Objective: A number of reports have suggested that the extracellular Calcium-Sensing Receptor (CaR), a pleiotropic GPCR calcium sensor involved in the systemic regulation of calcium absorption/excretion, may play a role in cardiac cell physiology and pathology. Interestingly, diverse signalling pathways have been described to be activated by this receptor in cardiac cells from different experimental models. Notwithstanding the recent abrupt proliferation of literature on the role of CaR in the heart, only scattered data are available regarding the effect of CaR agonists and modulators on second messenger dynamics of spontaneously beating cardiomyocytes. Methods: Here, by using single cell real-time imaging of calcium and cAMP levels we aimed at evaluating the impact of different CaR agonists and modulators on second messenger dynamics both in fibroblasts and spontaneously beating myocytes isolated from neonatal rat ventricles. Real time single cell imaging was performed on Ca2 + fluorophore (fluo4)-loaded or cAMP-probe - (H30) transfected cardiac cells. In parallel, the spontaneous electrical activity of beating cardiomyocytes was evaluated by conventional microelectrodes. Results: Stimulation of fluo-4 loaded cardiac cells with the calcimimetic NPS-R, or CaR agonists such as spermine and neomycin induced clear cytosolic [Ca2 +] peaks in fibroblasts, while reduced the frequency of Ca2 + oscillations in spontaneously-beating cardiomyocytes. Direct cAMP measurements in living cardiac myocytes demonstrated an apparent reduction of cAMP levels upon CaR stimulation with NPS-R and spermine. Accordingly, acute exposure to NPS-R and spermine significantly reduced spontaneous electrical activity frequency of neonatal rat cardiomyocytes in a pertussis-toxin sensitive manner. Conclusions: The data collected demonstrate that different physiological CaR-agonists and modulators activate the PLC/IP3 pathway in cardiac fibroblasts while decrease Ca2 + oscillation- and SEA- frequency in cardiomyocytes, via Gi mediated modulation of cAMP levels. Thanks to a multi technical approach on living cells, here we showed for the first time and in a straightforward manner that CaR-activation exert cell specific intracellular signalling pathways in neonatal cardiac cells. Given the quite wide use of calcimimetic and calcilytics in a number of human diseases associated with CaR abnormalities the implication of such findings spans from the basic molecular cardiology field to the clinics.
Signal transduction mechanisms of the calcium sensing receptor in neonatal rat cardiac fibroblasts and myocytes: A re-evaluation with real-time imaging and electrophysiological approaches
Ranieri, M.;Gerbino, A.;Carmone, C.;Debellis, L.;Caroppo, R.;Guerra, L.;Cotecchia, S.;
2018-01-01
Abstract
Objective: A number of reports have suggested that the extracellular Calcium-Sensing Receptor (CaR), a pleiotropic GPCR calcium sensor involved in the systemic regulation of calcium absorption/excretion, may play a role in cardiac cell physiology and pathology. Interestingly, diverse signalling pathways have been described to be activated by this receptor in cardiac cells from different experimental models. Notwithstanding the recent abrupt proliferation of literature on the role of CaR in the heart, only scattered data are available regarding the effect of CaR agonists and modulators on second messenger dynamics of spontaneously beating cardiomyocytes. Methods: Here, by using single cell real-time imaging of calcium and cAMP levels we aimed at evaluating the impact of different CaR agonists and modulators on second messenger dynamics both in fibroblasts and spontaneously beating myocytes isolated from neonatal rat ventricles. Real time single cell imaging was performed on Ca2 + fluorophore (fluo4)-loaded or cAMP-probe - (H30) transfected cardiac cells. In parallel, the spontaneous electrical activity of beating cardiomyocytes was evaluated by conventional microelectrodes. Results: Stimulation of fluo-4 loaded cardiac cells with the calcimimetic NPS-R, or CaR agonists such as spermine and neomycin induced clear cytosolic [Ca2 +] peaks in fibroblasts, while reduced the frequency of Ca2 + oscillations in spontaneously-beating cardiomyocytes. Direct cAMP measurements in living cardiac myocytes demonstrated an apparent reduction of cAMP levels upon CaR stimulation with NPS-R and spermine. Accordingly, acute exposure to NPS-R and spermine significantly reduced spontaneous electrical activity frequency of neonatal rat cardiomyocytes in a pertussis-toxin sensitive manner. Conclusions: The data collected demonstrate that different physiological CaR-agonists and modulators activate the PLC/IP3 pathway in cardiac fibroblasts while decrease Ca2 + oscillation- and SEA- frequency in cardiomyocytes, via Gi mediated modulation of cAMP levels. Thanks to a multi technical approach on living cells, here we showed for the first time and in a straightforward manner that CaR-activation exert cell specific intracellular signalling pathways in neonatal cardiac cells. Given the quite wide use of calcimimetic and calcilytics in a number of human diseases associated with CaR abnormalities the implication of such findings spans from the basic molecular cardiology field to the clinics.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
