Myosin Heavy Chain IIb Research Articles

Changes in expression of serine biosynthesis and integrated stress response genes during myogenic differentiation of C2C12 cells

Skeletal muscle is a highly metabolic and dynamic tissue that is formed through the complex and well-organised process of myogenesis. Although there is a good understanding about the role of the Muscle Regulatory Factors during myogenesis, little is known about the potential interplay of other metabolic proteins. The aim of this study was to determine the endogenous mRNA expression profile for a novel group of genes, recently associated with β2-adrenergic agonist (BA) induced muscle hypertrophy in pigs [1], during myogenic differentiation in C2C12 cells and their response to dibutyryl cyclic-AMP (dbcAMP). These genes included mitochondrial phosphoenolpyruvate carboxykinase (PCK2/PEPCK-M), genes involved in serine biosynthesis (Phosphoglycerate dehydrogenase, PHGDH; Phosphoserine aminotransferase-1, PSAT1; Phosphoserine phosphatase, PSPH) and those involved in an integrated stress response (Asparagine synthetase, ASNS; Sestrin-2, SESN2; and Activating transcription factor-5, ATF5).A coordinated peak in endogenous PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA expression was observed at day 2 of differentiation (P < 0.001) in C2C12 cells, which coincided with the peak in myogenin mRNA. Myotube hypertrophy was induced with dbcAMP (1 mM) treatment from day 0, thereby mimicking the in vivo BA response. Although dbcAMP treatment from day 0 induced larger myotubes and increased both myosin heavy chain-IIB (MyHC-IIB) and pyruvate carboxylase (PC) mRNA, the expression of PCK2, PHGDH, PSAT1 and ASNS mRNA were all unaffected. Treatment with dbcAMP from day 4 increased MyHC-IIB mRNA, however this was less dramatic compared to the response observed following treatment from day 0, but there was no effect on PC mRNA. There was also no effect of dbcAMP treatment from day 4 on PCK2, PHGDH, PSAT1 and ASNS mRNA.To conclude, the coordinated day 2 peak in endogenous expression of PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA may relate to a shift in biosynthetic demand required to initiate myogenic differentiation. However, dbcAMP had no effect on the expression of these genes in vitro suggesting that the effects observed in BA-treated pigs might be via other signalling pathways from the activation of the β2-adrenergic receptor, but independent of cAMP, or that there are species differences in the response.

Dietary mulberry leaf powder affects growth performance, carcass traits and meat quality in finishing pigs.

This study was conducted to evaluate the effect of mulberry leaves as an alternative source of protein on growth performance, carcass traits and meat quality in finishing pigs. A total of 180 Xiangcun Black pigs were randomly assigned to five treatment groups with six pens of six pigs per pen. The pigs were provided with a basal diet or a diet contained 3%, 6%, 9% or 12% of mulberry leaf powder during a 50-day experiment period. The results showed that dietary mulberry leaf powder had no negative effect on growth performance in Xiangcun Black pigs, except in the 12% mulberry group, where final body weight and average daily gain decreased (p<.05) and feed to gain ratio of the pigs increased (p<.05). Dietary mulberry inclusion decreased (quadratic, p<.05) the back fat thickness, fibre mean cross-sectional area (CSA) in the longissimus dorsi (LD) muscle and mRNA expression levels of myosin heavy chain (MyHC) IIb in LD and biceps femoris (BF) muscles, while increased (linear or quadratic, p<.05) the plasma concentration of albumin, levels of crude protein (CP), inosine monophosphate (IMP) and several amino acids in muscle tissues. When compared with the other groups, the 9% mulberry diet increased (p<.05) loin-eye area and contents of CP and IMP in muscles, while decreased (p<.05) plasma activity of cholinesterase and concentrations of uric acid and urea. The 6% mulberry diet had the lowest fibre mean CSA and shear force and increased total fibre number of the LD muscle, when compared with the other groups. These results suggest that including mulberry in the diet at <12% is an effective feed crop to improve meat quality and the chemical composition of muscle without negatively affecting growth performance.

