Background: Short-term fasting, often practiced in weight-category sports, alters whole-body metabolism. However, its impact on skeletal muscle calcium ion (Ca 2+ ) handling is not well understood. Furthermore, the effects of a fasted state on the muscle damage-recovery response following eccentric contractions (ECC) remain to be elucidated. We tested the hypothesis that a 48-hour fast would: 1) lower resting intracellular Ca 2+ concentration ([Ca 2+ ]i) in skeletal muscle, and 2) suppress the progression of inflammatory cell infiltration, an initial response to muscle damage, following ECC. Methods: Adult male Wistar rats were assigned to a control group (CON) or a 48-hour fasting group (48F). Resting [Ca 2+ ]i in the predominantly fast-twitch tibialis anterior (TA) muscle was evaluated via in vivo imaging using the Ca 2+ indicator Fura-2 AM, with the 340/380 nm fluorescence ratio serving as the index of [Ca 2+ ]i. ECC was induced by forced ankle plantar flexion synchronized with electrical stimulation (100 Hz, 0.7 s duration, every 3 s, 40 contractions, 5 sets). Twenty-four hours post-ECC, edema and inflammatory cell infiltration were assessed histologically. Results: Resting [Ca 2+ ]i was significantly lower in the 48F group compared to the CON group (Fura-2 ratio: CON 0.73 ± 0.060 vs. 48F 0.58 ± 0.072, p = 0.0028). No significant differences were observed between groups in the 30-min time course of resting intracellular Ca 2+ levels or in spontaneous Ca 2+ fluctuations measured at rest, indicating that basal Ca 2+ homeostasis was preserved despite the reduced baseline [Ca 2+ ]i. Peak torque during ECC was significantly attenuated by fasting (CON: 16.15 ± 2.25 N vs. 48F: 10.88 ± 1.50 N, p =0.033); however, the total force integral over 5 sets was not different (p = 0.1664). Twenty-four hours post-ECC, inflammatory cell infiltration was markedly suppressed in the 48F group (CON: 16.40 ± 10.60% vs. 48F: 3.80 ± 2.49%, p = 0.0034), whereas no difference was observed in the extent of edema (CON: 19.60 ± 11.35% vs. 48F: 15.20 ± 13.52%, p = 0.5929). These results support that fasting inhibits the transition process from edema to cellular infiltration. Conclusion: In vivo evaluation revealed that 48-hour fasting maintains resting skeletal muscle [Ca 2+ ]i at lower levels. Furthermore, the damage-recovery response to ECC under this condition was characterized by attenuated inflammatory cell infiltration. These findings suggest that fasting modulates early muscle damage responses following ECC. Although the overall force integral during ECC was comparable between groups, the fasted condition was associated with altered Ca 2+ dynamics and reduced peak torque during ECC. The extent to which changes in Ca 2+ handling, contractile characteristics, or their interaction contribute to the attenuation of inflammatory infiltration requires further investigation. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

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.

BACKGROUND: Age-related skeletal muscle atrophy primarily manifests as a heterogeneous decline in the size and function of individual fibers. Intracellular calcium ion concentration ([Ca 2 + ] i ) is a pivotal second messenger and regulates numerous cellular phenomena via its temporal and spatial dynamics. Dysregulation of [Ca 2 + ] i homeostasis has been identified as a characteristic of old skeletal muscle, however, the spatiotemporal pattern of [Ca 2 + ] i dynamics remains unknown. PURPOSE: To characterize the spatiotemporal dynamics and regulatory mechanisms of [Ca 2 + ] i in aged skeletal muscle at rest and in response to twitch contractions. METHODS: Male young (Y; 6 months old, n = 6) and old (O; 26 months old, n = 4) C57BL/6J mice were subjected to in vivo [Ca 2 + ] i imaging. Under anesthesia, the tibialis anterior (TA) muscle was gently exteriorized and loaded with ratiometric Ca 2 + indicator Fura 2-AM (40 μM). [Ca 2 + ] i was measured from the fluorescence ratio (R: 340/380) at rest (30 min) and in response to twitch contractions (1 Hz, 4 ms pulse, -10V, 10 min). After imaging, the TA and white gastrocnemius muscles were sampled for biochemical and morphological analysis. RESULTS: Resting [Ca 2 + ] i was significantly