Myostatin coordinating the proliferation and differentiation of adipose and skeletal muscle cells and energy metabolism balance based on gene chip technology / A miostatina na coordenação da proliferação e diferenciação de células musculares adiposas e esqueléticas e no equilíbrio do metabolismo energético com base na tecnologia de chips genéticos / Msrn, basado en la tecnología de chips genéticos, coordina la fertilización y el metabolismo de las células musculares grasas y óseas

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.

RESUMO Os mioblastos se fundem eM fibras musculares multinucleadas para formar e promover o crescimento do músculo esquelético. A fim de analisar o papel da miostatina (MSTN) na gordura corporal, proliferação de células musculares esqueléticas e diferenciação e metabolismo energético, este estudo utilizará a tecnologia anti-RNA de chips genéticos para estudá-la. Os resultados mostraram que o gene MSTN regulava o crescimento e a proliferação de mioblastos e afetava o desenvolvimento do músculo esquelético, afetando a expressão de Cdc42, bnip2, p38 e outros genes; a eliminação ou sobrexpressão do gene MSTN conduziria a uma tendência de os genes adiposos sintetizarem a gordura até sua decomposição; após a eliminação do gene MSTN, os níveis de expressão de cpti-b, PPARG e outros genes nas células mostraram-se correspondentes após a sobrexpressão do gene MSTN, e a expressão relativa do gene PPARG diminuiu. Sugere-se que a eliminação ou sobrexpressão da MSTN possa afetar a acumulação de lipídeos, e o cpti-b e o PPARG podem regular diretamente o nível lipídico. Espera-se que esta experiência possa fornecer uma referência para o estudo do efeito da MSTN sobre a deposição de gordura.

RESUMEN Los mioblastos se funden en fibras musculares multinucleadas para formar y promover el crecimiento del músculo esquelético. A fin de analizar el papel de la miostatina (MSTN) en la grasa corporal, proliferación de células musculares esqueléticas y diferenciación y metabolismo energético, este estudio utilizará la tecnología anti-RNA de chips genéticos para estudiarla. Los resultados mostraron que el gen MSTN regulaba el crecimiento y la proliferación de mioblastos y afectaba el desarrollo del músculo esquelético, afectando la expresión de Cdc42, bnip2, p38 y otros genes; la eliminación o sobreexpresión del gen MSTN conduciría a una tendencia de que los genes adiposos sinteticen la grasa hasta su descomposición; después de la eliminación del gen MSTN, los niveles de expresión de cpti-b, PPARG y otros genes en las células se mostraron correspondientes después de la sobreexpresión del gen MSTN, y la expresión relativa del gen PPARG disminuyó. Se sugiere que la eliminación o sobreexpresión de la MSTN pueda afectar la acumulación de lipídos, y el cpti-b y el PPARG pueden regular directamente el nivel lipídico. Se espera que esta experiencia pueda proveer una referencia para el estudio del efecto de la MSTN sobre el depósito de grasa.

Sumoylation of histone deacetylase 1 regulates MyoD signaling during myogenesis

Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.

Regulation of muscle plasticity and trophism by fatty acids: A short review / Regulação da plasticidade e do trofismo muscular pelos ácidos graxos: uma breve revisão

Summary The skeletal muscle tissue has a remarkable ability to alter its plastic structural and functional properties after a harmful stimulus, regulating the expression of proteins in complex events such as muscle regeneration. In this context, considering that potential therapeutic agents have been widely studied, nutritional strategies have been investigated in order to improve the regenerative capacity of skeletal muscle. There is evidence of the modulatory action of fatty acids, such that oleic and linoleic acids, that are abundant in Western diets, on muscle function and trophism. Thus, fatty acids appear to be potential candidates to promote or impair the recovery of muscle mass and function during regeneration, since they modulate intracellular pathways that regulate myogenesis. This study is the first to describe and discuss the effect of fatty acids on muscle plasticity and trophism, with emphasis on skeletal muscle regeneration and in vitro differentiation of muscle cells.

