Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mechanical activation of noncoding-RNA-mediated regulation of disease-associated phenotypes in human cardiomyocytes


How common polymorphisms in noncoding genome regions can regulate cellular function remains largely unknown. Here we show that cardiac fibrosis, mimicked using a hydrogel with controllable stiffness, affects the regulation of the phenotypes of human cardiomyocytes by a portion of the long noncoding RNA ANRIL, the gene of which is located in the disease-associated 9p21 locus. In a physiological environment, cultured cardiomyocytes derived from induced pluripotent stem cells obtained from patients who are homozygous for cardiovascular-risk alleles (R/R cardiomyocytes) or from healthy individuals who are homozygous for nonrisk alleles contracted synchronously, independently of genotype. After hydrogel stiffening to mimic fibrosis, only the R/R cardiomyocytes exhibited asynchronous contractions. These effects were associated with increased expression of the short ANRIL isoform in R/R cardiomyocytes, which induced a c-Jun N-terminal kinase (JNK) phosphorylation-based mechanism that impaired gap junctions (particularly, loss of connexin-43 expression) following stiffening. Deletion of the risk locus or treatment with a JNK antagonist was sufficient to maintain gap junctions and prevent asynchronous contraction of cardiomyocytes. Our findings suggest that mechanical changes in the microenvironment of cardiomyocytes can activate the regulation of their function by noncoding loci.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: MeHA synthesis and schematic of dynamic stiffening.
Fig. 2: Asynchronous calcium flux in R/R iPS cell-derived cardiomyocytes after dynamic stiffening.
Fig. 3: Gap junction remodelling in R/R iPS cell-derived cardiomyocytes after dynamic stiffening.
Fig. 4: JNK-mediated dysfunction in R/R cardiomyocytes in response to a stiffened hydrogel.
Fig. 5: Proposed mechanism for the regulation of 9p21 in cardiomyocytes.

Similar content being viewed by others

Code availability

All custom written code in this study is available at It contains code for the calcium handling analysis, correlation coefficient analysis and sarcomere analysis.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information. Source data for the figures are available from the corresponding author upon reasonable request.


  1. The 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015).

    Article  Google Scholar 

  2. Schaub, M. A., Boyle, A. P., Kundaje, A., Batzoglou, S. & Snyder, M. Linking disease associations with regulatory information in the human genome. Genome Res. 22, 1748–1759 (2012).

    Article  CAS  Google Scholar 

  3. Helgadottir, A. et al. A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science 316, 1491–1493 (2007).

    Article  CAS  Google Scholar 

  4. McPherson, R. Chromosome 9p21.3 locus for coronary artery disease: how little we know. J. Am. Coll. Cardiol. 62, 1382–1383 (2013).

    Article  CAS  Google Scholar 

  5. Roberts, R. & Stewart, A. F. R. 9p21 and the genetic revolution for coronary artery disease. Clin. Chem. 58, 104–112 (2012).

    Article  CAS  Google Scholar 

  6. Kim, J. B. et al. Effect of 9p21.3 coronary artery disease locus neighboring genes on atherosclerosis in mice. Circulation 126, 1896–1906 (2012).

    Article  Google Scholar 

  7. Visel, A. et al. Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 464, 409–412 (2010).

    Article  CAS  Google Scholar 

  8. Jarinova, O. et al. Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus. Arterioscler. Thromb. Vasc. Biol. 29, 1671–1677 (2009).

    Article  CAS  Google Scholar 

  9. Holdt, L. M. & Teupser, D. Recent studies of the human chromosome 9p21 locus, which is associated with atherosclerosis in human populations. Arterioscler. Thromb. Vasc. Biol. 32, 196–206 (2012).

    Article  CAS  Google Scholar 

  10. Congrains, A., Kamide, K., Ohishi, M. & Rakugi, H. ANRIL: molecular mechanisms and implications in human health. Int. J. Mol. Sci. 14, 1278–1292 (2013).

    Article  CAS  Google Scholar 

  11. Holdt, L. M. et al. Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks. PLoS Genet. 9, e1003588 (2013).

    Article  CAS  Google Scholar 

  12. Sun, N. et al. Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. Sci. Transl. Med. 4, 130ra47 (2012).

    Article  Google Scholar 

  13. Larson, M. G. et al. Framingham Heart Study 100k project: genome-wide associations for cardiovascular disease outcomes. BMC Med. Genet. 8, S5 (2007).

    Article  Google Scholar 

  14. Lo Sardo, V. et al. Unveiling the role of the most impactful cardiovascular risk locus through haplotype editing. Cell 175, 1796–1810 (2018).

  15. Berry, M. F. et al. Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. Am. J. Physiol. Heart Circ. Physiol. 290, H2196–H2203 (2006).

