In this study, we found that elevated levels of miR-499 in the heart can lead to cardiomyocyte hypertrophy and cardiomyopathy in a dose-dependent manner. High levels of miR-499 led to spontaneous cardiac contractile dysfunction, while more modest levels of transgenic expression conferred susceptibility to pressure-induced dysfunction.
Our studies support an association between elevated cardiac miR-499 levels and cardiac dysfunction, particularly in the setting of pressure overload. Interestingly, in a study that analyzed microRNAs in human aortic valve stenosis, miR-499 levels were decreased 
, and it is possible that this decrement may regulate cardiac gene expression changes that are important in the physiologic response to cardiac pressure load. We suspect the failure of normal miR-499 down-regulation in our transgenic mice disrupted the normal response to cardiac pressure stress; similar theories have recently been put forward for cardiac transgenic mice expressing miR-133a 
. Interestingly, a recent study 
reported that miR-499 levels increase in human cardiac failure, and the findings in our transgenic mouse model may support a detrimental role for elevated miR-499 levels.
We identified a discrete set of altered transcripts in unmanipulated, normally functioning, miR-499 transgenic hearts, including natriuretic peptide precursor type B (Nppb
), ß-MyHC (Myh7
) and alpha 1 skeletal muscle actin (Acta1
). This suggests gene expression changes due to miR-499 that favor hypertrophy. These changes were confirmed in the second transgenic line. The limited magnitude of transcript changes by miR-499 suggests that the hypertrophic phenotype observed was not due to gross gene expression disruption. Although the Sox6 3′UTR could be targeted by miR-499, as we and others have demonstrated 
, Sox6 levels were not altered in the transgenic mice, suggesting that Sox6 was not targeted by increased miR-499 levels in vivo
, or that additional compensatory mechanisms may be involved in vivo
. It is possible that targets seen in luciferase assays may not necessarily translate into targeting in vivo
We present several lines of evidence that demonstrate that miR-499 levels fundamentally alter the immediate early gene response, which is known to be important in the cardiac stress response. First, Egr1 and Fos were the most diminished transcripts in TG-17 miR-499-altered hearts, suggesting a robust alteration in the immediate early gene expression program, while in TG-9 Egr1 was also altered (although Fos was not significantly changed). Second, inhibition of miR-499 in culture led to increases in induced Egr1 and Fos levels, which demonstrates that this regulation is present even outside of the context of the cardiac transgene. Pressure-induced stress, which is known to invoke the immediate early gene response, dramatically altered cardiac function in transgenic mouse hearts. We therefore hypothesize that miR-499 alters the cardiac response to stress in part by modulating the immediate early gene response.
The connection between miR-499 and the immediate early gene response is important since activation of this pathway is thought to precede further transcriptional responses to stress. Similar to TG-17 mice expressing miR-499, mice deficient in Egr1 are normal at baseline, but have an impaired response to cardiac stress 
, as do transgenic mice expressing the Egr1-repressor, Nab1 
. Interestingly, mice lacking the cardiac-specific microRNA miR-208a have decreased amounts of miR-499 
. Evaluation of genes reported to be dysregulated in miR-208a mutant mice revealed elevated levels of Egr1, Egr2, and Fos in the heart, suggesting a potential relationship between miR-499 and miR-208 in regulation of the immediate early gene response to cardiac stress. The observation that miR-499 transgenic mice develop or are predisposed to cardiac dysfunction may reflect the fact that the immediate early response genes hold a key position in the hierarchy of the cardiac transcriptional response to stress. It will be interesting to determine if levels of this non-coding RNA affect the progression of disease in the setting of pressure overload, such as in hypertension or aortic valve disease.