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Versican is increased with inflammation and fibrosis, and is upregulated in Duchenne muscular dystrophy. In fibrotic diaphragm muscles from dystrophic mdx mice, genetic reduction of versican attenuated macrophage infiltration and improved contractile function. Versican is also implicated in myogenesis. Here, we investigated whether versican modulated mdx hindlimb muscle pathology, where inflammation and regeneration are increased but fibrosis is minimal. Immunohistochemistry and qRT-PCR were used to assess how fiber type and glucocorticoids (α-methylprednisolone) modify versican expression. To genetically reduce versican, female mdx and male versican haploinsufficient (hdf) mice were bred resulting in male mdx-hdf and mdx (control) pups. Versican expression, contractile function, and pathology were evaluated in hindlimb muscles. Versican immunoreactivity was greater in slow versus fast hindlimb muscles. Versican mRNA transcripts were reduced by α-methylprednisolone in soleus, but not in fast extensor digitorum longus, muscles. In juvenile (6-wk-old) mdx-hdf mice, versican expression was most robustly decreased in soleus muscles leading to improved force output and a modest reduction in fatiguability. These functional benefits were not accompanied by decreased inflammation. Muscle architecture, regeneration markers, and fiber type also did not differ between mdx-hdf mice and mdx littermates. Improvements in soleus contractile function were not retained in adult (20-wk-old) mdx-hdf mice. In conclusion, soleus muscles from juvenile mdx mice were most responsive to pharmacological or genetic approaches targeting versican; however, the benefits of versican reduction were limited due to low fibrosis. Preclinical matrix research in dystrophy should account for muscle phenotype (including age) and the interdependence between inflammation and fibrosis. [ABSTRACT FROM AUTHOR]

Background: Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by gene mutations resulting in deficiency of the membrane-associated protein dysferlin. They manifest post-growth and are characterised by muscle wasting (primarily in the limb and limb-gridle muscles), inflammation, and replacement of myofibres with adipose tissue. The precise pathomechanism for dysferlinopathy is currently unclear; as such there are no treatments currently available. Glucocorticoids (GCs) are widely used to reduce inflammation and treat muscular dystrophies, but when administered to patients with dysferlinopathy, they have unexpected adverse effects, with accelerated loss of muscle strength. Methods: To investigate the mechanistic basis for the adverse effects of GCs in dysferlinopathy, the potent GC dexamethasone (Dex) was administered for 4–5 weeks (0.5–0.75 µg/mL in drinking water) to dysferlin-deficient BLA/J and normal wild-type (WT) male mice, sampled at 5 (Study 1) or 10 months (Study 2) of age. A wide range of analyses were conducted. Metabolism- and immune-related gene expression was assessed in psoas muscles at both ages and in quadriceps at 10 months of age. For the 10-month-old mice, quadriceps and psoas muscle histology was assessed. Additionally, we investigated the impact of Dex on the predominantly slow and fast-twitch soleus and extensor digitorum longus (EDL) muscles (respectively) in terms of contractile function, myofibre-type composition, and levels of proteins related to contractile function and metabolism, plus glycogen. Results: At both ages, many complement-related genes were highly expressed in BLA/J muscles, and WT mice were generally more responsive to Dex than BLA/J. The effects of Dex on BLA/J mice included (i) increased expression of inflammasome-related genes in muscles (at 5 months) and (ii) exacerbated histopathology of quadriceps and psoas muscles at 10 months. A novel observation was pronounced staining for glycogen in many myofibres of the damaged quadriceps muscles, with large pale vacuolated myofibres, suggesting possible myofibre death by oncosis. Conclusion: These pilot studies provide a new focus for further investigation into the adverse effects of GCs on dysferlinopathic muscles. [ABSTRACT FROM AUTHOR]

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow‐ to fast‐twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow‐twitch soleus and fast‐twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre‐type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders. [ABSTRACT FROM AUTHOR]

Calpain-3 (CAPN3) is a muscle-specific member of the calpain family of Ca2+-dependent proteases. It has been reported that CAPN3 can also be autolytically activated by Na+ ions in the absence of Ca2+, although this was only shown under non-physiological ionic conditions. Here we confirm that CAPN3 does undergo autolysis in the presence of high [Na+], but this only occurred if all K+ normally present in a muscle cell was absent, and it did not occur even in 36 mM Na+, higher than what would ever be reached in exercising muscle if normal [K+] was present. CAPN3 in human muscle homogenates was autolytically activated by Ca2+, with ~50% CAPN3 autolysing in 60 min in the presence of 2 µM Ca2+. In comparison, autolytic activation of CAPN1 required about 5-fold higher [Ca2+] in the same conditions and tissue. After it was autolysed, CAPN3 unbound from its tight binding on titin and became diffusible, but only if the autolysis led to complete removal of the IS1 inhibitory peptide within CAPN3, reducing the C-terminal fragment to 55 kDa. Contrary to a previous report, activation of CAPN3, either by raised [Ca2+] or Na+ treatment, did not cause proteolysis of the skeletal muscle Ca2+ release channel-ryanodine receptor, RyR1, in physiological ionic conditions. Treatment of human muscle homogenates with high [Ca2+] caused autolytic activation of CAPN1, accompanied by proteolysis of some titin and complete proteolysis of junctophilin (JP1, full length ~95 kDa), generating an equimolar amount of a diffusible ~75 kDa N-terminal JP1 fragment, but without any proteolysis of RyR1. [ABSTRACT FROM AUTHOR]

Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca2+) handling and glucose/glycogen metabolism-related proteins in predominantly slow-twitch, oxidative soleus and fast-twitch, glycolytic extensor digitorum longus (EDL) muscles of 10-month-old wild-type (WT) C57BL/6J and dysferlin-deficient BLAJ male mice. Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy. [ABSTRACT FROM AUTHOR]

Ca2+ is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca2+ is reported to modulate the rate of ATP resynthesis, regulate the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1a) following exercise, and drive the generation of reactive oxygen species (ROS). Due to the latter, mitochondrial Ca2+ overload is recognized as a pathophysiological event but the former events represent important physiological functions in need of tight regulation. Recently, we described the relationship between [Ca2+]mito and resting [Ca2+]cyto and other mitochondrial Ca2+-handling properties of skeletal muscle. An important next step is to understand the triggers for Ca2+ redistribution between intracellular compartments, which determine the mitochondrial Ca2+ load. These triggers in both physiological and pathophysiological scenarios can be traced to the coupled activity of the ryanodine receptor 1 (RyR1) and store-operated Ca2+ entry (SOCE) in the resting muscle. In this piece, we will discuss some issues regarding Ca2+ measurements relevant to mitochondrial Ca2+-handling, the steady-state relationship between cytoplasmic and mitochondrial Ca2+, and the potential implications for Ca2+ handling by muscle mitochondria and cellular function. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the fiber type-specific abundance of autophagy-related proteins after an overnight fast and following ingestion of a mixed meal in human skeletal muscle. Twelve overweight, healthy young male volunteers underwent a 3-h mixed meal tolerance test following an overnight fast. Blood samples were collected in the overnight-fasted state and throughout the 180-min postmeal period. Skeletal muscle biopsies were collected in the fasted state, and at 30 and 90 min after meal ingestion. Protein content of key autophagy markers and upstream signaling responses were measured in whole muscle and pooled single fibers using immunoblotting. In the fasted state, type I fibers displayed lower LC3B-I but higher LC3B-II abundance and higher LC3B-II/LC3B-I ratio compared with type II fibers (P < 0.05). However, there were no fiber type differences in p62/SQSTM1, unc-51 like autophagy activating kinase (ULK1), ATG5, or ATG12 (P > 0.05). Compared with the fasted state, there was a reduction in LC3B-II abundance, indicative of lower autophagosome content, in whole muscle and in both type I and type II fibers following meal ingestion (P < 0.05). This reduction in autophagosome content occurred alongside similar increases in p-AktS473 and p-mTORS2448 in both type I and type II muscle fibers (P < 0.05). In human skeletal muscle, type I fibers have a greater autophagosome content than type II fibers in the overnight-fasted state despite comparable abundance of other key upstream autophagy proteins. Autophagy is rapidly inhibited in both fiber types following the ingestion of a mixed meal. [ABSTRACT FROM AUTHOR]

