Redox modification of ryanodine receptors by mitochondria‐derived reactive oxygen species contributes to aberrant Ca²⁺ handling in ageing rabbit hearts

Abstract

Key points• Ageing is associated with increased risk of sudden cardiac death due to malignant arrhythmias.• Shortened refractoriness of Ca 2+ release due to increased activity of Ca 2+ release channels (RyRs) is recognized as an important contributor to cardiac-triggered arrhythmias. However, molecular mechanisms of RyR dysfunction and its contribution to arrhythmias in ageing remain to be examined.• Using ventricular myocytes isolated from old rabbit hearts we demonstrate that age-associated increase in rate of production of reactive oxygen species (ROS) by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of the channels and thus shortened refractoriness of Ca 2+ release in cardiomyocytes from the ageing heart. Mitochondria-specific scavenging of ROS in old myocytes restored the redox status of RyRs, reducing SR Ca 2+ leak and arrhythmogenic spontaneous Ca 2+ waves.• We conclude that increased ROS production by mitochondria contributes to age-associated increased risk of stress-induced arrhythmia and sudden cardiac death through thiolmodifications of RyRs.Abstract Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca 2+ ) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca 2+ handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5-9 months) and old (4-6 years) rabbit hearts to test the hypothesis that changes in Ca 2+ homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca 2+ handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca 2+ dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca 2+ load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca 2+ leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca 2+ transient amplitude, SR Ca 2+ load, and latency of pro-arrhythmic spontaneous Ca 2+ waves (SCWs) were decreased while RyR-mediated SR Ca 2+ leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, whic...

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