Effect Of Apelin On Heart And Cardiomyocyte Of Rat

Regulation of myocardial contractility by endogenous peptides is important in physiological and pathophysiological conditions and may be a crucial therapeutic target.An autocrine or paracrine system which potentially regulates heart function is the recently discovered angiotensin receptor like-1 (APJ) with its endogenous ligand apelin.Apelin and APJ are widely expressed throughout the cardiovascular system. Apelin is actively synthesized by cardiac myocytes, vascular endothelial and smooth muscle cells, and has specific receptors in the heart. Ex vivo studies using isolated perfused rat hearts have identified apelin as one of the most potent inotropic substances yet recognised. Moreover, apelin has been reported to increase left ventricular contractility in vivo following acute as well as chronic infusion in rodents.The APJ receptor and apelin have been implicated in the pathophysiology of human heart failure. Plasma concentration of apelin has been shown to decrease in patients with congestive heart failure and long-term cardiac resynchronization therapy could restore plasma levels of the peptide. Moreover, mechanical ventricular support increases APJ mRNA levels in patients with heart failure. Apelin has also been implicated in the pathophysiology of arrhythmias. Ellinor et al. demonstrated that plasma apelin levels decrease in patients with lone atrial fibrillation. Despite recent advances in our understanding of the cardiovascular effects of the apelin-APJ system in vivo, the direct effects of apelin on cardiomyocyte contractility remain unknown.Aim:1. To find Apelin's effect on whole heart and isolated myocyte of rat.2. To find the Dose-Effect relationship of Apelin's positive inotropic action.3. To find the differences between Apelin and other medicines.4. To find the specificity of Apelin's positive inotropic action.5. To find the signal pathway of intracellular transduction of Apelin.6. To find different pre-load's effect on Apelin's positive inotropic action.7. To find the role of PLC and PKC in the mechanism of Apelin's positive inotropic action.8. To find the role of SERCA, RyR, NCX and NHE in the mechanism of Apelin's positive inotropinc action.Material and Methods:Rats were decapitated and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique as described previously.Heart rate was maintained constant (304±1 beats/min) by atrial pacing using a Grass stimulator (model S88, 11 V, 0.5 ms). Contractile force (apicobasal displacement) was obtained by connecting a force displacement transducer (Grass Instruments, FT03) to the apex of the heart at an initial preload stretch of 2 g. A 60-minute equilibration period and a 5-minute control period was followed by addition of various drugs to the perfusate by an infusion pump at a rate of 0.5 mL/min for 30 minutes. Initially, we determined the concentration-dependent effect of apelin-16 (0.01 to 1 nmol/L) on cardiac contractility. Next, we compared the effect of apelin to the responses of endothelin-1, adrenomedullin, and theβ-adrenergic receptor agonist isoproterenol. To test the specificity of the effect of apelin, it was infused in the presence of various receptor antagonists. To define the role of endogenous nitric oxide in apelin-induced inotropic response, the peptide was infused in the presence of L-NAME. L-NAME at a concentration of 300μmol/L effectively inhibited nitric oxide synthase in the myocardium.For signal transduction studies, the concentration of U-73122 (100 nmol/L), staurosporine (10 nmol/L) and GF-109203X (90 nmol/L), MIA (1μmol/L) and zoniporide (1μmol/L), and KB-R7943 (250 nmol/L) were selected because these concentrations have been demonstrated to suppress phospholipase C and protein kinase C activity and inhibit Na+/H+ exchange and the reverse mode Ca²⁺/Na+ exchange, respectively.The perfusion technique and the composition of the Krebs-Henseleit bicarbonate buffer was similar to that described above, however, left ventricular contractility was assessed by measuring isovolumic left ventricular pressure. Isovolumic left ventricular pressure was measured using a fluid-filled balloon, which was placed in the left ventricular chamber and connected to a pressure transducer. The balloon was large