Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Michele Scorzeto,Marta Canato,Feliciano Protasi,Marta Giacomello,Carlo Reggiani,Bert Blaauw,Ger J M Stienen,Cecilia Paolini,Luana Toniolo,Makoto Kanzaki

doi:10.1371/journal.pone.0074919

Michele Scorzeto, Marta Canato + Show 8 more

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https://doi.org/10.1371/journal.pone.0074919

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Abstract

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca2+-binding protein inside SR, calsequestrin. Mitochondrial free Ca2+-concentration ([Ca2+]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca2+ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca2+]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca2+ concentration. The rise in [Ca2+]mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s−1. Accordingly, frequency-dependent increase in [Ca2+]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca2+]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca2+]mito transients were increased by inhibition of mitochondrial Na+/Ca2+ exchanger (mNCX). These results provide direct evidence for fast Ca2+ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca2+-handling and a dependence of mitochondrial Ca2+-handling on intracellular (SR) and external Ca2+ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca2+-leakage.

Highlights

In all excitable and non-excitable cells, mitochondria take up and release Ca2+ in a well regulated manner
These results provide direct evidence for fast Ca2+ accumulation inside the mitochondria, involvement of the mitochondrial Na+/Ca2+ exchanger (mNCX) in mitochondrial Ca2+-handling and a dependence of mitochondrial Ca2+-handling on intracellular (SR) and external Ca2+ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia
Antibody staining for Ryanodine receptor (RyR) and 4mtD3cpv showed a ‘‘sandwich’’ pattern (Fig. 1A and C) as expected on the basis of the position of the Ca2+ release units relative to the mitochondria, resolved by electron microscopy (EM, see Fig. 2)

Summary

Introduction

In all excitable and non-excitable cells, mitochondria take up and release Ca2+ in a well regulated manner This Ca2+ transport is crucial for the regulation of mitochondrial activity [1] and is involved in the cascade of events leading to apoptosis [2,3]. It is well established that the activity of a number of key metabolic enzymes and the F0-F1 ATPase (ATP synthase) itself is controlled by the Ca2+ concentration inside the mitochondrial matrix ([Ca2+]mito) [4,1]. This regulation is of special (patho)physiological interest in muscle tissue, where the high energy cost of contractile activity requires large (even 100 fold) and fast variations of aerobic.

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