Polydatin Post-treatment Alleviates Myocardial Ischemia/reperfusion Injury Through Promoting Autophagic Flux

BackgroundAcute myocardial infarction (AMI) is one of the main causes of morbidity and mortality. Reperfusion is immediately required to prevent myocardial loss after AMI. Although reperfusion can limit infarct size, it causes myocardial ischemia/reperfusion (I/R) injury. However, ischemic pre-and post-conditioning both can confer cardioprotection against I/R injury. Compared with pre-conditioning, ischemic post-conditioning is a more attractive approach for cardioprotection because it is more practical and no need to predict myocardial ischemia. Thus, a pharmacological intervention at the onset of reperfusion has generated interest as an alternative method to mimic the protective effects of ischemic post-conditioning. Pharmacologic post-treatment can be clinically controlled and requires no invasive implementation. It is more feasible and suitable for patients treated with thrombolytic agents compared with ischemic post-conditioning.Polydatin (PD), a resveratrol glycoside, has a stronger anti-oxidative effect than resveratrol which has a cardioprotective effect against I/R injury. PD has several cardiovascular pharmacological effects, such as alleviation of pressure overload-induced ventricular remodeling and myocardial hypertrophy. In addition, PD has specialized biological properties due to its conformational difference from RES. For example, PD has more potent anti-oxidant effects than RES, and enters cells via an active mechanism using glucose carriers, whereas RES passively penetrates cells. Prior studies indicate that RES and PD have cardioprotective effects if administered before ischemia, however, whether PD post-treatment can potentially protect against myocardial I/R injury has not been investigated.Autophagy is a controlled lysosomal-dependent catabolic process which is involved in the degradation of long-lived proteins as well as removing excess or damaged organelles such as mitochondria. Previous studies indicate that RES pre-treatment induced cardiomyocyte survival via autophagic induction, but the role of PD post-treatment on autophagy during myocardial I/R is unknown.ObjectivesThis study aimed to assess the effects of PD post-treatment on myocardial I/R injury and to clarify its underlying mechanisms involving autophagy and apoptosis.Methods1. In vivo studyAdult male wild-type C57BL/6 mice (6-8 weeks; 20-25 g) were subjected to ischemia for 30 min and reperfusion for 120 min, and drug was performed 15 min before reperfusion. According to our preliminary dose-response experiments, three dosage of PD (5,7.5 and 10 mg/kg) were performed and the TUNEL-stained cell was recorded. The main experiments were performed on the following groups:Sham group; Sham+NS group; Sham+PD group (7.5 mg/kg); Sham+PD+3-MA group; Sham+3-MA group. TTC and Evans blue dye were used to detect myocardial infarct size. Myocardial apoptosis was analyzed by TUNEL assay. TEM was used to observe autophagosomes’and mitochondrial ultrastructure. Cardiac function was evaluated by echocardiography measurement.2. In vitro studyNRCMs were isolated from 1-3 day Sprague-Dawley rats and subjected to 3 h ischemia and 3 h reperfusion. In the main experiment, NRCMs were exposed to 3 h hypoxia/3 h re-oxygenation with or without 10 μM PD,10 mM 3-methyladenine (3-MA),100 nM Bafilomycin A1 (Baf) and 50 μM MnTMPyP in most experiments. In some experiments, NRCMs were pretreated with 10 mM N-acetylcysteine (NAC) 1 h before being exposed to hypoxia/re-oxygenation. MTS was performed to detect myocardial cell viability. Myocardial apoptosis was analyzed by TUNEL assay. TEM was used to observe myocardial ultrastructure. Intracellular ROS was quantified with DCFH-DA and superoxide anion/superoxide-derived ROS was monitored with DHE.3. Statistical analysisAll analyses were performed using SPSS 17.0 software. All data are expressed as means ± standard error of the mean (SEM) and P< 0.05 was considered to be statistically significant. Statistical differences were evaluated using one-way ANOVA followed by Bonferroni’s or Dunnett’s T3 multiple comparison exact probability tests.Results1. PD treatment-induced protective effects involved in increasing autophagy and decreasing apoptosis in vivo and in vitro.In NRCMs, PD treatment dramatically improved cell survival, and there was no significant change between PD 10 and PD 100 groups (Fig. 1B). In mice, TUNEL assay indicated that different doses of PD decreased apoptosis, but there was no significant difference between the middle and high dose of PD groups (Fig.1C). Thus, 10 μM and 7.5 mg/kg PD were chosen for subsequent experiments.2. PD treatment-induced autophagy was accompanied by decreased apoptosis in vivo and in vitroIn mice exposed to I/R, PD treatment increased the LC3 Ⅱ/Ⅰ ratio, LC3 Ⅱ and decreased cleaved caspase-3 expression. Co-treatment with 3-MA, an autophagy inhibitor via its inhibitory effect on class Ⅲ PI3K activity, causes the opposite effects (Fig.2A). Next, Ad-sh-Beclin 1 was used in NRCMs and Fig.2B showed that after 24 h infection, transduction efficiency was exceeded 90%. Meanwhile, Beclin 1 was reduced in the Ad-sh-Beclin 1 treatment group (Fig 2C). In NRCMs treated with Ad-sh-Beclin 1, LC3 Ⅱ/Ⅰ protein ratio and LC3 Ⅱ/GAPDH expression were reduced in H/R and H/R+PD groups (Fig.2D, E). MTS assay indicated that Ad-sh-Beclin 1 decreased the cell viability compared with the Ad-sh-NC groups (Fig 2F). These results showed that PD treatment-induced autophagy