Improved skeletal muscle Ca2+ regulation in vivo following contractions in mice overexpressing PGC-1α.

In skeletal muscle, resting intracellular Ca2+ concentration ([Ca2+]i) homeostasis is exquisitely regulated by Ca2+ transport across the sarcolemmal, mitochondrial, and sarcoplasmic reticulum (SR) membranes. Of these three systems, the relative importance of the mitochondria in [Ca2+]i regulation remains poorly understood in in vivo skeletal muscle. We tested the hypothesis that the capacity for Ca2+ uptake by mitochondria is a primary factor in determining [Ca2+]i regulation in muscle at rest and following contractions. Tibialis anterior muscle of anesthetized peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)-overexpressing (OE, increased mitochondria model) and wild-type (WT) littermate mice was exteriorized in vivo and loaded with the fluorescent probe fura 2-AM, and Rhod 2-AM Ca2+ buffering and mitochondrial [Ca2+] were evaluated at rest and during recovery from fatiguing tetanic contractions induced by electrical stimulation (120 s, 100 Hz). In addition, the effects of pharmacological inhibition of SR (thapsigargin) and mitochondrial [carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)] function were examined at rest. [Ca2+]i in WT remained elevated for the entire postcontraction recovery period (+6 ± 1% at 450 s), but in PGC-1α OE [Ca2+]i returned to resting baseline within 150 s. Thapsigargin immediately and substantially increased resting [Ca2+]i in WT, whereas in PGC-1α OE this effect was delayed and markedly diminished (WT, +12 ± 3; PGC-1α OE, +1 ± 2% at 600 s after thapsigargin treatment, P < 0.05). FCCP abolished this improvement of [Ca2+]i regulation in PGC-1α OE. Mitochondrial [Ca2+] accumulation was observed in PGC-1α OE following contractions and thapsigargin treatment. In the SR, PGC-1α OE downregulated SR Ca2+-ATPase 1 (Ca2+ uptake) and parvalbumin (Ca2+ buffering) protein levels, whereas mitochondrial Ca2+ uptake-related proteins (Mfn1, Mfn2, and mitochondrial Ca2+ uniporter) were upregulated. These data demonstrate a heretofore unappreciated role for skeletal muscle mitochondria in [Ca2+]i regulation in vivo following fatiguing tetanic contractions and at rest.

Sex differences in mitochondrial Ca2+ handling in mouse fast-twitch skeletal muscle in vivo.

We investigated sex differences in mitochondrial Ca2+ handling properties in mouse fast-twitch skeletal muscle. Changes in cytoplasmic Ca2+ concentration ([Ca2+]cyto) were measured in vivo using tibialis anterior muscles from male and female mice. The muscles were exposed to increasing concentrations of cyclopiazonic acid [CPA; sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor] (from 10 to 30 to 50 µM at 10 min intervals). Thirty minutes after treatment, [Ca2+]cyto was increased by 31.6 ± 2.0% and 13.5 ± 4.5% of initial [Ca2+]cyto in male and female muscles, respectively, and there was a significant difference between sexes. However, muscle preincubation for 5 min with 10 µM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (an inhibitor of mitochondria Ca2+ uptake) eradicated this difference between sexes with respect to the CPA-induced [Ca2+]cyto increase. Both intermyofibrillar mitochondrial number and volume, assessed in longitudinal fiber sections, were higher in females compared with males (mitochondria number: 13.1 ± 1.0 in males vs. 19.9 ± 2.3 in females; mitochondrial volume: 0.034 ± 0.004 µm3/µm3 fiber volume in males vs. 0.066 ± 0.008 µm3/µm3 fiber volume in females, both P < 0.05). There were no sex differences in the content of SR Ca2+-ATPase, mitochondrial Ca2+ uniporter, mitofusin (Mfn) 1, or Mfn2. These results suggest that 1) mitochondrial Ca2+ uptake ability is greater in female than male myocytes, and 2) this superior Ca2+ uptake ability of female myocytes is due, partly, to the higher intermyofibrillar mitochondrial content but not to the expression of mitochondrial proteins related to mitochondrial Ca2+ uptake.NEW & NOTEWORTHY This investigation presents evidence that female versus male fast-twitch muscle exhibits a greater mitochondrial calcium ion uptake capability that is partly conferred by the higher intermyofibrillar mitochondrial volume density.