Myosin Heavy Chain mRNA Expression Research Articles

Exercise microdosing for skeletal muscle health applications to spaceflight.

Findings from a recent 70-day bedrest investigation suggested intermittent exercise testing in the control group may have served as a partial countermeasure for skeletal muscle size, function, and fiber-type shifts. The purpose of the current study was to investigate the metabolic and skeletal muscle molecular responses to the testing protocols. Eight males (29 ± 2 yr) completed muscle power (6 × 4 s; peak muscle power: 1,369 ± 86 W) and V̇o2max (13 ± 1 min; 3.2 ± 0.2 L/min) tests on specially designed supine cycle ergometers during two separate trials. Blood catecholamines and lactate were measured pre-, immediately post-, and 4-h postexercise. Muscle homogenate and muscle fiber-type-specific [myosin heavy chain (MHC) I and MHC IIa] mRNA levels of exercise markers (myostatin, IκBα, myogenin, MuRF-1, ABRA, RRAD, Fn14, PDK4) and MHC I, IIa, and IIx were measured from vastus lateralis muscle biopsies obtained pre- and 4-h postexercise. The muscle power test altered (P ≤ 0.05) norepinephrine (+124%), epinephrine (+145%), lactate (+300%), and muscle homogenate mRNA (IκBα, myogenin, MuRF-1, RRAD, Fn14). The V̇o2max test altered (P ≤ 0.05) norepinephrine (+1,394%), epinephrine (+1,412%), lactate (+736%), and muscle homogenate mRNA (myostatin, IκBα, myogenin, MuRF-1, ABRA, RRAD, Fn14, PDK4). In general, both tests influenced MHC IIa muscle fibers more than MHC I with respect to the number of genes that responded and the magnitude of response. Both tests also influenced MHC mRNA expression in a muscle fiber-type-specific manner. These findings provide unique insights into the adaptive response of skeletal muscle to small doses of exercise and could help shape exercise dosing for astronauts and Earth-based individuals.NEW & NOTEWORTHY Declines in skeletal muscle health are a concern for astronauts on long-duration spaceflights. The current findings add to the growing body of exercise countermeasures data, suggesting that small doses of specific exercise can be beneficial for certain aspects of skeletal muscle health. This information can be used in conjunction with other components of existing exercise programs for astronauts and might translate to other areas focused on skeletal muscle health (e.g., sports medicine, rehabilitation, aging).

Acute Exercise Promptly Normalizes Myocardial Myosin Heavy-Chain Isoform mRNA Composition in Diabetic Rats: Implications for Diabetic Cardiomyopathy.

Background and Objectives: The acute effects of exercise on the myosin heavy-chain (MHC) isoform mRNA expression and the upstream transcription factors in diabetic and non-diabetic hearts remain unexplored. We aimed to determine the acute effect of a single exercise session on the expression of left ventricular MHC, MHC-α and MHC-β, and thyroid receptor (TR), TR-α1 and TR-β, isoform mRNA in diabetic and non-diabetic rats. Materials and Methods: Sprague-Dawley rats were assigned to four groups: non-diabetic control (CS), diabetic exercise (DIEX), sedentary diabetic (DIS), and non-diabetic exercise (CEX). Diabetes was induced via streptozotocin injection (55 mg/kg). DIEX and CEX rats performed an exercise session (60 min at 50 m/min and 0% grade) 6-7 weeks after diabetes induction. Results: MHC-α mRNA was lower in DIS (p = 0.03) and not different in DIEX (p = 0.1) relative to CS. DIS showed higher MHC-β mRNA than the non-diabetic rats, CS and CEX (p = 0.02 and p = 0.009, respectively). MHC-β mRNA in DIEX was normalized to non-diabetic levels in CS (p = 0.3). TR-α1 was higher in DIS and not different in DIEX relative to CS and CEX (p = 0.03 and p = 1.0, respectively). In CEX, exercise did not change MHC-α, MHC-β, and TR-α1 relative to CS (p = 1.0). TR-β was not different between groups. Conclusion: In conclusion, exercise appears to acutely normalize the myocardial MHC and TR isoform mRNA expression only in the diabetic heart. These responses may induce therapeutic mechanisms other than changing the MHC isoform composition.

