Slow Myosin Research Articles

PO-095 Effects of dynamic foot stimulation on GSK-3 beta signaling pathway in rat soleus muscle under hindlimb unloading

Objective It is known that under simulated microgravity GSK-3 beta phosphorylation (Ser 9) is decreased [Mirzoev et al., 2016] which is associated with the activation of its kinase activity. GSK-3 beta activation may lead to NFATc1 export from myonuclei resulting in a slow-to-fast myosin shift in soleus muscle and may cause a decrease in muscle oxidative capacity and protein synthesis [Glass, 2003, Theeuwes et al, 2017].
 It was demonstrated that GSK-3 beta Ser 9 phosphorylation is decreased in response to nitric oxide synthase inhibition [Martins et al., 2011]. Under unloading conditions, the content of NO as well as nitric oxide synthase is attenuated in rat soleus muscle [Tidball et al., 1998; Lomonosova et al, 2011]. Dynamic foot stimulation (DFS) of the soles of the feet results in an increase of neuromuscular activation [Muller et al., 2005] of the lower limb muscles and prevents nitric synthase content decrease during 7-day exposure to dry immersion [Moukhina et al, 2004; Shenkman et al., 2004]. The aim of our study was to analyze the effect of rat dynamic foot stimulation during early unloading on GSK-3 beta phosphorylation and some of its downstream targets.
 Methods Male Wistar rats were randomly assigned to vivarium control, 1-day unloading and 1-day
 unloading with DFS. The pressure stimulation protocol mimicked the normal animal walking (104 mm Hg pressure, 4 Hz frequency and 250 ms signal duration) for a total of 20 min followed by a 10-min rest interval for 4 hours.
 Results We found that 1-day unloading caused a significant decrease (p˂ 0.025) in NFATc1 nuclear content as well as slow myosin (MHC I (beta) isoform) mRNA expression. In the 1-day unloading group, the level of glycogen synthase 1 phosphorylation (Ser 641), which is a direct GSK-3 beta target, was significantly higher than that in the control group, although a decrease in GSK-3 beta Ser 9 phosphorylation in the 1-day unloaded group was not statistically significant compared to the control group. However, in the DFS-treated unloaded rats the level of GSK-3 beta phosphorylation was significantly higher than in the untreated unloaded rats. NFATc1 nuclear content, slow myosin mRNA expression and glycogen synthase 1 phosphorylation (Ser 641) did not differ from the control group. The glycogen content in soleus muscles of both unloaded groups was higher than in the control group, which is in accordance with previous studies [Henriksen and Tischler, 1988], but in DFS subjected unloaded group the glycogen content was higher compared to the untreated unloaded group.
 Conclusions Thus, we found that dynamic foot stimulation during 1-day hindlimb unloading leads to GSK-3 beta inactivation and prevention of both NFATc1 myonuclei export and the decrease in MyHC I beta expression.
 The study was supported by the Russian Foundation for Basic Research grant #17-29-1029 and Basic Research Program of the IBMP, RAS. The authors express their deep gratitude to Prof. Inessa B. Kozlovskaya for useful recommendations concerning the design of the study.

PO-104 The shifted balance of high-energy phosphates drives the AMPK dephosphorylation and reduced slow myosin expression during unloading

