Myosin Heavy Chain mRNA Research Articles

Vigeo Promotes Myotube Differentiation and Protects Dexamethasone-Induced Skeletal Muscle Atrophy via Regulating the Protein Degradation, AKT/mTOR, and AMPK/Sirt-1/PGC1α Signaling Pathway In Vitro and In Vivo.

Sarcopenia, a condition caused by an imbalance between muscle growth and loss, can severely affect the quality of life of elderly patients with metabolic, inflammatory, and cancer diseases. Vigeo, a nuruk-fermented extract of three plants (Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK)) has been reported to have anti-osteoporotic effects. However, evidence of the effects of Vigeo on muscle atrophy is not available. Here, in the in vivo model of dexamethasone (Dex)-induced muscle atrophy, Vigeo treatment significantly reversed Dex-induced decreases in calf muscle volume, gastrocnemius (GA) muscle weight, and histological cross-section area. In addition, in mRNA and protein analyses isolated from GA muscle, we observed that Vigeo significantly protected against Dex-induced mouse muscle atrophy by inhibiting protein degradation regulated by atrogin and MuRF-1. Moreover, we demonstrated that Vigeo significantly promoted C2C12 cell line differentiation, as evidenced by the increased width and length of myotubes, and the increased number of fused myotubes with three or more nuclei. Vigeo alleviated the formation of myotubes compared to the control group. Vigeo also significantly increased the mRNA and protein expression of myosin heavy chain (MyHC), MyoD, and myogenin compared to that in the control. Vigeo treatment significantly reduced the mRNA and protein expression of muscle degradation markers atrogin-1 and muscle RING Finger 1 (MuRF-1) in the C2C12 cell line in vitro. Vigeo also activated the AMP-activated protein kinase (AMPK)/silent information regulator 1 (Sirt-1)/peroxisome proliferator-activated receptor-γ co-activator-1α (PGC1α) mitochondrial biogenesis pathway and the Akt/mTOR protein synthesis signaling pathway in Dex-induced myotube atrophy. These findings suggest that Vigeo may have protective effects against Dex-induced muscle atrophy. Therefore, we propose Vigeo as a supplement or potential therapeutic agent to prevent or treat sarcopenia accompanied by muscle atrophy and degeneration.

LncRNA lncMGR regulates skeletal muscle development and regeneration by recruiting CDK9 and sponging miRNAs

Long non-coding RNAs (lncRNAs) play an essential role in vertebrate myogenesis and muscle diseases. However, the dynamic expression patterns, biological functions, and mechanisms of lncRNAs in skeletal muscle development and regeneration remain largely unknown. In this study, a novel lncRNA (named lncMGR) was differentially expressed during breast muscle development in fast- and slow-growing chickens. Functionally, lncMGR promoted myoblast differentiation, inhibited myoblast proliferation in vitro, and promoted myofiber hypertrophy and injury repair in vivo. Mechanistically, lncMGR increased the mRNA and protein expression of skeletal muscle myosin heavy chain 1 A (MYH1A) via both transcriptional and post-transcriptional regulation. Nuclear lncMGR recruited cyclin-dependent kinase 9 (CDK9) to the core transcriptional activation region of the MYH1A gene to activate MYH1A transcription. Cytoplasmic lncMGR served as a competitive endogenous RNA (ceRNA) to competitively absorb miR-2131-5p away from MYH1A and subsequently protected the MYH1A from miR-2131-5p-mediated degradation. Besides miR-2131-5p, cytoplasmic lncMGR could also sponge miR-143-3p to reconcile the antagonist between the miR-2131-5p/MYH1A-mediated inhibition effects and miR-143-3p-mediated promotion effects on myoblast proliferation, thereby inhibiting myoblast proliferation. Collectively, lncMGR could recruit CDK9 and sponge multiple miRNAs to regulate skeletal muscle development and regeneration, and could be a therapeutic target for muscle diseases.

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.

Role of p53 in Cisplatin-Induced Myotube Atrophy.