Palmitic Acid Inhibits Myogenic Activity and Expression of Myosin Heavy Chain MHC IIb in Muscle Cells through Phosphorylation-Dependent MyoD Inactivation.

Sarcopenia associated with aging and obesity is characterized by the atrophy of fast-twitch muscle fibers and an increase in intramuscular fat deposits. However, the mechanism of fast-twitch fiber-specific atrophy remains unclear. In this study, we aimed to assess the effect of palmitic acid (PA), the most common fatty acid component of human fat, on muscle fiber type, focusing on the expression of fiber-type-specific myosin heavy chain (MHC). Myotubes differentiated from C2C12 myoblasts were treated with PA. The PA treatment inhibited myotube formation and hypertrophy while reducing the gene expression of MHC IIb and IIx, specific isoforms of fast-twitch fibers. Consistent with this, a significant suppression of MHC IIb protein expression in PA-treated cells was observed. A reporter assay using plasmids containing the MHC IIb gene promoter revealed that the PA-induced reduction in MHC IIb gene expression was caused by the suppression of MyoD transcriptional activity through its phosphorylation. Treatment with a specific protein kinase C (PKC) inhibitor recovered the reduction in MHC IIb gene expression levels in PA-treated cells, suggesting the involvement of the PA-induced activation of PKC. Thus, PA selectively suppresses the mRNA and protein expression of fast-twitch MHC by modulating MyoD activity. This finding provides a potential pathogenic mechanism for age-related sarcopenia.

Relationship between Condition Index Values and Expression Levels of Gene and Protein in the Adductor Muscle of Diploid and Triploid Oysters Crassostrea gigas

Three proteins [myosin heavy chain (MHC), filamin-C fragment (FIL-C), and actin 2 (ACT2)] were identified in adductor muscle from diploid and triploid Pacific oysters (Crassostrea gigas) and the relationship between the condition index (CI) and mRNA expression of these genes was investigated, together with the mRNA expression of molluscan insulin-related peptide (MIP), C. gigas insulin receptor-related receptor (CIR), and insulin-like growth factor binding protein complex acid labile subunit (IGFBP-ALS). Monthly changes in the CI were similar to the changes in the tissue weight rate in both groups. ACT2 and MHC mRNA expression was statistically higher in the triploid than the diploid, while FIL-C mRNA expression was significantly higher in the diploid (p<0.05). The MIP, CIR, and IGFBP-ALS mRNA expression of the diploid oysters were all significantly higher in July than in other months (p<0.05). The MIP, CIR, and IGFBP-ALS mRNA expression in the triploid oysters was high in July, but there were no significant differences (p>0.05). Changes in the expression levels of the genes investigated in this study could be used as intrinsic indicators of the annual growth, maturity, and spawning period of cultured diploid and triploid C. gigas in Tongyeong, Korea.

Diet and Exercise Restores Muscle Fiber Phenotype Following Long Term High Fat/High Sugar Diet Yet mRNA Expression Remains Unchanged

