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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Using our own new modification of one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) we successfully separated myosin heavy chains from different chambers of rat heart. The key features of modification are changes in the content and buffer composition of the separating gel, and employment of constant current instead of voltage. These changes allow carrying out a safer and faster one-dimensional SDS-PAGE for cardiac myosin heavy chains using significantly fewer reagents. [ABSTRACT FROM AUTHOR]

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

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This study comprehensively explored muscle fibre characteristics and expression patterns of myosin heavy chain (MyHC) family in skeletal muscles of Large White and Mashen pigs exhibiting differences in muscle fibre development in postnatal muscle growth. Muscle fibre density and diameter were analysed using haematoxylin and eosin staining. The expression patterns of MyHCI, MyHCIIa, MyHCIIx and MyHCIIb were detected by quantitative real-time polymerase chain reaction. The muscle fibre diameter increased and density decreased gradually with aging. In individuals of the same age, the muscle fibre diameter was significantly lower and density was significantly higher in Mashen than in Large White pigs. The MyHCI expression increased, whereas that of MyHCIIa first increased and then decreased. The expression of MyHCI and MyHCIIa was significantly higher in Mashen. The expression of MyHCIIx and MyHCIIb decreased with increasing age; their expression was significantly lower in Mashen pigs. Muscle fibre diameter was negatively correlated with MyHCI expression and positively correlated with MyHCIIx and MyHCIIb expression (except the expression of MyHCI in psoas major). Muscle fibre diameter, muscle fibre density, and MyHCs expression follow certain patterns in skeletal muscles. These results provide valuable information for understanding the molecular mechanism responsible for pig growth performance and meat quality.

This study investigated the changes in myocardial myosin heavy chain (MHC) isoforms, MHC-α and MHC-β 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-β, thyroid receptor α1 (TR-α1) and β (TR-β) 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-β (%MHC-β) protein was not different between groups (CWI 34.7 ± 6.9%; Ex 32 ± 5.3%; CON 35.5 ± 10%; P = 0.7). MHC-β 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-β 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-β 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. [ABSTRACT FROM AUTHOR]

This review examines kinetic properties and distribution of the 11 isoforms of myosin heavy chain (MyHC) expressed in extraocular muscle (EOM) fibre types and the regulation and function of these MyHCs. Although recruitment and discharge characteristics of ocular motoneurons during fixation and eye movements are well documented, work directly linking these properties with motor unit contractile speed and MyHC composition is lacking. Recruitment of motor units according to Henneman's size principle has some support in EOMs but needs consolidation. Both neurogenic and myogenic mechanisms regulate MyHC expression as in other muscle allotypes. Developmentally, multiply‐innervated (MIFs) and singly‐innervated fibres (SIFs) are derived presumably from distinct myoblast lineages, ending up expressing MyHCs in the slow and fast ends of the kinetic spectrum respectively. They modulate the synaptic inputs of their motoneurons through different retrogradely transported neurotrophins, thereby specifying their tonic and phasic impulse patterns. Immunohistochemical analyses of EOMs regenerating in situ and in limb muscle beds suggest that the very impulse patterns driving various ocular movements equip effectors with appropriate MyHC compositions and speeds to accomplish their tasks. These experiments also suggest that satellite cells of SIFs and MIFs are distinct lineages expressing different MyHCs during regeneration. MyHC compositions and functional characteristics of orbital fibres show longitudinal variations that facilitate linear ocular rotation during saccades. Palisade endings on global MIFs are postulated to respond to active and passive tensions by triggering axon reflexes that play important roles during fixation, saccades and vergence. How EOMs implement Listings law during ocular rotation is discussed. [ABSTRACT FROM AUTHOR]

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

Background: Evaluation of improvements by nutrition support to severely ill patients requires sensitive methods to demonstrate activation of protein synthesis in various tissues from groups with a limited number of patients to be statistically efficient. This study examines effects of standard parenteral nutrition (PN) on abdominal muscle transcripts of amino acid (AA) transporters, myosin heavy chains (MHCs), and the insulin-like growth factor 1 and its receptor (IGF-1/IGF-1R) in patients aimed at major surgery.Methods: Twenty-two randomized patients received steady-state PN (0.16 gN/kg/d, 30 kcal/kg/d) or saline infusions for 12 hours before operation. Blood samples and muscle biopsies were obtained at operation start. Muscle messenger RNA (mRNA) levels of AA transporters (solute carrier family members SNAT2, LAT1, LAT3, LAT4, TAUT, PAT1, CD98), IGF-1, IGF-1R, MHC isoforms (MHC1, MHC2A, MHC2X), and LAT3 protein were quantified and related to concentrations of AA, IGF-1, insulin, and metabolic substrates in blood.Results: Muscle mRNA LAT3, LAT4, IGF-1R, and MHC2A increased by PN infusion, with correlations to specific AA transporters and MHC isoforms (P < .01-.05). TAUT and LAT3 correlated to slow (MHC1) and fast (MHC2A, MHC2X) isoforms (P < .001-.02). Muscle IGF-1 mRNA correlated to plasma essential AAs, whereas IGF-1R mRNA was related to LAT3, MHC2A, and serum IGF-1 (P < .001-.03).Conclusions: The results confirm that short-term preoperative PN activates transcription of AA transporters and myosin isoforms. Thus, combinations of methods on gene transcription and translation of muscle proteins can be applied to define efficient combinations of nutrition and hormones to catabolic patients in preoperative and postoperative settings. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study is to identify a possible damage to heart ventricles caused by supraphysiological doses of testosterone, voluntary physical activity or their combination.