    Article  CAS  Google Scholar 

  16. Lahtinen, A. M. et al. Common genetic variants associated with sudden cardiac death: the FinSCDgen study. PLoS ONE 7, e41675 (2012).

    Article  CAS  Google Scholar 

  17. Newton-Cheh, C. et al. A common variant at 9p21 is associated with sudden and arrhythmic cardiac death. Circulation 120, 2062–2068 (2009).

    Article  CAS  Google Scholar 

  18. Ondeck, M. G. & Engler, A. J. Mechanical characterization of a dynamic and tunable methacrylated hyaluronic acid hydrogel. J. Biomech. Eng. 138, 021003 (2016).

    Article  Google Scholar 

  19. Guvendiren, M. & Burdick, J. A. Stiffening hydrogels to probe short- and long-term cellular responses to dynamic mechanics. Nat. Commun. 3, 792 (2012).

    Article  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Young, J. L. & Engler, A. J. Hydrogels with time-dependent material properties enhance cardiomyocyte differentiation in vitro. Biomaterials 32, 1002–1009 (2012).

    Article  Google Scholar 

  22. Jacot, J. G., McCulloch, A. D. & Omens, J. H. Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes. Biophys. J. 95, 3479–3487 (2008).

    Article  CAS  Google Scholar 

  23. Fomovsky, G. M. & Holmes, J. W. Evolution of scar structure, mechanics, and ventricular function after myocardial infarction in the rat. Am. J. Physiol. Heart Circ. Physiol. 298, H221–H228 (2010).

    Article  CAS  Google Scholar 

  24. Sun, Y. & Weber, K. T. Infarct scar: a dynamic tissue. Cardiovasc. Res. 46, 250–256 (2000).

    Article  CAS  Google Scholar 

  25. Stauffer, B. L., Sobus, R. & Sucharov, C. C. Sex differences in cardiomyocyte connexin43 expression. J. Cardiovasc. Pharmacol. 58, 32–39 (2011).

  26. Kostin, S. Zonula occludens-1 and connexin 43 expression in the failing human heart. J. Cell. Mol. Med. 11, 892–895 (2007).

    Article  Google Scholar 

  27. Harvey, P. A. & Leinwand, L. A. The cell biology of disease: cellular mechanisms of cardiomyopathy. J. Cell Biol. 194, 355–365 (2011).

    Article  CAS  Google Scholar 

  28. Bupha-Intr, T., Haizlip, K. M. & Janssen, P. M. L. Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro. Am. J. Physiol. Heart Circ. Physiol. 296, H806–H814 (2009).

    Article  CAS  Google Scholar 

  29. Monteiro da Rocha, A. et al. Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived cardiomyocytes. J. Mol. Cell. Cardiol. 99, 197–206 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Folkersen, L. et al. Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants. PLoS ONE 4, e7677 (2009).

    Article  Google Scholar 

  32. Almontashiri, N. A. M. et al. Interferon-γ activates expression of p15 and p16 regardless of 9p21.3 coronary artery disease risk genotype. J. Am. Coll. Cardiol. 61, 143–147 (2013).

    Article  CAS  Google Scholar 

  33. Roberts, R. et al. Identifying genes for coronary artery disease: an idea whose time has come. Can. J. Cardiol. 23, 7A–15A (2007).

    Article  Google Scholar 

  34. Samani, N. J. & Schunkert, H. Chromosome 9p21 and cardiovascular disease: the story unfolds. Circ. Cardiovasc. Genet. 1, 81–84 (2008).

    Article  CAS  Google Scholar 

  35. Choi, B. Y. et al. The tumor suppressor p16INK4a prevents cell transformation through inhibition of c-Jun phosphorylation and AP-1 activity. Nat. Struct. Mol. Biol. 12, 699–707 (2005).

    Article  CAS  Google Scholar 

  36. Yan, J. et al. c-Jun N-terminal kinase activation contributes to reduced connexin43 and development of atrial arrhythmias. Cardiovasc. Res. 97, 589–597 (2013).

    Article  CAS  Google Scholar 

  37. Warn-Cramer, B. J., Cottrell, G. T., Burt, J. M. & Lau, A. F. Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase. J. Biol. Chem. 273, 9188–9196 (1998).

    Article  CAS  Google Scholar 

  38. Petrich, B. G. et al. Targeted activation of c-Jun N-terminal kinase in vivo induces restrictive cardiomyopathy and conduction defects. J. Biol. Chem. 279, 15330–15338 (2004).

