Inhibiting miR-205 Alleviates Cardiac Ischemia/Reperfusion Injury by Regulating Oxidative Stress, Mitochondrial Function, and Apoptosis

Yuerong Xu, Wangang Guo, Di Zeng, Yexian Fang, Runze Wang, Dong Guo, Bingchao Qi, Yugang Xue, Feng Xue, Zuolin Jin, Yan Li, Mingming Zhang, Yuerong Xu, Wangang Guo, Di Zeng, Yexian Fang, Runze Wang, Dong Guo, Bingchao Qi, Yugang Xue, Feng Xue, Zuolin Jin, Yan Li, Mingming Zhang

Abstract

Background: miR-205 is important for oxidative stress, mitochondrial dysfunction, and apoptosis. The roles of miR-205 in cardiac ischemia/reperfusion (I/R) injury remain unknown. The aim of this research is to reveal whether miR-205 could regulate cardiac I/R injury by focusing upon the oxidative stress, mitochondrial function, and apoptosis.

Methods: Levels of miR-205 and Rnd3 were examined in the hearts with I/R injury. Myocardial infarct size, cardiac function, oxidative stress, mitochondria function, and cardiomyocyte apoptosis were detected in mice with myocardial ischemia/reperfusion (MI/R) injury. The primary neonatal cardiomyocytes underwent hypoxia/reoxygenation (H/R) to simulate MI/R injury.

Results: miR-205 levels were significantly elevated in cardiac tissues from I/R in comparison with those from Sham. In comparison with controls, levels of Rnd3 were significantly decreased in the hearts from mice with MI/R injury. Furthermore, inhibiting miR-205 alleviated MI/R-induced apoptosis, reduced infarct size, prevented oxidative stress increase and mitochondrial fragmentation, and improved mitochondrial functional capacity and cardiac function. Consistently, overexpression of miR-205 increased infarct size and promoted apoptosis, oxidative stress, and mitochondrial dysfunction in mice with MI/R injury. In cultured mouse neonatal cardiomyocytes, downregulation of miR-205 reduced oxidative stress in H/R-treated cardiomyocytes. Finally, inhibiting Rnd3 ablated the cardioprotective effects of miR-205 inhibitor in MI/R injury.

Conclusions: We conclude that inhibiting miR-205 reduces infarct size, improves cardiac function, and suppresses oxidative stress, mitochondrial dysfunction, and apoptosis by promoting Rnd3 in MI/R injury. miR-205 inhibitor-induced Rnd3 activation is a valid target to treat MI/R injury.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Copyright © 2021 Yuerong Xu et al.

