MEF2-regulated Gtl2-Dio3 noncoding RNAs in cardiac muscle and disease
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The MEF2 transcription factor is a central regulator of skeletal and cardiac muscle development. Recently, we showed that MEF2A regulates skeletal muscle regeneration through direct regulation of the Gtl2-Dio3 microRNA mega-cluster. In addition to their expression in skeletal muscle, temporal expression analysis of selected Gtl2-Dio3 microRNAs revealed high enrichment in cardiac muscle. Therefore, I investigated the role of selected microRNAs from the Gtl2-Dio3 noncoding RNA locus in the heart. First, I found that Gtl2-Dio3 microRNAs are expressed at higher levels in perinatal hearts compared to adult, suggesting they function in cardiac maturation shortly after birth. I also demonstrated that these microRNAs are dependent on MEF2A in the perinatal heart and in neonatal cardiomyocytes. To determine the specific role in cardiac muscle, I overexpressed selected microRNA mimics in neonatal rat ventricular myocytes (NRVMs). My results showed that miR-410 and miR-495 stimulate cell cycle re-entry and proliferation of terminally differentiated NRVMs. Subsequent target prediction analyses revealed a number of candidate target genes known to function in the cell cycle and/or in cardiac muscle. One of these was Cited2, a cofactor required for proper cardiac development. Subsequently, I showed that Cited2 is a direct target of these miRNAs and that siRNA knockdown of Cited2 in NRVMs resulted in robust cardiomyocyte proliferation. This phenotype was associated with reduced expression of Cdkn1c/p57/Kip2, a cell cycle inhibitor, and increased expression of Vegfa, a growth factor with proliferation-promoting effects. Given the exciting possibility of manipulating the expression of these microRNAs to repair the damaged heart by stimulating cardiomyocyte proliferation, I then investigated whether they were regulated in cardiac disease and function in pathological signaling. Toward this end, I examined expression of miR-410, miR-495, miR-433, as well as the Gtl2 lncRNA in various cardiomyopathies. Interestingly, the microRNAs and lncRNA were dynamically regulated in mouse models of cardiac disease including myocardial infarction and chronic angiotensin II stimulation. Furthermore, I showed for the first time that the Gtl2 lncRNA and miRNAs are differentially regulated in myocardial infarction, indicating that the complex regulation of the Gtl2-Dio3 noncoding RNA locus may be important for response to cardiac injury. Lastly, I showed that inhibiting select Gtl2-Dio3 microRNAs in pathological signaling attenuated cardiomyocyte hypertrophy in vitro. Therefore, differential targeting of the Gtl2-Dio3 noncoding RNAs could provide new therapeutic strategies to control the response of the heart to cardiac diseases with diverse etiologies.
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