Slow-twitch Muscle Research Articles

Neuromuscular junction pathology is correlated with differential motor unit vulnerability in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, neuromuscular neurodegenerative disease for which there is no cure. The disease is characterized by a selective decrease in fast-muscle power (e.g., tongue pressure, grip strength) accompanied by a selective loss of fast-twitch muscle fibers. However, the relationship between neuromuscular junction (NMJ) pathology and fast-twitch motor unit vulnerability has yet to be explored. In this study, we used a cross-model comparison of two mouse models of SBMA to evaluate neuromuscular junction pathology, glycolytic-to-oxidative fiber-type switching, and cytoskeletal alterations in pre- and postsynaptic termini of tibialis anterior (TA), gastrocnemius, and soleus hindlimb muscles. We observed significantly increased NMJ and myofiber pathology in fast-twitch, glycolytic motor units of the TA and gastrocnemius compared to slow-twitch, oxidative motor units of the soleus, as seen by decreased pre- and post-synaptic membrane area, decreased pre- and post-synaptic membrane colocalization, increased acetylcholine receptor compactness, a decrease in endplate area and complexity, and deficits in neurofilament heavy chain. Our data also show evidence for metabolic dysregulation and myofiber atrophy that correlate with severity of NMJ pathology. We propose a model in which the dynamic communicative relationship between the motor neuron and muscle, along with the developmental subtype of the muscle, promotes motor unit subtype specific vulnerability, metabolic alterations, and NMJ pathology.

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

BackgroundA common polymorphism (R577X) in the ACTN3 gene results in the complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~ 16% of the world’s population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0 + 20% stretch did not cause eccentric damage. In contrast, L0 + 30% stretch produced a significant ~ 40% deficit in maximum force; here, we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this.MethodsSingle fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA.ResultsWe show that the absence of α-actinin-3 does not affect the visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically stretched. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar branching.ConclusionsThe absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0 + 30%, but not L0 + 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3-deficient humans have been reported to have a faster decline in muscle function with increasing age, that is, as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibre breakages.

93. The Effects of Subnormothermic Plasma and Red Blood Cell Based Perfusion on the Function and Viability of Limb Allografts

Purpose: Vascularized composite allotranplantation (VCA) is an evolving area of modern transplantation medicine to reconstruct composite tissue defects such as abdominal wall, face, and limb. One of the challenges in VCA is the narrow window of tolerable ischemia time which limits the number of available donors and imposes a single center transplantation approach. Current standard of care, static cold storage, is only effective for short periods and may result in severe tissue damage and acute rejection when prolonged. Ex-situ perfusion has the potential to overcome these challenges by extending allowable pre-transplantation ischemia time. However, optimum perfusate composition, preservation temperature, and hemodynamic parameters of perfusion are still debated. Over the past decade, the benefits of addition of fresh plasma in hemorrhagic shock resuscitation and attenuation of subsequent reperfusion injury have been recognized. Whether these observations are due to high or low molecular weight (HMW and LMW, respectively) plasma proteins require further investigation. This study investigated the effects of the addition of plasma on the viability of perfused limb allografts with prolonged ischemia time. Methods: All limb transplantations were performed orthotopically between male Lewis rats (200-250 g). In group 1, amputated limbs were transplanted immediately to determine the effects of surgical trauma. In group 2, amputated limbs were preserved at 4°C simulating the clinical standard of care. In other groups, amputated limbs were perfused for 6 hours at 20° to 22°C using one of the following perfusates groups and transplanted to another recipient (in less than 90 minutes): RBC only (G3), RBC + whole plasma (G4), RBC + LMW plasma (G5), RBC + HMW plasma (G6). At 3 months post-transplantation, surviving recipients had end-point assessment using electromyography where compound muscle action potentials (CMAP) and extensor digitorum longus (EDL) muscle force generation were compared to contralateral healthy limb. Muscle and nerve histology samples were collected at the end of EMG testing. Results: Recipients in the group perfused with RBC and HMW plasma proteins had severe reperfusion injury and did not survive beyond 3 days. Among surviving recipients, the addition of whole plasma did not appear to improve amplitude and latency on gastrocnemius and EDL muscles compared to immediately transplanted limbs. Groups perfused with RBC only and RBC with LMW plasma demonstrated excellent amplitude and latencies on both muscle groups, with no significant difference compared to immediately transplanted limbs (p>0.05). The EDL muscle in these groups also generated similar tetanic and twitch forces compared to the immediate transplantation group (p>0.05). Conclusion: Ex-situ perfusion at 20° to 22°C for 6 hours using RBC and LMW plasma appears to protect both fast and slow twitch muscles, and improve axonal regeneration and myelin sheath thickness to a degree comparable with the immediately transplanted group.