increased in old skeletal muscle compared to young (Y: 1.49 ± 0.02, O: 1.64 ± 0.05, p = 0.012). The spatial distribution of [Ca 2 + ] i within muscle fibers in aged muscle exhibited a distinct localized accumulation along the longitudinal muscle axis. This spatial heterogeneity was quantitatively characterized by a significant increase in the coefficient of variation of [Ca 2 + ] i in the fiber (Y: 4.46 ± 0.24%, O: 6.91 ± 0.51%, p = 0.0008). No statistical differences in the amplitude of temporal [Ca 2 + ] i changes at rest were observed between young and aged muscle (Y: 0.18 ± 0.05%, O: 0.14 ± 0.08%, p = 0.254). Twitch contraction-induced [Ca 2 + ] i elevation was observed in both young and old muscle (Y: 6.3 ± 1.9%, O: 8.5 ± 2.5%, main effect of contraction: p < 0.0001), while muscle torque was relatively decreased in aged muscle (main effect of age: p = 0.013). This result suggests that old muscle displayed higher [Ca 2 + ] i per unit of relative muscle force during the twitch contractions (main effect of age: p = 0.032). Levels of major Ca 2 + -handling proteins located on sarcoplasmic reticulum (RyR: p = 0.565, SERCA1: p = 0.258, CSQ1: p = 0.466) and mitochondria (MCU: p = 0.957, MICU1: p = 0.798, MICU2: p = 0.953, COXIV: p = 0.125), and SR-mitochondrial tethering protein (Mfn1: p = 0.869, Mfn2: p = 0.785) were maintained with aging. The muscle section revealed an abnormal sarcoplasmic reticulum with tubular aggregates arranged longitudinally in 91.4 % of aged muscle fibers. CONCLUSION: Our findings reveal novel aspects of [Ca 2 + ] i dysregulation in old skeletal muscle. We demonstrated that old muscle exhibited a chronically elevated basal [Ca 2 + ] i with a unique spatially heterogeneous distribution within individual fibers. This distinct, longitudinally localized, [Ca 2 + ] i pattern in concert with the emergence of tubular aggregates could potentially underlie the mechanisms promoting muscle atrophy in aging. Funding information: This study was supported in part by JSPS KAKENHI Grant (No.20H04074, 21J14283). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: Muscle glycogen is a preferential substrate for energy production over glucose and its levels are decreased with muscle contractions. Intramyocyte glucose concentration ([Glu] i ) regulates multiple processes involved in glucose metabolism: However, its dynamics during muscle contractions remain to be elucidated. Purpose: In this investigation, we resolved in vivo [Glu] i dynamics during contractions. We tested the hypothesis that, regardless of contraction intensity or type, [Glu] i is increased with muscle contractions, but that this increase does not necessarily relate to a post-contraction glycogen concentration. METHOD: Glucose-sensitive fluorescent protein (Pglu-700μDelta6) was transfected and expressed in the spinotrapezius muscle of male Wistar rats by electroporation of plasmid 7 days prior to the imaging experiment. Rats were divided into two groups: 1) control (CONT, n = 16), and 2) glucose transport inhibition (cytochalasin B: CB, n = 15). On the day of the experiment, the muscle was exposed and subjected to the [Glu] i imaging during twitch (1 Hz, ~ 10 V, 10 min) or tetanic (100 Hz for 700 ms, 50 times, 5 sets with 5 min intervals) contractions. [Glu] i was measured from the fluorescence ratio (R: 535 nm/480 nm) and normalized to the pre-contractions value (R/R pre ). In the CB group, CB (1 mM) was applied from 20 min before contractions. Intramuscular glycogen levels were measured by biochemical analysis from the muscle isolated post-contractions. Results: [Glu] i was significantly increased by twitch contractions (+12.3 ± 1.9%, p < 0.05 vs pre), and following 30 min remained higher than pre-contractions (+7.9 ± 1.7%, p < 0.05 vs pre). With tetanic contractions, [Glu] i was increased within the 1st set (+36.7 ± 6.6%, p < 0.01 vs pre), and remained elevated throughout the 5-set bout (post-5 set: 25.8 ± 8.2%, p < 0.01 vs pre) without significant recovery or additional increase among sets. The contractions-induced [Glu] i increase was significantly blunted by CB (post-twitch: +4.5 ± 2.0%; post-1 set tetanic: +4.5 ± 2.4%, p < 0.05 vs pre). CB treatment precipitated a decrease in relative muscle