Sprint interval training (SIT) causes fragmentation of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor 1 (RyR1), 24 h post‐exercise, potentially signalling mitochondrial biogenesis by increasing cytosolic [Ca2+]. Yet, the time course and skeletal muscle fibre type‐specific patterns of RyR1 fragmentation following a session of SIT remain unknown. Ten participants (n = 4 females; n = 6 males) performed a session of SIT (6 × 30 s 'all‐out' with 4.5 min rest after each sprint) with vastus lateralis muscle biopsy samples collected before and 3, 6 and 24 h after exercise. In whole muscle, full‐length RyR1 protein content was significantly reduced 6 h (mean (SD); −38 (38)%; P < 0.05) and 24 h post‐SIT (−30 (48)%; P < 0.05) compared to pre‐exercise. Examining each participant's largest response in pooled samples, full‐length RyR1 protein content was reduced in type II (−26 (30)%; P < 0.05) but not type I fibres (−11 (40)%; P > 0.05). Three hours post‐SIT, there was also a decrease in sarco(endo)plasmic reticulum Ca2+ ATPase 1 in type II fibres (−23 (17)%; P < 0.05) and sarco(endo)plasmic reticulum Ca2+ ATPase 2a in type I fibres (−19 (21)%; P < 0.05), despite no time effect for either protein in whole muscle samples (P > 0.05). PGC1A mRNA content was elevated 3 and 6 h post‐SIT (5.3‐ and 3.7‐fold change from pre, respectively; P < 0.05 for both), but peak PGC1A mRNA expression was not significantly correlated with peak RyR1 fragmentation (r2 = 0.10; P > 0.05). In summary, altered Ca2+‐handling protein expression, which occurs primarily in type II muscle fibres, may influence signals for mitochondrial biogenesis as early as 3–6 h post‐SIT in humans. Key points: Sprint interval training (SIT) has been shown to cause fragmentation of the sarcoplasmic reticulum calcium‐release channel, ryanodine receptor 1 (RyR1), 24 h post‐exercise, which may act as a signal for mitochondrial biogenesis.In this study, the time course was examined of RyR1 fragmentation in human whole muscle and pooled type I and type II skeletal muscle fibres following a single session of SIT.Full‐length RyR1 protein content was significantly lower than pre‐exercise by 6 h post‐SIT in whole muscle, and fragmentation was detectable in type II but not type I fibres, though to a lesser extent than in whole muscle.The peak in PGC1A mRNA expression occurred earlier than RyR1 fragmentation.The increased temporal resolution and fibre type‐specific responses for RyR1 fragmentation provide insights into its importance to mitochondrial biogenesis in humans. [ABSTRACT FROM AUTHOR]

Major stores of glucose are found as glycogen in skeletal muscle and liver. Skeletal muscle is a heterogenous tissue, with cellular metabolic and contractile distinctions dependent on whether the cell (fibre) is slow-twitch (Type I) or fast-twitch (Type II). We hypothesised that proteins important for glycogen metabolism would be differentially abundant between these diverse fibres. We further hypothesised that the cellular location of these proteins would be different in muscle samples between control (CON) and individuals with type 2 diabetes (T2D). We dissected individual muscle fibre segments from vastus lateralis skeletal muscle biopsy samples from CON and T2D and used cell-type-specific approaches to address muscle heterogeneity. We measured glycogen and glycogen-related proteins by immunoblotting techniques. A lower proportion of Type I fibres was found in muscle in T2D compared with CON. AMPK-β2, glycogen branching enzyme (GBE), glycogen debranching enzyme (GDE), and glycogen phosphorylase (GP) were differentially localized between fibre types and in fibres from CON and T2D individuals. A key novel finding was that the majority of glycogen is loosely bound or cytosolic in location in human skeletal muscle. The proportion of this diffusible pool of glycogen was significantly lower in Type I fibres in T2D compared to CON. A hyperinsulinaemic, euglycaemic clamp in people with type 2 diabetes had no effect on the proportion of diffusible glycogen. We identify cell-type as an important consideration when assessing glycogen metabolism in muscle. Our findings demonstrate varying glucose handling abilities in specific muscle fibre types in type 2 diabetes. A model is presented to provide an overview of the cell-specific differences in glycogen metabolism in type 2 diabetes. [ABSTRACT FROM AUTHOR]

Aim: Subcellular fractionation is often used to determine the subcellular localization of proteins, including whether a protein translocates to the nucleus in response to a given stimulus. Examining nuclear proteins in skeletal muscle is difficult because myonuclear proteins are challenging to isolate unless harsh treatments are used. This study aimed to determine the most effective method for isolating and preserving proteins in their native state in skeletal muscle. Methods: We compared the ability of detergents, commercially available kit‐based and K+‐based physiological methodologies for isolating myonuclear proteins from resting samples of human muscle by determining the presence of marker proteins for each fraction by western blot analyses. Results: We found that following the initial pelleting of nuclei, treatment with 1% Triton‐X 100, 1% CHAPS or 0.5% Na‐deoxycholate under various ionic conditions resulted in the nuclear proteins being either resistant to isolation or the proteins present behaving aberrantly. The nuclear proteins in brain tissue were also resistant to 1% Triton‐X 100 isolation. Here, we demonstrate aberrant behaviour and erroneous localization of proteins using the kit‐based method. The aberrant behaviour was the activation of Ca2+‐dependent protease calpain‐3, and the erroneous localization was the presence of calpain‐3 and troponin I in the nuclear fraction. Conclusion: Our findings indicate that it may not be possible to reliably determine the translocation of proteins between subcellular locations and the nucleus using subcellular fractionation techniques. This study highlights the importance of validating subcellular fractionation methodologies using several subcellular‐specific markers and solutions that are physiologically relevant to the intracellular milieu. [ABSTRACT FROM AUTHOR]

There are limited and equivocal data regarding potential fiber type-specific differences in the human skeletal muscle response to sprint interval training (SIT), including how this compares with moderate-intensity continuous training (MICT). We examined mixed-muscle and fiber type-specific responses to a single session (study 1) and to 12 wk (study 2) of MICT and SIT using Western blot analysis. MICT consisted of 45 min of cycling at ∼70% of maximal heart rate, and SIT involved 3 × 20-s "all-out" sprints interspersed with 2 min of recovery. Changes in signaling proteins involved in mitochondrial biogenesis in mixed-muscle and pooled fiber samples were similar after acute MICT and SIT. This included increases in the ratios of phosphorylated to total acetyl-CoA carboxylase and p38 mitogen-activated protein kinase protein content (main effects, P < 0.05). Following training, mitochondrial content markers including the protein content of cytochrome c oxidase subunit IV and NADH:ubiquinone oxidoreductase subunit A9 were increased similarly in mixed-muscle and type IIa fibers (main effects, P < 0.05). In contrast, only MICT increased these markers of mitochondrial content in type I fibers (interactions, P < 0.05). MICT and SIT also similarly increased the content of mitochondrial fusion proteins optic atrophy 1 (OPA1) and mitofusin 2 in mixed-muscle, and OPA1 in pooled fiber samples (main effects, P < 0.02). In summary, acute MICT and SIT elicited similar fiber type-specific responses of signaling proteins involved in mitochondrial biogenesis, whereas 12 wk of training revealed differential responses of mitochondrial content markers in type I but not type IIa fibers.NEW & NOTEWORTHY We examined mixed-muscle and fiber type-specific responses to a single session and to 12 wk of moderate-intensity continuous training (MICT) and sprint interval training (SIT) in humans. Both interventions elicited generally similar responses, although the training-induced increases in type I fiber-specific markers of mitochondrial content were greater in MICT than in SIT. These findings advance our understanding of the potential role of fiber type-specific changes in determining the human skeletal muscle response to intermittent and continuous exercise. [ABSTRACT FROM AUTHOR]