Objective Mechanical unloading of postural muscles results in both muscle atrophy and a slow to fast fibre type transition. The cause of such changes is a reduction in slow-type MyHC isoform expression and an increase in fast-type MyHC isoform expression. It is believed that calcineurin/NFAT signalling pathway and AMP-activated protein kinase (AMPK) are involved in the regulation of slow-type MyHC isoform. Previously we showed that AMPK phosphorylation is significantly decreased in rat soleus at the early stage of mechanical unloading. We found, that stimulation of AMPK activity at the early stage of gravitational unloading prevents a decline of slow MyHC expression. We assumed that a decrease in AMPK activity in rat soleus at the early stage of gravitational unloading could be associated with changes in the ratio of intracellular high-energy phosphates (ATP/ADP). The aim of the study was to investigate the impact of high-energy phosphates ratio changes on AMPK activity and slow-type MyHC isoform expression in rat soleus muscle at the early stages of gravitational unloading.
 Methods To verify the hypothesis, we used administration of β-guanidinopropionic acid (βGPA), before (6 day) and during 24-h hindlimb suspension. The content of phospho-AMPK, phospho-ACC, phospho-PKD, HDAC4/5 in rat soleus was determined by Western-blotting. The amount of MyHCI(β) pre-mRNA and mRNA was evaluated by RT-PCR. 
 Results After 24-h HS we observed a decrease (p<0.05) in phospho-AMPK content vs. control group, but in HS+ βGPA group didn’t differ from the control. After 24-h unloading we found a significant increase in the content of nuclear HDAC4 in the HS group, but in the HS+ βGPA group the content of nuclear HDAC4 didn’t differ from the control group. 24-h unloading resulted in a decrease in MyHCI(β) pre-mRNA and mRNA expression vs. the control group. The expression level of MyHCI(β) pre-mRNA and mRNA in HS+ βGPA group didn’t differ from the control.
 Conclusions Thus βGPA administration prevents a decline in AMPK phosphorylation. Therefore, we can conclude that at the early stage of gravitational unloading an accumulation of high-energy phosphates (ATP, ADP and creatine phosphate) takes place and leads to reduced AMPK activity and a slow to fast myosin fibre type transition. The study was supported by Russian Science Foundation grant # 18-15-00107.

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

DMD CLINICAL THERAPIES II: P.132Identification of developmental myosin positive fibres acts both as a clinical biomarker for muscle disease and an important component of the process to confirm ezutromid target engagement

The expression of developmental and neonatal myosin heavy chains can be detected in newly formed regenerating skeletal myofibres 2-3 days after injury and remain for 2-3 weeks. These myosin isoforms are transiently expressed during regeneration and replaced when adult fast and slow myosins become prevalent. Therefore, the presence of developmental myosin heavy chain (MHCd) provides a very specific biomarker of regenerating fibres in pathologic skeletal muscle including DMD. Utrophin is also expressed in regenerating myofibres, following a similar restricted expression profile to MHCd. Summit have utilised novel automated immunohistochemistry imaging algorithms, in partnership with Flagship Biosciences, to quantify expression of developmental myosin heavy chain (MHCd) and utrophin in DMD patient muscle biopsies. This technology was utilised in the current Phase 2 open label study of ezutromid administered to 40 ambulatory patients with DMD where key secondary endpoints are from muscle biopsies; collected at baseline (n=40) and treatment Week 24 (n=25) or Week 48 (n=15). Compared to baseline, the Week 24 biopsies demonstrated a statistically significant decrease in the number of fibres expressing MHCd; the average reduction was -2.61% (95% CI -4.33, -0.90) from mean baseline of 11.37%; a relative reduction of 23%. This suggests a significant decrease in the number of fibres undergoing the natural repair process following damage. Additional functionality of the algorithms developed allows for assessment of expression heterogeneity across fibres and quantification of MHCd expression and utrophin expression in the same fibre. The individual fibre specific data analyses quantifying MHCd and utrophin comparing baseline to both Week 24 biopsies and Week 48 biopsies will be presented including those profiles that potentially distinguish a utrophin profile specific to ezutromid modulation.

