Myosin Heavy Chain Isoforms Research Articles

Impacts of whole-body vibration on denervated skeletal-muscle atrophy in rats.

Whole-body vibration has been considered as a countermeasure against muscle atrophy. However, its effects on muscle atrophy are poorly understood. We evaluated the effects of whole-body vibration on denervated skeletal muscle atrophy. Whole-body vibration was performed on rats from Day 15 to 28 after denervation injury. Motor performance was evaluated using an inclined-plane test. Compound muscle action potentials of the tibial nerve were examined. Muscle wet weight and muscle fiber cross-sectional area were measured. Myosin heavy chain isoforms were analyzed in both muscle homogenates and single myofibers. Whole-body vibration resulted in a significantly decreased inclination angle and muscle weight, but not muscle fiber cross-sectional area of fast-twitch gastrocnemius compared to denervation only. In denervated gastrocnemius, a fast-to-slow shift was observed in myosin heavy chain isoform composition following whole-body vibration. There were no significant changes in muscle weight, muscle fiber cross-sectional area, and myosin heavy chain isoform composition in denervated slow-twitch soleus. These results imply that whole-body vibration does not promote recovery of denervation-induced muscle atrophy.

Birth trauma simulated by vaginal distention induced possible irreversible changes in the composition of the fiber types in the external urethral sphincter of rats.

The external urethral sphincter (EUS) is crucial in urinary continence development. Understanding the morphological features of the EUS in female rats after vaginal distention (VD), using a model of birth trauma, would aid in evaluating its functional and metabolic properties. Our recent study demonstrated that the EUS in female rats expresses one slow (type 1) and two fast (types 2A and 2B) myosin heavy chain (MHC) isoforms. Our preliminary experiment revealed that type 2B isoform expression was markedly reduced in the EUS 4 weeks after VD. Here, we aimed to examine the expression patterns of these three types of MHC isoforms, and an embryonic MHC, a marker of regeneration fibers, in the EUS of rats 3 days and 1, 2, and 8 weeks after VD using immunofluorescence staining. Hence, type 2B fibers were selectively damaged early in post-VD and did not recover fully later. Muscle regeneration in the sphincter peaked 1 week after trauma using a marker of immature fibers, embryonic myosin heavy chain. Electron microscopy revealed that the EUS of female rats was composed of mitochondria-rich muscle fibers. Myoblasts or immature muscle fibers were discovered in the sphincter layer 1 week after trauma. These results suggest that myogenesis after VD may not contribute to restoring normal fiber composition in a female rat's EUS.

Effects of Creatine Supplementation on the Myostatin Pathway and Myosin Heavy Chain Isoforms in Different Skeletal Muscles of Resistance-Trained Rats

Creatine has been used to maximize resistance training effects on skeletal muscles, including muscle hypertrophy and fiber type changes. This study aimed to evaluate the impact of creatine supplementation on the myostatin pathway and myosin heavy chain (MyHC) isoforms in the slow- and fast-twitch muscles of resistance-trained rats. Twenty-eight male Wistar rats were divided into four groups: a sedentary control (Cc), sedentary creatine supplementation (Cr), resistance training (Tc), and resistance training combined with creatine supplementation (Tcr). Cc and Tc received standard commercial chow; Cr and Tcr received a 2% creatine-supplemented diet. Tc and Tcr performed a resistance training protocol on a ladder for 12 weeks. Morphology, MyHC isoforms, myostatin, follistatin, and ActRIIB protein expressions were analyzed in soleus and white gastrocnemius portion samples. The results were analyzed using two-way ANOVA and Tukey’s test. Tc and Tcr exhibited higher performance than their control counterparts. Resistance training increased the ratio between muscle and body weight, the cross-sectional area, as well as the interstitial collagen fraction. Resistance training alone increased MyHC IIx and follistatin while reducing myostatin (p < 0.001) and ActRIIB (p = 0.040) expressions in the gastrocnemius. Resistance training induced skeletal muscle hypertrophy and interstitial remodeling, which are more evident in the gastrocnemius muscle. The effects were not impacted by creatine supplementation.

High sensitivity top-down proteomics captures single muscle cell heterogeneity in large proteoforms.

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single-cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we detected multiple isoforms of myosin heavy chain (~223kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems.