This study reports that in rat skeletal muscle the proteins specifically responsible for mitochondrial dynamics, mitofusin-2 (MFN2) and mitochondrial dynamics protein 49 (MiD49), are higher (p < 0.05) in oxidative soleus (SOL) muscle compared with predominantly glycolytic extensor digitorum longus (EDL) muscle, but not seen for optic atrophy 1 (OPA1; p = 0.06). Markers of mitochondrial content, complex I component, NADH:Ubiquinone oxidoreductase subunit A9 (NDUFA9) and complex IV protein, cytochrome C oxidase subunit IV (COXIV; p < 0.05) were also higher in SOL compared with EDL muscle; however, there was no difference in mitochondrial content between muscles, as measured using a citrate synthase assay (p > 0.05). SOL and EDL muscles were compared between age-matched sedentary rats that were housed individually with (RUN) or without (SED) free-access to a running wheel for 12 weeks and showed no change in mitochondrial content, as examined by the abundances of NDUFA9 and COXIV proteins, as well as citrate synthase activity, in either muscle (p > 0.05). Compared to SED animals, MiD49 and OPA1 were not different in either EDL or SOL muscles, and MFN2 was higher in SOL muscles from RUN rats (p < 0.05). Overall, these findings reveal that voluntary wheel running is an insufficient stimulus to result in a significantly higher abundance of most markers of mitochondrial content or dynamics, and it is likely that a greater stimulus, such as either adding resistance to the wheel or an increase in running volume by using a treadmill, is required for mitochondrial adaptation in rat skeletal muscle. [ABSTRACT FROM AUTHOR]

Little is known about the molecular responses to power resistance exercise that lead to skeletal muscle remodeling and enhanced athletic performance. We assessed the expression of titin-linked putative mechanosensing proteins implicated in muscle remodeling: muscle ankyrin repeat proteins (Ankrd 1, Ankrd 2, and Ankrd 23), muscle-LIM proteins (MLPs), muscle RING-finger protein-1 (MuRF-1), and associated myogenic proteins (MyoD1, myogenin, and myostatin) in skeletal muscle in response to power resistance exercise with or without a postexercise meal, in fed, resistance-trained men. A muscle sample was obtained from the vastus lateralis of seven healthy men on separate days, 3 h after 90 min of rest (Rest) or power resistance exercise with (Ex þ Meal) or without (Ex) a postexercise meal to quantify mRNA and protein levels. The levels of phosphorylated HSP27 (pHSP27-Ser15) and cytoskeletal proteins in muscle and creatine kinase activity in serum were also assessed. The exercise increased (P = 0.05) pHSP27-Ser15 (-6-fold) and creatine kinase (-50%), whereas cytoskeletal protein levels were unchanged (P > 0.05). Ankrd 1 (-15-fold) and MLP (-2-fold) mRNA increased, whereas Ankrd 2, Ankrd 23, MuRF-1, MyoD1, and myostatin mRNA were unchanged. Ankrd 1 (-3-fold, Ex) and MLPb (-20-fold, Ex þ Meal) protein increased, but MLPa, Ankrd 2, Ankrd 23, and the myogenic proteins were unchanged. The postexercise meal did not affect the responses observed. Power resistance exercise, as performed in practice, induced subtle early responses in the expression of MLP and Ankrd 1 yet had little effect on the other proteins investigated. These findings suggest possible roles for MLP and Ankrd 1 in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise. [ABSTRACT FROM AUTHOR]

Skeletal muscle is one of the largest functional tissues in the human body; it is highly plastic and responds dramatically to anabolic and catabolic stimuli, including weight training and malnutrition, respectively. Excessive loss of muscle mass, or atrophy, is a common symptom of many disease states with severe impacts on prognosis and quality of life. TNF-like weak inducer of apoptosis (TWEAK) and its cognate receptor, fibroblast growth factor-inducible 14 (Fn14) are an emerging cytokine signaling pathway in the pathogenesis of muscle atrophy. Upregulation of TWEAK and Fn14 has been described in a number of atrophic and injured muscle states; however, it remains unclear whether they are contributing to the degenerative or regenerative aspect of muscle insults. The current review focuses on the expression and apparent downstream outcomes of both TWEAK and Fn14 in a range of catabolic and anabolic muscle models. Apparent changes in the signaling outcomes of TWEAK-Fn14 activation dependent on the relative expression of both the ligand and the receptor are discussed as a potential source of divergent TWEAK-Fn14 downstream effects. This review proposes both a physiological and pathological model of TWEAK-Fn14 signaling. Further research is needed on the switch between these states to develop therapeutic interventions for this pathway. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs) are important regulators of cellular homeostasis and exert their effect by directly controlling protein expression. We have previously reported an age-dependent negative association between microRNA-99b (miR-99b-5p) expression and muscle protein synthesis in human muscle in vivo. Here we investigated the role of miR-99b-5p as a potential negative regulator of protein synthesis via inhibition of mammalian target for rapamycin (MTOR) signaling in human primary myocytes. Overexpressing miR-99b-5p in human primary myotubes from young and old subjects significantly decreased protein synthesis with no effect of donor age. A binding interaction between miR-99b-5p and its putative binding site within the MTOR 3=-untranslated region (UTR) was confirmed in C2C12 myoblasts. The observed decline in protein synthesis was, however, not associated with a suppression of the MTOR protein but of its regulatory associated protein of mTOR complex 1 (RPTOR). These results demonstrate that modulating the expression levels of a miRNA can regulate protein synthesis in human muscle cells and provide a potential mechanism for muscle wasting in vivo. [ABSTRACT FROM AUTHOR]

Elevated MMP2 abundance and activity in mdx mice are alleviated by prenatal taurine supplementation. Am J Physiol Cell Physiol 318: C1083-C1091, 2020. First published March 25, 2020; doi:10.1152/ajpcell.00437. 2019.--Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder that leads to early death. The mdx mouse is a naturally occurring mutant model for DMD. It lacks dystrophin and displays peak muscle cell necrosis at ~28 days (D28), but in contrast to DMD, mdx mice experience muscle regeneration by D70. We hypothesized that matrix metalloproteinase-2 (MMP2) and/or MMP9 play key roles in the degeneration/regeneration phases in mdx mice. MMP2 abundance in muscle homogenates, measured by calibrated Western blotting, and activity, measured by zymogram, were lower at D70 compared with D28 in both mdx and wild-type (WT) mice. Importantly, MMP2 abundance was higher in both D28 and D70 mdx mice than in age-matched WT mice. The higher MMP2 abundance was not due to infiltrating macrophages, because MMP2 content was still higher in isolated muscle fibers where most macrophages had been removed. Prenatal supplementation with the amino acid taurine, which improved muscle strength in D28 mdx mice, produced approximately twofold lower MMP2 activity, indicating that increased MMP2 abundance is not required when muscle damage is attenuated. There was no difference in MMP9 abundance between age-matched WT and mdx mice (P > 0.05). WT mice displayed decreased MMP9 abundance as they aged. While MMP9 may have a role during age-related skeletal muscle growth, it does not appear essential for degeneration/regeneration cycles in the mdx mouse. Our findings indicate that MMP2 plays a more active role than MMP9 in the degenerative phases of muscle fibers in D28 mdx mice. [ABSTRACT FROM AUTHOR]