Chemotherapy-induced sarcopenia is an unfavorable prognostic factor implicated in the development of postoperative complications and reduces the quality of life of patients with cancer. Skeletal muscle loss due to cisplatin use is caused by mitochondrial dysfunction and activation of muscle-specific ubiquitin ligases Atrogin-1 and muscle RING finger 1 (MuRF1). Although animal studies suggest the involvement of p53 in age-, immobility-, and denervation-related muscle atrophy, the association between cisplatin-induced atrophy and p53 remains unknown. Herein, we investigated the effect of a p53-specific inhibitor, pifithrin-alpha (PFT-α), on cisplatin-induced atrophy in C2C12 myotubes. Cisplatin increased the protein levels of p53, phosphorylated p53, and upregulated the mRNA expression of p53 target genes PUMA and p21 in C2C12 myotubes. PFT-α ameliorated the increase in intracellular reactive oxygen species production and mitochondrial dysfunction, and also reduced the cisplatin-induced increase in the Bax/Bcl-2 ratio. Although PFT-α also reduced the cisplatin-induced increase in MuRF1 and Atrogin-1 gene expression, it did not ameliorate the decrease in myosin heavy chain mRNA and protein levels and muscle-specific actin and myoglobin protein levels. We conclude that cisplatin increases muscle degradation in C2C12 myotubes in a p53-dependent manner, but p53 has minimal involvement in the reduction of muscle protein synthesis.

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.

Detection of intracellular sodium ions based on phenotype-specific activation of NaA43 DNAzyme

The intracellular sodium ion is one of the crucial elements for regulating physiological functions such as action potential and muscle contractions. However, detecting sodium ions in live cells is challenging because false signals may arise from the abundant sodium ions in the extracellular environment when introducing the detection agents. To minimize it, we report a DNAzyme-based detection of sodium ions in live cells via activation by endogenous mRNA. The substrate strand of DNAzyme first hybridizes to a blocking strand that prevents undesired cleavage of DNAzyme when delivered. Once entering cells, an endogenous mRNA biomarker binds to the toehold region of the blocking strand and displaces it, allowing the proper formation of the DNAzyme, which specifically catalyzes the cleavage of the substrate strand in the presence of intracellular Na+ and produces fluorescence signals. Using differentiating skeletal muscle cells as the model system, we demonstrated the successful delivery and phenotype-specific detection of intracellular sodium ions only in differentiated myotubes with highly-expressed myosin heavy chain mRNA. Moreover, using a drug cocktail to increase the permeability of the cell membrane, elevated levels of intracellular sodium ion was observed. This platform offers a broad and promising strategy for detecting intracellular metal ions, suggesting a great potential for understanding its role in cell/tissue physiology.

A Phytomolecule Icariin Protects from Sarcopenia Partially by Suppressing Myosin Heavy Chain Degradation in Orchiectomized Rats.

Treatments are lacking for sarcopenia, which is an age-related disease characterized by loss of skeletal muscle mass, strength, and/or physical performance. Icariin is a phytomolecule fromherbal Epimedium, a traditional Chinese medicine widely used to treat musculoskeletal disorders for thousands of years. Here the effects of icariin against sarcopenia are investigated and the underlying mechanism is elucidated. A classic rat model of bilaterally orchiectomized (ORX) is used to induce sarcopenia. After administration for 8 weeks, compared to the control group, the forelimb grip strength, the specific tetanic forces of the soleus (SOL) and extensor digitorum longus muscle (EDL) are higher, and the fiber cross-sectional areas (CSAs) of the gastrocnemius and tibialis anterior muscle are larger in the icariin group. In addition, icariin promotes mRNA and protein expressions of myosin heavy chain (MyHC) both in SOL and EDL. Mechanistically, icariin significantly suppresses the mRNA and protein expressions of FOXO3a, atrogin-1, and MuRF-1, which are related to the degradation of myosin heavy chain. Collectively, icariin protects from sarcopenia in ORX rats characterized by enhancing grip strength and skeletal muscle contraction, as well as increasing skeletal muscle CSA by inhibiting the ubiquitination degradation of the MyHC in skeletal muscle fibers.

Psoralea corylifolia L. seed extract attenuates dexamethasone-induced muscle atrophy in mice by inhibition of oxidative stress and inflammation