force in the tetanic (CONT: 68.1 ± 4.5%, CB: 49.8 ± 1.3%, p < 0.01), but not in the twitch contractions. Both CONT and CB exhibited decreased muscular glycogen with twitch contractions (CONT: 19.4 ±1.5 µmol/g, CB: 18.4 ±1.5 µmol/g) and tetanic contractions (CONT: 13.4 ± 1.3 µmol/g, CB: 10.3 ± 1.3 µmol/g) compared to the non-contracting muscle (29.9 ± 2.1 µmol/g, p < 0.01). Conclusion: In this investigation, we demonstrated that, while glucose is taken up and accumulates within the contracting myocyte, it does not appear to contribute to the suppression of muscle glycogen reduction in either twitch or tetanic contractions. Thus, whilst intramyocyte glucose levels are not associated with the reduction of glycogen during muscle contractions observed herein they may be crucial for maintenance of tetanic tension. This study was supported by JSPS KAKENHI Grant (No. 20K20620). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: Skeletal muscle mitochondria repeatedly divide and fuse in response to changing energy supply and demand. These dynamic processes of mitochondrial biogenesis form a catenated network of interconnected mitochondria. However, the characteristics of this behavior during skeletal muscle myogenesis and regeneration are unknown. We have previously developed a photothermal microscopy (PTM) method for intramyocyte localization of cytochrome c using histochemical sections for bright field observation. As cytochrome c is a heme protein bound to the inner mitochondrial membrane PTM facilitates resolution of whole-cell mitochondrial remodeling during muscle regeneration. Purpose: This investigation used PTM to resolve structural changes in the mitochondrial network of gastrocnemius myocytes during regeneration following eccentric contractions (ECCs) induced damage. The reliability of this PTM mitochondrial network characterization was assessed via parallel analysis of PTM and transmission electron microscopy (TEM) images. METHOD: Under anesthesia, 300 ECCs were applied to the gastrocnemius of male Wistar rats (13 weeks old) using electrical stimulation and motor drive-induced muscle lengthening. Seven days into post-ECC recovery (i.e., ECC 7-day), the muscles were perfused-fixed in situ, dissected and embedded in Epon and compared with an ECC non-load control group (CONT). Transverse sections were prepared for optical (1 μm) and electron (1 nm) microscopy. The volume density of mitochondria was evaluated with respect to the distance from the nucleus using PTM and TEM and determination of the network index Form Factor, (FF). PTM used a laser with 515 and 638 nm wavelengths for the PT pump and probe beams, respectively. Results: Seven days after ECC, many regenerated muscle fibers were observed with small diameters and central nuclei, characteristic of the recovery process after muscle damage. Using PTM, across the entire myocyte, a contiguous network of mitochondria, arranged predominantly along the myofibrils, was observed in CONT muscle. In marked contrast, the network index FF was significantly and substantially lower in regenerating muscle (CONT: 15.5 ± 1.8, ECC: 0.6 ± 0.0, p<0.001) evidencing a far more randomized morphology in regenerating (ECC 7-day) than normal (CONT) muscle. Furthermore, in regenerating muscle, mitochondria were aggregated strongly around the central nuclei. Similarly, the mitochondrial volume density in the close proximity of the central nuclei was significantly higher in TEM observations. Specifically, compared to normal muscle, the volume density of mitochondria was significantly higher in the region within 0.1 μm from the nucleus in regenerating muscle (CONT: 4.1±0.2%, ECC: 9.0±0.2%, p<0.0001) and 0.2 μm (CONT: 1.4±0.0%, ECC: 7.6±0.2%, p<0.0001). Conclusion: Muscle histochemical imaging with PTM allows high resolution mitochondrial quantification. During muscle regeneration 7 days post-ECC, PTM analyses revealed a characteristic morphology in which the mitochondria were concentrated preferentially around the nucleus rather than forming a diffuse interconnected network distributed across the entire myocyte characteristic of CONT. This work was supported by JSPS KAKENHI Grant Numbers 23K18417, 24K02827. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract: Muscle damage induced by eccentric contraction (ECC) is triggered by prolonged local accumulation of calcium ions (Ca 2+ ) in the cytoplasm. The lack of homeostasis in the regulation of intracellular Ca 2+ concentration ([Ca 2+ ]i) observed after ECC has been linked to a decrease in the function of the ryanodine receptor (RyR) which forms the Ca 2+ release channel in the sarcoplasmic reticulum. In contrast, transient and periodic Ca 2+ events mediated via RyR are essential for skeletal muscle differentiation and regeneration. However, the role of RyR in the dynamics of Ca 2+ events that signal the muscle regeneration process after ECC-induced muscle damage has not been elucidated. Purpose: This study tested the hypothesis that pharmacological inhibition of RyR during muscle regeneration suppresses [Ca 2+ ]i and delays myogenesis and growth of regenerating muscle cells. Methods: Tibialis anterior (TA) muscles of adult male Wistar rats either without ECC (CONT) or 5 (ECC5D) or 9 days (ECC9D) after ECC were imaged in vivo for [Ca 2+ ]i and subsequent in vitro histologic analysis. In addition an RyR inhibition group was treated with dantrolene (DAN) during the regeneration phase (intraperitoneal administration, 6-8 days) (ECC9D+DAN). TA muscles were loaded with the Ca 2+ indicator Fura2-AM and [Ca 2+ ]i distribution was evaluated at rest in each group by means of the 340/380 nm ratio. For muscle histology, satellite cell activation (Pax7, MyoD), inflammatory response (M2 macrophages), and muscle fiber cross-sectional area were evaluated by immunofluorescence and H&E staining. Results: Compared to control, [Ca 2+ ]i was significantly transiently decreased in ECC5D and recovered in ECC9D (Fura2 ratio values, CONT: 0.72 ± 0.05; ECC5D: 0.55 ± 0.07; ECC9D: 0.63 ± 0.06). The [Ca 2+ ]i of ECC9D+DAN (0.58 ± 0.08) was significantly lower than that of CONT. In the ECC9D+DAN group, TA muscle weight (right/left ratio, i.e., ECC leg/untreated leg) was lower than in the ECC9D group (ECC9D: 0.80 ± 0.06; ECC9D+DAN: 0.66 ± 0.06, p < 0.05). Furthermore, the proportion of muscle fibers with a cross-sectional area of less than 1000 μm 2 increased with dantrolene treatment (ECC9D: 48.0 ± 19.7, ECC9D+DAN: 65.8 ± 10.9%, p < 0.05). There were no significant differences between the two groups in the results of satellite cell activation (Pax7, MyoD) or inflammatory response (M2 macrophages). Conclusion: These findings support that decreased [Ca 2+ ]i consequent to RyR inhibition, 6 to 8 days post-ECC, delays the growth of regenerated skeletal muscle rather than preventing myogenesis per se. This study was supported in part by JSPS KAKENHI Grant (No. 20H04074, 23K24734). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Oxidative stress and reactive oxygen species (ROS) have been linked to muscle atrophy and weakness. Diabetes increases the oxidative status in all tissues, including muscle tissues, but the role of lipid ROS on diabetes-induced muscle atrophy is not fully understood. Deuterium reinforced polyunsaturated fatty acids (D-PUFA) are more resistant to ROS-initiated chain reaction of lipid peroxidation than regular hydrogenated PUFA (H-PUFA). In this study, we tested the hypothesis that D-PUFA would protect muscle atrophy induced by diabetes driven by an accumulation of lipid hydroperoxides (LOOH). C57BL/6J mice were dosed with H-PUFA or D-PUFA for four weeks through dietary supplementation (10 mg/day) and then injected with streptozotocin (STZ) to induce insulin-deficient diabetes. After two weeks, muscles tissues were analyzed for individual muscle mass, force generating capacity and cross-sectional area. Skeletal muscle fibers from diabetic mice exhibited increased total ROS and LOOH. This was abolished by the D-PUFA supplementation regardless of accumulated iron. D-PUFA were found to be protective against muscle atrophy and weakness from STZ-induced diabetes. Prevention of muscle atrophy and weakness by D-PUFA might be independent of ACSL4/LPCAT3/15-LOX pathway. These findings provide novel insights into the role of LOOH in the mechanistic link between oxidative stress and diabetic myopathy and suggest a novel therapeutic approach to diabetes-associated muscle weakness.

