PROJECT SUMMARY Ischemic heart failure (HF) secondary to chronic post myocardial infraction (MI), is a leading cause of death and disability worldwide. The high morbidity and mortality rates in ischemic HF are caused, in large part, by the low regenerative capacity (and therefore repair) of the mammalian adult heart. Several tissue-engineering, cell- and gene-based therapies have been attempted for induction of cardiac regeneration following MI. Since cardiomyocyte (CM) proliferation and cardiac regeneration are highly robust processes in Zebrafish and Newts, efforts to mimic the pro-proliferative mechanisms of these lower phylogenetic organisms in the adult mammalian heart have been attempted using standard gene therapy vectors. Those approaches however, have encountered major challenges that are related to short-term, low, or uncontrolled delivery of the target gene. At best, this has resulted in poor CM proliferation. In other cases, these strategies have led to highly undesirable outcomes, including exacerbation of pathological hypertrophy and inflammation. We have recently shown that modified mRNA (modRNA) delivery is a safe, efficient, transient, non-immunogenic, local, and dose-controlled cardiac gene delivery platform. We have found that the glycolytic enzyme Pyruvate Kinase Muscle Isozyme M2 (Pkm2), which is highly expressed in regenerative fetal and neonatal CMs but not in adult CMs is an effective vehicle for re-awakening cardiac regeneration when delivered at the time of MI. Selective Pkm2 modRNA delivery to distinct cell populations led to a significant increase in their respective rates of proliferation. Proliferation of non-CMs such as myofibroblasts may exacerbate pathological remodeling by impairing diastolic function, disrupting myocardial conduction, and promoting arrhythmias. To circumvent this, we have designed a unique CM-specific modRNA-based approach for improving post-MI cardiac repair and regeneration. This proposal will identify the optimal therapeutic widow for modRNA-mediated delivery of Pkm2 to CMs following the phase of active healing for effective reversal of electro-mechanical dysfunction, will determine the molecular mechanisms by which local and transient delivery of Pkm2 promotes CM proliferation via β-catenin and downstream signaling, and finally determine the altered conduction properties and risk of arrhythmias associated with efficient integration of newly-formed Pkm2-dependent CMs with the host myocardium post MI. The highly translatable nature of our proposed modRNA platform and our integrative, multi-disciplinary experimental plan are ultimately geared towards clinical applicability for ischemic heart failure.