Mitochondrial redox state and Ca²⁺ sparks in permeabilized mammalian skeletal muscle

Abstract

Intact skeletal muscle fibres from adult mammals exhibit neither spontaneous nor stimulated Ca 2+ sparks. Mechanical or chemical skinning procedures have been reported to unmask sparks. The present study investigates the mechanisms that determine the development of Ca 2+ spark activity in permeabilized fibres dissected from muscles with different metabolic capacity. Spontaneous Ca 2+ sparks were detected with fluo-3 and single photon confocal microscopy; mitochondrial redox potential was evaluated from mitochondrial NADH signals recorded with two-photon confocal microscopy, and Ca 2+ load of the sarcoplasmic reticulum (SR) was estimated from the amplitude of caffeine-induced Ca 2+ transients recorded with fura-2 and digital photometry. In three fibre types studied, there was a time lag between permeabilization and spark development. Under all experimental conditions, the delay was the longest in slow-twitch oxidative fibres, intermediate in fast-twitch glycolytic-oxidative fibres, and the shortest in fast-twitch glycolytic cells. The temporal evolution of Ca 2+ spark frequencies was bell-shaped, and the maximal spark frequency was reached slowly in mitochondria-rich oxidative cells but quickly in mitochondria-poor glycolytic fibres. The development of spontaneous Ca 2+ sparks did not correlate with the SR Ca 2+ content of the fibre, but did correlate with the redox potential of their mitochondria. Treatment of fibres with scavengers of reactive oxygen species (ROS), such as superoxide dismutase (SOD) and catalase, dramatically and reversibly reduced the spark frequency and also delayed their appearance. In contrast, incubation of fibres with 50 µM H 2 O 2 sped up the development of Ca 2+ sparks and increased their frequency. These results indicate that the appearance of Ca 2+ sparks in permeabilized skeletal muscle cells depends on the fibre's oxidative strength and that misbalance between mitochondrial ROS production and the fibre's ability to fight oxidative stress is likely to be responsible for unmasking Ca 2+ sparks in skinned preparations. They also suggest that under physiological and pathophysiological conditions the appearance of Ca 2+ sparks may be, at least in part, limited by the fine-tuned equilibrium between mitochondrial ROS production and cellular ROS scavenging mechanisms. Skeletal muscle depends on ATP supply to meet its energy demands. There are three major sources of ATP in muscle: creatine phosphate, anaerobic glycolysis and oxidative phosphorylation. The relative contribution of each ATP source varies among muscle fibre types. Type I (slow-twitch, oxidative) and type IIa (fast-twitch, glycolytic-oxidative) fibres are rich in mitochondria. They rely for their ATP production on oxidative phosphorylation. In contrast, type IIb (fast-twitch, glycolytic) fibres, are mitochondria-poor and have a E. V. Isaeva and V. M. Shkryl contributed equally to this work. very effective glycolytic ATP synthesis. Thus, muscle mitochondrial content is a reflection of the relative importance of mitochondria to th...

Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle – pivotal roles in Ca2+ and reactive oxygen species signaling

“…Another elegant study identified a subset of mitochondria that displayed a rise in [Ca 2+ ] m upon caffeine stimulation even when global [Ca 2+ ] c transients were buffered by BAPTA, further supporting a local SR-mitochondrial Ca 2+ transfer (Shkryl and Shirokova, 2006). Furthermore, mitochondria were found to attenuate spontaneous [Ca 2+ ] c sparks and transients in proportion to their abundance in fibers (Isaeva et al, 2005).…”

“…Mitochondria occupy ,40% of the cell volume in cardiomyocytes and ,20% in skeletal muscle fibers (Isaeva et al, 2005) and are mostly located among the myofibrils. In ventricular cardiomyocytes, intermyofibrillar mitochondria fill the space parallel with the myofibrils, whereas in skeletal muscle fibers, mitochondria are localized close to the CRUs (see Fig.…”

Pretreatment with insulin-like growth factor I protects skeletal muscle cells against oxidative damage via PI3K/Akt and ERK1/2 MAPK pathways

“…23 It was found that prolonged IGF-I treatment in mdx mice can induce muscle fibers from a fast-twitch phenotype toward the slow-twitch phenotype without a change in fiber cross areas. 22 Compared with fasttwitch muscle, slow-twitch muscle fibers are rich in mitochondria, 24 have higher oxidative capacities, 25 and therefore use ATP in a more efficient way and produce less ROS, which would cause less or no damage to muscle cells. Our finding that IGF-I is able to prevent muscle cell from H 2 O 2 -induced cell death and recover cell viability may offer a rational explanation for IGF-I conferring protection on dystrophic muscle from contraction-induced damage.…”

Cross-talk between calcium and reactive oxygen species signaling

“…In contrast, mitochondria-derived ROS, generated from diazoxide (an ATP-sensitive K + channel opener)-induced mitochondrial depolarization, elevates Ca 2+ spark frequency and enhances the coupling of sparks to Ca 2+ -sensitve K + channels in smooth muscle cells [63] . In permeabilized rat skeletal muscle fibers, 50 µmol/L H 2 O 2 was also found to increase Ca 2+ spark frequency [64] . More direct evidence is needed to confirm the regulation of local and global Ca 2+ signals by mitochondria-derived ROS in physiological and pathological conditions.…”