Ethnopharmacological relevanceThe seeds of Psoralea corylifolia (PCS), also called “Boh-Gol-Zhee” in Korean, have been used in traditional medicine. PCS is effective for the treatment of vitiligo, cancer, inflammatory diseases, neurodegenerative diseases, kidney diseases, and musculoskeletal diseases. Aim of the studyIn this study, we validated the beneficial effects of PCS extract on dexamethasone (DEX)-induced muscle atrophy in mice. Materials and MethodsDEX (20 mg/kg/day, 10 days) was intraperitoneally injected into C57BL/6 male mice to induce muscular atrophy. Oral administration of PCS extract (200 or 500 mg/kg/day) was started 2 days before DEX injection and continued for 12 days. ResultsPCS extract inhibited DEX-induced decrease in body and muscle weight, grip strength, and cross-sectional area of the tibialis anterior. PCS extract significantly increased the mRNA and protein expression levels of myosin heavy chain 1, 2A, and 2X in DEX-administered mice. DEX administration significantly increased the levels of muscle atrophy factors atrogin-1, muscle RING-finger protein-1, and myostatin, which were inhibited by the PCS extract. Additionally, PCS extract increased the expression of muscle regeneration factors, such as myoblast determination protein 1, myogenin, and embryonic myosin heavy chain, and muscle synthesis markers, such as protein kinase B and mammalian target of rapamycin signaling molecules. PCS extract also significantly decreased the DEX-induced production of 4-hydroxynonenal, an oxidative stress marker. Furthermore, PCS extract recovered superoxide dismutase 2, glutathione peroxidase, and catalase activities, which were significantly reduced by DEX administration. Moreover, DEX-induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells and expression of cytokines, such as tumor necrosis factor α and monocyte chemoattractant protein-1, significantly decreased after PCS extract administration. ConclusionsHere, we demonstrated that PCS extract administration protected against DEX-induced muscle atrophy. This beneficial effect was mediated by suppressing the expression of muscle degradation factors and increasing the expression of muscle regeneration and synthesis factors. This effect was probably due to the inhibition of oxidative stress and inflammation. These results highlight the potential of PCS extract as a protective and therapeutic agent against muscle dysfunction and atrophy.

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.

Correlation between Meat Quality and Muscle Fiber Types of Tibetan and Large White Pigs

This research was designed to explore the differences of meat quality and their causes of pigs in different regions, In this study, natural raised Tibetan pigs in different regions (Linzhi of Tibet, Gannan of Gansu, and Aba of Sichuan) and large white pigs were selected, the meat quality of their longissimus muscle (pH, color, cooking loss, shear force), myoglobin concentration, hydroxyproline content, relative expression of different Myosin Heavy Chain (MYHC) mRNA were determined, and correlation analysis were adopted. The results showed that the pH3h of Tibetan pigs was significantly lower than that of large white pigs 6.0 (P<0.05), and there was no significant difference among different regions (5.7~5.8),The redness value (a*) and myoglobin content of Tibetan pigs were significantly higher than those of large white pigs (2.12±0.87, 0.004 μmol/g). Among them, Aba Tibetan pigs had the highest a* (8.10±0.99). The shear force and hydroxyproline content of Tibetan pigs were significantly higher than those of large white pigs (41.81±0.83、5.92±0.52%) (P<0.05), the proportion of type IIa muscle fibers in Tibetan pigs was significantly higher and the proportion of type IIb muscle fibers is significantly lower than their of large white pigs (P<0.05); correlation analysis shown that a* was significantly negatively correlated with L* and type IIb muscle fibers (P<0.01), and significantly positively correlated with myoglobin content, type I and type IIa muscle fibers (P<0.01). Shear force and hydroxyproline content were extremely significantly correlated (P<0.01). In conclusion, the color of Tibetan pig is redder and the tenderness was worse than that of large white pig, the great differences between Tibetan pigs and large white pigs and the differences among Tibetan pigs from different regions in meat quality are highly related to their muscle fiber type.

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.

Preliminary Investigation of In vitro, Bidirectional Vocal Fold Muscle-Mucosa Interactions.

Oversimplified clinical dogma suggests that laryngeal diseases fall into two broad, mutually exclusive diagnostic categories-mucosal injury or neuromuscular/functional disorders. Extensive investigation in the lower airway as well as other organ systems suggest complex interactions between tissue types underlying both tissue health and pathological states. To date, no such relationship has been described in the vocal folds, likely the most bioactive organ in the body. We hypothesize interactions between the vocal fold muscle and mucosa likely contribute to aberrant phonatory physiology and warrant further investigation to ultimately develop novel therapeutic strategies. Primary culture of myoblasts from rat thyroarytenoid muscle and fibroblasts from the vocal fold mucosa were established. Co-culture and conditioned media experiments were performed to established bidirectional interactions between cell types. Transforming Growth Factor (TGF)-β was employed to stimulate a fibrotic phenotype in culture. In addition to quantitative PCR, standard migration and proliferation assays were performed as well as immunocytochemistry. Bidirectional cell-cell interactions were observed. Without TGF-β stimulation, myoblast conditioned media inhibited fibroblast migration, but enhanced proliferation. Conversely, fibroblast conditioned media increased both myoblast proliferation and migration. Myoblast conditioned media decreased TGF-β-mediated gene expression and of particular interest, ACTA2 mRNA expression. In both co-culture and in response to fibroblast conditioned media, myosin heavy chain (Myh2) mRNA expression decreased in myoblasts. These data are the first to describe interactions between cell types within the vocal fold. The implications for these interactions in vivo warrant further investigation to develop and refine optimal treatment strategies.

