AMPK regulates connexin 43 transcriptionally and post-transcriptionally in cardiac fibroblasts: Implication of miR-125b

Abstract

Cardiac fibroblasts (CFs) are crucial components of the fibrotic response following myocardial infarction (MI). We have previously shown that AMPK is a key regulator of CF properties and fibrosis. Our data suggest that AMPK exerts anti-fibrotic effects by regulating connexin 43 (Cx43) expression, a protein known to modulate CF activity and cardiac fibrosis. The present study sought to decipher the molecular mechanisms underlying Cx43 regulation by AMPK in CFs. Cultured human CFs (HCFs) were transfected with a specific siRNA targeting AMPK. Transcriptional and post-transcriptional regulation of Cx43 was assessed by luciferase reporter assay, actinomycin and proteasome inhibitor experiments. Its post-transcriptional regulation was further investigated using microRNAs (miRs) array, mimics and antimiRs. In cultured HCFs, AMPK deletion is associated with a drastic reduction of Cx43 protein expression. We demonstrated that proteasome is not involved in this regulation. Using luciferase reporter assay, we showed that AMPK deficiency decreases Cx43 promoter activity by 40%, indicating that AMPK contributes to the transcriptional regulation of Cx43. In addition, we provided evidence that AMPK can also affect Cx43 mRNA stability, suggesting the potential implication of miRs in the control of CX43 expression. Accordingly, the analysis of miRs profiling with microarrays revealed that several miRs are dysregulated in AMPK-depleted HCFs. Among them, in silico analysis identified miR-125b as a Cx43 regulator. We then confirmed that transfection of miR-125b mimic down-regulates Cx43 in HCFs. Importantly, inhibition of miR-125b expression using antimiR prevents Cx43 loss in absence of AMPK. Collectively, our data demonstrate that AMPK regulates Cx43 in CFs, transcriptionally and post-transcriptionally, by a mechanism involving miR-125b. Further investigations are needed to define whether miR-125b might be a pro-fibrotic biomarker.