Experimental Study On The Anti-Myocardial Apoptosis Mechanism Of Levocarnitine

With increased knowledge on the pathogenesis of ischemic heart disease, moreand more attention have been paid to the role of myocardial energy metabolismplayed in this process, especially the deterioration effect of cardiac function, whichhave been gain widely attention. Levocarnitine (LC) is a classical drug to improve theenergy metabolism, and its main role is to shuttle long-chain fatty acids and activatedacetate across the inner mitochondrial membrane, thus maintain the normal functionof tricarboxylic acid cycle and energy metabolism. Furthermore, LC is also aneffective oxygen free radical scavenger to improve mitochondrial function throughreducing the level of free radicals and calcium overload, as well as to promote themetabolic adjustments and anti-apoptotic effect. However, the detail mechanism ofhow LC to improve the myocardial cell metabolism and to inhibit the myocardial cellapoptosis under ischemia and hypoxia conditions is still poorly understood. Therefore,in this study, we establish the ischemic heart failure animal model of by ligation of therat left anterior descending (LAD) coronary artery, and to explore the effect of LCintervention on rats serum oxidative stress enzyme change with heart failure aftermyocardial infarction, as well as to discuss the mechanism of its impact onnon-infarcted myocardium apoptosis. In addition, when H9c2rat cardiomyocytes wasinduced by LC pretreatment of hydrogen peroxide (H2O2), the oxidative stress wasinhibited and thus led to the change of energy metabolism. Therefore, we investigatethe effect of LC on ischemic myocardial energy metabolism and cardiomyocyteapoptosis from the whole cell level, and this study will be further supplement themechanism pathogenesis of ischemic heart disease, as well as to supply the theoreticbasis on optimize heart failure drug treatment.Methods:The ischemia reperfusion after myocardial infarction animal model wasconstructed by ligation LAD in rats. After four weeks, the live rat model was dividedinto four groups, which including control group (Sham operation, SO), model group(Myocardial infarction, MI), low dosage LC treated model group (MI-LC1) and highdosage LC treated model group (MI–LC2). All the groups were intervened withSaline and LC intraperitoneal injection therapy, after2week and4week,the administration was terminated, and the following indicators were measured: heart rate(HR), left ventricular end systolic pressure (LVSP), left ventricular end-diastolicpressure (LVEDP) and maximum ascending/declining rate of ventricular pressure,(±dp/dtmax). Then the heart hypertrophy index and left ventricular mass index wasmeasured, plasma B-type brain natriuretic peptide (BNP) level was measured byELISA method, serum superoxide dismutase (SOD) and malondialdehyde (MDA)levels were detected by commercial kits, the protein expression of Bcl-2/Bax andCaspase-3in non-infarcted tissue was measured by immunohistochemical method,and western blot assay was used to detect the apoptosis expression of Bcl-2/Bax andCaspase-3in cardiomyocyte and the protein expression change of mitochondrialcytochrome pathway related molecule cytochrome C (Cyt C).The myocardial apoptosis was induced by H2O2stimulation in H9c2cell line,and then different concentrations of LC intervention was used, which include theblank group, L-carnitine group separately,H2O2group, LC protection group of high,medium and low levels. The following indicators were measured: cell viability wasmeasured by CCK-8assay, cardiomyocytes oxidative balance related enzymes such asreactive oxygen species (ROS), lactate dehydrogenase (LDH), MDA, SOD andGSH-PX level was detected by ELISA. JC-1method was used to detect mitochondrialmembrane potential (△Ψm) as well as intracellular ATP levels; The apoptosis levelof cardiomyocytes was detected by PI/AV double staining flow cytometry; theexpression of Bcl-2/Bax, Caspase-3and Cyt C was measured by Western Blot assay.Results1. Data derived from animal experiment: the MI model group rats lost its originalgeometry, formed myocardial infarct scar, fibrous tissue proliferation, increased in theLVEDP level, reduced the LVSP and±dp/dt max level, left ventricular mass index andserum BNP level significantly increased when compared with the SO group. However,compared with the MI model group, the LC treated group had reduced the LVEDP,increased the LVSP and±dp/dt max, fibrous tissue proliferation is inhibited, leftventricular mass index and serum BNP level significantly decline.Detection of enzymes related to oxidative balance system: The MI model grouprats serum MDA level increased when compared with the SO group, while the serumSOD level decreased, furthermore, the above mentioned indicators is antagonized inthe LC treated group, thus indicated that LC played a role in the anti-xoidative stress process.Compared with the MI model group, the expression of Caspase-3and Cyt Cis significantly reduced in the LC treated group, meanwhile, the Bcl-2expression issignificantly increased, and it can be inferred that LC may effectively inhibit theexpression of pro-apoptotic protein Bax. Therefore, LC may inhibit myocardialapoptosis by both regulating the imbalance of Bcl-2/Bax and reducing the expressionof Caspase-3and Cyt C.All the above mentioned suggested that that high LC concentration has aprotective trend better than the low concentration group.2. The vitro cell culture experiment results: when H9c2cell was treated withH2O2, compared with the blank group, the ROS, LDH and MDA level were increasedin the H2O2intervened group, while the SOD and GSH-Px concentration is decreased.However, in the LC pretreated group, compared with the H2O2intervened group, theROS, LDH and MDA level is decreased, and ROS, LDH and MDA concentration isincreased, and its effect is in dose-dependent manner. Furthermore, the cardiomyocytemitochondrial membrane potential (△Ψm) is increased in the LC pretreated groupwhen compared with the H2O2intervened group, and it is suggested that theintracellular ATP level is inhibited by LC.The PI/AV double staining flow cytometry experiment showed that comparedwith the blank group, the cardiomyocyte apoptosis rate was significantly increased inthe H2O2intervened group; while after the addition of LC, the apoptosis rate ofcardiomyocyte was decreased. Western Blot showed that compared with the blankgroup, the expression of Caspase-3and Bax protein was increased, the expression ofBcl-2protein wsa decreased, and also the marker protein Cyt C in the mitochondrialapoptosis pathway was increased. However, in the LC pretreated group, theexpression of Caspase-3and Bax protein was inhibited, Cyt C protein expression issignificantly decreased, while the Bcl-2protein expression is increased whencompared with the H2O2intervened group. And the greater the concentration of LC,the effect is more better.Conclusions:1.The oxidative stress enzyme imbalance is involved in the pathogenesis ofschemic heart disease, and LC can improve myocardial apoptosis through itsantioxidant effects. 2.LC can decrease mitochondrial membrane potential of the cardiomyocyte, andincrease in the ATP level, thus playing a positively regulation in the energymetabolism of cardiomyocyte.3.The cardioprotection effects afforded by L-cartine from one side are due to itsdown-regulation of Cyt C protein expression through the mitochondrial pathwayinhibition of cardiomyocyte apoptosis;another side by its down regulation of thepro-apoptotic molecules Bax and Caspase-3expression and upregulation of theanti-apoptotic molecule Bcl-2expression.4.L-carnitine to regulate the action of myocardial energy metabolism is of certaindose of relevance.