Obesity affects roughly 42% of the US population. High fat/high sugar diets (HFHS) often referred as “western diet” contributes to this prevalence. Diet‐induced obesity (DIO) results in impaired metabolic responses and associated disease states (i.e., Type 2 Diabetes). Metabolic impairments in DIO are a result of changes in muscle metabolism and muscle fiber phenotype. This is determined by both mRNA expression and protein isoform‐content of myosin heavy chain (MHC). Fast muscle fiber phenotype (i.e., type IIb in mice) is characterized by lower capacity for lipid utilization, implicated in the pathogenesis of Type 2 Diabetes. Regular exercise expresses mRNA in a fiber type specific manner, shifting MHC proportions under healthy circumstances. However, diet and exercise‐driven fiber type shifts in DIO are less understood. We aimed to determine the impact of exercise and diet on fiber‐type proportions in mice with DIO. We hypothesized exercise shifts mouse gastroc muscle phenotype induced by HFHS diet away from IIb fast fiber types. 49 C57BL/6 mice were split into 4 groups: 1) Lean Control (n = 9) fed standard chow and water for 24 wks, 2) HFHS (n = 10), fed HFHS diet (60% of calories from fat, high sugar/fructose: 42 g/L in drinking water) for 24 wks, 3) HFHS Control (n = 10) fed HFHS diet for 12 wks followed by standard chow and water for 12 wks (i.e., simulating traditional diet approach), and 4) HFHS + exercise group (n = 10) fed HFHS diet for 24 wks and performed aerobic exercise (30 min of treadmill running 5 d/wk) in the last 12 wks. Gastroc muscles were collected, homogenized, and analyzed for MHC mRNA expression of MYH 1 (type IIx), MYH 2 (type IIa), MYH 4 (type IIb), and protein isoforms via q‐PCR and SDS‐PAGE respectively. Intensity of bands corresponding to MHC isoforms were quantified using Image J. mRNA expression was calculated using comparative CT analysis (2‐∆∆CT). Paired sample t‐tests were conducted to test differences between MHC isoforms across groups. Proportions of MHC IIb isoform increased (91 ± 3%) in HFHS compared to Control (81 ± 6.2%, p&gt;0.05), HFHS Control (77 ± 3%, p=0.004), and HFHS + exercise groups (79 ± 5%, p=0.057). Additionally, MHC IIa/x proportions in the HFHS (8 ± 3%) compared to Control (17 ± 5.9%, p&gt;0.05), HFHS Control (20 ± 2%, p=0.004), and HFHS + exercise (18 ± 4%, p=0.054) groups were reduced. There were no differences in mRNA expression data. These data suggest performing aerobic exercise with HFHS diet or switching to a standard diet restores muscle fiber phenotype in mouse gastroc. Yet, this was not reflected in mRNA expression data. Changes in MHC isoforms are likely better detected at the protein level rather than mRNA expression. We conclude that exercise and dietary intervention may be a good strategy to shift MHC isoforms away from extreme fast fiber phenotype with lower capacity for lipid utilization. Future research should aim to determine single muscle fiber phenotype shifts with long‐term diet and exercise in humans with obesity to better understand regulation of muscle fiber phenotypes impact on human metabolism.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Colon-26 Tumor-Derived Factors.

Preclinical models and in vitro experiments have provided valuable insight into the regulation of cancer-induced muscle wasting. Colon-26 (C26) tumor cells induce cachexia in mice, and conditioned media (CM) from these cells promotes myotube atrophy and catabolic signaling. While mechanical stimuli can prevent some effects of tumor-derived factors on myotubes, the impact of mechanical signaling on tumor-derived factor regulation of myosin heavy chain (MyHC) expression is not well understood. Therefore, we examined the effects of stretch-induced mechanical signaling on C2C12 myotube growth and MyHC expression after C26 CM exposure. C26 CM was administered to myotubes on day 5 of differentiation for 48 h. During the last 4 or 24 h of C26 CM exposure, 5% static uniaxial stretch was administered. C26 CM suppressed myotube growth and MyHC protein and mRNA expression. Stretch for 24 h increased myotube size and prevented the C26 CM suppression of MyHC-Fast protein expression. Stretch did not change suppressed MyHC mRNA expression. Stretch for 24 h reduced Atrogin-1/MAFbx, MuRF-1, and LC3B II/I ratio and increased integrin β1D protein expression and the myogenin-to-MyoD protein ratio. Stretch in the last 4 h of CM increased ERK1/2 phosphorylation but did not alter the CM induction of STAT3 or p38 phosphorylation. These results provide evidence that in myotubes pre-incubated with CM, the induction of mechanical signaling can still provide a growth stimulus and preserve MyHC-Fast protein expression independent of changes in mRNA expression.