Background and Objectives: Endurance exercise induces cardiac remodeling. Also, myh6 gene is affected by cardiac remodeling. This study was performed with the objective of determining the effect of endurance activity on cardiac myh6 gene expression. Methods: In this study, 14 rats (weight, 231±24g), after familiarization with endurance trainings, were randomly divided into two groups of control and experimental (7 animals each). The experimental group performed a training program (30m/min, 50min/session, and 6 session/week, for 14 weeks) on treadmill. Forty-eight hours after the end of the last session, the animals of the experimental and control groups were anesthetized and anatomized, then, the heart and subsequently the left ventricle, were removed. Real time PCR and sonography methods were, respectively, used to assess the expression levels of myh6 gene and structural changes in left ventricle. Data were analyzed using t-test. Results: In this study, endurance exercise significantly increased heart weight to body surface area ratio (p=0.002), also final diastolic diameter increased (p=0.008) in experimental group compared to control group. The results showed 124 fold and significant (p=0.011) increase in myh6 gene expression in the experimental group compared to control group. Conclusion: According to the results of this study, endurance exercise along with structural and functional changes in the left ventricle, induces changes at the gene level and thereby increases heart contractility power.

We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+). We demonstrate that such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serial-section-labeling, to identify damaged fibers. The quadriceps femoris muscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21 ± 0.21%, 98.34 ± 1.06%, 0.07 ± 0.01%, and 0.53 ± 0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively. All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.

Different forms of myosin heavy chains (MyHCs), coded by a large family of sarcomeric MYH genes, are expressed in striated muscles. The generation of specific anti‐MyHC antibodies has provided a powerful tool to define the fiber types present in skeletal muscles, their functional properties, their response to conditions that affect muscle plasticity and their changes in muscle disorders. Cardiomyocyte heterogeneity has been revealed by the serendipitous observation that different MyHCs are present in atrial and ventricular myocardium and in heart conduction tissue. Developmental MyHCs present in embryonic and fetal/neonatal skeletal muscle are re‐expressed during muscle regeneration and can be used to identify regenerating fibers in muscle diseases. MyHC isoforms provide cell type‐specific markers to identify the signaling pathways that control muscle cell identity and are an essential reference to interpret the results of single‐cell transcriptomics and proteomics. Skeletal muscles consist of different fiber types that can be identified by immunohistochemistry using antibodies that recognize distinct myosin isoforms. In human skeletal muscle antimyosin staining reveals three major fiber types, type 1, 2A, and 2X, and a number of hybrid fibers with mixed myosin composition. [ABSTRACT FROM AUTHOR]

BACKGROUND: Owing to the advantages of low sensitization and natural three-dimensional structure, good biocompatibility and cell affinity, acellular heart scaffold materials are of great current interest in cardiac tissue engineering. OBJECTIVE: To investigate the cytocompatibility of an acellular heart scaffold of neonatal rats. METHODS: In order to construct the seed cell-scaffold complex, passage 3 bone marrow mesenchymal stem cells (BMSCs) of Sprague-Dawley neonatal rats were cultured with an acellular heart scaffold of Sprague-Dawley neonatal rats for 7 and 14 days. Hematoxylin eosin staining and scanning electron microscopy were used to observe the growth of BMSCs in the scaffold. The cell-scaffold complex was induced in myocardial tissue lysate for 14 days. BMSCs with planar orientation differentiation for 14 and 20 days were used as control group. RT-PCR was used to detect the expression of myosin heavy chain α-MHC and zinc finger transcription factor GATA-4 in BMSCs. RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining showed the acellular heart scaffold contained a large number of eosinophilic fibrous structures, and the cell number of cell-scaffold complex after co-culture for 14 days was higher than that after co-culture for 7 days. Under the scanning electron microscope, a large amount of cells adhered to the fiber surface of the acellular scaffold at 14 days of co-culture. (2) BMSCs with planar orientation differentiation for 14 and 20 days had the bamboo-like and myotube-like structures. In the cell-scaffold complex with planar orientation differentiation for 14 days, the expression of α-MHC and GATA-4 could be detected, and their expression levels fulfilled the requirement for the presence of bamboo-like cells and myotube-like structure. These results indicate that the acellular heart scaffold exhibits good cytocompatibility. [ABSTRACT FROM AUTHOR]