Intrauterine growth restriction (IUGR) affects vascular reactivity in older rats, but at present the causative factors for this change are unknown. Therefore, we investigated downstream events associated with vascular reactivity, specifically, Ca2+-regulated force production and shifts in contractile protein content. The mesenteric artery from male and female 1-year-old Wistar-Kyoto rats was examined using two distinct experimental growth restriction models. Uterine ligation surgery restriction or a sham surgery was conducted at day 18 of pregnancy, whilst a food restriction diet (40% control diet) began on gestational day 15. Extracellular vascular reactivity was studied using intact mesenteric arteries, which were subsequently chemically permeabilized using 50 μM β-escin to examine Ca2+-activated force. Peak contractile responses to a K+-induced depolarization and phenylephrine were significantly elevated due to an increase in maximum Ca2+-activated force in the male surgery restricted group. No changes in contractile forces were reported between female experimental groups. Sections of mesenteric artery were examined using western blotting, revealing IUGR increased the relative abundance of the voltage-gated Ca2+ channel, inositol-1,4,5-trisphosphate receptor and myosin light chain kinase, in both male growth restricted groups, whereas no changes were seen in females. These findings demonstrate for the first time in 1-year-old rats that changes in vascular reactivity due to IUGR are caused by a change in Ca2+-activated force and shifts in important contractile protein content. These changes affect the Wistar-Kyoto rat in a sex-specific and maternal insult-dependent manner. [ABSTRACT FROM AUTHOR]

After this article [[1]] was published, concerns were raised by the corresponding author that demarcation lines to indicate that lanes 7 and 8 in the myosin heavy chain SDS-PAGE gels in Fig 7A and 7B are noncontiguous were omitted. Graph: Fig 7 Myofibre myosin heavy chain (MHC) composition of soleus and EDL muscle from WT and BLAJ mice aged 10 months (n = 6).Representative MHC gels (A, B) for soleus and EDL muscles, loaded with a pooled sample (Mixed) used to generate the Calibration curve (see Methods), with (C, D) showing percentage of different MHC in soleus and EDL muscles. [Extracted from the article]

A substantial intracellular localization of matrix metalloproteinase 2 (MMP2) has been reported in cardiomyocytes, where it plays a role in the degradation of the contractile apparatus following ischemia-reperfusion injury. Whether MMP2 may have a similar function in skeletal muscle is unknown. This study determined that the absolute amount of MMP2 is similar in rat skeletal and cardiac muscle and human muscle (~10 –18 nmol/kg muscle wet wt) but is ~50- to 100-fold less than the amount of calpain-1. We compared mechanically skinned muscle fibers, where the extracellular matrix (ECM) is completely removed, with intact fiber segments and found that ~30% of total MMP2 was associated with the ECM, whereas ~70% was inside the muscle fibers. Concordant with whole muscle fractionation, further separation of skinned fiber segments into cytosolic, membranous, and cytoskeletal and nuclear compartments indicated that ~57% of the intracellular MMP2 was freely diffusible, ~6% was associated with the membrane, and ~37% was bound within the fiber. Under native zymography conditions, only 10% of MMP2 became active upon prolonged (17 h) exposure to 20 M Ca2+, a concentration that would fully activate calpain-1 in seconds to minutes; full activation of MMP2 would require ~1 mM Ca2+. Given the prevalence of intracellular MMP2 in skeletal muscle, it is necessary to investigate its function using physiological conditions, including isolation of any potential functional relevance of MMP2 from that of the abundant protease calpain-1. [ABSTRACT FROM AUTHOR]

Dysferlinopathies are a form of muscular dystrophy caused by gene mutations resulting in deficiency of the protein dysferlin. Symptoms manifest later in life in a muscle specific manner, although the pathomechanism is not well understood. This study compared the impact of dysferlin-deficiency on in vivo and ex vivo muscle function, and myofibre type composition in slow (soleus) and fast type (extensor digitorum longus; EDL) muscles using male dysferlin-deficient (dysf-/-) BLAJ mice aged 10 months, compared with wild type (WT) C57Bl/6J mice. There was a striking increase in muscle mass of BLAJ soleus (+25%) (p<0.001), with no strain differences in EDL mass, compared with WT. In vivo measures of forelimb grip strength and wheel running capacity showed no strain differences. Ex vivo measures showed the BLAJ soleus had faster twitch contraction (-21%) and relaxation (-20%) times, and delayed post fatigue recovery (ps<0.05); whereas the BLAJ EDL had a slower relaxation time (+11%) and higher maximum rate of force production (+25%) (ps<0.05). Similar proportions of MHC isoforms were evident in the soleus muscles of both strains (ps>0.05); however, for the BLAJ EDL, there was an increased proportion of type IIx MHC isoform (+5.5%) and decreased type IIb isoform (-5.5%) (ps<0.01). This identification of novel differences in the impact of dysferlin-deficiency on slow and fast twitch muscles emphasises the importance of evaluating myofibre type specific effects to provide crucial insight into the mechanisms responsible for loss of function in dysferlinopathies; this is critical for the development of targeted future clinical therapies. [ABSTRACT FROM AUTHOR]

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca2+-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca2+-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca2+ sensor of Ca2+ dynamics in the junctional space. The sensor was used to assess the basal Ca2+-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca2+ handling under defined ionic conditions. [ABSTRACT FROM AUTHOR]

Effects of regular use of cold-water immersion (CWI) on fiber type-specific adaptations in muscle K+ transport proteins to intense training, along with their relationship to changes in mRNA levels after the first training session, were investigated in humans. Nineteen recreationally active men (24 ± 6 yr, 79.5 ± 10.8 kg, 44.6 ± 5.8 ml·kg-1·min-1) completed six weeks of sprint-interval cycling, either without (passive rest; CON) or with training sessions followed by CWI (15 min at 10°C; COLD). Muscle biopsies were obtained before and after training to determine abundance of Na+, K+-ATPase isoforms (α1-3, β1-3) and phospholemman (FXYD1) and after recovery treatments (+0 h and +3 h) on the first day of training to measure mRNA content. Training increased ( P < 0.05) the abundance of α1 and β3 in both fiber types and β1 in type-II fibers and decreased FXYD1 in type-I fibers, whereas α2 and α3 abundance was not altered by training ( P > 0.05). CWI after each session did not influence responses to training ( P > 0.05). However, α2 mRNA increased after the first session in COLD (+0 h, P < 0.05) but not in CON ( P > 0.05). In both conditions, α1 and β3 mRNA increased (+3 h; P < 0.05) and β2 mRNA decreased (+3 h; P < 0.05), whereas α3, β1, and FXYD1 mRNA remained unchanged ( P > 0.05) after the first session. In summary, Na+,K+-ATPase isoforms are differently regulated in type I and II muscle fibers by sprint-interval training in humans, which, for most isoforms, do not associate with changes in mRNA levels after the first training session. CWI neither impairs nor improves protein adaptations to intense training of importance for muscle K+ regulation. NEW & NOTEWORTHY Although cold-water immersion (CWI) after training and competition has become a routine for many athletes, limited published evidence exists regarding its impact on training adaptation. Here, we show that CWI can be performed regularly without impairing training-induced adaptations at the fiber-type level important for muscle K+ handling. Furthermore, sprint-interval training invoked fiber type-specific adaptations in K+ transport proteins, which may explain the dissociated responses of whole-muscle protein levels and K+ transport function to training previously reported. [ABSTRACT FROM AUTHOR]

