Browsing by Author "Ward, Christopher W."
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- The activity and content of calpains in maturing dystrophic muscle membranesWang, Qiong (Virginia Tech, 2005-05-11)Increased calcium-activated calpain proteolysis in the sarcolemma membrane is thought to be a primary mechanism in the pathophysiology of Duchenne Muscular Dystrophy (DMD). However, few studies have tested this possibility prior to the overt signs of the dystrophy. The purpose of this study was to test the hypothesis that there is greater calpain content and total relative calpain activity in membranes obtained from dystrophic (mdx; mdx:utrophin-deficient (mdx:utrn-/-)) compared to wildtype (wt) mouse skeletal muscles during maturation at ages 7- and 21-d,and at a post-maturation age of 35-d. Calpain activity was determined as the calcium-dependent cleavage of the flurogenic substrate SLY-AMC, and content was determined by Western analysis with an anti-calpain antibody. There were several intriguing findings: 1. There was an inverse relationship between calpain content and relative activity in the whole muscle in both wt and mdx mice from age 7- to 35-d: calpain content decreased, and relative calpain activity increased as the mice aged. This suggests a similar role for calpain in both genotypes, which might relate to specific maturation processes, possibly up to age 21-d. Although the inverse relation was evident at 35-d, the targets for calpain in mdx compared to wt likely differed. 2. The increased relative calpain activity in the membrane fraction of mdx mice at age 35-d (26.73 Arbitrary Units, (AU)) compared to that of age 7- (4.9AU; p<0.05) and 21-d (8.74AU; p<0.05) is temporally related to degeneration and regeneration processes, and may also indicate activation of apoptosis, in mdx muscles at this age. 3. At age 7-d, there were no significant differences in either calpain content or relative calpain activity in all subcellular fractions for wt and mdx mice. This result might suggest similar calpain distribution and activities that are related to the regulation of muscle maturation and differentiation in both genotypes. (Note:data were not obtained for the mdx:utrn-/- mice at age 7-d because of insufficient animals). 4. At age 21-d, there was greater relative calpain activity in the myofibrillar supernatant fraction in mdx (15.13AU) than wt mice (1.18AU; p<0.05). This could indicate calpain's role in the initiation of myofibrillar protein turnover and the proteolysis of submembranous networks in the mdx muscles. 5. At age 21-d, greater calpain content in the mdx (1.40ìg) compared to wt (0.23 ìg; p<0.05) membrane fraction might suggest a broader distribution of calpain along membranes that contributes to the onset of dystrophy in the mdx muscles. 6. At age 35-d, there was greater calpain content in the mdx:utrn-/- compared to the wt membrane (0.48ìg vs 0.13 ìg), cytosolic (0.88ìg vs 0.30ìg), and myofibrillar supernatant (0.49ìg vs 0.17ìg; p<0.05 ) fractions This increased content and broad distribution across several subcellular fractions may reflect degeneration and regeneration processes, and potentially activation of apoptosis, in the mdx:utrn-/- muscles. These data suggest that calpain activity contributes to dystrophic pathophysiology mainly in the membrane fraction of mdx skeletal muscles at age ~21-d, but appears to contribute later at 35-d and in more subcellular fractions in mdx:utrn-/- skeletal muscles.
