Slow-twitch Soleus Research Articles

Elevated GLUT4 and glycogenin protein abundance correspond to increased glycogen content in the soleus muscle of mdx mice with no benefit associated with taurine supplementation.

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.

Crush injury to motor nerves in the G93A transgenic mouse model of amyotrophic lateral sclerosis promotes muscle reinnervation and survival of functionally intact nerve-muscle contacts

Selective survival of small motor nerve fibers and their neuromuscular contacts in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS) suggests that smaller regenerated nerve fibers are more able to sustain reformed nerve-muscle connections as functionally intact motor units (MUs). The sciatic nerve was crushed unilaterally in SOD1G93A transgenic mice at 40 days of age and contractile forces of reinnervated muscles and their MUs were recorded at 90 days in order to determine the capacities of the nerves to regenerate and to form and retain functional neuromuscular connections. Reduced MU numbers in fast-twitch tibialis anterior, extensor digitorum longus and medial gastrocnemius muscles and the lesser reductions in slow-twitch soleus muscle of SOD1G93A transgenic mice were reversed in reinnervated muscles: there were more reinnervated MUs and their contractile forces and the muscle forces and weights increased. In line with the contrasting ability of only small not large nerve fibers to sprout to form enlarged MUs in the SOD1G93A transgenic mouse, the smaller regenerating nerve fibers formed enlarged MUs that were better able to survive. Because nerve fibers with and without muscle contacts were severed by the sciatic nerve crush injury, the conditioning lesion is untenable as the explanation for improved maintenance of reinnervated neuromuscular junctions. Elevated neurotrophic factor expression in axotomized motoneurons and/or denervated Schwann cells and the synapse withdrawal from axotomized motoneurons are other factors that, in addition to reduced size of nerve fibers reinnervating muscles, may account for increased survival and size of reinnervated MUs in ALS.

Changes in contractile and metabolic parameters of skeletal muscle as rats age from 3 to 12 months.

Laboratory rats are considered mature at 3months 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, 12months). 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.

Effects of rapid or slow body weight reduction on intramuscular protein degradation pathways during equivalent weight loss on rats.

The purpose of this study was to compare the effects of short-term fasting-induced rapid weight loss with those of slower but equivalent body weight loss induced by daily calorie restriction on muscle protein degradation pathways and muscle protein content. Male Fischer rats were subjected to either 30 % calorie restriction for 2 weeks to slowly decrease body weight (Slow) or 3-day fasting to rapidly decrease body weight by a comparable level of that of the Slow group (Rapid). The final body weights were about 15 % lower in both the Slow and Rapid groups than in the Con group (p<0.001). The total protein content and wet weight of fast-twitch plantaris muscle, but not slow-twitch soleus muscle, were significantly lower in the Rapid group compared with the control rats fed ad libitum. Substantial increases in the expression ratio of autophagosomal membrane proteins (LC3-II/-I ratio) and polyubiquitinated protein concentration, used as biomarkers of autophagy-lysosome and ubiquitin-proteasome activities, respectively, were observed in the plantaris muscle of the Rapid group. Moreover, the LC3-II/-I ratio and polyubiquitinated protein concentration were negatively correlated with the total protein content and wet weight of plantaris muscle. These results suggest that short-term fasting-induced rapid body weight loss activates autophagy-lysosome and ubiquitin-proteasome systems more strongly than calorie restriction-induced slower weight reduction, resulting in muscular atrophy in fast-twitch muscle.

Aging in Rats Differentially Affects Markers of Transcriptional and Translational Capacity in Soleus and Plantaris Muscle.