Introduction. The group of autosomal dominant disorders - Epstein syndrome, Sebastian syndrome, Fechthner syndrome and May-Hegglin anomaly - are characterised by thrombocytopenia with giant platelets, inclusion bodies in granulocytes and variable levels of deafness, disturbances of vision and renal function impairment. A common genetic background of these disorders are mutations in MYH9 gene, coding for the nonmuscle myosin heavy chain IIA. Differential diagnosis is important for the adequate treatment strategy. The aim of this case report was to present a patient with MYH9 disorder in Serbia. Case report. A 16-year-old boy was referred to our hospital with the diagnosis of resistant immune thrombocytopenia for splenectomy. Thrombocytopenia was incidentally discovered at the age of five. The treatment with corticosteroids on several occasions was unsuccessful. Although the platelet count was below 10 × 109/L, there were no bleeding symptoms. Besides thrombocytopenia with giant platelets, on admission the patient also suffered sensorineuronal hearing loss and proteinuria. The diagnosis was confirmed with immunofluorescence and genetic analyses. Conclusion. Early recognition of MYH9-related diseases is essential to avoid unnecessary and potentially harmful treatments for misdiagnosed immune thrombocytopenia, and also for timely and proper therapy in attempt to delay end-stage renal failure and improve quality of life. [Projekat Ministartsva nauke Republike Srbije, br. 175056 i br. 15079]

Hibernation is a biological phenomenon enabling animals to endure hypothermia. This study was designed to find the changes in myosin heavy chain (MHC) isoforms and their relationship to calcium sensitivity in frog ventricular muscle during hibernation. MHC profile was determined by SDS-4% polyacrylamide gel electrophoresis in active and hibernating frogs for different intervals of time (10, 20, 30, and 60 days). Calcium sensitivity of Triton-skinned ventricular muscle fiber bundles was determined. Compared with active and hibernated frogs for 10 and 20 days, hibernated frogs for 30 and 60 days expressed significant amount ( p < 0.05) of MHC type β (15. 3 ± 3.8%, 35.6 ± 1.5%, respectively). This change in myosin heavy chain profile seen in long period of hibernation was associated with significant decrease ( p < 0.05) in sensitivity of skinned myofilaments to Ca where skinned ventricular muscle taken from hibernating frogs for 30 and 60 days p value were 5.52 and 5.50, respectively, while active frog showed p = 5.67. In conclusion, expression of increased amount of β-MHC in hibernated frog for 30 days and more was associated with a decrease in contractile responses of skinned ventricular myocardium to calcium. [ABSTRACT FROM AUTHOR]

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

The enzymatic dissociation of short muscles from mice, such as flexor digitorum brevis, has allowed a great accumulation of physiological, pharmacological and biochemical knowledge about skeletal muscle. However, this body of knowledge has been restricted to the types of fibers present in these muscles. Information about the other fiber types has been limited and has been primarily obtained by the manual isolation of fibers from other species, typically rats, via a difficult and time-consuming procedure. In this report, the author describes a technique for the enzymatic dissociation of long muscles, such as soleus or extensor digitorum longus (EDL), which can be applied to study a wider spectrum of fiber types and larger quantities of cells. Additionally, the kinetics of Ca2+ transients obtained in soleus and EDL fibers are compared in this report. The usefulness of this methodology for other physiological, biochemical and molecular biology experiments is also discussed. This methodology introduces the possibility of using the whole spectrum of fiber types to study normal muscle biology and the pathophysiology of muscle diseases.

The present contribution provides an overview of stereological methods applied in the skeletal muscle research at the Institute of Anatomy of the Medical Faculty in Ljubljana. Interested in skeletal muscle plasticity we studied three different topics: (i) expression of myosin heavy chain isoforms in slow and fast muscles under experimental conditions, (ii) frequency of satellite cells in young and old human and rat muscles and (iii) capillary supply of rat fast and slow muscles. We analysed the expression of myosin heavy chain isoforms within slow rat soleus and fast extensor digitorum longus muscles after (i) homotopic and heterotopic transplantation of both muscles, (ii) low frequency electrical stimulation of the fast muscle and (iii) transposition of the fast nerve to the slow muscle. The models applied were able to turn the fast muscle into a completely slow muscle, but not vice versa. One of the indicators for the regenerative potential of skeletal muscles is its satellite cell pool. The estimated parameters, number of satellite cells per unit fibre length, corrected to the reference sarcomere length (Nsc/Lfib) and number of satellite cells per number of nuclei (myonuclei and satellite cell nuclei) (Nsc/Nnucl) indicated that the frequency of M-cadherin stained satellite cells declines in healthy old human and rat muscles compared to young muscles. To access differences in capillary densities among slow and fast muscles and slow and fast muscle fibres, we have introduced Slicer and Fakir methods, and tested them on predominantly slow and fast rat muscles. Discussing three different topics that require different approach, the present paper reflects the three decades of the development of stereological methods: 2D analysis by simple point counting in the 70's, the disector in the 80's and virtual spatial probes in the 90's. In all methods the interactive computer assisted approach was utilised.

