Morphology and expression of genes related to skeletal muscle growth in juveniles of pirarucu (Arapaima gigas, Arapaimatidae, Teleostei)

Skeletal muscle growth in the pirarucu (Arapaima gigas) is highly interesting to fish farmers because it provides information about how the mechanism in muscle mass increase, characteristic of the species, is regulated. Pirarucu has specific muscle growth that highlights the species's significance and commercial value. Current research evaluates the morphology and the growth-related gene expression in the red and white skeletal muscles of the pirarucu. Muscle samples were collected from the lateral anterior region and frozen in liquid nitrogen. Histological sections were performed and stained by HE for morphological analysis. Red and white muscle samples were used to determine MyoD, myogenin, and myostatin genes expression by Real-time Polymerase Chain Reaction. Although MyoD and myogenin were not statistically different in the two types of muscles, myostatin was significantly higher in the white rather than in the red muscle. Results show the muscle growth characteristics of the species and may be helpful for improving aquaculture management programs.

Differential expression of myogenic regulatory factor MyoD in pacu skeletal muscle (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei) during juvenile and adult growth phases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Quantitative expression of myogenic regulatory factors MyoD and myogenin in pacu (Piaractus mesopotamicus) skeletal muscle during growth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MicroRNA transcriptome profiles during swine skeletal muscle development

Background: MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult. Results: Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate. Conclusion: These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

Lack of Smad3 signaling leads to impaired skeletal muscle regeneration.

Smad3 is a key intracellular signaling mediator for both transforming growth factor-β and myostatin, two major regulators of skeletal muscle growth. Previous published work has revealed pronounced muscle atrophy together with impaired satellite cell functionality in Smad3-null muscles. In the present study, we have further validated a role for Smad3 signaling in skeletal muscle regeneration. Here, we show that Smad3-null mice had incomplete recovery of muscle weight and myofiber size after muscle injury. Histological/immunohistochemical analysis suggested impaired inflammatory response and reduced number of activated myoblasts during the early stages of muscle regeneration in the tibialis anterior muscle of Smad3-null mice. Nascent myofibers formed after muscle injury were also reduced in number. Moreover, Smad3-null regenerated muscle had decreased oxidative enzyme activity and impaired mitochondrial biogenesis, evident by the downregulation of the gene encoding mitochondrial transcription factor A, a master regulator of mitochondrial biogenesis. Consistent with known Smad3 function, reduced fibrotic tissue formation was also seen in regenerated Smad3-null muscle. In conclusion, Smad3 deficiency leads to impaired muscle regeneration, which underscores an essential role of Smad3 in postnatal myogenesis. Given the negative role of myostatin during muscle regeneration, the increased expression of myostatin observed in Smad3-null muscle may contribute to the regeneration defects.

EXPRESSION OF GENES RELATED TO MYOSTATIN SIGNALING DURING RAT SKELETAL MUSCLE LONGITUDINAL GROWTH

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve 40: 992-999, 2009

Potential Role of Growth Hormone in Impairment of Insulin Signaling in Skeletal Muscle, Adipose Tissue, and Liver of Rats Chronically Treated with Arginine

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85 alpha/55 alpha determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85 alpha in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55 alpha total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect. (Endocrinology 150: 2080-2086, 2009)

Growth characteristics of skeletal muscle tissue in Oreochromis niloticus larvae fed on a lysine supplemented diet

The effect of lysine amino acid supplementation on the growth characteristics and morphological pattern of skeletal muscle tissue in Nile tilapia Oreochromis niloticus larvae was evaluated. There were four treatments (T) with increasing levels of lysine supplement (T1 = 0.0%: T2 =1.1%; T3 = 1.7%; T4 = 4.0%) and one treatment with a commercial diet (T5). In all treatments, morphological and histochemical muscle tissue analyses were similar. Two distinct layers were identified: a superficial red layer, more developed in the lateral line region, formed by fibres with intense to moderate NADH-TR reaction and strong acid-stable mATPase activity, and a deep white one, most of the Muscle mass, formed by fibres with weak NADH-TR reaction and strong alkali-stable mATPase activity. There was an intermediate layer between these two layers with fibres exhibiting either weak acid-stable or acid-labile mATPase activity. Body mass increase was significantly higher in T5 than in the lysine treatments (T1-T4). There was no difference in number and diameters of muscle fibres between lysine treatments. In T5, muscle fibre diameter and number were higher. The frequency of red fibres with diameters <= 8 mu m was higher in the lysine treatments, and with diameters between 16 and 24 mu m, was higher in T5. Most white fibre diameters in T5 were significantly larger than 24 mu m and in T1-T4 were between 8 and 16 mu m. Cell proliferation was higher in the lysine treatments and muscle growth in T5 was mainly by fibre hypertrophy. (c) 2005 the Fisheries Society of the British