Alterations in transcriptional and translational mechanisms occur during skeletal muscle aging and such changes may contribute to age-related atrophy. Herein, we examined markers related to global transcriptional output (i.e., myonuclear number, total mRNA and RNA pol II levels), translational efficiency [i.e., eukaryotic initiation and elongation factor levels and muscle protein synthesis (MPS) levels] and translational capacity (ribosome density) in the slow-twitch soleus and fast-twitch plantaris muscles of male Fischer 344 rats aged 3, 6, 12, 18, and 24 months (n = 9–10 per group). We also examined alterations in markers of proteolysis and oxidative stress in these muscles (i.e., 20S proteasome activity, poly-ubiquinated protein levels and 4-HNE levels). Notable plantaris muscle observations included: (a) fiber cross sectional area (CSA) was 59% (p < 0.05) and 48% (p < 0.05) greater in 12 month vs. 3 month and 24 month rats, respectively, suggesting a peak lifetime value near 12 months and age-related atrophy by 24 months, (b) MPS levels were greatest in 18 month rats (p < 0.05) despite the onset of atrophy, (c) while regulators of ribosome biogenesis [c-Myc and upstream binding factor (UBF) protein levels] generally increased with age, ribosome density linearly decreased from 3 months of age and RNA polymerase (Pol) I protein levels were lowest in 24 month rats, and d) 20S proteasome activity was robustly up-regulated in 6 and 24 month rats (p < 0.05). Notable soleus muscle observations included: (a) fiber CSA was greatest in 6 month rats and was maintained in older age groups, and (b) 20S proteasome activity was modestly but significantly greater in 24 month vs. 3/12/18 month rats (p < 0.05), and (c) total mRNA levels (suggestive of transcriptional output) trended downward in older rats despite non-significant between-group differences in myonuclear number and/or RNA Pol II protein levels. Collectively, these findings suggest that plantaris, not soleus, atrophy occurs following 12 months of age in male Fisher rats and this may be due to translational deficits (i.e., changes in MPS and ribosome density) and/or increases in proteolysis rather than increased oxidative stress and/or alterations in global transcriptional mechanisms.

Skeletal Myosin-Binding Protein C Modulates Actomyosin Contractility in an Isoform-Dependent Manner

Myosin-binding protein C (MyBP-C) is a sarcomeric protein localized to discrete regions (C-zones) of myosin thick filaments in all striated muscles. Unlike cardiac muscle with its specific MyBP-C isoform, mass spectrometry shows that rat slow-twitch soleus (SOL) muscle expresses two different slow MyBP-C isoforms (sMyBP-C), having one (sMyBP-C1) or two N-terminal inserts (sMyBP-C2), while fast-twitch extensor digitorum longus (EDL) expresses these same sMyBP-C isoforms and a fast MyBP-C isoform (fMyBP-C). Do these MyBP-C isoforms differentially regulate skeletal actomyosin contractility in a muscle-specific manner? To address this, we characterized Ca2+-regulated motion of SOL and EDL native actin-thin filaments (NTFs) over their corresponding native myosin-thick filaments, having the physiological complement of MyBP-C in the C-zone. At low calcium (pCa7.5) where NTFs should be inhibited, motion occurred within the C-zone of both SOL and EDL native thick filaments. Whereas, at high Ca2+ (pCa5), SOL and EDL NTFs had initial fast velocities followed by ∼30% slower velocities within the C-zone. These data suggest that one or more of these MyBP-C can sensitize NTFs to Ca2+ and modulate contractility at high Ca2+. To determine how each isoform contributes to these modulatory capacities, we bacterially-expressed N-terminal fragments (sMyBP-C1, sMyBP-C2, fMyBP-C) and assessed their individual impact on SOL or EDL NTF motility over their respective monomeric myosins. For the EDL where all three MyBP-Cs are expressed, at pCa7.5 sMyBP-C1 was most effective at activating NTFs, while at pCa5 all three reduced velocity by ∼50%. For SOL, where only sMyBP-C isoforms are expressed, at pCa7.5 sMyBP-C1 activated NTFs while at pCa5 sMyBP-C2 reduced velocity ∼40%. Our data suggest that the distinctive mixture of MyBP-C isoforms expressed in the SOL and EDL is matched to the muscle's physiological demands. Each MyBP-C isoform possesses specific modulatory capacities that may be called upon under different contractile states. Specifically, to fine-tune actomyosin contractility within the C-zone by Ca2+-sensitizing the thin filament and/or modulating velocity at maximal activation.

Changes in FOXO and proinflammatory cytokines in the late stage of immobilized fast and slow muscle atrophy.

