Rebecca J Zaunbrecher1,2,3, Ashley N Abel2,3, Kevin Beussman4,2,3, Andrea Leonard4,2,3, Marion von Frieling-Salewsky5, Paul A Fields6,2,3, Lil Pabon6,2,3, Hans Reinecke6,2,3, Xiulan Yang6,2,3, Jesse Macadangdang1,2,3, Deok-Ho Kim1,2,3, Wolfgang A Linke5,7, Nathan J Sniadecki4,2,3, Michael Regnier1,2,3, Charles E Murry1,6,2,3,8. 1. Department of Bioengineering (R.J.Z., J.M., D.-H.K., M.R., C.E.M.), University of Washington, Seattle. 2. Center for Cardiovascular Biology (R.J.Z., A.N.A., K.B., A.L., P.A.F., L.P., H.R., X.Y., J.M., D.-H.K., N.J.S., M.R., C.E.M.), University of Washington, Seattle. 3. Institute for Stem Cell and Regenerative Medicine (R.J.Z., A.N.A., K.B., A.L., P.A.F., L.P., H.R., X.Y., J.M., D.-H.K., N.J.S., M.R., C.E.M.), University of Washington, Seattle. 4. Department of Mechanical Engineering (K.B., A.L., N.J.S.), University of Washington, Seattle. 5. Institute of Physiology II, University of Muenster, Germany (M.v.F.-S., W.A.L.). 6. Department of Pathology (P.A.F., L.P., H.R., X.Y., C.E.M.), University of Washington, Seattle. 7. Deutsches Zentrum für Herz-Kreislaufforschung, Partner Site Goettingen, Germany (W.A.L.). 8. Department of Medicine/Cardiology (C.E.M.), University of Washington, Seattle.
Abstract
BACKGROUND: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (TTN) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. METHODS: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z-/-) and A-band (TTN-A-/-) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. RESULTS: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z-/--CMs had sarcomeres and visibly contracted, whereas TTN-A-/--CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z-/--CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z-/--CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. CONCLUSIONS: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.
BACKGROUND: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (TTN) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. METHODS: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z-/-) and A-band (TTN-A-/-) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. RESULTS: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z-/--CMs had sarcomeres and visibly contracted, whereas TTN-A-/--CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z-/--CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z-/--CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. CONCLUSIONS: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to humancardiac disease.
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