FNDC5 expression closely correlates with muscle fiber types in porcine longissimus dorsi muscle and regulates myosin heavy chains (MyHCs) mRNA expression in C2C12 cells.

BackgroundIrisin (a glycosylated protein) is cleaved from fibronectin type III domain-containing protein 5 (FNDC5), which is expressed mainly in animal muscle tissues and has multiple metabolic regulatory activities. However, their roles in controlling myofiber types in skeletal muscle remain unclear.MethodologyTwo different commercial hybridized pigs, LJH (a crossed pig containing Chinese native pig genotypes) and DLY (Duroc × Landrace × Yorkshire) were selected to analyze FNDC5 mRNA expression and the mRNA composition of four adult myosin heavy chain (MyHC) isoforms (IIIaIIxIIb) in the longissimus dorsi (LD) muscle. C2C12 myoblasts were cultured to investigate the effects of FNDC5 on the four MyHCs mRNA expressive levels, using small interfering RNA for depletion and a eukaryotic expression vector carrying FNDC5 for overexpression. ZLN005 (a small molecule activator of FNDC5’s upstream control gene PGC1α) or recombinant human irisin protein were also used.ResultsIn LD muscle, LJH pigs had the higher FNDC5 mRNA level, and MyHC I or IIa proportion than DLY pigs (P < 0.05). For C2C12 cells in vitro, small interfering RNA (si-592) silencing of FNDC5 expression markedly reduced MyHC IIa mRNA levels (P < 0.05), while FNDC5 overexpression significantly increased MyHC IIa mRNA levels (P < 0.05). Exogenous irisin increased the mRNA levels of PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), FNDC5, MyHCI, MyHCIIa, NRF1 (nuclear respiratory factor 1), VEGF (vascular endothelial growth factor), and TFAM (mitochondrial transcription factor A,) (P < 0.05), and the enzyme activities of SDH (succinate dehydrogenase), CK (creatine kinase), and MDH (malate dehydrogenase) in C2C12 myotubes (P < 0.05). These results showed that FNDC5 mRNA expression had a significant association with the characteristics of myofiber types in porcine muscle, and participated in regulating MyHCs mRNA expression of C2C12 myogenic differentiation cells in vitro. FNDC5 could be an important factor to control muscle fiber types, which provides a new direction to investigate pork quality via muscle fiber characteristics.

Early-senescent bone marrow mesenchymal stem cells promote C2C12 cell myogenic differentiation by preventing the nuclear translocation of FOXO3

AimsMouse bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells with self-renewal and differentiation abilities. Since the effects of senescent BMSCs on C2C12 cells are not fully clear, the present study aimed to elucidate these effects. Main methodsSenescence-associated β-galactosidase staining and western blotting were performed to confirm the senescence of BMSCs. Immunofluorescence and western blotting were used to assess myoblast differentiation in each group. The role of the AKT/P70 signaling pathway and forkhead box O3 (FOXO3) nuclear translocation was explored by western blotting. BMSC-derived exosomes were injected into the tibialis anterior of mice, and RT-qPCR was used to assess the role of exosomes in promoting muscle differentiation. Key findingsConditioned medium (CM) from early-senescent BMSCs promoted myogenic differentiation in vitro, which was detected as enhanced expression of myosin heavy chain (MHC), myogenin (MYOG), and myogenic differentiation 1 (MyoD). The AKT signaling pathway was found to be regulated by CM, which inhibited FOXO3 nuclear translocation. RT-qPCR analysis results showed that MHC, MyoD, and MYOG mRNA expression increased in the tibialis anterior of mice after exosome injection. SignificanceThe present study demonstrated that early-senescent BMSCs accelerated C2C12 cell myogenic differentiation, and the transcription factor, FOXO3, was the target of senescent cells. Collectively, our results suggest that the AKT/P70 signaling pathway mediates the effect of BMSCs on neighboring cells.

