Estrogen effects on skeletal muscle insulin-like growth factor 1 and myostatin in ovariectomized rats.

Previous work showed that estrogen replacement attenuates muscle growth in immature rats. The present study examined muscle insulin-like growth factor-1 (IGF-1) and myostatin expression to determine whether these growth regulators might be involved in mediating estrogen's effects on muscle growth. IGF-1 and myostatin message and protein expression in selected skeletal muscles from 7-week-old sham-ovariectomized (SHAM) and ovariectomized rats that received continuous estrogen (OVX/E2) or solvent vehicle (OVX/CO) from an implant for 1 week or 5 weeks was measured. In the 1-week study, ovariectomy increased IGF-1 mRNA expression in fast extensor digitorum longus and gastrocnemius muscles; the increase was reversed by estrogen replacement. A similar trend was observed in the slow soleus muscle, although the change was not statistically significant. In contrast to mRNA, muscle IGF-1 protein expression was not different between SHAM and OVX/ CO animals in the 1-week study. One week of estrogen replacement significantly decreased IGF-1 protein level in all muscles examined. Myostatin mRNA expression was not different among the 1-week treatment groups. One week of estrogen replacement significantly increased myostatin protein in the slow soleus muscle but not the fast extensor digitorum longus and gastrocnemius muscles. There was no treatment effect on IGF-1 and myostatin expression in the 5-week study; this finding suggested a transient estrogen effect or upregulation of a compensatory mechanism to counteract the estrogen effect observed at the earlier time point. This investigation is the first to explore ovariectomy and estrogen effects on skeletal muscle IGF-1 and myostatin expression. Results suggest that reduced levels of muscle IGF-1 protein may mediate estrogen's effect on growth in immature, ovariectomized rats. Increased levels of muscle myostatin protein may also have a role in mediating estrogen's effects on growth in slow but not fast skeletal muscle.

Basal, but not overload-induced, myonuclear addition is attenuated by NG-nitro-L-arginine methyl ester (L-NAME) administration.

The purpose of this study was to examine the effect of blocking nitric oxide synthase (NOS) activity via NG-nitro-L-arginine methyl ester (L-NAME) on myonuclear addition in skeletal muscle under basal and overloaded conditions. Female Sprague-Dawley rats (approx. 220 g) were placed into 1 of the following 4 groups (n = 7-9/group): 7-day skeletal muscle overload (O), sham operation (S), skeletal muscle overload with L-NAME treatment (OLN), and sham operation with L-NAME treatment (SLN). Plantaris muscles were overloaded via bilateral surgical ablation of the gastrocnemius muscles and L-NAME (0.75 mg/mL) was administered in the animals' daily drinking water starting 2 days prior to surgery and continued until sacrifice. Myonuclear addition was assessed as subsarcolemmal incorporation of nuclei labeled with 5-bromo-2'-deoxyuridine (approx. 25 mg.(kg body mass)-1.day-1) delivered via osmotic pump during the overload period. As expected, muscle wet mass, total protein content, fiber cross-sectional area, and myonuclear addition were significantly higher (p

Episodic hypoxia exacerbates respiratory muscle dysfunction in DMD(mdx) mice.

Many patients with Duchenne muscular dystrophy (DMD) are eventually diagnosed with sleep-disordered breathing (SDB). SDB is associated with reduced ventilation, decreased arterial oxygen tension, and increased respiratory muscle recruitment during sleep, factors that could be especially detrimental to respiratory muscles in DMD. To assess whether SDB impacts dystrophin-deficient respiratory muscle function and fibrosis, diaphragm strength, and collagen content were evaluated in dystrophic mice (Dmd(mdx)) exposed to experimental SDB. Diurnal exposure to episodic hypoxia resulted in a 30% reduction in diaphragm strength without affecting collagen content. Episodic hypoxia secondary to SDB can exacerbate respiratory muscle dysfunction in DMD.

Satellite cells express distinct patterns of myogenic proteins in immature skeletal muscle.