Succinate induces skeletal muscle fiber remodeling via SUNCR1 signaling.

The conversion of skeletal muscle fiber from fast twitch to slow‐twitch is important for sustained and tonic contractile events, maintenance of energy homeostasis, and the alleviation of fatigue. Skeletal muscle remodeling is effectively induced by endurance or aerobic exercise, which also generates several tricarboxylic acid (TCA) cycle intermediates, including succinate. However, whether succinate regulates muscle fiber‐type transitions remains unclear. Here, we found that dietary succinate supplementation increased endurance exercise ability, myosin heavy chain I expression, aerobic enzyme activity, oxygen consumption, and mitochondrial biogenesis in mouse skeletal muscle. By contrast, succinate decreased lactate dehydrogenase activity, lactate production, and myosin heavy chain IIb expression. Further, by using pharmacological or genetic loss‐of‐function models generated by phospholipase Cβ antagonists, SUNCR1 global knockout, or SUNCR1 gastrocnemius‐specific knockdown, we found that the effects of succinate on skeletal muscle fiber‐type remodeling are mediated by SUNCR1 and its downstream calcium/NFAT signaling pathway. In summary, our results demonstrate succinate induces transition of skeletal muscle fiber via SUNCR1 signaling pathway. These findings suggest the potential beneficial use of succinate‐based compounds in both athletic and sedentary populations.

Age-related changes in histone modification in rat gastrocnemius muscle.

Changes in histone acetylation and methylation status with aging affect gene expression and phenotype in several tissues; however, age-related changes in histone modification in the skeletal muscle have not been elucidated yet. This study investigated age-related global changes in histone modification in rat gastrocnemius muscle. Male Wistar rats (n = 28) were assigned to one of four age groups (n = 7 per group) corresponding to different life stages: 3 months old (3-mo; young), 6 months old (adult), 12 months old (12-mo; middle-aged), and 24 months old (24-mo; old). The gastrocnemius muscle was removed and global histone modification (acetylation and tri-methylation) at K9 and K27 was evaluated by western blotting. Relative muscle mass decreased in the 12- and 24-mo rats accompanied with reduction in type IIb myosin heavy chain isoforms and Myh4 (MHC IIB) mRNA expression. Histone H3 acetylation decreased in an age-dependent manner, with lower levels in 12- and 24-mo groups than in the 3-mo group. K9 and K27 acetylation decreased with age. Although there was no significant change in K27 tri-methylation, K9 tri-methylation showed an age-dependent decline. Histone modification status (acetylation at K9 and K27 and tri-methylation at K9) was positively associated with relative gastrocnemius muscle weight, the percentage of type IIb myosin heavy chain isoform, myosin heavy chain type IIb protein expression, and the level of Myh4 mRNA. Thus, global histone H3 methylation and acetylation decrease with age, and the latter might be associated with age-related muscle atrophy of rat gastrocnemius muscle.

Myoblast maturity on aligned microfiber bundles at the onset of strain application impacts myogenic outcomes

