Abstract
Calsarcin-1 deficient mice develop dilated cardiomyopathy (DCM) phenotype in pure C57BL/6 genetic background (Cs1-ko) despite severe contractile dysfunction and robust activation of fetal gene program. Here we performed a microRNA microarray to identify the molecular causes of this cardiac phenotype that revealed the dysregulation of several microRNAs including miR-301a, which was highly downregulated in Cs1-ko mice compared to the wild-type littermates. Cofilin-2 (Cfl2) was identified as one of the potential targets of miR-301a using prediction databases, which we validated by luciferase assay and mutation of predicted binding sites. Furthermore, expression of miR-301a contrastingly regulated Cfl2 expression levels in neonatal rat ventricular cardiomyocytes (NRVCM). Along these lines, Cfl2 was significantly upregulated in Cs1-ko mice, indicating the physiological association between miR-301a and Cfl2 in vivo. Mechanistically, we found that Cfl2 activated serum response factor response element (SRF-RE) driven luciferase activity in neonatal rat cardiomyocytes and in C2C12 cells. Similarly, knockdown of miR301a activated, whereas, its overexpression inhibited the SRF-RE driven luciferase activity, further strengthening physiological interaction between miR-301a and Cfl2. Interestingly, the expression of SRF and its target genes was strikingly increased in Cs1-ko suggesting a possible in vivo correlation between expression levels of Cfl2/miR-301a and SRF activation, which needs to be independently validated. In summary, our data demonstrates that miR-301a regulates Cofilin-2 in vitro in NRVCM, and in vivo in Cs1-ko mice. Our findings provide an additional and important layer of Cfl2 regulation, which we believe has an extended role in cardiac signal transduction and dilated cardiomyopathy presumably due to the reported involvement of Cfl2 in these mechanisms.
Highlights
We have earlier reported that mice with Calsarcin-1 null mutation in a mixed genetic background do not exhibit any overt basal cardiac hypertrophy phenotype, show accelerated hypertrophic cardiomyopathy in response to pathological biomechanical stress [13]
Through a microRNA microarray we identified miR-301a is significantly downregulated in Calsarcin-1 deficient mice that present dilated cardiomyopathy (DCM) phenotype in a pure C57BL/6 background
Lygate et al ascertained that mitochondrial miR-301a regulates Cofilin-2 in the heart creatine kinase knockout mice do not display any cardiac phenotype in pure C57BL/6 genetic background [40], which earlier reportedly led to LV dysfunction and hypertrophy [41]
Summary
Several forms of cardiomyopathies including dilated (DCM), hypertrophic (HCM), and ischemic cardiomyopathy (ICM), can lead to heart failure which is linked to poor prognosis [1, 2]. Calsarcin-1 deficient mice are sensitized to Calcineurin signaling and show a massive hypertrophic cardiomyopathy when stressed biomechanically in a mixed genetic background without displaying a baseline hypertrophy phenotype [13]. We performed a microRNA microarray on cardiac RNA extracted from Calsarcin-1 deficient mice in a pure C57BL/6 background, which display a strict DCM phenotype. Through this screen, we identified miR-301a as the most downregulated microRNA that we found targets Cfl, a major regulator of actin dynamics, in vitro and in vivo. We believe that our findings provide an important layer in Cfl regulation, which needs further in vivo validations for exploiting the therapeutic potential of miR-301/Cfl interactions in cardiac signal transduction and DCM
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