Arginine promotes skeletal muscle fiber type transformation from fast-twitch to slow-twitch via Sirt1/AMPK pathway

This study investigated the effect of arginine on skeletal muscle fiber type transformation in mice and in C2C12 myotubes. Our data showed that dietary supplementation of arginine in mice significantly up-regulated the slow myosin heavy chain (MyHC), troponin I-SS, sirtuin1 (Sirt1) and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) protein expressions, as well as significantly down-regulated the fast MyHC protein expression. In C2C12 myotubes, arginine significantly increased the protein level of slow MyHC and the number of slow MyHC-positive cells, as well as significantly decreased the protein level of fast MyHC and the number of fast MyHC-positive cells. We also showed that arginine increased the activities of succinic dehydrogenase and malate dehydrogenase and decreased the activity of lactate dehydrogenase in mice and in C2C12 myotubes. Here we found that AMP-activated protein kinase (AMPK) was activated by arginine in mice and in C2C12 myotubes. However, inhibition of AMPK activity by compound C significantly attenuated the effects of arginine on slow MyHC and fast MyHC expressions in C2C12 myotubes. Finally, we showed that inhibition of Sirt1 expression by EX527 attenuated arginine-induced increase in the protein levels of phospho-AMPK and slow MyHC, the mRNA level of nitric oxide synthase (NOS) and the contents of NOS and NO, as well as decrease in fast MyHC protein level. Together, our findings indicated that arginine promotes skeletal muscle fiber type switching from fast-twitch to slow-twitch via Sirt1/AMPK pathway.

Changing muscle function with sustained glial derived neurotrophic factor treatment of rabbit extraocular muscle

Recent microarray and RNAseq experiments provided evidence that glial derived neurotrophic factor (GDNF) levels were decreased in extraocular muscles from human strabismic subjects compared to age-matched controls. We assessed the effect of sustained GDNF treatment of the superior rectus muscles of rabbits on their physiological and morphological characteristics, and these were compared to naïve control muscles. Superior rectus muscles of rabbits were implanted with a sustained release pellet of GDNF to deliver 2μg/day, with the contralateral side receiving a placebo pellet. After one month, the muscles were assessed using in vitro physiological methods. The muscles were examined histologically for alteration in fiber size, myosin expression patterns, neuromuscular junction size, and stem cell numbers and compared to age-matched naïve control muscles. GDNF resulted in decreased force generation, which was also seen on the untreated contralateral superior rectus muscles. Muscle relaxation times were increased in the GDNF treated muscles. Myofiber mean cross-sectional areas were increased after the GDNF treatment, but there was a compensatory increase in expression of developmental, neonatal, and slow tonic myosin heavy chain isoforms. In addition, in the GDNF treated muscles there was a large increase in Pitx2-positive myogenic precursor cells. One month of GDNF resulted in significant extraocular muscle adaptation. These changes are interesting relative to the decreased levels of GDNF in the muscles from subjects with strabismus and preliminary data in infant non-human primates where sustained GDNF treatment produced a strabismus. These data support the view that GDNF has the potential for improving eye alignment in subjects with strabismus.

Damaged muscle fibers might masquerade as hybrid fibers - a cautionary note on immunophenotyping mouse muscle with mouse monoclonal antibodies.

We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+). We demonstrate that, such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serialsection- labeling, to identify damaged fibers. The quadriceps femorismuscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21±0.21%, 98.34±1.06%, 0.07±0.01%, and 0.53±0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively. All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.

Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.

Myosin binding protein-C slow (sMyBP-C) comprises a family of accessory proteins in skeletal muscles that bind both myosin and actin filaments. Herein, we examined the role of sMyBP-C in adult skeletal muscles using in vivo gene transfer and clustered regularly interspaced short palindromic repeats technology to knock down all known sMyBP-C variants. Our findings, confirmed in two different skeletal muscles, demonstrated efficient knockdown (KD) of sMyBP-C (>70%) resulting in notably decreased levels of thick, but not thin, filament proteins ranging from ∼50% for slow and fast myosin to ∼20% for myomesin. Consistent with this, A bands were selectively distorted, and sarcomere length was significantly reduced. Contrary to earlier in vitro studies showing that addition of recombinant sMyBP-C slows down the formation of actomyosin crossbridges, our work demonstrates that KD of sMyBP-C in intact myofibers results in decreased contraction and relaxation kinetics under no-load conditions. Similarly, KD muscles develop markedly reduced twitch and tetanic force and contraction velocity. Taken together, our results show that sMyBP-C is essential for the regular organization and maintenance of myosin filaments into A bands and that its structural role precedes its ability to regulate actomyosin crossbridges.-Geist, J., Ward, C. W., Kontrogianni-Konstantopoulos, A. Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.

