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. 2022 Aug 24:2022:9729018.
doi: 10.1155/2022/9729018. eCollection 2022.

Silencing RNA for MMPs May Be Utilized for Cardioprotection

Marta Banaszkiewicz  1 , Anna Krzywonos-Zawadzka  1 , Agnieszka Olejnik  1 , Agnieszka Noszczyk-Nowak  2 , Iwona Bil-Lula  1
Affiliations

Silencing RNA for MMPs May Be Utilized for Cardioprotection

Marta Banaszkiewicz et al. Cardiovasc Ther. .

Abstract

Ischemia/reperfusion (I/R) injury is accompanied by an increase of matrix metalloproteinase 2 (MMP-2) activity, which degrades heart contractile proteins. The aim of the study was to investigate the effect of MMP-2 small interfering RNA (MMP-2 siRNA) administration on I/R heart. Isolated rat hearts perfused by the Langendorff method were subjected to I/R in the presence or absence of MMP-2 siRNA. The hemodynamic parameters of heart function were monitored. Lactate dehydrogenase (LDH) activity was measured in coronary effluents. Activity and concentration of MMPs in the hearts were measured. Concentration of troponin I (TnI) in coronary effluents was examined as a target for MMP-2 degradation. Recovery of heart mechanical function was reduced after I/R; however, administration of MMP-2 siRNA resulted in restoration of proper mechanical function (p < 0.001). LDH activity was decreased after the use of MMP-2 siRNA (p = 0.02), providing evidence for reduced cardiac damage. Both MMP-2 and MMP-9 syntheses as well as their activity were inhibited in the I/R hearts after siRNA administration (p < 0.05). MMP-2 siRNA administration inhibited TnI release into the coronary effluents (p < 0.001). The use of MMP-2 siRNA contributed to the improvement of heart mechanical function and reduction of contractile proteins degradation during I/R; therefore, MMP-2 siRNA may be considered a cardioprotective agent.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Experimental protocol for I/R and aerobic control with or without MMP-2 siRNA/scrambled RNA and siPORT mixture infusion. The hearts were perfused with MMP-2 siRNA/scrambled RNA and siPORT mixture for the last 10 minutes of aerobic stabilization and the first 10 minutes of reperfusion. I/R: ischemia-reperfusion.
Figure 2
Figure 2
MMP-2 gene expression (a), concentration (b), and activity (c) in heart homogenates and MMP-9 gene expression (d), concentration (e), and activity (f) in the heart tissue. Representative zymogram of MMP-2/MMP-9 activity in heart homogenates (HT10-80 and PRP were used as standards for pro-/active MMP-2 and MMP-9) (g). Graph presenting the site of siRNA binding to the fragment of MMP-9 mRNA sequence (h). Aero: aerobic control group; I/R: ischemia/reperfusion; MMP-2: matrix metalloproteinase 2; MMP-9: matrix metalloproteinase 9; PRP: platelet-rich plasma.
Figure 3
Figure 3
An influence of MMP-2 siRNA on heart function: heart rate (a). RPP calculated as the product of the heart rate and left ventricular developed pressure (heart rate x left ventricular developed pressure/1000) (b). Recovery of heart mechanical function (difference between RPP at 25th and 77th min of perfusion expressed as a percentage of RPP recovery) (c). Correlation between heart mechanical function recovery, heart rate and MMP-2 activity (d); MMP-9 activity (e) in heart homogenates; straight line: standard curve; dotted line: 95% confidence band. Aero: aerobic control group; bpm: beats per minute; HR: heart rate; I/R: ischemia-reperfusion group; MMP-2: matrix metalloproteinase 2; MMP-9: matrix metalloproteinase 9; RPP: rate pressure product. Mean ± SEM, nAero = 3; nAero+MMP−2 siRNA = 2; nI/R = 5; nI/R+scrRNA = 5; nI/R+MMP−2 siRNA = 4. ∗p < 0.05 vs. Aero, #p < 0.05 vs. I/R, and $p < 0.05 vs. I/R+scrRNA.
Figure 4
Figure 4
LDH activity in coronary effluents (a). Correlation between LDH activity in coronary effluents and recovery of heart mechanical function and heart rate (b) and correlation between LDH activity in coronary effluents and MMP-2 and MMP-9 activity in heart homogenates (c). Straight line: standard curve; dotted line: 95% confidence band. Aero: aerobic control group; I/R: ischemia/reperfusion; HR: heart rate; LDH: lactate dehydrogenase; MMP-2: matrix metalloproteinase 2; MMP-9: matrix metalloproteinase 9.
Figure 5
Figure 5
TnI (a) and MLC1 (b) release into coronary effluents. Correlation between LDH activity and TnI (c) and MLC1 (d) release. Correlation between MMP-2 activity and TnI (e) and MLC1 (f) release into extracellular space. Correlation between MMP-9 synthesis in rat hearts and TnI (g) and MLC1 (h) release into coronary effluents. Straight line: standard curve; dotted line: 95% confidence band. Aero: aerobic control group; I/R: ischemia/reperfusion; LDH: lactate dehydrogenase; MLC1: myosin light chain 1; MMP-2: matrix metalloproteinase 2; TnI: troponin I.
Figure 6
Figure 6
TIMP-4 level in rat hearts (a). Correlation between TIMP-4 level and MMP-2 (b) and MMP-9 (c) synthesis/activity in rat hearts. Correlation between TIMP-4 level in rat hearts and HR/RPP (d). Straight line: standard curve; dotted line: 95% confidence band. Aero: aerobic control group; I/R: ischemia/reperfusion; MMP-2: matrix metalloproteinase 2; TIMP-4: endogenous tissue inhibitor of matrix metalloproteinase 4.
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