MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

BACKGROUND: Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. METHODS: We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. RESULTS: We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. CONCLUSIONS: Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

A New Autosomal Myh11-CreERT2 Smooth Muscle Cell Lineage Tracing and Gene Knockout Mouse Model-Brief Report.

BACKGROUND: The Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre-driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the existing Myh11-CreERT2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Here, we describe a new autosomal Myh11-CreERT2 mouse (referred to as Myh11-CreERT2-RAD), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice. METHODS: A Myh11-CreERT2-RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ERT2 after the Myh11 start codon. Myh11-CreERT2-RAD mice were crossed with 2 different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses. RESULTS: Myh11-CreERT2-RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreERT2-RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreERT2 mice. Labeling was equivalent in both male and female Cre+ mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence. CONCLUSIONS: We generated and validated the function of an autosomal Myh11-CreERT2-RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.

Effects of age and diet consistency on the expression of myosin heavy-chain isoforms on jaw-closing and jaw-opening muscles in a rat model.

BACKGROUND: Skeletal craniofacial morphology can be influenced by changes in masticatory muscle function, which may also change the functional profile of the muscles. OBJECTIVES: To investigate the effects of age and functional demands on the expression of Myosin Heavy-Chain (MyHC) isoforms in representative jaw-closing and jaw-opening muscles, namely the masseter and digastric muscles respectively. METHODS: Eighty-four male Wistar rats were divided into four age groups, namely an immature (n = 12; 4-week-old), early adult (n = 24; 16-week-old), adult (n = 24; 26-week-old) and mature adult (n = 24; 38-week-old) group. The three adult groups were divided into two subgroups each based on diet consistency; a control group fed a standard (hard) diet, and an experimental group fed a soft diet. Rats were sacrificed, and masseter and digastric muscles dissected. Real-time quantitative polymerase chain reaction was used to compare the mRNA transcripts of the MyHC isoforms-Myh7 (MyHC-I), Myh2 (MyHC-IIa), Myh4 (MyHC-IIb) and Myh1 (MyHC-IIx)-of deep masseter and digastric muscles. RESULTS: In the masseter muscle, hypofunction increases Myh1 (26, 38 weeks; p < .0001) but decreases Myh4 (26 weeks; p = .046) and Myh2 (26 weeks; p < .0001) expression in adult rats. In the digastric muscle, hypofunction increases Myh1 expression in the mature adult rats (38 weeks; p < .0001), while Myh2 expression decreases in adult rats (26 weeks; p = .021) as does Myh4 (26 weeks; p = .001). Myh7 expression is increased in the digastric muscle of mature adult rats subjected to hypofunction (38 weeks; p = <.0001), while it is very weakly expressed in the masseter. CONCLUSION: In jaw-opening and jaw-closing muscles, differences in myosin expression between hard- and soft-diet-fed rats become evident in adulthood, suggesting that long-term alteration of jaw function is associated with changes in the expression of MyHC isoforms and potential fibre remodelling. This may give insight into the role of function on masticatory muscles and the resultant craniofacial morphology.

Changes in myocardial myosin heavy chain isoform composition with exercise and post-exercise cold-water immersion.

This study investigated the changes in myocardial myosin heavy chain (MHC) isoforms, MHC-α and MHC-ß composition in young healthy rodents following endurance training, with and without post-exercise cold-water immersion (CWI). Male rats were either trained on a treadmill for 10 weeks with (CWI) or without (Ex) regular CWI after each running session, or left sedentary (CON). Left ventricular mRNA of MHC-α, MHC-ß, thyroid receptor α1 (TR-α1) and ß (TR-ß) were analyzed using rt-PCR and semiquantitative PCR analysis. MHC isoform protein composition was determined using SDS-PAGE electrophoresis. MHC-α isoform protein was predominant in all groups. The relative expression of MHC-ß (%MHC-ß) protein was not different between groups (CWI 34.7 ± 6.9%; Ex 32 ± 5.3%; CON 35.5 ± 10%; P = 0.7). MHC-ß mRNA was reduced in Ex (0.7 ± 0.3-fold) compared to CWI (1.3 ± 0.2-fold; P < 0.001) and CON (1.01 ± 0.2-fold; P = 0.03). TRα1 mRNA was lower in CWI (0.4 ± 0.05-fold) than Ex (1.02 ± 0.3-fold) and CON (1.01 ± 0.2-fold) (P < 0.001 for both). CWI exhibited greater %MHC-ß mRNA (56.8 ± 4.1%) than Ex (44.4 ± 7.7%; P = 0.001) and CON (48.5 ± 7.8%; P = 0.03). Neither exercise nor post-exercise CWI demonstrated a distinct effect on myocardial MHC protein isoform composition. However, CWI increased the relative expression of MHC-ß mRNA compared with Ex and CON. Although this implicates a potential negative long-term impact of post-exercise CWI, future studies should include measures of cardiac function to better understand the effect of such isoform mRNA shifts following regular use of CWI.