Differential MMP-2 and MMP-9 Activity and Collagen Distribution in Skeletal Muscle from pacu (Piaractus mesopotamicus) During Juvenile and Adult Growth Phases

Here, we evaluated collagen distribution and matrix metalloproteinases (MMPs) MMP-2 and MMP-9 activities in skeletal muscle of pacu (Piaractus mesopotamicus) during juvenile and adult growth phases. Muscle samples from juvenile and adult fishes were processed by histochemistry for collagen system fibers and for gelatin-zymography for MMP-2 and MMP-9 activities analysis. Picrosirius staining revealed a myosept, endomysium, and perimysium-like structures in both growth phases and muscle types, with increased areas of collagen fibers in adults, mainly in red muscle. Reticulin staining showed that reticular fibers in the endomysium-like structure were thinner and discontinuous in the red muscle fibers. The zymography revealed clear bands of the pro-MMP-9, active-MMP-9, intermediate-MMP-2, and active-MMP-2 forms in red and white muscle in both growth phases. MMP-2 activity was more intense in juvenile than adult muscle fibers. Comparing the red and white muscle types, MMP-2 activity was significantly higher in red muscle in adult phase only. The activity of MMP-9 forms was similar in juvenile red and white muscles and in the adult red muscle, without any activity in adult white muscle. In conclusion, our results show that, in pacu, the higher activities of MMP-2 and -9 are associated with the rapid muscle growth in juvenile age and in adult fish, these activities are related with a different red and white muscle physiology. This study may contribute to the understanding muscle growth mechanisms and may also contribute to analyse red and the white muscle parameters of firmness and softness, respectively, of the commercial product. Anat Rec, 292:387-395, 2009. (C) 2009 Wiley-Liss, Inc.

Growth hormone attenuates skeletal muscle changes in experimental chronic heart failure

Objective: This study evaluated the effects of growth hormone (GH) on morphology and myogenic regulatory factors (MRF) gene expression in skeletal muscle of rats with ascending aortic stenosis (AAS) induced chronic heart failure.Design: Male 90-100 g Wistar rats were subjected to thoracotomy. AAS was created by placing a stainless-steel clip on the ascending aorta. Twenty five weeks after surgery, rats were treated with daily subcutaneous injections of recombinant human GH (2 mg/kg/day; AAS-GH group) or saline (AAS group) for 14 days. Sham-operated animals served as controls. Left ventricular (LV) function was assessed before and after treatment. IGF-1 serum levels were measured by ELISA. After anesthesia, soleus muscle was frozen in liquid nitrogen. Histological sections were stained with HE and picrosirius red to calculate muscle fiber cross-sectional area and collagen fractional area, respectively. MRF myogenin and MyoD expression was analyzed by reverse transcription PCR.Results: Body weight was similar between groups. AAS and AAS-GH groups presented dilated left atrium, left ventricular (LV) hypertrophy (LV mass index: Control 1.90 +/- 0.15; AAS 3.11 +/- 0.44; AAS-GH 2.94 +/- 0.47 g/kg; p < 0.05 AAS and AAS-GH vs. Control), and reduced LV posterior wall shortening velocity. Soleus muscle fiber area was significantly lower in AAS than in Control and AAS-GH groups; there was no difference between AAS-GH and Control groups. Collagen fractional area was significantly higher in MS than Control; AAS-GH did not differ from both Control and AAS groups. Serum IGF-1 levels decreased in AAS compared to Control. MyoD mRNA was significantly higher in AAS-GH than AAS; there was no difference between AAS-GH and Control groups. Myogenin mRNA levels were similar between groups.Conclusion: In rats with aortic stenosis-induced heart failure, growth hormone administration increases MyoD gene expression above non-treated animal levels, preserves muscular trophism and attenuates interstitial fibrosis. These results suggest that growth hormone may have a potential role as an adjuvant therapy for chronic heart failure. (C) 2009 Elsevier Ltd. All rights reserved.

