Dihydromyricetin Ameliorates Cardiac Ischemia/Reperfusion Injury through Sirt3 Activation

Abstract

During myocardial infarction, quickly opening the occluded coronary artery is a major method to save the ischemic myocardium. However, it also induces reperfusion injury, resulting in a poor prognosis. Alleviating the reperfusion injury improves the prognosis of the patients. Dihydromyricetin (DHM), a major component in the Ampelopsis grossedentata, has numerous biological functions. This study aims to clarify the effects of DHM under the ischemia/reperfusion (I/R) condition. We elucidated the role of Sirt3 in the cardiomyocyte response to DHM based on the hearts and primary cardiomyocytes. Cardiac function, mitochondrial biogenesis, and infarct areas were examined in the different groups. We performed Western blotting to detect protein expression levels after treatments. In an in vitro study, primary cardiomyocytes were treated with Hypoxia/Reoxygenation (H/R) to simulate the I/R. DHM reduced the infarct area and improved cardiac function. Furthermore, mitochondrial dysfunction was alleviated after DHM treatment. Moreover, DHM alleviated oxidative stress indicated by decreased ROS and MnSOD. However, the beneficial function of DHM was abolished after removing the Sirt3. On the other hand, the mitochondrial function was improved after DHM intervention in vitro study. Interestingly, Sirt3 downregulation inhibited the beneficial function of DHM. Therefore, the advantages of DHM are involved in the improvement of mitochondrial function and decreased oxidative stress through the upregulation of Sirt3. DHM offers a promising therapeutic avenue for better outcome in the patients with cardiac I/R injury.

Figure 1

DHM alleviated MI/R injury. (a, b) Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB). (c) The infarct area in each group. (d, e) Quantitative analysis of infarct area and AAR/LV. Mean ± SEM (n=12), ^∗P<0.05 vs WT+sham group; ^#P<0.05 vs Sirt3^−/− + sham group; ^§P < 0.05 vs MI/R group; ^$P<0.05 vs Sirt3^−/−+MI/R group; ^&P<0.05 vs MI/R +DHM group.

Figure 2

DHM improved cardiac function after MI/R. (a, b) LVEF, LVFS was measured by echocardiography. (c, d) The maximal ± LV dp / dt was obtained by hemodynamic evaluation. Mean ± SEM (n=12), ^∗P<0.05 vs WT+sham group; ^#P<0.05 vs Sirt3^−/− + sham group; ^§P < 0.05 vs MI/R group; ^$P<0.05 vs Sirt3^−/−+MI/R group; ^&P<0.05 vs MI/R +DHM group.

Figure 3

DHM attenuated the mitochondrial dysfunction after MI/R. (a) The morphology of mitochondria (m). (b, c) ATP content and citrate synthase (CS) activity. (d) Mitochondrial DNA content. (e) ROS levels. (f) Manganese superoxide dismutase (MnSOD). (g) Caspase-3 activity. (h-k) Representative bands shown for Sirt3, TFAM, and NRF-2 proteins are provided. Mean ± SEM (n=12), ^∗P<0.05 vs WT+sham group; ^#P<0.05 vs Sirt3^−/− + sham group; ^§P < 0.05 vs MI/R group; ^$P<0.05 vs Sirt3^−/−+MI/R group; ^&P<0.05 vs MI/R +DHM group.

Figure 4

DHM improved mitochondrial function after H/R. (a, b) Representative blots of Sirt3. (c) The morphology of mitochondria (yellow arrow) in cardiomyocytes. (d) JC-1 aggregates label normal mitochondria with polarized inner mitochondrial membranes (Red). JC-1 monomers represent DYm dissipation (Green). (e) Quantification of the DYm (n=50 in each group). Scale bars= 20 um. Mean ± SEM, ^∗P<0.05 vs Con; ^#P<0.05 vs Ad-sh-Sirt3; ^§P < 0.05 vs H/R; ^$P<0.05 vs Ad-sh-Sirt3+H/R; ^&P<0.05 vs H/R+DHM.