The Effect of Continuous Light on Growth and Muscle-Specific Gene Expression in Atlantic Salmon (Salmo salar L.) Yearlings.

Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species, however, the molecular mechanisms underlying this phenomenon are currently unknown. The effect of a continuous lighting (LD 24:0), compared with the usual hatchery lighting (HL) regime, on the growth rate and gene expression of myogenic regulatory factors (MRFs: MyoD1 paralogs, Myf5, and MyoG) myosin heavy chain (MyHC), and MSTN paralogs in the white muscles of hatchery-reared Atlantic salmon yearlings was evaluated over a 6-month period (May–October). The levels of gene expression were determined using real-time PCR. Continuous lighting was shown to have a positive effect on weight gain. MyHC, MyoD1c, MyoD1b, and MSTN1a/b mRNA expression was influenced by the light regime applied. In all the studied groups, a significant positive correlation was observed between the expression levels of MRFs and MSTN paralogs throughout the experiment. The study demonstrated seasonal patterns regarding the simultaneous expression of several MRFs. MyoD1a, MyoG, and MyHC mRNA expression levels were elevated in the mid-October, but MyoD1b/c, and Myf5 mRNA levels decreased by the end of this month. In general, the findings showed that constant lighting affected the regulatory mechanisms of muscle growth processes in salmon.

Sodium acetate, propionate, and butyrate reduce fat accumulation in mice via modulating appetite and relevant genes

ObjectivesAcetate, propionate, and butyrate, three of the most common short-chain fatty acids (SCFAs), can be produced when some non-digestible carbohydrates enter the large intestine and undergo bacterial fermentation. The aim of this study was to investigate the effects of these three SCFAs on appetite regulation and lipid metabolism and to determine the extent that appetite contributes to the beneficial influences of SCFAs. MethodsIn a 35-d study, 48 C57BL/6J male mice were randomly allocated to six groups: control; 5% sodium acetate; 5% sodium propionate; 5% sodium butyrate; pair fed 1; and pair fed 2. ResultsThe study showed that dietary supplementation of sodium acetate reduced serum triacylglycerol, free fatty acids, glucose, and interleukin (IL)-6 levels (P < 0.05), increased serum glucagon-like peptide 1, and leptin levels (P < 0.05), downregulated the mRNA expressions of fatty acid synthase, peroxisome proliferator-activated receptor, and lipoprotein lipase (P < 0.05), and upregulated the mRNA expressions of fasting-induced adipose factor, nuclear respiratory factor 1, mitochondrial transcription factor A, tumor necrosis factor receptor superfamily member 9, cytochrome-C oxidase IV and free fatty acid receptor 2 (P < 0.05). Sodium propionate also reduced serum IL-1β level (P < 0.05), increased serum peptide YY level (P < 0.05), downregulated the mRNA expressions of acetyl-coenzyme A carboxylase and sterol regulatory element-binding protein 1c (P < 0.05), and upregulated the mRNA expression of transmembrane protein 26 (P < 0.05). Additionally, sodium butyrate decreased average daily feed intake (P < 0.05) downregulated the mRNA expression of myosin heavy-chain (MyHc) Ⅱb (P < 0.05), and upregulated the mRNA expressions of lipase hormone-sensitive, MyHC Ⅱa and carnitine palmitoyltransferase-1α (P < 0.05). Moreover, the metabolic benefits of SCFAs were partly attributed to the reduction of feed intake. ConclusionTaken together, SCFAs could reduce appetite and fat accumulation via modulating relevant genes and hormones, which might further illustrate the potential mechanisms that underlay the effects of SCFAs on lipid homeostasis and control of body weight.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Procyanidin B2 Promotes Skeletal Slow-Twitch Myofiber Gene Expression through the AMPK Signaling Pathway in C2C12 Myotubes.