Acute fatigue induces paradoxical increases in velocity and power in a sex and myosin isoform dependent manner

OBJECTIVE or our study was to identify effects of acute fatigue on myofibrillar components and their function that persist beyond modifications to intracellular metabolic perturbations. Our HYPOTHESIS was that acute fatigue would induce differential phosphorylation in regulatory proteins in the sarcomere that would reduce tension and increase velocity and power. We used the following METHODS to test that hypothesis. Seven healthy, competitive athletes were recruited to participate (3 male, 4 female), entailing one session of unilateral fatiguing knee extension exercise to the point of task failure. Immediately after, bilateral percutaneous needle muscle biopsy was performed on the vastus lateralis muscle, providing a “fatigued” and “not fatigued” sample. Tissue was separated for phosphoproteomic analysis (western blot and isoelectric separation) and single fiber contractile mechanics (isotonic load clamps; isometric tension, velocity and power). Following mechanical assessment, myosin heavy chain (MHC) isoform was determined for each fiber via SDS-PAGE. Our RESULTS found no effect of fatigue on isometric tension across all fiber types. However, maximum shortening velocity was increased along with contractile power, consistent with our hypotheses. Surprisingly, our results varied by sex and fiber type, such that velocity was increased primarily in MHC IIA/X fibers in males, whereas females demonstrated a blunted response to fatigue. While baseline variation in regulatory protein phosphorylation (Myosin Binding Protein C; MyBP-C) were significantly correlated with power output in contractile power in fast isoforms (MHC IIA, MHC IIA/X) we did not detect significant alterations in phosphorylation of this protein with fatigue. We CONCLUDE that fatigue causes paradoxical increased in velocity and power that are particularly evident in males. While our measurement of MyBP-C phosphorylation did not detect effects of fatigue, other potential sarcomeric proteins may explain this paradoxical response to acute fatigue. Wu Tsai Human Performance Alliance This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Platinum-based anticancer drugs-induced downregulation of myosin heavy chain isoforms in skeletal muscle of mouse

Cisplatin, a platinum-based anticancer drug used frequently in cancer treatment, causes skeletal muscle atrophy. It was predicted that the proteolytic pathway is enhanced as the mechanism of this atrophy. Therefore, we investigated whether a platinum-based anticancer drug affects the expression of the major proteins of skeletal muscle, myosin heavy chain (MyHC). Mice were injected with cisplatin or oxaliplatin for four consecutive days. C2C12 myotubes were treated using cisplatin and oxaliplatin. Administration of platinum-based anticancer drug reduced quadriceps mass and muscle strength compared to the control group. Protein levels of all MyHC isoforms were reduced in the platinum-based anticancer drug groups. However, only Myh2 (MyHC-IIa) gene expression in skeletal muscle of mice treated with platinum-based anticancer drugs was found to be reduced. Treatment of C2C12 myotubes with platinum-based anticancer drugs reduced the protein levels of all MyHCs, and treatment with the proteasome inhibitor MG-132 restored this reduction. The expression of Mef2c, which was predicted to act upstream of Myh2, was reduced in the skeletal muscle of mice treated systemically with platinum-based anticancer drug. Degradation of skeletal muscle MyHCs by proteasomes may be a factor that plays an important role in muscle mass loss in platinum-based anticancer drug-induced muscle atrophy.

Depressed myocardial cross-bridge cycling kinetics in a female guinea pig model of diastolic heart failure.

Cardiac hypertrophy is associated with diastolic heart failure (DHF), a syndrome in which systolic function is preserved but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling are poorly understood. Accordingly, chronic pressure overload was induced by surgically banding the thoracic ascending aorta (AOB) in ∼400 g female Dunkin Hartley guinea pigs (AOB); Sham-operated age-matched animals served as controls. Guinea pigs were chosen to avoid the confounding impacts of altered myosin heavy chain (MHC) isoform expression seen in other small rodent models. In vivo cardiac function was assessed by echocardiography; cardiac hypertrophy was confirmed by morphometric analysis. AOB resulted in left ventricle (LV) hypertrophy and compromised diastolic function with normal systolic function. Biochemical analysis revealed exclusive expression of β-MHC isoform in both sham control and AOB LVs. Myofilament function was assessed in skinned multicellular preparations, skinned single myocyte fragments, and single myofibrils prepared from frozen (liquid N2) LVs. The rates of force-dependent ATP consumption (tension-cost) and force redevelopment (Ktr), as well as myofibril relaxation time (Timelin) were significantly blunted in AOB, indicating reduced cross-bridge cycling kinetics. Maximum Ca2+ activated force development was significantly reduced in AOB myocytes, while no change in myofilament Ca2+ sensitivity was observed. Our results indicate blunted cross-bridge cycle in a β-MHC small animal DHF model. Reduced cross-bridge cycling kinetics may contribute, at least in part, to the development of DHF in larger mammals, including humans.