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 a 3-min 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, whereas S-Cool decreased several antioxidant-related genes.NEW & NOTEWORTHY This in vivo model successfully characterized the effects of cooling on cytosolic and mitochondrial [H2O2] in mouse tibialis anterior skeletal muscle. Cooling decreased [H2O2] down to ∼13°C, but the effect was reversed at still lower temperatures. Sustained cooling decreased mRNA levels of antioxidant-related genes (Sod2, Cat, and Ucp3), whereas intermittent cooling increased Nrf mRNA expression. These results help elucidate the mechanistic bases for skeletal muscle adaptation to cooling.

Background: Mitochondria constitute their interconnected network through dynamic mitochondrial processes such as fission and fusion in response to changes in their intracellular environment, such as energy demand. Transmission electron microscopy is the standard method for evaluating mitochondrial morphology. We have developed a simple method to evaluate mitochondrial morphology using tissue sections for bright-field observation. In this study, we attempted to image light-absorbing molecules (cytochrome c) in mitochondria using photothermal (PT) microscopy. Purpose: This method can observe an entire myocyte, so this study attempted to evaluate the entire mitochondrial network of immature (regenerating) myocytes during the skeletal muscle regeneration process. Methods: The gastrocnemius muscle of anesthetized male Wistar rats (13 weeks old) was subjected to 300 controlled eccentric contractions (ECC) using electrical stimulation. 7 days after ECC loading, in situ perfusion fixation was performed, and transverse sections (1 μm) from red (deep regions) and white (superficial regions) were prepared after Epon embedding. The sections were observed by PT microscopy, and quantitative analysis of mitochondrial morphology was performed. Mitochondrial content was expressed as a percentage of the fiber total area in transverse sections. The PT imaging system uses lasers with wavelengths of 515 nm and 638 nm constituting the PT pump and the probe beams, respectively. Results: After 7 days of ECC, many myofibers with small fiber diameters and central nuclei, which are characteristic of the recovery process after muscle damage, were observed. The cross-sectional area of the regenerating muscle (superficial region 389.9 ± 15.2 μm2, deep region 366.2 ± 17.5 μm2) was significantly smaller than that of the normal muscle (superficial region 2211.1 ± 127.3 μm2, deep region 1396.1 ± 72.9 μm2). Mitochondrial content of the regenerating muscle (superficial region 26.1 ± 1.1%, deep region 22.7 ± 0.8%) was significantly lower than that of the normal muscle (superficial region 31.8 ± 1.9%, deep region 34.9 ± 1.8%). In normal muscle, a linear network of mitochondria along myofibrils was observed. On the other hand, no spatially distributed mitochondria were observed in the cytoplasm of the regenerating muscle. Furthermore, a mitochondrial network was formed around the periphery of the central nucleus. Conclusion: In muscle tissue imaging using PT microscopy, myofibers in the regenerative process have less mitochondrial content than mature muscle fibers and lack the mitochondrial network along myofibrils that characterizes mature muscle. On the other hand, the central nucleus of immature muscle was surrounded by highly developed mitochondria. The mitochondrial morphological characteristics of regenerating muscle fibers may offer a unique window through which to better understand mitochondrial design and function and their plasticity during maturation. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) KAKENHI Grant (No. JP20H04074, 21K19703). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hydrogen peroxide (H2O2) and calcium ions (Ca2+) are functional regulators of skeletal muscle contraction and metabolism. Although H2O2 is one of the activators of the type-1 ryanodine receptor (RyR1) in the Ca2+ release channel, the interdependence between H2O2 and Ca2+ dynamics remains unclear. This study tested the following hypotheses using an in vivo model of mouse tibialis anterior (TA) skeletal muscle. 