Embryonic Myosin Heavy Chain Research Articles

Intrinsic transient tracheal occlusion training and myogenic remodeling of rodent parasternal intercostal fibers.

It is recognized that diaphragm muscle plasticity occurs with mechanical overloads, yet less is known about synergistic parasternal intercostal muscle fiber remodeling. We conducted overload training with intrinsic transient tracheal occlusion (ITTO) exercises in conscious animals. We hypothesized that ITTO would yield significant fiber hypertrophy and myogenic activation that would parallel diaphragm fiber remodeling. Sprague-Dawley rats underwent placement of a tracheal cuff and were randomly assigned to receive daily 10 min sessions of conscious ITTO or observation (sham) over 2 wk. After training, fiber morphology, myosin heavy chain (MHC) isoform composition, cross-sectional area, proportion of Pax7-positive nuclei, and presence of embryonic MHC (eMHC) were quantified. Type IIx/b fibers were 20% larger after ITTO training than with sham training (ITTO: 4,431 +/– 676 μm2, sham: 3,689 +/– 400 μm2, p < 0.05), and type I fibers were more prevalent after ITTO (p < 0.01). Expression of Pax7 was increased in ITTO parasternals and diaphragm (p < 0.05). In contrast, the proportion of eMHC-positive fibers was increased only in ITTO parasternals (1.2% [3.4%–0.6%], sham: 0% [0.6%–0%], p < 0.05). Although diaphragm and parasternal type II fibers hypertrophy to a similar degree, myogenic remodeling appears to differ between the two muscles.

Laminin-111 improves muscle repair in a mouse model of merosin-deficient congenital muscular dystrophy

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a severe and fatal muscle-wasting disease with no cure. MDC1A patients and the dy(W-/-) mouse model exhibit severe muscle weakness, demyelinating neuropathy, failed muscle regeneration and premature death. We have recently shown that laminin-111, a form of laminin found in embryonic skeletal muscle, can substitute for the loss of laminin-211/221 and prevent muscle disease progression in the dy(W-/-) mouse model. What is unclear from these studies is whether laminin-111 can restore failed regeneration to laminin-α2-deficient muscle. To investigate the potential of laminin-111 protein therapy to improve muscle regeneration, laminin-111 or phosphate-buffered saline-treated laminin-α2-deficient muscle was damaged with cardiotoxin and muscle regeneration quantified. Our results show laminin-111 treatment promoted an increase in myofiber size and number, and an increased expression of α7β1 integrin, Pax7, myogenin and embryonic myosin heavy chain, indicating a restoration of the muscle regenerative program. Together, our results show laminin-111 restores muscle regeneration to laminin-α2-deficient muscle and further supports laminin-111 protein as a therapy for the treatment of MDC1A.

P.9.16 The cause of developmental myopathy due to the embryonic myosin heavy chain Thr178Ile mutation

Embryonic myosin heavy chain (MyHC), encoded by MYH3, is expressed during fetal development and its expression declines rapidly after birth. The essential role of embryonic MyHC for fetal development has been highlighted by the identification of dominant MYH3 mutations associated with distal arthrogryposis (DA) syndromes. DA is a clinically and genetically heterogeneous group of disorders characterized by flexion of the joints with congenital contractions. It has been hypothesized that the congenital contractions result from reduced fetal movement and severe myopathy during a period of fetal development. Due to lack of muscle biopsy from embryos and suitable animal models, it is not yet established how MYH3 mutations cause DA. Here we address the pathogenesis of developmental myopathy caused by heterozygocity for the Thr178Ile MYH3 mutation, using cultured myotubes from a DA patient as an experimental disease model. Both mutant and wild-type alleles were expressed equally at the mRNA level. An increased expression level of both embryonic and fetal MyHCs in cultured myotubes from the patient compared to healthy controls was observed. Cultured myotubes from patient showed delayed myofibrylogenesis and reduction of cell diameters. The results suggest that developmental myopathy caused by Thr178Ile MYH3 mutation is associated with expression of mutant isoform and abnormal muscle development. Consequently, the expression of mutated embryonic MyHC may cause structural and/or functional abnormalities leading to dysfunctions during fetal development.

