| BackgroudMyocardial ischemia reperfusion (ischaemia/reperfusion, I/R) injury, refers to the coronary arteries (coronary) block after a certain time, then restore blood flow reperfusion, is associated with injurried structure and function of and increased infartion of heart. Currently, some treatment can not prevent the injury effectively. Therefore, it is point to the critical need to identify effective therapeutic strategies.Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an allosteric tetrameric enzyme responsible for the metabolism or detoxification of acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal (4HNE), as well as mitochondrial oxidative ATP generation and reactive oxygen species (ROS) generation. ALDH2 activity can affect the cellular response to oxidative stress both in vitro and in vivo. Accumulating evidence shows that ALDH2, especially ALDH2 activation or overexpression is closely related to cardiovascular diseases. However, the exact mechanism of ALDH2-mediated is not fully elucidated.Autophagy is triggered by energy depletion, oxidative stresses, protein aggregates and damaged organelles. Despite many studies demonstrating the benefits of autophagy, excessive autophagy can induce cell death in myocardial I/R injury. ALDH2 has a dual regulatory paradox in cardioprotecting against ischemia/reperfusion (I/R) injury via autophagy.Mitophagy, a selective form of autophagy, is a specific process for degradation of dysfunctional or damaged mitochondria to maintain a healthy mitochondria population and mitochondrial quality Starving, hypoxemia, and ROS may trigger mitophagy, found associated with several forms of neurodegeneration and cardiovascular diseases.Mitophagy can be regulated by the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/Parkin pathway. Upon mitochondrial damage, PINK1, mitochondria-localized serine/threonine kinase, globally accumulates on depolarized mitochondria and recruits Parkin from the cytosol to mitochondria. Parkin, an E3 ubiquitin ligase, catalyzes the polyubiquitination of several substrates and triggers whole-mitochondrial engulfinent by autophagosomes and subsequent degradation via the autophagosome lysosome pathway.Nevertheless, the precise role of mitophagy in myocardial I/R injury is unclear. Here, we explored the potential involvement and function of mitophagy in the ALDH2-elicited cardioprotective effect in myocardial I/R injury in vivo and in vitro.ObjectiveThe objectives of this experiment is to investigate:1. The role of oxidative stress in PINKl/Parkin depenfent mitopahgy induced by I/R.2. The role and mechanism of ALDH2 in PINK1/Parkin depenfent mitopahgy pathway induced by I/R.3. The regulation and mechanism of ALDH2 to I/R induced myocardium injury.MethodsFor vivo study1. Animal model:Male Wistar rats (n=48),6-7 weeks old(l 80-200g) were divided into 4 groups (n=12/group):sham, I/R, I/R+Alda-1, and I/R+Daidzin. Rats were administered 100 mg/kg Alda-1 or 100 mg/Kg Daidzin through intramyocardial injection into the left ventricular myocardium 5 minutes before 30 min ischemia followed by 120 min reperfusion. Sham rats underwent the same operation, except that the suture was placed around LAD but not tied. At the end of reperfusion, the rats were killed.2. TTC were performed to measure myocardial infarction size of rats induced by I/R.3. TUNEL assay was performed to show apoptosis in myocardium of rats induced by I/R4. Measurement ofALDH2 Activity:cardiomyocytes mitochondria of rats were extracted.5. Western blot (WB):Total and mitochondria proteins with the same concentration were extracted from cardiomyocytes of rats. The protein expression of PINK1、Parkin、 COX IV、ALDH2、4HNE and β-actin were detected.For vitro study1. Cell culture:H9C2 cells were incubated with the Alda-1 or Daidzin for 30 minutes at 37℃ (95% O2/5% CO2) before a 120 minutes exposure to hypoxia (1%02/5% CO2/94% N2), followed by 60 minutes reoxygenation (95% O2/5% CO2). Furthermore, H9C2 cells were treated with hypoxia /reoxygenation, (hypoxia /reoxygenation, H/R) in the absence or presence of Alda-1 with CCCP.2. DCFH-DA assay were was performed to measure ROS in H9C2 cells induced by H/R.3. MitoSOX Red Mitochondrial Superoxide Indicator was performed to measure MitoSOX in H9C2 cells induced by H/R.4. CCK-8 assay were performed to assess cell viability in H9C2 cells induced by H/R.5. JC-1 assay was performed to measure mitochondrial membrane potential in H9C2 cells induced by H/R.6. TUNEL assay was performed to show apoptosis in H9C2 cells induced by