    Article  CAS  Google Scholar 

  39. Zhang, X. et al. AMPK suppresses connexin43 expression in the bladder and ameliorates voiding dysfunction in cyclophosphamide-induced mouse cystitis. Sci. Rep. 6, 19708 (2016).

    Article  CAS  Google Scholar 

  40. Harismendy, O. et al. 9p21 DNA variants associated with coronary artery disease impair interferon-γ signaling response. Nature 470, 264–268 (2011).

    Article  CAS  Google Scholar 

  41. Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/Î2-catenin signaling under fully defined conditions. Nat. Protocols 8, 162–175 (2013).

    Article  CAS  Google Scholar 

  42. Severs, N. J., Bruce, A. F., Dupont, E. & Rothery, S. Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc. Res. 80, 9–19 (2008).

    Article  CAS  Google Scholar 

  43. Chun, Y. W. et al. Differential responses of induced pluripotent stem cell-derived cardiomyocytes to anisotropic strain depends on disease status. J. Biomech. 48, 3890–3896 (2015).

    Article  Google Scholar 

  44. Almontashiri, N. A. M. et al. 9p21.3 coronary artery disease risk variants disrupt TEAD transcription factor-dependent transforming growht factor β regulation of p16 expression in human aortic smooth muscle cells.Circulation 132, 1969–1978 (2015).

    Article  CAS  Google Scholar 

  45. Motterle, A. et al. Functional analyses of coronary artery disease associated variation on chromosome 9p21 in vascular smooth muscle cells. Hum. Mol. Genet. 21, 4021–4029 (2012).

    Article  CAS  Google Scholar 

  46. Petrich, B. G. et al. c-Jun N-terminal kinase activation mediates downregulation of connexin43 in cardiomyocytes. Circ. Res. 91, 640–647 (2002).

    Article  CAS  Google Scholar 

  47. Clements, R. T., Feng, J., Cordeiro, B., Bianchi, C. & Sellke, F. W. p38 MAPK-dependent small HSP27 and αB-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest. Am. J. Physiol. Heart Circ. Physiol. 300, H1669–H1677 (2011).

    Article  CAS  Google Scholar 

Download references


We thank M. Ondeck, C. Happe, K. Vincent, X. Ma and A. McCulloch for technical support and helpful discussions regarding cardiomyocyte differentiation, functional assays and pacing; F. Urnov for design of the TALE nucleases. National Institutes of Health (NIH) grants R01AG045428 (A.J.E.), UL1RR025774 and U01HL107436 (Scripps Translational Science Institute, E.J.T. and K.K.B.), U54GM114833 (A.T.) and F32HL126406 (J.K.P.) supported this work. NIH grants T32HL105373 (E.J.T.) and T32AR060712 (A.K.) and the ARCS/Roche Foundation Scholar Award Program in the Life Science (A.K.) provided trainee support. National Science Foundation grant 1463689 (A.J.E.) and the graduate fellowship program (A.K.) also provided support. UC San Diego Frontiers of Innovation Scholars Program award 2-U1041 also provided trainee support (S.K.T.).

Author information

Authors and Affiliations



A.K., A.T., E.J.T., K.K.B. and A.J.E. conceived various aspects of the project. V.L.S. and W.C.F. generated iPS cell lines from peripheral blood mononuclear cells and performed TALEN gene editing. A.K., S.K.T., K.C.W., D.S.C., Y.-H.H. and K.P.T. differentiated cells and performed all cell culture experiments. Signalling analyses were performed by A.K. and J.K.P. Experiments were designed and the manuscript was written by A.K., A.T., E.J.T., K.K.B. and A.J.E. with input from the other authors.

Corresponding author

Correspondence to Adam J. Engler.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods, figures, tables and video captions.

Reporting Summary

Supplementary Video 1

Representative calcium imaging of R/R cardiomyocytes cultured on soft hydrogels.

Supplementary Video 2

Representative calcium imaging of N/N cardiomyocytes cultured on soft hydrogels.

Supplementary Video 3

Representative calcium imaging of R/R cardiomyocytes cultured on stiffened hydrogels.

Supplementary Video 4

Representative calcium imaging of N/N cardiomyocytes cultured on stiffened hydrogels.

Supplementary Video 5

Representative calcium imaging of R/R KO cardiomyocytes cultured on soft hydrogels.

Supplementary Video 6

Representative calcium imaging of R/R KO cardiomyocytes cultured on stiffened hydrogels.

Supplementary Dataset 1

Primers used for qPCR.

Supplementary Dataset 2

Raw data for figures where data was normalized.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kumar, A., Thomas, S.K., Wong, K.C. et al. Mechanical activation of noncoding-RNA-mediated regulation of disease-associated phenotypes in human cardiomyocytes. Nat Biomed Eng 3, 137–146 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing