Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca 2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 ± 2 and 10 ± 1 μmol (l fibre volume) −1 , quantitatively accounting for the maximal SR Ca 2+ content of each. Soleus type II [SOL (II)] fibres (∼20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast-twitch muscle sarco(endo)plasmic reticulum Ca 2+ -ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca 2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca 2+ content was inversely correlated with CSQ1 content. The relative SR Ca 2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (∼20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca 2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results indicating that at least part of the Ca 2+ leakage occurred through SERCA. It is concluded that CSQ1 plays an important role in EDL muscle fibres by providing a large total pool of releasable Ca 2+ in the SR whilst maintaining free [Ca 2+ ] in the SR at sufficiently low levels that Ca 2+ leakage through the high density of SERCA1 pumps does not metabolically compromise muscle function.

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 (ϳ10-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 ϳ70% 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 ϳ50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (ϳ15-60%) and decreased GBE diffusibility (ϳ20%). Amylase treatment, which breaks ␣-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. glycogen storage disease; glycogen enzymes; single fibers ONE OF THE MAIN FUEL SOURCES in skeletal muscle for both short-term and prolonged, repetitive contractions is glycogen, a branched polymer of glucose comprising of ␣-1,4-glycosidic bonds with ␣-1,6-glycosidic linkages at branch points (6). Glycogen synthesis is initiated by the autoglucosylation of glycogenin and elongated by the activities of glycogen synthase (GS, ␣-1,4-glycosidic links) and glycogen branching enzyme (GBE, ␣1,6-glycosidic shorter branches). GBE excises a segment of existing oligosaccharide on glycogen by cleaving an ␣-1,4-glycosidic linkage and reforms an ␣-1,6-glycosidic linkage (2, 7). Muscle glycogen is important for providing glucose as a source of ATP for energy-requiring events like muscle contraction. Glucose is mobilized from glycogen by the concerted action of glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE) acting in reverse to GS and GBE (6). Glycogen granules consist of several tiers of glucose moieties, with 30 -45% of the glucose being present in the outer tiers and thereby branched with ␣-1,4-glycosidic links and requiring GP for utiliz...

We hypothesised that normal skeletal muscle stimulated intensely either in vitro or in situ would exhibit reactive oxygen species (ROS)-mediated contractile apparatus changes common to many pathophysiological conditions. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were bubbled with 95% O2 and stimulated in vitro at 31°C to give isometric tetani (50 Hz for 0.5 s every 2 s) until maximum force declined to ≤30%. Skinned superficial slow-twitch fibers from the SOL muscles displayed a large reduction (∼41%) in maximum Ca2+-activated specific force (Fmax), with Ca2+-sensitivity unchanged. Fibers from EDL muscles were less affected. The decrease in Fmax in SOL fibers was evidently due to oxidation effects on cysteine residues because it was reversed if the reducing agent DTT was applied prior to activating the fiber. The GSH∶GSSG ratio was ∼3-fold lower in the cytoplasm of superficial fibers from stimulated muscle compared to control, confirming increased oxidant levels. The presence of Tempol and L-NAME during in vitro stimulation prevented reduction in Fmax. Skinned fibers from SOL muscles stimulated in vivo at 37°C with intact blood supply also displayed reduction in Fmax, though to a much smaller extent (∼12%). Thus, fibers from muscles stimulated even with putatively adequate O2 supply display a reversible oxidation-induced decrease in Fmax without change in Ca2+-sensitivity, consistent with action of peroxynitrite (or possibly superoxide) on cysteine residues of the contractile apparatus. Significantly, the changes closely resemble the contractile deficits observed in a range of pathophysiological conditions. These findings highlight how readily muscle experiences ROS-related deficits, and also point to potential difficulties when defining muscle performance and fatigue.

Heat shock proteins (HSPs) are essential for normal cellular stress responses. Absolute amounts of HSP72, HSP25, and αB-crystallin in rat extensor digitorum longus (EDL) and soleus (SOL) muscle were ascertained by quantitative Western blotting to better understand their respective capabilities and limitations. HSP72 content of EDL and SOL muscle was only ∼1.1 and 4.6 μmol/kg wet wt, respectively, and HSP25 content approximately twofold greater (∼3.4 and ∼8.9 μmol/kg, respectively). αB-crystallin content of EDL muscle was ∼4.9 μmol/kg but in SOL muscle was ∼30-fold higher (∼140 μmol/kg). To examine fiber heterogeneity, HSP content was also assessed in individual fiber segments; every EDL type II fiber had less of each HSP than any SOL type I fiber, whereas the two SOL type II fibers examined were indistinguishable from the EDL type II fibers. Sarcolemma removal (fiber skinning) demonstrated that 10-20% of HSP25 and αB-crystallin was sarcolemma-associated in SOL fibers. HSP diffusibility was assessed from the extent and rate of diffusion out of skinned fiber segments. In unstressed SOL fibers, 70-90% of each HSP was readily diffusible, whereas ∼95% remained tightly bound in fibers from SOL muscles heated to 45°C. Membrane disruption with Triton X-100 allowed dispersion of HSP72 and sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps but did not alter binding of HSP25 or αB-crystallin. The amount of HSP72 in unstressed EDL muscle is much less than the number of its putative binding sites, whereas SOL type I fibers contain large amounts of αB-crystallin, suggesting its importance in normal cellular function without upregulation.

Larkins NT, Murphy RM, Lamb GD. Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers. Am J Physiol Cell Physiol 303: C654 -C665, 2012. First published July 3, 2012; doi:10.1152/ajpcell.00180.2012.-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 ␣B-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, Ͼ80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ϳ95% of HSP25 and ␣B-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to Ն44°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 Ca 2ϩ -ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on ␣B-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. oxidation; stress response; skinned fiber; chaperones HEAT SHOCK PROTEINS (HSPs) are highly conserved and ubiquitously expressed proteins that act as protein chaperones and in stress situations bind and interact with various proteins to help preserve or restore their function (23,32). A great deal of work in skeletal muscle has focused on the increase in HSP protein and/or mRNA expression levels occurring in hours to days after various stressful stimuli, such as eccentric exercise (11,17,26,27,39), oxidative stress (50), heat (4, 35), and glycogen depletion (12). The present study focuses instead on the acute responses of HSPs to stresses, examining the effects of heat, contraction, and o...