Rationale Mammalian cardiomyocytes withdraw from the cell cycle during early post-natal development, which significantly limits the capacity of the adult mammalian heart to regenerate following injury. The regulatory mechanisms which govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Objective Given the potential of microRNAs (miRNAs) to influence large gene networks and modify complex developmental and disease phenotypes, we searched for miRNAs that were regulated during the postnatal switch to terminal differentiation. Methods and Results Microarray analysis revealed subsets of miRNAs that were up- or down-regulated in cardiac ventricles from mice at 1- and 10-days of age (P1 and P10). Interestingly, miR-195 (a member of the miR-15 family) was the most highly up-regulated miRNA during this period, with expression levels almost 6-fold higher in P10 ventricles relative to P1. Precocious over-expression of miR-195 in the embryonic heart was associated with ventricular hypoplasia and ventricular septal defects in βMHC-miR-195 transgenic mice. Using global gene profiling and Argonaute-2 immunoprecipitation approaches, we show that miR-195 regulates the expression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1), which we identify as a highly conserved direct target of miR-195. Finally, we demonstrate that knock-down of the miR-15 family in neonatal mice using locked nucleic acid (LNA)-modified antimiRs is associated with an increased number of mitotic cardiomyocytes and de-repression of Chek1. Conclusions These findings suggest that up-regulation of the miR-15 family during the neonatal period may be an important regulatory mechanism governing cardiomyocyte cell cycle withdrawal and binucleation.

Changes in the cardiac metabolic milieu during the perinatal period redirect mitochondrial substrate preference from carbohydrates to fatty acids. Mechanisms responsible for this mitochondrial plasticity are unknown. Here we found that PINK1-Mfn2-Parkin mediated mitophagy directs this metabolic transformation in mice. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary for Parkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressing mitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expression of Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrial maturation essential to survival. Five week old Mfn2 AA hearts retained a fetal mitochondrial transcriptional signature without normal increases in fatty acid metabolism and mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels and cardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed. Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondria undergo perinatal Parkin-mediated mitophagy and replacement by mature adult mitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocyte mitochondrial plasticity and metabolic transitioning of perinatal hearts.

Abstractβ-adrenergic receptor (βAR) blockade is standard therapy for cardiac failure and ischemia. G-protein coupled receptor kinases (GRKs) desensitize βAR, suggesting that genetic GRK variants might modify outcomes in these syndromes. Re-sequencing of GRK2 and GRK5 revealed a nonsynonymous polymorphism of GRK5, common in African Americans (AA), substituting leucine (L) for glutamine (Q) at position 41. GRK5-L41 more effectively uncoupled isoproterenol-stimulated responses than GRK5-Q41 in transfected cells and transgenic mice, and like pharmacological βAR blockade, GRK5-L41 protected against experimental catecholamine-induced cardiomyopathy. Human association studies showed a pharmacogenomic interaction between GRK5-L41 and β-blocker treatment on mortality outcome in independent cohorts of AA cardiac failure (P=0.036) and ischemia (P=0.023). In 375 prospectively followed AA heart failure subjects, GRK5-L41 was protective against death/cardiac transplant (single allele: RR=0.28, 95% CI=0.12-0.66; two alleles: RR=0.08, 95% CI=0.04-0.19; P=0.004). The gain-of-function GRK5-L41 polymorphism facilitates βAR desensitization during catecholamine excess, imparting "genetic β-blockade" and improving survival in heart failure.

Rationale: MicroRNA (miR)-133a regulates cardiac and skeletal muscle differentiation and plays an important role in cardiac development. Because miR-133a levels decrease during reactive cardiac hypertrophy, some have considered that restoring miR-133a levels could suppress hypertrophic remodeling. Objective: To prevent the "normal" downregulation of miR-133a induced by an acute hypertrophic stimulus in the adult heart. Methods and Results: miR-133a is downregulated in transverse aortic constriction (TAC) and isoproterenolinduced hypertrophy, but not in 2 genetic hypertrophy models. Using MYH6 promoter-directed expression of a miR-133a genomic precursor, increased cardiomyocyte miR-133a had no effect on postnatal cardiac development assessed by measures of structure, function, and mRNA profile. However, increased miR-133a levels increased QT intervals in surface electrocardiographic recordings and action potential durations in isolated ventricular myocytes, with a decrease in the fast component of the transient outward K ؉ current, I to,f , at baseline. Transgenic expression of miR-133a prevented TAC-associated miR-133a downregulation and improved myocardial fibrosis and diastolic function without affecting the extent of hypertrophy. I to,f downregulation normally observed post-TAC was prevented in miR-133a transgenic mice, although action potential duration and QT intervals did not reflect this benefit. miR-133a transgenic hearts had no significant alterations of basal or post-TAC mRNA expression profiles, although decreased mRNA and protein levels were observed for the I to,f auxiliary KChIP2 subunit, which is not a predicted target. Conclusions: These results reveal striking differences between in vitro and in vivo phenotypes of miR expression, and further suggest that mRNA signatures do not reliably predict either direct miR targets or major miR effects. (Circ Res. 2010;106:166-175.)