Tumor Necrosis Factor-α Mediates Inflammation-induced Early-Stage Left Ventricular Systolic Dysfunction.

Elevated systemic inflammation contributes to pathogenesis of heart failure with preserved ejection fraction (HFpEF), but molecular mechanisms are poorly understood. Although left ventricular (LV) diastolic dysfunction is the main cause of HFpEF, subclinical systolic dysfunction also contributes. We have previously shown that rats with collagen-induced arthritis (CIA) have systemic inflammation, LV diastolic dysfunction, and that increased circulating TNF-α contributes to inflammation-induced HFpEF pathogenesis, but does not mediate LV diastolic dysfunction in CIA rats. Contribution of systemic inflammation to dysfunction of the active process of LV diastolic and systolic function are unknown. In the present study, we used the CIA rat model to investigate the effects of systemic inflammation and TNF-α blockade on systolic function, and mRNA expression of genes involved in active diastolic relaxation and of myosin heavy chain (MyHC) isoforms. Collagen inoculation and TNF-α blockade did not affect LV mRNA expression of genes that mediate active LV diastolic function. Collagen-induced inflammation impaired LV global longitudinal strain ( P = 0.03) and velocity ( P = 0.04). This impairment of systolic function was prevented by TNF-α blockade. Collagen inoculation decreased mRNA expression of α-MyHC ( Myh6, P = 0.03) and increased expression of β-MyHC ( Myh7, P = 0.0002), a marker, which is upregulated in failing hearts. TNF-α blockade prevented this MyHC isoform-switch. These results show that increased circulating TNF-α changes the relative expression of MyHC isoforms, favoring β-MyHC, which may underlie changes in contractile function that impair systolic function. Our results indicate that TNF-α initiates early-stage LV systolic, rather than LV diastolic dysfunction.

Expression of neurotransmitters, vasculogenesis markers and myosin heavy chain isoforms in the masseter muscle of senescence-accelerated mouse prone 8 mice.

Learning and memory deficits and pathologic changes in the hippocampus caused by toothlessness and soft diet feeding are related to reduced masseter muscle (MM) function. Myosin heavy chain (MyHC) isoform expression in the MM also changes under different chewing conditions. The neurotransmitter calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor A (VEGF-A) are involved in MM formation. However, the relationship between CGRP, VEGF-A and MyHC isoforms in the MM in the senescence-accelerated mouse prone 8 (SAMP8) strain, a model of learning and memory deficits, remains unclear. Changes in CGRP, VEGF-A, vasculogenesis marker, and MyHC isoform mRNA expression in the MMs of aging SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice was investigated through quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. qRT-PCR revealed obviously high CGRP levels in the SAMP8 mouse MM (p<0.001). MyHC-IId/x mRNA expression in the MM was higher in 24-week-old SAMP8 mice than 24-week-old SAMR1 mice (p<0.001) but lower in slow-MyHC SAMP8 mice than SAMR1 mice (p<0.001). CGRP mRNA was observed on the muscle fibers of the SAMP8 mouse MM but not the SAMR1 mouse MM through in situ hybridization. Principal component analysis (PCA) revealed strong positive contributions of SAMP8-MyHC-IId/x, SAMP8-CGRP, SAMR1-MyHC-emb, SAMR1-CGRP, SAMR1-VEGF-A, SAMR1-CD31, SAMP8-VEGF-A, and SAMP8-CD31 in the MM at 12 and 24 weeks. CGRP is also key for the MyHC-IId/x and slow-MyHC patterns in the MMs of SAMP8 mice.