The force and stiffness of myosin motors in the isometric twitch of a cardiac trabecula and the effect of the extracellular calcium concentration.

Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07pNnm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07±0.09pNnm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8pNnm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61±4kPa to 93±9kPa, induced by raising [Ca2+ ]o from 1 to 2.5mm at sarcomere length ∼2.2μm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08±0.01 to 0.12±0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14±0.52pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.

P-246 - Distinct proteasomal activity for fast and slow twitch skeletal muscle during aging

Skeletal muscle alterations during aging lead to dysfunctional metabolism, correlating with frailty and early mortality. The loss of proteostasis is a hallmark of aging. Whether proteostasis loss plays a role in muscle aging remains elusive. To address this question we collected muscles, Soleus (SOL, type I) and Extensor digitorum longus (EDL, type II), from young (4 months) and old (25 months) C57BL/6 mice and evaluated the proteasomal system. Initial work showed decreased 26 S activity in old SOL. EDL displayed lower proteasomal activity in both ages compared to any of the SOL ages. Moreover, in order to understand if during aging there is the so-called “fiber switch from fast-to-slow”, we performed western blots against sMHC and fMHC (slow and fast myosin heavy chain, respectively). Preliminary results suggest that young SOL is composed by slow twitch fibers but also contains fast twitch fibers, while young EDL seems to be mostly composed by fast twitch fibers that level down during aging, suggesting the switch. As a conclusion, EDL seems to have less proteasomal activity, however, if this is a contributor or a consequence to the muscle fiber switch during aging still needs further investigation.

Arginine Promotes Slow Myosin Heavy Chain Expression via Akirin2 and the AMP-Activated Protein Kinase Signaling Pathway in Porcine Skeletal Muscle Satellite Cells.

This study aimed to investigate the effect of arginine on the expression of slow myosin heavy chain (MyHC) I and its underlying mechanism in porcine skeletal muscle satellite cells. Our results showed that arginine upregulated the mRNA (1.54 ± 0.08; p < 0.01) and protein (2.01 ± 0.01; p < 0.001) levels of MyHC I. We also showed that arginine upregulated the expression of Akirin2 (1.35 ± 0.1; p < 0.05) and increased the NO content (1.56 ± 0.04; p < 0.001). Akirin2 siRNA abolished arginine-induced upregulation of MyHC I and the increase of the NO content. In addition, arginine significantly increased the phospho-AMP-activated protein kinase (AMPK)/AMPK level (1.33 ± 0.06; p < 0.05), the AMPK content (79.55 ± 0.13; p < 0.001), and the AMPKα2 mRNA level (2.03 ± 0.20; p < 0.01). AMPKα2 silencing or AMPK inhibitor Compound C abolished arginine-induced upregulation of MyHC I. Our results provide, for the first time, evidence for the involvement of Akirin2 and the AMPK signaling pathway in arginine-induced MyHC I expression in porcine skeletal muscle satellite cells.

Nuclear Magnetic Resonance Spectroscopy

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Soleus muscle stability in wild hibernating black bears.