Engineered skeletal muscle grafts may be employed in various applications including the treatment of volumetric muscle loss (VML) and pharmacological drug screening. To recapitulate the well-defined structure of native muscle, tensile strains have been applied to the grafts. In this study, we cultured C2C12 murine myoblasts on electrospun fibrin microfiber bundles for 7 days in custom-built bioreactor units and investigated the impact of strain regimen and delayed onset of tensile straining on myogenic outcomes. The substrate topography induced uniaxial alignment of cells in all (strained and unstrained) groups. The engineered grafts in strained groups were subjected to 10% strain amplitude for 6 h per day. We found that both static and cyclic uniaxial strains resulted in similar morphological and gene expression outcomes. However, relative to 0% strain groups, there were stark increases in myotube diameter, myosin heavy chain (MHC) coverage, and expression of key myogenic genes (Pax 7, Troponin, MHC I, MHC IIb, MHC IIx) only if strain was applied at Days 5–7 rather than Days 3–7. This finding suggests that a critical indicator of myogenic improvement under strain in our system is the phenotype of the cells at the onset of strain and suggests that this is a key parameter that should be considered in studies where myoblasts are subjected to biophysical stimulation to promote tissue formation. Statement of SignificanceThis is the first report on the impact of the timing of the initial application of mechanical strain for improving the myogenic outcomes of 3D engineered skeletal muscle grafts. In this work, immature skeletal myoblasts were grown on topographically aligned, electrospun fibrin microfiber bundles and we applied 10% uniaxial static or cyclic strain. We concluded that the maturity of myoblasts prior to strain application, rather than strain waveform, was the primary predictor of improved myogenic outcomes, including myogenic gene expression and myotube morphology. Elucidating the optimal conditions for strain application is a vital step in recapitulating physiological myogenic properties in tissue engineered skeletal muscle constructs, with applications for treating volumetric muscle loss, disease modeling, and drug testing.

Negative Impact of Icing Treatment on Qualitative Recovery of Injured Soleus Muscle in Rats

PURPOSE: Although the RICE (Rest, Ice, Compression, and Elevation) treatment has been recognized as the gold standard of first aid treatment for sports injuries, we and the others previously demonstrated that a transient icing treatment immediately after skeletal muscle injury impaired muscle regeneration. However, the information about the influence of icing treatment following skeletal muscle injury remains limited. This study was, therefore, to investigate the impact of icing as a first-aid treatment on qualitative recovery of damaged muscle in terms of fibrosis and myosin heavy chain (MyHC) profile. METHODS: Male Wistar rats (9-10 weeks of age) were randomly assigned to control (Con), injured, and injured with icing groups (Ice). Bupivacaine (BPVC) was injected into slow soleus muscles bilaterally in order to induce muscle injury in the two injury groups. Application of icing treatment (ice pack, 0°C for 20 min) to the icing group was carried out immediately after the BPVC injection. At 3 days-4 weeks after BPVC injection, soleus muscles were removed and analyzed. RESULTS: Compared with the Con group, a significant increase in fibrotic area was observed after 2 weeks following injury in the injured groups, but after 1 week following injury in the Ice group (P < 0.05). This area was also tended to be higher in Ice than in injured animals during 1-4 weeks of recovery period. In addition, the number of Tcf4-positive nuclei, a fibroblast marker, located in interstitial spaces in both injured groups markedly increased 1 week after BPVC injection. The numbers were tended to be more magnified in the Ice group than in the injured group, then their number in both injured groups gradually degreased thereafter. Injury-related de novo appearance of embryonic, neonatal, IId/x, and IIb MyHC isoforms was noted in both injured groups 1 week after BPVC injection. These MyHC isoforms were significantly decreased toward the undetectable level thereafter. However, the embryonic MyHC isoform was still detectable in icing, but not in injured, animals 4 weeks after BPVC injection. CONCLUSIONS: Our results suggested that icing treatment following skeletal muscle injury will have a negative impact on recovery process (fibrosis and normalization of MyHC profile) of regenerating muscle.

Impact of different temperature stimuli on the expression of myosin heavy chain isoforms during recovery from bupivacaine-induced muscle injury in rats.

