| Cardiac disease, which is seriously harmful to human health, remains the leadingcause of morbidity and mortality worldwide. The amount of patients dying from suchdisease is19million each year, ranking first in all kinds of lethal diseases. Myocardialinfarction is one of the most severe cardiovascular diseases; it occurs when coronaryblood flow to the heart is reduced, leading to deficient oxygen and nutrient supply tothe heart. The most effective means of therapy for myocardial ischemia is rapidreperfusion, which can attenuate myocardial necrosis, reduce cardiomyocytesapoptosis and restore contractile dysfunction. However, myocardialischemia/reperfusion (I/R) injury is responsible for additional myocardial damageincluding lethal ventricular arrhythmia. Oxidative stress is considered as a causativefactor in the pathogenesis of ischemic heart disease. Reactive oxygen species (ROS)play a major role in the pathogenesis of myocardial dysfunction in a variety ofconditions, including cardiac hypertrophy, myocardial infarction and myocardialischemia/reperfusion injury. The redox state of the cell is a consequence of the precisebalance between the levels of ROS and endogenous antioxidants. Elevation of ROS inexcess of the antioxidant-buffering capacity results in potentially cytotoxic oxidativestress. H2O2, a major oxidant molecule known to be generated during cardiac injuryand swiftly penetrating the cell membrane, has been widely used in the experiments tomimic situation with oxidative stress.Lysophosphatidic acid (LPA) is a serum-born phospholipid capable of activatingspecific G protein coupled receptors (GPCRs) to evoke multiple cellular responses. Inrecent year, the relationship between the accumulation of lipid metabolites andischemic cardiovascular disease has been paid more attentions. Moreover, numerousstudies show that the concentration of LPA increases following ischemic cardiovascular disease, and the LPA levels might relate to the severity and prognosisof cardiovascular disease. The effects of LPA have been implicated in numerouscardiovascular diseases, such as myocardial infarction, atherosclerosis and myocardialhypertrophy. Myocardial infarction is followed by a significant increase in LPAconcentration. LPA involves in both apoptosis and remodeling of cardiomyocytes indifferent period after acute myocardial infarction; our previous studies also found thatLPA could induce arrhythmias after acute myocardial infarction. It is reported thatLPA manifests different electrophysiological effects on different parts of thecardiomyocyte, which reminds us that LPA might play different roles in damaged andundamaged parts of the cells after myocardial infarction. Our previous work reportedLPAcould up-regulate L-type calcium current and prolong action potential duration inadult guinea pig myocytes, but its impact on electrophysiological properties ofneonatal rat cardiac myocytes after oxidative damage has yet to do further research,and relevant research has not been reported.In this study, primary cultured neonatal rat ventricular myocytes were used toexamine the effects of LPA on the L-type calcium current and action potentialduration in normal and H2O2-treated myocytes, and possible molecular mechanismswere explored.This study includes the following work:1. Effects of LPAon action potential duration in ventricular myocytesWhole cell patch clamp method was used to detect the effects of LPA on actionpotential duration (APD) in primary cultured neonatal rat ventricular myocytes. Theresults showed that LPA (5,10and20μmol/L) prolonged APD in ventricularmyocytes, and the effect increased gradually with the extension of time. APD wasshortened after oxidative damage. LPA stimulation for2h and4h prolonged the APDof oxidative damaged myocytes, while LPAstimulation for8h and24h shortened theAPD.2. Effects of LPAon L-type calcium current in ventricular myocytesWhole cell patch clamp and cell-attached patch clamp method were used to detect the effects of LPA on L-type calcium current (ICa-L) in primary cultured neonatal ratventricular myocytes. The results showed that the values for ICa-Lof thecardiomyocytes gradually increased following LPA stimulation, and thesingle-channel open probability of calcium current also increased. ICa-Ldecreased afteroxidative damage. LPA stimulation for2h and4h increased the ICa-Lof oxidativedamaged myocytes, while LPA stimulation for8h and24h decreased the ICa-L. LPAincreased single-channel open probability of calcium current in oxidative damagedmyocytes.3. Effects of LPA on expression of CaV1.2mRNA in ventricularmyocytesReal-time quantitative PCR method was used to detect the effect of LPA on geneexpression that encodes L-type calcium channel α1csubunit in primary culturedneonatal rat ventricular myocytes. The results showed that CaV1.2mRNA expressionwas unchanged after LPA (10μmol/L) stimulation for2h,4h,8h and24h inprimary cultured myocytes. CaV1.2mRNA expression decreased after oxidativedamage. CaV1.2mRNA expression was unchanged after LPA stimulation for2h inoxidative damaged myocytes, while CaV1.2mRNA expression decreased after LPAstimulation for4h,8h and24h.4. Effects of LPA on expression of CaV1.2protein in ventricularmyocytesWestern blot method was used to detect the effect of LPA on protein expression thatencodes L-type calcium channel α1csubunit in primary cultured neonatal ratventricular myocytes. The results showed that CaV1.2protein expression wasunchanged after LPA (10μmol/L) stimulation for2h,4h,8h and24h in primarycultured myocytes. CaV1.2protein expression decreased after oxidative damage.CaV1.2protein expression was unchanged after LPA stimulation for2h and4h inoxidative damaged myocytes, while CaV1.2protein expression decreased after LPAstimulation for8h and24h. 5. Effects of LPAon the activity of CaN in ventricular myocytesSpectrophotometer absorbance method was used to detect the effect of LPA on theactivity of CaN in primary cultured neonatal rat ventricular myocytes. The resultsshowed that LPA had no effect on the activity of CaN in primary cultured myocytes.The activity of CaN increased after oxidative damage, and LPA further enhanced theactivity of CaN in oxidative damaged myocytes.6. Effects of LPA on the translocation of NFAT in ventricularmyocytesCell immunofluorescent staining method was used to detect the effect of LPA on thetranslocation of NFAT in primary cultured neonatal rat ventricular myocytes. Theresults showed that LPA had no effect on the translocation of NFAT in primarycultured myocytes. Oxidative damage induced the translocation of NFAT in culturedneonatal ventricular myocytes, and LPAfurther increases the translocation of NFAT inoxidative damaged myocytes.7. FK-506inhibits theAPD and ICa-Lalteration induced by LPAWhole cell patch clamp, real-time PCR and western blot method were used to detectthe effects of FK-506on APD, ICa-L, CaV1.2mRNA and protein in primary culturedneonatal rat ventricular myocytes treated with LPA. FK-506could inhibit the LPAinduced decrease of APD, ICa-L, CaV1.2mRNA and protein in oxidative damagedmyocytes.This study is the first to explore the different effects of LPA in normal andoxidative damaged primary cultured neonatal ventricular myocytes. LPAincreased theL-type calcium current by increasing single-channel open probability, therebyprolonging the action potential duration. The increased Ca2+concentration inoxidative damaged myocytes activated CaN-NFAT signaling pathway, whichdown-regulated L-type calcium channel protein, thereby decreasing L-type calciumcurrent. LPA increased the activity of CaN-NFAT signaling pathway, which decreased L-type calcium current. LPA increased the single-channel open probability of calciumcurrent in oxidative damaged myocytes, while CaN-NFAT signaling pathway played amajor role in regulating L-type calcium channel protein with the extention oftreatment time, thus LPA ultimately decreased the L-type calcium current and actionpotential duration. |
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