Slow Myosin Heavy Chain Research Articles

Leucine promotes porcine myofibre type transformation from fast-twitch to slow-twitch through the protein kinase B (Akt)/forkhead box 1 signalling pathway and microRNA-27a.

Muscle fibre types can transform from slow-twitch (slow myosin heavy chain (MyHC)) to fast-twitch (fast MyHC) or vice versa. Leucine plays a vital effect in the development of skeletal muscle. However, the role of leucine in porcine myofibre type transformation and its mechanism are still unclear. In this study, effects of leucine and microRNA-27a (miR-27a) on the transformation of porcine myofibre type were investigated in vitro. We found that leucine increased slow MyHC protein level and decreased fast MyHC protein level, increased the levels of phospho-protein kinase B (Akt)/Akt and phospho-forkhead box 1 (FoxO1)/FoxO1 and decreased the FoxO1 protein level. However, blocking the Akt/FoxO1 signalling pathway by wortmannin attenuated the role of leucine in porcine myofibre type transformation. Over-expression of miR-27a decreased slow MyHC protein level and increased fast MyHC protein level, whereas inhibition of miR-27a had an opposite effect. We also found that expression of miR-27a was down-regulated following leucine treatment. Moreover, over-expression of miR-27a repressed transformation from fast MyHC to slow MyHC caused by leucine, suggesting that miR-27a is interdicted by leucine and then contributes to porcine muscle fibre type transformation. Our finding provided the first evidence that leucine promotes porcine myofibre type transformation from fast MyHC to slow MyHC via the Akt/FoxO1 signalling pathway and miR-27a.

Muscle fibre type composition in the lateral muscle of olive flounder Paralichthys olivaceus

In this paper, a combined-method study has been made on the lateral muscle of the teleost olive flounder Paralichthys olivaceus in just-hatched and adult stages. In just-hatched stage, both slow and fast muscle fibres were detected: (1) in situ hybridization analysis indicated that slow and fast myosin heavy chain genes were specifically expressed in the superficial and deep part of the myotomal muscle, respectively; (2) immunohistochemistry analysis showed that fibres in the deep part reacted with anti-fast myosin antibody F310; (3) western blot analysis detected a weak expression of slow myosin and a strong expression of fast myosin. In adult stage, the slow and fast muscle fibres had their own distribution characteristics: (1) hematoxylin/eosin staining showed the histological characteristics of the muscle fibre composition; (2) histochemical observations showed that the deep muscle fibres, and some fibres near the epidermis, contain alkali-stable myofibrillar ATPase activity; (3) immunohistochemistry analysis indicated that all the deep muscle fibres reacted with F310 antibody and some fibres in the superficial layer of muscle also reacted with F310; (4) western blot analysis showed that fast myosin was expressed both in the blended muscles (the mix of superficial and deep muscles) and deep muscles, while slow myosin was mainly expressed in the blended muscles. These findings suggested that both slow and fast muscle fibres existed in the musculature of the olive flounder in just-hatched and adult stages. Notably, the adult fast fibres also exist in the superficial layer of the muscle.

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

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.

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.

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.

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.