Dietary nitrate increases submaximal SERCA activity and ADP transfer to mitochondria in slow-twitch muscle of female mice.

Rapid oscillations in cytosolic calcium (Ca2+) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca2+, which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca2+ ATPase (SERCA) to sequester Ca2+ within the sarcoplasmic reticulum (SR) during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ∼25% increase in submaximal SERCA activity and SERCA efficiency (P = 0.053) in the soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased by ∼20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest that improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue.NEW & NOTEWORTHY We show that nitrate supplementation increased force production during fatigue and increased submaximal SERCA activity. This was also evident regarding the high-energy phosphate transfer from SERCA to mitochondria, as nitrate increased mitochondrial respiration supported by SERCA-derived ADP. Surprisingly, these observations were only apparent in muscle primarily expressing type I (soleus) but not type II fibers (EDL). These findings suggest that alterations in SERCA properties are a possible mechanism in which nitrate increases force during fatiguing contractions.

The Landscape of DNA Methylation Generates Insight Into Epigenetic Regulation of Differences Between Slow-Twitch and Fast-Twitch Muscles in Pseudocaranx dentex

Skeletal muscles of teleost are mainly composed of slow-twitch muscles (SM) and fast-twitch muscles (FM) differed in contractile properties, metabolic capacities, and regeneration rate. The transcriptional regulatory mechanisms that control different muscle types have been elucidated in teleost according to transcriptome between SM and FM. However, the differences between SM and FM were affected not only by genotype but also by complicated epigenetic effects, including DNA methylation, which usually regulates genes in transcription level. To determine the essential role of DNA methylation in the regulation of different muscle types, we analyzed whole-genome methylation profiles of pelagic migratory fish Pseudocaranx dentex with abundant and well-separated SM and integrated DNA methylation profiles with the previously obtained transcriptome data. A total of 4,217 differentially methylated genes (DMGs) were identified, of which 3,582 were located in the gene body and 635 in the promoter. These DMGs mainly participated in muscle metabolite and cell junction. Enriched cell junction pathway reflected different capillary distribution between SM and FM. Through comprehensive analysis of methylome and transcriptome, 84 differentially expressed genes (DEGs) showed significant methylation variation in promoters between SM and FM, indicating that their expression was regulated by DNA methylation. Hypomethylated and highly expressed oxygen storage protein Myoglobin (myg) in SM indicated demethylation of myg promoter could upregulate its expression, thus increasing O2 supplying and meeting oxygen demands of SM. Hypermethylated and lowly expressed tnn (Troponin) and rlc (myosin regulatory light chain) in SM may be associated with low mobility of myosin cross bridges, which lead to slower and less frequent muscle contraction in SM than in FM. In addition, hypomethylated and highly expressed lbx1 (Ladybird homeobox protein homolog 1) and epo (erythropoietin) may be related to increased satellite cell numbers, and Semaphorin/Plexin genes may be related to higher rate of neuromuscular connection reconstruction, which further promote high muscle regeneration efficiency in SM. Our study elucidated the potential DNA methylation mechanisms that regulate physiological characteristics differences between SM and FM, which could facilitate our understanding of skeletal muscle adaptation in pelagic migratory fishes and further enrich the theoretical basis for the study of physiological characteristics and adaptive evolution in teleost fishes.

Cardiac ventricular myosin and slow skeletal myosin exhibit dissimilar chemomechanical properties despite bearing the same myosin heavy chain isoform

