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Epac-induced ryanodine receptor type 2 activation inhibits sodium currents in atrial and ventricular murine
Haseeb Valli1, Shiraz Ahmad1, Sujan Sriharan1
1Physiological Laboratory, University of Cambridge, Cambridge, UK.
Abstract:
Acute RyR2 activation by exchange protein directly activated by cAMP (Epac) reversibly perturbs myocyte Ca2+ homeostasis, slows myocardial action potential conduction, and exerts pro-arrhythmic effects. Loose patch-clamp studies, preserving in vivo extracellular and intracellular conditions, investigated Na+ current in intact cardiomyocytes in murine atrial and ventricular preparations following Epac activation. Depolarising steps to varying test voltages activated typical voltage-dependent Na+ currents. Plots of peak current against depolarisation from resting potential gave pretreatment maximum atrial and ventricular currents of -20.23 ± 1.48 (17) and -29.8 ± 2.4 (10) pA/microm2 (mean ± SEM [n]). Challenge by 8-CPT (1 micromol/L) reduced these currents to -11.21 ± 0.91 (12) (P < .004) and -19.3 ± 1.6 (11) pA/microm2 (P < .04) respectively. Currents following further addition of the RyR2 inhibitor dantrolene (10 micromol/L) (-19.91 ± 2.84 (13) and -26.6 ± 1.7 (17)), and dantrolene whether alone (-19.53 ± 1.97 (8) and -27.6 ± 1.9 (14)) or combined with 8-CPT (-19.93 ± 2.59 (12) and -29.9 ± 2.5(11)), were indistinguishable from pretreatment values (all P >> .05). Assessment of the inactivation that followed by applying subsequent steps to a fixed voltage 100 mV positive to resting potential gave concordant results. Half-maximal inactivation voltages and steepness factors, and time constants for Na+ current recovery from inactivation in double-pulse experiments, were similar through all the pharmacological conditions. Intracellular sharp microelectrode membrane potential recordings in intact Langendorff-perfused preparations demonstrated concordant variations in maximum rates of atrial and ventricular action potential upstroke, (dV/dt)max . We thus demonstrate an acute, reversible, Na+ channel inhibition offering a possible mechanism for previously reported pro-arrhythmic slowing of AP propagation following modifications of Ca2+ homeostasis, complementing earlier findings from chronic alterations in Ca2+ homeostasis in genetically-modified RyR2-P2328S hearts.
Insights
Acute activation of RyR2 by Epac reversibly inhibits Na+ channels, slowing cardiac action potential conduction and causing arrhythmias. This study demonstrates a direct link between RyR2 activity, Na+ current, and cardiac electrophysiology.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Cardiac Electrophysiology
Background:
- Acute RyR2 activation by Epac disrupts myocyte Ca2+ homeostasis, leading to slowed cardiac conduction and pro-arrhythmic effects.
- Previous research suggests chronic alterations in Ca2+ homeostasis impact cardiac function, but acute mechanisms remain less understood.
Purpose of the Study:
- To investigate the acute effects of Epac-mediated RyR2 activation on Na+ current in cardiomyocytes.
- To determine if Na+ channel inhibition is a mechanism underlying Epac-induced slowing of action potential propagation.
Main Methods:
- Utilized loose patch-clamp studies on murine atrial and ventricular cardiomyocytes to measure Na+ currents.
- Administered 8-CPT (Epac activator) and dantrolene (RyR2 inhibitor) to assess their effects on Na+ current.
- Performed intracellular sharp microelectrode recordings in Langendorff-perfused hearts to measure action potential upstroke velocity.
Main Results:
- Epac activation with 8-CPT significantly reduced peak Na+ currents in both atrial and ventricular cardiomyocytes.
- The RyR2 inhibitor dantrolene abolished the inhibitory effect of 8-CPT on Na+ currents.
- Pharmacological manipulations of RyR2 and Ca2+ homeostasis did not alter Na+ current inactivation properties or recovery from inactivation.
Conclusions:
- Acute RyR2 activation by Epac leads to reversible Na+ channel inhibition in cardiomyocytes.
- This Na+ channel inhibition provides a potential mechanism for the observed slowing of cardiac action potential propagation and pro-arrhythmic effects.
- Findings complement previous studies on chronic Ca2+ homeostasis alterations in genetically modified hearts.