Evonik is more optimistic after operational successes

Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with domestication, breeding and rearing conditions. This study was aimed to comparatively elucidate the age-dependent myosin heavy chain (MyHC) isoform expression and transition pattern in domestic and wild pig (WP) skeletal muscle from birth until adulthood. Domestic pigs (DPs) of Large White breed raised in conventional production system were compared with WPs reared in a large hunting enclosure. Muscle samples for immuno/enzyme histochemistry were taken from the longissimus dorsi muscle within 24 h postmortem at 24 to 48 h, 21 to 23 days, 7 months and ~2 years postpartum. Based on the antibody reactivity to MyHCs (NCL-MHCs, A4.74, BF-F3) and succinate dehydrogenase activity, myofibers were classified into I, I/IIa, IIa, IIx and IIb types. In addition, foetal MyHC expression was determined with the use of F158.4C10 antibody. Maturation of the longissimus dorsi muscle in the WP was characterized by an accelerated transformation of the fast to slow MyHC during the first hours postpartum, followed by differentiation towards oxidative myofibers in which type I, IIa and IIx MyHCs predominated. In the DP, the transformation shifted towards glycolytic myofibers that expressed MyHC-IIb. The expression of foetal MyHC was higher in the DP than in the WP at 1 day of age, and the decline in the foetal MyHC during the first 3 weeks was more rapid in the WP than in the DP denoting an accelerated early postnatal muscle maturation in WP than DP piglets. All foetal MyHC-positive myofibers co-expressed IIa isoform, but not vice versa. The intense myofiber hypertrophy was evident from 3 weeks until 7 months of age. In this period, the myofiber cross-sectional area increased up to 10- and 20-fold in the WP and the DP, respectively. In the DP, the hypertrophy of all myofiber types was more pronounced than in the WP, particularly the hypertrophy of IIx and IIb myofibers. To summarize, the comparison between growing DP with wild ancestors showed that genetic selection and rearing conditions lead to substantial changes in the direction and intensity of postnatal MyHC transformation as evidenced by different proportion of individual myofiber types and differences in their hypertrophic potential.

Rapid decline in MyHC I(β) mRNA expression in rat soleus during hindlimb unloading is associated with AMPK dephosphorylation.

Inactivation of a skeletal muscle results in slow to fast myosin heavy chain (MyHC) shift. AMP-activated protein kinase (AMPK) can be implicated in the regulation of genes encoding the slow MyHC isoform. Here we report that AMPK dephosphorylation after 24h of mechanical unloading can contribute to histone deacetylase (HDAC) nuclear translocation; activation of AMPK prevents HDAC4 nuclear accumulation after 24h of unloading and AMPK dephosphorylation inhibits slow MyHC expression following 24h of unloading. Our data indicate that AMPK dephosphorylation during the first 24h of mechanical unloading has a significant impact on the expression of MyHC isoforms in rat soleus causing a decrease in MyHC I(β) pre-mRNA and mRNA expression as well as MyHC IIa mRNA expression. One of the key events that occurs during skeletal muscle inactivation is a change in myosin phenotype, i.e. increased expression of fast isoforms and decreased expression of the slow isoform of myosin heavy chain (MyHC). It is known that calcineurin/nuclear factor of activated T-cells and AMP-activated protein kinase (AMPK) can regulate the expression of genes encoding MyHC slow isoform. Earlier, we found a significant decrease in phosphorylated AMPK in rat soleus after 24h of hindlimb unloading (HU). We hypothesized that a decrease in AMPK phosphorylation and subsequent histone deacetylase (HDAC) nuclear translocation can be one of the triggering events leading to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with AMPK activator (AICAR) for 6days before HU as well as during 24h of HU. We discovered that AICAR treatment prevented a decrease in pre-mRNA and mRNA expression of MyHC I as well as MyHC IIa mRNA expression. Twenty-four hours of hindlimb suspension resulted in HDAC4 accumulation in the nuclei of rat soleus but AICAR pretreatment prevented this accumulation. The results of the study indicate that AMPK dephosphorylation after 24h of HU had a significant impact on the MyHC I and MyHC IIa mRNA expression in rat soleus. AMPK dephosphorylation also contributed to HDAC4 translocation to the nuclei of soleus muscle fibres, suggesting an important role of HDAC4 as an epigenetic regulator in the process of myosin phenotype transformation.

Novel phenotypic variant in the MYH7 spectrum due to a stop-loss mutation in the C-terminal region: a case report

BackgroundDefects of the slow myosin heavy chain isoform coding MYH7 gene primarily cause skeletal myopathies including Laing Distal Myopathy, Myosin Storage Myopathy and are also responsible for cardiomyopathies. Scapuloperoneal and limb-girdle muscle weakness, congenital fiber type disproportion, multi-minicore disease were also reported in connection of MYH7. Pathogeneses of the defects in the head and proximal rod region of the protein are well described. However, the C-terminal mutations of the MYH7 gene are less known. Moreover, only two articles describe the phenotypic impact of the elongated mature protein product caused by termination signal loss.Case presentationHere we present a male patient with an unusual phenotypic variant of early-onset and predominant involvement of neck muscles with muscle biopsy indicating myopathy and sarcoplasmic storage material. Cardiomyopathic involvements could not be observed. Sequencing of MYH7 gene revealed a stop-loss mutation on the 3-prime end of the rod region, which causes the elongation of the mature protein.ConclusionsThe elongated protein likely disrupts the functions of the sarcomere by multiple functional abnormalities. This elongation could also affect the thick filament degradation leading to protein deposition and accumulation in the sarcomere, resulting in the severe myopathy of certain axial muscles. The phenotypic expression of the detected novel MYH7 genotype could strengthen and further expand our knowledge about mutations affecting the structure of MyHCI by termination signal loss in the MYH7 gene.