Dimer procyanidin B2 [epicatechin-(4β-8)-epicatechin] (PB2) has attracted a lot of interest in nutrition and medicine because of its significant health-promoting abilities. However, the function of PB2 on different types of skeletal myofiber is still unclear. Here, we have found that PB2 significantly increased protein expression of the slow myosin heavy chain (MyHC) and decreased fast MyHC protein in C2C12 myotubes, accompanied by upregulation of mRNA expression of MyHC I, MyHC IIa, and Tnni1 and downregulation of MyHC IIx and MyHC IIb. We have also found that PB2 enhanced the activities of malate dehydrogenase and succinic dehydrogenase and reduced lactate dehydrogenase activity. PB2 promoted phosphorylation of AMPK and significantly increased mRNA expression of AMPKα1. The upstream factors of AMPK, such as phospho-LKB1, NRF1, and CaMKKβ, and the downstream factors of AMPK, including Sirt1 and PGC-1α, were also increased by PB2. Specific suppression of AMPK signaling by AMPKα1 siRNA or by AMPK inhibitor compound C significantly attenuated the PB2-induced upregulation of phospho-AMPK, PGC-1α, and slow MyHC and downregulation of fast MyHC. Our findings suggested that PB2 promotes skeletal slow-twitch myofiber gene expression through the AMPK signaling pathway in C2C12 myotubes.

Changes in biomarker levels and myofiber constitution in rat soleus muscle at different exercise intensities.

Although exercise affects the function and structure of skeletal muscle, our knowledge regarding the biomedical alterations induced by different intensities of exercise is incomplete. Here we report on the changes in biomarker levels and myofiber constitution in the rat soleus muscle induced by exercise intensity. Male adult rats at 7weeks of age were divided into 3 groups by exercise intensity, which was set based on the accumulated lactate levels in the blood using a treadmill: stationary control (0m/min), aerobic exercise (15m/min), and anaerobic exercise (25m/min). The rats underwent 30min/day treadmill training at different exercise intensities for 14days. Immediately after the last training session, the soleus muscle was dissected out in order to measure the muscle biomarker levels and evaluate the changes in the myofibers. The mRNA expression of citrate synthase, glucose-6-phosphate dehydrogenase, and Myo D increased with aerobic exercise, while the mRNA expression of myosin heavy-chain I and Myo D increased in anaerobic exercise. These results suggest that muscle biomarkers can be used as parameters for the muscle adaptation process in aerobic/anaerobic exercise. Interestingly, by 14days after the anaerobic exercise, the number of type II (fast-twitch) myofibers had decreased by about 20%. Furthermore, many macrophages and regenerated fibers were observed in addition to the injured fibers 14days after the anaerobic exercise. Constitutional changes in myofibers due to damage incurred during anaerobic exercise are necessary for at least about 2weeks. These results indicate that the changes in the biomarker levels and myofiber constitution by exercise intensity are extremely important for understanding the metabolic adaptations of skeletal muscle during physical exercise.

Leucine regulates slow-twitch muscle fibers expression and mitochondrial function by Sirt1/AMPK signaling in porcine skeletal muscle satellite cells.

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow-twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow-twitch muscle fibers and mitochondrial function-related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho-AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow-twitch muscle fibers and mitochondrial function-related gene expression. Finally, we showed that Sirt1 was required for leucine-induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow-twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.

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

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.

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.

Apigenin enhances skeletal muscle hypertrophy and myoblast differentiation by regulating Prmt7.