Meat homogenates and intestinal extracts were prepared from walleye pollack (Theragra chalcogramma) and white croaker (Pennahia argentata). Then, the proteolytic profiles of meat homogenate in the presence of the intestinal extracts were determined. The proteolytic activity in the intestinal extracts from walleye pollack was the maximum at 50°C and pH 8.53. The maximum activity was recognized also at 50°C when the intestinal extracts from walleye pollack were added to the meat homogenate prepared from white croaker, while it was at 60°C when the intestinal extracts from white croaker were added to the meat homogenate prepared from walleye pollack. These results suggest that the proteolytic profile is predominantly attributable to proteolytic enzymes and not to the substrate species of myosin heavy chain. These findings are useful for managing heating conditions in the industry in order to avoid modori and thus improve the quality of surimi-based products. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Thermally-induced gels were made from white croaker ( Pennahia argentata ) meat in the presence of its organ extracts by pre-heating at 40 and 65 °C for 20 min and subsequent heating at 85 °C for 20 min. The breaking strength of the gels decreased with increasing concentrations of the intestinal extracts accompanying decomposition of myosin heavy chains. However, no significant changes in the gel strength occurred when the kidney extract was added. The proteolytic activity in the intestinal extracts examined in the meat homogenate had a maximum at 60 °C and pH 8.90. These results suggest that the intestinal rather than kidney proteolytic activities are responsible for gel softening known as a modori phenomenon. Thus, the removal of intestinal tracts is essential to maintain a high quality of surimi-based products. [ABSTRACT FROM AUTHOR]

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. Fasttwitch (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 _-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. [ABSTRACT FROM AUTHOR]

Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the same over its length. We analyzed this in detail in three muscle types: the temporalis (closer) and digastricus (opener; both first brachial arch), and the medial gastrocne-mius (somite). Sections of the muscles were incubated with monoclonal antibodies against various MyHC isoforms, and the distribution of these isoforms within individual fibers was followed over a distance of approximately 1 mm. The staining intensity of a fiber was measured and compared with the other fibers in the section. In the temporalis, digastricus, and gastrocnemius, 46,11, and 15%, respectively, of their MyHC-I fibers showed a variation in the staining intensity over the length of their fibers, as well as 47,87, and 22%, respectively, of their MyHC-IIA fibers. Most variable fibers were found amongst those with an overall relative intermediate staining intensity, which are presumably hybrid fibers. We conclude that different parts of a muscle fiber can have different fiber type compositions and, thus, contractile properties. Some muscle parts might reach their maximum contraction peak sooner or later than a muscle part a few microns further away. Next to stimulation by the nerve and systemic influences, local influences might also have an impact on the MyHC expression of the fiber. [ABSTRACT FROM AUTHOR]

The article discusses hypoplastic left heart genetic determinants with latent right ventricular dysfunction in patients with a Fontan circulation. It says that whole genome sequencing was conducted on DNA and protein modeling of highly conserved amino acids substitutions. Results showed that compound heterozygosity for recessive MYH6 mutations and declined systemic right ventricular ejection fraction.

Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the same over its length. We analyzed this in detail in three muscle types: the temporalis (closer) and digastricus (opener; both first brachial arch), and the medial gastrocnemius (somite). Sections of the muscles were incubated with monoclonal antibodies against various MyHC isoforms, and the distribution of these isoforms within individual fibers was followed over a distance of approximately 1 mm. The staining intensity of a fiber was measured and compared with the other fibers in the section. In the temporalis, digastricus, and gastrocnemius, 46, 11, and 15%, respectively, of their MyHC-I fibers showed a variation in the staining intensity over the length of their fibers, as well as 47, 87, and 22%, respectively, of their MyHC-IIA fibers. Most variable fibers were found amongst those with an overall relative intermediate staining intensity, which are presumably hybrid fibers. We conclude that different parts of a muscle fiber can have different fiber type compositions and, thus, contractile properties. Some muscle parts might reach their maximum contraction peak sooner or later than a muscle part a few microns further away. Next to stimulation by the nerve and systemic influences, local influences might also have an impact on the MyHC expression of the fiber. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objectives We studied adaptation of diaphragm, oxygen consumption and carbon dioxide elimination to forced oral breathing (lasting for only 4 days) following reversible bilateral nasal obstruction performed on day 8 post-natal male rats. Methods Diaphragm myosin heavy chain (MHC) composition, oxygen consumption, carbon dioxide elimination and hormones level were analysed during nasal obstruction period. Results Diaphragm muscle showed significant increases in adult isoforms (MHC 1, 2a) in oral breathing group versus control. Reversible nasal obstruction was associated with a decrease of oxygen consumption and carbon dioxide elimination. Nasal obstruction period was associated with reduced growth of the olfactory bulbs and an initial decrease in lung growth. One day after implementing nasal obstruction, basal corticosterone levels had increased (by over 1000). Oral breathing was also associated with a lower level of thyroid hormone. Conclusions We conclude that a 4 day nasal obstruction period in young rats leads to hormonal changes and to Diaphragm myosin heavy chain structural adaptation. [ABSTRACT FROM AUTHOR]