1) Under resting conditions, elevated cytosolic H2O2 concentration ([H2O2](cyto)) leads to a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+](cyto)) through its effect on RyR1; and 2) in hypoxia (cardiac arrest) and muscle contractions (electrical stimulation), increased [H2O2](cyto) induces Ca2+ accumulation. Cytosolic H2O2 (HyPer7) and Ca2+ (Fura-2) dynamics were resolved by TA bioimaging in young C57BL/6J male mice under four conditions: 1) elevated exogenous H2O2; 2) cardiac arrest; 3) twitch (1 Hz, 60 s) contractions; and 4) tetanic (30 s) contractions. Exogenous H2O2 (0.1-100 mM) induced a concentration-dependent increase in [H2O2](cyto) (+55% at 0.1 mM; +280% at 100 mM) and an increase in [Ca2+](cyto) (+3% at 1.0 mM; +8% at 10 mM). This increase in [Ca2+](cyto) was inhibited by pharmacological inhibition of RyR1 by dantrolene. Cardiac arrest-induced hypoxia increased [H2O2](cyto) (+33%) and [Ca2+](cyto) (+20%) 50 min postcardiac arrest. Compared with the exogenous 1.0 mM H2O2 condition, [H2O2](cyto) after tetanic muscle contractions rose less than one-tenth as much, whereas [Ca2+](cyto) was 4.7-fold higher. In conclusion, substantial increases in [H2O2](cyto) levels evoke only modest Ca2+ accumulation via their effect on the sarcoplasmic reticulum RyR1. On the other hand, contrary to hypoxia secondary to cardiac arrest, increases in [H2O2](cyto) from muscle contractions are small, indicating that H2O2 generation is unlikely to be a primary factor driving the significant Ca2+ accumulation after, especially tetanic, muscle contractions.NEW & NOTEWORTHY We developed an in vivo mouse myocyte H2O2 imaging model during exogenous H2O2 loading, ischemic hypoxia induced by cardiac arrest, and muscle contractions. In this study, the interrelationship between cytosolic H2O2 levels and Ca2+ homeostasis during muscle contraction and hypoxic conditions was revealed. These results contribute to the elucidation of the mechanisms of muscle fatigue and exercise adaptation.

BACKGROUND: Across the lifespan, body temperature homeostasis is tightly regulated and impacts various physiological phenomena. In skeletal muscle, heat is produced via contraction-dependent (shivering) and independent (nonshivering) mechanisms, regulating the body temperature and muscle physiology including contractile properties. PURPOSE: In this study, we aimed to reveal the intracellular temperature dynamics in skeletal muscle in vivo during the contraction-relaxation cycle. Additionally, we tested the hypothesis that calcium ion (Ca2+) handling mediated by sarcoplasmic reticulum Ca2+ ATPase (SERCA) is involved in the thermogenesis process during contraction-relaxation cycle in skeletal muscle fibers. METHOD: The spinotrapezius muscle of anesthetized adult male Wistar rats (n = 18) was exteriorized and subjected to microinjection of fluorescent probe Cellular Thermoprobe for Fluorescence Ratio (49.3 μM) for intracellular temperature imaging in vivo. The fluorescence ratio (R: 580 nm / 515 nm) was measured in vivo during temperature increases induced by an external heater for thermoprobe calibration. R was also measured following Ca2+ injection (3.9 nL, 2.0 mM), and Ca2+ injection with SERCA inhibition by cyclopiazonic acid (CPA, 100 μM). RESULTS: The fluorescence ratio linearly increased with the increase of the muscle surface temperature from 25 °C to 40 °C (r2 = 0.97, P < 0.01). The intracellular temperature in muscle was increased and sustained following the Ca2+ injection and this thermogenetic response was significantly suppressed with SERCA inhibition (Ca2+: 38.3 ± 1.4 °C vs Ca2++CPA: 28.3 ± 2.8 °C, P < 0.01 at 1 min following injection). Importantly, elevated muscle temperature occurred predominantly in the muscle relaxation phase that followed the muscle shortening: That shortening occurred immediately and transiently after the Ca2+ injection. CONCLUSION: In this investigation, we demonstrated a novel technique for in vivo intracellular temperature imaging of skeletal muscle. Herein we demonstrate that substantial heat is generated concomitantly with SERCA mediated Ca2+ handling and muscle relaxation. This work was supported by JSPS KAKENHI Grant Numbers 20H04074, 19K22800. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Eccentric contractions (ECC) induce excessive intracellular calcium ion (Ca 2+ ) accumulation and muscle structural damage in localized regions of the muscle fibers. In this investigation, we present the novel hypothesis that the ryanodine receptor (RyR) plays a central role in evoking a Ca 2+ dynamics profile that is markedly distinguishable from other muscle adaptive responses.

