Abstract 19255: A Role for Mitochondria in Triggered Activity from Cardiomyocytes with Reduced Sarcoplasmic Reticulum Calcium Release

Abstract

Introduction: A prominent feature of systolic heart failure is the reduction of Ca2+ induced Ca2+ release from sacroplasimic reticulum (SR). Moreover, patients with heart failure are at high risk of arrhythmic sudden cardiac death. Hypothesis: The basis for increased arrhythmic risk with reduced SR Ca2+ release was investigated using embryonic stem cell derived ventricular-like cardiomyocytes (CMs) deficient for the expression of the ryanodine receptor (RYR-/-). Methods: Action potentials (APs) and L-type Ca2+ currents were recorded in the perforated current-clamp and whole cell voltage-clamp respectively. Changes in [Ca2+]i were determined by confocal imaging of Fluo-4/AM ESCs. APs were recorded from CMs differentiated for 19 days. Results: Compared with wild type CMs, L-type Ca2+ channels showed less current density and slightly slower activation/inactivation kinetics in RyR-/- CMs. AP and Ca2+ transient frequency were decreased. Ca2+ transient amplitudes were smaller in RyR-/- CMs and had prolonged transient duration at 50% amplitude. Ca2+ transients are comparable to those of WT cells after SR depletion by caffeine, indicating that the SR in RyR-/- CMs did not contribute significantly to Ca2+ release or removal. Despite a shorter AP duration at 90% repolarization (APD90: 187.7 ± 13.8 ms vs. 274.3 ± 36.3 ms), RyR-/- ventricular-like CMs displayed more delayed after-depolarizations (DADs, 75% vs. 0%, p<0.05) than WT CMs. Arrhythmia-inducing Ca2+ leak in RyR-/- CMs can come from IP3R mediated Ca2+ release or from mitochondrial Ca2+. In the presence of the IP3R blocker 2-APB, AP frequency decreased (from 1.3±0.1 to 0.3±0.1 Hz) supporting its role in pacemaker activity, but DADs remained. In the absence of [Ca2+]o, an increase in SR load after a caffeine-induced depletion of the SR indicated Ca2+ transfer from the mitochondria to the SR. Application of the mitochondrial NCX blocker (CGP) suppressed this transfer in WT CMs. Block of the mitochondrial Ca2+ uptake (Ru360) prevented DADs in RyR-/- CMs. Conversely application of PPT, an activator of the mitochondrial Ca2+ uniporter, enhanced DADs in RyR-/- ESCs. Conclusions: DADs may occur in the presence of reduced SR Ca2+ release. DADs in RyR-/- CMs depends on mitochondrial Ca2+ flux.