Mitochondrial Dna Oxidative Damage Contributes To Cardiomyocyte Ischemia/Reperfusion-injury In Rats: Cardioprotective Role Of Lycopene

Background:Ischemia/reperfusion(I/R)-injury is a worldwide problem that people are constantly looking for new intrinsic or exogenous methods to reduce this damage. Recent studies indicate that mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of I/R-injury. Mitochondrial DNA(mt DNA) is very susceptible to oxidative stress. Lycopene is a lipophilic antioxidant that is the red crystalline unsaturated hydrocarbon carotenoid pigment mainly found in tomatoes and various berries and fruits and can protect the mtDNA against oxidative damage. Our recent study suggested that lycopene could decrease I/R-injury in vitro by preventing mitochondrial dysfunction and alleviating oxidative stress. The purpose of this study was to further determine whether mt DNA oxidative damage is involved in I/R-injury and whether lycopene can protect cardiomyocyte from I/R-injury by alleviating mt DNA oxidative damage.Methods1. The rat hearts were suffered from regional ischemia for 30 minutes, followed by 24 hours reperfusion to establish myocardial I/R-injury models in vivo. The H9c2 cells were subjected to hypoxia for 6 hours and followed by reoxygenation for 12 hours to establish myocardial I/R-injury models in vitro. To found out wheather I/R-injury related to mtDNA damage, the 8-hydroxyguanine(8-OHdG) was detected through immunostaining, and the mt DNA content and mt DNA transcription levels were detected through real time PCR both in vivo and in vitro. For immunostaining, we used COX IV in vivo and Mitotracker Red in vitro to label mitochondria, respectively.2. We detected the mitochondrial membrane potential(ΔΨm) and ATP level of cardiomyocytes both in vivo and in vitro to ensure if mitochondrial dysfunction related to I/R-injury in our models.3. To explore wheather mt DNA damage alone could lead to mitochondrial dysfunction, we pre-incubated H9c2 cells with ethidium bromide(EtBr, 100 ng/ml) to deplete mt DNA, then the ΔΨm and ATP level and the cell viability of H9c2 cells were detected. The mt DNA content and mt DNA transcription levels were also detected at the same time.4. To found out whether lycopene can protect cardiomyocyte from I/R-injury by inhibiting mtDNA damage. Triphenyltetrazolium chloride(TTC) stain was utilized to measure the myocardial infarct size. Blood serum samples were collected to detect the enzymes and markers indicating of myocardial injury, such as lactate dehydrogenase(LDH), creatine kinase-MB(CK-MB) and cardiac troponin T. We analysed the levels of apoptosis through terminal deoxynucleotidyl transferase dUTP nick end labeling(Tunel) staining. The levels of cleaved caspase-3 protein were analysed with Western blot. Apoptosis was also examined with FITC-labeled Annexin V/propidiumiodide(PI) Apoptosis Detection kit(BD Biosciences, New Jersey, USA) following the manufacturer’s instructions. At the same, the 8-OHdG, mt DNA content, mt DNA transcription levels, ΔΨm and ATP levels of cardiomyocyte were also detected at the same time to ensure the protective effect of lycopene on mtDNA and mitochondrial function.5. To further explore the protective mechanism of lycopene on mt DNA, malondialdehyde(MDA), reactive oxygen species(ROS) and mitochondrial superoxide levels were determined. In vitro experiment, H9c2 cells incubated with H2O2 to further confirmed the protective role of lycopene on ROS-induced mitochondrial damage.6. We also detected the effects of lycopene on mitochondrial transcription factor A(Tfam), a key protein involved in mt DNA replication, transcription, and nucleoid organization, through Western blot and immunostaining.Results1. The number of 8-OHd G-positive cells significantly increased both in the I/R-injured heart and H9c2 cells. Most of the 8-OHdG co-localized with mitochondria. PCR results indicated that both I/R-treatment in vivo and in vitro decreased mt DNA copy number and mt DNA transcripts significantly as compared with control. These results suggested that I/R-injury is related to mtDNA damage.2. ATP concentrations were significantly decreased in the I/R groups both in vivo and in vitro compared with controls. In I/R-treated H9c2 cells the ΔΨm also decreased. These results proved that I/R caused cardiomyocytes mitochondrial dysfunction.3. To explore the relationship of mtDNA damage and mitochondrial dysfunction, we depleted mt DNA with EtBr, and found mt DNA copy number and mt DNA transcript levels were time-dependently decreased by EtBr. At the same time, we also found ΔΨm and ATP were significantly decreased in mt DNA-depleted cells, while mitochondrial superoxide anion generation was significantly increased. In addition, mtDNA depletion significantly reduced the cell viability with in a time-dependent manner. These results indicating that mt DNA depletion alone was sufficient to induce cardiomyocyte mitochondrial dysfunction and cell death.4. A significant reduction in infarct size and myocardial enzymes(Troponin T, LDH, and CK-MB) released in the plasma occurred with lycopene pre-treatment compared with the vehicle-treated I/R group. We also found lycopene pre-treatment significantly reduced Tunel-positive cardiomyocytes both in vivo and in vitro. Pre-treatment with lycopene aslo reduced cleaved caspase-3 expression as compared to I/R group. The results of the Annexin V/PI analysis with flow cytometry confirmed that lycopene reduced H/R-induced cell death. At the same time, we noticed lycopene signicantly prevented I/R indued 8-OHd G-positive cells increase and mt DNA copy number and mtDNA transcripts decrease. Lycopene also prevented I/R indued ΔΨm and ATP decrease both in vivo and in vitro.5. Mt DNA is very susceptible to oxidative stress. In our experiments, we found that pre-treatment with lycopene significantly reduced the elevated MDA production and ROS generation induced by I/R both in vivo and in vitro. We also found pre-treatment with lycopene decreased H2O2 induced mt DNA damage and mitochondrial dysfunction.6. To further explore the underlying mechanism for mtDNA damage, we measured Tfam, a key protein involved in mtDNA replication, transcription, and nucleoid organization. The protein expression of Tfam in mitochondria were significantly decreased in I/R-injured rat hearts compared with controls. This decrease was prevented by lycopene pre-treatment.Conclusions:In conclusion, this study suggested that mtDNA oxidative damage is involved in myocardial I/R-injury. Lycopene has a pharmacological potential in the treatment myocardial I/R-injury by protecting cardiomyocyte mt DNA from oxidative damage. The protective role of lycopene on mt DNA may be due to the reduction in mitochondrial ROS and stabilization of mitochondrial Tfam.