Cardioprotective Effect Of Apelin On Cardiac Performance Against Ischemia Reperfusion

Apelin is a peptide ligand for a G-protein coupled receptor (APJ receptor). Apelin/APJsystem is widely expressed in brain, heart, stomach, lung and the vascular system. Apelinregulates cardiovascular function, producing vasodilatory and positive inotropic effect.There is an increasing body of evidence to suggest that Apelin protects the heart againstischemia/reperfusion (I/R) induced infarction, but the mechanism is still controversial.While irreversible injuries inducing cardiomyocytes necrosis or apoptosis occurredduring I/R, myocardial contractile dysfunction resulting from I/R is also a commonclinical problem in patients with some heart diseases and therapies. But the effect ofApelin on cardiac dysfunction induced by I/R was not well investigated. During I/R, cardiac contractile dysfunction is attributed to the impairment of calcium handlingactivities of the cardiomyocyte. Ca2+enters the cardiomyocyte through the L-type Ca2+channels (LTCC) triggering further release of Ca2+via the ryanodine receptor (RyR) fromthe sarcoplasmic reticulum (SR), which lead to a large increase in cytosolic free calciumconcentration, known as the intracellular [Ca2+] transient ([Ca2+]i). The elevatedintracellular calcium concentration, which stimulates contraction of the myofilaments, isremoved mainly to the SR by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)and out of the myocytes by the Na+/Ca2+exchanger (NCX) to initiate relaxation.Abnormalities in function of Ca2+handling has been suggested to explain contractiledysfunction of the heart following I/R in the heart.On the other hand, the increase in reactive oxygen species (ROS) within the first fewminutes of reperfusion has been proposed to explain the I/R-induced contractile changes inthe heart. Furthermore, SR Ca2+uptake and release activities have been reported to bedepressed by ROS in I/R hearts. In fact, exposure of the heart to different species of ROShas been shown to cause structural and functional alterations in the heart that are similarto those seen in I/R; these changes have been demonstrated to be due to abnormalities inCa2+handling by SR and sarcolemma.Therefore it is likely that, during I/R, over release of ROS impaired the SR Ca2+handling activities in the cardiomyocytes, inducing the contractile dysfunction. Apelincould increase SERCA activity and calcium transient in normal cardiomyocytes, but theeffects of Apelin on ROS and SR in I/R heart have not been investigated.Survival kinases, such as phosphatidylinositol-3-kinase (PI3K)/Akt and PKC may acton downstream mitochondrial targets to open mitochondrial ATP-sensitive potassium(mitoKATP) channels and to affect cellular survival, reducing both necrosis and apoptosis.The mitoKATPchannels may modulate ROS production, which may lead to a re-activationof a pool of PKCs. In particular, in concert with mitoKATPchannels, theintra-mitochondrial protein kinase C (PKC) activation may take part to the so-called“memory-associated protection”. In previous reports, PI3K/Akt pathway was involved inthe cardioprotective effect of Apelin against I/R. We supposed that these survival kinases and downstream targets might play a role in the effect of Apelin on ROS production andSR function in I/R.The present study was therefore undertaken for three purposes to examine:1)whether Apelin can improve the cardiac dysfunction induced by I/R;2) if Apelin canprevent abnormalities in ROS production and SR function in I/R hearts;3) the probablyunderlying mechanisms.Methods1. Heart perfusionThe hearts of male Sprague-Dawley rats were rapidly excised, cannulated to theLangendorff’s apparatus and perfused under a constant pressure of80mmHg withKrebs-Henseleit (K-H) medium containing (mmol/L)120NaCl,25NaHCO3,4.7KCl,1.2KH2PO4,1.2MgSO4,1.25CaCl2, and11glucose (37°C, pH7.4). The left ventricularend-diastolic pressure (LVEDP) was adjusted at5mmHg. Such a volume of balloon wasmaintained throughout the experiment. The coronary flow, left ventricular systolicpressure (LVSP) and left ventricular developed pressure (LVDP) were recorded andmaximum derivatives of the ventricular pressure (±dp/dtmax) were calculated continuouslyby the Acknowledge software.2. Determination of myocardial infarct sizeInfarct size was determined by2,3,5-triphenyltetrazolium chloride (TTC) staining.3. Determination of oxidative stress, redox state and nitrotyrosine content in isolatedhearts.4. Isolation of SR vesicles.5. Measurement of calcium uptake by SERCA.6.3H-Ryanodine binding assay.7. Isolation of adult rat ventricular myocytes and treatment with simulated I/R.8. Measurement of [Ca2+]i transients and cell shortening in the single cardiomyocyte.9. Measurement of ROS Generation in cardiomyocytes.10. Immunoprecipitation with anti-SERCA or anti-RyR antibody.11. Tissue Biochemistry. Perchloric acid extractions were performed on frozen ventricle tissue.12. NMR Measurements. All NMR data were acquired on a spectrometer equipped withan Aspect3000series computer and a9.4-T, vertical-bore superconducting magnet.Relative metabolite levels within each spectrum were determined by integration of theareas under each resonance peak of interest using an analysis subroutine within the NMRdedicated software.Results1. Cardioprotection effects of Apelin-13. A significant reduction in the LDH release inthe100nmol/L and1μmol/L groups was observed.2. Apelin improved cardiac dysfunction of the I/R hearts.3. Apelin attenuated ischemic and reperfused contracture.4. Apelin ameliorated myocardial oxidative stress caused by I/R.5. Apelin-13restored the activities of SERCA and RyR during I/R.6. Apelin attenuated the tyrosine nitration of SERCA and maintained theS-glutathiolation of SERCA and S-glutathiolation of RyR in I/R rat hearts.7. Effect of Apelin-13on ROS Generation during I/R in Cardiomyocytes. To examinethe ROS generation during simulated I/R, we examined the fluorescence of DCFH-loadedcells. The ROS level was found to be increased after reperfusion and Apelin-13reducedthis increase.8. Apelin-13improved the impairment of Ca2+homeostasis and cell shorteninginduced by I/R.9. Tissue lactate content was similar, whereas alanine content was higher in controlhearts perfused with pyruvate versus glucose controls.10. Effects of Apelin on Tissue Metabolites. Apelin treatment did not affect tissuelactate levels under any of the experimental conditions, including ischemia. In contrast,Apelin treatment raised tissue alanine content.11. Exogenous Apelin was effective in preserving ATP levels at ischemia when glucose was available.12. Apelin treatment delayed the onset of contracture.Conclusion: Apelin protects SR function and cardiac performance during I/R byattenuating oxidation of SERCA and RyR and preserving ATP content.