Resumo O tecido muscular esquelético possui a notável capacidade plástica de alterar suas propriedades estruturais e funcionais após um estímulo lesivo, regulando a expressão de proteínas durante eventos complexos como a regeneração muscular. Nesse contexto, considerando que possíveis agentes terapêuticos vêm sendo amplamente estudados, estratégias nutricionais têm sido investigadas na perspectiva de melhorar a capacidade regenerativa do músculo esquelético. Há evidências da ação modulatória dos ácidos graxos, como os ácidos oleico e linoleico, que são abundantes nas dietas ocidentais, sobre a função muscular e o trofismo. Nesse sentido, os ácidos graxos parecem ser potenciais candidatos para promover ou prejudicar a recuperação da massa e a função muscular durante a regeneração, uma vez que modulam vias intracelulares reguladoras da miogênese. Este trabalho é o primeiro a descrever e discutir o efeito dos ácidos graxos sobre a plasticidade e o trofismo muscular, com ênfase na regeneração do músculo esquelético e na diferenciação de células musculares in vitro.

The maintenance ability and Ca²⁺ availability of skeletal muscle are enhanced by sildenafil

Sildenafil relaxes vascular smooth muscle cells and is used to treat pulmonary artery hypertension as well as erectile dysfunction. However, the effectiveness of sildenafil on skeletal muscle and the benefit of its clinical use have been controversial, and most studies focus primarily on tissues and organs from disease models without cellular examination. Here, the effects of sildenafil on skeletal muscle at the cellular level were examined using mouse primary skeletal myoblasts (the proliferative form of skeletal muscle stem cells) and myotubes, along with single-cell Ca2+ imaging experiments and cellular and biochemical studies. The proliferation of skeletal myoblasts was enhanced by sildenafil in a dose-independent manner. In skeletal myotubes, sildenafil enhanced the activity of ryanodine receptor 1, an internal Ca2+ channel, and Ca2+ movement that promotes skeletal muscle contraction, possibly due to an increase in the resting cytosolic Ca2+ level and a unique microscopic shape in the myotube membranes. Therefore, these results suggest that the maintenance ability of skeletal muscle mass and the contractility of skeletal muscle could be improved by sildenafil by enhancing the proliferation of skeletal myoblasts and increasing the Ca2+ availability of skeletal myotubes, respectively.

Current Concepts in Stem Cell Therapy for Cardiovascular Diseases: What We Know and Don't Know / 한양의대학술지

Medical therapies and mechanical interventions for the treatment of myocardial infarction (MI) and ischemic heart failure have seen great progress. However, current therapies only slow the progression to heart failure, but do not stimulate regeneration to recover the loss of functional myocytes. Stem cell-based therapy is a novel modality that can potentially be used for the treatment of ischemic cardiac injury and heart failure wherein cardiac tissue is regenerated thereby improving cardiac function and reducing the morbidity and mortality of patients. Many preclinical and clinical trials have employed various types of stem cells including bone marrow-derived mononuclear cells, skeletal myoblasts, resident cardiac stem cells, mesenchymal stem cells, and endothelial progenitor cells, and these studies have suggested great potential for the clinical application of stem cell therapy for cardiac disease. Here we will review and summarize the current evidence indicating the potential of stem cell therapy, focusing on the clinical trials conducted to date.

High passage numbers induce resistance to apoptosis in C2C12 muscle cells

Cell lines with high passage numbers exhibit alterations in cell morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (<20) or high (>60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.

TGF-β1-induced synthesis of collagen fibers in skeletal muscle-derived stem cells / 华中科技大学学报（医学）（英德文版）

