Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB026

Poster Communications

Effects of ESI-05 induced Epac inhibition on action potential duration are modified by intracellular Ca2+ buffering and abolished by pyruvate in isolated rat ventricular myocytes

H. M. Kirton1, C. Peers1, D. S. Steele1

1. Biomedical Sciences, University of Leeds, Leeds, United Kingdom.

We have shown previously that the Epac2 inhibitor ESI-05 can induce arrhythmias in vivo and prolong the action potential (AP) in ventricular myocytes via a mechanism that involves increased mitochondrial reactive oxygen species (ROS) production and ranolazine sensitive activation of the late sodium current (INalate) (1). Here we describe further work showing that aspects of the experimental conditions can markedly influence the effects of ESI-05 in rat ventricular myocytes. In current clamp mode, AP's were elicited by 10 ms depolarizing pulses every 10 s. Patch electrodes contained (mM) KCl 130, NaCl 10, EGTA 5 or 0.1, HEPES 5, MgATP 5, CaCl2 1, and MgCl2 1; pH 7.2. The perfusate contained (mM) NaCl 120, KCl 5, CaCl2 1, MgCl2 1, HEPES 10, glucose 10 and pyruvate 0 or 5; pH 7.4. Results are presented as mean ± S.E.M. and statistical analysis was performed using Student's t-tests where p < 0.05 was considered statistically significant. One aspect that commonly varies among studies is the level of EGTA within the patch pipette. While milimolar EGTA will abolish Ca2+ cycling, effects that persist under these conditions can be assumed to be Ca2+ independent, providing useful mechanistic information. In the present study, the APD90 was more than doubled (to 221% ± 21, n=10, p<0.001) by ESI-05 with 5 mM EGTA in the pipette. This effect was blocked entirely when 5 mM pyruvate was present in the perfusate (n=13, p>0.05), which is sometimes included due to its beneficial effect on cell viability. Pyruvate has antioxidant properties and its ability to block the effects of ESI-05 is consistent with our previous finding that antioxidants prevent ESI-05 induced activation of INalate and AP prolongation (1). These experiments were repeated after reducing the level of EGTA to 0.1 mM, which is sufficient to allow Ca2+ cycling and contraction. Under these conditions, the mean increase in APD90 induced by ESI-05 was similar (261% ± 83, n=6). However, the response was more variable and some cells exhibited a much greater prolongation, leading to early afterdepolarisations (EADs). As with 5 mM EGTA, this effect was inhibited by pyruvate, suggesting a ROS dependent mechanism. However, ranolazine failed to prevent AP prolongation (n=8) or EADs, suggesting that effects on INalate may not dominate when Ca2+ buffering more closely approximates to the physiological state.

Where applicable, experiments conform with Society ethical requirements