Satellite cells are the myogenic cells lying between the myofiber sarcolemma and basal lamina. The objective of this study was to determine the expression patterns of MyoD, myogenin, and Pax7 within the satellite cell population in the growing rat soleus and extensor digitorum longus (EDL) muscles. Secondly, the expression of the myogenic markers was also studied within the interstitial cell compartment and myonuclei. It was discovered that the soleus contained a higher number of Pax7, MyoD, or myogenin-positive nuclei compared with the EDL. Similarly, myogenin was expressed at a lower level in the myonuclei of the soleus compared with the EDL, and myogenin was expressed at a higher level in the interstitial compartment of the soleus compared with the EDL. When interstitial nuclei, myonuclei, and double-labeled nuclei were used in the estimate of the satellite cell population, it was discovered that approximately of 13% of the myofibers in a transverse section of the soleus muscle and 4.1% of EDL myofibers exhibit a labeled satellite cell nucleus. Overall, results from this study suggest that expression patterns of these markers vary predictably among muscles with different growth dynamics and phenotypic characteristics.

Effect of oestrogen on myofibre size and myosin expression in growing rats.

This study examined the effect of oestrogen deprivation and replacement on plantaris muscle size and myosin heavy chain (MHC) isoform composition in rats during a period of physiological growth. Seven-week-old female Sprague-Dawley rats were assigned to one of the three treatment groups: (1) control animals (Sham); (2) ovariectomized animals without oestrogen replacement (OVX/CO); and (3) ovariectomized animals with 17beta-oestradiol replacement (OVX/E2). OVX/CO and OVX/E2 animals were pair-fed with Sham animals to rule out the potentially confounding effects of differences in food intake and weight gain. Rats were killed 4 weeks after surgery and the plantaris muscle was removed for analysis. Ovariectomy had no effect on muscle fibre size, but reduced the relative amount of type IIx MHC. This was reversed with oestrogen replacement, suggesting that the reduction in type IIx MHC expression was an oestrogen-mediated effect. Oestrogen replacement reduced type IIb MHC expression and fast muscle fibre size. Changes in fast fibre size and type IIb MHC expression were not seen with ovariectomy, indicating that these changes were not simply due to the presence of oestrogen in the ovariectomized, oestrogen-replaced animals. These results suggest that another ovarian hormone may counteract the effect of oestrogen on fast fibre size and type IIb MHC expression in intact animals.

Effects of ovariectomy and estrogen on skeletal muscle function in growing rats.

This study examined the effect of estrogen replacement on soleus muscle size and contractile function in ovariectomized rats during physiological growth. Seven week old female Sprague-Dawley rats were assigned to one of three treatment groups: (1) control animals (SHAM), (2) ovariectomized animals without estrogen replacement (OVX/CO), and (3) ovariectomized animals with 17 beta-estradiol replacement (OVX/E2). OVX/CO and OVX/E2 animals were pair-fed to SHAM animals to rule out the potentially confounding effect of differences in food intake. Rats were sacrificed 4 weeks after surgery and the soleus muscle was removed for analysis. Estrogen replacement reduced body weight, relative body weight gain, and soleus muscle fiber size despite all groups having a similar food intake. Ovariectomy alone had no effect on any of these parameters suggesting that estrogen may inhibit skeletal muscle growth when it is the only ovarian hormone present. Neither ovariectomy nor estrogen replacement affected maximal specific isometric force. Estrogen replacement increased half relaxation time. Ovariectomy resulted in a reduction in time to peak tension that was reversed with estrogen replacement. This reduction was not accompanied by a change in myosin heavy chain composition implying that calcium handling may have been altered. Results from this study suggest that estrogen affects skeletal muscle growth and twitch kinetics.

Continuous myofiber remodeling in uninjured extraocular myofibers: myonuclear turnover and evidence for apoptosis.

Unlike normal mature limb skeletal muscles, in which satellite cells are quiescent unless the muscle is injured, satellite cells in mammalian adult extraocular muscles (EOM) are chronically activated. This is evidenced by hepatocyte growth factor, the myogenic regulatory factor, Pax-7, and the cell-cycle marker, Ki-67, localized to the satellite cell position using serial sections and the positional markers laminin and dystrophin. Bromodeoxyuridine (brdU) labeling combined with dystrophin immunostaining showed brdU-positive myonuclei, presumably the result of fusion of activated satellite cells into existing myofibers. One new myonucleus was added to every 1000 myofibers in cross-section using a 12-hour brdU-labeling paradigm. The EOM thus appear to retain a stable nuclear population by an opposing process of apoptosis that results in myonuclear removal as visualized by terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL). Activated caspase-3 was present in localized cytoplasmic domains extending from 10 to 210 microm within individual myofibers, suggesting segmental cytoplasmic reorganization. Understanding the cellular mechanisms that maintain this process of continuous myonuclear addition and removal in normal adult EOM may suggest new hypotheses to explain the preferential involvement or sparing of these muscles in skeletal muscle disease.