Limited information exists regarding the impact of different temperature stimuli on myosin heavy chain (MyHC) expression in skeletal muscle during recovery from injury. Therefore, this experiment investigated the impact of both cold and heat exposure on the MyHC isoform profile in the rat soleus during recovery from injury. Male Wistar rats were randomly divided into control, bupivacaine-injected (BPVC), BPVC with icing, and BPVC with heat stress groups. Muscle injury was induced by intramuscular injection of bupivacaine into soleus muscles of male Wistar rats. Icing treatment (0°C for 20 min) was performed immediately after the injury. Intermittent heat stress (42°C for 30 min on alternating days) was carried out during 2-14 days after bupivacaine injection. In response to injury, a transient increase in developmental, IId/x, and IIb MyHC isoforms, as well as various types of hybrid fibers, followed by the recovery of the MyHC profile toward the control level, was noted in the regeneration of the soleus. The restoration of the MyHC profile in the regenerating muscle at whole-muscle and individual myofiber levels was partially delayed by icing but facilitated by heat stress. In addition, the application of repeated heat stress promoted the recovery of soleus muscle mass toward the control level following injury. We conclude that compared with acute and immediate cold (icing) treatment, chronic and repeated heat stress may be a more appropriate treatment for the enhancement of both normalization of the MyHC profile and restoration of muscle mass following injury. NEW & NOTEWORTHY Cold exposure (icing), but not heat exposure, has been well accepted as a first-aid treatment for accidental and/or sports-related injuries. However, recent evidence suggests the negative impact of icing treatment on skeletal muscle regeneration following injury. Here, we demonstrated that acute/immediate icing treatment delayed the restoration of the myosin heavy chain (MyHC) profile, but intermittent hyperthermia, repeated for several days, facilitated the recovery of both muscle mass and the MyHC profile in the regeneration of skeletal muscle following injury.

AMPK/FOXO1 signaling pathway is indispensable in visfatin-regulated myosin heavy chain expression in C2C12 myotubes

ObjectiveFew studies have addressed the effects of visfatin on skeletal muscle remodeling. The aim of the study was to investigate the effects of visfatin on the expressions of myosin heavy chain (MHC) and its isoforms, the major indicator of fiber types and contractile properties of skeletal muscle. Materials and methodsLevels of MHC, MHC I, MHC IIa, MHC IIb, adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), p-AMPK and forkhead box protein O1 (FOXO1) were tested in visfatin-treated C2C12 myotubes. C2C12 myotubes were treated with visfatin combined with AMPK inhibitor or AMPK activator to investigate the role of AMPK in visfatin-mediated MHC expression. FOXO1 was overexpressed or knocked down in C2C12 myotubes to explore the role of FOXO1 in visfatin-mediated MHC expression. ResultsCompared with the vehicle group, treatment with 5 μg/ml visfatin increased the levels of total MHC and its isoforms, MHC I, MHC IIa and MHC IIb, by 1.93, 1.84, 1.80, and 1.92 folds, respectively (all p = 0,001). Visfatin suppressed AMPK phosphorylation and decreased FOXO1 expression in C2C12 myotubes. The effects of visfatin on MHC I and MHC IIa expression were canceled by AMPK activator AICAR. FOXO1 overexpression minimized the visfatin-induced upregulation of MHC I, MHC IIa and MHC IIb. The effect of AMPK activator AICAR on MHC and its isoforms expression was minimized by knockdown of FOXO1. ConclusionsThe findings revealed that visfatin promoted expressions of MHC and its isoforms in C2C12 myotubes via suppressing AMPK/FOXO1 signaling pathway.

Effect of electroacupuncture on degradation of myosin heavy chain of gastrocnemius muscle in diabetes rats

