Isoprotenerol is a well-established agonist of the beta-adrenergic receptor. It is known to act on cardiomyocytes in an inotropic manner, increasing the contractility of cells via the production and downstream signalling of cyclic AMP (cAMP). Recent developments in this field have led to the discovery of new regulators of excitation-contraction coupling in cardiomyocytes that involve both cAMP/protein kinase A (PKA) and cAMP/guanine nucleotide exchange factor directly activated by cAMP (Epac) signalling pathways. Availability of cAMP- analogues (including selective activators of PKA and Epac) will help to improve our understanding of the contribution of these signalling pathways in health and in disease. The aim of this work was to compare the effect of different cAMP-analogues with Isoprotenerol on calcium transients in isolated perfused cardiomyocytes. Adult male Wistar rat hearts were extracted, perfused in Langendorff apparatus and cardiac myocytes were isolated using enzymatic digestion. The cardiomyocytes were perfused and field stimulated at 0.5Hz at 30°C. In this study we examined the effects of Isoprotenerol at different concentrations on calcium transients and compared the changes with those of three cAMP analogues; an activator of both PKA and Epac (8-Br-cAMP-AM, 8-Br), an activator of PKA (6-Bnz-cAMP-AM, 6-Bnz) and an activator of Epac (8-CPT-2′-O-Me-cAMP-AM, CPT). Data are shown as mean±SE. Exposure to Isoprotenerol at 10, 100 and 200nM concentrations produced an increase in contractility and a dose dependent increase in the amplitude of the calcium transients. Isoprotenerol at 100 and 200nM but not at 10nM caused arrhythmias. At 10nM, Isoprotenerol perfused for 3 mins produced a 2.02±0.31 (n=9) fold increase in the transient amplitude. The three cAMP analogues 8-Br (5μM, n=7), 6-Bnz (10μM, n=6) and CPT (10μM, n=7), each perfused for 5 minutes, induced an increase in transient amplitude that was similar to that of 10nM Isoprotenerol. However the time taken to reach peak increase in transients was different for each analogue and was significantly less (p<0.05, Kruskal-Wallis one-way ANOVA statistical test) than for Isoprotenerol (188±18msec vs. 383±31msec, 435±65msec and 379±82msec for 8-Br, 6-Bnz and CPT respectively). This work has identified selected concentrations of cAMP analogues that target PKA, Epac or both which can produce similar increase in calcium transients to isoproterenol. Furthermore, the effects of cAMP-analogues take longer to establish as they need to partition across the membrane and then diffuse to reach their respective targets. This is in contrast to the relatively fast beta-adrenergic stimulation where cAMP levels are elevated to cope with the increased demand of the fight or flight response.
|Journal||Proceedings of The Physiological Society|
|Publication status||Published - 2015|