Daisuke HOSHINO (星野 太佑)

This study aimed to characterize the spatial distribution and ultrastructural changes of mitochondria in regenerating muscle following eccentric contractions (ECC)-induced injury, utilizing photothermal microscopy (PTM) and transmission electron microscopy (TEM). ECC was applied to the gastrocnemius muscles of male rats (13 wk old, 284.8 ± 8.9 g), and regenerating muscles were harvested 7 days post injury. PTM, featuring a high-sensitivity optical system, was used to visualize the wide-range and three-dimensional distribution of mitochondria within the white gastrocnemius muscle region. Concurrently, TEM was used for quantitative analysis of mitochondrial ultrastructural morphology, including cristae density. In regenerating muscle, the regular lattice-like network observed in normal tissue was disrupted and replaced by fragmented, randomly distributed mitochondria. Notably, both PTM and TEM analyses revealed a high concentration of mitochondria specifically around "central nuclei," a hallmark of regenerating muscle (i.e., within 0.1-1.0 µm: normal 1.8 ± 2.0% vs. regeneration 5.5 ± 3.6%, P < 0.0001, by TEM data). Detailed morphological analysis further demonstrated that mitochondria in the immediate vicinity of the central nucleus (<0.1 µm) had significantly lower cristae density (inner and outer membranes ratio, 1.10 ± 0.43) compared with those in distal regions (>2.0 µm) (1.80 ± 0.65, P < 0.0001), indicating that they may be structurally immature. In conclusion, during the muscle regeneration process, mitochondria specifically localize around the central nucleus. Given their low cristae density, these potentially represent newly synthesized (biogenesis-derived) mitochondria. This perinuclear accumulation is thought to function as a critical energy source for the nuclear transcriptional and translational activities required for muscle differentiation while also serving as a hub for organelle coordination during the regeneration process.NEW & NOTEWORTHY Using PTM and TEM, this study reveals that mitochondrial networks fragment and cluster around central nuclei during muscle regeneration. These perinuclear mitochondria are structurally immature, exhibiting low cristae density. This localization suggests a potential bioenergetic hub that may support the transcriptional and translational demands of muscle differentiation. Thus, this accumulation likely plays a key role in metabolic and organelle coordination during functional muscle repair.

Intracellular calcium ion concentration ([Ca2+]i) regulation in skeletal muscle may vary with sex and muscle fiber type, but the precise nature of its response to temperature changes and pharmacological caffeine stimulation is not fully understood. This study aimed to elucidate sex-dependent and muscle fiber type-specific characteristics of muscle cooling, caffeine stimulation, and their combined effects. We investigated the effects of cooling (30 °C to 0 °C) and caffeine stimulation (1.25-80 mM) separately and in combination (cooling + 1.25 mM caffeine) in fast-twitch (plantaris, PLA) and slow-twitch (soleus, SOL) muscles of male and female C57BL/6J mice. [Ca2+]i dynamics were analyzed using in vivo Fura-2 bioimaging under isoflurane anesthesia. The temperature threshold for the onset of [Ca2+]i accumulation was significantly higher in SOL than in PLA, with no significant difference between sexes (males: PLA 2.3 ± 0.9 °C, SOL 4.5 ± 2.2 °C; females: PLA 2.3 ± 0.8 °C, SOL 4.3 ± 1.3 °C). Conversely, the [Ca2+]i response to caffeine was significantly higher in females than in males at high concentrations (80 mM). Furthermore, the combined stimulation of cooling and caffeine had a greater effect on females than on males. Our findings also indicate that the phosphorylation response of ryanodine receptors to caffeine was significantly higher in females than in males. In conclusion, while no sex differences were observed in the [Ca2+]i response to cooling, clear sex-dependent differences (males < females) were observed in the response to caffeine.

While post-exercise cooling is known to enhance skeletal muscle adaptations, the effects of pre-exercise cooling remain unclear. This study investigated whether local muscle cooling immediately before exercise augments training-induced mitochondrial biogenesis in male C57BL/6 mice. Pre-exercise cooling involved 6 cycles of 6 min at 13°C followed by 4 min at 35°C on one hindlimb for 60 min before treadmill running. Acutely, this pre-cooling protocol increased exercise-induced PGC-1α mRNA expression by ∼13 % (p < 0.05) compared to exercise alone; this effect was abolished when cooling and exercise were separated by 3 h. Chronically, over 4 weeks, combined pre-cooling and exercise training significantly enhanced mitochondrial enzyme activities: citrate synthase (both main effects) and β-hydroxyacyl-CoA dehydrogenase (+23.9 % compared with exercise alone). However, mitochondrial structural protein levels remained unchanged. These results indicate that pre-exercise cooling promotes mitochondrial enzymatic adaptations in skeletal muscle, potentially offering a practical strategy to optimize the outcomes of endurance training.