- Cardioascular Responses to Exercise: an Evaluation of the Effectiveness of a Brief Exposure to Cpap in Obstructive Sleep Apnea PatientsWalker, Eric III (Virginia Tech, 1997-08-01)In order to clarify the effects of a single night of CPAP titration on various cardiovascular, gas exchange, and perceptual measures, we conducted submaximal ramping exercise tests to an intensity of ~75% of the heart rate reserve in five male subjects. Means and standard deviation for their age and BMI were 57.0±14.7 years and 30.5±7.2, respectively. The baseline exercise test was administered immediately after the patients arose from bed, following an overnight PSG diagnostic evaluation. The exercise test was repeated within ~2 weeks of completion of an overnight CPAP evaluation trial. Patients reported experiencing improved sleep quality (50%) after the CPAP titration, based on comparison of morning questionnaire responses from the diagnostic PSG vs. CPAP titration. Statistical significance was not attained (p>0.05) upon analysis of the following parameters at 60% of the individuals maximum workload although there were changes in the mean values of the variables from the diagnostic PSG vs CPAP titration. The following changes were noted: heart rate increased by 6%, systolic blood pressure decreased by 6%, and the rate pressure product decreased by 5.8%. Respiratory variables changed as follows: VO2 decreased by 5.3% and VE decreased by 8.5%. The perceptual measure rate of perceived exertion (RPE) decreased by 17.5%. These preliminary findings demonstrate that self-reports of sleep quality in patients with diagnosed OSA improved after a single night of CPAP titration, even in a setting wherein the total time of CPAP sleep and reduction of apneas, hypopneas, and hypoxemic episodes are highly variable. Additionally, sleep structure revealed a marked increase in slow wave (53.2%) and REM (30.4%) sleep with CPAP titration in comparison to the diagnostic PSG. It was concluded that CPAP titration effectively improves sleep structure and patient ratings of sleep quality, but does not have significant effects on cardiorespiratory responses to submaximal endurance exercise.
- The effect of fatigue on the caffeine sensitivity of skeletal muscle sarcoplasmic reticulumWard, Christopher W. (Virginia Tech, 1993)Several studies have shown that the loss in tension during fatigue can be virtually reversed by exposure of the muscle to agents which evoke Ca²⁺ release from the SR. The purpose of this study was to determine whether the SR Ca²⁺ release mechanism of fatigued muscle is less sensitive to caffeine than that of rested muscle. Following a fatigue bout of electrically evoked tetanic pulses, the functioning of the SR of chemically skinned muscle fibers was determined by the sensitivity of the SR to increasing concentrations of caffeine. Measurements of tension and rate of tension development were made at the maximal Ca²⁺ activated contracture(pCa4.5), the maximal caffeine(25mM) activated contracture and at the caffeine threshold for contraction. All tension and rate values were normalized per cross sectional area and expressed as percents of the maximal calcium activated values. Results of the maximal Ca²⁺ and caffeine data suggest that the both control and fatigue fibers are similar in maximal tension and Ca²⁺ loading characteristics. While no differences were found between rested or fatigued maximal Ca²⁺ or caffeine contractures, significant difference was found at the caffeine threshold (p<.05) with the fatigued muscle tending to contract at a higher caffeine concentration. This suggests that fatigued muscle is less sensitive to the caffeine stimulus for Ca²⁺ release from the SR.
- The Effects of Lactate on Whole Muscle Function and Sarcoplasmic Reticulum FunctionSpangenburg, Espen E. (Virginia Tech, 1997-04-24)Numerous studies have attributed the decrease in force production of skeletal muscle during exercise to a increase in lactate concentration ([lactate]). This notion is based on the high negative correlation between plasma lactate and force during fatigue and recovery. These experiments attempted to determine if lactate directly effects force production in skeletal muscle. Mouse extensor digitorum longus muscles (EDL) were isolated and incubated in a buffered Ringers solution at a pH 7.2 and exposed at three minute intervals to a final concentration of 10, 20, 30, 50mM lactate. At 21° C, tetanic force production (Po, 250ms, 110Hz) decreased to 99.3 ± 1.0, 97.1 ± 1.2, 94.9 ± 1.1* and 93.1 ± 1.3*% of initial and the rate of force development (+dP/dt) was reduced to 99.4 ± 0.7, 96.8 ± 0.5, 93.5 ± 0.6*, and 89.3 ± 1.2*% of initial (*p<0.05 vs untreated muscles). At 37° C the effects of lactate were augmented. Po was reduced to 89.7 ± 1.1, 81.0 ± 2.4, 73 ± 3.9*, and 61.6 ± 5.4*% and +dP/dt was reduced to 79.4 ± 1.8*, 65.9 ± 2.8*, 55.4 ± 4.0*, and 44.3 ± 5.0*% of initial (*p<0.05 vs control muscles). The next phase was to determine if the changes in Po and +dP/dt were due to alterations in the sacroplasmic reticulum (SR) Ca2+ exchange. The SR of EDL homogenates were actively loaded with Ca2+ and release was initiated by 25 mM AgNO3. The rate of Ca2+ release was significantly reduced by 31% (2.48 ± 1.21 vs 1.72 ± 0.24 mmol·mg-1·min-1) in the presence of 25 mM lactate. These results indicate that exposure to increased [lactate], independent of the H+, decreases force production of whole muscle, effects that are greater at 37° C than 21° C. Also increased lactate reduces the rate of SR Ca2+ release. These results suggest that lactate depresses whole muscle force production by altering Ca2+ release of the SR. They also support the idea that increased lactate concentrations disrupt normal muscle function leading to the development of fatigue.