The myosin II motors are ATP-powered force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta (β-MyHC) is primarily expressed in the ventricular myocardium and in slow-twitch muscle fibers, such as M. soleus. M. soleus–derived myosin II (SolM-II) is often used as an alternative to the ventricular β-cardiac myosin (βM-II); however, the direct assessment of biochemical and mechanical features of the native myosins is limited. By employing optical trapping, we examined the mechanochemical properties of native myosins isolated from the rabbit heart ventricle and soleus muscles at the single-molecule level. We found purified motors from the two tissue sources, despite expressing the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. We demonstrate βM-II was approximately threefold faster in the actin filament–gliding assay than SolM-II. The maximum actomyosin (AM) detachment rate derived in single-molecule assays was also approximately threefold higher in βM-II, while the power stroke size and stiffness of the “AM rigor” crossbridge for both myosins were comparable. Our analysis revealed a higher AM detachment rate for βM-II, corresponding to the enhanced ADP release rates from the crossbridge, likely responsible for the observed differences in the motility driven by these myosins. Finally, we observed a distinct myosin light chain 1 isoform (MLC1sa) that associates with SolM-II, which might contribute to the observed kinetics differences between βM-II and SolM-II. These results have important implications for the choice of tissue sources and justify prerequisites for the correct myosin heavy and light chains to study cardiomyopathies.

Maternal vitamin D deficiency affects the morphology and function of glycolytic muscle in adult offspring rats.

BackgroundFetal stage is a critical developmental window for the skeletal muscle, but little information is available about the impact of maternal vitamin D (Vit. D) deficiency (VDD) on offspring lean mass development in the adult life of male and female animals.MethodsFemale rats (Wistar Hannover) were fed either a control (1000 IU Vit. D3/kg) or a VDD diet (0 IU Vit. D3/kg) for 6 weeks and during gestation and lactation. At weaning, male and female offspring were randomly separated and received a standard diet up to 180 days old.ResultsVitamin D deficiency induced muscle atrophy in the male (M‐VDD) offspring at the end of weaning, an effect that was reverted along the time. Following 180 days, fast‐twitch skeletal muscles [extensor digitorum longus (EDL)] from the M‐VDD showed a decrease (20%; P < 0.05) in the number of total fibres but an increase in the cross‐sectional area of IIB (17%; P < 0.05), IIA (19%; P < 0.05) and IIAX (21%; P < 0.05) fibres. The fibre hypertrophy was associated with the higher protein levels of MyoD (73%; P < 0.05) and myogenin (55% %; P < 0.05) and in the number of satellite cells (128.8 ± 14 vs. 91 ± 7.6 nuclei Pax7 + in the M‐CTRL; P < 0.05). M‐VDD increased time to fatigue during ex vivo contractions of EDL muscles and showed an increase in the phosphorylation levels of IGF‐1/insulin receptor and their downstream targets related to anabolic processes and myogenic activation, including Ser 473Akt and Ser 21/9GSK‐3β. In such muscles, maternal VDD induced a compensatory increase in the content of calcitriol (two‐fold; P < 0.05) and CYP27B1 (58%; P < 0.05), a metabolizing enzyme that converts calcidiol to calcitriol. Interestingly, most morphological and biochemical changes found in EDL were not observed in slow‐twitch skeletal muscles (soleus) from the M‐VDD group as well as in both EDL and soleus muscles from the female offspring.ConclusionsThese data show that maternal VDD selectively affects the development of type‐II muscle fibres in male offspring rats but not in female offspring rats and suggest that the enhancement of their size and fatigue resistance in fast‐twitch skeletal muscle (EDL) is probably due to a compensatory increase in the muscle content of Vit. D in the adult age.

Poor hygiene of housing conditions influences energy metabolism in a muscle type-dependent manner in growing pigs differing in feed efficiency

The ability of pigs to cope with inflammatory challenges may by modified by selection for residual feed intake (RFI), a measure of feed efficiency. In the current study, we evaluated skeletal muscle metabolic responses to degraded hygiene conditions in pigs divergently selected for RFI. At 82 d of age, low RFI and high RFI pigs were housed in either poor or good hygiene conditions. After a 6-week challenge, the poor hygiene conditions induced a decrease in growth performance (P < 0.001) and in plasma IGF-I concentrations (P < 0.003) in both lines. In the slow-twitch oxidative semispinalis muscle, poor hygiene conditions induced a shift towards a more oxidative metabolism and an activation of the AMPK pathway in pigs of both RFI lines. In the fast-twitch glycolytic longississimus muscle, poor hygiene conditions were associated to a less glycolytic metabolism in the HRFI line only. Poor hygiene conditions also increased the protein level of lipidation of microtubule-associated protein 1 light-chain 3β (LC3-II) in both RFI lines, suggesting an activation of the autophagy pathway. Altogether, the data revealed muscle-type specific metabolic adaptations to poor hygiene conditions, which may be related to different strategies to fuel the activated immune system.

Reversal of diabetic-induced myopathy by swimming exercise in pregnant rats: a translational intervention study.