Role of Akirin1 in the regulation of skeletal muscle fiber-type switch.

Akirin1 is a highly conserved ubiquitously expressed nuclear protein. Owing to its strong nuclear localization signal and protein-protein interaction properties, Akirin1 has been speculated to regulate transcription of target genes as a cofactor. Previous studies have reported Akirin1 as a downstream target of myostatin, a potent negative regulator of myogenesis. Mice lacking myostatin displayed enhanced Akirin1 gene expression. Further, in vitro evidence has shown Akirin1 overexpression leads to hypertrophy in C2 C 12 myotubes. In this study, we used Akirin1 knockout mice as a model system to further elucidate the function of Akirin1 in fully differentiated skeletal muscle. Akirin1 knockout mice did not show any obvious phenotypic difference when compared with wild type. However, promoter-reporter assay suggested that Akirin1 regulated the transcription of muscle-specific RING finger 1 (MuRF-1), an important E3 ubiquitin ligase in skeletal muscle. Furthermore, ablation of Akirin1 resulted in increased type IIa and decreased type I muscle fibers, which was further supported by an increase in Myh2 and decrease in Myh7 gene expression. Also, histochemical studies for succinate dehydrogenase activity revealed a less oxidative muscle in the absence of Akirin1. Together, our study suggests a novel role of Akirin1 in maintaining the muscle fiber type and regulation of the metabolic activity of the skeletal muscle.

Hypertrophic Cardiomyopathy: Genetics, Pathogenesis, Clinical Manifestations, Diagnosis, and Therapy.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by left ventricular hypertrophy unexplained by secondary causes and a nondilated left ventricle with preserved or increased ejection fraction. It is commonly asymmetrical with the most severe hypertrophy involving the basal interventricular septum. Left ventricular outflow tract obstruction is present at rest in about one third of the patients and can be provoked in another third. The histological features of HCM include myocyte hypertrophy and disarray, as well as interstitial fibrosis. The hypertrophy is also frequently associated with left ventricular diastolic dysfunction. In the majority of patients, HCM has a relatively benign course. However, HCM is also an important cause of sudden cardiac death, particularly in adolescents and young adults. Nonsustained ventricular tachycardia, syncope, a family history of sudden cardiac death, and severe cardiac hypertrophy are major risk factors for sudden cardiac death. This complication can usually be averted by implantation of a cardioverter-defibrillator in appropriate high-risk patients. Atrial fibrillation is also a common complication and is not well tolerated. Mutations in over a dozen genes encoding sarcomere-associated proteins cause HCM. MYH7 and MYBPC3, encoding ß-myosin heavy chain and myosin-binding protein C, respectively, are the 2 most common genes involved, together accounting for ≈50% of the HCM families. In ≈40% of HCM patients, the causal genes remain to be identified. Mutations in genes responsible for storage diseases also cause a phenotype resembling HCM (genocopy or phenocopy). The routine applications of genetic testing and preclinical identification of family members represents an important advance. The genetic discoveries have enhanced understanding of the molecular pathogenesis of HCM and have stimulated efforts designed to identify new therapeutic agents.

Nonmuscle myosin II isoforms interact with sodium channel alpha subunits.