- Effects of Reduced Muscle Glycogen on Sarcoplasmic Reticulum (SR), Muscle and Exercise PerformanceBatts, Timothy W. (Virginia Tech, 2002-04-19)Fatigue during exercise is associated with reduced muscle glycogen. However, evidence linking glycogen content to fatigue is lacking. In this study we examined whether reduced muscle glycogen content limited SR function or muscle performance. Two groups of female Sprague-Dawley rats were fasted for 24 hr and exercised for 90 min to reduce muscle glycogen; rats fasted after exercise formed the low glycogen (LG) group. Rats in the high glycogen (HG) group were allowed free access to food and a 5% sucrose solution. The LG group had 42% less muscle glycogen and 90% less glycogen associated with the sarcoplasmic reticulum (SR) than the HG group. Notably, time to exhaustion during a subsequent treadmill run (21 m/min at 10% grade) was markedly lower in the LG group (35 vs. 166.75 min). Despite less glycogen, the LG group had a higher SR Ca2+ uptake rate (45%) and Ca2+-stimulated ATPase activity (51%) possibly due to a 33% greater SERCA content. Surprisingly, in situ gastrocnemius initial twitch and tetanic forces were not different between groups although the rates of relaxation were higher in the LG group. The force responses to fatigue-inducing stimulus trains (20 Hz for 333 ms every 1 sec for 30 min) also were similar for both groups as were twitch and tetanic forces in the fatigued state. These results suggest that despite reduction in exercise performance, reduced muscle glycogen does not limit muscle performance or SR function.
- Glycogen extraction from skeletal muscle sarcoplasmic reticulum: structural and functional implicationsLees, Simon J. (Virginia Tech, 2003-03-27)In this investigation, skeletal muscle sarcoplasmic reticulum (SR) was purified from female Sprague Dawley rats (200-250 g). SR samples were subjected to two different biochemical glycogen-extraction protocols. The results suggest that both amylase and removal of EDTA (No-EDTA) from the homogenization and storage buffers reduced the amount of glycogen associated with the SR. Both of these treatments failed to impair SR calcium (Ca2+) handling when assayed under conditions where exogenous ATP was added and utilized for SR Ca2+ transport. In fact, these treatments seemed to cause a small increase in both SR Ca2+-uptake and release rates under these assay conditions. As expected, glycogen phosphorylase content was reduced as a result of glycogen extraction in the presence of amylase, however this was not the case for No-EDTA samples. Interestingly, many other proteins differed in content after glycogen extraction. These treatments resulted in a greater recovery of the sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase (SERCA) and a substantial loss of glycogen phosphorylase and glycogen debranching enzyme (AGL) in amylase-treated samples. Creatine kinase (CK) and pyruvate kinase (PK) contents were increased as a result of both glycogen-extraction conditions. It was imperative to consider these altered protein contents while analyzing the data and assessing the effects of glycogen extraction on SR Ca2+ handling. After normalizing to SERCA content, only No-EDTA samples had higher adenosine triphosphate (ATP)-supported SR Ca2+-uptake rates compared to control samples. For endogenously synthesized ATP-supported SR Ca2+-uptake experiments, normalizing data to protein content (either CK and SERCA or PK and SERCA) revealed that amylase-treated samples had lower SR Ca2+-uptake rates, compared to control samples. Although not significant, SR Ca2+-uptake rates for No-EDTA samples were also lower than control samples. These data suggest that changes in endogenously supported SR Ca2+-uptake due to glycogen extraction affected the source of ATP synthesis (either PK or CK), the effectiveness of energy utilization for Ca2+ transport (SERCA), or altered the metabolic channeling properties.
