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. / Ph. D.

Duchenne's muscular dystrophy (DMD) is a debilitating disease that affects approximately 1 in 3500 boys, with many DMD patients dying before the age of 20 due to cardio-respiratory complications. DMD is the result of defects in the gene that encodes dystrophin, an integral muscle membrane protein. Although the genetic defect has been identified, the relation between the absence of expressed dystrophin and the mechanisms leading to its onset are still unclear. One possibility is that disrupted calcium (Ca²⁺) handling by the sarcoplasmic reticulum (SR) leads to an increased cytosolic Ca²⁺ concentration that activates proteolytic and apoptotic pathways that initiate muscle fiber death. However, little is known about the role of disrupted SR function in the onset of DMD. The purpose of this study was to test the hypothesis that altered calcium cycling by the SR could contribute to elevated cytosolic Ca²⁺ levels in the early stages of DMD, and thereby account for the onset of disease pathogenesis. Rates of SR Ca²⁺ uptake and release were determined in quadriceps muscles obtained from maturing dystrophic and control mice prior to the overt signs of the disease at ages ~9 and 21 days. In addition, the content of several key Ca²⁺ handling proteins, including two isoforms of the sarco(endo)plasmic reticulum ATPase pump (SERCA 1 & 2), ryanodine receptor type 1 (RyR1), parvalbumin, and calsequestrin were determined by Western analysis. Two dystrophic mouse models were used, the mdx mouse which lacks dystrophin, and the mdx:utrophin-deficient (mdx:utrn^-/-) mouse which also lacks utrophin, a protein homolog of dystrophin. The rate of SR Ca²⁺ uptake in quadriceps muscles of mdx/utrn^-/- mice aged 21 days was 73.1% and 61.3% higher than age-matched control and mdx muscles, respectively (p < 0.05). There was no difference in SR Ca²⁺ release rates between the genotypes at either age. There were significant increases in the content of each of the calcium handling proteins with age (p < 0.05), but no significant differences were detected between genotypes at either age. These data demonstrate the Ca²⁺ release rates of dystrophic SR are not compromised, but suggest the increased uptake rates of mdx:utrn^-/- SR may be an adaptation to increased cytosolic calcium levels, and/or be due to changes in intrinsic SERCA function and/or regulation. The role of increased SR Ca²⁺ uptakes rates in onset of DMD pathogenesis can not be directly determined from the present data; therefore it is suggested that future studies directly assess cytosolic Ca²⁺ concentration and examine the role of SERCA regulatory proteins in intact fibers obtained from mdx:utrn^-/- muscles at age 21 days. / Master of Science

Fatigue and skeletal muscle weakness are problems associated with congestive heart failure. Research does not support the theory that the affected cardiac function is responsible for the fatigue. During skeletal muscle fatigue, calcium handling is altered. Thus, the fatigue associated with congestive heart failure could be attributed to altered calcium handling. The main proteins involved in calcium release are the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The main proteins involved in calcium uptake are the fast and slow isoforms of sarco(endo)plasmic reticulum calcium ATPase (SERCA 1 and SERCA 2 respectively). Calsequestrin (Csq) and calmodulin (CaM) play regulatory roles in calcium handling. Changes in the levels of these proteins could explain alterations in calcium handling and subsequent muscle function. The purpose of this study was to use a genetic model of heart failure, the SHHF rat, to examine the levels of regulatory calcium handling proteins to determine if changes in the amounts of RyR, DHPR, SERCA1, SERCA2, Csq and CaM are altered in congestive heart failure. A significant decrease was found in the amounts of RyR, DHPR, and SERCA 1 of the SHHF gastrocnemius and diaphragm samples in comparison to the control. There was no significant difference found in the amounts of CaM or SERCA 2 between the two groups. Csq was not found to be statistically different between the two groups of the gastrocnemius samples. However, there was an increase in Csq in the SHHF diaphragm samples in comparison to the control. In conclusion, the calcium handling proteins are affected in the genetic model of heart failure. These changes could explain previous reports of altered calcium handling within the skeletal muscles of congestive heart failure animals. / Master of Science

No description available.