Cl- channel protein 1 (ClC-1) may be important for excitability and contractility in skeletal muscle, but ClC-1 abundance has not been examined in human muscle. The aim of the present study was to examine ClC-1 abundance in human skeletal muscle, including fiber type specific differences and the effect of exercise training. A commercially available antibody was tested with positive and negative control tissue, and it recognized specifically ClC-1 in the range from 100 to 150 kDa. Abundance of ClC-1 was 38% higher (P 0.01) in fast twitch Type IIa muscle fibers than in slow twitch Type I. Muscle ClC-1 abundance did not change with 4 wk of training consisting of 30 min cycling at 85% of maximal heart rate (HRmax) and 3 x 30-s all out sprints or during a 7-wk training period with 10 –12 x 30 s uphill cycling and 4 –5 x ~4 min cycling at 90%–95% of HRmax. ClC-1 abundance correlated negatively (P < 0.01) with maximal oxygen consumption (r = – 0.552) and incremental exercise performance (r = – 0.546). In addition, trained cyclists had lower (P 0.01) ClC-1 abundance than lesser trained individuals. The present observations indicate that a low abundance of muscle ClC-1 may be beneficial for exercise performance, but the role of abundance and regulation of ClC-1 in skeletal muscle of humans with respect to exercise performance and trainability need to be elucidated. NEW & NOTEWORTHY Abundance of the Cl channel protein 1 (ClC-1) chloride channel may be important for excitability and contractility in human skeletal muscle and may therefore have implications for fatigue development. In this study, we confirmed ClC-1 specificity for a commercially available antibody, and this study is first to our knowledge to determine ClC-1 protein abundance in human muscle by Western blotting. We observed that abundance of ClC-1 was higher in fast compared with slow twitch fibers and lower in trained individuals than in recreationally active. [ABSTRACT FROM AUTHOR]

Laboratory rats are sedentary if housed in conditions where activity is limited. Changes in muscle characteristics with chronic inactivity were investigated by comparing sedentary rats with rats undertaking voluntary wheel running for either 6 or 12 weeks. EDL (type II fibers) and soleus (SOL) muscles (predominantly type I fibers) were examined. When measured within 1-2 h post-running, calcium sensitivity of the contractile apparatus was increased, but only in type II fibers. This increase disappeared when fibers were treated with DTT, indicative of oxidative regulation of the contractile apparatus, and was absent in fibers from rats that had ceased running 24 h prior to experiments. Specific force production was ~ 10 to 25% lower in muscle fibers of sedentary compared to active rats, and excitability of skinned fibers was decreased. Muscle glycogen content was ~ 30% lower and glycogen synthase content ~ 50% higher in SOL of sedentary rats, and in EDL glycogenin was 30% lower. Na+, K+-ATPase α1 subunit density was ~ 20% lower in both EDL and SOL in sedentary rats, and GAPDH content in SOL ~ 35% higher. There were no changes in content of the calcium handling proteins calsequestrin and SERCA, but the content of CSQ-like protein was increased in active rats (by ~ 20% in EDL and 60% in SOL). These findings show that voluntary exercise elicits an acute oxidation-induced increase in Ca2+ sensitivity in type II fibers, and also that there are substantial changes in skeletal muscle characteristics and biochemical processes in sedentary rats. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is a pathological feature of Duchenne muscular dystrophy (DMD), a debilitating and fatal neuromuscular disorder characterized by progressive muscle wasting and weakness. Mitochondria are a source of cellular ATP involved in Ca2 regulation and apoptotic signaling. Ameliorating aberrant mitochondrial function has therapeutic potential for reducing DMD disease severity. The dystrophic mdx mouse exhibits peak muscle damage at 21–28 days, which stabilizes after 8 wk. The amino acid taurine is implicated in mitochondrial health and function, with endogenous concentrations low when measured during the cycle of peak muscle damage in mdx mice. Using whole soleus and extensor digitorum longus (EDL) muscle homogenates from 28- and 70-day mdx mice, we found that there was no change in native state mitochondrial complexes using blue native-PAGE. NADH:ubiquinone oxidotreductase subunit-A9 (NDUFA9) protein abundance was lower in soleus muscle of 28- and 70-day mdx mice and EDL muscle of 70-day mdx mice compared with same muscles in WT (C57/BL10ScSn) animals. There were agedependent increases in both NDUFA9 protein abundance and citrate synthase activity in soleus muscles of mdx and wild-type mice. There was no change in abundances of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49). Taurine administration essentially did not affect any measurements of mitochondria. Collectively, these findings suggest mitochondrial content and dynamics are not reduced in the mdx mouse regardless of disease severity. We also elucidate that taurine affords no significant benefit to mitochondrial content or dynamics in the mdx mouse at either 28 or 70 days. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) patients and the dystrophic mdx mouse have an elevated demand for ATP requiring processes, including Ca2+ regulation and skeletal muscle regeneration. As a key substrate for cellular ATP production, altered glycogen metabolism may contribute significantly to dystrophic pathology and explain reports of mild glucose intolerance. We compare the soleus and extensor digitorum longus (EDL) muscles of the mdx mouse during active muscle necrosis (at 28 days) and at 70 days where pathology is stable. We further investigate the impact of taurine (tau) on dystrophic glycogen metabolism to identify if the benefit seen with tau in a previous study (Barker et al. 2017) was in part owed to altered glycogen handling. The soleus muscle of 28- and 70-day-old mdx mice had elevated glucose transporter type 4 (GLUT4), glycogenin protein abundances and glycogen content compared to WT (C57BL10/ScSn) controls. Mdx tau mice exhibited modestly reduced glycogen compared to their respective mdx group. The EDL muscle of 28 days mdx tau mice had a ~70% increase in glycogenin protein abundance compared to the mdx but 50% less glycogen content. A twofold greater phosphorylated glycogen synthase (p-GS) and glycogen phosphorylase (p-GP) protein abundance was observed in the 70-day-old mdx soleus muscle than in the 28-day-old mdx soleus muscle. Glycogen debranching enzyme (GDE) protein abundance was elevated in both 28- and 70-day-old mdx soleus muscles compared to WT controls. We identified an increase in proteins associated with glucose uptake and utilization specific to the predominantly slow-twitch soleus muscle of mdx mice regardless of age and that taurine affords no obvious benefit to glycogen metabolism in the mdx mouse. [ABSTRACT FROM AUTHOR]

Summary: The contractile properties of vastus lateralis muscle fibres were examined in prostate cancer (PrCa) patients undergoing androgen deprivation therapy (ADT) and in age‐ and activity‐matched healthy male subjects (Control). Mechanically‐skinned muscle fibres were exposed to a sequence of heavily Ca2+‐buffered solutions at progressively higher free [Ca2+] to determine their force‐Ca2+ relationship. Ca2+‐sensitivity was decreased in both type I and type II muscle fibres of ADT subjects relative to Controls (by −0.05 and −0.04 pCa units, respectively, P < .02), and specific force was around 13% lower in type I fibres of ADT subjects than in Controls (P = .02), whereas there was no significant difference in type II fibres. Treatment with the reducing agent dithiothreitol slightly increased specific force in type I and type II fibres of ADT subjects (by ~2%‐3%, P < .05) but not in Controls. Pure type IIx fibres were found frequently in muscle from ADT subjects but not in Controls, and the overall percentage of myosin heavy chain IIx in muscle samples was 2.5 times higher in ADT subjects (P < .01). The findings suggest that testosterone suppression can negatively impact the contractile properties by (i) reducing Ca2+‐sensitivity in both type I and type II fibres and (ii) reducing maximum specific force in type I fibres. [ABSTRACT FROM AUTHOR]