Figures

Figure 1
Figure 1
miR-205 inhibitor alleviates, while miR-205 mimic administration aggravates cardiac MI/R injury in mice. (a) Relative expression of miRNA-205. (b, c) Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) release after myocardial I/R injury in mice. (d–g) Left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular end systolic diameter (LVESD), and left ventricular end diastolic diameter (LVEDD) measured by echocardiography. (h) Representative images of infarct size as stained by Evans Blue and TTC. (i, j) Quantitative analysis of infarct size and AAR/LV at 3 h after I/R injury in mice. n = 6 in each group. The columns and errors bars represent means and SD. ∗p < 0.05 vs. Sham, †p < 0.05 vs. MI/R+NC, ‡p < 0.05 vs. MI/R+miR-205 inhibitor, &p < 0.05 vs. MI/R+Control mimic.
Figure 2
Figure 2
miR-205 inhibitor improves, while miR-205 mimic administration aggravates mitochondrial dysfunction and oxidative stress in mice that underwent MI/R injury. (a) Mitochondria morphological defects (magnification: upper panel ×9900; middle panel ×20500; lower panel ×43000). (b, c) ATP content and citrate synthase (CS) activity in the ischemic myocardium in mice subjected to MI/R injury. (d) Sensitivity of the mitochondrial permeability transition pore (mPTP) opening to calcium as evidenced by mCRC measurement. (e) ROS levels assessed by EPR spectroscopy. (f) Mitochondrial MnSOD activity. (g, h) Western blot analysis of Rnd3 expression. n = 6 in each group. ∗p < 0.05 vs. Sham, †p < 0.05 vs. MI/R+NC, ‡p < 0.05 vs. MI/R+miR-205 inhibitor, &p < 0.05 vs. MI/R+Control mimic.
Figure 3
Figure 3
Inhibiting miR-205 improves, while miR-205 overexpression administration aggravates apoptosis in mice that underwent cardiac MI/R injury. (a, b) Representative images of TUNEL staining and percentage of TUNEL-positive nuclei, scale bars = 50 μm. (c–e) Western blot analysis of cleaved caspase-3 and cleaved caspase-9 expression. n = 6 in each group. ∗p < 0.05 vs. Sham, †p < 0.05 vs. MI/R+NC, ‡p < 0.05 vs. MI/R+miR-205 inhibitor, &p < 0.05 vs. MI/R+Control mimic.
Figure 4
Figure 4
Inhibiting RND3 ablated the cardioprotective effects of miRNA-205 inhibitor. (a, b) Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) release. (c–f) Left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular end systolic diameter (LVESD), and left ventricular end diastolic diameter (LVEDD). (g) Representative images of infarct size as stained by Evans Blue and TTC. (h, i) Infarct size. AAR/LV had no statistical difference between groups 3 h after I/R injury. n = 6 in each group. The columns and errors bars represent means and SD. ∗p < 0.05 vs. MI/R, †p < 0.05 vs. MI/R+AAV9-sh-Rnd3, ‡p < 0.05 vs. MI/R+miR-205 inhibitor.
Figure 5
Figure 5
Inhibiting RND3 ablated the cardioprotective effects of miRNA-205 inhibitor in mitochondrial dysfunction and oxidative stress in mice that underwent MI/R injury. (a) Mitochondria morphological defects (magnification: upper panel ×9900; middle panel ×20500; lower panel ×43000). (b, c) ATP content and citrate synthase (CS) activity in the ischemic myocardium in the isolated mitochondrial in mice subjected to cardiac I/R injury. (d) Sensitivity of the mitochondrial permeability transition pore (mPTP) opening to calcium as evidenced by mCRC measurement. (e) ROS levels assessed by EPR spectroscopy. (f) Mitochondrial MnSOD activity. (g, h) Western blot analysis of Rnd3 expression. n = 6 in each group. ∗p < 0.05 vs. MI/R, †p < 0.05 vs. MI/R+AAV9-sh-Rnd3, ‡p < 0.05 vs. MI/R+miR-205 inhibitor.
Figure 6
Figure 6
Inhibiting RND3 ablated the cardioprotective effects of miRNA-205 inhibitor in apoptosis in mice that underwent cardiac MI/R injury (a, b) Representative images of TUNEL staining and percentage of TUNEL-positive nuclei, scale bars = 50 μm. (c–e) Western blot analysis of cleaved caspase-3 and cleaved caspase-9 expression. n = 6 in each group. ∗p < 0.05 vs. MI/R, †p < 0.05 vs. MI/R+AAV9-sh-Rnd3, ‡p < 0.05 vs. MI/R+miR-205 inhibitor.
Figure 7
Figure 7
Inhibiting miR-205 improves H/R-induced oxidative stress, while inhibiting RND3 ablated the cardioprotective effects of miR-205 inhibitor in primary cardiomyocytes. (a, b) Representative images of mitochondrial ROS in primary cardiomyocytes, scale bars = 50 μm. (c, d) Representative images of JC-1 and the ratio of aggregated (red) and monomeric (green) in neonatal mice cardiomyocytes, scale bars = 20 μm. ∗p < 0.05 vs. Con, †p < 0.05 vs. H/R+NC, ‡p < 0.05 vs. H/R+miR-205 inhibitor, &p < 0.05 vs. H/R+Control mimic. (e, f) Representative images of mitochondrial ROS in neonatal mice cardiomyocytes, scale bars = 50 μm. (g, h) Representative images of JC-1 and the ratio of aggregated (red) and monomeric (green) in neonatal mice cardiomyocytes, scale bars = 20 μm. The number of cardiomyocytes was counted (n = 50 in each group). ∗p < 0.05 vs. H/R, †p < 0.05 vs. H/R+AAV9-sh-Rnd3, ‡p < 0.05 vs. H/R+miR-205 inhibitor.

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