The role of Store-Operated Cyclic AMP Signalling (SOcAMPS) in cardiac physiology and pathology: an in vitro study on neonatal rat cardiomyocytes.

Background and Aims: Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a recently identified mechanism of cross-talk between Ca2+ and cAMP signals. In this process, depletion of Ca2+ in the endoplasmic reticulum (ER) leads to increases in cAMP levels, independently of cytosolic Ca2+ changes. Expression and functionality of STIM1 (Stromal Interaction Molecule 1), a transmembrane ER Ca2+ sensor protein, is necessary for SOcAMPS to occur. Interestingly, recent reports have demonstrated a critical role for STIM1 in the development of cardiac hypertrophy, a process notoriously controlled both by Ca2+ and cAMP signaling. Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat cardiomyocytes and its potential role in cardiac cell hypertrophy. Methods: To monitor changes in cAMP levels, real time imaging experiments were performed on neonatal rat cardiomyocytes transiently transfected with an EPAC-based fluorescent probe for [cAMP], EPAC H30. Fura-2 and Fluo-4 were used to monitor cytosolic Ca2+ levels and an ER/SR targeted probe, D1ERcameleon, was used to measure ER [Ca2+]. Long term incubation (48h) of cardiomyocytes with angiotensin II (1 μM) and aldosterone (1 μM) was used to induce "in vitro" cell hypertrophy. Increases in cell size and/or sarcomere alignment were monitored microscopically after labeling with phalloidin-TRITC. Results: To verify the existence of SOcAMPS in neonatal rat cardiomyocytes, cells were stimulated in Ca2+-free Ringer's solutions with the low affinity membrane permeant Ca2+ chelator TPEN (1mM), able to induce a reduction of SR Ca2+ levels ([Ca2+]SR) without affecting cytosolic [Ca2+]. SR Ca2+ measurements demonstrated that under these experimental conditions, 1 mM TPEN led to a reduction in intraluminal [Ca2+] that was 50,5±2,4% (8 exp, 11 cells, p<0.001) of the maximal store depletion. Parallel experiments performed with the EPAC H30 cAMP sensor showed increases in [cAMP] that were 26,5±3% (13 exp, 13 cells, p<0.001) of the maximum delta ratio. In the presence of 5 μM Forskolin (FRSK) the TPEN-induced cAMP augmentation resulted 63,7±3,9% of the maximal response (16 exp, 19 cells, p<0.001). Also depletion of SR by the Ca2+ ionophore ionomycin (10 μM) was found to induce significant cAMP increases both in the absence and presence of FRSK. The participation of STIM1 in the observed phenomenon was proven by the 47 % reduction of the TPEN+FRSK induced [cAMP] signal after transfection of cells with a shRNA against STIM1 (6 exp, p<0,01). To evaluate the putative role of SOcAMPS in cardiac hypertrophy, cAMP measurements were performed on angio+aldo treated cells and compared to control cardiomyocytes. Under these experimental conditions a 20% increase of the TPEN+FRSK induced response was observed in hypertrophic myocytes (16 exp, p<0,01). Conclusions: These data straightforwardly establish, for the first time, the existence of SOcAMPS in the neonatal cardiomyocyte cell model. Also, a significantly increased SOcAMP signalling was shown to exist in hypertrophic cardiomyocytes. Further experiments to ascertain whether a causeand- effect relationship exists between SOcAMPS and cardiac cell hypertrophy are in progress.