Gestational diabetes mellitus (GDM) plus rectus abdominis muscle (RAM) myopathy predicts long-term urinary incontinence (UI). Atrophic and stiff RAM are characteristics of diabetes-induced myopathy (DiM) in pregnant rats. This study aimed to determine whether swimming exercise (SE) has a therapeutic effect in mild hyperglycemic pregnant rats model. We hypothesized that SE training might help to reverse RAM DiM. Mild hyperglycemic pregnant rats model was obtained by a unique subcutaneous injection of 100 mg/kg streptozotocin (diabetic group) or citrate buffer (non-diabetic group) on the first day of life in Wistar female newborns. At 90 days of life, the rats are mated and randomly allocated to remain sedentary or subjected to a SE protocol. The SE protocol started at gestational day 0 and consisted of 60 min/day for 6 days/week in a period of 20 days in a swim tunnel. On day 21, rats were sacrificed, and RAM was collected and studied by picrosirius red, immunohistochemistry, and transmission electron microscopy. The SE protocol increased the fiber area and diameter, and the slow-twitch and fast-twitch fiber area and diameter in the diabetic exercised group, a finding was also seen in control sedentary animals. There was a decreased type I collagen but not type III collagen area and showed a similar type I/type III ratio compared with the control sedentary group. In conclusion, SE during pregnancy reversed the RAM DiM in pregnant rats. These findings may be a potential protocol to consider in patients with RAM damage caused by GDM.

L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens.

L-Arginine (L-Arg), the precursor of nitric oxide (NO), plays an important role in muscle function. Fast-twitch glycolytic fibres are more susceptible to age-related atrophy than slow-twitch oxidative fibres. The effect of L-Arg/NO on protein metabolism of fast- and slow-twitch muscle fibres was evaluated in chickens. In Exp. 1, 48 chicks at 1 day old were divided into 4 groups of 12 birds and subjected to 4 treatments: basal diet without supplementation or supplemented with 1% L-Arg, and water supplemented with or without L-nitro-arginine methyl ester (L-NAME, 18.5 mM). In Exp. 2, 48 chicks were divided into 4 groups of 12 birds fed with the basal diet and subjected to the following treatments: tap water (control), tap water supplemented with L-NAME (18.5 mM), or molsidomine (MS, 0.1 mM), or 18.5 mM L-NAME + 0.1 mM MS (NAMS). The regulatory effect of L-Arg/NO was further investigated in vitro with myoblasts obtained from chicken embryo pectoralis major (PM) and biceps femoris (BF). In vivo, dietary L-Arg supplementation increased breast (+14.94%, P < 0.05) and thigh muscle mass (+23.40%, P < 0.05); whereas, MS treatment had no detectable influence. However, L-NAME treatment blocked the beneficial influence of L-Arg on muscle development. L-Arg decreased (P < 0.05) protein synthesis rate, phosphorylated mTOR and ribosomal protein S6 kinase beta-1 (p70S6K) levels in breast muscle, which was recovered by L-NAME treatment. In vitro, L-Arg or sodium nitroprusside (SNP) reduced protein synthesis rate, suppressed phosphorylated mTOR/p70S6K and decreased atrogin-1 and muscle RING finger 1 (MuRF1) in myoblasts from PM muscle (P < 0.05). L-NAME abolished the inhibitory effect of L-Arg on protein synthesis and the mTOR/p70S6K pathway. However, myoblasts from BF muscle showed the weak influence. Moreover, blocking the mTOR/p70S6K pathway with rapamycin suppressed protein synthesis of the 2 types of myoblasts; whereas, the protein expression of atrogin-1 and MuRF1 levels were restricted only in myoblasts from PM muscle. In conclusion, L-Arg/NO/mTOR/p70S6K pathway enhances protein accumulation and muscle development in fast-twitch glycolytic muscle in chickens. L-Arg/NO regulates protein turnover in a muscle fibre specific way, which highlights the potential clinical application in fast-twitch glycolytic muscle fibres.

