Titin cleavage in living cardiomyocytes induces sarcomere disassembly but does not trigger cell proliferation

Adult mammalian hearts have limited regenerative capacity due to the inability of cardiomyocytes to proliferate, a major clinical hurdle in contemporary cardiology. The presence of highly organized, contractile sarcomeres has long been considered an impediment for cardiomyocyte division. Indeed, sarcomere disassembly is a crucial step to complete the cell cycle in the few situations where cardiomyocytes have been observed to proliferate. However, whether sarcomere disassembly can per se trigger cell cycle re-entry remains unknown, a possibility that we have tested here. In this study, we have engineered a system to induce sarcomere disassembly in living murine cardiomyocytes based on the specific cleavage of the structural protein titin by tobacco etch virus protease. Although isolated neonatal cardiomyocytes with disassembled sarcomeres remain viable and retain low-amplitude contractile activity, our results show no evidence of increased cardiomyocyte proliferation in targeted cells, as indicated by the analyses of markers of DNA synthesis and cytokinesis. We obtain equivalent results when titin is cleaved in cardiomyocytes stimulated with mitogenic factors in vitro and in the adult myocardium in vivo. These findings suggest that the removal of sarcomere structural barriers is necessary, but not sufficient, for cardiomyocyte proliferation, which implies that additional factors are required for cardiomyocytes to undergo cell division.