Sarcolipin (SLN), a small molecular weight, hydrophobic protein found in skeletal muscle, is a known regulator of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pumps. Earlier in vitro reconstitution experiments have shown that SLN uncouples ATP hydrolysis from Ca2+ transport by the SERCA pumps and increases the amount of heat released per mol of ATP hydrolyzed by inducing an increased rate of ???slippage??? during the reaction cycle of SERCA pumps. In order to determine whether SLN causes slippage of SERCA activity by uncoupling ATP hydrolysis from Ca2+ transport under more physiological conditions, comparisons were made between skeletal muscle Ca2+ ATPase activity and Ca2+ uptake in homogenates from soleus muscle of wild-type (WT) and Sln-null (KO) mice under conditions in which a Ca2+ gradient was preserved across the sarcoplasmic reticulum (SR) vesicles. Ca2+ ATPase activity, measured in the absence of the Ca2+ ionophore, A23187, was 15-25% lower in KO muscles, compared with WT, consistent with the proposal that SLN increases ???slippage??? and reduces the extent of back-inhibition of the Ca2+ ATPase. Ca2+ uptake, measured in homogenates without oxalate, was not different (p>0.05) in SR vesicles from WT and KO mice, indicating that the calculated Ca2+ transport efficiency (coupling ratio) in KO mice was increased by about 20% (P<0.04). The basal oxygen consumption (VO2) of soleus muscles isolated from WT and KO mice and the contribution of energy utilized by SERCA was also compared. Surprisingly, basal VO2 was not lower in the soleus of KO mice, but the contribution of energy utilized by SERCA pumps was about 7% lower (P<0.0001). It was also found that uncoupling protein 3 (UCP-3) was expressed at a higher (P<0.03) concentration in soleus muscle of KO compared to WT. Thus UCP-3 could, potentially, provide compensation, resulting in higher basal VO2 in KO mice than expected. These data show that at physiological SLN:SERCA ratios, SLN uncouples ATP hydrolysis from SR Ca2+ uptake in skeletal muscle, resulting in a lower contribution of Ca2+ handling to basal VO2. Thus, SLN is a key regulator of both ATP utilization in Ca2+ handling and of overall energy metabolism in skeletal muscle. To further examine the role of SLN in adaptive thermogenesis, obesity and glucose intolerance, KO and WT mice were placed on a high fat diet (HFD; 42% of kcal derived from fat) for an eight week period. Whole body metabolism, weight gain, glucose tolerance and insulin tolerance were measured before and after the HFD. Fat pads, liver, pancreas, hindlimb muscles and plasma samples were collected from standard chow fed control and HFD WT and KO mice. KO mice gained more weight (P<0.05) and became more obese (P<0.05) than WT mice after consuming the HFD. The comprehensive laboratory animal monitoring system (CLAMS) revealed no differences in whole body metabolic rate (ml O??2/kg/hr) between KO and WT mice pre diet; however, daily metabolic rate was lower (P<0.05) in KO mice compared with WT mice after the HFD which may explain the increased obesity in KO mice. Western blotting analyses revealed SLN protein content to be 3.8 fold higher (P<0.05) in WT soleus post HFD compared to control. Phospholamban (PLN), a homologue of SLN, was found to be 2.1 fold higher (P<0.05) in brown adipose tissue (BAT) in both WT and KO mice post HFD. Protein contents of other Ca2+ handling proteins (SERCA1a, SERCA2a, PLN and calsequestrin) within fast (white gastrocnemius) and slow (soleus) twitch muscle were not different between KO and WT mice following the HFD. Collectively, these results suggest that PLN and SLN could play a role in adaptive diet-induced thermogenesis. On the other hand, compared with chow fed control mice, the metabolic cost of Ca2+ handling in soleus muscle was significantly reduced post HFD in both WT and KO mice, although to a greater extent (P<0.05) in KO mice than WT mice. Moreover, there were no differences in resting energy expenditure of soleus muscles between WT and KO mice following the HFD. These observations can be accounted for by diet-induced increases in sympathetic nervous system activity in KO mice and other adaptive responses leading to increased energy expenditure of soleus in both WT and KO mice. Therefore, differences in whole body metabolic rate and obesity between high fat fed WT and KO mice do not appear to be due to adaptive thermogenesis mechanisms in skeletal muscle involving SLN. Interestingly, soleus and EDL muscle weights increased proportionately to body weight in high fat fed WT mice but not KO mice. Therefore, lower lean body tissue mass may explain the lower whole body metabolic rate and increased susceptibility to obesity in KO mice compared with WT mice. With increased obesity, KO mice became extremely glucose intolerant (P<0.05) post HFD compared to WT mice who also demonstrated glucose intolerance (P<0.05) compared to the pre-HFD values. Surprisingly, the insulin tolerance test responses were not different between KO and WT mice post HFD suggesting that KO mice did not develop greater whole body insulin resistance despite being more obese than WT mice. Blood serum analysis showed that non-esterified fatty acids (NEFA) and LDL cholesterol levels were also increased more (P<0.05) in KO mice compared to the WT mice post HFD. Overall, it is concluded that SLNs impact on Ca2+ handling influences not only ATP consumption by SERCA pumps in resting soleus muscle via uncoupling of ATP hydrolysis from SR Ca2+ uptake but also blunts the negative effect of high fat feeding by increasing resistance to diet-induced obesity and glucose intolerance in mice through mechanisms which are currently unidentified.

