Hydrogen peroxide and sarcoplasmic reticulum dysfunction in rat ventricular myocytes

Greensmith, David; Nirmalan, Mahesh; Eisner, David

Abstract

Reactive oxygen species (ROS) are mediators of myocardial systolic and diastolic dysfunction in disease states such as sepsis. An understanding of ROS induced cellular dysfunction is essential to understand the underlying mechanisms. We have previously presented the effects of the free radical generating system H2O2 on [Ca2+]i handling and contractility in the rat ventricular myocyte (Greensmith et al (2008)). Key findings were significant diastolic and systolic dysfunction, the latter explainable by reduced sarco endoplasmic reticulum calcium ATPase (SERCA) activity and so a reduction in sarcoplasmic reticulum (SR) Ca content. The apparent importance of SR dysfunction led us to perform a more complete investigation into the effects of H2O2 on SERCA and ryanodine receptor (RyR) function using Ca waves and Ca sparks. Male Wistar Rats were killed by cervical dislocation in accordance with the Home Office Animal (Scientific Procedures) Act 1986, and ventricular myocytes isolated by enzymatic digestion. Membrane currents and [Ca2+]i were measured using voltage clamp via perforated patch and Fluo-3 respectively. Ca sparks were observed using confocal microscopy. All experiments were performed at 37 °C. Data shown is mean ± SEM. 500 µM H2O2 caused a progressive decrease in Ca spark frequency and amplitude. By 1 minute, mean Ca spark frequency was reduced by 29 % (Control, 0.089 ± 0.01; H2O2, 0.064 ± 0.01 sparks μm-1s-1, n=7, p <0.005) whilst mean amplitude was reduced by 5.8 % (Control, 1.7 ± 0.02; H2O2, 1.6 ± 0.02 F/F0, n=7, p <0.005). In parallel experiments 500 µM H2O2 reduced SR Ca content by 27 % (Control, 130 ± 18; H2O2, 95 ± 17 μmol / l, n=5, p <0.05). 100 µM H2O2 caused a progressive decrease in Ca wave frequency, with a mean decrease of 59 % (Control, 4.8 ± 0.7; H2O2, 1.9 ± 0.5 waves, n=17, p <0.005). Ca wave duration was increased by 23 % (Control, 1.168 ± 0.047; H2O2, 1.442 ± 0.104 s, n=12, p <0.05), however no change in the degree of sarcolemmal Ca efflux evoked by Ca waves was observed (Control, 17 ± 1.4; H2O2, 17 ± 2.0 μmol / l, n=12, p = 0.6). In these experiments, SR Ca content was reduced by 16 % (Control, 156 ± 6 (n=10); H2O2, 131 ± 10 (n=6) μmol / l, p <0.05). The reduction in Ca spark frequency suggests a decrease in the open probability of the RyR. SR Ca content is decreased in these experiments, explaining the decrease in Ca spark amplitude. The fact that Ca wave magnitude is unaltered suggests the decreased RyR open probability is dependent on luminal Ca rather than a direct oxidative effect on the RyR. The increase in Ca wave duration supports a H2O2 impairment of SERCA activity.

• Presentation Conference Type: Conference Abstract
• Conference Name: Physiology 2010
• Start Date: Jul 5, 2010
• End Date: Jul 8, 2010
• Acceptance Date: Jul 5, 2010
• Online Publication Date: Jul 5, 2010
• Publication Date: Jul 5, 2010
• Deposit Date: Jun 24, 2025
• Peer Reviewed: Peer Reviewed
• Volume: 19
• Article Number: PC206
• Series Number: 19
• Book Title: Proceedings of The Physiological Society
• Publisher URL: https://www.physoc.org/abstracts/hydrogen-peroxide-and-sarcoplasmic-reticulum-dysfunction-in-rat-ventricular-myocytes/