Pharmacological Modulation of Mitochondrial Ca2+ Content Regulates Sarcoplasmic Reticulum Ca2+ Release via Oxidation of the Ryanodine Receptor by Mitochondria-Derived Reactive Oxygen Species.

Shanna Hamilton,György Csordás,Bum-Rak Choi,Jin O-Uchi,Radmila Terentyeva,Tae Yun Kim,Peter Bronk,Richard T Clements,Dmitry Terentyev

doi:10.3389/fphys.2018.01831

Abstract

In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca2+ uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca2+ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca2+-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca2+ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca2+ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na+/Ca2+ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca2+ accumulation monitored by mitochondrial-targeted Ca2+ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca2+ imaging showed that accelerated Ca2+ accumulation reduced Ca2+ transient amplitude and promoted generation of spontaneous Ca2+ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca2+ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca2+ imaging demonstrated that enhancement of mitochondrial Ca2+ accumulation further perturbed global Ca2+ handling, increasing the number of cells exhibiting spontaneous Ca2+ waves, shortening RyR refractoriness and decreasing SR Ca2+ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca2+ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca2+ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca2+ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca2+ accumulation can be protective.

Highlights

Sudden cardiac death remains the leading global cause of mortality, and over half of patients with heart failure (HF) die suddenly due to the development of ventricular arrhythmia (Benjamin et al, 2018)
Mitochondria are in close spatial proximity to the sarcoplasmic reticulum (SR) (Dorn and Scorrano, 2010; Eisner et al, 2013; Lu et al, 2013; Seidlmayer et al, 2016; LopezCrisosto et al, 2017; Csordás et al, 2018), and it is well established that during higher workload, there is an elevation of cystolic Ca2+ concentration in ventricular myocytes (VMs) that transpires to a small and slow rise in mitochondrial Ca2+ concentration ([Ca2+]m), leading to enhanced energy production (Brandes and Bers, 1997; Luongo et al, 2015)
We have previously reported that the rat model hypertrophy induced by thoracic aortic banding (TAB) is highly arrhythmogenic, with incidences of non-sustained ventricular tachycardia and fibrillation (VT/VF) occurring in 100% of TAB hearts exposed to 50 nmol/L ISO (Kim et al, 2017)

Summary

Introduction

Sudden cardiac death remains the leading global cause of mortality, and over half of patients with heart failure (HF) die suddenly due to the development of ventricular arrhythmia (Benjamin et al, 2018). Arrhythmogenesis in the failing heart is often linked to enhanced Ca2+-dependent triggered activity, in the form of early and delayed afterdepolarizations (Landstrom et al, 2017). These abnormal electrical activities arise in part as a consequence of untimely and dysregulated Ca2+ release from the sarcoplasmic reticulum (SR), through SR Ca2+ release channel, the ryanodine receptor (RyR). Mitochondrial efflux mainly occurs via the mitochondrial Na/Ca2+/Li+-exchanger (NCLX) (Palty et al, 2010; Boyman et al, 2013; Luongo et al, 2017)

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