Sex Differences in Skeletal Muscle Force Production, Fatiguability and Recovery in Slow and Fast Twitch Muscles

Sex differences in skeletal muscle fatiguability have been well documented during isometric contractions in humans but whether this sexual dimorphism occurs in skeletal muscle at the cellular level is not well understood. Therefore, we aimed to determine if sex differences persisted in skeletal muscle function in isolated tissue. We hypothesized that sexual dimorphisms would not be apparent in twitch and tetanic contraction characteristics or fatigue and recovery of slow and fast twitch muscles. Soleus (SOL) and extensor digitorum longus (EDL) muscles were isolated from male and female CD‐1 mice. Muscle force production at optimal length was measured during 5 protocols: 1) force‐frequency relationship (1‐120Hz), 2) twitch contraction (1Hz), 3) non‐fatiguing contraction protocol (1 contraction/90sec, for 22.5 mins at submaximal (SOL‐25Hz; EDL‐50Hz) and maximal (SOL‐80Hz; EDL‐120Hz) stimulus frequencies), 4) fatiguing contraction protocols at submaximal and maximal stimulus frequencies using contraction frequencies of 15, 30, 45 and 60 contractions per min (cpm) over 5 mins and 5) recovery protocols (1contraction/90sec for 30 mins). The force‐frequency relationship revealed that females generated significantly larger force compared to males at stimulation frequencies of 10‐120Hz (SOL) and 30‐120Hz (EDL). Female SOL and EDL produced significantly higher forces during twitch contractions compared to males (SOL: male 27.8+/‐2.0 mN/mm2 vs. female 34.7+/‐2.4 mN/mm2; EDL: male 32.5+/‐2.1 mN/mm2 vs. female 41.2+/‐2.2 mN/mm2). No significant differences in time to peak contraction or time to half relaxation during the twitch were found between males and females. There were no differences in fatigue rates between male and female SOL or EDL during the non‐fatiguing contraction protocols. Fatiguing contraction protocols revealed that females were less fatiguable than males at submaximal stimulus frequencies at 30 and 45cpm (SOL) and at maximal stimulus frequencies at 45cpm (EDL). No significant differences in fatigue between males and females were found in the remaining 13 fatiguing contraction protocols. Significant differences between males and females in recovery protocols were found in only 4 of the 16 recovery protocols. Female SOL had less recovery of force compared to males at 25Hz at 30 and 45cpm, and at 80Hz at 45 and 60cpm. Collectively, these data demonstrate that there are sexual dimorphisms in absolute force production during twitch and a range of tetanic contractions in slow and fast twitch muscles. Although there were specific contraction parameters that showed enhanced fatigue resistance of females and less recovery of female slow twitch muscles, these findings were not systematic and not present in the vast majority of the fatigue/recovery protocols. Our data show that force production and fatiguability at the in vitro, cellular level cannot explain the in vivo observations in the literature and the lack of sexual dimorphisms present during the majority of fatigue/recovery protocols in vitro indicate that systemic influences may be driving these documented differences at the whole‐body level.

Apocynin Attenuates Diabetes-Induced Skeletal Muscle Dysfunction by Mitigating ROS Generation and Boosting Antioxidant Defenses in Fast-Twitch and Slow-Twitch Muscles.

In response to diabetes mellitus, skeletal muscle is negatively affected, as is evident by reduced contractile force production, increased muscle fatigability, and increased levels of oxidative stress biomarkers. Apocynin is a widely used NADPH oxidase inhibitor, with antioxidant and anti-inflammatory potential. It has been effective for amelioration of a variety of disorders, including diabetic complications. Therefore, the present study was conducted to evaluate the effects and action mechanisms of apocynin in slow- and fast-twitch diabetic rat muscles. Male Wistar rats were rendered diabetic by applying intraperitoneally a single dose of streptozotocin (45 mg/kg). Apocynin treatment (3 mg/kg/day) was administered over 8 weeks. Fasting blood glucose (FBG), insulin tolerance and body weight gain were measured. Both slow (soleus) and fast (extensor digitorum longus, EDL) skeletal muscles were used for muscle function evaluation, oxidative stress markers, and evaluating gene expression using qRT-PCR. Treatment with apocynin significantly reduced FBG levels and enhanced insulin tolerance. Apocynin also prevented muscle contractile dysfunction in EDL muscle but had no significant effect on this parameter in soleus muscles. However, in both types of muscles, apocynin mitigated the oxidative stress by decreasing ROS levels and increasing total glutathione levels and redox state. Concomitantly, apocynin also statistically enhanced Nrf-2 and GLU4 mRNA expression and downregulated NOX2, NOX4, and NF-κB mRNA. Collectively, apocynin exhibits properties myoprotective in diabetic animals. These findings indicate that apocynin predominantly acts as an antioxidant in fast-twitch and slow-twitch muscles but has differential impact on contractile function.

