Background. Ca2+ is commonly known as a crucial molecule in heart tissue. It triggers signal transduction, cellular contractility, and activates enzymes. Moreover, Ca2+ is considered to be an important second messenger in cardiovascular tissue. Recently, Ca2+ ions were shown to act as a first extracellular messengers via special calcium-sensing receptors (CaR). These receptors are G protein-coupled and are present in many organs and tissues throughout the entire body including the cardiovascular system. They are supposed to activate several signaling cascades in cardiomyocytes. In the past few years, the involvement of CaR in heart diseases was shown in many publications. CaR are involved in ischemia-reperfusion injury, hypertension, hypertrophy and heart failure development. However, underlying mechanisms remained elusive so far. Thus in present study we aimed to investigate on the role of CaR in physiological heart function and during the development of cardiac hypertrophy.Materials and Methods. Experiments were performed on isolated cardiomyocytes and heart muscle strips. Load-free cell shortening was measured with a special cell-edge detection system. Contraction force of the heart muscle strips was measured using tensometry equipment. Cells and tissue material were exposed to Western Blot and RT-PCR analysis. Ca2+-transients were detected in isolated cardiac myocytes loaded with FURA-2AM by ION Optix system. To determine protein kinase C (PKC) activity under CaR stimulation, special nonradioactive PKC activity assay was used. Hypertrophy was modulated in vivo and in vitro. As in vivo model rats with NO-deficiency were used. Hypertrophy in vitro was provoked by adding ET-1 and PE to the cardiomyocytes culture for 24 h. Polyamines putrescine and spermine and synthetic CaR agonist gadolinium (Gd) were used to activate CaR and NPS2390 and siRNA vs CaR to antagonize the receptor.Results. CaR in physiological heart function. RT-PCR and Western Blot analysis confirmed the presence of the receptor in adult rat cardiomyocytes. Differences in CaR expression between right and left ventricular and atrial tissues were detected. Cells and heart muscle strips shortening as well as relaxation velocity of cardiomyocytes increased under CaR activation. Inhibition of the receptor lead to a reduced contraction force of the heart strips and decreased load-free cell shortening of cardiomyocytes, and also to a deceleration of the relaxation velocity. Significant effect of CaR inhibition was observed just under physiological Ca2+-concentrations. The effect of CaR agonists and inhibitors on atrial strips was less pronounced. Systolic Ca2+ and Ca2+-uptake to the sarcoplasmic reticulum (SR) were enhanced under CaR activation. Putrescine increased the activity of PKC and the levels of phosphorylated phospholamban (PLB). Moreover, inhibition of PKC abolished the effect of CaR activation on load-free cell shortening and relaxation velocity of cardiomyocytes. Effect of CaR was also abolished under downregulated RAMP1. CaR and hypertrophy. Hypertrophy developed in vivo was accompanied by increased mRNA levels of CaR and RAMP1. Interestingly, the mRNA of ETB receptors was also enhanced in one month L-NAME treated rats, whereas ETA and ECE showed no differences.ET-1 and PE chronic stimulation decreased shortening and relaxation velocity of the cells. However, the effect of ET-1 was potentiated by NPS2390 and siRNA vs CaR. Enhanced CaR protein expression was shown under ET-1 exposure for 24 hours, but not under PE stimulation. Inhibition of ETB1 and ETA receptors cancelled ET-1 remodeling effect and enhanced CaR protein expression. Conclusion. Cardiomyocytes express CaR under physiological conditions and this contributes to the basal electromechanical coupling of cardiomyocytes under nearly physiological Ca2+concentrations. Activation of the receptor with direct agonists is able to further increase contractility. It is also leading to the enhanced relaxation velocity which is explained by PKC activation, PLB phosphorylation and enhanced SERCA activity. To be functionally expressed on cardiomyocytes membrane, CaR require the activity of RAMP1.During the development of pressure induced hypertrophy, ET-1 via ETA or ETB1 receptors is able to upregulate CaR expression. Elevated levels of CaR are supposed to compensate a loss of the heart function during hypertrophy development.
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