Growth hormone influences atrophy pathways in skeletal muscle of heart failure rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of growth-related factors in skeletal muscle of Pirarucu (Arapaima gigas) during growth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Studies on regulation of skeletal muscle differentiation by growth factors

Skeletal muscle differentiation involves sequential events in which proliferating undifferentiated myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes. The process of fusion is accompanied by the disappearance of proteins associated with cell proliferation and the coordinate induction of a battery of muscle-specific gene products, which includes the muscle isoenzyme of creatine kinase, nicotinic acetylcholine receptor, and contractile proteins such as alpha-actin. The molecular events associated with myogenesis are particularly amenable to experimental analysis because the events which occur in vivo can be recapitulated in vitro using established muscle cell lines. Initiation of myogenic differentiation in vitro can be achieved by removing serum from the culture medium. Myogenesis, therefore, can be considered to be regulated through a repression-type of mechanism by components in serum. The objectives of this project were to identify the components involved in regulation of myogenesis and approach the mechanism(s) whereby these components achieve their regulatory function. Initially, the effects of a series of polypeptide growth factors on myogenesis were examined. Among them TGF$\beta$ and FGF were found to be potent inhibitors of myogenic differentiation which did not affect cell proliferation. The inhibitory effects of these growth factors on differentiation requires their persistent presence in the culture medium. After myoblasts have undergone fusion, they become refractory to the inhibitory effects of TGF$\beta$, FGF, and serum. When fusion is inhibited by the presence of EGTA, a Ca$\sp{2+}$ chelator, muscle-specific genes are expressed reversibly upon removal of inhibitory growth factors. Subsequent exposure of biochemically differentiated cells to serum or TGF$\beta$ leads to down-regulation of muscle-specific genes. Stimulation with serum also leads to reentry of myocytes into the cell cycle, whereas fused myotubes are irreversibly and terminally differentiated. Measurement of levels of TGF$\beta$ receptors reveals that under non-fusing conditions, TGF$\beta$ receptor levels in biochemically differentiated myocytes remained as high as in undifferentiated myoblasts, while during terminal differentiation, TGF$\beta$ receptors decreased at least five-fold. Thus, down-regulation of TGF$\beta$ receptors is coupled to irreversible differentiation, but not reversible differentiation in the absence of fusion. The possible involvement of second messenger systems, such as cAMP and protein kinase C, in the pathway(s) by which TGF$\beta$, FGF, or serum factors transduce their signals from the cell surface to the nucleus was also examined. The results showed that myogenic differentiation is subject to negative regulation through cAMP elevation-dependent and cAMP elevation-independent pathways and that serum mitogens, TGF$\beta$ and FGF inhibit differentiation through a mechanism independent of cAMP-elevation or protein kinase C activation. ^

Capillary growth, ultrastructure remodelling and exercise training in skeletal muscle of essential hypertensive patients

AIM The aim was to elucidate whether essential hypertension is associated with altered capillary morphology and density and to what extent exercise training can normalize these parameters. METHODS To investigate angiogenesis and capillary morphology in essential hypertension, muscle biopsies were obtained from m. vastus lateralis in subjects with essential hypertension (n = 10) and normotensive controls (n = 11) before and after 8 weeks of aerobic exercise training. Morphometry was performed after transmission electron microscopy, and protein levels of several angioregulatory factors were determined. RESULTS At baseline, capillary density and capillary-to-fibre ratio were not different between the two groups. However, the hypertensive subjects had 9% lower capillary area (12.7 ± 0.4 vs. 13.9 ± 0.2 μm(2)) and tended to have thicker capillary basement membranes (399 ± 16 vs. 358 ± 13 nm; P = 0.094) than controls. Protein expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 and thrombospondin-1 were similar in normotensive and hypertensive subjects, but tissue inhibitor of matrix metalloproteinase was 69% lower in the hypertensive group. After training, angiogenesis was evident by 15% increased capillary-to-fibre ratio in the hypertensive subjects only. Capillary area and capillary lumen area were increased by 7 and 15% in the hypertensive patients, whereas capillary basement membrane thickness was decreased by 17% (P < 0.05). VEGF expression after training was increased in both groups, whereas VEGF receptor-2 was decreased by 25% in the hypertensive patients(P < 0.05). CONCLUSION Essential hypertension is associated with decreased lumen area and a tendency for increased basement membrane thickening in capillaries of skeletal muscle. Exercise training may improve the diffusion conditions in essential hypertension by altering capillary structure and capillary number.