Depotentiation of intact rat cardiac muscle unmasks an Epac‐dependent increase in myofilament Ca²⁺ sensitivity

Abstract

Recently, a family of guanine nucleotide exchange factors have been identified in many cell types as important effectors of cyclic adenosine 3',5'-monophospahte (cAMP) signalling that is independent of protein kinase A (PKA). In the heart, investigation of exchange protein directly activated by cAMP (Epac) has yielded conflicting results. Since cAMP is an important regulator of cardiac contractility, this study aimed to examine whether Epac activation modulates excitation-contraction coupling in ventricular preparations from rat hearts. The study used 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (cpTOME), an analogue of cAMP that activates Epac, but not PKA. In isolated myocytes, cpTOME increased Ca(2+) spark frequency from about 7 to 32/100 μm(3)/s (n = 10), P = 0.05 with a reduction in the peak amplitude of the sparks. Simultaneous measurements of intracellular Ca(2+) and isometric force in multicellular trabeculae (n = 7, 1.5 mmol/L [Ca(2+)]o) revealed no effect of Epac activation on either the amplitude of Ca(2+) transients (Control 0.7 ± 0.1 vs cpTOME 0.7 ± 0.1; 340/380 fura-2 ratio, P = 0.35) or on peak stress (Control 24 ± 5 mN/mm(2) vs cpTOME 23 ± 5 mN/mm(2), P = 0.20). However, an effect of Epac in trabeculae was unmasked by lowering extracellular [Ca(2+)]o. In these depotentiated trabeculae, activation of the Epac pathway increased myofilament Ca(2+) sensitivity, an effect that was blocked by addition of KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) inhibitor. This study suggests that Epac activation may be a useful therapeutic target to increase the strength of contraction during low inotropic states.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…EPAC activation increased the frequency of these sparks in a PLCdependent manner, but in turn decreased the amplitude which further supports a role of EPAC on RyRs (820,821). Interestingly in a multicellular cardiac preparation, these effects were only observed in the absence of near-physiological extracellular Ca 2ϩ levels, suggesting this pathway might only be fully activated under conditions of stress where the driving force of Ca 2ϩ entry is from intracellular stores (504). Moreover, these changes in intracellular Ca 2ϩ influx are consistently found to be blocked by the CaMKII inhibitor (KN93), and RyR2 Ser2815 phosphorylation was again greater after EPAC activation explaining the increased Ca 2ϩ leak and decreased Ca 2ϩ transient (820).…”

“…Indeed, EPAC activation was also observed to increase phosphorylation of cardiac myosin binding protein-C Ser282 (cMyBP-C) and cardiac troponin I (cTn1) at a PKA-independent site (154). These phosphorylation events were unaffected by inhibition of Rap1, but rather were determined to be sensitive to inhibition of PLC, PKC, and CaMKII (154,504). Thus, during low ionotropic states, increased Ca 2ϩ sensitivity by selective EPAC activation could be potentially useful to increase contraction strength (504).…”

“…These phosphorylation events were unaffected by inhibition of Rap1, but rather were determined to be sensitive to inhibition of PLC, PKC, and CaMKII (154,504). Thus, during low ionotropic states, increased Ca 2ϩ sensitivity by selective EPAC activation could be potentially useful to increase contraction strength (504).…”

β-Adrenergic induced SR Ca 2+ leak is mediated by an Epac-NOS pathway

“…Epac activation has also been reported to increase myofilament Ca 2+ sensitivity via CaMKII-dependent phosphorylation of myofilament proteins, resulting in stronger contraction for a given Ca 2+ transient [18, 44]. To the extent that this Epac myofilament effect is beneficial in HF, it would need assessment when targeting the Epac induced SR Ca 2+ leak therapeutically.…”

Hypertonic saline inhibits airway smooth muscle contraction by inhibiting Ca2+ sensitization

“…Intracellular Ca 2+ was measured using Fura‐2AM as described previously . Briefly, single ASMCs were incubated with 2.5 μmol/L Fura‐2AM for 15 minutes at room temperature in a specified chamber and then superfused with oxygenated PSS solution for 10 minutes.…”