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.)

Background-Much has been learned about transcriptional control of cardiac gene expression in clinical and experimental congestive heart failure (CHF), but less is known about dynamic regulation of microRNAs (miRs) in CHF and during CHF treatment. We performed comprehensive microarray profiling of miRs and messenger RNAs (mRNAs) in myocardial specimens from human CHF with (nϭ10) or without (nϭ17) biomechanical support from left ventricular assist devices in comparison to nonfailing hearts (nϭ11). Methods and Results-Twenty-eight miRs were upregulated Ͼ2.0-fold (PϽ0.001) in CHF, with nearly complete normalization of the heart failure miR signature by left ventricular assist device treatment. In contrast, of 444 mRNAs that were altered by Ͼ1.3-fold in failing hearts, only 29 mRNAs normalized by as much as 25% in post-left ventricular assist device hearts. Unsupervised hierarchical clustering of upregulated miRs and mRNAs with nearest centroid analysis and leave-1-out cross-validation revealed that combining the miR and mRNA signatures increased the ability of RNA profiling to serve as a clinical biomarker of diagnostic group and functional class. Conclusions-These results show that miRs are more sensitive than mRNAs to the acute functional status of end-stage heart failure, consistent with important functions for regulated miRs in the myocardial response to stress. Combined miR and mRNA profiling may have superior potential as a diagnostic and prognostic test in end-stage cardiomyopathy.

Rationale Mitochondria interact via actions of outer and inner membrane fusion proteins. The role of mitochondrial fusion on functioning of the heart, where mitochondria comprise ~30% of cardiomyocyte volume and their inter-myofilament spatial arrangement with other mitochondria is highly ordered, is unknown. Objective Model and analyze mitochondrial fusion defects in Drosophila melanogaster heart tubes with tincΔ4Gal4-directed expression of RNAi for Mitochondrial Assembly Regulatory Factor (MARF) and Optic atrophy 1 (Opa1). Methods and Results Live imaging analysis revealed that heart tube-specific knockdown of MARF or Opa1 increases mitochondrial morphometric heterogeneity and induces heart tube dilation with profound contractile impairment. Sarcoplasmic reticular structure was unaffected. Cardiomyocyte expression of human mitofusin (mfn) 1 or 2 rescued MARF RNAi cardiomyopathy, demonstrating functional homology between Drosophila MARF and human mitofusins. Suppressing mitochondrial fusion increased compensatory expression of nuclear-encoded mitochondrial genes, indicating mitochondrial biogenesis. The MARF RNAi cardiomyopathy was prevented by transgenic expression of superoxide dismutase 1. Conclusions Mitochondrial fusion is essential to cardiomyocyte mitochondrial function and regeneration. Reactive oxygen species are key mediators of cardiomyopathy in mitochondrial fusion-defective cardiomyocytes. Postulated mitochondrial-ER interactions mediated uniquely by mfn2 appear dispensable to functioning of the fly heart.

Rationale MicroRNA-499 and other members of the myomiR family regulate myosin isoforms in pressure overload hypertrophy. miR-499 expression varies in human disease but results of mouse cardiac miR-499 overexpression are inconsistent, either protecting against ischemic damage or aggravating cardiomyopathy after pressure overload. Likewise, there is disagreement over direct and indirect cardiac mRNAs targeted in vivo by miR-499. Objectives 1. Define the associations between regulated miR-499 level in clinical and experimental heart disease and modulation of its predicted mRNA targets. 2. Determine the consequences of increased cardiac miR-499 on direct mRNA targeting, indirect mRNA modulation, and on myocardial protein content and post-translational modification. Methods and Results miR-499 levels were increased in failing and hypertrophied human hearts, and associated with decreased levels of predicted target mRNAs. Likewise, miR-499 is increased in Gq-mediated murine cardiomyopathy. Forced cardiomyocyte expression of miR-499 at levels comparable to human cardiomyopathy induced progressive murine heart failure and exacerbated cardiac remodeling after pressure overloading. Genome-wide RISC- and RNA-sequencing identified 67 direct, and numerous indirect, cardiac mRNA targets including Akt and MAPKs. Myocardial proteomics identified alterations in protein phosphorylation linked to the miR-499 cardiomyopathy phenotype, including of HSP90 and PP1α. Conclusions miR-499 is increased in human and murine cardiac hypertrophy and cardiomyopathy, is sufficient to cause murine heart failure, and accelerates maladaptation to pressure overloading. The deleterious effects of miR-499 reflect the cumulative consequences of direct and indirect mRNA regulation, modulation of cardiac kinase and phosphatase pathways, and higher order effects on post-translational modification of myocardial proteins.