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.

SARS-CoV-2 induced the novel coronavirus disease (COVID-19) outbreak, the most significant medical challenge in the last century. COVID-19 is associated with notable increases in morbidity and death worldwide. Preexisting conditions, like cardiovascular disease (CVD), diabetes, hypertension, and obesity, are correlated with higher severity and a significant increase in the fatality rate of COVID-19. COVID-19 induces multiple cardiovascular complexities, such as cardiac arrest, myocarditis, acute myocardial injury, stress-induced cardiomyopathy, cardiogenic shock, arrhythmias and, subsequently, heart failure (HF). The precise mechanisms of how SARS-CoV-2 may cause myocardial complications are not clearly understood. The proposed mechanisms of myocardial injury based on current knowledge are the direct viral entry of the virus and damage to the myocardium, systemic inflammation, hypoxia, cytokine storm, interferon-mediated immune response, and plaque destabilization. The virus enters the cell through the angiotensin-converting enzyme-2 (ACE2) receptor and plays a central function in the virus’s pathogenesis. A systematic understanding of cardiovascular effects of SARS-CoV2 is needed to develop novel therapeutic tools to target the virus-induced cardiac damage as a potential strategy to minimize permanent damage to the cardiovascular system and reduce the morbidity. In this review, we discuss our current understanding of COVID-19 mediated damage to the cardiovascular system.

Modified mRNA (modRNA) is a new technology in the field of somatic gene transfer that has been used for the delivery of genes into different tissues, including the heart. Our group and others have shown that modRNAs injected into the heart are robustly translated into the encoded protein and can potentially improve outcome in heart injury models. However, the optimal compositions of the modRNA and the reagents necessary to achieve optimal expression in the heart have not been characterized yet. In this study, our aim was to elucidate those parameters by testing different nucleotide modifications, modRNA doses, and transfection reagents both in vitro and in vivo in cardiac cells and tissue. Our results indicate that optimal cardiac delivery of modRNA is with N1-Methylpseudouridine-5'-Triphosphate nucleotide modification and achieved using 0.013 μg modRNA/mm/500 cardiomyocytes (CMs) transfected with positively charged transfection reagent in vitro and 100 μg/mouse heart (1.6 μg modRNA/μL in 60 μL total) sucrose-citrate buffer in vivo. We have optimized the conditions for cardiac delivery of modRNA in vitro and in vivo. Using the described methods and conditions may allow for successful gene delivery using modRNA in various models of cardiovascular disease.

Myocardial infarction (MI) and heart failure (HF) are the leading causes of death in the United States and in most other industrialized nations. MI leads to a massive loss of cardiomyocytes (CMs), which are replaced with non-CM cells, leading to scarring and, in most cases, HF. The adult mammalian heart has a low intrinsic regenerative capacity, mainly because of cell-cycle arrest in CMs. No effective treatment promoting heart regeneration is currently available. Recent efforts to use DNA-based or viral gene therapy approaches to induce cardiac regeneration post-MI or in HF conditions have encountered major challenges, mostly because of the poor and uncontrolled delivery of the introduced genes. Modified mRNA (modRNA) is a safe, non-immunogenic, efficient, transient, local, and controlled nucleic acid delivery system that can overcome the obstacles to DNA-based or viral approaches for cardiac gene delivery. We here review the use of modRNA in cardiac therapy, to induce cardioprotection and vascular or cardiac regeneration after MI. We discuss the current challenges in modRNA-based cardiac treatment, which will need to be overcome for the application of such treatment to ischemic heart disease.

The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are also utilized in cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.