Laboratory rats are considered mature at 3 months despite that musculoskeletal growth is still occurring. Changes in muscle physiological and biochemical characteristics during development from 3 months, however, are not well understood. Whole muscles and single skinned fibres from fast-twitch extensor digitorum longus (EDL) and predominantly slow-twitch soleus (SOL) muscles were examined from male Sprague-Dawley rats (3, 6, 9, 12 months). Ca2+ sensitivity of contractile apparatus decreased with age in both fast- (~ 0.04 pCa units) and slow-twitch (~ 0.07 pCa units) muscle fibres, and specific force increased (by ~ 50% and ~ 25%, respectively). Myosin heavy chain composition of EDL and SOL muscles altered to a small extent with age (decrease in MHCIIa proportion after 3 months). Glycogen content increased with age (~ 80% in EDL and 25% in SOL) and GLUT4 protein density decreased (~ 35 and 20%, respectively), whereas the glycogen-related enzymes were little changed. GAPDH protein content was relatively constant in both muscle types, but COXIV protein decreased ~ 40% in SOL muscle. Calsequestrin (CSQ) and SERCA densities remained relatively constant with age, whereas there was a progressive ~ 2-3 fold increase in CSQ-like proteins, though their role and importance remain unclear. There was also ~ 40% decrease in the density of the Na+, K+-ATPase (NKA) α1 subunit in EDL and the α2 subunit in SOL. These findings emphasise there are substantial changes in skeletal muscle function and the density of key proteins during early to mid-adulthood in rats, which need to be considered in the design and interpretation of experiments. [ABSTRACT FROM AUTHOR]

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule. [ABSTRACT FROM AUTHOR]

Key points Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres., These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE)., Following 12 weeks of high-intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres., We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria., Abstract Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High-intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin-2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70-100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults. [ABSTRACT FROM AUTHOR]

Key points A classic unresolved issue in human integrative physiology involves the role of exercise intensity, duration and volume in regulating skeletal muscle adaptations to training., We employed counterweighted single-leg cycling as a unique within-subject model to investigate the role of exercise intensity in promoting training-induced increases in skeletal muscle mitochondrial content., Six sessions of high-intensity interval training performed over 2 weeks elicited greater increases in citrate synthase maximal activity and mitochondrial respiration compared to moderate-intensity continuous training matched for total work and session duration., These data suggest that exercise intensity, and/or the pattern of contraction, is an important determinant of exercise-induced skeletal muscle remodelling in humans., Abstract We employed counterweighted single-leg cycling as a unique model to investigate the role of exercise intensity in human skeletal muscle remodelling. Ten young active men performed unilateral graded-exercise tests to measure single-leg [ABSTRACT FROM AUTHOR]

Young adults typically adapt to intense exercise training with an increased skeletal muscle Na+,K+-ATPase (NKA) content, concomitant with reduced extracellular potassium concentration [K+] during exercise and enhanced exercise performance. Whether these changes with longitudinal training occur in older adults is unknown and was investigated here. Fifteen older adults (69.4 ± 3.5 years, mean ± SD) were randomized to either 12 weeks of intense interval training (4 × 4 min at 90-95% peak heart rate), 3 days/week (IIT, n = 8); or no exercise controls (n = 7). Before and after training, participants completed an incremental cycle ergometer exercise test until a rating of perceived exertion of 17 (very hard) on a 20-point scale was attained, with measures of antecubital venous [K+]v. Participants underwent a resting muscle biopsy prior to and at 48-72 h following the final training session. After IIT, the peak exercise work rate (25%), oxygen uptake (16%) and heart rate (6%) were increased (P < 0.05). After IIT, the peak exercise plasma [K+]v tended to rise (P = 0.07), while the rise in plasma [K+]v relative to work performed (nmol.L -1.J -1) was unchanged. Muscle NKA content increased by 11% after IIT (P < 0.05). Single fiber measurements, increased in NKA α2 isoform in Type II fibers after IIT (30%, P < 0.05), with no changes to the other isoforms in single fibers or homogenate. Thus, intense exercise training in older adults induced an upregulation of muscle NKA, with a fiber-specific increase in NKA α2 abundance in Type II fibers, coincident with increased muscle NKA content and enhanced exercise performance. [ABSTRACT FROM AUTHOR]

High-force eccentric exercise results in sustained increases in cytoplasmic Ca2+ levels ([Ca2+]cyto), which can cause damage to the muscle. Here we report that a heavy-load strength training bout greatly alters the structure of the membrane network inside the fibres, the tubular (t-) system, causing the loss of its predominantly transverse organization and an increase in vacuolation of its longitudinal tubules across adjacent sarcomeres. The transverse tubules and vacuoles displayed distinct Ca2+-handling properties. Both t-system components could take up Ca2+ from the cytoplasm but only transverse tubules supported store-operated Ca2+ entry. The retention of significant amounts of Ca2+ within vacuoles provides an effective mechanism to reduce the total content of Ca2+ within the fibre cytoplasm. We propose this ability can reduce or limit resistance exercise-induced, Ca2+-dependent damage to the fibre by the reduction of [Ca2+]cyto to help maintain fibre viability during the period associated with delayed onset muscle soreness. Heavy-load eccentric exercise causes an increase in cytoplasmic Ca2+ that can damage muscles. Here the authors show that the t-tubule system remodels into vacuoles that can sequester calcium from the cytoplasm and are not responsive to store-operated Ca2+ entry, thereby potentially protecting muscles against elevated [Ca2+]. [ABSTRACT FROM AUTHOR]

Physical training increases skeletal muscle Na+,K+-ATPase content (NKA) and improves exercise performance, but the effects of inactivity per se on NKA content and isoform abundance in human muscle are unknown. We investigated the effects of 23-day unilateral lower limb suspension (ULLS) and subsequent 4-wk resistance training (RT) on muscle function and NKA in 6 healthy adults, measuring quadriceps muscle peak torque; fatigue and venous [K+] during intense one-legged cycling exercise; and skeletal muscle NKA content ([³H]ouabain binding) and NKA isoform abundances (immunoblotting) in muscle homogenates (α1-3, β1-2) and in single fibers (α1-3, β1). In the unloaded leg after ULLS, quadriceps peak torque and cycling time to fatigue declined by 22 and 23%, respectively, which were restored with RT. Whole muscle NKA content and homogenate NKA α1-3 and β1-2 isoform abundances were unchanged with ULLS or RT. However, in single muscle fibers, NKA α3 in type I (-66%, P = 0.006) and β1 in type II fibers (-40%, P = 0.016) decreased after ULLS, with other NKA isoforms unchanged. After RT, NKA α1 (79%, P = 0.004) and β1 (35%, P = 0.01) increased in type II fibers, while α2 (76%, P = 0.028) and α3 (142%, P = 0.004) increased in type I fibers compared with post-ULLS. Despite considerably impaired muscle function and earlier fatigue onset, muscle NKA content and homogenate α1 and α2 abundances were unchanged, thus being resilient to inactivity induced by ULLS. Nonetheless, fiber type-specific downregulation with inactivity and upregulation with RT of several NKA isoforms indicate complex regulation of muscle NKA expression in humans. [ABSTRACT FROM AUTHOR]

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (⩽100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into Ca2+ oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro. [ABSTRACT FROM AUTHOR]

The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and β-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, 60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total β2-AMPK that was phosphorylated in whole muscle homogenates measured by immuno-precipitation. These findings suggest that, in rat skeletal muscle, β2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of β2-AMPK in skeletal muscle. [ABSTRACT FROM AUTHOR]

Cytoplasmic lipid droplets provide a reservoir for triglyceride storage and are a central hub for fatty acid trafficking in cells. The protein perilipin 5 (PLIN5) is highly expressed in oxidative tissues such as skeletal muscle and regulates lipid metabolism by coordinating the trafficking and the reversible interactions of effector proteins at the lipid droplet. PLIN5 may also regulate mitochondrial function, although this remains unsubstantiated. Hence, the aims of this study were to examine the role of PLIN5 in the regulation of skeletal muscle substrate metabolism during acute exercise and to determine whether PLIN5 is required for the metabolic adaptations and enhancement in exercise tolerance following endurance exercise training. Using muscle-specific Plin5 knockout mice (Plin5MKO), we show that PLIN5 is dispensable for normal substrate metabolism during exercise, as reflected by levels of blood metabolites and rates of glycogen and triglyceride depletion that were indistinguishable from control (lox/lox) mice. Plin5MKO mice exhibited a functional impairment in their response to endurance exercise training, as reflected by reduced maximal running capacity (20%) and reduced time to fatigue during prolonged submaximal exercise (15%). The reduction in exercise performance was not accompanied by alterations in carbohydrate and fatty acid metabolism during submaximal exercise. Similarly, mitochondrial capacity (mtDNA, respiratory complex proteins, citrate synthase activity) and mitochondrial function (oxygen consumption rate in muscle fiber bundles) were not different between lox/lox and Plin5MKO mice. Thus, PLIN5 is dispensable for normal substrate metabolism during exercise and is not required to promote mitochondrial biogenesis or enhance the cellular adaptations to endurance exercise training. [ABSTRACT FROM AUTHOR]