Eccentric contractions (ECC) are accompanied by the 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. We tested the hypothesis that repeated ECC suppresses Ca2+ accumulation via adaptations in Ca2+ regulation. 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. 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). Five 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 (MCU) 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). Fourteen 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 sarcoplasmic reticulum Ca2+ regulation.NEW & NOTEWORTHY We demonstrated a reduced [Ca2+]i profile with suppressed muscle damage after a repeated bout of ECC in vivo: the ECC-induced immediate [Ca2+]i increase was suppressed and the persistence of increased [Ca2+]i with localized accumulation was diminished after repeated ECC. This effect occurred consonant with the upregulation of the mitochondrial Ca2+ uniporter complex and better preservation of SERCA1 and MICU1. These findings suggest that the mechanistic bases for repeated bout protection involve adaptation of Ca2+ regulation.

Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin-proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X-prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl-cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine-sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched-chain amino acid levels, and enhanced mTORC2-mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation.

Intracellular Ca2+ concentration ([Ca2+]i) is considered important in the regulation of skeletal muscle mass. This study tested the hypothesis that chronic repeated cooling and/or caffeine ingestion would acutely increase [Ca2+]i and hypertrophy muscles potentially in a fiber-type-dependent manner. Control rats and those fed caffeine were subjected to repeated bidiurnal treatments of percutaneous icing, under anesthesia, to reduce the muscle temperature below ∼5°C. The predominantly fast-twitch tibialis anterior (TA) and slow-twitch soleus (SOL) muscles were evaluated after 28 days of intervention. The [Ca2+]i elevating response to icing was enhanced by caffeine loading only in the SOL muscle, with the response present across a significantly higher temperature range than in the TA muscle under caffeine-loading conditions. In both the TA and SOL muscles, myofiber cross-sectional area (CSA) was decreased by chronic caffeine treatment (mean reductions of 10.5% and 20.4%, respectively). However, in the TA, but not the SOL, CSA was restored by icing (+15.4 ± 4.3% vs. noniced, P < 0.01). In the SOL, but not TA, icing + caffeine increased myofiber number (20.5 ± 6.7%, P < 0.05) and satellite cell density (2.5 ± 0.3-fold) in cross sections. These contrasting muscle responses to cooling and caffeine may reflect fiber-type-specific [Ca2+]i responses and/or differential responses to elevated [Ca2+]i.

Eccentric contractions (ECC) accompany the intracellular calcium ion (Ca2+) accumulation and induce skeletal muscle damage. ECC-induced muscle damage is reduced in repeated bouts of ECC. PURPOSE: We examined the hypothesis that repeated ECC bouts cause remodeling of Ca2+ handling, resulting in a lower accumulation of intracellular Ca2+ concentration ([Ca2+]i) and reduced muscle damage. METHODS: Male Wistar rats were divided into two groups: 1) single bout (SB), and 2) repeated bouts (RB). Tibialis anterior (TA) muscles were subjected to ECC (40 times, 5 sets) once (SB) or twice 2 weeks apart (RB). Under anesthesia, the TA muscle was loaded with Ca2+ indicator Fura2-AM and the 340/380 nm ratio was evaluated as [Ca2+]i changes with ECC. The expression of Ca2+ handling proteins in TA muscle were also measured by western blots. Muscle damage area was assessed post-4 days ECC from Hematoxylin-Eosin staining. RESULTS: The ECC protocols both increased [Ca2+]i, but with the RB evincing a suppressed [Ca2+]i elevation compared with SB (Time: p < 0.01, p = 0.0357). 5 hours post-ECC, a [Ca2+]i elevation was sustained with regional and heterogeneous distribution in SB, but not in RB (Ratio; SB: 1.79 ± 0.04, RB: 1.57 ± 0.05, p < 0.01). The protein expression level of MCU and MICU2, components of the mitochondria Ca2+ uniporter complex, were significantly increased in RB compared to SB (p < 0.01). Mitochondrial content evaluated from COXIV expression and Ca2+ handling proteins on the sarcoplasmic reticulum, ryanodine receptor (RyR) and Ca2+ ATPase (SERCA) were not differentially expressed in SB and RB (p > 0.05). The muscle damage area was significantly decreased in RB compared with SB (SB: 63.4 ± 4.8%, RB: 5.7 ± 2.1%, p < 0.01). CONCLUSION: Skeletal muscle following 14 days of ECC demonstrated upregulated mitochondrial Ca2+ regulating proteins and suppressed ECC-induced [Ca2+]i accumulation and subsequent muscle damage. These findings suggest that the muscle damage-regeneration processes following the initial bout of ECC provide a protective mechanism mediated principally by enhanced mitochondrial Ca2+ uptake that suppresses ECC-induced muscle damage.

