Abstract
Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under β-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.
Highlights
Contraction of the heart is a high-energy demanding process
Our results suggest that increased RyR2 activity disturbs mito-Ca2+ homeostasis and elevates mito-reactive oxygen species (ROS) emission, in a vicious feedback cycle exacerbates diastolic sarcoplasmic reticulum (SR) Ca2+ leak by increasing RyR2 oxidation, driving spontaneous Ca2+ release that is detrimental in cardiac disease
We first sought to demonstrate that low-dose caffeine (200 μmol/L) could reproduce the leaky SR phenotype observed in cardiac disease, using cultured ventricular myocytes (VMs) isolated from healthy rat hearts
Summary
Contraction of the heart is a high-energy demanding process. Most of the energy required for this process in the form of ATP is generated by mitochondria, which occupy ~ 35% of myocyte volume [56]. In ventricular myocytes (VMs), release of C a2+ from the sarcoplasmic reticulum (SR) by ryanodine receptors (RyR2s) is critical in initiating muscle contraction and a major determinant of its strength [24]. During SR C a2+ release, local transfer of C a2+ to the mitochondrial matrix activates enzymes in the tricarboxylic acid cycle and drives the electron transport chain (ETC) to accelerate ATP production [52], thereby providing a link between Ca2+-dependent contraction and mitochondria metabolic output. A mito-ROS-mediated increase in activity of RyR2 has been linked to the increased propensity for aberrant Ca2+ leak from the sarcoplasmic reticulum (SR), leading to diminished systolic C a2+ transients and increased incidence of pro-arrhythmic diastolic Ca2+ waves [18]. The role of increased RyR2-mediated Ca2+ release on SR-mitochondria Ca2+ transfer and mito-ROS emission remains incompletely understood
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