was accompanied by decreased apoptosis in vivo and in vitro.3. PD post-treatment limits infarct size and preserves heart function during I/RTo examine the efficacy of PD post-treatment, myocardial cell ultrastructures after I/R were examined with TEM. The sham group showed numerous dense and tight mitochondria, and neatly arranged and intact myocardial fibrils in the cytoplasm. I/R-treated group had loose and swollen mitochondria with ruptured or disappearing swollen myocardial fibrils. Myocardial cells treated with PD had less pathological changes:less ruptured mitochondria and myocardial fibrils, and more highly visible vacuoles with/without content. I/R induced mitochondrial swelling, and PD treatment reduced this damage as evidenced by decreased mitochondrial mean area (Fig.3A). The infarct size was～59% of the IS/AAR in I/R group, whereas in I/R+PD group the infarct size was reduced ～29% compared with I/R mice (59±4.5% vs.30± 4.0%; Fig.3B, C). In addition, the infarct size in I/R+PD+3-MA group was～12% larger than that in I/R+PD group (42±2.8% vs.30±4.0%), but 19% smaller than in I/R+ 3-MA group (42±2.8% vs.61± 4.0%; Fig.3B, C). There was no significant difference in AAR/LV among the groups (Fig.3B, D). Thus, PD treatment reduced the infarct size, which was partially abrogated by co-treatment with 3-MA. To assess heart function after PD treatment in I/R mice, M-mode echocardiographic images were obtained from the mid-papillary muscle region of the left ventricle. Echocardiography of I/R mice revealed a significant decrease in LVFS and EF after surgery. In contrast, I/R+PD group had significantly enhanced LVFS and EF (Fig. 3G, H). LVFS and EF were markedly lower in I/R+PD+3-MA group compared with I/R+PD group, but higher than that in I/R+3-MA group (Fig.3G, H). Therefore, PD treatment may improve heart function in mice subjected to I/R and these effects was partially abolished by 3-MA.4. PD treatment upregulated autophagy with smooth process of autophagic flux in NRCMsTo monitor autophagic flux, tandem fluorescent mRFP-GFP-LC3 was performed on NRCMs (Ad-LC3-NRCMs). Ad-LC3-NRCMs in normal culture had basal autophagy with few autolysosomes (Fig.4A, control, red dots in mRFP and Fig.4B) and few autophagosomes (Fig.4A, control, yellow dots in merged image and Fig.4B). Ad-LC3-NRCMs subjected to H/R had accumulated autophagosomes and almost no autolysosomes (Fig.4A, H/R, and Fig.4B), suggesting that autophagosome clearance was inhibited and autophagic flux was blocked or impaired [18]. PD-treated Ad-LC3-NRCMs subjected to H/R had markedly increased abundance of autolysosomes with few autophagosomes (Fig.4A, H/R+PD, and Fig.4B), indicating consumption of autophagosomes and smooth or intact autophagic flux. In addition, co-treatment with PD and 3-MA decreased autolysosomes compared with PD treatment only. We assessed cumulative autophagic flux by measuring protein expression of LC3 Ⅱ/Ⅰ, LC3 Ⅱ in the presence and absence of Baf, which is a cell-permeable lysosomal inhibitor by inhibiting vacuolar H+-ATPase and autophagosome-lysosome fusion to prevent the final digestion step of autophagy. Data showed that the ratio of LC3 Ⅱ/Ⅰ and LC3 Ⅱ were unchanged after Baf treatment in NRCMs exposed to H/R. Therefor, inhibition of autophagic degradation led to autophagosome accumulation via the blockage of autophagosome-lysosome fusion. However, LC3 Ⅱ/Ⅰ and LC3 Ⅱ accumulated in NRCMs co-treated Baf and PD, indicating autophagic flux (Fig.4C). Thus, autophagosomes accumulated may be due to impaired autophagy during H/R, and PD treatment induced a significant clearance of autophagosomes with a smooth autophagic flux. TUNEL assay data indicated that PD post-treatment decreased apoptosis of NRCMs exposed to H/R, and PD and Baf co-treatment partly abolished the effect of PD. Thus, PD-induced autophagic flux reduced myocardial death (Fig.4D).5. PD treatment decreased damaged mitochondria and cellular ROSTEM images revealed that the ultrastructure of mitochondria was swelling, rupture and loss of cristae in H/R group, while PD post-treatment significantly ameliorated the H/R-induced mitochondrial damaged. In addition, TEM reveal that PD treatment groups had more autophagosomes, indicating a shift from early autophagic vacuoles to late autolysosomes compared with the H/R group (Fig.5A). We next investigated the effect of PD on the ΔΨm and noted that was dramatically reduced in H/R treated cells and PD treatment partially restored ΔΨm. After co-treatment with PD and Baf, ΔΨm was decreased compared with PD alone group, indicating that PD-induced autophagic flux can alleviate the mitochondrial damage (Fig.5B, C). As damaged mitochondria are the major source of ROS, we investigated effects of PD treatment on cellular ROS production in NRCMs. Exposing NRCMs to H/R induced a significant increase in ROS compared with control, and PD treatment markedly inhibited ROS production (Fig.6A, B). In addition, intracellular superoxide anion/superoxide-derived ROS were involved in H/R, and PD post-treatment reduced ROS production (Fig.6C, D, E). However, the effect of PD treatment on ATm and ROS generation was blocked by co-treated with PD and Baf, suggesting that ATm and ROS generation is involved in autophagic flux (Fig.6A-E).ConclusionsThis paper provides the evidences that PD post-treatment alleviates myocardial I/R injury via inhibiting apoptosis and promoting autophagic flux which degrades damaged mitochondria and ROS level. Thus, PD post-treatment may be a potential pharmacological intervention against myocardial I/R injury.