Estrogen effect on some enzymes in female rats after downhill running

The study investigates the effect of administered estrogen on plasma creatine kinase (CK) and lactate dehydrogenase (LD) levels in female ovariectomized rats after downhill running. Rats ovariectomized before sexual maturity were subcutaneously implanted with pellets containing 17 beta-estradiol or placebo. Three weeks later they were subjected to a 90-min intermittent downhill running protocol. Blood samples were obtained from the jugular vein immediately after and 72 h after exercise for determination of plasma CK, LD and 17 beta-estradiol levels. A two-way analysis of variance was used for data evaluation. Plasma CK and LD levels were significantly lower (p<0.05) in the estrogen-supplemented, ovariectomized animals which suggests that less muscle damage occurred compared to the controls immediately and 72 h after exercise. Estrogens may have a protective effect on muscle tissue possibly due to their antioxidant and membrane stabilizing properties.     

Electrical stimulation influences satellite cell proliferation and apoptosis in unloading-induced muscle atrophy in mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.     

Effect of training on skeletal muscle injury from downhill running in rats

Experiments were conducted to test the hypothesis that injury to skeletal muscle in rats resulting from prolonged downhill running is prevented to a greater extent by prior downhill training than by either uphill or level training. Changes in plasma creatine phosphokinase (CPK) activity and glucose-6-phosphate dehydrogenase (G-6-PDase) activity in the soleus (S), vastus intermedius (VI), and medial head of triceps brachii (TM) muscles were evaluated as markers of muscle injury 48 h after 90 min of intermittent downhill running (16 m . min -1). Prior to this acute downhill run, groups of rats were trained by either downhill (-16 degrees), level (0 degrees), or uphill (+16 degrees) running (16 m . min -1) for 30 min/day. Training duration was either 5 days or 1 day. A training effect (i.e., reduced muscle injury) was indicated if muscle G-6-PDase or plasma CPK activity in a trained group following the 90-min downhill run was not different from that of nonexercised control animals and/or if it was lower than that of nontrained runners. A significant training effect was achieved in all three muscles with 5 days of either downhill or level training, but only in S after 5 days of uphill training. Elevation of plasma CPK activity was prevented by 5 days of training on all three inclines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ground reaction forces during downhill and uphill running

We investigated the normal and parallel ground reaction forces during downhill and uphill running. Our rationale was that these force data would aid in the understanding of hill running injuries and energetics. Based on a simple spring-mass model, we hypothesized that the normal force peaks, both impact and active, would increase during downhill running and decrease during uphill running. We anticipated that the parallel braking force peaks would increase during downhill running and the parallel propulsive force peaks would increase during uphill running. But, we could not predict the magnitude of these changes. Five male and five female subjects ran at 3m/s on a force treadmill mounted on the level and on 3 degrees, 6 degrees, and 9 degrees wedges. During downhill running, normal impact force peaks and parallel braking force peaks were larger compared to the level. At -9 degrees, the normal impact force peaks increased by 54%, and the parallel braking force peaks increased by 73%. During uphill running, normal impact force peaks were smaller and parallel propulsive force peaks were larger compared to the level. At +9 degrees, normal impact force peaks were absent, and parallel propulsive peaks increased by 75%. Neither downhill nor uphill running affected normal active force peaks. Combined with previous biomechanics studies, our normal impact force data suggest that downhill running substantially increases the probability of overuse running injury. Our parallel force data provide insight into past energetic studies, which show that the metabolic cost increases during downhill running at steep angles.     

Glycogen depletion and resynthesis in the rat after downhill running

To study the effect of downhill running on glycogen metabolism, 94 rats were exercised by running for 3 h on the level or down an 18 degrees incline. Muscle and liver glycogen concentrations were measured before exercise and 0, 48 and 52 h postexercise. Rats were not fed during the first 48 h of recovery but ingested a glucose solution 48 h postexercise. Downhill running depleted glycogen in the soleus muscle and liver significantly more than level running (P less than 0.01). The amount of glycogen resynthesized in the soleus muscle and liver in fasting or nonfasting rats was not altered significantly by downhill running (P greater than 0.05). On every day of recovery the rats were injected with dexamethasone, which induced similar increases in glycogen concentration in the soleus muscle and liver after the 52nd h of the postexercise period in the case of downhill and level running. The glycogen depletion and repletion results indicated that, under our experimental conditions, downhill running in the rat, a known model of eccentric exercise, affected muscle glycogen metabolism differently from eccentric cycling in humans.     

Energy cost of walking and running at extreme uphill and downhill slopes.