To explore the effect of electroacupuncture（EA）on the expression of muscle-specific ring finger protein 1（MuRF1/Trim63），F-box only protein 32（Fbxo32），myosin heavy chain-IIa（Myh2），myosin heavy chain-IIb（Myh4）and myosin heavy chain-I（Myh7）in diabetes rats. Thirty-six male Wistar rats were equally randomized into control, model and EA groups. The diabetes model was established by intraperitoneal injection of 0.1% Streptozocin (STZ) solution (50 mg/kg). After that, EA (2 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36), "Yinlingquan" (SP9) and "Shenshu" (BL23) for 10 min, once a day, 6 times a week for 2 weeks. The fasting blood glucose (FBG) and fasting serum insulin (FINS) contents were assayed by using ELISA, and the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. The body weight, and wet weight of bilateral gastrocnemius muscles were measured. The cross-sectional area (CSA) of the gastrocnemius muscle was measured after H.E. stai-ning. The expression of MuRF1, Fbxo32, Myh2, Myh4 and Myh7 mRNAs in the gastrocnemius tissue was tested using quantitative real time-PCR. Compared with the control group, the FBG and HOMA-IR were significantly higher (P<0.05), and the FINS, body weight were significantly lower (P<0.05) after intravenous injection of STZ for 1, 2, 3, 4, 5 weeks respectively. Following EA treatment and compared with the model group, the FBG and HOMA-IR were significantly down-regulated (P<0.05), and the FINS and body weight were considerably increased (P<0.05). Following modeling and compared with the control group, the wet weight of gastrocnemius muscle, CSA, and expression levels of Myh2, Myh4 and Myh7 mRNAs were obviously decreased, and the expression of MuRF1 and Fbxo32 mRNA was obviously increased in the model group (P<0.05). After EA treatment, the gastrocnemius muscle wet weight, CSA, expression levels of Myh2, Myh4 and Myh7 mRNA were significantly up-regulated (P<0.05), and the expression levels of MuRF1 and Fbxo32 mRNA were markedly down-regulated in comparison with those of the model group (P<0.05). EA treatment can delay the atrophy of gastrocnemius muscle (skeletal muscle) in diabetes rats possibly by improving the degradation of myosin heavy chain via regulating the expression of muscular MuRF1, Fbxo32, Myh2, Myh4 and Myh7 mRNAs.

Ferulic acid regulates muscle fiber type formation through the Sirt1/AMPK signaling pathway.

Ferulic acid (FA) is a polyphenolic compound of plant cell walls. Several recent studies demonstrated that polyphenolic compounds could regulate muscle fiber type formation. In this study, we investigated the effect of FA on muscle fiber type formation and also explored its molecular mechanism in mouse C2C12 myotubes. Real-time quantitative PCR showed that FA significantly increased the mRNA abundances of myosin heavy chain (MyHC) I, MyHC IIa, sirtuin1 (Sirt1), peroxisome proliferator-activated receptor gamma coactivator-1-α (PGC-1α) and myocyte enhancer factor 2C (MEF2C), but decreased the mRNA abundance of MyHC IIb. FA increased succinate dehydrogenase (SDH) activity and decreased lactate dehydrogenase (LDH) activity. Western blot analysis showed that FA significantly increased the protein level of slow-MyHC, but significantly decreased the protein level of fast-MyHC, which were attenuated by AMP-activated protein kinase (AMPK) inhibitor Compound C, AMPKα2 siRNA or Sirt1 inhibitor EX527. We also showed that EX527 attenuated FA-induced increase in the phosphorylation levels of AMPK and liver kinase B1 (LKB1), indicating that AMPK serves as a downstream target of Sirt1. Our present study provides the first in vitro evidence that FA regulates muscle fiber type formation via the Sirt1/AMPK signal pathway.

Effects of active immunization against porcine Sox6 on meat quality and myosin heavy chain isoform expression in growing-finishing pigs

A feeding trial for 91 days was conducted to investigate effects of active immunization against porcine Sox6 (pSox6) on meat quality and myosin heavy chain (MyHC) isoform expression in growing-finishing pigs. Twenty-four castrated Duroc × Landrace × Yarkshire pigs were randomly divided into three groups: (1) Control group; (2) 1 mg/head pSox6 active immunity group; (3) 4 mg/head pSox6 active immunity group (4 mg/head group). The results showed that pigs in 4 mg/head group had a greater a* (Redness) and a higher marbling score, while no significant effect was observed in L* (Lightness), b* (Yellowness), intramuscular fat and cooking loss. Muscle succinic dehydrogenase activity in pSox6 active immunization groups was significantly increased, and muscle lactate dehydrogenase activity was significantly reduced. Meanwhile, active immunization against pSox6 upregulated the mRNA expression of MyHC I, while no effect was observed on the mRNA expressions of MyHC IIa, MyHC IIx, MyHC IIb. In addition, pigs in the 4 mg/head group exhibited lower Sox6 mRNA level and higher MyHC I protein level, while no significant influence was observed on MyHC IIb protein level. Together, our data imply that active immunization against pSox6 could improve the pork quality and promote the MyHC I expression in growing-finishing pigs.