EMX2 activates slow myosin heavy chain 2 gene expression in embryonic muscle fibers

Avian myogenesis is partly characterized by commitment of distinct myoblast cell lineages to the formation of specific muscle fiber types. Previous studies have identified the transcription factor EMX2 as a regulator of slow myosin heavy chain 2 (MyHC2) gene expression in fast/slow primary muscle fibers. We report here the interaction of EMX2 with the slow MyHC2 transcriptional regulatory region in fast/slow embryonic muscle fibers. Promoter activity and electromobility shift assays localized the site of interaction of EMX2 with the slow MyHC2 gene within a defined binding site located between 3336 and 3326bp from the 3′ end of the cloned slow MyHC2 DNA containing the transcriptional regulatory region. Using clonally-derived myoblasts stably committed to the formation of fast/slow muscle fibers, we also report the effect of altered EMX2 gene expression on genome-wide gene expression within these myoblasts. Increased EMX2 gene expression in fast/slow myoblasts caused altered gene expression of 1185 genes, indicating that EMX2 plays a central role in the gene expression profile of embryonic myoblasts.

Slow-Myofiber Commitment by Semaphorin 3A Secreted from Myogenic Stem Cells.

Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cell-membrane receptor (neuropilin2-plexinA3) → myogenin-myocyte enhancer factor 2D → slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropilin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fast-myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreERT2 -Sema3Afl °x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease. Stem Cells 2017;35:1815-1834.

Histone deacetylase activity modulates exercise-induced skeletal muscle plasticity in zebrafish (Danio rerio).

Aerobic exercise has a positive impact on animals by enhancing skeletal muscle function and locomotor performance. Responses of skeletal muscle to exercise involve changes in energy metabolism, calcium handling, and the composition of contractile protein isoforms, which together influence contractile properties. Histone deacetylases (HDAC) can cause short-term changes in gene expression and may thereby mediate plasticity in contractile properties of skeletal muscle in response to exercise. The aim of this project was to determine (in zebrafish, Danio rerio) the traits that mediate interindividual differences in sustained and sprint performance and to determine whether inhibiting class I and II HDACs mediates exercise-induced changes in these traits. High sustained performers had greater aerobic metabolic capacity [citrate synthase (CS) activity], calcium handling capacity [sarco/endoplasmic reticulum ATPase (SERCA) activity], and slow contractile protein concentration [slow myosin heavy chain (MHC)] compared with low performers. High sprint performers had lower CS activity and slow MHC concentrations compared with low performers, but there were no significant differences in lactate dehydrogenase activity or fast MHC concentrations. Four weeks of aerobic exercise training increased sustained performance, CS activity, SERCA activity, and slow MHC concentration. Inhibiting class I and II HDACs increased slow MHC concentration in untrained fish but not in trained fish. However, inhibiting HDACs reduced SERCA activity, which was paralleled by a reduction in sustained and sprint performance. The regulation of muscle phenotypes by HDACs could be a mechanism underlying the adaptation of sustained locomotor performance to different environmental conditions, and may therefore be of therapeutic and ecological significance.

Obesity-induced decreases in muscle performance are not reversed by weight loss.