The present study aimed to examine the changes in the expression of Forkhead box protein O (FOXO) and proinflammatory cytokines in both slow-twitch soleus and fast-twitch plantaris muscles following cast immobilization. Male C57BL/6 mice were subjected to cast immobilization for 7 and 21 days. Cast immobilization increased FOXO3a mRNA and total protein expression in both the soleus and plantaris muscles. Although FOXO3a phosphorylation tended to increase in response to cast immobilization in both muscles, a significant increase was evident after 21 days of immobilization only in the soleus muscle. The degree of the response of FOXO3a was very different between the soleus and plantaris muscles; however, the kinetics of FOXO3a in both muscles were similar. Thus, the regulation of muscle atrophy by FOXO might act via a common mechanism in both slow-twitch soleus and fast-twitch plantaris muscles. Gene expression of proinflammatory cytokines tended to increase in response to cast immobilization, and a significant increase was evident after 21 days of immobilization in the soleus muscle. However, in the plantaris muscle, proinflammatory cytokine gene expression remained unchanged throughout the immobilization period; nevertheless, immobilization induced greater reduction in muscle fiber cross-sectional area in the plantaris than in the soleus muscle. Thus, these observations indicate that regulation of muscle atrophy by proinflammatory cytokines might contribute to muscle fiber type-specific mechanisms.

A Reduction in Selenoprotein S Amplifies the Inflammatory Profile of Fast-Twitch Skeletal Muscle in the mdx Dystrophic Mouse.

Excessive inflammation is a hallmark of muscle myopathies, including Duchenne muscular dystrophy (DMD). There is interest in characterising novel genes that regulate inflammation due to their potential to modify disease progression. Gene polymorphisms in Selenoprotein S (Seps1) are associated with elevated proinflammatory cytokines, and in vitro SEPS1 is protective against inflammatory stress. Given that SEPS1 is highly expressed in skeletal muscle, we investigated whether the genetic reduction of Seps1 exacerbated inflammation in the mdx mouse. F1 male mdx mice with a heterozygous Seps1 deletion (mdx:Seps1−/+) were generated. The mdx:Seps1−/+ mice had a 50% reduction in SEPS1 protein expression in hindlimb muscles. In the extensor digitorum longus (EDL) muscles, mRNA expression of monocyte chemoattractant protein 1 (Mcp-1) (P = 0.034), macrophage marker F4/80 (P = 0.030), and transforming growth factor-β1 (Tgf-β1) (P = 0.056) were increased in mdx:Seps1−/+ mice. This was associated with a reduction in muscle fibre size; however, ex vivo EDL muscle strength and endurance were unaltered. In dystrophic slow twitch soleus muscles, SEPS1 reduction had no effect on the inflammatory profile nor function. In conclusion, the genetic reduction of Seps1 appears to specifically exacerbate the inflammatory profile of fast-twitch muscle fibres, which are typically more vulnerable to degeneration in dystrophy.

Osteoprotegerin and β2-Agonists Mitigate Muscular Dystrophy in Slow- and Fast-Twitch Skeletal Muscles

Our recent work showed that daily injections of osteoprotegerin (OPG)-immunoglobulin fragment complex (OPG-Fc) completely restore the function of fast-twitch extensor digitorum longus muscles in dystrophic mdx mice, a murine model of Duchenne muscular dystrophy. However, despite marked improvements, OPG-Fc was not as effective in preventing the loss of function of slow-twitch soleus and diaphragm muscles. Because β2-agonists enhance the function of slow- and fast-twitch dystrophic muscles and because their use is limited by their adverse effects on bone and cardiac tissues, we hypothesized that OPG-Fc, a bone and skeletal muscle protector, acts synergistically with β2-agonists and potentiates their positive effects on skeletal muscles. We observed that the content of β2-adrenergic receptors, which are mainly expressed in skeletal muscle, is significantly reduced in dystrophic muscles but is rescued by the injection of OPG-Fc. Most important, OPG-Fc combined with a low dose of formoterol, a member of a new generation of β2-agonists, histologically and functionally rescued slow-twitch dystrophic muscles. This combination of therapeutic agents, which have already been tested and approved for human use, may open up new therapeutic avenues for Duchenne muscular dystrophy and possibly other neuromuscular diseases.

Can inorganic phosphate explain sag during unfused tetanic contractions of skeletal muscle?

