MMP-9 gene ablation and TIMP-4 mitigate PAR-1-mediated cardiomyocyte dysfunction: A plausible role of dicer and miRNA

Although matrix metalloproteinase-9 (MMP-9) is involved in cardiomyocytes contractility dysfunction, tissue inhibitor of metalloproteinase-4 (TIMP-4) mitigates the effect of MMP-9, and proteinase-activated receptor-1 (PAR-1, a G-protein couple receptor, GPCR) is involved in the signaling cascade of MMP-9-mediated cardiac dysfunction, the mechanism(s) are unclear. To test the hypothesis that induction of dicer and differential expression of microRNAs (miRNAs) contribute, in part, to the down regulation of sarcoplasmic reticulum calcium ATPase isoform 2a (serca-2a) in MMP-9 and PAR-1-mediated myocytes dysfunction, ventricular cardiomyocytes were isolated from C57BL/6J mice and treated with 3 ng/ml of MMP-9, 12 ng/ml of TIMP-4, and 10 and 100 μM of PAR-1 antagonist with MMP-9. Specific role of MMP-9 was determined by using MMP-9 knock out (MMP-9KO) and their corresponding control (FVB) mice. Ion Optics video-edge detection system and Fura 2-AM loading were used for determining the contractility and calcium release from cardiomyocytes. Quantitative and semi-quantitative PCR were used to determine the expression of dicer, TIMP-4 and serca-2a. miRNA microarrays were used for assessing the expression of different miRNAs between MMP-9KO and FVB cardiomyocytes. The results suggest that MMP-9 treatment attenuates the voltage-induced contraction of primary cardiomyocytes while TIMP-4, an inhibitor of MMP-9, reverses the inhibition. MMP-9 treatment is also associated with reduced Ca²⁺ transients. This effect is blocked by a PAR-1 antagonist, suggesting that PAR-1 mediates this effect. The effect is not as great at high concentrations (100 μM) perhaps due to mild toxicity. The PAR-1 antagonist effect did not affect calcium transients unlike TIMP-4. Interestingly, we show that MMP-KO myocytes contract more rapidly and release more Ca²⁺ than FVB. The relevant RNA species serca-2a is induced and dicer is inhibited. There is selective inhibition of miR-376b and over-expression of miR-1, miR-26a, miR-30d, and miR-181c in MMP-9KO that are implicated in regulation of G-PCR and calcium handling.