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. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of N-nitro- l-arginine methyl ester and thyroxine on the distribution of cardiac myosin heavy chains in mice. Myocardial hypertrophy was induced in mice by intraperitoneal injection with l-thyroxine and oral administration of N-nitro- l-arginine methyl ester ( l-NAME). Mice were randomly allocated into five groups: control, high and low doses of thyroxine and L-NAME. Heart weight divided by body weight was calculated and used as indicator for cardiac hypertrophy. Myosin heavy chains (MHCs) were separated using 4 % polyacrylamide gel electrophoresis. Hypertrophy was induced in mice treated with thyroxine and a high dose of l-NAME and was accompanied by a shift toward α-MHC in thyroxine-treated mice and β-MHC in l-NAME-treated mice. There was no difference with respect to MHC between a low dose of l-NAME and a high dose; however, low doses of l-NAME did not result in cardiac hypertrophy. In conclusion, l-NAME treatment changes the MHC distribution from α- toward β-MHC and this transition in the MHCs occurs before heart hypertrophy. Further investigations are needed to determine whether l-NAME treatment causes this transition via a direct or indirect effect on the heart muscles. [ABSTRACT FROM AUTHOR]

Using the real-time reverse-transcription polymerase chain reaction (RT-PCR) and low percentage SDS-gel electrophoresis, changes in the gene expression and isoform composition of a giant sarcomeric protein titin (connectin) in cardiac muscle and changes in its isoform composition in skeletal muscle m. soleus of chronically alcoholized rats were studied. The decreased content of titin protein in examined muscles and decreases in titin gene expression in the myocardium of chronically alcoholized rats were found, which indicates the development of the pathological process. [ABSTRACT FROM AUTHOR]

Introduction: The molecular adaptations specifically induced by different muscle contraction types have only been partially elucidated. We previously demonstrated that eccentric contractions in human quadriceps elicited proteome modifications that suggest a muscle fiber typology adaptation. We address this question in a more systematic way by examining here the effects of different running modes on the mouse muscle proteome and the muscle fiber typology. Methods: Male adult mice (C57BL6) were randomly divided into downhill running (DHR) (quadricipital eccentrically biased contractions), uphill running (UHR) (quadricipital concentrically biased contractions), and untrained control (CONT) groups. Running groups performed five training sessions on an inclined treadmill for 75 to 135 min⋅d-1, and the quadriceps muscles were dissected 96 h after the last session. Muscle protein extracts of DHR and UHR groups (n = 4/group) were subjected to a two-dimensional difference in gel electrophoresis (2D-DIGE) analysis coupled with mass spectrometry. The assessment of fiber type, size, and number was performed on the rectus femoris of the three groups (n = 6/group) using myosin heavy chain immunohistochemistry. Results: In the proteomic analysis, eight spots identified as the fast myosin heavy chain isoforms exhibited a lower abundance in DHR compared with UHR (P < 0.05, t-test). In contrast, adenosine triphosphate (ATP) synthase subunit a and tubulin ß were more expressed in DHR (P < 0.05). A significant higher proportion of Type I and Ha fibers was found for DHR compared with UHR or CONT groups (P < 0.05, one-way ANOVA). Conclusions: Our data suggest that the eccentrically biased contractions in mice induced specific adaptations in protein expression and muscle fiber composition, which may reflect a more oxidative muscle phenotype. The differences in stress placed on the muscle between both trainings may be responsible for some unique adaptations resulting from the eccentrically biased training [ABSTRACT FROM AUTHOR]

The expression of myosin heavy chain (MyHC) isoforms in the myofibers of domestic and wild pig was studied to characterize muscle tissue differences related to species domestication and selection. Muscle samples were obtained from longissimus muscle of five two years old wild and domestic Large White pigs. Four different MyHC isoforms (MyHC-I, MyHC-IIa, MyHC-IIb, MyHC-IIx) were determined in the myofibers of both, domestic and wild pig, and allowed the distinction of I, IIa, IIx/b and IIb myofiber types. Oxidative types I and IIa and type IIx/b myofibers were notably more abundant in longissimus muscle of wild than domestic pig. On the contrary, the number of glycolytic IIb myofibers prevailed in domestic pig. The cross sectional areas (CSA) of different MyHC myofiber types were 2 to 3 times smaller in wild than in domestic pig. In wild pig, CSA was more homogeneous between myofiber types with no difference between CSA of types I, IIx/b and IIb myofibers, whereas IIx/b and IIb myofibers exhibited greater CSA in domestic pigs. Type IIa myofibers were the smallest ones in both, domestic and wild pig. The presence of MyHC-IIb isoform was clearly established in the myfibers of wild pigs denoting that its expression is not just the result of the intensive selection for growth efficiency and muscularity. On the other hand, it is evident that domestication and breeding goals in pigs resulted in the hypertrophy of all myofiber types, in particular of those in which MyHC-IIb isoform is expressed. [ABSTRACT FROM AUTHOR]

Abstract: Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation–contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development. [Copyright &y& Elsevier]