Sodium channels play pivotal roles in health and diseases due to their ability to control cellular excitability. The pore-forming α-subunits (sodium channel alpha subunits) of the voltage-sensitive channels (i.e., Nav1.1-1.9) and the nonvoltage-dependent channel (i.e., Nax) share a common structural motif and selectivity for sodium ions. We hypothesized that the actin-based nonmuscle myosin II motor proteins, nonmuscle myosin heavy chain-IIA/myh9, and nonmuscle myosin heavy chain-IIB/myh10 might interact with sodium channel alpha subunits to play an important role in their transport, trafficking, and/or function. Immunochemical and electrophysiological assays were conducted using rodent nervous (brain and dorsal root ganglia) tissues and ND7/23 cells coexpressing Nav subunits and recombinant myosins. Immunoprecipitation of myh9 and myh10 from rodent brain tissues led to the coimmunoprecipitation of Nax, Nav1.2, and Nav1.3 subunits, but not Nav1.1 and Nav1.6 subunits, expressed there. Similarly, immunoprecipitation of myh9 and myh10 from rodent dorsal root ganglia tissues led to the coimmunoprecipitation of Nav1.7 and Nav1.8 subunits, but not Nav1.9 subunits, expressed there. The functional implication of one of these interactions was assessed by coexpressing myh10 along with Nav1.8 subunits in ND7/23 cells. Myh10 overexpression led to three-fold increase ( P < 0.01) in the current density of Nav1.8 channels expressed in ND7/23 cells. Myh10 coexpression also hyperpolarized voltage-dependent activation and steady-state fast inactivation of Nav1.8 channels. In addition, coexpression of myh10 reduced ( P < 0.01) the offset of fast inactivation and the amplitude of the ramp currents of Nav1.8 channels. These results indicate that nonmuscle myosin heavy chain-IIs interact with sodium channel alpha subunits subunits in an isoform-dependent manner and influence their functional properties.

[Gene screening and phenotype analysis in a pedigree with familial hypertrophic cardiomyopathy from Yunnan Province].

Objective: To identify the disease-causing mutations in a pedigree with familial hypertrophic cardiomyopathy (HCM) from Yunnan province, and analyze the relationship between the genotype and the phenotype. Methods: The blood samples and the clinical data of the HCM family members were collected.The coding exons and their flanking intronic regions of 28 previously reported genes related to HCM were screened in the proband by high-throughput sequencing. The mutations in proband were confirmed and detected in all family members as well as in 159 healthy controls by Sanger sequencing.The relationship between the genotype and the phenotype was analyzed in this pedigree. Results: Two missense mutations of Arg1045His and Ala26Val in ß myosin heavy chain gene(MYH7) were identified. Genetic screening showed that the mother and brother of the proband carried Arg1045His mutation.Both mutations were absent in other family members and in 159 healthy controls.Disease onset age was less than 50 years old in this pedigree, chest pain, exertional dyspnea and syncope were the major symptoms, and all accompanied by severe left ventricular hypertrophy and left ventricular outflow tract stenosis.The grandma of the proband suffered sudden cardiac death. The proband had the worst symptoms and the earliest disease onset in this pedigree. Conclusions: We find a pedigree with familial HCM from Yunnan province carrying MYH7 Arg1045His and Ala26Val mutations. The study suggests that Arg1045His mutation in MYH7 gene caused HCM is malignant with early onset, severe ventricular hypertrophy and poor prognosis. Arg1045His and Ala26Val double-mutant might have dosage effects and aggravate the clinical phenotype of the patient.

Does the injection of platelet-rich plasma induce changes in the gene expression and morphology of intact Thoroughbred skeletal muscle?

Platelet-rich plasma (PRP) therapy is promising for treating skeletal muscle injuries in human athletes by promoting muscle regeneration. It might also be useful for treating muscle injuries in equine athletes. In the present study, muscle regeneration induced by injection of PRP into intact muscle of Thoroughbred was investigated. Autologous PRP and saline were injected twice into intact left and right gluteus medius muscles of seven clinically healthy Thoroughbreds. Muscle samples were collected from the injection sites by needle biopsy at 2 and 7 days after PRP injection. Immunohistochemical staining to identify the types of myosin heavy chains (MHCs) and satellite cells was performed to compare morphological changes among intact (pre-injection), saline-, and PRP-injected muscles. The expression of marker genes related to muscle regeneration (MHC-I, MHC-II, and embryonic MHC [MHC-e]), satellite cell activity (CK, Pax7, MyoD, and myogenin), and proinflammatory and promyogenic cytokines (IL-6, IGF-1, and HGF) was analyzed and compared between saline- and PRP-injected muscles. There were no obvious morphological differences among the three treatments. There were no significant differences in gene expression associated with satellite cell activity between saline and PRP injection at 7 days after injection. MHC genes showed significantly higher expression levels with PRP than with saline, including MHC-e at 2 days and MHC-I at 7 days after injection. It is suggested that injection of PRP into intact skeletal muscle does not induce specific morphological changes, but upregulate the expression of genes related to muscle regeneration.