Obesity can affect muscle phenotypes, and may thereby constrain movement and energy expenditure. Weight loss is a common and intuitive intervention for obesity, but it is not known whether the effects of obesity on muscle function are reversible by weight loss. Here we tested whether obesity-induced changes in muscle metabolic and contractile phenotypes are reversible by weight loss. We used zebrafish (Danio rerio) in a factorial design to compare energy metabolism, locomotor capacity, muscle isometric force and work-loop power output, and myosin heavy chain (MHC) composition between lean fish, diet-induced obese fish, and fish that were obese and then returned to lean body mass following diet restriction. Obesity increased resting metabolic rates (P<0.001) and decreased maximal metabolic rates (P=0.030), but these changes were reversible by weight loss, and were not associated with changes in muscle citrate synthase activity. In contrast, obesity-induced decreases in locomotor performance (P=0.0034), and isolated muscle isometric stress (P=0.01), work-loop power output (P<0.001) and relaxation rates (P=0.012) were not reversed by weight loss. Similarly, obesity-induced decreases in concentrations of fast and slow MHCs, and a shift toward fast MHCs were not reversed by weight loss. Obesity-induced changes in locomotor performance and muscle contractile function were not reversible by weight loss. These results show that weight loss alone may not be a sufficient intervention.

Akirin2 promotes slow myosin heavy chain expression by CaN/NFATc1 signaling in porcine skeletal muscle satellite cells.

The objective of this study was to evaluate the effect of Akirin2 on slow myosin heavy chain (slow MyHC, MyHC I) gene expression and its molecular mechanisms. In this study, we showed that the protein expression of Akirin2 in pig slow oxidative Psoas major muscle is higher than that in fast glycolytic tibialis anterior muscle, suggesting that Akirin2 may play a role in myofiber typing. Knockdown of Akirin2 decreased the MyHC I expression and the calcineurin (CaN) activity, and also decreased the expressions of NFATc1 and MCIP1.4. Conversely, overexpression of Akirin2 got the opposite results. Furthermore, inhibition of CaN or knockdown of NFATc1 attenuated Akirin2 overexpression-induced upregulation of MyHC I. Together, these results demonstrate that Akirin2 promotes MyHC I expression via CaN/NFATc1 signaling pathway in porcine skeletal muscle satellite cells.

Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

Dynamics of myosin heavy chain isoform transition in the longissimus muscle of domestic and wild pigs during growth: a comparative study

Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with domestication, breeding and rearing conditions. This study was aimed to comparatively elucidate the age-dependent myosin heavy chain (MyHC) isoform expression and transition pattern in domestic and wild pig (WP) skeletal muscle from birth until adulthood. Domestic pigs (DPs) of Large White breed raised in conventional production system were compared with WPs reared in a large hunting enclosure. Muscle samples for immuno/enzyme histochemistry were taken from the longissimus dorsi muscle within 24 h postmortem at 24 to 48 h, 21 to 23 days, 7 months and ~2 years postpartum. Based on the antibody reactivity to MyHCs (NCL-MHCs, A4.74, BF-F3) and succinate dehydrogenase activity, myofibers were classified into I, I/IIa, IIa, IIx and IIb types. In addition, foetal MyHC expression was determined with the use of F158.4C10 antibody. Maturation of the longissimus dorsi muscle in the WP was characterized by an accelerated transformation of the fast to slow MyHC during the first hours postpartum, followed by differentiation towards oxidative myofibers in which type I, IIa and IIx MyHCs predominated. In the DP, the transformation shifted towards glycolytic myofibers that expressed MyHC-IIb. The expression of foetal MyHC was higher in the DP than in the WP at 1 day of age, and the decline in the foetal MyHC during the first 3 weeks was more rapid in the WP than in the DP denoting an accelerated early postnatal muscle maturation in WP than DP piglets. All foetal MyHC-positive myofibers co-expressed IIa isoform, but not vice versa. The intense myofiber hypertrophy was evident from 3 weeks until 7 months of age. In this period, the myofiber cross-sectional area increased up to 10- and 20-fold in the WP and the DP, respectively. In the DP, the hypertrophy of all myofiber types was more pronounced than in the WP, particularly the hypertrophy of IIx and IIb myofibers. To summarize, the comparison between growing DP with wild ancestors showed that genetic selection and rearing conditions lead to substantial changes in the direction and intensity of postnatal MyHC transformation as evidenced by different proportion of individual myofiber types and differences in their hypertrophic potential.