H/R.7. Measurement of ALDH2 Activity:H9C2 cells mitochondria were extracted.8. Western blot (WB):Total and mitochondria proteins with the same concentration were extracted to detecte the protein expression of PINK1、Parkin、COX Ⅳ、ALDH2、 4HNE and p-actin.9. Imunofluorescence staining of COX IV and PINK1, COX IV and Parking were used to show the co-localization.Results1. ALDH2 reduced the myocardial infarct size and cardiomyocyte apoptosis in I/R rats.Compared with I/R alone, I/R+Alda-1 reduced the infarct size and I/R+Daidzin increased the size. I/R-induced cardiomyocyte apoptosis was alleviated with I/R+Alda-1.Howerever, it had increasing trend, but no statistical significance with I/R+Daidzin.2. ALDH2 inhbited 4HNE accumulation and mitophagy in I/R rats.I/R damaged ALDH2 activity, which was significantly alleviated and aggravated, respectively, with Alda-1 and Daidzin treatment, but with no change in ALDH2 expression.Western blot showed that I/R increased PINK1 and Parkin levels. ALDH2 activation with Alda-1 suppressed the I/R-elevated expression of PINK 1 and Parkin. However, ALDH2 inhibition with Daidzin could not promoted their expression significantly.I/R treatment significantly enhanced 4HNE accumulation, which was accentuated and mitigated by ALDH2 inhibition and activation, respectively.3. ALDH2 inhbited oxidative and apoptosis in H/R-treated H9C2 cellsLevels of ROS were higher with H/R than with ALDH2 activation in cells. However, it had the lower trend with ALDH2 inhibition. ALDH2 activation with Alda-1 also suppressed the I/R-elevated mitochondrial superoxide levels but ALDH2 inhibition with Daidzin promoted the levels. As well, ALDH2 activation significantly alleviated the decreased mitochondrial membrane potential. In line with the effects on myocardial I/R injury, ALDH2 activation reduced H/R-induced apoptosis but ALDH2 inhibition could not promoted it significantly.4. ALDH2 inhibited 4HNE accumulation in H/R-treated H9C2 cellsH/R significantly decreased ALDH2 activity, which was rescued by ALDH2 activation with no change in ALDH2 expression. Ratio of cell viability were lower with H/R than with ALDH2 activation, but higher than ALDH2 inhibition in cells. 4HNE protein adduct formation was significantly increased in response to H/R. Although ALDH2 inhibition significantly promoted H/R-increased 4HNE protein adduct content, Alda-1 further alleviated the increase.5. ALDH2 inhibitd mitophagy in H/R-treated H9C2 cells.H/R increased the co-localization of PINK1 or Parkin with COX IV, which suggests the activation of mitophagy with H/R. ALDH2 activation reduced the H/R induced co-localization. As expected, A1DH2 activation also reduced these protein expressions which induced by H/R. However, Daidzin induced the co-co-localization and expression but no significantly.6. ALDH2 alleviated CCCP-induced oxidative stress and apoptosis in H/R-treated H9C2 cellsLevels of ROS and mitochondrial superoxide levels were higher with H/R than with ALDH2 activation in cells, but lower than with CCCP. However, ALDH2 activation also suppressed the CCCP-elevated trends. As well, ALDH2 activation significantly alleviated the decreased mitochondrial membrane potential which damaged by CCCP in H/R treated H9C2 cells. Futhermore, ALDH2 activation reduced CCCP-induced apoptosis in H/R treated H9C2 cells significantly.7. ALDH2 alleviated CCCP-induced 4HNE accumulation in H/R-treated H9C2 cellsCCCP significantly decreased ALDH2 activity, which was rescuecPby ALDH2 activation and with no change in ALDH2 expression in H/R treated H9C2 cells. Ratio of cell viability were lower with CCCP than with ALDH2 activation.4HNE protein adduct formation was significantly increased in CCCP-induced. However, ALDH2 further alleviated the increase. 8. ALDH2 alleviated CCCP-induced mitophagy pathwasy activation inH/R-treated H9C2 cells CCCP increased the co-localization of PINK1 or Parkin with COX IVsignificantly in H/R treated H9C2 cells. ALDH2 activation reduced the CCCPinduced co-localization. As expected, A1DH2 activation also reduced these proteinexpressions which induced by CCCP.Conclusion1. I/R and H/R could activate the PINK1/Parking depenfent mitopahgy signaling pathway through the oxidative stress.2. ALDH2 mediated the activation of PINK1/Parkin depenfent mitopahgy signaling pathway induced byI/R and H/R.3. ALDH2 activation could ameliorate I/R and H/R induced myocardium injury, may through inhibiting the activation of PINK1/Parkin depenfent mitopahgy signaling... |
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