We test the hypothesis that cytosolic inorganic phosphate (Pi) can account for the contraction‐induced reductions in twitch duration which impair summation and cause force to decline (sag) during unfused tetanic contractions of fast‐twitch muscle. A five‐state model of crossbridge cycling was used to simulate twitch and unfused tetanic contractions. As Pi concentration ([Pi]) was increased from 0 to 30 mmol·L−1, twitch duration decreased, with progressive reductions in sensitivity to Pi as [Pi] was increased. When unfused tetani were simulated with rising [Pi], sag was most pronounced when initial [Pi] was low, and when the magnitude of [Pi] increase was large. Fast‐twitch extensor digitorum longus (EDL) muscles (sag‐prone, typically low basal [Pi]) and slow‐twitch soleus muscles (sag‐resistant, typically high basal [Pi]) were isolated from 14 female C57BL/6 mice. Muscles were sequentially incubated in solutions containing either glucose or pyruvate to create typical and low Pi environments, respectively. Twitch duration was greater (P < 0.05) in pyruvate than glucose in both muscles. Stimuli applied at intervals approximately three times the time to peak twitch tension resulted in sag of 35.0 ± 3.7% in glucose and 50.5 ± 1.4% in pyruvate in the EDL (pyruvate > glucose; P < 0.05), and 3.9 ± 0.3% in glucose and 37.8 ± 2.7% in pyruvate in the soleus (pyruvate > glucose; P < 0.05). The influence of Pi on crossbridge cycling provides a tenable mechanism for sag. Moreover, the low basal [Pi] in fast‐twitch relative to slow‐twitch muscle has promise as an explanation for the fiber‐type dependency of sag.

A short-term statin treatment changes the contractile properties of fast-twitch skeletal muscles.

BackgroundCumulative evidence indicates that statins induce myotoxicity. However, the lack of understanding of how statins affect skeletal muscles at the structural, functional, and physiological levels hampers proper healthcare management. The purpose of the present study was to investigate the early after-effects of lovastatin on the slow-twitch soleus (Sol) and fast-twitch extensor digitorum longus (EDL) muscles.MethodsAdult C57BL/6 mice were orally administrated with placebo or lovastatin [50 mg/kg/d] for 28 days. At the end of the treatment, the isometric ex vivo contractile properties of the Sol and EDL muscles were measured. Subtetanic and tetanic contractions were assessed and contraction kinetics were recorded. The muscles were then frozen for immunohistochemical analyses. Data were analyzed by two-way ANOVA followed by an a posteriori Tukey’s test.ResultsThe short-term lovastatin treatment did not induce muscle mass loss, muscle fiber atrophy, or creatine kinase (CK) release. It had no functional impact on slow-twitch Sol muscles. However, subtetanic stimulations at 10 Hz provoked greater force production in fast-twitch EDL muscles. The treatment also decreased the maximal rate of force development (dP/dT) of twitch contractions and prolonged the half relaxation time (1/2RT) of tetanic contractions of EDL muscles.ConclusionsAn early short-term statin treatment induced subtle but significant changes in some parameters of the contractile profile of EDL muscles, providing new insights into the selective initiation of statin-induced myopathy in fast-twitch muscles.

The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3.

Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intronic one-base mutation in the iron-sulfur cluster assembly (ISCU) gene, resulting in aberrant splicing. The incorrectly spliced transcripts contain a 100 or 86 bp intron sequence encoding a non-functional ISCU protein, which leads to defects in several Fe-S containing proteins in the respiratory chain and the TCA cycle. The symptoms in HML are restricted to skeletal muscle, and it has been proposed that this effect is due to higher levels of incorrectly spliced ISCU in skeletal muscle compared with other energy-demanding tissues. In this study, we confirm that skeletal muscle contains the highest levels of incorrect ISCU splice variants compared with heart, brain, liver and kidney using a transgenic mouse model expressing human HML mutated ISCU. We also show that incorrect splicing occurs to a significantly higher extent in the slow-twitch soleus muscle compared with the gastrocnemius and quadriceps. The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) was identified as a potential candidate for the slow fiber specific regulation of ISCU splicing since this factor was expressed at higher levels in the soleus compared to the gastrocnemius and quadriceps. We identified an interaction between SRSF3 and the ISCU transcript, and by overexpressing SRSF3 in human myoblasts we observed increased levels of incorrectly spliced ISCU, while knockdown of SRSF3 resulted in decreased levels. We therefore suggest that SRSF3 may participate in the regulation of the incorrect splicing of mutant ISCU and may, at least partially, explain the muscle-specific symptoms of HML.