Pyruvate administration leads to the accumulation of intracellular lactate in adipocytes and affects inflammatory cytokine production and whole-body glucose metabolism. Therefore, the purpose of this study was to determine whether pyruvate administration improves the dysfunction of glucose metabolism induced by high-fat diet (HFD) intake. In an acute experiment, intraperitoneal injection of male mice with sodium pyruvate (1 g/kg body weight) increased pyruvate and lactate concentrations in blood and epididymal white adipose tissue (eWAT). In a chronic experiment, male mice were divided into three groups: normal diet, HFD with saline administration (HFD + SAL), and HFD with sodium pyruvate administration (HFD + PYR); the HFD + PYR group was injected with pyruvate five times a week for 8 weeks. Insulin concentrations in the basal state and during an oral glucose tolerance test were significantly lower in the HFD + PYR group than in the HFD + SAL group. The mRNA expression of inflammatory cytokines (tumor necrosis factor-alpha and interleukin 6) and a M2 macrophage polarity marker in eWAT was significantly higher in the HFD + PYR group than in the other groups. These results suggest that chronic pyruvate administration partially improves whole-body glucose metabolic dysfunction in HFD-fed mice, accompanied by increased mRNA expression of inflammatory cytokines and a M2 macrophage marker in the eWAT.

Changes in intracellular hydrogen peroxide concentration ([H2O2]) constitute an important signal controlling cellular adaptations. In response to cooling, decreases in [H2O2] and changes in antioxidant-related gene expression have been observed in skeletal muscle. However, the specific temperature dependence of cooling-induced [H2O2] changes and their quantitative relationship to induced gene expression are unknown. This investigation tested the hypothesis that differences in muscle cytosolic and mitochondrial [H2O2] changes during cooling/rewarming determine the pattern of H2O2-related gene expression. H2O2-sensitive cytosolic (HyPer7) and mitochondrial (MLS-HyPer7) fluorescent proteins were expressed into tibialis anterior (TA) muscle of male C57BL/6J mice. The temperature dependence of [H2O2] was determined via in vivo imaging during 3-minute cooling protocol from 35°C to 0°C. Two cooling patterns (6 bouts of intermittent cooling, I-Cool vs. sustained cooling, S-Cool; both to 13°C) were applied over 60 min. Three hours after cooling, the muscles were removed and gene expression was evaluated using real-time PCR. The decrease in [H2O2] was observed in both cytosolic and mitochondrial compartments from 35°C to 13°C, but was of greater magnitude in the cytosol; in contrast, further cooling from 12°C to 0°C induced a rebound increase especially in cytosolic [H2O2]. I-Cool increased the mRNA level of Nrf2 (+15%, p < 0.001). S-Cool decreased the mRNA levels of Sod2, Cat, and Ucp3 (i.e., -20%, -23%, and -30%, respectively, p<0.05). In conclusion, the greatest decrease in temperature-dependent [H2O2] occurred at 13°C in the cytosolic and mitochondrial compartments of muscle fibers and I-Cool increased Nrf2 mRNA expression, while S-Cool decreased several antioxidant-related genes.

Eccentric contractions (ECC) are accompanied by accumulation of intracellular calcium ions ([Ca2+]i) and induce skeletal muscle damage. Suppressed muscle damage in repeated bouts of ECC is well characterized, however, whether it is mediated by altered Ca2+ profiles remains unknown. PURPOSE: We tested the hypothesis that repeated ECC suppresses Ca2+ accumulation via adaptions in Ca2+ regulation. METHODS: Male Wistar rats were divided into two groups: ECC single bout (ECC-SB) and repeated bout (ECC-RB). Tibialis anterior (TA) muscles were subjected to ECC (40 times, 5 sets) once (ECC-SB), or twice 14 days apart (ECC-RB). Under anesthesia, the TA muscle was loaded with Ca2+ indicator Fura-2 AM and the 340/380 nm ratio was evaluated as [Ca2+]i. Ca2+ handling proteins were measured by western blots. RESULTS: ECC induced [Ca2+]i increase in both groups, but ECC-RB evinced a markedly suppressed [Ca2+]i (Time: P < 0.01, Group: P = 0.0357). 5 hours post-ECC, in contrast to the localized [Ca2+]i accumulation in ECC-SB, ECC-RB exhibited lower and more uniform [Ca2+]i (P < 0.01). In ECC-RB mitochondria Ca2+ uniporter complex components, MCU and MICU2, were significantly increased pre-second ECC bout (P < 0.01) and both SERCA1 and MICU1 were better preserved after contractions (P < 0.01). CONCLUSION: 14 days after novel ECC skeletal muscle mitochondrial Ca2+ regulating proteins were elevated. Following subsequent ECC [Ca2+]i accumulation and muscle damage were suppressed and SERCA1 and MICU1 preserved. These findings suggest that tolerance to a subsequent ECC bout is driven, at least in part, by enhanced mitochondrial and SR Ca2+ regulation.