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

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

Effects of temporal muscle detachment and coronoidotomy on facial growth in young rats

This study analyzed the effects of unilateral detachment of the temporal muscle and coronoidotomy on facial growth in young rats. Thirty one-month-old Wistar rats were distributed into three groups: detachment, coronoidotomy and sham-operated. Under general anesthesia, unilateral detachment of the temporal muscle was performed for the detachment group, unilateral coronoidotomy was performed for the coronoidotomy group, and only surgical access was performed for the sham-operated group. The animals were sacrificed at three months of age. Their soft tissues were removed, and the mandible was disarticulated. Radiographic projections-axial views of the skulls and lateral views of hemimandibles-were taken. Cephalometric evaluations were performed, and the values obtained were submitted to statistical analyses. There was a significant homolateral difference in the length of the premaxilla, height of the mandibular ramus and body, and the length of the mandible in all three groups. However, comparisons among the groups revealed no significant differences between the detachment and coronoidotomy groups for most measurements. It was concluded that both experimental detachment of the temporal muscle and coronoidotomy during the growth period in rats induced asymmetry of the mandible and affected the premaxilla.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Growth, carcass characteristics, chemical composition and fatty acid profile of the longissimus dorsi muscle in goat kids fed diets with castor oil

The objective in this study was to determine growth, carcass characteristics, chemical composition and fatty acid profile of the longissimus dorsi of crossbred Boer x Saanen kids fed castor oil. Twenty-four kids (12 males and 12 females) were assigned in a randomized complete block design with two treatments and twelve replications. Blocks were defined according to weight, gender and initial age of animals for the evaluation of performance. The experimental treatments consisted of two diets containing 900 g concentrate/kg: a control diet (without addition of oil) and another containing castor oil at 30 g/kg (on a dry matter basis). After they reached an average body weight of 25 kg, males were slaughtered for the evaluation of carcass characteristics, chemical composition and fatty acid profile of the longissimus dorsi muscle. The addition of castor oil in the diet did not affect the intake of dry matter, crude protein and neutral detergent fiber; the average daily gain; and feed conversion, but increased the ether extract intake. No difference was observed for the carcass characteristics, chemical composition of the meat, concentration of C18:2 cis-9, trans-11 (CLA) and total concentration of saturated, monounsaturated and polyunsaturated fatty acids and their relations; however, there was increase in the concentrations of C18:2 trans-10, cis-12 (CLA) and C20:4 omega-6. The addition of castor oil to the diet of crossbred Boer x Saanen kids containing a high content of concentrate did not promote benefit to the characteristics evaluated.

Local pain and spreading hyperalgesia induced by intramuscular injection of nerve growth factor are not reduced by local anesthesia of the muscle

Injections with local anesthesia for therapeutic and diagnostic purposes are common clinical practice. This double-blind placebo controlled study explores the rational of local anesthetic blocks for the detection of muscle pain as the primary generator in spreading hyperalgesic conditions.

Effects of Initial Fat Thickness, Hip Height, and Concentration of Dietary Energy on Growth of Area of the Longissimus dorsi Muscle and Subcutaneous Fat of Yearling Steers

Steers were sorted into four groups based on hip height and fat cover at the start of the finishing period. Each group of sorted steers was fed diets containing 0.59 or 0.64 Mcal NEg per lb. of diet dry matter. Steers with less initial fat cover (0.08 in.) compared with those with more (0.17) had less carcass fat cover 103 days later. The steers with less fat cover accumulated fat at a faster rate, but this was not apparent prior to 80 days. Accretion of fat was best predicted by an exponential growth equation, and was not affected by the two concentrations of energy fed in this study. Steers with greater initial height accumulated fat cover at a slower rate than shorter steers. This difference was interpreted to mean that large-frame steers accumulate subcutaneous fat at a slower rate than medium-frame steers. Increase in area of the ribeye was best described by a linear equation. Initial fat cover, hip height, and concentrations of energy in the diet did not affect rate of growth of this muscle. Predicting carcass fat cover from the initial ultrasound measurement of fat thickness found 46 of the 51 carcasses with less than 0.4 in. of fat cover. Twelve carcasses predicted to have less than 0.4 in. of fat cover had more than 0.4 in. Five carcasses predicted to have more than 0.4 in. actually had less than that. Accurate initial measurements of initial fat thickness with ultrasound might be a useful measurement to sort cattle for specific marketing grids.

Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

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.

Modulation of replicative senescence of skeletal muscle satellite cells by insulin -like growth factor-I (IGF-I)

Growth and regeneration of postnatal skeletal muscle requires a population of mononuclear myogenic cells, called satellite cells to add/replace myonuclei, which are postmitotic. Wedged between the sarcolemma and the basal lamina of the skeletal muscle fiber, these cells function as the stem cells of mature muscle fibers. Like other normal diploid cells, satellite cells undergo cellular senescence. Investigations of aging in both rodents and humans have shown that satellite cell self-renewal capacity decreases with advanced age. As a consequence, this could be a potential reason for the characteristically observed age-associated loss in skeletal muscle mass (sarcopenia). This provided the rationale that any intervention that can further increase the proliferative capacity of these cells should potentially be able to either delay, or even prevent sarcopenia. ^ Using clonogenicity assays to determine a cell's proliferation potential, these studies have shown that IGF-I enhances the doubling potential of satellite cells from aged rodents. Using a transgenic model, where the mice express the IGF-I transgene specifically in their striated muscles, some of the underlying biochemical mechanisms for the observed increase in replicative life span were delineated. These studies have revealed that IGF-I activates the PI3/Akt pathway to mediate downregulation of p27KIP1, which consequently is associated with an increase in cyclin E-cdk2 kinase activity, phosphorylation of pRb, and upregulation of cyclin A protein. However, the beneficial effects of IGF-I on satellite cell proliferative potential appears to be limited as chronic overexpression of IGF-I in skeletal muscles did not protect against sarcopenia in 18-mo old mice, and was associated with an exhaustion of satellite cell replicative reserves. ^ These results have shown that replicative senescence can be modulated by environmental factors using skeletal muscle satellite cells as a model system. A better understanding of the molecular basis for enhancement of proliferative capacity by IGF-I will provide a rational basis for developing more effective counter-measures against physical frailty. However, the implications of these studies are that these beneficial effects of enhanced proliferative potential by IGF-I may only be over a short-term period, and other alternative approaches may need to be considered. ^

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development.

Factors that regulate cellular migration during embryonic development are essential for tissue and organ morphogenesis. Scatter factor/hepatocyte growth factor (SF/HGF) can stimulate motogenic and morphogenetic activities in cultured epithelial cells expressing the Met tyrosine kinase receptor and is essential for development; however, the precise physiological role of SF/HGF is incompletely understood. Here we provide functional evidence that inappropriate expression of SF/HGF in transgenic mice influences the development of two distinct migratory cell lineages, resulting in ectopic skeletal muscle formation and melanosis in the central nervous system, and patterned hyperpigmentation of the skin. Committed TRP-2 positive melanoblasts were found to be situated aberrantly within defined regions of the transgenic embryo, including the neural tube, which overproduced SF/RGF. Our data strongly suggest that SF/HGF possesses physiologically relevant scatter activity, and functions as a true morphogenetic factor by regulating migration and/or differentiation of select populations of premyogenic and neural crest cells during normal mammalian embryogenesis.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line.

In the present study we used the mutant muscle cell line NFB4 to study the balance between proliferation and myogenic differentiation. We show that removal of serum, which induced the parental C2C12 cells to withdraw from the cell cycle and differentiate, had little effect on NFB4 cells. Gene products characteristic of the proliferation state, such as c-Jun, continued to accumulate in the mutant cells in low serum, whereas those involved in differentiation, like myogenin, insulin-like growth factor II (IGF-II), and IGF-binding protein 5 (IGFBP-5) were undetectable. Moreover, NFB4 cells displayed a unique pattern of tyrosine phosphorylated proteins, especially in low serum, suggesting that the signal transduction pathway(s) that controls differentiation is not properly regulated in these cells. Treatment of NFB4 cells with exogenous IGF-I or IGF-II at concentrations shown to promote myogenic differentiation in wild-type cells resulted in activation of myogenin but not MyoD gene expression, secretion of IG-FBP-5, changes in tyrosine phosphorylation, and enhanced myogenic differentiation. Similarly, transfection of myogenin expression constructs also enhanced differentiation and resulted in activation of IGF-II expression, showing that myogenin and IGF-II cross-activate each other's expression. However, in both cases, the expression of Jun mRNA remained elevated, suggesting that IGFs and myogenin cannot overcome all aspects of the block to differentiation in NFB4 cells.