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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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.
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.
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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.).
The authors declare no competing interests.
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Supplementary methods, figures, tables and video captions.
Representative calcium imaging of R/R cardiomyocytes cultured on soft hydrogels.
Representative calcium imaging of N/N cardiomyocytes cultured on soft hydrogels.
Representative calcium imaging of R/R cardiomyocytes cultured on stiffened hydrogels.
Representative calcium imaging of N/N cardiomyocytes cultured on stiffened hydrogels.
Representative calcium imaging of R/R KO cardiomyocytes cultured on soft hydrogels.
Representative calcium imaging of R/R KO cardiomyocytes cultured on stiffened hydrogels.
Primers used for qPCR.
Raw data for figures where data was normalized.
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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). https://doi.org/10.1038/s41551-018-0344-5
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