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. 2020 Sep 17:11:1083.
doi: 10.3389/fphar.2020.01083. eCollection 2020.

Astragaloside IV Derivative (LS-102) Alleviated Myocardial Ischemia Reperfusion Injury by Inhibiting Drp1Ser616 Phosphorylation-Mediated Mitochondrial Fission

Li Chen  1   2 , Xiao-Yi Chen  1 , Qian-Long Wang  1 , Si-Jin Yang  2 , Hua Zhou  1 , Li-Sheng Ding  3 , Lin-Sen Qing  3 , Pei Luo  1
Affiliations

Astragaloside IV Derivative (LS-102) Alleviated Myocardial Ischemia Reperfusion Injury by Inhibiting Drp1Ser616 Phosphorylation-Mediated Mitochondrial Fission

Li Chen et al. Front Pharmacol. .

Abstract

Our previous studies showed that Astragaloside IV derivative (LS-102) exhibited potent protective function against ischemia reperfusion (I/R) injury, but little is known about the mechanisms. Mitochondrial fission regulated by dynamin-related protein1 (Drp1) is a newly recognized determinant of mitochondrial function. This study aimed to investigate the protection of LS-102 on mitochondrial structure and function by regulating the activity of Drp1 using models of H9c2 cardiomyocyte injury induced by hypoxia-reperfusion (H/R), and rat heart injury induced by I/R. The results showed that LS-102 significantly decreased apoptosis, levels of ROS, CK, LDH, and calcium, upregulating MMP, and the Bax/Bcl-2 ratio in cardiomyocytes during I/R injury. Furthermore, LS-102 prevented I/R-induced mitochondrial fission by decreasing Drp1's mitochondrial localization through decreasing the phosphorylation of Drp1 at Ser616 (Drp1Ser616) and increasing the phosphorylation of Drp1 at Ser637 (Drp1Ser637) in H9c2 cells. Importantly, we also robustly confirmed Drp1Ser616 as a novel GSK-3β phosphorylation site. GSK-3β-mediated phosphorylation at Drp1Ser616 may be associated with mitochondrial fission during I/R of cardiomyocytes. In conclusion, LS-102 exerts cardio protection against I/R-induced injury by inhibiting mitochondrial fission via blocking GSK-3β-mediated phosphorylation at Ser616 of Drp1.

Keywords: Drp1 phosphorylation; GSK-3β; astragalosidic acid; mitochondrial fission; myocardial ischemia reperfusion.