Decreased vascular smooth muscle contractility in Hutchinson\u2013Gilford Progeria Syndrome linked to defective smooth muscle myosin heavy chain expression

Children with Hutchinson–Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.

Regulation of Cyclic Nucleotide Phosphodiesterase 1 by Endoplasmic Reticulum Stress Contributes to Homocysteine‐induced Cardiac Hypertrophy

Objectives Mechanistic understanding of the role of homocysteine in the development of cardiac hypertrophy is inadequate at present. This study aimed to elucidate the association of cyclic nucleotide phosphodiesterase 1 (PDE1) and endoplasmic reticulum (ER) stress with cardiomyocyte hypertrophy induced by homocysteine. Methods H9c2 cardiomyocytes were allocated to control group and homocysteine-treated group in the presence or absence of ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or PDE1 inhibitor Lu AF58027 for 72h. In another set of experiment, H9c2 cells were transfected with specific siRNA targeting PDE1 subtypes prior to homocysteine exposure. The cell surface area was measured by phase contrast microscope and image analysis software and ultrastructural changes of the cells were observed under transmission electron microscopy. Quantitative polymerase chain reaction and western blot were employed to detect the mRNA and protein expression of the hypertrophic markers beta-smooth muscle myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP), PDE1 subtypes (PDE1A, 1B, and 1C), and ER stress molecules (GRP78, phosphor-PERK, PERK, phosphor-IRE1, IRE1, and ATF6). The intracellular levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) were measured by enzyme linked immunosorbent assay. Results Homocysteine exposure resulted in an enlargement in cell size and an increase in β-MHC and ANP expression. Homocysteine induced ER stress in H9c2 cells, shown by increased expression of GRP78 and enhanced phosphorylation of PERK and IRE1, as well as dilatation and degranulation of rough ER. The expression of PDE1A and PDE1C was significantly upregulated by homocysteine. Inhibition of ER stress or PDE1 suppressed homocysteine-induced upregulation of β-MHC and ANP and alleviated cell enlargement. In addition, the protein expression of PDE1A and PDE1C in homocysteine-exposed H9c2 cells was downregulated by the ER stress inhibitor TUDCA. Silencing of PDE1C significantly inhibited homocysteine-induced cell hypertrophy whereas knockdown of PDE1A showed minor effect. Both cAMP and cGMP levels were lowered in H9c2 cells subjected to homocysteine exposure, while only cAMP content could be restored by Lu AF58027 and TUDCA. Conclusions ER stress mediates homocysteine-induced cardiomyocyte hypertrophy via upregulating PDE1 expression. Cyclic nucleotide, in particular cAMP, is the downstream mediator of the ER stress-PDE1 signaling axis in homocysteine-induced hypertrophy.

Association Between Soluble Epoxide Hydrolase and TRPC3 Channels in Pathological Cardiac Hypertrophy

Objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Although previous studies have shown a positive correlation between homocysteine levels and the incidence of cardiac hypertrophy and remodeling, the molecular mechanisms by which homocysteine promotes the development of cardiac hypertrophy remain inadequately understood. In this study, we investigated the role of soluble epoxide hydrolase (sEH) and transient receptor potential isoform 3 (TRPC3) channels in homocysteine-induced cardiac hypertrophy with further elucidating the association between sEH and TRPC3. Methods H9c2 cardiomyocytes were exposed to different concentrations of homocysteine ranging from 25 to 200 μmol/L for different duration. The cell surface area was measured by phase contrast microscope and image analysis software. The mRNA and protein expressions of sEH, TRPC3 channels, and hypertrophic markers beta-smooth muscle myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) were detected by quantitative polymerase chain reaction and western blot. Results Homocysteine treatment dose-dependently and time-dependently increased the expression of β-MHC and ANP in H9c2 cardiomyocytes. Both mRNA and protein levels of sEH and TRPC3 channels were unregulated by homocysteine. Inhibition of sEH with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) significantly suppressed homocysteine-induced upregulation of β-MHC and ANP and attenuated the increase of cell size. Pyr3, a selective TRPC3 channel blocker, also exerted a strong anti-hypertrophic effect against homocysteine. Treatment with TPPU significantly suppressed the expression of TRPC3 channels in homocysteine-exposed H9c2 cells. Conclusions: Both sEH and TRPC3 channels are involved in homocysteine-induced hypertrophy of cardiomyocytes. TRPC3 channels may act as a downstream mediator for sEH activation induced by homocysteine to promote cardiac hypertrophy.

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.

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.

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.