AMPK Mediates Muscle Mass Change But Not the Transition of Myosin Heavy Chain Isoforms during Unloading and Reloading of Skeletal Muscles in Mice.

5′AMP-activated protein kinase (AMPK) plays an important role in the regulation of skeletal muscle mass and fiber-type distribution. However, it is unclear whether AMPK is involved in muscle mass change or transition of myosin heavy chain (MyHC) isoforms in response to unloading or increased loading. Here, we checked whether AMPK controls muscle mass change and transition of MyHC isoforms during unloading and reloading using mice expressing a skeletal-muscle-specific dominant-negative AMPKα1 (AMPK-DN). Fourteen days of hindlimb unloading reduced the soleus muscle weight in wild-type and AMPK-DN mice, but reduction in the muscle mass was partly attenuated in AMPK-DN mice. There was no difference in the regrown muscle weight between the mice after 7 days of reloading, and there was concomitantly reduced AMPKα2 activity, however it was higher in AMPK-DN mice after 14 days reloading. No difference was observed between the mice in relation to the levels of slow-type MyHC I, fast-type MyHC IIa/x, and MyHC IIb isoforms following unloading and reloading. The levels of 72-kDa heat-shock protein, which preserves muscle mass, increased in AMPK-DN-mice. Our results indicate that AMPK mediates the progress of atrophy during unloading and regrowth of atrophied muscles following reloading, but it does not influence the transition of MyHC isoforms.

Apigenin inhibits sciatic nerve denervation-induced muscle atrophy.

Apigenin (AP) has been reported to elicit anti-inflammatory effects. In this study, we investigated the effect of AP on sciatic nerve denervation-induced muscle atrophy. Sciatic nerve-denervated mice were fed a 0.1% AP-containing diet for 2 weeks. Muscle weight and cross-sectional area (CSA), and the expression of atrophic genes and inflammatory cytokines in the gastrocnemius were analyzed. Denervation significantly induced muscle atrophy. However, values for muscle weight and CSA were greater in the denervated muscle of the AP mice than the controls. AP suppressed the expression of MuRF1, but upregulated both myosin heavy chain (MHC) and MHC type IIb. AP also significantly suppressed expression of tumor necrosis-alpha in the gastrocnemius and soleus muscles, and interleukin-6 expression in the soleus muscle. AP appears to inhibit denervation-induced muscle atrophy, which may be due in part to its inhibitory effect on inflammatory processes within muscle. Muscle Nerve 58: 314-318, 2018.

Estrogen‐related Receptor α Overexpression Drives a Metabolic and Gene Expression Program Mimicking Aerobic Exercise in Skeletal Muscle

