Abstract
The role of mitochondrial Ca2+ transport in regulating intracellular Ca2+ signaling and mitochondrial enzymes involved in energy metabolism is widely recognized in many tissues. However, the ability of skeletal muscle mitochondria to sequester Ca2+ released from the sarcoplasmic reticulum (SR) during the muscle contraction-relaxation cycle is still disputed. To assess the functional cross-talk of Ca2+ between SR and mitochondria, we examined the mutual relationship connecting cytosolic and mitochondrial Ca2+ dynamics in permeabilized skeletal muscle fibers. Cytosolic and mitochondrial Ca2+ transients were recorded with digital photometry and confocal microscopy using fura-2 and mag-rhod-2, respectively. In the presence of 0.5 mM slow Ca2+ buffer (EGTA (ethylene glycolbis(2-aminoethylether)-N,N,N',N'-tetraacetic acid)), application of caffeine induced a synchronized increase in both cytosolic and mitochondrial [Ca2+]. 5 mM fast Ca2+ buffer (BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)) nearly eliminated caffeine-induced increases in [Ca2+]c but only partially decreased the amplitude of mitochondrial Ca2+ transients. Confocal imaging revealed that in EGTA, almost all mitochondria picked up Ca2+ released from the SR by caffeine, whereas only about 70% of mitochondria did so in BAPTA. Taken together, these results indicated that a subpopulation of mitochondria is in close functional and presumably structural proximity to the SR, giving rise to subcellular microdomains in which Ca2+ has preferential access to the juxtaposed organelles.
Highlights
Available morphological data, revealing a close proximity of mitochondria to the sarcoplasmic reticulum (SR) in skeletal muscle (e.g. Ref. 12), place the organelles on the side of the SR/t-tubule junctions opposite to that where SR Ca2ϩ release takes place
We evaluated the functional proximity of the SR Ca2ϩ release sites and mitochondrial Ca2ϩ uptake sites in fast- and slow-twitch skeletal muscles of rat by comparing caffeine-induced cytosolic and mitochondrial Ca2ϩ transients in the presence of slow (EGTA) and fast (BAPTA) Ca2ϩ buffers
Cytosolic and Mitochondrial [Ca2ϩ] Transients in 0.5 mM EGTA— Fig. 1A show cytosolic and mitochondrial Ca2ϩ transients recorded at the same time from extensor digitorum longus (EDL) muscle fiber at 1 Hz
Summary
Preparation of Skeletal Muscle Fibers and Solutions—Rats (SpragueDawley, 175–200 g) were killed by cervical dislocation under deep anesthesia induced by intraperitoneal injection of sodium pentobarbital (100 –200 mg/kg of body weight). Segments were first loaded with 5 M mag-rhod-2 AM for 20 min at room temperature and washed, permeabilized with saponin (as in Ref. 4), and immersed into one of the “internal solutions.”. Both solutions contained a low concentration of the fluorescent Ca2ϩ indicator fura-2 (2 M; potassium salt) and 20 M N-benzyl-p-toluene sulfonamide to minimize the contraction of fast-twitch EDL fibers. Throughout the experimental protocol, the permeabilized fiber preloaded with mag-rhod-2 was continuously perfused with internal solution containing fura-2 at a rate of ϳ0.5 ml/min. Imaging Local Changes in [Ca2ϩ] within Individual Mitochondria— Mitochondria labeled with mag-rhod-2 (as described above) were imaged with a confocal laser scanning microscope (Radiance 2000; BioRad) mounted on a Zeiss Axiovert 100 inverted microscope equipped with a ϫ63, 1.2 NA, water immersion lens (Zeiss Inc., Oberkochen, Germany). Student’s t test was used for comparing paired observations. p Ͻ 0.05 was considered significant
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