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle

Mechanical unloading of skeletal muscles leads to the development of atrophic processes and a decrease in the total number of satellite cells (SCs) that are involved in muscle regeneration. In vitro studies revealed an increased differentiation of myoblasts derived from rat soleus muscle after an unloading-induced decrease in AMP-activated protein kinase (AMPK). AMPK is necessary for the activation of SCs and also participates in the regulation of myoblast proliferation and differentiation. It can be assumed that a decrease in the activity of AMPK after mechanical unloading can contribute to the acceleration of myoblast differentiation. The main purpose of this study was to elucidate a possible role of AMPK in the regulation of differentiation of myoblasts isolated from rat soleus muscle after mechanical unloading. To test this hypothesis, a specific AMPK activator, AICAR, was used to prevent a decrease in AMPK activity during differentiation of myoblasts isolated from rat soleus muscle after 7-day unloading. Immunocytochemistry, PCR-RT and Western blotting were used to assess changes during myoblast differentiation. In differentiating myoblasts derived from the unloaded soleus muscle there was a significant decrease in AMPK (Thr172) and ACC (Ser 79) phosphorylation levels, an increase in myotube differentiation index, myoblast fusion factors and the expression of myogenic regulatory factors (MRF). Furthermore, there was a decrease in the expression of slow myosin heavy chains (MyHC) and an increase in the expression of fast MyHC isoforms. AICAR treatment of differentiating myoblasts obtained from the unloaded soleus muscle prevented a decrease in AMPK and ACC phosphorylation, returned the expression levels of MRF and fast isoforms of MyHC to the control levels as well as maintained the expression of slow MyHC. Thus, abnormally accelerated differentiation of myoblasts isolated from atrophied rat soleus muscle can be compensated by maintaining the control levels of AMPK activity using AICAR.

Altered coronary artery function, arteriogenesis and endothelial YAP signaling in postnatal hypertrophic cardiomyopathy.

Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular genetic disease caused largely by sarcomere protein mutations. Gaps in our understanding exist as to how maladaptive sarcomeric biophysical signals are transduced to intra- and extracellular compartments leading to HCM progression. To investigate early HCM progression, we focused on the onset of myofilament dysfunction during neonatal development and examined cardiac dynamics, coronary vascular structure and function, and mechano-transduction signaling in mice harboring a thin-filament HCM mutation. Methods: We studied postnatal days 7-28 (P7-P28) in transgenic (TG) TG-cTnT-R92Q and non-transgenic (NTG) mice using skinned fiber mechanics, echocardiography, biochemistry, histology, and immunohistochemistry. Results: At P7, skinned myofiber bundles exhibited an increased Ca2+-sensitivity (pCa50 TG: 5.97 ± 0.04, NTG: 5.84 ± 0.01) resulting from cTnT-R92Q expression on a background of slow skeletal (fetal) troponin I and α/β myosin heavy chain isoform expression. Despite the transition to adult isoform expressions between P7-P14, the increased Ca2+- sensitivity persisted through P28 with no apparent differences in gross morphology among TG and NTG hearts. At P7 significant diastolic dysfunction was accompanied by coronary flow perturbation (mean diastolic velocity, TG: 222.5 ± 18.81mm/s, NTG: 338.7 ± 28.07mm/s) along with localized fibrosis (TG: 4.36% ± 0.44%, NTG: 2.53% ± 0.47%). Increased phosphorylation of phospholamban (PLN) was also evident indicating abnormalities in Ca2+ homeostasis. By P14 there was a decline in arteriolar cross-sectional area along with an expansion of fibrosis (TG: 9.72% ± 0.73%, NTG: 2.72% ± 0.2%). In comparing mechano-transduction signaling in the coronary arteries, we uncovered an increase in endothelial YAP expression with a decrease in its nuclear to cytosolic ratio at P14 in TG hearts, which was reversed by P28. Conclusion: We conclude that those early mechanisms that presage hypertrophic remodeling in HCM include defective biophysical signals within the sarcomere that drive diastolic dysfunction, impacting coronary flow dynamics, defective arteriogenesis and fibrosis. Changes in mechano-transduction signaling between the different cellular compartments contribute to the pathogenesis of HCM.

Gαi2 regulates the adult myogenesis of masticatory muscle satellite cells

Although similar to trunk and limb skeletal muscles, masticatory muscles are believed as unique in both developmental origins and myogenesis. Gαi2 has been demonstrated to promote muscle hypertrophy and muscle satellite cell differentiation in limb muscles. However, the effect of Gαi2 on masticatory muscles is still unexplored. This study aimed to identify the role of Gαi2 in the proliferation and differentiation of masticatory muscle satellite cells, further exploring the metabolic mechanism of masticatory muscles. The proliferation rate, myotube size, fusion index of masticatory muscle satellite cells and Pax7, Myf5, MyoD, Tcf21 and Musculin expressions were significantly decreased by Gαi2 knockdown, while in cells infected with AdV4-Gαi2, the proliferation rate, myotube size, fusion index and Tbx1 expression were significantly increased. Masticatory muscle satellite cells also displayed phenotype transformation as Gαi2 changed. In addition, Gαi2 altered myosin heavy chain (MyHC) isoforms of myotubes with less MyHC-2A expression in siGαi2 group and more MyHC-slow expression in AdV4-Gαi2 group. In conclusion, Gαi2 could positively affect the adult myogenesis of masticatory muscle satellite cells and maintain the superiority of MyHC-slow. Masticatory muscle satellite cells may have their unique Gαi2-regulated myogenic transcriptional networks, although they may share some common characteristics with trunk and limb muscles.

