Cyclic stretching boosts microRNA‐499 to regulate Bcl‐2 via microRNA‐208a in atrial fibroblasts

MicroRNAs that modulate transcription can regulate other microRNAs and are also up‐regulated under pathological stress. MicroRNA‐499 (miR‐499), microRNA‐208a (miR‐208a) and B‐cell lymphoma 2 (Bcl‐2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl‐2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts‐adult atrial (HCF‐aa) was investigated. Using negative pressure, HCF‐aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up‐regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl‐2 was up‐regulated by longer stretching times. Pre‐treatment with antagomir‐499 reversed the miR‐208a down‐regulation, whereas Bcl‐2 expression could be suppressed by miR‐208a overexpression. In the HCF‐aa under stretching for 1 h, miR‐499 overexpression decreased pri‐miR‐208a luciferase activity; this inhibition of pri‐miR‐208a luciferase activity with stretching was reversed when the miR‐499‐5p binding site in pri‐miR‐208a was mutated. The addition of antagomir‐208a reversed the Bcl‐2‐3′UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre‐treatment with miR‐499 or antagomir‐208a inhibited cellular apoptosis in stretched HCF‐aa. In hearts with volume overload, miR‐499 overexpression inhibited myocardial miR‐208a expression, whereas Bcl‐2 expression could be suppressed by the addition of miR‐208a. In conclusion, miR‐208a mediated the regulation of miR‐499 on Bcl‐2 expression in stretched HCF‐aa and hearts with volume overload.