The aim of this study was to investigate the mechanism of deposition of extracellular matrix induced by TGF-β1 in skeletal muscle-derived stem cells (MDSCs). Rat skeletal MDSCs were obtained by using preplate technique, and divided into four groups: group A (control group), group B (treated with TGF-β1, 10 ng/mL), group C (treated with TGF-β1 and anti-connective tissue growth factor (CTGF), both in 10 ng/mL), and group D (treated with anti-CTGF, 10 ng/mL). The expression of CTGF, collagen type-I (COL-I) and collagen type-III (COL-III) in MDSCs was examined by using RT-PCR, Western blot and immunofluorescent stain. It was found that one day after TGF-β1 treatment, the expression of CTGF, COL-I and COL-III was increased dramatically. CTGF expression reached the peak on the day 2, and then decreased rapidly to a level of control group on the day 5. COL-I and COL-III mRNA levels were overexpresed on the day 2 and 3 respectively, while their protein expression levels were up-regulated on the day 2 and reached the peak on the day 7. In group C, anti-CTGF could partly suppress the overexpression of COL-I and COL-II induced by TGF-β1 one day after adding CTGF antibody. It was concluded that TGF-β1 could induce MDSCs to express CTGF, and promote the production of COL-I and COL-III. In contrast, CTGF antibody could partially inhibit the effect of TGF-β1 on the MDSCs by reducing the expression of COL-I and COL-III. Taken together, we demonstrated that TGF-β1-CTGF signaling played a crucial role in MDSCs synthesizing collagen proteins in vitro, which provided theoretical basis for exploring the methods postponing skeletal muscle fibrosis after nerve injury.

Insulin promotes proliferation of skeletal myoblast cells through PI3K/Akt and MEK/ERK pathways in rats / 生理学报

The present study was to explore the effects of insulin on proliferation of skeletal myoblast cells in rats. Separated and cultured primary skeletal myoblast cells from rats were treated by insulin. By means of the incorporation of (3)H-TdR, BrdU assay and MTT assay, the proliferation of skeletal myoblast cells was detected. Western blot was used to check the phosphorylation of Akt and ERK of myoblast cells. The results showed that insulin significantly promoted the incorporation of (3)H-TdR into cultured skeletal myoblast cells in a dose-dependent manner. MTT assay and BrdU assay also showed insulin promoted the proliferation of skeletal myoblast cells. The promotion of skeletal myoblast cells proliferation by insulin was inhibited by PI3K inhibitor wortmannin or MEK inhibitor U0126, and the same phenomenon was shown in L6 and C2C12 cells. Also, insulin increased the phosphorylation of Akt and ERK in myoblast cells. These results suggest that insulin may promote proliferation of skeletal myoblast cells through PI3K/Akt and MEK/ERK pathways.

Transplantation of autologous myoblasts transfected with VEGF165 improves heart function after myocardial infarction in rabbits / 中南大学学报(医学版)

OBJECTIVE@#To determine the improvements of post-infarction heart function after transplantation of autologous skeletal myoblasts transfected with VEGF165 in rabbits.@*METHODS@#Myocardium infarction was induced in rabbits by left anterior descending coronary artery ligation. At 2 weeks, 1.75×10(7) autologous skeletal myoblasts transfected with pcDNA3.1-VEGF165 were infused into the region of MI via direct intramuscular injection; pcDNA3.1 served as a control.@*RESULTS@#The DAPI-labeled and Desmin-positive immunostained skeletal myofibers were found throughout the infracted areas and border zones, and the density of blood capillary in the MI region transplanted by myoblasts with VEGF165 was increased (measured 4 weeks later and compared with controls). Heart function was examined by the Buxco system and demonstrated that maximum dp/dt [(1607.23±102.67) mmHg/s vs (1217.77±89.91) mmHg/s] and minimum dp/dt [(-1535.09 ± 81.34) mmHg/s vs (1174.58 ± 91.5) mmHg/s] were improved in the heart transplanted with the transfected myoblasts(P<0.05).@*CONCLUSION@#Autologous skeletal myoblasts transfected with VEGF165 could ameliorate the blood supply in the MI region, and aid recovery of heart function more quickly in post-infarction hearts. This suggests an effective treatment for myocardium infarction.

Mioblasto esquelético: um justo abandono?: [revisão] / Skeletal myoblast: fair abandonment?: [review]