Proteome network analysis of skeletal muscle in lignan-enriched nutmeg extract-fed aged mice

Sarcopenia, characterized by reduced muscle mass and fiber number leading to muscular atrophy, has been associated with serious socioeconomic challenges among the elderly in developed countries. Therefore, preventing sarcopenia could be a promising strategy for achieving a healthy aging society. Nutmeg (Myristica fragrans) has been used as a spice to increase flavor and prevent putrefaction of food. Nutmeg contains various bioactive components that improve muscle activity. To determine the potential effect of lignan-enriched nutmeg extract (LNX) on sarcopenia, LNX (100 mg/kg body weight)-fed aged mice were subjected to forced exercise. Herein, aged (22-month-old) mice fed LNX for three weeks exhibited a shortened and thickened soleus muscle. The ratio of the soleus muscle mass (%) to body weight was significantly increased in LNX-fed aged mice. The relative increase in muscle mass in LNX-fed aged mice improved exercise activities, including rotarod, swimming, and grip strength test results. Proteome profiles of the soleus muscle of LNX-fed mice were used to analyze protein–protein interaction network. Several myosin heavy chain isoforms were found to interact with actin, ACTA1, which functions as a hub protein. Furthermore, the expression of myogenic proteins, such as MYH1, MYH4, and ACTA1, was dose-dependently increased in vivo. In result, our functional proteomic analysis revealed that feeding LNX restored muscle proteins in aged mice.

Atrial cardiomyocytes from a canine model of atrial fibrillation demonstrate reduced force development and altered expression of contractile proteins.

Atrial fibrillation (AF) is the most common cardiac rhythm disturbance and associates with worse outcomes in cardiac contractile-related diseases including cardiomyopathy. As most studies surrounding AF focus on the electrophysiology of the disease, there is little known about cardiomyocyte contractile function in AF. We used atrial tissue from an established atrial tachypacing canine model of AF. Whole protein lysates were collected from the atria to conduct mass spectrometry analysis showing dysregulation of numerous molecular pathways, including contractility. Skinned atrial cardiomyocytes were isolated from the tissue for force calcium and passive tension experiments. Force calcium experiments showed a significant reduction in the maximum force of contraction of AF cardiomyocytes. To explain this, myosin heavy chain (MHC) isoform expression was evaluated because isoform switching is known to alter force of contraction. Decreased percent beta expression was found, explaining the reduction of force due to MHC alpha's capacity for reduced force of contraction. AF cardiomyocytes showed increased calcium sensitivity of force development, which may translate to prolonged contraction with slower relaxation in vivo. A significant reduction in S100A1 was seen in the AF samples via mass spectrometry, which is associated with increased calcium sensitivity. Prolonged contraction along with reduced force of each contraction could support the development of atrial clots. Atrial cardiomyocytes from the AF hearts demonstrated a significant reduction in the passive tension. Evaluation of titin isoform expression, which is known to alter passive tension, showed increased titin degradation, supporting the capacity for atrial dilation in vivo. Discerning the role of contractility in the pathology of atrial fibrillation could provide novel avenues of AF treatment, especially in instances where it is associated with contractile diseases.

Stretch activated force production increases during fatigue in fast-contracting skeletal muscle fibers.

