Differential impact of mitochondrial positioning on mitochondrial Ca2+ uptake and Ca2+ spark suppression in skeletal muscle

Rossi AE, Boncompagni S, Wei L, Protasi F, Dirksen RT. Differential impact of mitochondrial positioning on mitochondrial Ca2+ uptake and Ca2+ spark suppression in skeletal muscle. Am J Physiol Cell Physiol 301: C1128-C1139, 2011. First published August 17, 2011; doi:10.1152/ajpcell.00194.2011.-Muscle contraction requires ATP and Ca2+ and, thus, is under direct control of mitochondria and the sarcoplasmic reticulum. During postnatal skeletal muscle maturation, the mitochondrial network exhibits a shift from a longitudinal (longitudinal mitochondria) to a mostly transversal orientation as a result of a progressive increase in mitochondrial association with Ca2+ release units (CRUs) or triads (triadic mitochondria). To determine the physiological implications of this shift in mitochondrial disposition, we used confocal microscopy to monitor activity-dependent changes in myoplasmic (fluo 4) and mitochondrial (rhod 2) Ca2+ in single flexor digitorum brevis (FDB) fibers from 1- to 4-mo-old mice. A robust and sustained Ca2+ accumulation in triadic mitochondria was triggered by repetitive tetanic stimulation (500 ms, 100 Hz, every 2.5 s) in FDB fibers from 4-mo-old mice. Specifically, mitochondrial rhod 2 fluorescence increased 272 +/- 39% after a single tetanus and 412 +/- 45% after five tetani and decayed slowly over 10 min following the final tetanus. Similar results were observed in fibers expressing mitochondrial pericam, a mitochondrial-targeted ratiometric Ca2+ indicator. Interestingly, sustained mitochondrial Ca2+ uptake following repetitive tetanic stimulation was similar for triadic and longitudinal mitochondria in FDB fibers from 1-mo-old mice, and both mitochondrial populations were found by electron microscopy to be continuous and structurally tethered to the sarcoplasmic reticulum. Conversely, the frequency of osmotic shock-induced Ca2+ sparks per CRU density decreased threefold (from 3.6 +/- 0.2 to 1.2 +/- 0.1 events.CRU-1.min(-1).100 mu m(-2)) during postnatal development in direct linear correspondence (r(2) = 0.95) to an increase in mitochondrion-CRU pairing. Together, these results indicate that mitochondrion-CRU association promotes Ca2+ spark suppression but does not significantly impact mitochondrial Ca2+ uptake.