enough so that negligible pressure resulted, when the balloon alone was filled up to the maximum volume used. The following parameters were obtained: peak systolic left ventricular pressure, left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (DP), maximum and minimum values of the first derivative of isovolumic pressure (dP/dtmax, dP/dtmin), time from peak systolic pressure to 60% relaxation (RT 60%), time from peak systolic pressure to 90% relaxation (RT 90%). A 20-minute equilibration was followed by addition of apelin (1 nmol/L) or vehicle to the perfusate for 30 minute. During the first 10 minutes of infusion the left ventricular balloon was flaccid, thereafter the balloon was inflated in 10μL steps to achieve a LVEDP of 1, 5, 10 and 15 mmHg. Parameters of left ventricular contractility were obtained when a new steady-state was reached. Myocytes were isolated using standard procedures.We used two different Ca²⁺ indicators and protocols to measure intracellular Ca²⁺. The first: Freshly isolated myocytes were placed on laminin-coated glass coverslips and allowed to attach for 30 minutes before they were loaded with fura 2-AM. The free [Ca²⁺]_i of loaded cardiac myocytes were measured as the fluorescence ratio (360/380nm). The second: Cells were loaded during 30 minutes with 5μmol/l indo-1/AM and 0.01% pluronic befor each experiment. The field stimulation (0.5Hz) was elicited and indo-1 fluorescence was measured in dual emission mode, excited at 340nm with xenon lamp flashes (100 W). Dual wavelength emission was measured at 410 and 516 nm, respectively. Shortening of myocytes was simultaneously measured with E[Ca²⁺]_i. Myocytes were perfused with Tyrode's solution at 22°C and 1 ml/min flow rate. Myocytes were equilibrated at 22°C for about 20 minutes before use. All experimental protocols were carried out at room temperature. The contractileshortening of ventricular myocyte was measured by a video-based motion edge-detection system and an inverted microscope.Caffeine-induced Ca²⁺ (C[Ca²⁺]_i) transient amplitude was used as a measurement of SR Ca²⁺ content.SR Ca²⁺ content was measured by another way since this was the most important observation in the current study. RC causes complete depletion of calcium from SR and calcium released remains confined to the cytoplasm.The activity of Ca²⁺-ATPase was determined with a kit (Jiancheng, Nanjing, China) by measuring the inorganic phosphate (Pi) liberated from ATP hydrolysis.Results:1. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1±1.5 pmol/L).2. Moreover, preload-induced increase in dP/dtmax was significantly augmented (P<0.05) in the presence of apelin.3. Inhibition of phospholipase C (PLC) with U-73122 and suppression of protein kinase C (PKC) with staurosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect (P<0.001).4. In addition, zoniporide, a selective inhibitor of NHE isoform-1, and KB-R7943, a potent inhibitor of the reverse mode NCX, significantly suppressed the response to apelin (P<0.001).5. Compared with control, treatment with apelin caused a 55.7±13.9% increase in sarcomere fraction shortening (FS) and a 43.6±4.56% increase in amplitude of E[Ca²⁺]_i transients (n=14,P<0.05).6. SR Ca²⁺ content measured by caffeine-induced Ca²⁺ (C[Ca²⁺]_i) transient was decreased 8.41±0.92% in response to apelin (n=14,P<0.05).7. NCX function was increased since half-decay time (T50) of C[Ca²⁺]_i was decreased 16.22±1.36% in response to apelin.8. Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity was also increased by apelin.9. These responses can be partially or completely blocked by chelerythrine chloride (CHE), a PKC inhibitor.10. In addition, to confirm our data, we used indo-1 as another Ca²⁺ indicator and rapid cooling(RC) as another way to measure SR Ca²⁺ content, and we observed the similar results. Conclusions:1. In the whole rat heart level, Apelin has dose-dependent positive inotropic action. PLC, PKC and NHE, NCX are involved.2. In the isolated myocyte level, 1nmol/l Apelin has positive inotropic action.3. The increase of amplitude of E[Ca²⁺]_i is one of reasons of Apelin's positive inotropic action. Some other reason is remained, and is PKC dependent.4. Apelin decreases SR Ca²⁺ content, with a PKC dependent way, and NCX, SERCA are two key proteins.