Background- m6A methylation is the most prevalent internal post-transcriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known. Methods- To determine the role of m6A methylation in the heart we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase METTL3 both in culture and in vivo. We generated cardiac-restricted gain and loss of function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function. Results- We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, while increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for maintenance of cardiac homeostasis. Conclusions- Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

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.

Background In the heart acute injury induces a fibrotic healing response that generates collagen rich scarring that is at first protective but if inappropriately sustained can worsen heart disease. The fibrotic process is initiated by cytokines, neuroendocrine effectors and mechanical strain that promote resident fibroblast differentiation into contractile and extracellular matrix producing myofibroblasts. The mitogen-activated protein kinase (MAPK) p38α (Mapk14 gene) is known to influence the cardiac injury response, but its direct role in orchestrating programmed fibroblast differentiation and fibrosis in vivo is unknown. Methods A conditional Mapk14 allele was used to delete the p38α encoding gene specifically in cardiac fibroblasts or myofibroblasts using 2 different tamoxifen-inducible Cre recombinase expressing gene-targeted mouse lines. Mice were subjected to ischemic injury or chronic neurohumoral stimulation and monitored for survival, cardiac function and fibrotic remodeling. Antithetically, mice with fibroblast-specific transgenic overexpression of activated MAPK kinase 6 (MKK6), a direct inducer of p38, were generated to investigate if this pathway can directly drive myofibroblast formation and the cardiac fibrotic response. Results In mice loss of Mapk14 blocked cardiac fibroblast differentiation into myofibroblasts and ensuing fibrosis in response to ischemic injury or chronic neurohumoral stimulation. A similar inhibition of myofibroblast formation and healing was also observed in a dermal wounding model with deletion of Mapk14. Transgenic mice with fibroblast-specific activation of MKK6-p38 developed interstitial and perivascular fibrosis in the heart, lung and kidney due to enhanced myofibroblast numbers. Mechanistic experiments show that p38 transduces cytokine and mechanical signals into myofibroblast differentiation through the transcription factor serum-response factor (SRF) and the signaling effector calcineurin. Conclusions These findings suggest that signals from diverse modes of injury converge on p38α MAPK within the fibroblast to program the fibrotic response and myofibroblast formation in vivo, suggesting a novel therapeutic approach with p38 inhibitors for future clinical application.