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy.

Role of the JP45-calsequestrin complex on calcium entry in slow twitch skeletal muscles.

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La3+- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca2+ concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca2+ concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.

Exercise training increases protein O-GlcNAcylation in rat skeletal muscle.

Protein O‐GlcNAcylation has emerged as an important intracellular signaling system with both physiological and pathophysiological functions, but the role of protein O‐GlcNAcylation in skeletal muscle remains elusive. In this study, we tested the hypothesis that protein O‐GlcNAcylation is a dynamic signaling system in skeletal muscle in exercise and disease. Immunoblotting showed different protein O‐GlcNAcylation pattern in the prototypical slow twitch soleus muscle compared to fast twitch EDL from rats, with greater O‐GlcNAcylation level in soleus associated with higher expression of the modulating enzymes O‐GlcNAc transferase (OGT), O‐GlcNAcase (OGA), and glutamine fructose‐6‐phosphate amidotransferase isoforms 1 and 2 (GFAT1, GFAT2). Six weeks of exercise training by treadmill running, but not an acute exercise bout, increased protein O‐GlcNAcylation in rat soleus and EDL. There was a striking increase in O‐GlcNAcylation of cytoplasmic proteins ~50 kDa in size that judged from mass spectrometry analysis could represent O‐GlcNAcylation of one or more key metabolic enzymes. This suggests that cytoplasmic O‐GlcNAc signaling is part of the training response. In contrast to exercise training, postinfarction heart failure (HF) in rats and humans did not affect skeletal muscle O‐GlcNAcylation level, indicating that aberrant O‐GlcNAcylation cannot explain the skeletal muscle dysfunction in HF. Human skeletal muscle displayed extensive protein O‐GlcNAcylation that by large mirrored the fiber‐type‐related O‐GlcNAcylation pattern in rats, suggesting O‐GlcNAcylation as an important signaling system also in human skeletal muscle.

Inhibition of Akt2 phosphorylation abolishes the calorie restriction-induced improvement in insulin-stimulated glucose uptake by rat soleus muscle.

Calorie restriction (CR; ∼60%-65% of ad libitum consumption) can enhance insulin-stimulated glucose uptake (ISGU) in predominantly slow-twitch skeletal muscles (e.g., soleus) by an incompletely understood mechanism. We used an Akt inhibitor (MK-2206) to eliminate CR's effect on insulin-stimulated Akt2 phosphorylation in isolated rat soleus muscles. We found long-term CR-enhanced ISGU was abolished by eliminating the CR effect on Akt2 phosphorylation, suggesting the CR-induced benefit on ISGU in the predominantly slow-twitch soleus relies on enhanced Akt2 phosphorylation.

Role of the JP45-Calsequestrin Complex on Calcium Entry in Slow Twitch Skeletal Muscles

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La3+- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca2+ concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca2+ concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.

Effect of resistance exercise training on expression of Hsp70 and inflammatory cytokines in skeletal muscle and adipose tissue of STZ-induced diabetic rats.

Impairment of adipose tissue and skeletal muscles accrued following type 1 diabetes is associated with protein misfolding and loss of adipose mass and skeletal muscle atrophy. Resistance training can maintain muscle mass by changing both inflammatory cytokines and stress factors in adipose tissue and skeletal muscle. The purpose of this study was to determine the effects of a 5-week ladder climbing resistance training program on the expression of Hsp70 and inflammatory cytokines in adipose tissue and fast-twitch flexor hallucis longus (FHL) and slow-twitch soleus muscles in healthy and streptozotocin-induced diabetic rats. Induction of diabetes reduced body mass, while resistance training preserved FHL muscle weight in diabetic rats without any changes in body mass. Diabetes increased Hsp70 protein content in skeletal muscles, adipose tissue, and serum. Hsp70 protein levels were decreased in normal and diabetic rats by resistance training in the FHL, but not soleus muscle. Furthermore, resistance training decreased inflammatory cytokines in FHL skeletal muscle. On the other hand, Hsp70 and inflammatory cytokine protein levels were increased by training in adipose tissue. Also, significant positive correlations between inflammatory cytokines in adipose tissue and skeletal muscles with Hsp70 protein levels were observed. In conclusion, we found that in diabetic rats, resistance training decreased inflammatory cytokines and Hsp70 protein levels in fast skeletal muscle, increased adipose tissue inflammatory cytokines and Hsp70, and preserved FHL muscle mass. These results suggest that resistance training can maintain skeletal muscle mass in diabetes by changing inflammatory cytokines and stress factors such as Hsp70 in skeletal muscle and adipose tissue.

