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

Abstract

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.

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Code availability

All custom written code in this study is available at https://github.com/englea52/Englerlab. 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.

References

  1. 1.

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

  2. 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).

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 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).

  7. 7.

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

  8. 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).

  9. 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).

  10. 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).

  11. 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).

  12. 12.

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

  13. 13.

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

  14. 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. 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).

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 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).

  21. 21.

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

  22. 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).

  23. 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).

  24. 24.

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

  25. 25.

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

  26. 26.

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

  27. 27.

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

  28. 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).

  29. 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).

  30. 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).

  31. 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).

  32. 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).

  33. 33.

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

  34. 34.

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

  35. 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).

  36. 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).

  37. 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).

  38. 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).

  39. 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).

  40. 40.

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

  41. 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).

  42. 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).

  43. 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).

  44. 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).

  45. 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).

  46. 46.

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

  47. 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).

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Acknowledgements

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

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.

Correspondence to Adam J. Engler.

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The authors declare no competing interests.

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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.

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Further reading

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.