A doença isquêmica do miocárdio é causa crescente de insuficiência cardíaca e considerada, há muito, situação irreversível devido ao fato de que os cardiomiócitos adultos são células terminalmente diferenciadas e sem capacidade proliferativa. Para a reversão da insuficiência cardíaca, seria necessário reposição dos miócitos danificados e restauração do fluxo sanguíneo. Nos últimos anos, especial atenção tem sido dada à capacidade potencial da terapia celular em reparar o miocárdio injuriado devido à sua habilidade de diferenciação in vitro em cardiomiócitos e células endoteliais. As células tronco são células indiferenciadas, capazes de autorrenovação, proliferação e diferenciação em múltiplas linhagens. O objetivo final de sua utilização é a regeneração tecidual. As células-tronco hematopoiéticas foram as primeiras a serem identificadas como potencial fonte celular. Em seguida, diversos outros tipos celulares foram reconhecidos, assim como as células progenitoras parcialmente diferenciadas capazes de proliferação e diferenciação em múltiplas linhagens. O objetivo deste artigo é descrever em detalhes os mioblastos esqueléticos, células que surgiram nos anos 90 como potencial fonte celular para a regeneração cardíaca, mas que, com os resultados dos primeiros estudos, mostraram limitações proibitivas ao seu uso mais extenso, principalmente relacionadas a questões de segurança.

Myocardial ischemia is an increasingly frequent cause of heart failure and has long been considered irreversible, as adult cardiomyocytes are terminallydifferentiated cells with no proliferative capacity. In order to reverse heart failure, damaged myocytes would have to be replaced and blood flow restored.In recent years, special attention has been paid to the potential ability of cell therapy to repair an injured myocardium, due to the in vitro ability to distinguish between the develop of cardiomyocytes and endothelial cells. Stem cells are undifferentiated cellscapable of self-renewal , proliferation and differentiation into multiple lineages. The ultimate goal of their use is tissue regeneration. Hematopoietic stem cells were the first to be identified as a potential cell source, followed by several other cell types, as well as partially differentiated progenitor cells capable of proliferation and differentiation intomultiple lineages. This paper offers a detailed description of skeletal myoblasts - cells that were viewed during the 1990s as a potential cell source for cardiac regeneration but which were subject to prohibitive constraints on more extensive use, shown through the findings of early studies and related mainly to security issues.

Regulatory effect of glucagon-like peptide-1 on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the regulatory effect of glucagon-like peptide-1 (GLP-1) on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism.</p><p><b>METHODS</b>L6 cells cultured in DMEM high glucose culture medium containing 10% FBS were divided into control group (C, without addition), GLP-1 group (G, added with 10 nmol/L GLP-1), PI3K inhibitor group (W, added with 50 nmol/L PI3K specific inhibitor wortmannin), and GLP-1 + PI3K inhibitor group (GW, added with 10 nmol/L GLP-1 and 50 nmol/L wortmannin) according to the random number table. Cell proliferation activity was detected with MTT assay at post culture hour (PCH) 24, 48, 72 (denoted as absorbance value). At PCH 24, the change in cell cycle was evaluated with flow cytometer, the expression level of proliferating cell nuclear antigen (PCNA) was determined with immunohistochemical staining, the protein levels of phosphorylated PI3K (p-PI3K) and p-Akt were determined with Western blotting. Data were processed with multi-group analysis of variance.</p><p><b>RESULTS</b>(1) The cell proliferation activity at PCH 48, 72 in G group was respectively 0.660 +/- 0.120, 0.870 +/- 0.240, all significantly higher than those in C group (0.530 +/- 0.060, 0.700 +/- 0.100, with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). The cell proliferation activity in W group at each time point was lower than that in C group. The cell proliferation activity in GW group at PCH 48, 72 was respectively 0.510 +/- 0.080, 0.740 +/- 0.160, all lower than those in G group (with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). (2) The percentage of S phase cell in G group at PCH 24 [(15.7 +/- 0.4)%] was significantly higher than that in C group [(13.6 +/- 0.6)%] and GW group [(10.1 +/- 0.6)%], while that in W group [(6.8 +/- 1.2)%] was lower than that in C group (with F values all equal to 15.39, P values all below 0.01). (3) PCNA level in G group at PCH 24 [(51.24 +/- 1.18)%] was markedly higher than that in C group [(36.72 +/- 1.56)%] and GW group [(25.90 +/- 1.22)%], and while in W group [(21.70 +/- 0.09)%] was lower than that in C group (with F values equal to 783.80, P values all below 0.05). (4) The protein level of p-Akt in G group at PCH 24 was significantly higher than that in the other 3 groups, while that in W group was lower than that in C group (with F values equal to 94.43, P values all below 0.01). There was no obvious difference in protein level of p-PI3K at PCH 24 among G, GW, and C groups ( F = 20.94, P > 0.05). The protein level of p-PI3K at PCH 24 in W group was lower than that in C group (F = 20.94, P < 0.05).</p><p><b>CONCLUSIONS</b>GLP-1 can promote cell proliferation of skeletal myoblast by accelerating the progression of cell cycle and increasing the synthesis of DNA, which can be attributed to PI3K/Akt signal pathway.</p>

