PROJECT SUMMARY Ischemic heart disease (IHD) is the single largest cause of death worldwide. A heart attack or myocardial infarction (MI) results from limitation of coronary blood flow to the heart, causing ischemia and ultimately irreversible death of cardiomyocytes. The size of a myocardial infarct correlates with the degree of deterioration of heart function, compromise of contractile reserve, and overtime the likelihood of mortality from heart failure (HF). Prompt restoration of arterial perfusion with thrombolytic and antiplatelet therapy during percutaneous coronary intervention has led to a decline in acute mortality from MI. However, the prevalence of HF among survivors has augmented, because irreversible cardiomyocyte death results in a residual inducible ischemia and permanent scarring. A major pathologic problem is the failure of human adult cardiomyocytes to regenerate themselves endogenously following a MI. This is compounded by a lack of adjunctive treatments, pharmacologic or cellular, that can be administered in conjunction with reperfusion, or after to stimulate regeneration of heart muscle. Effective promotion of endogenous cardiomyocyte regeneration in the ischemic heart with concomitant reduction of scar size would potentially offer a powerful new treatment of MI and its adverse pathophysiologic consequences. Inhibition of a specific combination of four MicroRNAs (miR); miR-99, miR-100, let-7a and let-7c, is a critical regulator of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. The sequences and target proteins of these miRs are conserved in humans. In vivo, adeno-associated virus (AAV) delivery of inhibitors of these miRs into the hearts of mice with left coronary artery ligation increases the expression of the beta subunit of farnesyl-transferase and SWI/SNF-related matrix associated actin-dependent regulator of chromatin subfamily a, member 5. Cardiac regeneration was confirmed by the expression of proliferation and cytokinesis markers, labeled uridine incorporation into DNA, together with scar tissue regression and heart functional improvement. A disadvantage of viral delivery is the high prevalence of anti-AAV antibodies in humans that could neutralize their effect. As an alternative, JAAN Biotherapeutics will investigate whether two synthetic, specific, potent and nuclease resistant nucleic acid miR inhibitors (anti-miRs) to miR-99/100 and let-7a/c can reduce ischemic injury in an experimental murine model of ischemic reperfusion and constitute an innovative approach to regenerate human cardiomyocytes. [The research in Phase I will define whether the anti-miRs proliferate normal heart muscle, provide information on an effective dose and sustainability of effect, and establish any proliferative or off-target side effects in remote tissues (Aim 1). Aim 2 will study whether the anti- miRs administered after a 60 min cardiac ischemic injury in mice either at the time of reperfusion or 2 days after ischemia can reduce scar size, increase cardiac function and regenerate cardiac muscle. Aim 1 will provide safety information and Aim 2 is critical to confirm efficacy of the anti-miRs in the murine ischemic heart. These studies are pivotal for future preclinical and clinical study design.]