Kathleen M. McCormick

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 17β-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.

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‐α), 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‐α significantly improved the hypercapnic ventilatory response and diaphragm muscle maximal isometric force. Long‐term TNF‐α 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‐α influences the progressive deterioration of diaphragm muscle in mdx mice. Muscle Nerve 28: 336–343, 2003

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 17β‐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.

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.

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.

TGFβ2 activation status during cardiac morphogenesis

Transforming growth factor beta (TGFβ) is secreted as a biologically inactive complex by many cell types in vitro, but little is known of TGFβs activation status in vivo. This study examined the in vivo expression of active and total (active + acid‐activatable) TGFβ2 in embryonic chicken hearts during cardiac morphogenesis (Hamburger‐Hamilton stage 10–24). The concentration of TGFβ2 was measured by an enzyme‐linked immunoassay that recognized active TGFβ2. Whole heart homogenates were either left untreated to measure active TGFβ2 or treated with acid before assay to measure total (active + acid‐activatable) TGFβ2. Total TGFβ2 concentration increased more than 16‐fold between stage 10/11 and stage 24. Active TGFβ2 concentration was highest at stage 14/15, but overall remained relatively constant varying at most by 2.8‐fold. When expressed relative to total TGFβ2, the amount of active TGFβ2 progressively declined from 70% in stage 10/11 hearts to 7% in stage 24 hearts. The distribution of active and total TGFβ2 was examined by immunostaining with an antibody against active TGFβ2. Before immunostaining, sections were either treated with acid or left untreated to determine the distribution of total and active TGFβ2, respectively. Active TGFβ2 immunostaining was first detected in the endothelium, myocardium, and cardiac jelly of stage 14 hearts. Acid treatment had no effect on the distribution or intensity of immunostaining at this stage. Faint, active TGFβ2 immunostaining was restricted to the ventricular myocardium in stage 18 hearts. Acid treatment resulted in a marked increase in staining intensity in the ventricle, but no staining was observed in the atrium or outflow tract. In stage 24 hearts, faint active TGFβ2 staining was detected in the ventricle before acid treatment. After acid treatment, patches of intense punctate stain were found in all regions of the embryonic heart. Increases in TGFβ2 concentration and immunostaining intensity after acidification suggest that a significant amount of TGFβ2 is in the latent form. Stage‐dependent differences in activation status suggest that activation may be a developmentally regulated process in the chick heart and support the notion that activation is an important step in regulating TGFβ actions in vivo.

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.