The distal arthrogryposis (DA) syndromes are a group of disorders characterized by non-progressive congenital contractures of the limbs. Mutations that cause distal arthrogryposis syndromes have been reported in six genes, each of which encodes a component of the contractile apparatus of skeletal myofibers. However, these reports have usually emanated from gene discovery efforts and thus potentially bias estimates of the frequency of pathogenic mutations at each locus. We characterized the spectrum of pathogenic variants in a cohort of 153 cases of DA1 (n = 48) and DA2B (n = 105). Disease-causing mutations in 56/153 (37%) kindreds including 14/48 (29%) with DA1 and 42/105 (40%) with DA2B were distributed nearly equally across TNNI2, TNNT3, TPM2, and MYH3. In TNNI2, TNNT3, and TPM2 the same mutation caused DA1 in some families and DA2B in others. We found no significant differences among the clinical characteristics of DA by locus or between each locus and DA1 or DA2B. Collectively, the substantial overlap between phenotypic characteristics and spectrum of mutations suggests that DA1 and DA2B should be considered phenotypic extremes of the same disorder. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Thirty-four untrained women participated in a 6-week program to investigate slow-speed versus 'normal' speed resistance-training protocols. Subjects were divided into: slow-speed (SS), normal-speed/traditional-strength (TS), normal-speed/traditional muscular endurance (TE), and non-exercising control (C) groups. Leg press, squats, and knee extensions were performed 2 days/week for the first week and 3 days/week for the remaining 5 weeks (~2 min rest). The SS group performed 6-10 repetitions maximum (6-10RM) for each set with 10 s concentric (con) and 4 s eccentric (ecc) contractions. The TS and TE groups performed sets of 6-10RM and 20-30RM, respectively, at 'normal' speed (1-2 s/con and ecc contractions). TE and SS trained at the same relative intensity (~40-60% 1RM), whereas TS trained at ~80-85% 1RM. Pre- and post-training muscle biopsies were analyzed for fiber-type composition, cross-sectional area (CSA), and myosin heavy chain (MHC) content. The percentage of type IIX fibers decreased and IIAX increased in all three training groups. However, only TS showed an increase in percentage of type IIA fibers. CSA of fiber types I, IIA, and IIX increased in TS. In SS, only the CSA of IIA and IIX fibers increased. These changes were supported by MHC data. No significant changes for any parameters were found for the C group. In conclusion, slow-speed strength training induced a greater adaptive response compared to training with a similar resistance at 'normal' speed. However, training with a higher intensity at 'normal' speed resulted in the greatest overall muscle fiber response in each of the variables assessed. [ABSTRACT FROM AUTHOR]

The article reports on research conducted to investigate the classification of hybrid fast fibers in resistance trained human skeletal muscle using histochemical and immunohistochemical methods. Researchers evaluated 16 women who participated in a six week progressive resistance training program with two methods of classifying fiber types. They found that the best approach to determine muscle fiber type composition used both histochemical and immunohistochemical methods.

Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil ( Meriones unguiculatus) were investigated after 12-day spaceflight on board of Russian space vehicle 'Foton-M3.' In m. psoas and m. soleus in the gerbils from 'Flight' group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in 'Flight' group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from 'Flight' group were observed. In skeletal muscles of the gerbils from 'Flight' group the relative content of titin N2A-isoform was reduced (by 1.2-1.7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from 'Flight' group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made. [ABSTRACT FROM AUTHOR]

Objective: The aims of this study were to examine whether a passive stretch stimulus by means of a functional appliance induces changes in the fiber composition of masticatory muscles and whether these changes are similar to the changes in stretched limb muscle fi bers by using RT-PCR, western blot, and immunohistochemical assays. Methods: Five male New Zealand White rabbits were fi tted with a prefabricated inclined plane on the maxillary central incisors to force the mandible forward (- 2 mm) and downward (- 4 mm). Further, 1 hind limb was extended and constrained with a cast so that the extensor digitorum longus (EDL) was stretched when the animal used the limb. The animals were sacrifi ced aft er 1 week and the masseter, lateral pterygoid, and EDL were processed and compared with those from control animals (n = 3). Results: The stretched EDL had a significantly higher percentage of slow fibers, whereas the stretched masticatory muscles did not show changes in the composition of the major contractile proteins aft er 7 days. Conclusions: Th e transition of fi ber phenotypes in response to a stretch stimulus may take longer in the masticatory muscles than in the limb muscles. [ABSTRACT FROM AUTHOR]

