Serum response factor mRNA induction in the hypertrophying chicken patagialis muscle

Gene expression in the stretched chicken patagialis (Pat) muscle has not been extensively examined. This study's purpose was to determine the Pat muscle's expression pattern of serum response factor (SRF), skeletal alpha-actin, and MyoD mRNAs after 3 days (onset of stretch), 6 days (end of first week of rapid growth), and 14 days (slowed rate of stretch-induced growth) of stretch. SRF mRNA demonstrated two species (B1 and B2), with B2 being more prevalent in the predominantly fast-twitch Pat muscle, compared with the slow-tonic muscle. Stretch overload increased B1 and B2 SRF mRNA concentrations, and the increase in B1 SRF mRNA concentration was greater at day 6 compared with days 3 or 14. MyoD mRNA concentration was greater in 3-day-stretched Pat muscles, compared with days 6 or 14. Skeletal alpha-actin mRNA concentration was not changed during the study. Gel mobility shift assays demonstrated that SRF binding with serum response element 1 of the skeletal alpha-actin promoter had no altered binding patterns from 6-day-stretched Pat nuclear extracts. It appears that SRF and MyoD mRNAs are induced in the stretch-overloaded Pat muscle but at different time points.     

RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation

Serum response factor (SRF) plays a central role during myogenesis, being required for the expression of striated alpha-actin genes. As shown here, the small GTPase RhoA-dependent activation of SRF results in the expression of muscle-specific genes, thereby promoting myogenic differentiation in myoblast cell lines. Co-expression of activated V14-RhoA and SRF results in an approximately 10-fold activation of the skeletal alpha-actin promoter in replicating myoblasts, while SRFpm1, a dominant negative SRF mutant, blocks RhoA dependent skeletal alpha-actin promoter activity. Serum withdrawal further potentiates RhoA- and SRF-mediated activation of alpha-actin promoter to about 30-fold in differentiated myotubes. In addition, the proximal SRE1 in the skeletal alpha-actin promoter is sufficient to mediate RhoA signaling via SRF. Furthermore, SRFpm1 and to a lesser extent dominant negative N19-RhoA inhibit myoblast fusion, postreplicative myogenic differentiation, and expression of direct SRF targets such as skeletal alpha-actin and indirect targets such as myogenin and alpha-myosin heavy chain. Moreover, RhoA also stimulates the autoregulatable murine SRF gene promoter in myoblasts, and the expression level of SRF is reduced in myoblasts overexpressing N19-RhoA. Our study supports the concept that RhoA signaling via SRF serves as an obligatory muscle differentiation regulatory pathway.     

Expression of fibroblast growth factor family during postnatal skeletal muscle hypertrophy

The potential role of the fibroblast growth factor (FGF) family during stretch-induced postnatal skeletal muscle hypertrophy was analyzed by using an avian wing-weighting model. After 2 or 11 days of weighted stretch, anterior latissimus dorsi (ALD) muscles were, on average, 34 (P < 0.01) and 85% (P < 0.01) larger, respectively, than unweighted ALD control muscles. By using quantitative RT-PCR, FGF-1 mRNA expression was found to be significantly decreased in ALD muscles stretched for 2 or 11 days. In contrast, FGF-4 and FGF-10 mRNA expression was significantly increased 2 days after initiation of stretch. FGF-2, FGF-10, fibroblast growth factor receptor 1, and FREK mRNA expression was significantly increased at 11 days poststretch. Increases in FGF-2 and FGF-4 protein could be detected throughout the myofiber periphery after 11 days of stretch. On a cellular level, FGF-2 and FGF-4 proteins were differentially localized. This differential expression pattern and protein localization of the FGF family in response to stretch-induced hypertrophy suggest distinct roles for individual FGFs during the postnatal hypertrophy process.     

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.     

Contractile properties of aged avian muscle after stretch-overload

The effects of ageing on muscle contractile adaptations to stretch-overload was examined in the anterior latissimus dorsi (ALD) muscle of 12 old (90 weeks of age) and 12 young adult (10 weeks of age) Japanese quails. A weight corresponding to 12% of the birds' body weight was attached to one wing for 30 days, while the contralateral wing served as the intra-animal control. In vitro contractile measurements were made at 25 degrees C by indirect stimulation of the ALD by its nerve (pulse 0.2 ms). Compared with young adult twitch characteristics, aged muscles had significantly greater contraction time (149 +/- 9 ms vs. 174 +/- 16 ms). Stretch-overload increased contraction time to 162 +/- 7 ms in young muscles and 215 +/- 14 ms in old muscles. Ageing and overload resulted in a greater fusing of twitches at stimulation frequencies of 5 and 10 Hz which resulted in a leftward shift of the force-frequency curve at these frequencies, relative to young adult control muscles. Maximal shortening velocity (Vmax) decreased from 2.6 +/- 0.3 to 1.2 +/- 0.1 muscle lengths/s in young muscles after overload. Vmax in old control muscles was similar to young muscles after stretch, but stretch further decreased Vmax in old muscles to 0.8 muscle lengths/s. Maximal tetanic force and specific force were similar in young and old muscles, both before and after stretch. These data indicate that ageing induces a slowing of both twitch contractile characteristics and shortening velocity in the ALD, without affecting maximal force capabilities.     

