Prompt coronary catheterization and revascularization have dramatically improved the outcome of myocardial infarction, but also have resulted in a growing number of survived patients with permanent structural damage of the heart, which frequently leads to heart failure. Finding new treatments for this condition is a largely unmet clinical need 1, especially because of the incapacity of cardiomyocytes to replicate after birth and thus achieve regeneration of the lost contractile tissue 2. Here we show that expression of human microRNA-199a in infarcted pig hearts is capable of stimulating cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, the treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. At longer follow-up, however, persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however this therapy needs to be tightly dosed.

A single administration of synthetic miRNA-lipid formulations is sufficient to stimulate cardiac repair and restoration of cardiac function.

Summary Loss of functional cardiomyocytes is a major determinant of heart failure after myocardial infarction. Previous high throughput screening studies have identified a few microRNAs (miRNAs) that can induce cardiomyocyte proliferation and stimulate cardiac regeneration in mice. Here, we show that all of the most effective of these miRNAs activate nuclear localization of the master transcriptional cofactor Yes-associated protein (YAP) and induce expression of YAP-responsive genes. In particular, miR-199a-3p directly targets two mRNAs coding for proteins impinging on the Hippo pathway, the upstream YAP inhibitory kinase TAOK1, and the E3 ubiquitin ligase β-TrCP, which leads to YAP degradation. Several of the pro-proliferative miRNAs (including miR-199a-3p) also inhibit filamentous actin depolymerization by targeting Cofilin2, a process that by itself activates YAP nuclear translocation. Thus, activation of YAP and modulation of the actin cytoskeleton are major components of the pro-proliferative action of miR-199a-3p and other miRNAs that induce cardiomyocyte proliferation.

In contrast to prenatal life, cardiomyocyte proliferation in mammals is rapidly blunted after birth; as a consequence, clinically significant cardiac regeneration does not occur in adulthood. Thus, the modulation of cardiomyocyte proliferation by gene transfer offers an invaluable opportunity to both understand the mechanisms regulating renewal of these cells in the fetus and identify novel strategies for myocardial repair.In this Chapter, we report an exhaustive protocol to isolate, culture, and manipulate the properties of neonatal ventricular rat cardiomyocytes by small RNA transfection or transduction with viral vectors based on the adeno-associated virus, which exhibit exquisite tropism for these cells. We also provide techniques to assess DNA synthesis and cell proliferation.