Key points Current methods do not allow a quantitative description of Ca2+ movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre., Here we present a fluorescence-based method that allows determination of the t-system [Ca2+] transients and derivation of t-system Ca2+ fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca2+-handling properties between fast- and slow-twitch fibres from rat muscle are resolved for the first time using this new technique., The method can be used to study Ca2+ handling of the t-system and allows direct comparisons of t-system Ca2+ transients and Ca2+ fluxes between groups of fibres and fibres from different strains of animals., Abstract The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca2+ gradient and exchanges Ca2+ between the extracellular and intracellular environments. Little is known of the Ca2+-handling properties of the t-system as the small Ca2+ fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca2+]t-sys, allowing confocal measurements of Ca2+-dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca2+] transients in the t-system ([Ca2+]t-sys ( t)). Furthermore, t-system Ca2+-buffering power was determined so that t-system Ca2+ fluxes could be derived from [Ca2+]t-sys ( t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca2+ induced a robust store-operated Ca2+ entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca2+]t-sys to < 0.1 m m. The rapid activation of SOCE upon Ca2+ release was consistent with the presence of STIM1L in both fibre types. Abruptly introducing internal solutions with 1 m m Mg2+ and [Ca2+]cyto (28 n m-1.3 μ m) to Ca2+-depleted fibres generated t-system Ca2+ uptake rates dependent on [Ca2+]cyto with [Ca2+]t-sys reaching final plateaus in the millimolar range. For the same [Ca2+]cyto, t-system Ca2+ fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca2+ signals indicated that both membrane compartments accumulated Ca2+ at similar rates and that SOCE was activated early during SR Ca2+ depletion. [ABSTRACT FROM AUTHOR]

Intrauterine growth restriction (IUGR), defined as a birth weight below the 10th centile, may be caused by maternal undernutrition, with evidence that IUGR offspring have an increased risk of cardiovascular disease (CVD) in adulthood. Calcium ions (Ca2+) are an integral messenger for several steps associated with excitation-contraction coupling (ECC); the cascade of events from the initiation of an action potential at the surface membrane, to contraction of the cardiomyocyte. Any changes in Ca2+ storage and release from the sarcoplasmic reticulum (SR), or sensitivity of the contractile apparatus to Ca2+ may underlie the mechanism linking IUGR to an increased risk of CVD. This study aimed to explore the effects of maternal nutrient restriction on cardiac function, including Ca2+ handling by the SR and force development by the contractile apparatus. Juvenile Long Evans hooded rats born to Control (C) and nutrient restricted (NR) dams were anaesthetized for collection of the heart at 10–12 weeks of age. Left ventricular bundles from male NR offspring displayed increased maximum Ca2+-activated force, and decreased protein content of troponin I (cTnI) compared to C males. Furthermore, male NR offspring showed a reduction in rate of rise of the caffeine-induced Ca2+ force response and a decrease in the protein content of ryanodine receptor (RYR2). These physiological and biochemical findings observed in males were not evident in female offspring. These findings illustrate a sex-specific effect of maternal NR on cardiac development, and also highlight a possible mechanism for the development of hypertension and hypertrophy in male NR offspring. [ABSTRACT FROM AUTHOR]

Inhibition of nitric oxide synthase (NOS) significantly attenuates the increase in skeletal muscle glucose uptake during contraction/exercise, and a greater attenuation is observed in individuals with Type 2 diabetes compared with healthy individuals. Therefore, NO appears to play an important role in mediating muscle glucose uptake during contraction. In this study, we investigated the involvement of neuronal NOSµ (nNOSµ), the main NOS isoform activated during contraction, on skeletal muscle glucose uptake during ex vivo contraction. Extensor digitorum longus muscles were isolated from nNOSµ and nNOSµ+/+ mice. Muscles were contracted ex vivo in a temperature-controlled (30°C) organ bath with or without the presence of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and the NOS substrate L-arginine. Glucose uptake was determined by radioactive tracers. Skeletal muscle glucose uptake increased approximately fourfold during contraction in muscles from both nNOSµ-/- and nNOSµ+/+ mice. L-NMMA significantly attenuated the increase in muscle glucose uptake during contraction in both genotypes. This attenuation was reversed by L-arginine, suggesting that L-NMMA attenuated the increase in muscle glucose uptake during contraction by inhibiting NOS and not via a nonspecific effect of the inhibitor. Low levels of NOS activity (~4%) were detected in muscles from nNOSµ-/- mice, and there was no evidence of compensation from other NOS isoform or AMP-activated protein kinase which is also involved in mediating muscle glucose uptake during contraction. These results indicate that NO regulates skeletal muscle glucose uptake during ex vivo contraction independently of nNOSµ. [ABSTRACT FROM AUTHOR]

Background Skeletal muscle undergoes significant atrophy in Type 2 diabetic patients and animal models. We aimed to determine if atrophy of Zucker rat skeletal muscle was due to the activation of intracellular damage pathways induced by excess reactive oxygen species production (specifically those associated with the peroxidation of lipid membranes) and calpain activity. 14 week old obese Zucker rats and littermate lean controls were injected with 1% Evan's Blue Dye. Animals were anaesthetised and extensor digitorum longus and soleus muscles were dissected, snap frozen and analysed for ROS-mediated F2-isoprostane production and calpain activation/autolysis. Contralateral muscles were histologically analysed for markers of muscle membrane permeability and atrophy. Results Muscle mass was lower in extensor digitorum longus and soleus of obese compared with lean animals, concomitant with reduced fibre area. Muscles from obese rats had a higher proportional area of Evan's Blue Dye fluorescence, albeit this was localised to the interstitium/external sarcolemma. There were no differences in F2-isoprostane production when expressed relative to arachidonic acid content, which was lower in the obese EDL and soleus muscles. There were no differences in the activation of either μ-calpain or calpain-3. Conclusions This study highlights that atrophy of Zucker rat skeletal muscle is not related to sarcolemmal damage, sustained hyperactivation of the calpain proteases or excessive lipid peroxidation. As such, establishing the correct pathways involved in atrophy is highly important so as to develop more specific treatment options that target the underlying cause. This study has eliminated two of the potential pathways theorised to be responsible. [ABSTRACT FROM AUTHOR]