The costs of walking (Cw) and running (Cr) were measured on 10 runners on a treadmill inclined between -0.45 to +0.45 at different speeds. The minimum Cw was 1.64 +/- 0.50 J. kg(-1). m(-1) at a 1.0 +/- 0.3 m/s speed on the level. It increased on positive slopes, attained 17.33 +/- 1.11 J. kg(-1). m(-1) at +0.45, and was reduced to 0.81 +/- 0.37 J. kg(-1). m(-1) at -0.10. At steeper slopes, it increased to reach 3.46 +/- 0.95 J. kg(-1). m(-1) at -0.45. Cr was 3.40 +/- 0.24 J. kg(-1). m(-1) on the level, independent of speed. It increased on positive slopes, attained 18.93 +/- 1.74 J. kg(-1). m(-1) at +0.45, and was reduced to 1.73 +/- 0.36 J. kg(-1). m(-1) at -0.20. At steeper slopes, it increased to reach 3.92 +/- 0.81 J. kg(-1). m(-1) at -0.45. The mechanical efficiencies of walking and running above +0.15 and below -0.15 attained those of concentric and eccentric muscular contraction, respectively. The optimum gradients for mountain paths approximated 0.20-0.30 for both gaits. Downhill, Cr was some 40% lower than reported in the literature for sedentary subjects. The estimated maximum running speeds on positive gradients corresponded to those adopted in uphill races; on negative gradients they were well above those attained in downhill competitions.     

Elevated small GTPase activation influences the cell proliferation signaling control in Niemann-Pick type C fibroblasts

Niemann-Pick type C (NPC) disease is characterized at the cellular level by the intracellular accumulation of free cholesterol. We have previously identified a similar phenotype in cystic fibrosis (CF) cell models that results in the activation of the small GTPase RhoA. The hypothesis of this study was that NPC cells would also exhibit an increase in small GTPase activation. An examination of the active, GTP-bound form of GTPases revealed a basal increase in the content of the active-form Ras and RhoA small GTPases in NPC fibroblasts compared to wt controls. To assess whether this increase in GTP-bound Ras and RhoA manifests a functional outcome, the expression of the proliferation control proteins p21/waf1 and cyclin D were examined. Consistent with increased GTPase signaling, p21/waf1 expression is reduced and cyclin D expression is elevated in NPC fibroblasts. Interestingly, cell growth rate is not altered in NPC fibroblasts compared to wt cells. However, NPC sensitivity to statin treatment is reversed by addition of the isoprenoid geranylgeranyl pyrophosphate (GGPP), a modifier of RhoA. It is concluded that Ras and RhoA basal activation is elevated in NPC fibroblasts and has an impact on cell survival pathways.     

Estrogen stimulation of ovarian surface epithelial cell proliferation

Summary Ovarian cancer is the leading cause of gynecological cancer mortality, and 85–90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17β-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor α demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17β-estradiol depends on the expression of the estrogen receptor α in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor α in OSE growth.     

Myostatin negatively regulates satellite cell activation and self-renewal

Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

下坡跑后大鼠腓肠肌成肌调节因子的变化

目的:探讨成肌调节因子(MyoD)在肌肉损伤修复过程中的动态表达,为促进运动肌肉损伤的再生修复提供实验依据。方法:将健康雄性2月龄SD大鼠80只,随机分为对照组(n=10)和下坡运动组(n=70),下坡运动组再分为运动后即刻组、12h、24h、48h、72h、7d和14d组,各运动组动物均进行持续性下坡跑,分别在运动结束后8个时间点麻醉,下腔静脉取血,分离血清,取双侧腓肠肌。常规检测CK、LDH的活性。采用免疫组织化学染色法以及计算机图像分析技术定量统计MyoD因子表达情况。结果:血清CK、LDH在运动后即刻显著上升,后逐渐下降至正常水平。成肌调节因子MyoD在正常骨骼肌中即有表达,各运动组大鼠腓肠肌MyoD因子表达较对照组均有增加,48h组大鼠腓肠肌MyoD免疫阳性细胞核数明显多于对照组(P0.05),后随时间逐渐下降。结论:离心运动后即刻MyoD的表达水平开始上升,48h达到峰值,随后逐渐下降至正常水平。提示成年早期大鼠(2月龄)已具备较成熟的肌肉再生修复能力。     

Glutathione influences the proliferation as well as the extent of mitochondrial activation in rat splenocytes.

The time-dependent changes of mitochondrial membrane potential and mass have been investigated on rat splenic lymphocytes stimulated with Con A in the presence and absence of reduced glutathione (GSH). Rhodamine-123 (Rh-123) and nonyl acridine orange (NAO) were used as specific dyes to monitor the membrane potential and mass of mitochondria, respectively. The percentage of cells showing blast transformation and the level of Rh-123 or NAO uptake were analyzed by flow cytometry. Present results demonstrate that a large number of cells showed activated mitochondria already at 24 hr after Con A stimulation and the activation of these organelles was not related to blast transformation. The addition of GSH into the culture medium increased the number of cells responding to mitogenic stimulation. In parallel it augmented the percentage of lymphocytes with activated mitochondria and also prevented their depolarization.     

c-Flip overexpression affects satellite cell proliferation and promotes skeletal muscle aging

