Measuring cardiomyocyte cell-cycle activity and proliferation in the age of heart regeneration. American journal of physiology. Heart and circulatory physiology During the past two decades, the field of mammalian myocardial regeneration has grown dramatically, and with this expanded interest comes increasing claims of experimental manipulations that mediate bona fide proliferation of cardiomyocytes. Too often, however, insufficient evidence or improper controls are provided to support claims that cardiomyocytes have definitively proliferated, a process that should be strictly defined as the generation of two de novo functional cardiomyocytes from one original cardiomyocyte. Throughout the literature, one finds inconsistent levels of experimental rigor applied, and frequently the specific data supplied as evidence of cardiomyocyte proliferation simply indicate cell-cycle activation or DNA synthesis, which do not necessarily lead to the generation of new cardiomyocytes. In this review, we highlight potential problems and limitations faced when characterizing cardiomyocyte proliferation in the mammalian heart, and summarize tools and experimental standards, which should be used to support claims of proliferation-based remuscularization. In the end, definitive establishment of de novo cardiomyogenesis can be difficult to prove; therefore, rigorous experimental strategies should be used for such claims. 10.1152/ajpheart.00666.2021

Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration. Circulation BACKGROUND:The adult mammalian heart has limited regenerative capacity, mostly attributable to postnatal cardiomyocyte cell cycle arrest. In the last 2 decades, numerous studies have explored cardiomyocyte cell cycle regulatory mechanisms to enhance myocardial regeneration after myocardial infarction. Pkm2 (Pyruvate kinase muscle isoenzyme 2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. The role of Pkm2 in cardiomyocyte proliferation, heart development, and cardiac regeneration is unknown. METHODS:We investigated the effect of Pkm2 in cardiomyocytes through models of loss (cardiomyocyte-specific Pkm2 deletion during cardiac development) or gain using cardiomyocyte-specific Pkm2 modified mRNA to evaluate Pkm2 function and regenerative affects after acute or chronic myocardial infarction in mice. RESULTS:Here, we identify Pkm2 as an important regulator of the cardiomyocyte cell cycle. We show that Pkm2 is expressed in cardiomyocytes during development and immediately after birth but not during adulthood. Loss of function studies show that cardiomyocyte-specific Pkm2 deletion during cardiac development resulted in significantly reduced cardiomyocyte cell cycle, cardiomyocyte numbers, and myocardial size. In addition, using cardiomyocyte-specific Pkm2 modified RNA, our novel cardiomyocyte-targeted strategy, after acute or chronic myocardial infarction, resulted in increased cardiomyocyte cell division, enhanced cardiac function, and improved long-term survival. We mechanistically show that Pkm2 regulates the cardiomyocyte cell cycle and reduces oxidative stress damage through anabolic pathways and β-catenin. CONCLUSIONS:We demonstrate that Pkm2 is an important intrinsic regulator of the cardiomyocyte cell cycle and oxidative stress, and highlight its therapeutic potential using cardiomyocyte-specific Pkm2 modified RNA as a gene delivery platform. 10.1161/CIRCULATIONAHA.119.043067