Through its upregulation and/or translocation, heat shock protein 72 (HSP72) is involved in protection and repair of key proteins after physiological stress. In human skeletal muscle we investigated HSP72 protein after eccentric (ECC1) and concentric (CONC) exercise and repeated eccentric exercise (ECC2; 8 wk later) and whether it translocated from its normal cytosolic location to membranes/myofibrils. HSP72 protein increased ~ 2-fold 24 h after ECC1, with no apparent change after CONC or ECC2. In resting (nonstressed) human skeletal muscle the total pool of HSP72 protein was present almost exclusively in the cytosolic fraction, and after each exercise protocol the distribution of HSP72 protein remained unaltered. Overall, the amount of HSP72 protein in the cytosol increased 24 h after ECC1, matching the fold increase that was measured in total HSP72 protein. To better ascertain the capabilities and limitations of HSP72, using quantitative Western blotting we determined the HSP72 protein content to be 11.4 p,mol/kg wet weight in resting human vastus lateralis muscle, which is comprised of Type I (slow-twitch) and Type II (fast-twitch) fibers. HSP72 protein content was similar in individual Type I or II fiber segments. After physiological stress, HSP72 content can increase and, although the functional consequences of increased amounts of HSP72 protein are poorly understood, it has been shown to bind to and protect protein pumps like SERCA and Na+-K+-ATPase. Given no translocation of cytosolic HSP72, these findings suggest eccentric contractions, unlike other forms of stress such as heat, do not trigger tight binding of HSP72 to its primary membrane-bound target proteins, in particular SERCA. [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHSPs) are a subgroup of the highly conserved family of HSPs that are stress inducible and confer resistance to cellular stress and injury. This study aimed to quantitatively examine whether type of contraction (concentric or eccentric) affects sHSPs, HSP27 and αB-crystallin, localization, and phosphorylation in human muscle. Vastus lateralis muscle biopsies from 11 healthy male volunteers were obtained pre- and 3 h, 24 h, and 7 days following concentric (CONC), eccentric (ECC1), and repeated bout eccentric (ECC2) exercise. No changes were apparent in a control group (n = 5) who performed no exercise. Eccentric exercise induced muscle damage, as evidenced by increased muscle force loss, perceived muscle soreness, and elevated plasma creatine kinase and myoglobin levels. Total HSP27 and αB-crystallin amounts did not change following any type of exercise. Following eccentric exercise (ECC1 and ECC2) phosphorylation of HSP27 at serine 15 (pHSP27-Serl5) was increased approximately 3- to 6-fold at 3 h, and pαB-crystallin-Ser59 increased ~ 10-fold at 3 h. Prior to exercise most of the sHSP and psHSP pools were present in the cytosolic compartment. Eccentric exercise resulted in partial redistribution of HSP27 (~23%) from the cytosol to the cytoskeletal fraction (~28% for pHSP27-Serl5 and ~7% for pHSP27-Ser82), with subsequent full reversal within 24 h. αB-crystallin also showed partial redistribution from the cytosolic to cytoskeletal fraction (~18% of total) 3 h post-ECCl, but not after ECC2. There was no redistribution or phosphorylation of sHSPs with CONC. Eccentric exercise results in increased sHSP phosphorylation and translocation to the cytoskeletal fraction, but the sHSP translocation is not dependent on their phosphorylation. [ABSTRACT FROM AUTHOR]

Western blotting has been used for protein analyses in a wide range of tissue samples for >30 years. Fundamental to Western blotting success are a number of important considerations, which unfortunately are often overlooked or not appreciated. Firstly, lowly expressed proteins may often be better detected by dramatically reducing the amount of sample loaded. Single cell (fibre) Western blotting demonstrates the ability to detect proteins in small sample sizes, 5-10 μg total mass (1-3 μg total protein). That is an order of magnitude less than often used. Using heterogeneous skeletal muscle as the tissue of representation, the need to undertake Western blotting in sample sizes equivalent to single fibre segments is demonstrated. Secondly, incorrect results can be obtained if samples are fractionated and a proportion of the protein of interest inadvertently discarded during sample preparation. Thirdly, quantitative analyses demand that a calibration curve be used. This is regardless of using a loading control, which must be proven to not change with the intervention and also be appropriately calibrated. Fourthly, antibody specificity must be proven using whole tissue analyses, and for immunofluorescence analyses it is vital that only a single protein is detected. If appropriately undertaken, Western blotting is reliable, quantitative, both in relative and absolute terms, and extremely valuable. [ABSTRACT FROM AUTHOR]

Membrane phospholipid (PL) composition has been shown to affect cellular function by altering membrane physical structure. The sarcolemma plasma membrane (SL) is integral to skeletal muscle function and health. Previous studies assessing SL PL composition have demonstrated contamination from transverse (t)-tubule, sarcoplasmic reticulum, and nuclear membranes. This study assessed the possibility of isolating SL by mechanically skinning skeletal muscle fiber segments for the analysis of SL PL composition. Mechanically skinned SL from rat extensor digitorum longus (EDL) muscle fibers underwent Western blot analysis to assess contamination from t-tubule, sarcoplasmic reticulum, nuclear and mitochondrial membranes. The results indicate that isolated SL had minimal nuclear and mitochondrial membrane contamination and was void of contamination from sarcoplasmic reticulum and t-tubule membranes. After performing both high-performance thin layer chromatography and gas chromatography, we found that the SL obtained by mechanical skinning had higher sphingomyelin and total fatty acid saturation and lower phosphatidylcholine when compared to previous literature. Thus, by avoiding the use of various chemical treatments and membrane fractionation, we present data that may truly represent the SL and future studies can use this technique to assess potential changes under various perturbations and disease conditions such as insulin resistance and muscular dystrophy. [ABSTRACT FROM AUTHOR]

Key points Most human experiments examine proteins at the whole muscle level, and knowledge of fibre type specificity is mainly obtained in animal muscles. We used a new methodology and provide novel findings at the single fibre level., Potassium changes across muscle cell membranes may be important for development of fatigue during exercise. The Na+-K+ pump with the regulatory phospholemman (FXYD1) subunit and the ATP-dependent K+ channel Kir6.2 play crucial roles in potassium regulation., We show that in humans Na+-K+ pump α2 is expressed to a greater extent in type II than in type I fibres and Kir6.2 is expressed primarily in type I fibres. During exercise, phosphorylation of FXYD1 serine 68 is increased only in type II fibres, indicating differences in Na+-K+ pump activity., These results show that human Kir6.2 and Na+-K+ pump subunit expression and FXYD1 phosphorylation are fibre type specific, which may influence exercise-induced potassium regulation and fatigue development., Abstract The aim of the present study was to examine fibre type-specific Na+-K+ pump subunit expression and exercise-induced alterations in phospholemman (FXYD1) phosphorylation in humans. Segments of human skeletal muscle fibres were dissected and fibre typed, and protein expression was determined by Western blotting. The protein expression of the Na+-K+ pump α2 isoform was lower in type I than in type II fibres (0.63 ± 0.04 a.u. vs. 1.00 ± 0.07 a.u., P < 0.001), while protein expression of the Na+-K+ pump α1 and β1 isoforms was not different. Protein expression of the ATP-dependent potassium channel Kir6.2 was higher in type I compared with type II fibres. In both type I ( P < 0.01) and type II fibres ( P < 0.001) the AB_FXYD1 signal was lower after exercise compared with rest, indicating an increase in unspecific FXYD1 phosphorylation. The FXYD1 serine 68 phosphorylation was higher ( P < 0.001) after exercise compared with rest in type II fibres (1.90 ± 0.25 vs. 1.00 ± 0.08) and not changed in type I fibres. Total FXYD1 was not expressed in a fibre type-specific manner. Expression of phosphofructokinase was lower ( P < 0.001) in type I than in type II fibres, whereas citrate synthase and 3-hydroxyacyl-CoA dehydrogenase were more abundant ( P < 0.001) in type I fibres. In conclusion, FXYD1 phosphorylation at serine 68 increased after an acute bout of intense exercise in human type II fibres, while AB_FXYD1 signal intensity was lower in both type I and type II fibres, indicating fibre type-specific differences in FXYD1 phosphorylation on serine 63, serine 68 and threonine 69. This, together with the observation of a higher abundance of the Na+-K+ pump α2 isoform protein in type II fibres, is likely to have importance for the exercise-induced human Na+-K+ pump activity in the different fibre types. [ABSTRACT FROM AUTHOR]

To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (∼10- to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed ∼70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a ∼50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (∼15-60%) and decreased GBE diffusibility (∼20%). Amylase treatment, which breaks α-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, v80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ~95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to α44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca2+- ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding. [ABSTRACT FROM AUTHOR]