Stretch activation (FSA) is the delayed increase in fiber tension (force per cross-sectional area) following a rapid stretch and can improve muscle performance during repetitive cyclical contractions. Historically considered minimal in skeletal muscle, our recent work showed the ratio of stretch activation to calcium-activated tension (FSA/F0) increased from 10 to 40% with greater inorganic phosphate (Pi) levels in soleus muscle fibers (Straight et al., 2019). Given Pi increases with muscle fatigue, we hypothesize that FSA helps maintain force generation during fatigue. To test this, FSA, induced by a stretch of 0.5% fiber length, was examined during control (pCa 4.5, pH 7.0, Pi 5 mM), high calcium fatigue (pCa 4.5, pH 6.2, Pi 30 mM) and low calcium fatigue (pCa 5.1, pH 6.2, Pi 30 mM) in fibers expressing myosin heavy chain (MHC) I, IIA, IIX and IIB isoforms from soleus and extensor digitorum longus muscles of C57BL/6NJ mice. F0 of all MHC isoforms decreased from control to high calcium fatigue to low calcium fatigue, as expected. In MHC IIX and IIB fibers, FSA occurred under all conditions and FSA/F0 increased from control (17-20%) to high calcium fatigue (32-35%) to low calcium fatigue (42-44%). In MHC IIA fibers, FSA/F0 was similar to MHC IIX and IIB fibers in control and high calcium fatigue, however MHC I fiber values were much less (5% and 18%). For MHC I and IIA fibers, no discernable FSA was apparent in low calcium fatigue. These results show that FSA is a significant modulator of force production under fatiguing conditions in fast-contracting muscle fibers. This mechanism could play an important physiological role during cyclical contractions, when the antagonistic muscle rapidly stretches the agonist muscle, by reducing the effect of fatigue on force production.

Time-dependent unloading effects on muscle and bone and involvement of FNDC5/irisin axis

The identification of biomarkers and countermeasures to prevent the adverse effects on the musculoskeletal system caused by the absence of mechanical loading is the main goal of space biomedical research studies. In this study, we analyzed over 4 weeks of unloading, the modulation in the expression of key proteins in Vastus lateralis, Gastrocnemius and cortical bone in parallel with the modulation of irisin serum levels and its precursor FNDC5 in skeletal muscle of hind limb unloaded (HU) mice. Here we report that Atrogin-1 was up-regulated as early as 1- and 2-week of unloading, whereas Murf-1 at 2- and 3-weeks, along with a marked modulation in the expression of myosin heavy chain isoforms during unloading. Since HU mice showed reduced irisin serum levels at 4-weeks, as well as FNDC5 decrease at 3- and 4-weeks, we treated HU mice with recombinant irisin for 4 weeks, showing that unloading-dependent decline of myosin heavy chain isoforms, MyHCIIα and MyHCIIx, and the anti-apoptotic factor Bcl2, were prevented. In parallel, irisin treatment inhibited the increase of the senescence marker p53, and the pro-apoptotic factor Bax. Overall, these results suggest that the myokine irisin could be a possible therapy to counteract the musculoskeletal impairment caused by unloading.

Temperature Fluctuations Modulate Molecular Mechanisms in Skeletal Muscle and Influence Growth Potential in Beef Steers.

Our investigation elucidated the effects of severe temperature fluctuations on cellular and physiological responses in beef cattle. Eighteen Red Angus beef steers with an average body weight of 351 ± 24.5 kg were divided into three treatment groups: 1) Control (CON), exposed to a temperature-humidity index (THI) of 42 for 6 h without any temperature changes; 2) Transport (TP), subjected to a one-mile trailer trip with a THI of 42 for 6 h; and 3) Temperature swing (TS), exposed to a one-mile trailer trip with a THI shift from 42 to 72-75 for 3 h. Our findings indicate that TS can induce thermal stress in cattle, regardless of whether the overall temperature level is excessively high or not. Behavioral indications of extreme heat stress in the cattle were observed, including extended tongue protrusion, reduced appetite, excessive salivation, and increased respiratory rate. Furthermore, we observed a pronounced overexpression (P < 0.05) of heat shock proteins (HSPs) 20, 27, and 90 in response to the TS treatment in the longissimus muscle (LM). Alterations in signaling pathways associated with skeletal muscle growth were noted, including the upregulation (P < 0.01) of Pax7, Myf5, and myosin heavy chain (MHC) isoforms. In addition, an increase (P < 0.05) in transcription factors associated with adipogenesis was detected (P < 0.05), such as PPARγ, C/EBPα, FAS, and SCD in the TS group, suggesting the potential for adipose tissue accumulation due to temperature fluctuations. Our data illustrated the potential impacts of these temperature fluctuations on the growth of skeletal muscle and adipose tissue in beef cattle.

FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism.

