| Objective:1. To observe progression of myocardial apoptosis in diabetic rat at several timepoints and aimed to explore its possible mechanism involved in diabeticcardiomyopathy.2. To explore whether myocardial apoptosis of diabetic rat is mediated bymitochondrial cytochrome c mediated caspase-3 activation pathway.3. To determine beneficial effect of antioxidant--alpha lipoic acid (α-LA) onmitochondrial oxidative damage derived from high levels of glucose and decreasethe number of apoptotic cardiomyocytes.Methods:1. Diabetic rat model was induced by a single tail intravenous injection ofstreptozotocin with the dose of 45mg/kg body weight.2. Experimental animals were randomly divided into 3 groups: normal control,diabetes control andα-LA. Rats in each group were scarified at three time points:4 weeks, 8 weeks and 12 weeks from the onset of diabetes. Diabetic rats inα-LAgroup were administrated byα-LA (100mg/kg) by intraperitoneal injection (ip)per day.3. Before sacrifice, animals were anesthetized with chloral hydrate (100mg/kg,ip),the carotid artery was cannulated to measure arterial systolic pressure anddiastolic pressure, and then advanced into the left ventricle to evaluateventricular pressures(LVSP, LVEDP) and the rate of pressure rise anddelay(+dp/dtmax).4. HE and Masson staining displayed the derangement of cardiac structure.5. The mitochondrion of cardiac tissue was extracted by differentialcentrifugation, the content of MDA in the myocardium mitochondrion wasassayed by thiobarbituric acid, the content of GSH, the activities of SOD in themyocardium mitochondrion were examined by spectophotometer respectively.6. Expression of caspase-9,-3 mRNA, protein and release of mitochondrialcytochrome c in cardiac tissues were determined by SQ-RT-PCR,immunohistochemical technique and Western blotting assay, respectively.7. The number of apoptotic cardiomyocytes was detected by TUNEL assay. Result:1. The incidence of STZ-induced diabetes was 90％.2. Compared to control group, DM andα-LA groups showed significant decreasein body weight and increased plasma glucose levels. The levels of body weightand plasma glucose showed no difference between DM andα-LA groups.3. Compared to control group, the number of apoptotic cardiomyocytes indiabetes rats were maintained at the high value from 4 weeks to 12 weeks afterSTZ treatment, 10 folds greater than that in control group. Diabetic rats treatedwithα-LA showed a significant reduction in the number of apoptotic cells ascompared with DM group, and had a 27.3％, 43.8％, 47.6％decline in apoptoticcells at 4 weeks, 8 weeks, 12 weeks, respectively.4. The content of GSH in the myocardial mitochondrion of diabetic rats showed35％, 40％, 72％decline compared as control group at 4 weeks, 8 weeks, 12weeks after STZ treatment respectively. At 4 weeks, there were no difference inthe activities of SOD in the myocardial mitochondrion between control anddiabetes groups, but at 8 weeks, 12 weeks, the activities of SOD in DM showed asignificant reduction, 57％, 74％decline compared as control group. The contentof MDA in the myocardial mitochondrion obviously increased 2.26 fold, 3.28fold, 5.54 fold than that of corresponding control at 3 time points. Antioxidativetreatment withα-LA can significantly increase the content of GSH, the activitiesof SOD and decrease the content of MDA. So we drew a conclusion thatantioxidantα-LA can attenuate the oxidative damage in myocardiummitochondrion of diabetes.5. Elevation of Cyt c in cytosol was in accordance with the decline inmitochondrial Cyt c content. Cyt c in cytosol increased gradually from 4 weeks to12 weeks after STZ treatment and accordingly decreased in Cyt c inmitochondrion. High glucose could up-regulate the caspase-9, -3 mRNAexpressions, which were gradually increased from 4 weeks to 12 weeks. Thecontent protein of caspase-9, -3 was in accordance with their mRNA expressions.Antioxidantα-LA could reduce change of index above-mentioned.6. Compared to control group, diabetic rats showed swollen myocardial cell,swelling cellular nucleus, disappearance of transverse striation, widen interspace,compensatory hypertrophy, muscle fiber reduction, cardiac muscle fibersconfused rank, verge of muscle bundle coarse, Severity myolysis and myorrhexis.All the change exacerbated step by step from 4 weeks to 12 weeks after STZtreatment. Collagen content in DM group was 13.39％±1.17％, 18.91％±1.53％,22.20％±1.55％respectively, significantly higher than that of the control group(8.54％±1.56％, 8.01％±1.02％, 8.37％±1.185％, P＜0.01), resulting from Massonstaining. The distribution of a great amount of collagen in the myocardial interstitial tissue interconnected reticulation. Antioxidantα-LA could reversederangement of structure above-mentioned to some degree.7. There were no differences in SBP and DBP among three groups. Arrhythmiaoccurred in DM group more often. DM group had constant higher cardiac massindex than corresponding control andα-LA groups, which suggested that cardiachypertrophy existed in untreated diabetic rats at these three time points.-dp/dtmax in diabetes group was decreased in 4-, 8-, 12-weeks as compared withcorresponding controls (P＜0.05) and had a downward trend, so LVEDP wasincreased. At 12 weeks, cardiac function was decreased further, +dp/dtmax andLVSP were decreased significantly, while LVEDP increased. Antioxidantα-LAcould improve index above-mentioned.Conclusion:Hyperglycemia could induce myocardial apoptosis, which played an importantrole in the development of diabetic cardiomyopathy. Under diabetic conditions,myocardial apoptosis was mediated, at least in part, by activation of thecytochrome c-activated caspase-3 pathway, which may be triggered bymitochondrial oxidative stress. Antioxidantα-LA could attenuate mitochondrialoxidative damage by enhance SOD activity and increase GSH content, whichresulted in a decline in cardiomyocyte apoptosis dependent on mitochondrion. Weconcluded thatα-LA may play a preventive role in diabetic cardiomyopathy.
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