Diseases of the human cardiovascular system are the main cause of death in developed countries. Therefore, searching for new risk factors thereof is of particular interest. Upon comparing epidemiological data with data of transcriptome of cardiomyocytes and comparative physiology of vertebrate ontogenesis, we have come to the conclusion that one such factor may be gastroenteritis. This disease includes at once several stimuli able to cause functional and metabolic alterations in the heart: tachycardia, hormonal and ionic misbalance, and outflow of resources from the cardiovascular system. Using the model of rat neonatal gastroenteritis caused by the widespread human and animal enteropathogen Cryptosporidium parvum ( Apicomplexa, Sporozoa), we studied the change of expression of α- and β-myosin heavy chains after the developed cryptosporidiosis. Online data obtained by methods of immunocytochemistry, quantitative morphometry, and polymerase chain reaction not only have confirmed our suggestion, but also have shown that moderate 4-day-long cryptosporidiosis is sufficient for producing a significant (1.7- to 4.5-fold) shift in the ratio of myosin isoforms toward the β-isoform beta at the level of mRNA and at the level of protein (2.5-6 times). The reciprocity of the changes, as well as their clear similarity at the level of mRNA and of protein, indicates that the cryptosporidial gastroenteritis involves all the main chains of a complex network of regulation of expression of the myosin heavy chains. A shift of the ratio of myosin isoforms toward the β-isoform that has an ATPase activity several times lower than the α-isoform is the commonly accepted indicator of human heart failure; therefore, the cryptosporidial gastroenteritis can be considered a novel risk factor for decrease of the heart's contractile ability. Our data may be of interest for clinical and preventive medicine. [ABSTRACT FROM AUTHOR]

Abstract: Objective: We studied adaptation of diaphragm and orofacial muscles as well as hormonal responses to forced oral breathing (lasting for only 4 days) following reversible bilateral nasal obstruction performed on day 8 post-natal male rats. Design: Muscle myosin heavy chain (MHC) composition and hormone levels were analysed during two periods: 1 and 3 days after obstruction (days 9 and 11 post-natal), and following 3 months recovery with nasal breathing (90 days, adult). Results: Diaphragm muscle showed significant increases in adult isoforms (MHC 1, 2a) in oral breathing group versus control. We observed increases in MHC neonatal and adult type 1 isoforms in muscles involved with oral breathing, masseter superficialis and anterior digastric. No changes were observed in the levator nasolabialis muscle involved with nasal breathing. Reversible nasal obstruction was associated with reduced growth of the olfactory bulbs lasting into adulthood, and an initial decrease in lung growth followed by recovery at 90 days. Adrenal hypertrophy was observed after 1 day of nasal obstruction and lasted into adulthood. The “stress” hormone response was variable, increased (over 1000%) during the obstruction but normal by adulthood. An increase in plasma testosterone was observed during the obstruction, and a decrease in thyroid hormone levels throughout. Conclusions: Very short term nasal obstruction, i.e. forced oral breathing, leads to long term hormonal changes and respiratory muscle fibre adaptation. [Copyright &y& Elsevier]

We developed a new method for the quantitative determination of myosin heavy chain (MyHC) isoforms taking advantage of immunochemical differences and based on the ELISA principle. In the present paper we compare analysis of MyHC isoforms using the SDS-PAGE and the ELISA methods in the same samples of adult female inbred Lewis strain euthyroid, hyperthyroid and hypothyroid rats. In all thyroid states, the same composition and corresponding changes of MyHC isoforms were determined using both methodological approaches in the slow soleus and the fast extensor digitorum longus muscles. Our results showed that ELISA can be used for a "semi-quantitative" or "comparative" measurement of MyHC isoforms in multiple muscle samples, but that it is neither more exact nor faster compared to SDS-PAGE. [ABSTRACT FROM AUTHOR]

Abstract: We have previously shown that mitochondrial protein synthesis regulates myoblast differentiation, partly through the control of c-Myc expression, a cellular oncogene regulating myogenin expression and myoblast withdrawal from the cell cycle. In this study we provide evidence of the involvement of Calcineurin in this regulation. In C2C12 myoblasts, inhibition of mitochondrial protein synthesis by chloramphenicol decreases Calcineurin expression. Conversely, stimulation of this process by overexpressing the T3 mitochondrial receptor (p43) increases Calcineurin expression. Moreover, expression of a constitutively active Calcineurin (ΔCN) stimulates myoblast differentiation, whereas a Calcineurin antisense has the opposite effect. Lastly, ΔCN expression or stimulation of mitochondrial protein synthesis specifically increases slow myosin heavy chain expression. In conclusion, these data clearly suggest that, partly via Calcineurin expression, mitochondrial protein synthesis is involved in muscle development through the control of myoblast differentiation and probably the acquisition of the contractile and metabolic phenotype of muscle fibres. [Copyright &y& Elsevier]

Ossabaw swine fed excess kilocalorie diet develop metabolic syndrome (MS) characterized by obesity, hypertension, insulin resistance, and glucose intolerance with/without dyslipidemia. The purpose of this study was to test the hypothesis that MS would have a detrimental effect on skeletal muscle structure and cause changes in the expression of myosin heavy chains (MHCs). Adult male Ossabaw swine were fed for 24 wk high-fructose or high-fat/cholesterol/fructose diets to induce normolipidemic MS (MetS) or dyslipidemic MS (DMetS), respectively, and were compared with the lean swine on control diet. MetS swine showed mild MS, lacking increases in total and low density lipoprotein (LDL) cholesterol, both of which were highly upregulated in DMetS swine. There was an ∼1.2-fold increase in the cross-sectional areas of muscle fibers in MetS and DMetS groups compared with control for biceps femoris and plantaris muscles. In plantaris muscles, DMetS diet caused an ∼2-fold decrease in slow MHC mRNA and protein expression and an ∼1.2- to 1.8-fold increase in the number of intramyocellular lipid (IMCL) droplets without large changes in the size of the droplets. There was a trend to the decrease in slow MHC expression in muscles of swine on MetS diet. The number of IMCL droplets in muscle fibers of the MetS group was comparable to controls. These data correlate well with the data on total plasma cholesterol (control = 60, MetS = 70, and DMetS = 298 mg/dl) and LDL (control = 29, MetS = 30, and DMetS = 232 mg/dl). We conclude that structural changes observed in skeletal muscle of obese Ossabaw swine correlate with those previously reported for obese humans. [ABSTRACT FROM AUTHOR]