Dog Faces Are Faster than Wolf Faces

Humans domesticated dogs with attention to the facial expressions that dogs produce, selecting for a suite of facial movement. Domestic dogs and humans are adept at accurately understanding one another’s facial expressions and movement around the eyes in dogs is highly valued by humans. This unique, mutual ability to accurately process facial expressions is part of the dog‐human bond. An accurate understanding of how dog and wolf faces differ from one another is fundamental to understanding the processes of animal domestication, evolution of the dog, and the origins of human behavior since the Upper Paleolithic. While we know that dogs have facial musculature that wolves do not regularly possess, our understanding of evolutionary muscle physiology between these two species is incomplete. The present study was designed to assess differential distribution of slow‐twitch (type I) and fast‐twitch (type II) myosin fibers in select facial muscles of dogs and wolves. We tested the hypothesis that dog facial muscle physiology would be more similar to humans than to wolves in terms of myosin fiber type distribution. We used samples from the orbicularis oris muscle (OOM) and the zygomaticus major muscle (ZM) from a range of domestic dog breeds (Canis familiaris) and gray wolves (Canis lupus) that were part of previous studies. Samples were processed for myosin immunohistochemistry (IHC) and we selected 3‐5 sections from each specimen stained for identification of each fiber type in both the OOM and ZM muscles. We created composite images of stained sections and assessed fiber type percentages using ImageJ. Percentage of fast‐twitch fibers in dog samples ranged from 66%‐95% and wolves averaged around 25%. Slow‐twitch fiber percentages in dogs averaged only 10% while wolves averaged 29%. The dog range of type II fibers mirrors the range found in human facial muscles, indicating that the domestication process in dogs also involved selection for rapid facial movement, similar to the movement seen in human faces.

Aerobic exercise training-induced follistatin-like 1 secretion in the skeletal muscle is related to arterial stiffness via arterial NO production in obese rats.

Follistatin‐like 1 (FSTL1), which is mainly secreted from skeletal muscle and myocardium, upregulates protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) phosphorylation in vascular endothelial cells. It is unclear whether skeletal muscle‐ and myocardium‐derived FSTL1 secretion induced by aerobic exercise training is involved in the reduction of arterial stiffness via arterial NO production in obese rats. This study aimed to clarify whether aerobic exercise training‐induced FSTL1 secretion in myocardium and skeletal muscle is associated with a reduction in arterial stiffness via arterial Akt‐eNOS signaling pathway in obese rats. Sixteen Otsuka Long‐Evans Tokushima Fatty (OLETF) obese rats were randomly divided into two groups: sedentary control (OLETF‐CON) and eight‐week aerobic exercise training (treadmill for 60min at 25m/min, 5days/week, OLETF‐AT). Eight Long‐Evans Tokushima Otsuka (LETO) rats were used as a healthy sedentary control group. In OLETF‐CON, serum FSTL1, arterial Akt and eNOS phosphorylation, and arterial nitrite/nitrate (NOx) levels were significantly lower, and carotid‐femoral pulse wave velocity (cfPWV) was significantly greater than those in LETO. These parameters were improved in the OLETF‐AT compared to the OLETF‐CON. In the OLETF‐AT, FSTL1 levels in slow‐twitch fiber‐rich soleus muscle were significantly greater than those in the OLETF‐CON, but not in myocardium, fast‐twitch fiber‐rich tibialis anterior muscle, and adipose tissue. Serum FSTL1 levels were positively correlated with soleus FSTL1, arterial eNOS phosphorylation, and NOx levels and negatively correlated with cfPWV. Thus, aerobic exercise training‐induced FSTL1 secretion in slow‐twitch fiber‐rich muscles may be associated with a reduction in arterial stiffness via arterial NO production in obese rats.

