| ObjectiveTo investigate the protective effects of L-carnitine preconditioning on myocardial ischemia-reperfusion injury (MIRI) in rats and explore its effects as an ingredient of cardiac arresting solution in the process of cardiopulmonary bypass(CPB) in dog and in rheumatic heart disease patients. Methods1. Thirty six wistar rats were randomly divided into three groups (n = 12 for each group): (1) L-carnitne treated group (L-CN group), L-carnitne (200mg/kg per day) was injected into the abdominal cavity for 3 days before operation; (2) Myocardial ischemia-reperfusion group (IR group), equal volume saline was given for 3 days before operation; (3) Sham operative group (S group), the same pretreatment was given as the IR group. Rats in the L-CN and IR group were subjected to left anterior descending coronary (LAD) occlusion for 30 min, followed by reperfusion for 3 h. The S group was identical to the IR group except that LAD was not ligated. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and cardiac Troponin I (cTnI) level in the serum were measured before and after operation. At 3 h after reperfusion, myocardial tissues were removed for the evaluation of ①histological changes; ②apoptotic protein expression in myocardium detected by immunohistochemistry method; ③myocytes apoptosis detect by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL); and ④ultrastructure changes of myocardial tissues examined by electron microscope.2. Twenty male healthy mongrel dogs, 15 to 20kg, were randomly allocated into two groups: L-CN group (n=10, 12g/L L-carnitine was put into the 4°C ST. Thomas II cardioplegia) and control group (n=10, identical to the L-carnitine group except that normal saline was administered instead of L-carnitine), the other conditions were the same. The cardioplegia solutions were infused for the antegrade intermittent coronary perfusion when the ascending aorta was cross-clamped, and were perfused for 4 times in all and repeated for every 30 min, but the concentration of the postassium chloride would be reduced by half in last perfusion. In both groups, cTnI and creatine kinase MB isozyme (CK-MB) levels in coronary venous sinus serum were measured just before the ascending aorta was cross-clamped and after the cross-clamp was removed, and myocardial tissue from left heart ventricle was available for the examination of the contents of MDA, adenosine triphosphate (ATP), the activity of SOD. The expression of poly-(adenosine-ribose) polymerase (PARP) protein in the myocardial tissues was also examined by western blot analysis.3. Sixty nine patients undergoing heart valve replacement with CPB were divided into 3 groups: test group I (n = 22, 12 g/L L-camitine was put in the St. Thomas II cold crystal cardiac arresting liquid ), test group II (n = 24, 6 g/L L-camitine was put into the St. Thomas II cold crystal cardiac arresting liquid ), and control group (n = 23, identical to the test group I except that normal saline was administered instead of L-carnitine). Before operation, 20 min after the beginning of shunt, after the finish of shunt, and 8 hours, one day, 3 days, and 7 days after operation venous blood was drawn to test the serum cTnI, aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and CK-MB isozyme and the plasma matrix metalloproteinases (MMPs). Heart color ultrasonography was conducted to test the cardiac index (CI) and left heart ejecting fraction (EF) one day before operation and 7 days after operation. Before shunt and by the end of intracardiac procedure a small pieces of myocardial tissue was taken to examine the (1) expression of apoptotic protein (Bcl-2/Bax) by immunohistochemistry and myocytes apoptosis detect by TUNEL methods; (2)ultrastructure changes of myocardial tissues examined by electron microscope, and (3) the activity of SOD, glutathion peroxidase (GSH-PX) and nitric oxide synthase (iNOS) as well as the concentrations of MDA and nitric oxide (NO) were determined in the extracted mitochondrion. The amounts of vasoactive drugs, such as dopamine and dobutamine, used postoperatively, and the postoperative cardiac auto-rebeating rate were also recorded. ResultsPart 1: (1) The average episodes of ventricular tachycardia(VT) and ventricular fibrillation(VF) in L-CN group were significantly less than that in IR group (P < 0.01). (2) The serum levels of MDA, cTnI were significantly less in L-CN group than that in IR group (P < 0.01) and the serum levels of SOD was significantly higher in L-CN group than that in IR group (P < 0.01). In