We have developed a high-speed 3D color planar lightwave circuit digital holographic microscope (PLC-DHM) by applying our new thermo-optical phase control method and switching the three-wavelength laser. In this study, we experimentally demonstrate that this high-speed 3D color PLC-DHM successfully captures the cell structure of mammalian skeletal muscle with a thickness of 40 um and discuss its effectiveness for application in live cell imaging.

Skeletal muscle generates heat via contraction-dependent (shivering) and independent (nonshivering) mechanisms. While this thermogenic capacity of skeletal muscle undoubtedly contributes to the body temperature homeostasis of animals and impacts various cellular functions, the intracellular temperature and its dynamics in skeletal muscle in vivo remain elusive. We aimed to determine the intracellular temperature and its changes within skeletal muscle in vivo during contraction and following relaxation. In addition, we tested the hypothesis that sarcoplasmic reticulum Ca2+ ATPase (SERCA) generates heat and increases the myocyte temperature during a transitory Ca2+-induced contraction-relaxation cycle. The intact spinotrapezius muscle of anesthetized adult male Wistar rats (n = 18) was exteriorized and loaded with the fluorescent probe Cellular Thermoprobe for Fluorescence Ratio (49.3 μM) by microinjection over 1 s. The fluorescence ratio (i.e., 580 nm/515 nm) was measured in vivo during 1) temperature increases induced by means of an external heater, and 2) Ca2+ injection (3.9 nL, 2.0 mM). The fluorescence ratio increased as a linear function of muscle surface temperature from 25 °C to 40 °C (r2 = 0.97, P < 0.01). Ca2+ injection (3.9 nL, 2.0 mM) significantly increased myocyte intracellular temperature: An effect that was suppressed by SERCA inhibition with cyclopiazonic acid (CPA, Ca2+: 38.3 ± 1.4 °C vs Ca2++CPA: 28.3 ± 2.8 °C, P < 0.01 at 1 min following injection). While muscle shortening occurred immediately after the Ca2+ injection, the increased muscle temperature was maintained during the relaxation phase. In this investigation, we demonstrated a novel model for measuring the intracellular temperature of skeletal muscle in vivo and further that heat generation occurs concomitant principally with SERCA functioning and muscle relaxation.

AIM:Cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (Cmah) is an enzyme, which converts Neu5Ac to the sialic acid Neu5Gc. Neu5Gc is thought to increase inflammatory cytokines, which are, in part, produced in senescent cells of adipose tissues. Cellular senescence in adipose tissues induces whole-body aging and impaired glucose metabolism. Therefore, we hypothesized that Cmah deficiency would prevent cellular senescence in adipose tissues and impaired glucose metabolism. METHODS:Wild-type (WT) and Cmah knockout (KO) mice aged 24-25 months were used. Whole-body metabolism was assessed using a metabolic gas analysis system. We measured blood glucose and insulin concentrations after oral glucose administration. The size of the lipid droplets in the liver was quantified. Markers of cellular senescence and senescence-associated secretory phenotypes were measured in adipose tissues. RESULTS:Cmah KO had significantly increased VO2 and energy expenditure (P < 0.01). Unlike glucose, the insulin concentration after oral glucose administration was significantly lower in the Cmah KO group than in the WT group (P < 0.001). Lipid droplets in the liver were significantly lower in the Cmah KO group than in the WT group (P < 0.05). The markers of cellular senescence and senescence-associated secretory phenotypes in the adipose tissues were significantly lower in the Cmah KO group than in the WT group (P < 0.05). CONCLUSIONS:Cmah deficiency blunted cellular senescence in adipose tissues and improved whole-body glucose metabolism. These characteristics in aged Cmah KO mice might be associated with higher energy expenditure. Geriatr Gerontol Int 2023; 23: 958-964.