Apigenin, a natural flavone abundant in various plant-derived foods including parsley and celery, has been shown to prevent inflammation and inflammatory diseases. However, the effect of apigenin on skeletal muscle hypertrophy and myogenic differentiation has not previously been elucidated. Here, we investigated the effects of apigenin on quadricep muscle weight and running distance using C57BL/6 mice on an accelerating treadmill. Apigenin stimulated mRNA expression of MHC (myosin heavy chain) 1, MHC2A, and MHC2B in the quadricep muscles of these animals. GPR56 (G protein-coupled receptor 56) and its ligand collagen III were upregulated by apigenin supplementation, together with enhanced PGC-1α, PGC-1α1, PGC-1α4, IGF1, and IGF2 expression. Prmt7 protein expression increased in conjunction with Akt and mTORC1 activation. Apigenin treatment also upregulated FNDC5 (fibronectin type III domain containing 5) mRNA expression and serum irisin levels. Furthermore, apigenin stimulated C2C12 myogenic differentiation and upregulated total MHC, MHC2A, and MHC2B expression. These events were attributable to an increase in Prmt7-p38-myoD expression and Akt and S6K1 phosphorylation. We also observed that Prmt7 regulates both PGC-1α1 and PGC-1α4 expression, resulting in a subsequent increase in GPR56 expression and mTORC1 activation. Taken together, these findings suggest that apigenin supplementation can promote skeletal muscle hypertrophy and myogenic differentiation by regulating the Prmt7-PGC-1α-GPR56 pathway, as well as the Prmt7-p38-myoD pathway, which may contribute toward the prevention of skeletal muscle weakness.

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.

Deletion of Kvβ2 Alters Murine Skeletal Muscle Morphology and Phenotype

IntroductionMammalian skeletal muscle expresses various potassium channels that regulate myogenesis. Recent evidence points to the role of Kv1.5 in cell cycle progression in myoblasts as pharmacological inhibition of this channel in L6E9 cells inhibited G0/G1 transition. Additionally, skeletal muscle also expresses voltage‐gated potassium channel beta subunits 1–3 (Kvβ, 2, 3) of aldo‐keto reductases (AK6) family, although their role in skeletal myogenesis is not yet known. Given that Kvβ subunits binds to and regulate members of Kv1 family including Kv1.5, it is plausible that Kvβ subunits play a significant role in skeletal myogenesis.Hypothesiswe hypothesized that Kvβ2 modulates skeletal myogenesis.MethodsAge matched, 8–12 week old, wild type (Wt) and Kvβ2 knockout mice (KO) mice were used in this study. Transcript expression analysis of key transcription factors, muscle growth regulators and contractile genes was conducted utilizing real time quantitative PCR (qPCR). Myofiber size was estimated in biceps femoris muscle cross‐sections using Hematoxylin and eosin staining. Protein expression was assessed by Western blots in lysates isolated from Biceps femoris muscle.ResultsBody weight of KO mice recorded at 6, 11 or 16 weeks of age, was significantly (p &lt; 0.05) less than that of age matched Wt mice. Body weight normalized to tibia length in 16 week old KO mice was nearly 30% less than that of age matched Wt mice. Biceps femoris muscle cross‐sections in KO mice revealed significantly smaller myofiber area when compared to that of Wt mice (p &lt; 0.05). Myosin heavy chain mRNA expression profiles were also significantly different between the KO and Wt mice. when compared with that of Wt mice, gastrocnemius and biceps femoris of KO mice showed marginal (p &lt; 0.1) to significantly (p &lt; 0.05) higher expression of myosin heavy chain IIA and IIX isoforms, while myosin heavy chain I mRNA expression was not different between the groups. Soleus muscle of KO mice showed significantly higher myosin heavy chain I expression, while the expression of myosin heavy chain 2B was profoundly (p &lt; 0.05) decreased. Interestingly, the expression of major myogenic regulatory factors was unchanged between the two genotypes, with the exception that MyoD expression showed a consistently decreasing trend in all three muscles of KO mice when compared with that of Wt. In C2C12 cells, Kvβ2 mRNA expression declined steadily during the course of differentiation reaching to approximately 10% of that observed before differentiation (p &lt; 0.05).ConclusionOur data suggest that Kvβ2 subunit is essential for skeletal myogenesis. Kvβ2 appear to effect muscle fitness by altering myosin heavy chain composition, suggesting a significant role of Kvβ subunits in skeletal muscle size and phenotype determination.