Role of microRNA-582 in cardiac signal transduction, hypertrophy and heart failure
Final Report Abstract
In a microRNA microarray screen performed in a cardiomyopathy model (Muscle LIM protein (MLP) knockout mice) we identified miR-582 to be highly upregulated. For this microRNA no cardiac function was known so far. We also observed differential regulation of miR-582 in several mouse models of cardiac hypertrophy as well as in vitro in stressed cardiomyocytes. These findings led to the hypothesis that this microRNA might play an important role during the development or progression of cardiac muscle diseases. Interestingly, microRNA-582 is localized in an intron of the cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D) gene. In mice, ablation of PDE4D leads to the development of cardiac hypertrophy. Of note, the PDE4D-isoform PDE4D7 is also significantly upregulated in MLP knockout mice, along with microRNA-582. One major aim of this proposal was thus to shed light on the function of miR-582 under stress conditions in different mouse models. Yet, in contrast to our initial hypothesis, we could not observe any changes in echocardiographic or morphometric cardiac parameters in miR-582-KO mice at baseline nor after transverse aortic constriction (TAC)-surgery or in miR-582-KO or TG582 mice crossbred with Calsarcin 1 (CS1)-KO mice, another mouse model for dilated cardiomyopathy. However, on gene expression level we observed an inhibition of the induction of hypertrophic gene markers in female CS1-KO/miR-582-KO mice. The same effect was found in miR-582-KO mice after TAC-surgery: the induction of hypertrophic gene markers like Nppa and Nppb by TAC-surgery was attenuated in miR-582-KO mice, pointing to a potential protective role of the knockout of miR-582 in cardiomyopathy, that only becomes visible on gene expression level and is not able to improve cardiac function on the organ level. Another important goal was to analyze target genes for miR-582. We identified seven potential target genes in silico and measured their expression in vitro in neonatal rat cardiomyocytes (NRVCM) after overexpression and inhibition of miR-582 respectively and could not find a significant regulation for any of them. The same holds true for heart tissue obtained from miR-582-KO animals (8 weeks and 1 year of age) and TG582 animals. Additionally, we further analyzed the host gene of miR-582, PDE4D, and especially the isoform PDE4D7, which we found to be coregulated with miR-582. We could show, that PDE4D7 overexpression induces the expression of hypertrophic marker genes and an increase of cell size of cardiomyocytes. This effect is even more pronounced after induction of hypertrophy by PE (phenylephrine). Localization of PDE4D7 in cardiomyocytes seemed not be altered after induction of hypertrophy by PE. Taken together, our work showed that miR-582, though regulated under a variety of cardiac stress conditions, on its own is not sufficient to affect the development or progression of cardiomyopathies beyond altered gene expression level. Nevertheless, it provides the basis for ongoing work on the analysis of PDE4D7 in cardiac disease.