Aerobic exercise is considered one of the most effective interventions to prevent obesity and type 2 diabetes (T2D). Exercise increases skeletal muscle insulin sensitivity by stimulating mitochondrial respiration and fatty acid oxidation through coordinated regulation of metabolic genes. Transcriptional pathways that regulate exercise‐induced metabolic gene programs present an attractive class of therapeutic targets to combat obesity and related metabolic diseases. We and others have shown that the Estrogen‐related Receptor α (ERRα) nuclear receptor regulates mitochondrial biogenesis, respiration and fatty acid oxidation and is required for metabolic reprogramming in response to exercise training. The objective of these studies is to determine whether ERRα over‐expression is sufficient to mediate exercise‐induced adaptations in muscle. We generated a conditional ERRα transgenic mouse line that expresses the Flag‐tagged human ERRα in skeletal muscle‐specific manner when crossed with Mck‐Cre recombinase mice (ERRαMOE). We examined gene expression (trancript and protein) and characterized the metabolic phentoype of the ERRαMOE mice. ERRαMOE mice exhibited a reddening of the superficial gastrocnemius and quadriceps, characteristic of oxidative muscles, accompanied by a reciprocal change in myosin heavy chain (MHC) IIb (−35%) and IIa (+80%) expression. Mitochondrial content was higher in skeletal muscle and heart, but not in non‐muscle tissues, based on mtDNA quantitation and citrate synthase protein expression. Consistently, basal (+32%) and maximal (+70%) O2 consumption rates were increased in FDB fibers isolated from ERRα‐MOE compared to WT. Signaling through the AMP‐activated kinase (AMPK) pathway, which mediates exercise‐dependent metabolic reprogramming, was unchanged in the basal state; however, maximal activation by AICAR was enhanced in ERRα‐overexpressing myotubes. We assessed systemic glucose homeostasis in adult chow fed mice. Although fasting glucose (6 hr) was slightly lower in ERRαMOE, no significant difference in glucose tolerance was observed. However, the results of insulin tolerance tests showed that ERRαMOE mice were more insulin sensitive than WT. In future studies we will investigate whether ERRα activation in skeletal muscle protects against high fat diet‐induced insulin resistance. Based on the results from these studies, ERRα over‐expression mimics the effects of exercise on skeletal muscle mitochondrial respiratory capacity and improves whole body insulin sensitivity.Support or Funding InformationSupported by Diabetes and Metabolic Research Institute (DMRI) and Shared Resource Pilot Awards from the City of Hope and NIH U24DK097748This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential impact of tongue exercise on intrinsic lingual muscles.

Aging is associated with muscle fiber hypotrophy and decreased percentages of rapidly contracting myosin heavy chain (MyHC) type IIb muscle fibers. Tongue exercise programs used to treat dysphagia target age-related decline in tongue muscle function, but the impact of exercise on the intrinsic tongue muscles is unknown. We hypothesized that exercise would induce muscle fiber hypertrophy and increase the percentage of MyHC IIa fibers in the intrinsic tongue. Animal model. Eight old and eight young-adult rats underwent 8 weeks of tongue exercise training, and 8 animals of each age group served as controls. Longitudinal, transverse, and verticalis muscle samples from the anterior, middle, and posterior regions of the tongue were sectioned and stained to determine muscle fiber diameter and MyHC composition. MyHC fiber type distribution was altered by exercise, and the effects differed by muscle and region of the tongue. In the exercise groups, as compared to the control groups, the anterior transverse and middle superior longitudinal muscles had significantly reduced percentages of MyHC IIx positive fibers and higher percentages of rapidly contracting fatigable MyHC IIb positive muscle fibers, whereas the middle transverse and posterior longitudinal muscles had increased percentages of the less rapidly contracting and more fatigue-resistant MyHC IIa fibers. The impact of exercise did not differ with age, as there was no significant interaction between age and exercise. Tongue exercise had no significant effect on muscle fiber diameter. The impact of exercise varied among the tongue muscles, which may indicate different functional contributions to the tongue exercise task. NA Laryngoscope, 128:2245-2251, 2018.