This study shows that forcing c-Flip overexpression in undifferentiated skeletal myogenic cells in vivo results in early aging muscle phenotype. In the transgenic mice, adult muscle histology, histochemistry and biochemistry show strong alterations: reduction of fibers size and muscle mass, mitochondrial abnormalities, increase in protein oxidation and apoptosis markers and reduced AKT/GSK3β phosphorylation. In the infant, higher levels of Pax-7, PCNA, P-ERK and active-caspase-3 were observed, indicating enhanced proliferation and concomitant apoptosis of myogenic precursors. Increased proliferation correlated with NF-κB activation, detected as p65 phosphorylation, and with high levels of embryonic myosin heavy chain. Reduced regenerative potential after muscle damage in the adult and impaired fiber growth associated with reduced NFATc2 activation in the infant were also observed, indicating that the satellite cell pool is prematurely compromised. Altogether, these data show a role for c-Flip in modulating skeletal muscle phenotype by affecting the proliferative potential of undifferentiated cells. This finding indicates a novel additional mechanism through which c-Flip might possibly control tissue remodeling.     

Angiotensins II and IV modulate adrenocortical cell proliferation in ovariectomized rats.

Effects of angiotensins II (AngII), angiotensin IV (AngIV, 3-8 fragment of angiotensin II) and losartan (an antagonist of angiotensin receptor type 1) on the proliferation of adrenocortical cells in ovariectomized rats have been studied. The incorporation of bromodeoxyuridine (BrdU) into cell nuclei was used as an index of cell proliferation. AngIV decreased BrdU labeling index mainly in the reticularis zone and losartan (Los) was able to partially reverse this inhibitory effect of AngIV. AngII had no effect on the adrenocortical cell proliferation when given alone, however Los given simultaneously diminished BrdU incorporation into nuclei of glomerulosa and reticularis zones as compared with AngII. These findings suggest that AngII and AngIV modulate adrenocortical cell proliferation in ovariectomized rats.     

An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats

Summary Satellite cells were traced autoradiographically during the regeneration of skeletal muscle in young Sprague-Dawley rats. Approximately 31% of the satellite cells in uninjured muscles appeared labelled after three injections of tritiated thymidine; none of the myonuclei were labelled in the same muscles. Four to six days after transplanting the radioactive muscles to non-radioactive littermates, regenerating myotube nuclei in the host appeared labelled. Thus, this study confirms that satellite cells in young rats can differentiate into multinucleated myotubes following muscle injury.Supported by NIH grant No. 5 S01-RR05356-13I wish to acknowledge the excellent technical assistance of Ms. Amy Erisman     

Characteristics of B cell proliferation and activation in murine AIDS

A syndrome characterized by lymphadenopathy, hypergammaglobulinemia, and immunodeficiency develops in C57BL/6 mice inoculated with LP-BM5 murine leukemia viruses. By studying the number and antigenic specificity of B cells activated in the course of this disease, we found that a series of reproducible changes in the humoral immune system were induced by retroviral infection. The rate of B cell proliferation and the proportion of B cells activated to secrete Ig increased by nearly 10-fold at 4 wk post inoculation. B cells producing antibodies reactive with a panel of three conventional Ag and five autoantigens were stimulated simultaneously and proportionally to secrete, demonstrating that such activation was polyclonal in nature. At 12 wk post infection, the number of Ig-secreting B cells continued to rise and significant hypergammaglobulinemia developed. At 16 wk post infection, immunostimulation gave way to immunosuppression, as evidenced by a slight decline in the number of Ig-secreting lymphocytes and a sharp reduction in the concentration of serum antibody. At this time, the B cell repertoires of infected mice diverged markedly from those of uninfected animals. These changes are comparable to those found in some patients infected with HIV, and provide a useful model to study the association between retroviral infection and regulatory abnormalities of the humoral immune system.     

The turkey myogenic satellite cell: optimization of in vitro proliferation and differentiation

Myogenic satellite cells were isolated from the pectoralis major muscle of young growing tom turkeys. These cells were capable of proliferating and forming large multinucleated myotubes in vitro. Of 36 media-sera combinations evaluated, McCoy's 5A medium containing 15% chicken serum (CS) promoted the greatest level of proliferation and subsequent myotube formation when cells were induced to differentiate (P less than 0.05). Myotube formation was maximized following exposure of cultures to Dulbecco's Modified Eagle's Medium (DMEM) containing 1% horse serum (HS; DMEM-1% HS) for 4 days. Satellite cells grown under these conditions generally resulted in cultures containing greater than 90% fused nuclei. Cells plated in the presence of DMEM-10% HS resulted in greater attachment and larger cultures (and consequently a greater fused nuclei number) when transferred to growth media than similarly grown cultures plated in McCoy's 5A medium-10% CS, regardless of substrata tested (P less than 0.05). The greatest proliferation and myotube formation was seen in cultures grown in gelatin-coated wells. Proliferation was maximized in McCoy's 5A medium containing 18% CS, although this was not significantly different than the proliferation with media containing 15% CS (P greater than 0.05). Our results (1) document that the postnatal myogenic satellite cell can be isolated from the turkey in sufficient quantities for biological studies and (2) identify culture conditions which optimize proliferation and differentiation of these cells in vitro.