High-phosphorus diet maximizes and low-dose calcitriol attenuates skeletal muscle changes in long-term uremic rats.

Although disorders of mineral metabolism and skeletal muscle are common in chronic kidney disease (CKD), their potential relationship remains unexplored. Elevations in plasma phosphate, parathyroid hormone, and fibroblastic growth factor 23 together with decreased calcitriol levels are common features of CKD. High-phosphate intake is a major contributor to progression of CKD. This study was primarily aimed to determine the influence of high-phosphate intake on muscle and to investigate whether calcitriol supplementation counteracts negative skeletal muscle changes associated with long-term uremia. Proportions and metabolic and morphological features of myosin-based muscle fiber types were assessed in the slow-twitch soleus and the fast-twitch tibialis cranialis muscles of uremic rats (5/6 nephrectomy, Nx) and compared with sham-operated (So) controls. Three groups of Nx rats received either a standard diet (0.6% phosphorus, Nx-Sd), or a high-phosphorus diet (0.9% phosphorus, Nx-Pho), or a high-phosphorus diet plus calcitriol (10 ng/kg 3 day/wk ip, Nx-Pho + Cal) for 12 wk. Two groups of So rats received either a standard diet or a high-phosphorus diet (So-Pho) over the same period. A multivariate analysis encompassing all fiber-type characteristics indicated that Nx-Pho + Cal rats displayed skeletal muscle phenotypes intermediate between Nx-Pho and So-Pho rats and that uremia-induced skeletal muscle changes were of greater magnitude in Nx-Pho than in Nx-Sd rats. In uremic rats, treatment with calcitriol preserved fiber-type composition, cross-sectional size, myonuclear domain size, oxidative capacity, and capillarity of muscle fibers. These data demonstrate that a high-phosphorus diet potentiates and low-dose calcitriol attenuates adverse skeletal muscle changes in long-term uremic rats.

The Loss of the Transmembrane Protein MG23 Affects the Fast-Twitch Feature of EDl Muscle

We previously identified the 23-kDa transmembrane protein MG23 in skeletal muscle. MG23 is abundantly expressed in skeletal muscle, and weakly detected in various tissues. Immunochemical and electron-microscopic analyses suggested that MG23 forms a bowl-shaped homo-oligomer, and is distributed throughout the outer nuclear membrane and sarco/endoplasmic reticulum. In planar lipid bilayer membranes, purified recombinant MG23 behaved as voltage-sensitive cation-conducting channels which are equally permeable to Ca2+ and K+. Based on the observations, MG23 may take part in rapid cationic flux across intracellular membrane systems, while the physiological role of MG23 remains to be investigated. To survey MG23 function in vivo, we generated MG23-knockout (KO) mice. MG23-KO mice were healthy and apparently normal in growth and reproduction. In MG23-KO mice, fast-twitch EDL muscle exhibited reduced wet weight and slow relaxation after contraction. Although MG23 is unlikely to contribute directly to transcriptional machinery, gene-chip analysis detected aberrant gene expression in MG23-KO EDL muscle; for example, the expression levels of Ryr1, Cacna1s and Jph1 were obviously reduced, while Sln expression was >10-fold facilitated. On the other hand, slow-twitch soleus muscle from MG23-KO mice was apparently normal in contraction and gene expression. Our observations may imply that MG23 is a critical membranous component for the development and/or maintenance of fast-twitch properties in muscle cells.