Skeletal muscle growth and expression of skeletal muscle alpha-actin mRNA and insulin-like growth factor I mRNA in pigs during feeding and withdrawal of ractopamine

Sixty crossbred barrows were used to study the effect of ractopamine (a phenethanolamine/beta-adrenergic agonist) treatment and its withdrawal on muscle growth and on the relative abundance of skeletal muscle alpha-actin (sk-alpha-actin) mRNA and of liver and longissimus muscle IGF-I mRNA at 4 wk. Ractopamine was fed (20 ppm) for periods of 2, 4, and 6 wk (six pigs per group). Additional pigs (four per group) were fed ractopamine (20 ppm) for 6 wk and then slaughtered 1, 3, and 7 d after withdrawal of ractopamine. Ractopamine increased (P < .05) longisimus muscle weight and protein content, although protein concentrations were not different. The increased muscle weight and protein content attained by feeding ractopamine for 6 wk was retained when ractopamine was withdrawn. The RNA and DNA concentrations did not change, whereas total DNA and RNA content per muscle was 18 and 26.7% greater, respectively, in ractopamine-treated pigs at 4 wk, but there were no differences at 2 or 6 wk or among the withdrawal groups. The relative abundance of sk-alpha-actin mRNA in the longissimus muscle was 41 and 62% greater (P < .05) in treated animals at 2 and 4 wk but was similar to that in controls at 6 wk and during the withdrawal period. The relative abundance of IGF-I mRNA in liver and longissimus muscle was not altered with ractopamine treatment for 4 wk. These results indicate that the ractopamine-enhanced muscle growth may result from increased myofibrillar gene expression at the pretranslational level, which is maximal with short-term treatment of ractopamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulsatile stretch stimulates vascular endothelial growth factor (VEGF) secretion by cultured rat cardiac myocytes

Evidence has accumulated that vascular endothelial growth factor (VEGF) is expressed in the heart, and its expression is markedly increased in response to hypoxia. Recently, it was shown that pulsatile myocardial stretch in vivo markedly enhanced VEGF mRNA level in the heart. To investigate whether pulsatile mechanical stretch really stimulates VEGF expression by cardiac myocytes, using an in vitro preparation, we examined the secretion of VEGF into the culture media from cardiac myocytes subjected to pulsatile stretch. We found that pulsatile mechanical stretch induced rapid secretion of VEGF by cultured rat cardiac myocytes and mRNA expression of VEGF and VEGF receptors in the cardiac myocytes. We also found that the stretch-induced secretion of VEGF was at least in part mediated by TGF-beta. These data provide the direct evidence that mechanical overload itself can induce VEGF secretion by cardiac myocytes, which may play a role in ameliorating the relative myocardial hypoxia.     

Pulsatile stretch stimulates superoxide production and activates nuclear factor-kappa B in human coronary smooth muscle

There is increasing evidence that oxidative stress is of pathophysiological importance in cardiovascular disease. Mechanical forces such as pulsatility may also contribute. Using human coronary artery smooth muscle cells (HCAS), we tested the hypothesis that stretch-induced cell proliferation is associated with oxidative stress. Stretch induced DNA synthesis in HCAS, and this was prevented by the antioxidants N-acetylcysteine and pyrrolidinedithiocarbamate (PDTC). Pulsatile stretch also increased superoxide production from HCAS in a time- and stretch dependent manner. Stretch-induced superoxide production was inhibited by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor, and p-chloromercuriphenylsulfonic acid, an NADH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. In electrophoretic mobility shift assays, tumor necrosis factor-alpha activated nuclear factor-kappa B (NF-kappa B) with a peak at approximately 3 hours, whereas pulsatile stretch showed sustained activation during stimulation for up to 24 hours. The sustained activation of NF-kappa B was abolished by cotreatment with N-acetylcysteine or PDTC. Furthermore, treatment of HCAS with antisense p65 and p50 oligodeoxynucleotides of NF-kappa B inhibited stretch-induced DNA synthesis. We propose that pulsatile stretch increases oxidative stress and, in turn, promotes DNA synthesis via NF-kappa B in cultured human coronary artery smooth muscle cells.