Contraction-induced injury run amok: an introduction.

Skeletal muscle has an amazing capacity to adapt to increased levels of physical activity. Adaptation is often preceded by contraction-induced injury. In most cases, the damage is repaired quickly, the muscle adapts, and becomes stronger and less fatigable. Diseased or deconditioned muscle is an exception; the response to increased functional demand, and the associated injury can be incomplete or even maladaptive. When and why is an adaptive response limited? This question will be addressed in the symposium papers following this brief introduction. The papers will discuss cellular, molecular, and immunological mechanisms that may be involved in impaired muscle adaptation.

Targeting the immune system to improve ventilatory function in muscular dystrophy.

Skeletal muscle is a unique tissue whose function is dependent in great part on its ultrastructure. Repeated intense muscular contractions, especially those resulting in muscle lengthening, can lead to alterations in muscle structure (i.e., muscle damage) and subsequent decline in contractile force. The damage-induced decline in contractile force can have a significant impact on exercise performance during an athletic performance. In some disease conditions such as Duchenne muscular dystrophy (DMD), the muscles are more vulnerable to contraction-induced damage than normal muscle. In the case of the respiratory muscles, for example, the diaphragm, the consequences of muscle weakness secondary to damage are profound in that respiratory failure leading to premature death often ensues. In normal skeletal muscle, damage is followed by an inflammatory response involving multiple cell types that subsides after several days. This transient inflammatory response is a normal homeostatic reaction to muscle damage. In contrast, a persistent inflammatory response is observed in dystrophic skeletal muscle that leads to an altered extracellular environment, including an increased presence of inflammatory cells (e.g., macrophages) and elevated levels of various inflammatory cytokines (e.g., TNF-alpha, TGF-beta). The signals that lead to successful muscle repair in healthy muscle may promote muscle wasting and fibrosis in dystrophic muscle. Preliminary data indicate that immunosuppression in dystrophic (mdx) mice has beneficial effects on some indices of muscle dysfunction, thereby indicating that targeted immunosuppression may offer some promise in delaying the pathological progression of this insidious muscular disease.

Ventilatory dysfunction in mdx mice: impact of tumor necrosis factor-alpha deletion.

Muscular dystrophy is associated with inflammation and fiber necrosis in the diaphragm that may alter ventilatory function. The purpose of this study was to determine to what extent in vivo ventilatory function in dystrophic (mdx) mice was compromised and to assess the impact of deletion of tumor necrosis factor-alpha (TNF-alpha), a known proinflammatory cytokine, on ventilatory function, diaphragm contractility, and myosin heavy chain (MHC) distribution in 10-12-month-old mdx mice. Although the resting ventilatory pattern did not significantly differ between control and mdx mice, the ventilatory response to hypercapnia in mdx mice was significantly attenuated. Elimination of TNF-alpha significantly improved the hypercapnic ventilatory response and diaphragm muscle maximal isometric force. Long-term TNF-alpha deletion also altered the myosin heavy chain isoform profile of the diaphragm. These data indicate that a blunted ventilatory response to hypercapnia exists in mdx mice, and that TNF-alpha influences the progressive deterioration of diaphragm muscle in mdx mice.

High-efficiency adenovirus-mediated in vivo gene transfer into neonatal and adult rodent skeletal muscle.

Several methodological limitations have emerged in the use of viral gene transfer into skeletal muscle. First, because the nuclei of mature muscle fibers do not undergo division, the use of strategies involving replicative integration of exogenous DNA is greatly limited. Another important limitation concerns the maturation-dependent loss in muscle fiber infectivity with adenoviral vectors. In this study, we investigated the possibility that high-titer infections with recombinant adenovirus, expressing a foreign marker gene under the control of a strong viral promoter, can significantly improve the efficiency of gene transfer in vivo into neonatal and adult rat skeletal muscle. High-titer (2 x 10(10) plaque forming units) intramuscular injection of replication-defective adenovirus vector, expressing green fluorescent protein (GFP) under the control of cytomegalovirus promoter, resulted in GFP expression in 99 +/- 0.34% of fibers in the adult soleus muscle and in approximately 85 +/- 1.44% of fibers in the adult tibialis anterior muscle. Interestingly, reduction in injected adenoviral dose significantly reduced the number of GFP-positive fibers in the adult tibialis anterior muscle, but not in the soleus muscle. However, in neonates, adenoviral infection resulted in GFP expression in 96-99% of the fibers in the tibialis anterior and the gastrocnemius muscles regardless of administered adenoviral dose.