A novel TNNI2 mutation causes Freeman–Sheldon syndrome in a Chinese family with an affected adult with only facial contractures

Distal arthrogryposes (DAs), a clinically and genetically heterogeneous group of disorders characterized by congenital contractures with predominant involvement of the hands and feet, can be classified into at least 12 different forms. These autosomal dominant disorders are of variable expressivity and reduced penetrance. Mutations in sarcomeric protein genes, including troponin I2 (TNNI2), troponin T3 (TNNT3), tropomyosin 2 (TPM2), embryonic myosin heavy chain 3 (MYH3), and myosin binding protein C1 (MYBPC1), have been identified in distal arthrogryposis type 1 (DA1, MIM 108120), type 2B (DA2B, MIM 601680) and type 2A (DA2A)/Freeman–Sheldon syndrome (FSS, MIM 193700). However, mutations causing FSS have only been reported in MYH3. Herein we describe a Chinese DA family whose members meet classical strict criteria for FSS, as well as one member of the family who has isolated facial features consistent with FSS. No disease-causing mutation was found in MYH3. Segregation of microsatellite markers flanking the TNNI2 and TNNT3 genes at 11p15.5 was compatible with linkage. Subsequent sequencing of TNNI2 revealed a novel mutation, c.A493T (p.I165F), located in the C-terminal region, which is critical for proper protein function. This mutation was found to cosegregate with the FSS phenotype in this family, and assessment using SIFT and PolyPhen-2 predicted a damaging effect. To the best of our knowledge, we report the first TNNI2 mutation in classical FSS and describe an atypical adult FSS case with only facial contractures resulting from somatic mosaicism. We infer that DA1, DA2B and FSS represent a phenotypic continuum of the same disorder and provide further genetic evidence for this hypothesis.

Protein Turnover and Cellular Stress in Mildly and Severely Affected Muscles from Patients with Limb Girdle Muscular Dystrophy Type 2I

Patients with Limb girdle muscular dystrophy type 2I (LGMD2I) are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. Our aim was to investigate if wasting could be caused by impaired regeneration in the proximal compared to distal muscles. Biopsies were simultaneously obtained from proximal and distal muscles of the same patients with LGMD2I (n = 4) and healthy subjects (n = 4). The level of past muscle regeneration was evaluated by counting internally nucleated fibers and determining actively regenerating fibers by using the developmental markers embryonic myosin heavy chain (eMHC) and neural cell adhesion molecule (NCAM) and also assessing satellite cell activation status by myogenin positivity. Severe muscle histopathology was occasionally observed in the proximal muscles of patients with LGMD2I whereas distal muscles were always relatively spared. No difference was found in the regeneration markers internally nucleated fibers, actively regenerating fibers or activation status of satellite cells between proximal and distal muscles. Protein turnover, both synthesis and breakdown, as well as cellular stress were highly increased in severely affected muscles compared to mildly affected muscles. Our results indicate that alterations in the protein turnover and myostatin levels could progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.

Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.

Over-expression of Neuron-derived Orphan Receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury

We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

Passive Repetitive Stretching for a Short Duration within a Week Increases Myogenic Regulatory Factors and Myosin Heavy Chain mRNA in Rats' Skeletal Muscles

Stretching is a stimulation of muscle growth. Stretching for hours or days has an effect on muscle hypertrophy. However, differences of continuous stretching and repetitive stretching to affect muscle growth are not well known. To clarify the difference of continuous and repetitive stretching within a short duration, we investigated the gene expression of muscle-related genes on stretched skeletal muscles. We used 8-week-old male Wistar rats (N = 28) for this study. Animals medial gastrocnemius muscle was stretched continuously or repetitively for 15 min daily and 4 times/week under anesthesia. After stretching, muscles were removed and total RNA was extracted. Then, reverse transcriptional quantitative real-time PCR was done to evaluate the mRNA expression of MyoD, myogenin, and embryonic myosin heavy chain (MyHC). Muscles, either stretched continuously or repetitively, increased mRNA expression of MyoD, myogenin, and embryonic MyHC more than unstretched muscles. Notably, repetitive stretching resulted in more substantial effects on embryonic MyHC gene expression than continuous stretching. In conclusion, passive stretching for a short duration within a week is effective in increasing myogenic factor expression, and repetitive stretching had more effects than continuous stretching for skeletal muscle on muscle growth. These findings are applicable in clinical muscle-strengthening therapy.