The proportions of the various muscle fiber types are important in the regulation of skeletal muscle metabolism, as well as animal meat production. Four-and-a-half LIM domain protein 3 (FHL3) is highly expressed in fast glycolytic muscle fibers and differentially regulates the expression of myosin heavy chain (MyHC) isoforms at the cellular level. Whether FHL3 regulates the transformation of muscle fiber types in vivo and the regulatory mechanism is unclear. In this study, muscle-specific FHL3 transgenic mice were generated by random integration, and lentivirus-mediated gene knockdown or overexpression in muscles of mice or pigs was conducted. Functional analysis showed that overexpression of FHL3 in muscles significantly increased the proportion of fast-twitch myofibers and muscle mass but decreased muscle succinate dehydrogenase (SDH) activity and whole-body oxygen consumption. Lentivirus-mediated FHL3 knockdown in muscles significantly decreased muscle mass and the proportion of fast-twitch myofibers. Mechanistically, FHL3 directly interacted with the Yin yang 1 (YY1) DNA-binding domain, repressed the binding of YY1 to the fast glycolytic MyHC2b gene regulatory region, and thereby promoted MyHC2b expression. FHL3 also competed with EZH2 to bind the repression domain of YY1 and reduced H3K27me3 enrichment in the MyHC2b regulatory region. Moreover, FHL3 overexpression reduced glucose tolerance by affecting muscle glycolytic metabolism, and its mRNA expression in muscle was positively associated with hemoglobin A1c (HbA1c) in patients with type 2 diabetes. Therefore, FHL3 is a novel potential target gene for the treatment of muscle metabolism-related diseases and improvement of animal meat production.

THRIFTY-A High-throughput Single Muscle Fiber Typing Method Based on Immunofluorescence Detection.

Skeletal muscle consists of a mixture of fiber types with different functional and metabolic characteristics. The relative composition of these muscle fiber types has implications for muscle performance, whole-body metabolism, and health. However, analyses of muscle samples in a fiber type-dependent manner are very time consuming. Therefore, these are often neglected in favor of more time-efficient analyses on mixed muscle samples. Methods such as western blot and myosin heavy chain separation by SDS-PAGE have previously been utilized to fiber type-isolated muscle fibers. More recently, the introduction of the dot blot method significantly increased the speed of fiber typing. However, despite recent advancements, none of the current methodologies are feasible for large-scale investigations because of their time requirements. Here, we present the protocol for a new method, which we have named THRIFTY (high-THRoughput Immunofluorescence Fiber TYping), that enables rapid fiber type identification using antibodies towards the different myosin heavy chain (MyHC) isoforms of fast and slow twitch muscle fibers. First, a short segment (<1 mm) is cut off from isolated muscle fibers and mounted on a customized gridded microscope slide holding up to 200 fiber segments. Second, the fiber segments attached to the microscope slide are stained with MyHC-specific antibodies and then visualized using a fluorescence microscope. Lastly, the remaining pieces of the fibers can either be collected individually or pooled together with fibers of the same type for subsequent analyses. The THRIFTY protocol is approximately three times as fast as the dot blot method, which enables not only time-sensitive assays to be performed but also increases the feasibility to conduct large-scale investigations into fiber type specific physiology. Graphical Overview Graphical overview of the THRIFTY workflow. Cut off a small segment (0.5 mm) of an individually dissected muscle fiber and mount it onto the customized microscope slide containing a printed grid system. Using a Hamilton syringe, fixate the fiber segment by applying a small droplet of distilled water on the segment and let it fully dry (1A). The remaining large segment of the fiber should be placed in the corresponding square on a black A4 paper (1B). Once the microscope slide has been fully mounted with fiber segments, submerge the slide in a polypropylene slide mailer (illustrated as a Coplin jar in the figure) containing acetone to permeabilize the fiber segments. Thereafter, incubate the slide with primary antibodies targeting MyHC-I and MyHC-II. Following washes in PBS solution, incubate the slides with fluorescently labeled secondary antibodies, wash again, and mount with a cover glass and antifade reagent (2). Identification of fiber type can be performed using a digital fluorescence microscope (3), whereafter the remaining pieces of the fiber segments (large) are pooled together according to their fiber type or individually collected for experiments on single fibers (4). Image modified from Horwath et al. (2022).

Evaluation of Bovine Myosin Heavy Chain Isoforms and Muscle Fiber Cross-Sectional Area on the Eating Quality of 11 Different Beef Muscles

Objective: The objective of this study was to investigate the contribution of muscle fiber type and size on the eating quality of 11 different beef muscles.