dult skeletal muscle fibers can be categorized into slow-oxidative and fast-glycolytic subtypes based on specialized metabolic and contractile properties. The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism, and cell differentiation, and has been shown to be involved in regulating muscle fiber type specification. However, to date, the mechanism behind FoxO1-mediated fiber type diversity is still unclear. In this article, FoxO1 being expressed preferentially in fast twitch fiber enriched muscles is reported. Moreover, the autors also detected that FoxO1 expression decreased in both fast and slow muscles from mice undergoing endurance exercise which induced a fast-to-slow fiber type transition. Using C2C12 myoblast, constitutively active FoxO1 mutant altered the proportion of muscle fiber type composition toward a fast-glycolytic phenotype and attenuated calcineurin phosphatase activity. In addition, a transcriptionally inactive FoxO1 by resveratrol triggered the expression of genes related to slow-oxidative muscle but not sufficient to induce a complete slow fiber transformation. Taken together, these results suggest that FoxO1 up-regulates fast fiber-type formation and down-regulates muscle oxidative capacity at least in part through inhibition of the calcineurin pathway. [ABSTRACT FROM AUTHOR]

In vascular smooth muscle cells, Axl is a key receptor tyrosine kinase, because it is upregulated in injury, increases migration and neointima formation, and is activated by reactive oxygen species. Reaction of glutathione with cysteine residues (termed "glutathiolation") is an important posttranslational redox modification that may alter protein activity and protein-protein interactions. To investigate the mechanisms by which reactive oxygen species increase Axl-dependent vascular smooth muscle cell function we assayed for glutathiolated proteins that associated with Axl in a redox-dependent manner. We identified glutathiolated nonmuscle myosin heavy chain (MHC)-IIB as a novel Axl interacting protein. This interaction was specific in that other myosins did not interact with Axl. The endogenous ligand for Axl, Gas6, increased production of reactive oxygen species in vascular smooth muscle cells and also increased the association of Axl with MHC-IIB. Antioxidants ebselen and N-acetylcysteine decreased the association of Axl with MHC-IIB in response to both Gas6 and reactive oxygen species. Blocking the Axl-MHC-IIB interaction with the specific myosin II inhibitor blebbistatin decreased phosphorylation of Axl and activation of extracellular signal-regulated kinase 1/2 and Akt. Association of MHC-IIB with Axl was increased in balloon-injured rat carotid vessels. Finally, expression of MHC-IIB was upregulated in the neointima of the carotid artery after balloon injury similar to upregulation of Axl protein expression, as shown in our previous studies. These results demonstrate a novel interaction between Axl and MHC-IIB in response to reactive oxygen species. This interaction provides a direct link between Axl and molecular motors crucial for directed cell migration, which may mediate increased migration in vascular dysfunction. [ABSTRACT FROM AUTHOR]

Abstract: Background: Recently, microRNA-208 (miR-208) encoded by the α-myosin heavy chain (MHC) gene, has been shown to be involved in pathological cardiac growth, fibrosis, and up-regulation of β-MHC expression. A recent study has also reported 2 additional myosin-expressed miRNAs (miR-208b and miR-499). The aim of this study was to determine whether miR-208, miR-208b, and miR-499 are expressed with MHC mRNA in human dilated cardiomyopathy (DCM), and whether these levels are related to left ventricular (LV) function and to clinical outcomes. Methods and Results: Endomyocardial biopsy tissues were obtained from 82 patients with DCM and 21 subjects without LV dysfunction as controls. Levels of miR-208, miR-208b, and miR-499 were higher in DCM patients than in controls. Levels of α-MHC mRNA were lower in patients with DCM than in controls, whereas β-MHC mRNA levels were higher in patients with DCM compared with controls. Levels of miR-208 were correlated with β-MHC mRNA levels and myocardial collagen volume, whereas levels of miR-208b and miR-499 showed no correlation. After a mean follow-up of 517 days, an increase in miR-208 levels was shown to be a strong predictor of clinical outcomes (RR 3.4, 95% CI 1.1–11.2). Conclusions: This study suggests that myocardial expression of miR-208 is associated with MHC mRNA expression and with poor clinical outcomes in patients with DCM. We conclude that miR-208 may therefore be involved in the progression of human DCM. [Copyright &y& Elsevier]