Contractile Properties of Human Fetal Skeletal Myofibrils

Congenital contractures such as clubfoot are present in ∼1/250 live births. Several congenital contractures syndromes are caused by mutations in genes that code for skeletal myofilament proteins, including MYH3 and MYH8. Because embryonic (MYH3) and perinatal (MYH8) myosin heavy chains are unique to the prenatal development of muscle, it is important to understand the contractile properties of human embryonic (HE) myosin and human fetal (HF) muscle to determine how mutations affect performance and development. However, information on HF skeletal muscle function is lacking. We previously reported HE myosin crossbridge cycling (measured by in vitro motility) was much slower that rabbit psoas (RP) myosin (Biophys. J. (2010), 98:542a). Here we characterized the contraction and relaxation properties of HF muscle using myofibril mechanics techniques. HF skeletal muscle and myofibrils were isolated from a 15.4 week gestation fetus. During maximal calcium activation (15°C) HF myofibrils produced much lower force (FMAX=5.9±1.2mN/mm2) as compared to myofibrils from human adult (HA) skeletal muscle (84±34mN/mm2) or RP muscle (220±40mN/mm2). Unlike control HA and RP fibrils, no striation pattern was apparent for HF myofibrils, suggesting that immature sarcomeres could explain the lower force production. HF myofibrils had slower kinetics of force development (kACT=0.66±0.1s−1) vs. HA (7.5±6.3s−1) and RP (5.7±0.5s−1) myofibrils. The initial (slow) phase of relaxation upon return to low calcium solution was slower and prolonged (kREL,SLOW=0.59±0.22s−1; tREL,SLOW=174±13ms) vs. HA (2.9±1.7s−1; 71±18ms) or RP (2.1±0.4s−1; 73±14ms) myofibrils. The larger, faster phase of relaxation was also slower (HF=1.5±0.2s−1 vs. HA=12±5s−1 and RP=21±4s−1). Our previous in vitro motility experiments indicated a similar inhibition of filament speed by increasing [ADP] for HE and RP myosin, but this was under low load, thus ongoing experiments will determine if ADP release is responsible for the slower kinetics of HF muscle. Funded by F31AR06300(A.R.), 5K23HD057331(A.B.), HD048895(M.B., M.R.).

Temporal myosin heavy chain isoform expression transitions faster in broiler chickens compared with Single Comb White Leghorns

Myosin heavy chain (MyHC), one of the major components in the contractile machinery of skeletal muscle fibers, is found in several isoforms during myogenesis. During chicken development, embryonic, neonatal, and adult MyHC isoforms are expressed. Broiler chickens have been selected for fast and large muscle growth, whereas Single Comb White Leghorn (SCWL) chickens have been selected for egg laying capabilities. This has led to an obvious difference in muscle growth and development with broilers being much larger than SCWL. The objective of this study was to determine if differences in muscle growth and development of SCWL and broilers are associated with differences in temporal expression of MyHC isoforms in skeletal muscle between the 2 breeds. Pectoralis major muscle (PM) was collected from SCWL and broilers at embryonic d 15, 17, and 19 and 1, 5, 11, 20, 27, and 33 d posthatch with n = 3 samples per time point and breed. Western blotting using 3 monoclonal antibodies (EB165, 2E9, and AB8) was performed to compare the expression patterns of embryonic/adult, neonatal, and adult isoforms of MyHC, respectively, for all time points in both SCWL and broiler chickens. Both broiler and SCWL chickens began expressing the neonatal MyHC isoform on d 5; however, SCWL chickens expressed the neonatal isoform much longer than broilers. The SCWL chickens had sustained expression of the neonatal MyHC isoform through d 27, whereas in broiler chickens the neonatal isoform was not expressed at d 20. Pectoralis major tissue from broiler chickens expressed the adult MyHC isoform as early as d 20, whereas the SCWL chickens began expressing the adult isoform later. The rate of transition to neonatal and adult MyHC isoforms in broilers and Leghorns is consistent with the faster maturation and growth of broilers relative to Leghorns. This relationship between faster growth of the PM and the rate of transition of MyHC isoforms within the fast skeletal muscle of the PM may indicate a selection marker for improvement of broiler PM growth.

