| BackgroundDiabetes, especially type2diabetes, is a worldwide major disease. It can induceischemia heart disease (IHD), which represents the main cause of dead diabetic patients.Although Percutaneous coronary intervention(PCI) has been highly developed, therecovery and prognosis of diabetic heart’s function after myocardial infarction(MI) aremuch worse than non-diabetic heart and the underlying mechanisms of increasingsensitivity of diabetic myocardial cells in response to ischemia/reperfusion injury have notbeen fully investigated.Mitochondria is an intra-cellular center for regulating energy metabolism and headquarters of apoptosis. A clinical research in2004found mitochondrial dysfunction in type2diabetic patients. Mitochondria self-renew through mitochondrial biogenesis plays an important role of maintaining integrity of mitochondria. The injury of this process is related with lots of diseases, including aging, neurodegenerat-ion and type2diabetes. Mitochondrial biogenesis dysfunction can impair enzyme activity and increase intra-myocellular lipid (IMCL) levels, eventually leading to ins-ulinresistence in type2diabetes. Thus, Mitochondrial biogenesis dysfunction has beenknown as one of the important causes of injury of type2diabetic heart.Adiponectin (APN) is an adipocytokine secreted from adipose tissue. The content ofAPN is rich in the plasma of humans, whereas it decreases significantly in obesity relateddiseases, including coronary disease and type2diabetes. A clinical research over18,000male patients in2004indicated there is a strong negative correlation between the level ofplasma APN and morbidity of MI. Recent researches strongly indicate that the decrease ofplasma APN of diabetic patients might be a key factor of increasing morbidity of diabeticIHD.Masato Iwabu’s recent research showed in skeletal muscles, APN downstreampathway is related with mitochondrial biogenesis. However, it is still unknown whethercardioprotection of APN is through mitochondrial biogenesis.Research aims:1. Whether high fat/high glucose(HGHF) could affect myocardial mitochondrialbiogenesis and function?2. Whether and how APN reversed myocardial mitochondrial biogenesis dysfunction inresponse to HGHF incubation?3. Whether APN improved the myocardial Hypoxia/reoxygenation(H/R) injury in responseto HGHF incubation via mitochondrial biogenesis?Research methods:1. Cultured neonatal rat ventricular myocytes (NRVMs) were randomized into controlgroup (5mmol/L glucose and20mmol/L mannitol), HGHF group (25mmol/L glucose and500μmol/Lsodium palmitate,18h incubation) and HGHF+APN group (25mmol/Lglucose, 500μmol/L sodium palmitate and3μg/mL globular APN,18h incubation). Cells wereharvested for evaluation of mRNA levels of Tfam, NRF-1, PGC-1α, protein levels ofPGC-1α, mitochondrial function and morphological changes after HGHF incubation.2. Cultured NRVMs were randomized into control group, HGHF group, HGHF+APNgroup, HGHF+APN+PGC-1α siRNA group and HGHF+APN+Scrambled siRNA group(the concentration of DMEM is as same as1). Cells were harvested for evaluation ofmitochondrial DNA level and mitochondrial membrane potential.3. Cultured NRVMs were randomized into control group, HGHF group, HGHF+APNgroup, HGHF+APN+PGC-1α siRNA group and HGHF+APN+Scrambled siRNAgroup(the concentration of DMEM is as same as1). Cells were harvested for evaluation ofcell apoptosis.Research results:1.We successfully isolated and cultured NRVMs with combination of pancreatic enzymeand collagenase. After5days of normal culture, a confuent monolayer of spontaneouslybeating NRVMs had formed. The beating rate of NRVMs was about from60to120perminute.2. We cultured NRVMs different time with HGHF treatment and found that both mRNAlevels of NRF-1and PGC-1α decreased in a time-dependent fashion. The highestdecrement of mRNA expression occurred at18h, suggesting after18h of incubation,mitochondrial biogenesis was down regulated most obviously.3. We made cell samples for transmission electron microscopy(TEM) after18h of HGHFincubation. The results showed that compared to Controls, HGHF treatment resulted in bigand round mitochondria, ambiguous, short and disappeared mitochondrial cristae inNRVMs.4. gAd was administered to NRVMs after18h of HGHF incubation. Compared to Controls,18hours of HGHF incubation significantly reduced the mRNA expression ofNRF-1,PGC-1a and Tfam(P<0.05). Moreover, treatment with gAd could reverse thedecrease in mRNA expression of NRF-1,PGC-1a and Tfam in response to HGHFtreatment(P<0.05), indicating that gAd can improve the mitochondrial biogenesis impairment in cultured cardiomyocytes in response to HGHF treatment.5. We inhibit the expression of PGC-1α with PGC-1α siRNA treatment in NRVMs, thentested the expression of mitochondrial DNA (mtDNA). The results showed that comparedto Controls, mtDNA levels in HGHF group were unchanged, but markedly increased aftertreatment with gAd. Treatment with PGC-1α siRNAcould attenuate this increase(P<0.05),indicating APN increased mitochondrial biogenesis through PGC-1α.6. We used JC-1assay kit and ATP assay kit to measure mitochondrial membrane potentialand ATP content of NRVMs, respectively. The results showed that compared to Controls,mitochondrial membrane potential and ATP content decreased in HGHF group, suggestingmitochondrial function was damaged; Treatment with gAd could improve mitochondrialmembrane potential and increase ATP content, suggesting mitochondrial function wasimproved; PGC-1α siRNA treatment could block the additional cardiomyocyte protectioncaused by treatment with gAd.7. We made H/R (Hypoxia for9h, reoxygenation for3h) experiment to NRVMs after18hof HGHF incubation, then measured the activity of Caspase-3and made the TUNELstaining experiment. The results showed that in contrast to Controls, the activity ofCaspase-3of H/R group was highly elevated (P<0.05). Treatment with gAd could blockthe additional cardiomyocyte apoptosis. But Caspase-3activity increased again aftertreatment with PGC-1α siRNA. TUNEL staining results were consistent with Caspase-3results. These results demonstrate that APN exerts cardioprotection to H/R NRVMs inresponse to HGHF treatment and inhibition of PGC-1α can attenuate this protective effect.Conclusion:HGHF treatment decreases myocardial mitochondrial biogenesis. APN improvesmitochondrial biogenesis dysfunction and attenuates H/R injury via upregulation ofPGC-1α in cardiomyocytes with HGHF treatment. |
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