1387–1398.

    Article  Google Scholar 

  28. Wang, S., et al. (2020). AAV Gene Therapy Prevents and Reverses Heart Failure in a Murine Knockout Model of Barth Syndrome. Circulation Research, 126(8), 1024–1039.

    Article  CAS  Google Scholar 

  29. Chen, J., et al. (2020). aYAP modRNA reduces cardiac inflammation and hypertrophy in a murine ischemia-reperfusion model. Life Sci Alliance, 3(1), e201900424.

    Article  Google Scholar 

  30. Chen, B., et al. (2018). Reactivation of Dormant Relay Pathways in Injured Spinal Cord by KCC2 Manipulations. Cell, 174(3), 521-535.e13.

    Article  CAS  Google Scholar 

  31. Suzuki-Hatano, S., Saha, M., Rizzo, S. A., Witko, R. L., Gosiker, B. J., Ramanathan, M., et al. (2019). AAV-Mediated TAZ gene replacement restores mitochondrial and cardioskeletal function in Barth Syndrome. Human Gene Therapy, 30(2), 139–154.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Prof. William T. Pu in Boston Children’s Hospital for providing the AAV-related plasmid and Cas9 genetic mice. Also they thank Prof. Long-Sheng Song in University of Iowa Carver College of Medicine for sharing the AutoTT software in T-Tubule analysis.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81700360).

Author information

Author notes

  1. Peng Yue and Shutao Xia contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, 610041, Sichuan, China

    Peng Yue, Gang Wu, Lei Liu, Kaiyu Zhou, Hongyu Liao, Jiawen Li, Xiaolan Zheng, Yimin Hua & Yifei Li

  2. State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Science, Hubei University, Wuhan, 430062, Hubei, China

    Shutao Xia & Donghui Zhang

  3. Department of Cardiology, Boston Children’s Hospital, Boston, MA, 02115, USA

    Yuxuan Guo

Authors
  1. Peng Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shutao Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gang Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kaiyu Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hongyu Liao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jiawen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xiaolan Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yuxuan Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yimin Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Donghui Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yifei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Donghui Zhang or Yifei Li.

Ethics declarations

Conflict of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Communicated by Shazina Saeed.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yue, P., Xia, S., Wu, G. et al. Attenuation of Cardiomyocyte Hypertrophy via Depletion Myh7 using CASAAV. Cardiovasc Toxicol 21, 255–264 (2021). https://doi.org/10.1007/s12012-020-09617-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12012-020-09617-y

Keywords

Search

Navigation