Influence of past injurious exercise on fiber type-specific acute anabolic response to resistance exercise in skeletal muscle

We investigated the influence of past injurious exercise on anabolic response of skeletal muscle fibers to resistance exercise (RE). Wistar rats were divided into exercise (E) and exercise-after-injury (I-E) groups. At age 10 wk, the right gastrocnemius muscle in each rat in the I-E group was subjected to strenuous eccentric contractions. Subsequently, RE was imposed on the same muscle of each rat at 14 wk of age in both groups. Peak joint torque and total force generation per body mass during RE were similar between the groups. Muscle protein synthesis (MPS) in the I-E group was higher than that in the E group 6 h after RE. Furthermore, levels of phospho-p70S6 kinase (Thr389) and phospho-ribosomal protein S6 (phospho-rpS6) (Ser240/244), a downstream target of p70S6 kinase, were higher in the I-E group than in the E group. For the anabolic response in each fiber type, the I-E group showed a higher MPS response in type IIb, IIa, and I fibers and a higher phospho-rpS6 response in type IIx, IIa, and I fibers than the E group. In the I-E group, the relative content of myosin heavy chain (MHC) IIa was higher and that of MHC IIb was lower than those in the E group. In addition, type IIa fibers showed a lower MPS response to RE than type IIb fibers in the I-E group. In conclusion, the past injurious exercise enhanced the MPS and phospho-rpS6 responses in type IIb, IIa, and I fibers and type IIx, IIa, and I fibers, respectively. NEW & NOTEWORTHY Past injurious exercise increased the muscle protein synthesis (MPS) response and mammalian target of rapamycin complex 1 (mTORC1) signaling activation to resistance exercise. In the responses of each fiber type, the past injurious exercise increased the MPS and phosphorylation ribosomal protein (Ser240/244) responses in type IIb, IIa, and I fibers and type IIx, IIa, and I fibers, respectively.

Fiber-type distribution and expression of myosin heavy chain isoforms in newborn heterozygous myostatin-knockout pigs.

To explore the effects of heterozygous myostatin-knockout (MSNT+/-) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs. The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN+/- pigs at birth than in wild-type (WT) pigs. MSTN+/- pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was~threefold higher in MSTN+/- pigs compared with WT pigs. MSTN+/- pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization.

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community.

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes.

ObjectiveThis study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula’s extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes.MethodsIn a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction.ResultsThe final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber I, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type I fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC I, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC I and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor γ coactivator-1α and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by 4 μg/mL and 20 μg/mL TCMF water extraction (p<0.05). Both 1 μg/mL and 5 μg/mL of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC I mRNA expression in porcine myocytes was decreased by 5 μg/mL TCMF water extraction (p<0.05). Porcine myocyte MyHC I and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by 5 μg/mL TCMF ethyl acetate extraction (p<0.05). MyHC I and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by 1 μg/mL TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by 5 μg/mL TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by 1 μg/mL in a TCMF petroleum ether extraction (p<0.05).ConclusionThese results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.

IGF1 stimulates greater muscle hypertrophy in the absence of myostatin in male mice.

Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue, we crossed myostatin null (Mstn-/-) mice with mice overexpressing Igf1 in skeletal muscle (Igf1+) to generate six genotypes of male mice; wild type (Mstn+/+ ), Mstn+/-, Mstn-/-, Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+ Overexpression of Igf1 increased the mass of mixed fibre type muscles (e.g. Quadriceps femoris) by 19% over Mstn+/+ , 33% over Mstn+/- and 49% over Mstn-/- (P < 0.001). By contrast, the mass of the gonadal fat pad was correspondingly reduced with the removal of Mstn and addition of Igf1 Myostatin regulated the number, while IGF1 regulated the size of myofibres, and the deletion of Mstn and Igf1+ independently increased the proportion of fast type IIB myosin heavy chain isoforms in T. anterior (up to 10% each, P < 0.001). The abundance of AKT and rpS6 was increased in muscles of Mstn-/-mice, while phosphorylation of AKTS473 was increased in Igf1+mice (Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+). Our results demonstrate that a greater than additive effect is observed on the growth of skeletal muscle and in the reduction of body fat when myostatin is absent and IGF1 is in excess. Finally, we show that myostatin and IGF1 regulate skeletal muscle size, myofibre type and gonadal fat through distinct mechanisms that involve increasing the total abundance and phosphorylation status of AKT and rpS6.