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

Abstract

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 microM 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 Ca(2+) 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 microM) 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 Ca(2+) 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.

Fig. 1

Percent (%) myocyte contraction and relaxation: a Adult cardiomyocytes were isolated from C57BL/6J mouse heart (control). Myocytes were treated with MMP-9 (3 ng/ml), prior to stimulation (MMP-9). To inhibit MMP-9, myocytes were pre-treated with TIMP-4 (12 ng/ml) prior to adding MMP-9 (TIMP-4 + MMP-9) and control myocytes treated with TIMP-4 (TIMP-4). Myocytes were stimulated at 1 Hz. b % Myocyte cell shortening, c Rate of systolic contraction; d Rate of diastolic relaxation. Each bar represents average ± SD from 5 to 7 sets of myocytes preparation. * P < 0.05 compared with untreated control

Fig. 2

Calcium (Ca²⁺) transients. a The ratio of FURA-2 fluorescence binding to calcium was recorded. Myocytes were treated with MMP-9 (3 ng/ml) or TIMP-4 (12 ng/ml), prior to stimulation (MMP-9 or TIMP-4). To inhibit MMP-9, myocytes were pre-treated with TIMP-4 (12 ng/ml) prior to adding MMP-9 (TIMP-4 + MMP-9). Myocytes were stimulated at 1 Hz. b Histographic presentation of Ca²⁺ transients. Each bar represents average ± SD from 5 to 7 sets of myocytes preparation. * P < 0.05 compared with untreated control

Fig. 3

Percent (%) myocyte contraction and relaxation: a Adult cardiomyocytes were isolated from C57BL/6J mouse heart (control). Myocytes were treated with MMP-9 (3 ng/ml) or TIMP-4 (12 ng/ml), prior to stimulation (MMP-9 or TIMP-4). To block PAR-1, myocytes were pre-treated with PAR-1 antagonist (10 and 100 µM), prior to adding MMP-9 (10 µM PAR + MMP-9) and (100 µM PAR + MMP-9). Myocytes were stimulated at 1 Hz. b % Myocyte cell shortening, c Rate of systolic contraction, d Rate of diastolic relaxation. Each bar represents average ± SD from 5 to 7 sets of myocytes preparation. * P < 0.05 compared with untreated control

Fig. 4

Calcium (Ca²⁺) transients. a The fluorescence ratio of FURA-2 and binding to calcium was recorded (control). Myocytes were treated with MMP-9 (3 ng/ml) prior to stimulation (MMP-9). To block PAR-1, myocytes were pre-incubated with PAR-1 antagonist (10 and 100 µM), prior to adding MMP-9 (10 µM PAR + MMP-9) and (100 µM PAR + MMP-9). Myocytes were stimulated at 1 Hz. b Histographic presentation of Ca²⁺ transients during systolic contraction. c Histographic presentation of Ca²⁺ transients during diasystolic relaxation. Each bar represents average ± SD from 5 to 7 sets of myocytes preparation. * P < 0.05 compared with untreated control

Fig. 5

Myocyte lengthening (micron, µM). a Myocytes from MMP-9KO and FVB mice were isolated. The cell lengthening and duration of lengthening were measured. Myocytes were stimulated at 1 Hz. b Time to contraction, c Time to relaxation, d Rate of systolic contraction, and e Rate of diastolic relaxation. Each bar represents average ± SD from 5 to 7 sets of myocytes preparation. * P < 0.05 compared with FVB myocytes

Fig. 6

Calcium (Ca²⁺) transients. a The fluorescence ratio of FURA-2 and binding to calcium was recorded. Myocytes from MMP-9KO and FVB mice were isolated. Calcium ratio at cell lengthening and the duration of lengthening was measured. Myocytes were stimulated at 1 Hz. b Rate of decay in calcium transients. c Histographic presentation of Ca²⁺ transients. Each bar represents average ± SD from 5 to 7 sets of myocytes preparation. * P < 0.05 compared with FVB myocytes

Fig. 7

Semi-quantitative RT-PCR analysis of dicer, TIMP-4, and serca-2a. The mRNA from FVB and MMP-9KO cardiomyocytes was isolated and amplified for dicer (a) and TIMP-4 (b) regions. GAPDH (c) was used as loading control. d The bar graph represents real-time PCR amplification of serca-2a in FVB and MMP-9KO cardiomyocytes. GAPDH was used as endogenous control. Each bar represents average ± SD from 5 to 7sets of myocytes preparation. * P < 0.05 compared with FVB myocytes

Fig. 8

Model showing effect of MMP-9 on contractile dysfunction: The active MMP-9 passes signal into cytoplasm through PAR-1 receptor and disturbs the intracellular balance of MMP-9/TIMP-4 axis. The high cytoplasmic MMP-9 causes contractile dysfunction in cardiomyocytes through altering the expression of dicer and serca-2a. Differential expression of selective miRNAs plays important role in contractility dysfunction