In vitro characterization of proliferation and differentiation of pig satellite cells

Skeletal muscle contains various muscle fiber types exhibiting different contractile properties based on the myosin heavy chain (MyHC) isoform profile. Muscle fiber type composition is highly variable and influences growth performance and meat quality, but underlying mechanisms regulating fiber type composition remain poorly understood. The aim of the present work was to develop a model based on muscle satellite cell culture to further investigate the regulation of adult MyHC isoforms expression in pig skeletal muscle. Satellite cells were harvested from the mostly fast-twitch glycolytic longissimus (LM) and predominantly slow-twitch oxidative rhomboideus (RM) muscles of 6-week-old piglets. Satellite cells were allowed to proliferate up to 80% confluence, reached after 7day of proliferation (D7), and then induced to differentiate. Kinetics of proliferation and differentiation were similar between muscles and more than 95% of the cells were myogenic (desmin positive) at D7 with a fusion index reaching 65±9% after 4day of differentiation. One-dimensional SDS polyacrylamide gel electrophoresis revealed that satellite cells from both muscles only expressed the embryonic and fetal MyHC isoforms in culture, without any of the adult MyHC isoforms that were expressed in vivo. Interestingly, triiodothyronine (T3) induced de novo expression of adult fast and α-cardiac MyHC in vitro making our culture system a valuable tool to study de novo expression of adult MyHC isoforms and its regulation by intrinsic and/or extrinsic factors.

G.P.42 Protein turnover and cellular stress in mildly and severely affected muscles from patients with limb girdle muscular dystrophy type 2I

Patients with limb girdle muscular dystrophy (LGMD2I) are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. We are interested in finding out if this is caused by impaired or uneven regeneration in the proximal compared to distal muscles. Biopsies were simultaneously obtained from proximal and distal muscles of the same moderately affected LGMD2I patients (N=5) and healthy subjects (N=4). The level of past muscle regeneration was evaluated by counting internally nucleated fibers and determining recent regeneration by using the developmental marker NCAM and embryonic myosin heavy chain (eMHC) as well as myogenin for satellite cell activation status. Markers for protein synthesis Akt/mTOR/p70S6K, for protein breakdown MAFbx and MURF, the negative regulator of muscle mass myostatin, and for cellular stress p90RSK, p38 and JNK were analysed by Western blotting. We found a significant increase in activity of key molecular signaling pathways, specifically myogenesis, protein synthesis and degradation in more severely affected muscles associated with marked atrophy increased degree of muscle histopathology. Level of muscle regeneration was surprisingly comparable in the proximal and distal muscles. Our results indicate that muscle mass maintenance and/or cellular stress negatively affects proximal muscles leading to progressively more muscle wasting in these muscles compared to distal muscles in patients with LGMD2I.

Muscle Fiber Type-Dependent Differences in the Regulation of Protein Synthesis

This study examined fiber type-dependent differences in the regulation of protein synthesis in individual muscle fibers found within the same whole muscle. Specifically, the in vivo SUrface SEnsing of Translation (SUnSET) methodology was used to measure protein synthesis in type 1, 2A, 2X and 2B fibers of the mouse plantaris muscle, in response to food deprivation (FD), and mechanical overload induced by synergist ablation (SA). The results show that 48 h of FD induced a greater decrease in protein synthesis in type 2X and 2B fibers compared to type 1 and 2A fibers. Type 2X and 2B fibers also had the largest FD-induced decrease in total S6 protein and Ser240/244 S6 phosphorylation, respectively. Moreover, only type 2X and 2B fibers displayed a FD-induced decrease in cross-sectional area (CSA). Ten days of SA also induced fiber type-dependent responses, with type 2B fibers having the smallest SA-induced increases in protein synthesis, CSA and Ser240/244 S6 phosphorylation, but the largest increase in total S6 protein. Embryonic myosin heavy chain (MHCEmb) positive fibers were also found in SA muscles and the protein synthesis rates, levels of S6 Ser240/244 phosphorylation, and total S6 protein content, were 3.6-, 6.1- and 2.9-fold greater than that found in fibers from control muscles, respectively. Overall, these results reveal differential responses in the regulation of protein synthesis and fiber size between fiber types found within the same whole muscle. Moreover, these findings demonstrate that changes found at the whole muscle level do not necessarily reflect changes in individual fiber types.