Impact of cell‐free hemoglobin on oxygen pressure dynamics in muscles comprised primarily of fast‐twitch fibers

Cell free hemoglobin (Hb), released due to hemolysis, reduces the bioavailability of nitric oxide (NO) and the microvascular PO2 (PO2mv, the pressure head that facilitates blood‐myocyte O2 flux) during contractions in mixed fiber‐type skeletal muscle. Despite the known impacts of cell‐free Hb on mixed fiber type muscles, its effect within muscles composed primarily of fast‐twitch fibers remains unknown. Thus, the purpose of this investigation was to test the hypothesis that cell free Hb would lower the PO2mv in the white (fast‐twitch) portion of the gastrocnemius muscle during muscle contractions with no effect at rest or during the rest‐contraction transition. Six young‐adult male Sprague‐Dawley rats had the white portion of the gastrocnemius muscle surgically exposed (100% type IIb + dx). PO2mv was measured at rest and during 180 s of electrically induced contractions (1 Hz, 6‐8 v) under control and following 50 mg of Hb infusion (tail‐artery catheter). Hb resulted in a 48% increase in mean arterial pressure at the onset of muscle contractions (control: 97.16 ± 6.5, Hb 143.40 ± 9.0 mmHg, p&lt;0.01), significantly shortened time‐delay (control 14.3 ± 2.0, Hb 4.8 ± 1.6 s, p&lt;0.02), and mean response time (control 24.9 ± 1.2, Hb 16.4 ± 2.0 s, p&lt;0.02). There were no between‐condition differences in steady‐state (control 10.4 ± 1.4, Hb 14.1 ± 1.4 mmHg, p&gt;0.05) or baseline PO2mv (control 16.4 ± 0.2, Hb 19.1 ± 0.01). Acute exposure to cell‐free Hb resulted in a faster fall in the PO2mv during the rest‐contraction transition. However, despite a significant increase in mean arterial pressure, there were no differences in the PO2mv observed during the contracting steady state. These results suggest that cell free Hb significantly increases vascular resistance within fast‐twitch muscle, which causes O2 delivery‐to‐utilization mismatch at the onset of muscle contractions. These findings provide a potential mechanism by which hemolytic diseases like sickle cell disease impair exercise tolerance. Given that slow‐twitch muscles rely more heavily on NO to support O2 delivery at rest and during contractions, it is likely that the effects observed in the present investigation will be exacerbated in highly oxidative muscles.

Skeletal muscle sympathetic denervation disrupts the neuromuscular junction postterminal organization: A single-cell quantitative approach

The sympathetic nervous system (SNS) regulates skeletal muscle motor innervation and stabilizes the NMJ in health, disease and aging. Previous studies using both chemical (6-hydroxydopamine, 6-OHDA) and microsurgically-induced sympathetic denervation examined the NMJ organization and transmission in the mouse; however, a detailed quantification of the postterminal on larger hindlimb muscles involved in gait mechanics and posture is lacking. The purpose of this study was to determine whether targets of the sympathetic neuron (SN) exhibiting different intrinsic composition such as the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles differ in their response to SN deprivation, and to develop a strategy to accurately quantify the impact of sympathectomy on the NMJ postterminal including those fibers located deeper in the muscle. This approach included muscle fixed ex vivo or through transcardial perfusion in mice treated with 6-OHDA or control ascorbic acid. We measured NMJ postterminal mean terminal total area, number of postterminal fragments, mean fragment area, and mean distance between fragments in free-floating alpha-bungarotoxin-stained in 1038 isolated muscle fibers. We found that muscle fiber sympathetic innervation plays a crucial role in the structural organization of the motorneuron-myofiber synapse postterminal and its deprivation leads to AChR cluster dispersion or shrinking as described in various neuromuscular diseases and aging.

Characterization of the Skeletal Muscle Proteome in Undernourished Old Rats.

Aging is associated with a progressive loss of skeletal muscle mass and function termed sarcopenia. Various metabolic alterations that occur with aging also increase the risk of undernutrition, which can worsen age-related sarcopenia. However, the impact of undernutrition on aged skeletal muscle remains largely under-researched. To build a deeper understanding of the cellular and molecular mechanisms underlying age-related sarcopenia, we characterized the undernutrition-induced changes in the skeletal muscle proteome in old rats. For this study, 20-month-old male rats were fed 50% or 100% of their spontaneous intake for 12 weeks, and proteomic analysis was performed on both slow- and fast-twitch muscles. Proteomic profiling of undernourished aged skeletal muscle revealed that undernutrition has profound effects on muscle proteome independently of its effect on muscle mass. Undernutrition-induced changes in muscle proteome appear to be muscle-type-specific: slow-twitch muscle showed a broad pattern of differential expression in proteins important for energy metabolism, whereas fast-twitch muscle mainly showed changes in protein turnover between undernourished and control rats. This first proteomic analysis of undernourished aged skeletal muscle provides new molecular-level insight to explain phenotypic changes in undernourished aged muscle. We anticipate this work as a starting point to define new biomarkers associated with undernutrition-induced muscle loss in the elderly.

