Effects of cytosolic NADH/NAD+ levels on sarcoplasmic reticulum Ca2+ release in permeabilized rat ventricular myocytes

Abstract

In the heart ischaemic conditions induce metabolic changes known to have profound effects on Ca2+ signalling during excitation-contraction coupling. Ischaemia also affects the redox state of the cell. However, the role of cytosolic redox couples, such as the NADH/NAD+ redox system, for the regulation of Ca2+ homeostasis has remained elusive. We studied the effects of NADH and NAD+ on sarcoplasmic reticulum (SR) Ca2+ release in permeabilized rat ventricular myocytes as well as on Ca2+ uptake by SR microsomes and ryanodine receptor (RyR) single channel activity. Exposure of permeabilized myocytes to NADH (2 mM; [Ca 2+ ]cyt =100 nM) decreased the frequency and the amplitude of spontaneous Ca2+ sparks by 62% and 24%, respectively. This inhibitory effect was reversed by NAD+ (2 mM) and did not depend on mitochondrial function. The inhibition of Ca 2+ sparks by NADH was associated with a 52% decrease in SR Ca2+ load. Some of the effects observed with NADH may involve the generation of superoxide anion (O2-·) as they were attenuated to just a transient decrease of Ca2+ spark frequency by superoxide dismutase (SOD). O2-· generated in situ from the xanthine/xanthine oxidase reaction caused a slowly developing decrease of Ca2+ spark frequency and SR Ca2+ load by 44% and 32%, respectively. Furthermore, in studies with cardiac SR microsomes NADH slowed the rate of ATP-dependent Ca2+ uptake by 39%. This effect also appeared to depend on O2-· formation. Single channel recordings from RyRs incorporated into lipid bilayers revealed that NADH (2 mM) inhibited the activity of RyR channels by 84%. However, NADH inhibition of RyR activitywas O2-· -independent. In summary, an increase of the cytoplasmic NADH/NAD+ ratio depresses SR Ca2+ release in ventricular cardiomyocytes. The effect appears to be mediated by direct NADH inhibition of RyR channel activity and by indirect NADH inhibition (O2-· mediated) of SR Ca2+ -ATPase activity with a subsequent decrease in SR Ca2+ content. © The Physiological Society 2004.