Conditional over-expression of PITX1 causes skeletal muscle dystrophy in mice.

SummaryPaired-like homeodomain transcription factor 1 (PITX1) was specifically up-regulated in patients with facioscapulohumeral muscular dystrophy (FSHD) by comparing the genome-wide mRNA expression profiles of 12 neuromuscular disorders. In addition, it is the only known direct transcriptional target of the double homeobox protein 4 (DUX4) of which aberrant expression has been shown to be the cause of FSHD. To test the hypothesis that up-regulation of PITX1 contributes to the skeletal muscle atrophy seen in patients with FSHD, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscle can be controlled by oral administration of doxycycline. After PITX1 was over-expressed in the skeletal muscle for 5 weeks, the mice exhibited significant loss of body weight and muscle mass, decreased muscle strength, and reduction of muscle fiber diameters. Among the muscles examined, the tibialis anterior, gastrocnemius, quadricep, bicep, tricep and deltoid showed significant reduction of muscle mass, while the soleus, masseter and diaphragm muscles were not affected. The most prominent pathological change was the development of atrophic muscle fibers with mild necrosis and inflammatory infiltration. The affected myofibers stained heavily with NADH-TR with the strongest staining in angular-shaped atrophic fibers. Some of the atrophic fibers were also positive for embryonic myosin heavy chain using immunohistochemistry. Immunoblotting showed that the p53 was up-regulated in the muscles over-expressing PITX1. The results suggest that the up-regulation of PITX1 followed by activation of p53-dependent pathways may play a major role in the muscle atrophy developed in the mouse model.

Ginsenoside Rb1 inhibits the carotid neointimal hyperplasia induced by balloon injury in rats via suppressing the phenotype modulation of vascular smooth muscle cells

This study aims to investigate the effects of ginsenoside Rb1 on vascular intimal hyperplasia in rats and explore the mechanisms. The rat vascular neointimal hyperplasia model was made by rubbing the endothelia of carotid artery with a balloon and Rb1 (10 and 30mg/kg/day) was given the day after surgery for 14 consecutive days. The neointimal hyperplasia level and the degree of vascular smooth muscle cells (VSMCs) proliferation were evaluated by histopathology and by calculating the proliferating cell nuclear antigen (PCNA) positive expression percentage; protein expressions of PCNA, phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2), smooth muscle α-actin (SM α-actin), and the mRNA expressions of proto-oncogene c-myc, SM α-actin, SM-emb (embryonic smooth muscle myosin heavy chain) and p38 MAPK were detected by immunohistochemistry and Real Time RT-PCR, respectively. Compared with the endothelia rubbing model group, Rb1 10 and 30mg/kg/day medication significantly ameliorated the neointimal hyperplasia (P<0.05), and decreased the positive expression percentage of PCNA(P<0.05). Rb1 medication also significantly decreased the elevated protein expression of pERK1/2 and the mRNA expression of c-myc(P<0.05), and tended to reduce the expression of p38 MAPK mRNA. Endothelial rubbing increased the SM-emb mRNA expression, but decreased the expression of SM α-actin mRNA which was reversed by Rb1 (P<0.05). The results indicate that Rb1 inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats via suppressing the VSMC proliferation, which may be involved in part the inhibition of pERK1/2 protein and related to its inhibition on VSMC phenotype modulation.

Selective estrogen receptor‐β activation stimulates skeletal muscle growth and regeneration

