Abstract
BackgroundMicroRNAs play an important role in cardiac remodeling. MicroRNA 499 (miR499) is highly enriched in cardiomyocytes and targets the gene for Calcineurin A (CnA), which is associated with mitochondrial fission and apoptosis. The mechanism regulating miR499 in stretched cardiomyocytes and in volume overloaded heart is unclear. We sought to investigate the mechanism regulating miR499 and CnA in stretched cardiomyocytes and in volume overload-induced heart failure.Methods & ResultsRat cardiomyocytes grown on a flexible membrane base were stretched via vacuum to 20% of maximum elongation at 60 cycles/min. An in vivo model of volume overload with aorta-caval shunt in adult rats was used to study miR499 expression. Mechanical stretch downregulated miR499 expression, and enhanced the expression of CnA protein and mRNA after 12 hours of stretch. Expression of CnA and calcineurin activity was suppressed with miR499 overexpression; whereas, expression of dephosphorylated dynamin-related protein 1 (Drp1) was suppressed with miR499 overexpression and CnA siRNA. Adding p53 siRNA reversed the downregulation of miR499 when stretched. A gel shift assay and promoter-activity assay demonstrated that stretch increased p53 DNA binding activity but decreased miR499 promoter activity. When the miR499 promoter p53-binding site was mutated, the inhibition of miR499 promoter activity with stretch was reversed. The in vivo aorta-caval shunt also showed downregulated myocardial miR499 and overexpression of miR499 suppressed CnA and cellular apoptosis.ConclusionThe miR499-controlled apoptotic pathway involving CnA and Drp1 in stretched cardiomyocytes may be regulated by p53 through the transcriptional regulation of miR499.
Highlights
MicroRNAs are approximately 22 nucleotides long, non-coding RNAs that act as negative regulators of gene expression by interacting with the 3’-untranslated regions of target mRNA and promoting mRNA degradation.[1]
The miR499-controlled apoptotic pathway involving Calcineurin A (CnA) and dynamin related protein 1 (Drp1) in stretched cardiomyocytes may be regulated by p53 through the transcriptional regulation of miR499
The aims of the current study were to investigate whether the expression of miR499 in cardiomyocytes can be regulated with mechanical stretch and a rat model of volume-overload induced heart failure caused by aorta-caval shunt, and to evaluate the molecular mechanism for regulating miR499 on gene and protein expression
Summary
MicroRNAs (miRNA) are approximately 22 nucleotides long, non-coding RNAs that act as negative regulators of gene expression by interacting with the 3’-untranslated regions of target mRNA and promoting mRNA degradation (gene silencing).[1]. The cardiomyocytes are enriched with mitochondria that provide the ATP for the requisite continuous cardiac mechanical and electrical work.[13] Constant mitochondrial fusion and fission are necessary for the maintenance of organelle fidelity.[14,15,16] Mitochondrial dysfunction, as evidenced by abnormal mitochondrial fission and reduced ATP production, is a characteristic of the initiation of apoptosis in cardiomyocytes.[17] Calcineurin and dynamin related protein 1 (Drp1) have been shown to be involved in mitochondrial fission during cardiac apoptosis.[17, 18] Calcineurin contains a heterodimer of a 61-kD calmodulin-binding catalytic subunit, calcineurin A (CnA) contains a 19-kD Ca2+-binding regulatory subunit, and calcineurin B is a cytosolic serine and threonine phosphatase.
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