Based on studies of fast skeletal muscles, hibernating black and brown bears resist skeletal muscle atrophy during months of reduced physical activity and not feeding. The present study examined atrophy sparing in the slow soleus muscle, known to be highly prone to disuse atrophy in humans and other mammals. We demonstrated histochemically that the black bear soleus is rich in slow fibers, averaging 84.0 ± 6.6%. The percentages of slow fibers in fall (87.3 ± 4.9%) and during hibernation (87.1 ± 5.6%) did not differ ( P = 0.3152) from summer. The average fiber cross-sectional area to body mass ratio (48.6 ± 11.7 µm2/kg) in winter hibernating bears was not significantly different from that of summer (54.1 ± 11.8 µm2/kg, P = 0.4186) and fall (47.0 ± 9.7 µm2/kg, P = 0.9410) animals. The percentage of single hybrid fibers containing both slow and fast myosin heavy chains, detected biochemically, increased from 2.6 ± 3.8% in summer to 24.4 ± 24.4% ( P = 0.0244) during hibernation. The shortening velocities of individual hybrid fibers remained unchanged from that of pure slow and fast fibers, indicating low content of the minority myosins. Slow and fast fibers in winter bears exhibited elevated specific tension (kN/m2; 22%, P = 0.0161 and 11%, P = 0.0404, respectively) and maintained normalized power. The relative stability of fiber type percentage and size, fiber size-to-body mass ratio, myosin heavy chain isoform content, shortening velocity, power output, and elevated specific tension during hibernation validates the ability of the black bear to preserve the biochemical and performance characteristics of the soleus muscle during prolonged hibernation.

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo.

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.

Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit.

The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them. In this study fast sarcomere-level mechanics has been applied to Ca2+ -activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano-kinetics of force generation by slow and fast myosin isoforms in situ. The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform-dependent functional diversity between skeletal muscles. These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle. The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4μm, temperature 12°C, 4% dextran T-500) from slow (soleus, containing the MHC-1 isoform) and fast (psoas, containing the MHC-2X isoform) skeletal muscle of the rabbit. The stiffness of the half-sarcomere was determined at the plateau of Ca2+ -activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56±0.04 and 1.70±0.37pNnm-1 for the slow and fast isoform, respectively, while the average strain per attached motor (s0 ) was similar (∼3.3nm) in both isoforms. Consequently the force per motor (F0 =εs0 ) was three times smaller in the slow isoform than in the fast isoform (1.89±0.43 versus 5.35±1.51pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5 ) was 0.47±0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29±0.08), so that F0 in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.

Effects of gravitational hindlimb unloading and plantar mechanical stimulation on calcineurin/NFATc1 signaling pathway and slow myosin expression in rat soleus muscle

Effects of gravitational hindlimb unloading and plantar mechanical stimulation on calcineurin/NFATc1 signaling pathway and slow myosin expression in rat soleus muscle

The Role of GSK-3β Phosphorylation in the Regulation of Slow Myosin Expression in Soleus Muscle during Functional Unloading

Skeletal muscle myosin phenotype (i.e., the predominance in the muscle of a particular isoform or isoforms of myosin heavy chains (MyHC)) determines the properties of muscle, such as contraction speed and fatigue. The aim of this study was to identify the functional relationship between the decrease of the nitric oxide (NO) content, the GSK-3β phosphorylation (leading to the GSK-3β activation), the NFATc1 amount in the muscle nuclei, and the MyHC I(β) isoform expression in the rat soleus muscle under gravitational unloading. Male Wistar rats were divided into five groups: the vivarium control group; the group of animals with a 7-day hind limb suspension receiving placebo; the group of animals with a hind limb suspension receiving a NO donor (L-arginine); the group of animals with a hind limb suspension receiving a NO donor and a NO-synthase inhibitor (L-NAME); and the group of animals with a hind limb suspension receiving a GSK-3β inhibitor. We have shown that a 7-day unloading leads to a NO content decrease in the soleus muscle, and this effect is prevented by L-arginine administration. In addition, administration of L-arginine blocks the GSK-3β phosphorylation decrease, NFATc1 export from the muscle nuclei, and MyHC I(β) expression decrease caused by unloading. The L-arginine effect in each case can be blocked by the NO-synthase inhibitor. Administration of the GSK-3β inhibitor prevents the unloading-induced NFATc1 export from the muscle nuclei and a decrease of the MyHC I(β) expression. The prevention of the MyHC I(β) expression decrease and the NFATc1 export from the nucleus by the selective GSK-3β inhibition confirms the hypothesis on the NO influence on the MyHC I(β) expression and the NFATc1 export from the nucleus via the GSK-3β phosphorylation decrease. Thus, the NO level decrease in the rat soleus muscle in unloading leads to the GSK-3β activation, which in turn, promotes the NFATc1 export from the nucleus and stabilization of the fast myosin phenotype.