- Increased structure-bound proteolytic activity in maturing dystrophic skeletal muscleDraper, Kati Elizabeth (Virginia Tech, 2004-04-02)Duchenne Muscular Dystrophy (DMD) is a severe X-linked progressive muscle wasting disease resulting from the absence of the membrane-associated protein dystrophin and the secondary components of the dystrophin-glycoprotein complex. Although the genetic basis of the disease has been known for over 15 years, the onset mechanism of the disease is not yet known and no treatment is yet available to significantly increase the lifespan of DMD patients. Increased levels of intracellular calcium have been noted in dystrophic muscle (Turner et al., 1991) and increased intracellular levels of calcium in skeletal muscle lead to increased levels of calcium-dependent proteolysis (Zeman et al., 1985). Increased levels of calpain, a calcium-dependent protease have been reported as early as age 4 weeks in mdx (dystrophin-deficient) mice (Spencer et al., 1995). Increased calpain activity has been demonstrated in mdx myotubes (Alderton et al., 2000a). There is also evidence of a role for calpain in DMD, but the contribution of calpain activity to the onset of DMD has not yet been determined. The purpose of this study was to test the hypothesis that increased calpain activity contributes to the onset of DMD in maturing (birth to weaning) dystrophic skeletal muscles and to determine if increased calpain activity was due to the relative distribution of calpain and calpastatin, calpain's endogenous inhibitor. Calpain activity was assessed in quadriceps and diaphragm muscle homogenate supernatant and pellet fractions from C57BL/6 control and mdx mice at ages 7, 14, and 21 days. Total calpain and calpastatin content were determined by Western analysis. In both the quadriceps and diaphragm samples, calpain activity in the supernatant increased with age. There was a significant increase (47.7%; p<0.05) in calciumdependent calpain activity in mdx quadriceps pellet compared to control at age 7 days. In the quadriceps at age 7 days, calpain activity in the pellet in the presence of calcium was significantly greater than at age 14 (61.2%) and 21 days (52.6%; p<0.05). In the diaphragm, there were no significant differences in pellet activity in either the presence or absence of calcium at any age between control and mdx samples. In both control and mdx diaphragms, pellet calpain activity in the absence compared with the presence of calcium was significantly greater at both age 7 (control, 46.4%; mdx, 45.4%) and 14 days (control, 42.4%; mdx, 43.6%; p<0.05). At age 21 days, both control and mdx calpain activities in the diaphragm supernatants in the presence of calcium were significantly greater than those at ages 7 (control, 66.7%; mdx, 72.1%) and 14 days (control, 39.9%; mdx 49.5%; p<0.05). In general, there were no differences in total calpain and calpastatin content that would account for the differences in calpain activity. There were similar patterns of calpain activity and total calpain and calpastatin content in both control and mdx samples, suggesting a similar pattern of development in control and mdx muscle from ages 7-21 days. The increase in calcium-dependent calpain activity in mdx quadriceps pellet compared to control at age 7 days may be due to differences in regulation and/or distribution of the calpain system early in mdx maturation compared to control. From the present study, the role of calpain in the onset of DMD appears to be minor if global calcium-dependent activity is evaluated.
- Leucine and exercise improve skeletal muscle function in the mdx mouseVoelker, Kevin Andrew (Virginia Tech, 2010-01-21)Duchene muscular dystrophy (DMD) is a lethal X-linked disease that afflicts approximately 1 in 3500 newborn males. Boys with DMD will become progressively weaker causing wheelchair dependence by their early teens and death by their mid to late twenties. Currently there is no cure for DMD, the exact mechanism of disease action remains elusive, and treatments to improve quality of life are limited. Two areas of DMD research that could begin to fill this void and provide simple, cost effective therapy aimed to improve quality of life are neutriceutical and exercise therapies. We hypothesized that leucine, a branched chain amino acid (BCAA) with anabolic properties, given to sedentary and exercised x-linked dystrophic mice (mdx) over 4 weeks would improve skeletal muscle function and decrease markers of skeletal muscle degradation. In sedentary mdx mice, leucine improved tetanic extensor digitorum longus (EDL) stress (p < 0.05), gastrocnemius mammalian target or rapamycin (mTOR) phosphorylation (p < 0.05), while decreasing the rate of real-time calpain activity in flexor digitorum brevis (FDB) fibers (p < 0.05) compared to sedentary mice given no leucine. In exercised mdx mice, leucine improved total running distance over the 4 week testing period by 40% (p < 0.02) and increased EDL stress at every frequency recorded (p < 0.05). Our data lead us to the conclusion that the BCAA leucine can increase EDL muscle stress in dystrophic animals, and that the effects of leucine treatment are enhanced when leucine supplementation is combined with exercise. Leucine supplementation should be explored further and in higher order species of muscular dystrophy to determine if its use could provide clinical improvements in DMD patients.