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Figures

FIGURE 1
FIGURE 1
Outline of the experimental protocol to assess the cardioprotective effect of LS-102 on the model of myocardial I/R-induced rat injury. (A) Molecular structure of LS-102. (B) Protocols of dose-dependent study and pharmacological inhibitors study. Rats were pretreated with vehicle or LS-102, followed by 30 min of ischemia and then 90 min of reperfusion. Rat hearts were collected for TTC and H&E staining, the rat serum samples were also collected to detect the activities of CK and LDH.
FIGURE 2
FIGURE 2
Oral-administration of LS-102 exhibited a dose-dependent relationship of cardioprotective effect in rat heart. (A) Representative image of TTC staining sections of the left ventricle. The viable tissue was stained as red area and the unstained pale areas were presumed to be infarcted tissue. (B) Bar chart of infarct size (%). (C) The activities of CK and LDH in serum were detected. (D) Representative high power views of I/R myocardial tissue slice showing neutrophil polymorphs flooding the infarcted area (thin arrow), infarcted cardiomyocytes appearing deeply eosinophilic with loss of cross striations (arrow head), prominent interstitial edema (thick arrow), RBCs in the interstitial spaces (open arrow). (E) Illustration of H&E staining of the myocardial tissue in different groups (200 × magnification). Sham and I/R, distilled water 8ml/kg; LS-102 2.5, LS-102 2.5 mg/kg; LS-102 5, LS-102 5 mg/kg; LS-102 10, LS-102 10 mg/kg. Data are shown as mean ± SEM, n = 8–10/group. ^^^P < 0.001, ^^P < 0.01, vs. Sham group, ***P < 0.001, **P < 0.01 vs. I/R group.
FIGURE 3
FIGURE 3
Effect of LS-102 on the PI3K/Akt signaling in rat heart. (A) Representative Western blot of total and phosphorylated PI3K and Akt. (B) Quantitative analysis of band intensity, values are expressed as mean ± S.E.M. of 4 hearts from each group. (C) Infarct size of a rat after the pharmacological blockage of PI3K and Akt. Sham and I/R, distilled water 8 ml/kg; LS-102, LS-102 10 mg/kg; LS-102+LY294002, LS-102 10 mg/kg+ PI3K inhibitor LY294002 0.25 mg/kg; LS-102+ IV, LS-102 10 mg/kg+Akt inhibitor IV 4mg/kg. Vehicle or LS-102 was given to rats 30 min before LAD ligation. Pharmacological inhibitors were given immediately after the administration of LS-102. Values are mean ± SEM of six rats from each group. ^^^P < 0.001, vs Sham group; *P < 0.05, ***P < 0.001, vs I/R group.
FIGURE 4
FIGURE 4
Effects of LS-102 on cell survival after H/R in H9c2 cells. (A) Effect of LS-102 on cell viability. (B) Cytotoxicity of LS-102 in H9c2 cells under normoxia condition. (C) Effects of LS-102 on CK and LDH activities in serum. (D) Representative DIC images of H9c2 cells. (E) Effect of LS-102 on H/R-induced H9c2 cell apoptosis. Flow cytometry was used to detect apoptotic cells which were calculated the sum of the Q2 and Q3 quadrant. (F) Using western blot analysis to determine the protein expression of Bcl-2, Bax, and Cleaved-Caspase-3. The ratio of Bcl-2 to Bax was calculated and normalized to the Normal group. Values are means ± SEM of three or four independent experiments. ^^^P < 0.001 vs. Normal group. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. H/R group.
FIGURE 5
FIGURE 5
Effects of LS-102 on the mitochondrial dysfunction induced by H/R in H9c2 cells. (A) Effect of LS-102 on mitochondrial viability, the content of ATP, and SOD activity. (B) The Seahorse XFp Extracellular Flux Analyzer was used to evaluate the effect of LS-102 on oxygen consumption rate (OCR). (C) Representative images of JC-1 fluorescent dye stained the mitochondrial membrane potential captured by confocal microscopy with ×40 objective (Olympus). Using the fluorescence microplate assay to calculate the red/green ratio. (D) The protein of cytochrome c was detected by Western blot. (E) Representative images of Fluo-4AM stained calcium captured by confocal microscopy with ×20 objective (Olympus). The relative mean of the calcium fluorescence intensity was analyzed by flow cytometry. (F) The content of the ROS labeled H2DCFDA probe was analyzed by flow cytometry. The values were the means ± SEM of three or four independent experiments. ^^P < 0.01, ^^^P < 0.001 vs. Normal group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. H/R group.
FIGURE 6
FIGURE 6
Effects of LS-102 on Drp1-dependent mitochondrial fission induced by H/R in H9c2 cells. (A) Representative images showing MitoTracker Red CM-H2Ros staining (MitoROS; red) were captured by confocal microscopy with ×40 objective (Olympus). (B) Bar graph showing the percentage of fragmented mitochondria (mitochondrial length of less than 1 μm). (C) Co-localization analysis of Drp1 on mitochondria using a confocal microscope with ×40 objective (Olympus). H9c2 cells were stained with MitoROS (red), anti-Drp1 antibody (green). Nucleus was trained with DAPI (blue). The yellow dots indicated Drp1(green) on the mitochondria (red) location. (D) Qualitative analysis of colocalization by Image J software. (E) Phosphorylation of Drp1 was detected by Western blot analysis and incubated with anti-phospho-Drp1(Ser637) or anti-phospho-Drp1(Ser637) antibodies, the loading control was α-Tubulin. (F) The protein expression of p-Drp1Ser616 and p-Drp1Ser637 after treatment of GSK-3β inhibitor AR-A014418 were determined by Western blot. (G) Co-localization analysis of Drp1 (green) with GSK-3β (red) were captured by confocal microscopy with × 40 objective (Olympus). (H) Bar chart showing yellow fluorescence intensity. (I,J) The protein expression of Drp1 with GSK-3β was examined by Co-immunoprecipitation (Co-IP) assay and Western blot. The relative densities of immunoreactive bands were quantified over α-Tubulin using image J software. Values are the means ± SEM of three or four independent experiments. ^^P < 0.05, ^^^P < 0.001 vs. Normal group. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. H/R group.
FIGURE 7
FIGURE 7
Effect of LS-102 on PI3K/Akt/GSK-3β pathway of H9c2 cells exposed to H/R. (A) The protein expression of PI3K, p-PI3K, Akt, p-Akt, GSK-3β, and p-GSK-3β was analyzed by Western blot. (B,C) PI3K inhibitor LY294002 (10 μM) altered the effect of LS-102 on cell viability, mitochondrial viability, and the cytochrome c release after H/R in H9c2 cells. Values were the means ± SEM of three independent experiments in H9c2 cells after H/R. ^^^P < 0.001 vs. Normal group. **P < 0.01 and ***P < 0.001 vs. H/R group.
FIGURE 8
FIGURE 8
Possiblemechanism of the cardioprotective effect of LS-102 in injury of H/R.
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