addition, the percentages of apoptotic cardiomyocytes in L-CN group were significantly lower than that in IR group. The expression of Fos and Jun protein in myocardial tissues was also decreased in L-CN group than that in IR group. (3) Electron microscope examination revealed some part of myocardial fiber broken or dissolved, the collapse of the mitochondria, and the disappearance of glycogen particles in the IR group.Part 2: The levels of cTnI and CK-MB in coronary venous sinus serum, and MDA in myocardial tissue were significantly lower in the test group than that in the control group after cross-clamp was removed ( P < 0.05 or P < 0.01), but the contents of ATP and SOD in myocardial tissue was significantly higher in the test group than that in the control group (P < 0.05). The postoperative cardiac auto-rebeating rate in the test group was significantly higher than that in the control group (P < 0.01). Western blot analysis showed that the intact 113-kd PARP was degraded in both groups, but the increasing quantity of 89-kd cleavage product in L-CN group was significantly less than that in control group. Part 3: 3.1 The Amounts of vasoactive drugs used after operation in the test two groupswere significantly less than in the control group (P < 0.01) without a significant difference between the 2 test groups. Since the end of CPB to 3 days after operation, the serum levels of cTnI, AST,LDH, CK and CK-MB isozyme were significantly lower in the 2 test groups than in the control group (P < 0.05 or P < 0.01), the serum level of cTnI in test group I was significantly lower than in the test group IT (P < 0.05). The postoperative cardiac auto-rebeating rate in the 2 test groups were significantly higher than that in the control group (P < 0.05 or P < 0.01). Heart color ultrasonogram showed that 7 days postoperatively the CI and EF in the 2 test groups were significantly higher than that in the control group (P < 0.05). Electron microscopy showed only slight swelling of mitochondria in the cardial cell and the myocardial fiber was intact by the end of operation in the 2 test groups without significant difference between these 2 groups, however, in the control group swelling of mitochondria with vesicle formation, fissure of part of mitochondrial ridges, and disappearance of glycogen particles were found.3.2 From the end of CPB to 3 days after operation, the serum level of cTnI in test group I were significant lower than in test group II ( P < 0.05) and control group(P < 0.01). 20 rnin after the initiation of CPB to 1 day after operation, plasma level of MMP-2 in test group I was significant lower than in control group (P < 0.05 or P < 0.01) .20 min after the beginning of shunt to the termination of CPB, plasma level of MMP-9 in test group I was significant lower than in control group (P < 0.05 or P < 0.01).3.3 The percentage of apoptotic cardiomyocytes at the end of CPB was significantly higher in control group than in the both test groups. The expression of Bax was increased at the end of CPB as compared to the baseline before CPB in all groups; while Bcl-2 expression was decreased only in control group. Bax expression was significantly lower whereas Bcl-2 expression signicantly higher in test groups than in control group at the end of CPB. Bcl-2 / Bax ratio were signicantly higher in test groups than in control group.3.4 Compared to the control group, the activity of SOD, GSH-PX in both test groups were significantly higher at the end of CPB (P < 0.05 or P < 0.01), but iNOS activity and the concentrations of MDA and NO were markedly reduced (P < 0.05 or P < 0.01). Conclusion1. L-CN preconditioning on MIRI in rats can decrease the levels of cTnI and MDA in serum and inhibit the expression of Fos and Jun proteins in myocardial tissue, and reduce the average episodes of ventricular tachycardia (VT) and ventricular fibrillation (VF),and also protect the ultrastructure of myocardium.2. Antegrade coronary perfusion of L-CN as an ingredient of cardiac arresting solution in the process of operation in dog improves energy metabolism, reduces the toxic metabolites of cardiomyocytes, and inhibits the apoposis of myocardial cells.3. L-CN can enhance the antioxidative capability in cardiomyocytes, decrease the levels of MMPs, cTnI, and cardiac enzymes in serum (or plasma), protect the ultrastructure (mitochondrion especially) of myocardium, and improve the postoperative cardiac function in rheumatic heart disease patients undergoing heart valve replacement with CPB.
|
PDF Full Text Request