Calcium-Dependent Interaction Occurs between Slow Skeletal Myosin Binding Protein C and Calmodulin

Myosin binding protein C (MyBP-C) is a multi-domain protein that participates in the regulation of muscle contraction through dynamic interactions with actin and myosin. Three primary isoforms of MyBP-C exist: cardiac (cMyBP-C), fast skeletal (fsMyBP-C), and slow skeletal (ssMyBP-C). The N-terminal region of cMyBP-C contains the M-motif, a three-helix bundle that binds Ca2+-loaded calmodulin (CaM), but less is known about N-terminal ssMyBP-C and fsMyBP-C. Here, we characterized the conformation of a recombinant N-terminal fragment of ssMyBP-C (ssC1C2) using differential scanning fluorimetry, nuclear magnetic resonance, and molecular modeling. Our studies revealed that ssC1C2 has altered thermal stability in the presence and absence of CaM. We observed that site-specific interaction between CaM and the M-motif of ssC1C2 occurs in a Ca2+-dependent manner. Molecular modeling supported that the M-motif of ssC1C2 likely adopts a three-helix bundle fold comparable to cMyBP-C. Our study provides evidence that ssMyBP-C has overlapping structural determinants, in common with the cardiac isoform, which are important in controlling protein–protein interactions. We shed light on the differential molecular regulation of contractility that exists between skeletal and cardiac muscle.

Exercise-Induced Hypertrophic and Oxidative Signaling Pathways and Myokine Expression in Fast Muscle of Adult Zebrafish

Skeletal muscle is a plastic tissue that undergoes cellular and metabolic adaptations under conditions of increased contractile activity such as exercise. Using adult zebrafish as an exercise model, we previously demonstrated that swimming training stimulates hypertrophy and vascularization of fast muscle fibers, consistent with the known muscle growth-promoting effects of exercise and with the resulting increased aerobic capacity of this tissue. Here we investigated the potential involvement of factors and signaling mechanisms that could be responsible for exercise-induced fast muscle remodeling in adult zebrafish. By subjecting zebrafish to swimming-induced exercise, we observed an increase in the activity of mammalian target of rapamycin (mTOR) and Mef2 protein levels in fast muscle. We also observed an increase in the protein levels of the mitotic marker phosphorylated histone H3 that correlated with an increase in the protein expression levels of Pax7, a satellite-like cell marker. Furthermore, the activity of AMP-activated protein kinase (AMPK) was also increased by exercise, in parallel with an increase in the mRNA expression levels of pgc1α and also of pparda, a β-oxidation marker. Changes in the mRNA expression levels of slow and fast myosin markers further supported the notion of an exercise-induced aerobic phenotype in zebrafish fast muscle. The mRNA expression levels of il6, il6r, apln, aplnra and aplnrb, sparc, decorin and igf1, myokines known in mammals to be produced in response to exercise and to signal through mTOR/AMPK pathways, among others, were increased in fast muscle of exercised zebrafish. These results support the notion that exercise increases skeletal muscle growth and myogenesis in adult zebrafish through the coordinated activation of the mTOR-MEF2 and AMPK-PGC1α signaling pathways. These results, coupled with altered expression of markers for oxidative metabolism and fast-to-slow fiber-type switch, also suggest improved aerobic capacity as a result of swimming-induced exercise. Finally, the induction of myokine expression by swimming-induced exercise support the hypothesis that these myokines may have been produced and secreted by the exercised zebrafish muscle and acted on fast muscle cells to promote metabolic remodeling. These results support the use of zebrafish as a suitable model for studies on muscle remodeling in vertebrates, including humans.

Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue

The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define “fatigability” in vivo of fast vs. slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin subfragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH, and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a preequilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs. pH 6.2), and Pi (zero, 15, or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1, but for slow S1, KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.