- Physiological Responses in OSA Patients to Ramping Exercise After CPAP TreatmentShifflett, D. Edward Jr. (Virginia Tech, 1999-04-15)Continuous positive airway pressure (CPAP) is the primary therapy administered for those afflicted with obstructive sleep apnea (OSA). We examined the effects of CPAP therapy on physiological variables during a ramped exercise. The five male, OSA patients had mean values and standard deviations for RDI=60.7 +/- 19.1, BMI=29.9 +/- 2.9, and age=56 +/- 16.1 yr. Subjects were examined before and after 4 wk of CPAP therapy. After 4 wk of CPAP therapy, patient responses to exercise showed a 17.6%, (p<0.05) improvement in rating of perceived exertion (RPE) at identical power outputs (60% of the individual's apparent functional capacity). Statistical significance was not attained (p>0.05) upon analysis of the following parameters at 60% of the individuals maximum workload although there was a trend showing a decrease in these variables: heart rate (6% improvement), VO2 (11.7% improvement) systolic blood pressure (4% improvement), and rate pressure product (8.6% improvement). This data shows that the decrease in RPE during 60% of the individual's maximum predicted HR reserve corresponded with an increase in sleep quality (mean increase of 40%, 3.2 units) as measured by the Pittsburgh Sleep Quality Index before and after 4 wk of CPAP therapy. It was concluded that the improvement in exercise tolerance could be attributed to the subjective feelings of improved sleep quality after 4 wk of CPAP therapy. Key Words: Obstructive sleep apnea---CPAP--- exercise---physiological responses.
- Political Roles of Presidential Children: FDR Through ClintonWarters, Tabitha Alissa (Virginia Tech, 1998-04-21)There are many facets of the institution of the presidency that warrant examination. Individual presidents, cabinets, staffs, wives...have all been studied in depth but one aspect of the presidency still remains fundamentally unexplored: the presidents' children and the political roles that each has had or has the potential to have. This thesis is based upon role analysis and the basic assumption that all presidential children from FDR through Clinton have performed political roles. Among the 32 presidential children studied, four roles were designated. First is the role of symbol. Symbols serve to display the presidential candidate or president as a person that is a good family man, loving father, and someone with high moral integrity. Surrogates serve to stand in for the president when the president cannot be present. The bulk of a surrogate's role takes place on the campaign trail. Informal advisors/confidant(e)s provide opinions and advice to the president. Lastly, skeletons tend to embarrass the president. If an individual presidential child performs several of these roles equally, they have been labeled as hybrids. Each of the 32 children from FDR through Clinton have been categorized in one of the above roles and their actions are analyzed in depth. Through the course of the thesis, three hypotheses are tested. The first two are whether or not the political roles of presidential children vary be age and by sex. The third hypothesis is whether or not there is an increased need for symbols and surrogates as 1960 as opposed to before.
- The role of the apparent rate constant of cross-bridge transition from the strong binding state (G app ) in skeletal muscle force productionWard, Christopher W. (Virginia Tech, 1996)Force regulation at the level of the actin-myosin cross-bridge (XB) can be described by a 2 state model in which the XB's cycle between a strongly bound (SB), force generating state and a weakly bound (WB), non-force generating state. This cycle can be characterized by the apparent rate constants for transition into the SB state (fapp) and returning to the WB state (gapp), Increases in XB force can be accounted for by an increase in fapp a decrease in gapp., or both. While effort towards understanding XB force regulation has focused on the notion that force production is primarily regulated by fapp the purpose of this investigation was to determine if gapp continues to force regulation at the XB and to determine whether gapp differs in,muscles with differing contractile characteristics.