Modulatory effect of insulin on scalded rat serum-induced apoptosis of skeletal myoblast / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the modulatory effect of insulin on apoptosis of skeletal myoblast (L6 cells) by serum of scalded rat and its mechanism.</p><p><b>METHODS</b>L6 cells cultured with DMEM medium containing 10% FBS were divided into control (C, added with 20% normal rat serum), serum from rat with scald injury (S, added with 20% serum from scalded rat), insulin (I, added with 20% normal rat serum and 100 nmol/L insulin), and serum of scalded rat + insulin (SI, added with 20% serum of scalded rat + 100 nmol/L insulin) groups according to the random number table. After being cultured for 48 hours, apoptosis was observed with Hoechst 33258 staining and its number counted, annexin V -FITC/PI double-labeling method was used to assess apoptosis rate, the protein levels of phosphorylated (p-) Akt, p-PI3K, Bax, Bcl-2, and active caspase-3 were determined by Western blotting. Data were processed with grouped or paired t test.</p><p><b>RESULTS</b>(1) The amount of apoptosis with typical morphological change in S group [(59.6 +/- 3.9) per visual field] was more than that in C, I, and SI groups [(4.9 +/- 2.6), (5.5 +/- 2.1), (19.7 +/- 2.3) per visual field, with t value respectively 28.53, 29.86, 21.53, P values all below 0.01]. (2) Apoptotic rate in S group was (18.5 +/- 1.8)%, which was markedly higher than that in C, I, and SI groups [(1.1 +/- 0.6)%, (1.5 +/- 0.3)%, (7.8 +/- 0.6)%, with t value respectively 22.41, 22.83, 13.92, P values all below 0.01]. (3) Compared with those in C group, the protein levels of Bax and active caspase-3 in S group were up-regulated (1.12 +/- 0.63 vs. 0.16 +/- 0.03, 2.15 +/- 0.51 vs. 0.21 +/- 0.03, with t value respectively 3.80, 10.69, P values all below 0.01), the protein level of p-Akt was lowered (0.20 +/- 0.03 vs. 0.42 +/- 0.07, t = -8.46, P < 0.01), and the protein levels of p-PI3K and Bcl-2 showed no statistical difference (0.19 +/- 0.03 vs. 0.26 +/- 0.09, 0.17 +/- 0.03 vs. 0.28 +/- 0.07, with t value respectively -2.73, - 1.14, P values all above 0.05). The protein levels of Bax (0.40 +/- 0.14) and active caspase-3 (0.83 +/- 0.18) in SI group were lowered (t = -3.23, P < 0.05; t = 6.66, P < 0.01) and the protein levels of p-Akt, Bcl-2, and p-PI3K in SI group were elevated (0.39 +/- 0.10, 0.78 +/- 0.03, 0.47 +/- 0.12, with t value respectively 4.07, 18.71, 5.05, P < 0.05 or P < 0.01) as compared with those in S group.</p><p><b>CONCLUSIONS</b>Serum from scalded rat can induce apoptosis in skeletal myoblast, and the effect can be inhibited by insulin through PI3K/Akt signal pathway.</p>

Regulation of Pax7 protein levels by caspase-3 and proteasome activity in differentiating myoblasts

The transcription factor Pax7 negatively regulates the activity of the muscle regulatory transcription factor MyoD, preventing muscle precursor cells from undergoing terminal differentiation. In this context, the ratio between Pax7 and MyoD protein levels is thought to be critical in allowing myogenesis to proceed or to maintain the undifferentiated muscle precursor state. We have previously shown that Pax7 is subject to rapid down regulation in differentiating myoblasts, via a proteasome-dependent pathway. Here we present evidence indicating that Pax7 is also subject to caspase-3-dependent regulation. Furthermore, simultaneous inhibition of caspase-3 and proteasome activity induced further accumulation of Pax7 protein in differentiating myoblasts. These results suggest that at early stages of muscle differentiation, Pax7 levels are regulated by at least two independent mechanisms involving caspase-3 and proteasome activity.

TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.

Hypoxia enhances the proliferation of skeletal myoblasts and possible mechanism / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the role of hypoxia in skeletal myoblasts proliferation and related mechanism.</p><p><b>METHODS</b>The numbers and proliferous indexes of skeletal myoblasts were detected by flow cytometer under 20%, 3%, and 10% oxygen concentration. Hypoxia inducing factor 1alpha (HIF-1alpha) mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). HIF-1alpha proteinum in endochylema and intranucelus were respectively detected by Western blot.</p><p><b>RESULTS</b>The numbers and proliferous indexes were higher (P < 0.05) in hypoxia group than those of control group. The expression HIF-1alpha mRNA had no difference in hypoxia and in normal groups. The level of HIF-1alpha proteinum in endochylema under normoxia was more than that in intranucelus and it was opposite under hypoxia.</p><p><b>CONCLUSION</b>Hypoxia can promote the proliferation of skeletal myoblasts. The possible mechanism of hypoxia promoting the proliferation of skeletal myoblasts might be that low oxygen concentration regulates HIF-la nuclear translocation.</p>

Recombinant proteins secreted from tissue-engineered bioartificial muscle improve cardiac dysfunction and suppress cardiomyocyte apoptosis in rats with heart failure / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tissue-engineered bioartificial muscle-based gene therapy represents a promising approach for the treatment of heart diseases. Experimental and clinical studies suggest that systemic administration of insulin-like growth factor-1 (IGF-1) protein or overexpression of IGF-1 in the heart exerts a favorable effect on cardiovascular function. This study aimed to investigate a chronic stage after myocardial infarction (MI) and the potential therapeutic effects of delivering a human IGF-1 gene by tissue-engineered bioartificial muscles (BAMs) following coronary artery ligation in Sprague-Dawley rats.</p><p><b>METHODS</b>Ligation of the left coronary artery or sham operation was performed. Primary skeletal myoblasts were retrovirally transduced to synthesize and secrete recombinant human insulin-like growth factor-1 (rhIGF-1), and green fluorescent protein (GFP), and tissue-engineered into implantable BAMs. The rats that underwent ligation were randomly assigned to 2 groups: MI-IGF group (n = 6) and MI-GFP group (n = 6). The MI-IGF group received rhIGF-secreting BAM (IGF-BAMs) transplantation, and the MI-GFP group received GFP-secreting BAM (GFP-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: S-IGF group (n = 6) and S-GFP group (n = 6). The S-IGF group underwent IGF-1-BAM transplantation, and S-GFP group underwent GFP-BAM transplantation. IGF-1-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats after two weeks of operation was performed. Four weeks after the treatment, hemodynamics was performed. IGF-1 was measured by radioimmunoassay, and then the rats were sacrificed and ventricular samples were subjected to immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of bax and Bcl-2. TNF-α and caspase 3 expression in myocardium was examined by Western blotting.</p><p><b>RESULTS</b>Primary rat myoblasts were retrovirally transduced to secrete rhIGF-1 and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted consistent levels of hIGF (0.4 - 1.2 µg×BAM(-1)×d(-1)). When implanted into syngeneic rat, IGF-BAMs secreted and delivered rhIGF. Four weeks after therapy, the hemodynamics was improved significantly in MI rats treated with IGF-BAMs compared with those treated with GFP-BAMs. The levels of serum IGF-1 were increased significantly in both MI and sham rats treated with IGF-BAM. The mRNA expression of bax was lower and Bcl-2 expression was higher in MI-IGF group than MI-GFP group (P < 0.05). Western blotting assay showed TNF-α and caspase 3 expression was lower in MI-IGF group than MI-GFP group after therapy.</p><p><b>CONCLUSIONS</b>rhIGF-1 significantly improves left ventricular function and suppresses cardiomyocyte apoptosis in rats with chronic heart failure. Genetically modified tissue-engineered BAMs provide a method delivering recombinant protein for the treatment of heart failure.</p>