Myosin content of individual human muscle fibers isolated by laser capture microdissection.

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle.

Reduced expression of MyHC slow isoform in rat soleus during unloading is accompanied by alterations of endogenous inhibitors of calcineurin/NFAT signaling pathway.

Under muscle disuse conditions decrease of expression of MyHC of slow type, and sometimes of type IIa, as well as upregulation of expression of IIb and IId/x isoforms were observed. Through dephosphorylation and entry of NFAT molecules to the nucleus calcineurin/NFATc1 signaling pathway promotes upregulation of the slow MyHC expression. We supposed that downregulation of calcineurin pathway took place during unloading. The study was aimed to analyze the states of the myonuclear NFAT inhibitors calsarcin I (CSI) and calsarcin II (CSII) (also referred to as myozenin II and I) and GSK3ß in rat soleus during hindlimb suspension (HS). Male Wistar rats were subjected to 3, 7 and 14 day of HS. We found that after 3 days of HS the content of CSII mRNA twofold increased in soleus as compared to the controls. This level was increased by more than fivefold (as compared to control) after 2 weeks of HS. The increase of CSII mRNA expression may be explained as the mechanism of stabilization of fast phenotype. We found that from the 3 day till 14 day of HS the content of MuRF-1 and MuRF-2 in the nuclear fraction fourfold to fivefold increased in HS soleus. We supposed that nuclear import of the MuRFs allows to promote CSII expression during unloading. We also observed the decline of the phosphorylated GSK3ß content in the nuclear extract of the soleus tissue. Thus decline of slow MyHC expression characteristic for the unloading conditions is accompanied with the increased expression and activation of the factors known to prevent NFAT accumulation in the myonuclei.

Polyamines, myosin heavy chains, and collagen specific amino acids after a repeated bout of eccentric exercise.

We investigated alternatives to commonly used biomarkers of exercise-induced tissue damage. Over 5 days following two bouts of 100 drop-to-vertical jumps (inter-bout rest period of 3 weeks), myosin heavy chain 1, hydroxylysine (HYL), hydroxyproline (HYP), spermine (SPM) and spermine synthase (SMS) were measured in the serum of 10 participants. HYL significantly increased from 5.92 ± 1.49 ng/mL to 6.48 ± 1.47 ng/mL at 24 h. A similar trend was observed for bout 2, but without reaching significance. SPM significantly increased only after bout 1 from 0.96 ± 0.19 ng/mL at pretest to a peak level of 1.12 ± 0.26 ng/mL at 24 h, while B2 increments remained non-significant. Myosin heavy chain 1, HYP and SMS values remained below the detection limit of the applied enzyme-linked immunosorbent assay (ELISA) kit. Though HYL and SM increased after the intervention, both markers showed a large standard deviation (SD) combined with small increments. Therefore, none of the investigated biomarkers provides a meaningful alternative to commonly used damage markers.

Genotype-phenotype relationships in Freeman-Sheldon syndrome.

Distal arthrogryposis (DA) syndromes are a group of disorders characterized by multiple congenital contractures. DA type 2A (DA2A or Freeman-Sheldon syndrome), caused by mutations in MYH3, is typically considered the most severe of the DA syndromes. However, there is wide phenotypic variability among individuals with DA2A. We characterized genotype-phenotype relationships in 46 families with DA2A. MYH3 mutations were found in 43/46 (93%) kindreds, with three mutations (p.T178I, p.R672C, and p.R672H) explaining 39/43 (91%) of cases. Phenotypic severity varied significantly by genotype (P=0.0055). Individuals with p.T178I were the most severely affected with both facial contractures and congenital scoliosis. Classification of individuals with DA2A into phenotypic groups of varying severity should facilitate providing families with more accurate information about natural history and suggests that individuals might benefit from personalized medical management motivated by MYH3 genotype.

Mechanical interaction of myosin and native thin filament in the disused rat soleus muscle.