Effect of Long-Term Supplementation with Acetic Acid on the Skeletal Muscle of Aging Sprague Dawley Rats

Mitochondrial function in skeletal muscle, which plays an essential role in oxidative capacity and physical activity, declines with aging. Acetic acid activates AMP-activated protein kinase (AMPK), which plays a key role in the regulation of whole-body energy by phosphorylating key metabolic enzymes in both biosynthetic and oxidative pathways and stimulates gene expression associated with slow-twitch fibers and mitochondria in skeletal muscle cells. In this study, we investigate whether long-term supplementation with acetic acid improves age-related changes in the skeletal muscle of aging rats in association with the activation of AMPK. Male Sprague Dawley (SD) rats were administered acetic acid orally from 37 to 56 weeks of age. Long-term supplementation with acetic acid decreased the expression of atrophy-related genes, such as atrogin-1, muscle RING-finger protein-1 (MuRF1), and transforming growth factor beta (TGF-β), activated AMPK, and affected the proliferation of mitochondria and type I fiber-related molecules in muscles. The findings suggest that acetic acid exhibits an anti-aging function in the skeletal muscles of aging rats.

2,3',4,4',5-Pentachlorobiphenyl attenuated fast-twitch fibers and fiber size of skeletal muscle via disturbing thyroid hormone signaling and mitochondrial dynamics.

Polychlorinated biphenyls (PCBs) affect multiple organs, and some of the effects are mediated by interfering with thyroid hormone (TH) signaling that regulates physiological processes in mammals. It remains unclear how PCBs affect skeletal muscle (SM). In our study, wistar rats were injected 2,3',4,4',5-pentachlorobiphenyl (PCB118) intraperitoneally at 0, 10, 100, and 1000 μg/kg/day for 13 weeks, and C2C12 myoblasts were treated PCB118 (0, 0.25, 25, and 50 nM) for 24 h or 48 h. We found that myocyte cross-sectional area (MCSA) was reduced, MyHC IIa and MyHC IIb mRNA levels significantly decreased, and muscle strength was weakened in PCB118-exposed rats. TH receptor α (TRα) and iodothyronine deiodinase type 2 (DIO2) were upregulated after PCB118 exposure both in vivo and in vitro. Transmission electron microscopy showed significant mitochondrial abnormalities in PCB118-treated rats, and the expression of mitochondrial regulators such as PTEN-induced kinase 1 (PINK1) and GTPase dynamin-related protein 1 (DRP1) were altered after PCB118 exposure. These results suggest that PCB118 could weaken muscle strength and attenuate fast-twitch fibers and fiber size of SM in rats. TH signaling, mitochondrial dynamics and mitophagy were also disturbed by PCB118, which may contribute to the alternations of SM structure and function.

LncRNA SMARCD3-OT1 Promotes Muscle Hypertrophy and Fast-Twitch Fiber Transformation via Enhancing SMARCD3X4 Expression.

Long noncoding RNA (lncRNA) plays a crucial part in all kinds of life activities, especially in myogenesis. SMARCD3 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3) is a member of the SWI/SNF protein complex and was reported to be required for cell proliferation and myoblast differentiation. In this study, we identified a new lncRNA named SMARCD3-OT1 (SMARCD3 overlapping lncRNA), which strongly regulated the development of myogenesis by improving the expression of SMARCD3X4 (SMARCD3 transcripts 4). We overexpressed and knockdown the expression of SMARCD3-OT1 and SMARCD3X4 to investigate their function on myoblast proliferation and differentiation. Cell experiments proved that SMARCD3-OT1 and SMARCD3X4 promoted myoblast proliferation through the CDKN1A pathway and improved differentiation of differentiated myoblasts through the MYOD pathway. Moreover, they upregulated the fast-twitch fiber-related genes and downregulated the slow-twitch fiber-related genes, which indicated that they facilitated the slow-twitch fiber to transform into the fast-twitch fiber. The animals’ experiments supported the results above, demonstrating that SMARCD3-OT1 could induce muscle hypertrophy and fast-twitch fiber transformation. In conclusion, SMARCD3-OT1 can improve the expression of SMARCD3X4, thus inducing muscle hypertrophy. In addition, SMARCD3-OT1 can facilitate slow-twitch fibers to transform into fast-twitch fibers.