Autologous skeletal myoblast transplantation improves hemodynamics dogs with chronic heart failure dogs / 南方医科大学学报

<p><b>OBJECTIVE</b>To test the effects of autologous skeletal myoblast transplantation on the hemodynamics in dogs with coronary microembolization-induced chronic heart failure (CHF).</p><p><b>METHODS</b>CHF models were successfully induced in 19 dogs and divided into ASMT group (n=9) and control group (n=10). The myoblasts were injected into the embolized region in the 9 dogs of the ASMT group, and saline was injected in the control dogs, and the hemodynamics of the dogs were evaluated 5 weeks after the injections.</p><p><b>RESULT</b>Compared with saline injection, ASMT significantly increased dP/dtmax, MAP and LVSP (P<0.05) and decreased LVEDP (P<0.05) 5 weeks after myoblast transplantation. Desmin and Brd-U immunofluorescent staining showed myoblast survival at the injected sites in the dogs.</p><p><b>CONCLUSION</b>ASMT provides mild improvements in the hemodynamics of dogs with CHF.</p>

Recombinant human growth hormone secreted from tissue-engineered bioartificial muscle improves left ventricular function in rat with acute myocardial infarction / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Experimental studies and preliminary clinical studies have suggested that growth hormone (GH) treatment may improve cardiovascular parameters in chronic heart failure (CHF). Recombinant human GH (rhGH) has been delivered by a recombinant protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. The present study aimed to examine a new method for delivery of rhGH using genetically modified bioartificial muscles (BAMs), and investigate whether the rhGH delivered by this technique improves left ventricular (LV) function in rats with CHF.</p><p><b>METHODS</b>Primary skeletal myoblasts were isolated from several Sprague-Dawley (SD) rats, cultured, purified, and retrovirally transduced to synthesize and secrete human rhGH, and tissue-engineered into implantable BAMs. Ligation of the left coronary artery or sham operation was performed. The rats that underwent ligation were randomly assigned to 2 groups: CHF control group (n = 6) and CHF treatment group (n = 6). The CHF control group received non-rhGH-secreting BAM (GFP-BAMs) transplantation, and the CHF treatment group received rhGH-secreting BAM (GH-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: sham control group (n = 6) and sham treatment group (n = 6). The sham control group underwent GFP-BAM transplantation, and the sham treatment group underwent GH-BAM transplantation. GH-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats with ligation of the left coronary artery or sham operation was performed. Eight weeks after the treatment, echocardiography was performed. hGH, insulin-like growth factor-1 (IGF-1) and TNF-alpha levels in rat serum were measured by radioimmunoassay and ELISA, and then the rats were killed and ventricular samples were subjected to immunohistochemistry.</p><p><b>RESULTS</b>Primary rat myoblasts were retrovirally transduced to secrete rhGH and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 1 to 2 microg of bioactive rhGH per day. When implanted into syngeneic rat, GH-BAMs secreted and delivered rhGH. Eight weeks after therapy, LV ejection fraction (EF) and fractional shortening (FS) were significantly higher in CHF rats treated with GH-BAMs than in those treated with GFP-BAMs ((65.0 +/- 6.5)% vs (48.1 +/- 6.8)%, P < 0.05), ((41.3 +/- 7.4)% vs (26.5 +/- 7.1)%, P < 0.05). LV end-diastolic dimension (LVEDD) was significantly lower in CHF rats treated with GH-BAM than in CHF rats treated with GFP-BAM (P < 0.05). The levels of serum GH and IGF-1 were increased significantly in both CHF and sham rats treated with GH-BAM. The level of serum TNF-alpha decreased more significantly in the CHF treatment group than in the CHF control group.</p><p><b>CONCLUSIONS</b>rhGH significantly improves LV function and prevents cardiac remodeling in rats with CHF. Genetically modified tissue-engineered bioartificial muscle provides a method delivering recombinant protein for the treatment of heart failure.</p>

p38 MAPK Participates in Muscle-Specific RING Finger 1-Mediated Atrophy in Cast-Immobilized Rat Gastrocnemius Muscle

Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces alfa2beta1 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.