There is increasing evidence suggesting that estrogens augment skeletal muscle regeneration processes after injury. To study the contribution of estrogen receptors α and β (ERα and ERβ) during muscle regeneration, skeletal muscles of ovariectomized (OVX) rats, as well as ERα- and ERβ-knockout (αErko and βErko) mice, were injured with a myotoxin (notexin). OVX rats were simultaneously treated with the ER-selective ligands genistein, ERα agonist 16α-LE2 (alpha), ERβ agonist 8β-VE2 (beta), or 17β-estradiol (E(2)). OVX rats showed significantly elevated serum creatine kinase (CK) activity after muscle injury compared to intact sham-treated animals. Treatment with ER ligands significantly reduced CK activity. TNF-α, IL-10, and MCP-1 expression served to characterize immune responses. Treatment with all ER ligands, but particularly E(2) and beta, reduced TNF-α, but elevated MCP-1 and IL-10 expression. PCNA and MyoD expression served to define satellite cell activation and proliferation and were found to be up-regulated by beta and E(2). To further study muscle regeneration responses, expression of the embryonic myosin heavy chain (MHC) was analyzed. Beta and E(2) but not alpha increased embryonic MHC expression compared to OVX. The absence of ERβ in βErko mice negatively affected CK activity levels and expression of satellite cell and muscle regeneration markers (MHC embryonic, MyoD, Pax7) compared with αErko and wild-type mice. In a classic Hershberger assay using male rats, beta stimulated muscle growth, accompanied by a strong induction of IGF-1 expression. Our data provide evidence that ERβ signaling is involved in the regulation of skeletal muscle growth and regeneration by stimulating anabolic pathways, activating satellite cells and modulating immune responses.

Angiotensin II Infusion Induces Marked Diaphragmatic Skeletal Muscle Atrophy

Advanced congestive heart failure (CHF) and chronic kidney disease (CKD) are characterized by increased angiotensin II (Ang II) levels and are often accompanied by significant skeletal muscle wasting that negatively impacts mortality and morbidity. Both CHF and CKD patients have respiratory muscle dysfunction, however the potential effects of Ang II on respiratory muscles are unknown. We investigated the effects of Ang II on diaphragm muscle in FVB mice. Ang II induced significant diaphragm muscle wasting (18.7±1.6% decrease in weight at one week) and reduction in fiber cross-sectional area. Expression of the E3 ubiquitin ligases atrogin-1 and muscle ring finger-1 (MuRF-1) and of the pro-apoptotic factor BAX was increased after 24 h of Ang II infusion (4.4±0.3 fold, 3.1±0.5 fold and 1.6±0.2 fold, respectively, compared to sham infused control) suggesting increased muscle protein degradation and apoptosis. In Ang II infused animals, there was significant regeneration of injured diaphragm muscles at 7 days as indicated by an increase in the number of myofibers with centralized nuclei and high expression of embryonic myosin heavy chain (E-MyHC, 11.2±3.3 fold increase) and of the satellite cell marker M-cadherin (59.2±22.2% increase). Furthermore, there was an increase in expression of insulin-like growth factor-1 (IGF-1, 1.8±0.3 fold increase) in Ang II infused diaphragm, suggesting the involvement of IGF-1 in diaphragm muscle regeneration. Bone-marrow transplantation experiments indicated that although there was recruitment of bone-marrow derived cells to the injured diaphragm in Ang II infused mice (267.0±74.6% increase), those cells did not express markers of muscle stem cells or regenerating myofibers. In conclusion, Ang II causes marked diaphragm muscle wasting, which may be important for the pathophysiology of respiratory muscle dysfunction and cachexia in conditions such as CHF and CKD.

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart.

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.

Impact of static magnetic fields on human myoblast cell cultures

Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches.

Potential sources of early-postnatal increase in myofibre number in pig skeletal muscle

In pigs, myogenesis is a biphasic phenomenon with the formation of primary and secondary fibres. Hyperplasia was reported to be accomplished around 90days of gestation. However, some studies suggest a substantial increase in the total fibre number (TFN) from birth to weaning by counting fibre number in the muscle cross sections. The aim of this study was to establish in which way TFN increases after birth and whether this increase is imputable to new (tertiary) myofibres and/or fibre elongation. The semitendinosus muscle of 128 piglets was examined at days 1 (n=63), 7 (n=12), 21 (n=12), and 28 (n=41) of age. TFN was increased at days 7, 21 and 28 of age when compared with day 1 (P<0.01). From day 1 to 28, TFN increased from 463×10(3) to 825×10(3). Microscopy of longitudinal and transversal serial sections revealed that at day 7 of age very small fibres expressing the embryonic myosin heavy chain (MyHC) isoform were apparent all over the muscle. In addition, intrafascicular terminations of normal-sized fibres expressed the embryonic MyHC isoform. These data suggest that the TFN in the pig muscle is not fixed at birth and its postnatal increase may be related to both elongation of existing muscle fibres and genesis of tertiary myofibres, mainly between birth and 3weeks of age.