The disuse of skeletal limb muscles occurs in a variety of conditions, yet our comprehension of the molecular mechanisms involved in adaptation to disuse remains incomplete. We studied the mechanical characteristics of actin-myosin interaction using an in vitro motility assay and isoform composition of myosin heavy and light chains by dint of SDS-PAGE in soleus muscle of both control and hindlimb-unloaded rats. 14 days of hindlimb unloading led to the increased maximum sliding velocity of actin, reconstituted, and native thin filaments over rat soleus muscle myosin by 24 %, 19 %, and 20 %, respectively. The calcium sensitivity of the "pCa-velocity" relationship decreased. There was a 26 % increase in fast myosin heavy chain IIa (MHC IIa), a 22 % increase in fast myosin light chain 2 (MLC 2f), and a 13 % increase in fast MLC 1f content. The content of MLC 1s/v, typical for slow skeletal muscles and cardiac ventricles did not change. At the same time, MLC 1s, typical only for slow skeletal muscles, disappeared. The maximum velocity of soleus muscle native thin filaments was 24 % higher compared to control ones sliding over the same rabbit myosin. Therefore, both myosin and native thin filament kinetics could influence the mechanical characteristics of the soleus muscle. Additionally, the MLC 1s and MLC 1s/v ratio may contribute to the mechanical characteristics of slow skeletal muscle, along with MHC, MLC 2, and MLC 1 slow/fast isoforms ratio.

Skeletal Muscle Function Is Altered in Male Mice on Low-Dose Androgen Receptor Antagonist or Estrogen Receptor Agonist.

In males, skeletal muscle function may be altered by shifts in either circulating testosterone or estrogen. We examined the effect of acute (2-week) exposures to 17α-ethinyl estradiol (EE2), an estrogen receptor (ER) agonist, or flutamide, an androgen receptor (AR) antagonist, on the contractile function of individual skeletal muscle fibers from slow-contracting soleus and fast-contracting extensor digitorum longus muscles from adult male mice. Single fiber specific tension (force divided by cross-sectional area) was decreased with flutamide treatment in all myosin heavy chain (MHC) fiber types examined (I, IIA, and IIB); similar effects were observed with EE2 treatment but only in the fastest-contracting MHC IIB fibers. The decreases in maximally Ca2+-activated specific tension were primarily a result of fewer strongly bound myosin-actin cross-bridges, with flutamide treatment also showing lower myofilament lattice stiffness. Myosin-actin cross-bridge kinetics were slower in MHC IIA fibers in flutamide-treated mice, but faster in EE2-treated mice, indicating that contractile velocity may be affected differently in this fiber type, which is commonly expressed in human skeletal muscle. Importantly, these effects were observed in the absence of outcomes previously used to evaluate ER agonists or AR antagonists in rodents including weight of reproductive organs or mammary gland morphology. Our findings indicate that substantial shifts in skeletal muscle function occur in male mice following acute exposures to low doses of a pharmacological ER agonist and an AR antagonist. These results suggest that countermeasures to maintain physical function may be needed early in situations that induce similar ER agonist and AR antagonist conditions.

Effects of sheep slaughter age on myogenic characteristics in skeletal muscle satellite cells.

OBJECTIVE: The objective of this study was to investigate the effects of sheep slaughter age on myogenic characteristics in skeletal muscle satellite cells (SMSCs). METHODS: Primary SMSCs were isolated from hind leg biceps femoris muscles of Wurank lambs (slaughtered at three months, Mth-3) and adults (slaughtered at fifteen months, Mth-15). SMSCs were selected by morphological observation and fluorescence staining. Myogenic regulatory factors (MRF) and myosin heavy chain (MyHC) expressions of SMSCs were analyzed on days 1, 3, 4, and 5. RESULTS: The expressions of myogenic factor 5 (Myf5), myogenic differentiation (MyoD), Myf6, and myogenin (MyoG) in Mth-15 were significantly higher in Mth-15 than in Mth-3 on days 1, 3, and 4 (p<0.05). However, MyoG expression in Mth-15 was significantly lower than in Mth-3 on day 5 (p<0.05). The expressions of MyHC I, MyHC IIa, and MyHC IIx in Mth-15 were significantly higher than in Mth-3 on days 1 and 3 (p<0.05), and MyHC IIb were significantly lower than in Mth-3 on days 3 and 4 (p<0.05). In contrast, the expression of MyHC IIx in Mth-15 was significantly lower and MyHC IIb was significantly higher than in Mth-3 on days 5 (p<0.05). CONCLUSION: The slaughter age altered the expression of MRFs and MyHCs in SMSCs while differentiation, which caused the variation of myogenic characteristics, and thus may affect the meat quality of Wurank sheep.

Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.