Cardiac hypertrophy is a fundamental response to an increased workload on the heart characterized by cardiac myocyte (CM) growth and left ventricular (LV) wall thickening. In a model of hypertension, e.g. chronic pressure overload, this process may become maladaptive, initially leading to impaired myocardial relaxation and LV filling, and subsequently to LV dilation, wall thinning, and contractile failure. Hemodynamic overload activates Gαq-mediated signaling responsible for transcriptional reactivation of fetal growth programs, activation of the mitogen-activated protein kinase (MAPK) cascade, and oxidative stress. The precise mechanism by which MAPK is activated in pressure overload, and the role oxidative stress plays in mediating this hypertrophic signaling are still under investigation. In CMs, the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) maintains calcium stores, and thus may regulate calcium-dependent MAPK signaling. Our laboratory showed that SERCA is activated in CMs by reversible oxidative post-translational modification (OPTM) of its most reactive cysteine site (C674). We hypothesized that OPTMs mediate the effects of hypertrophic stimuli in CMs via reversible oxidation of SERCA at C674. To test this hypothesis, we employed a reductionist model: isolated adult rat ventricular myocytes (ARVM) overexpressing wild-type (WT) or mutant SERCA, in which C674 is substituted with a redox-insensitive serine (C674S). Using alpha-adrenergic receptor (αAR) stimulation as a model of Gq-mediated hypertrophy, we found that C674S expression decreased both CM growth and MAPK activation. Furthermore, biotin switch revealed that αAR stimulation induced a reversible OPTM of SERCA at C674. We generated a transgenic mouse expressing a single-allele C674S SERCA2 knock-in mutation (SKI) to explore this mechanism further in the setting of pressure overload, a disease model of Gq-activation in vivo. SKI mice subjected to ascending aortic constriction (AAC) had decreased hypertrophy compared to WT. Ventricular myocytes isolated from adult SKI mice also had diminished MAPK activation in response to hypertrophic stimulation in vitro and decreased SERCA function at baseline. These findings led us to the conclusion that redox-activation of SERCA via reversible modification of C674 is critical for the complete transduction of hypertrophic stimuli to MAPK signaling and CM hypertrophy.

No description available.

No description available.

Phospholamban (PLN) is an integral membrane protein that regulates the sarco(endo)plasmic Ca2+-ATPase (SERCA2a) within the cardiac sarcoplasmic reticulum (CSR). SERCA2a regulates intracellular Ca2+- handling and thus plays a critical role in initiating cardiac contraction and relaxation. It is believed that dysregulation of SERCA2a is a contributing factor in human heart failure patients. Even though there have been substantial advancements in understanding heart failure pharmacological therapies, patient prognosis remains poor. Nitroxyl (HNO), a new candidate heart failure drug therapy, has been shown to enhance overall cardiovascular function in both healthy and failing hearts, at least in part, by increasing Ca²⁺ re-uptake into the CSR. Previous research has shown that activation of SERCA2a by HNO is PLN-dependent; however, the mechanism of action of HNO remains unknown. We propose that HNO, a thiol oxidant, modifies one or more of the three PLN cysteine residues (C36, C41, C46) affecting the regulatory potency of PLN toward SERCA2a. To test this hypothesis, a series of PLN mutants were constructed containing single, double and triple cysteine substitutions. Using the baculovirus expression insect cell system, each PLN cysteine mutant was expressed alone and co-expressed with SERCA2a in insect cells and isolated in cellular endoplasmic reticulum (ER) microsomes. Samples were treated with Angeli's salt (an HNO donor) to determine the role of each PLN cysteine residue in the mechanism of SERCA2a activation by nitroxyl. Using a standard phosphate activity assay and SDS-PAGE/immunoblot techniques, we determined that the PLN cysteine residues at positions 41 and 46 are important in HNO activation of SERCA2a. Both SERCA2a + 41C PLN and SERCA2a + 46C PLN microsomal samples showed a ΔK0.5 of ~0.33 μM and evidence of reversible HNO induced disulfide bond formation. These studies provide important new insight into the mechanism of action of HNO on cardiac SR and thereby help evaluate the drug as a candidate therapy for congestive heart failure. / Ph. D.

Numerous references have suggested that alterations in exercise capacity during congestive heart failure (CHF) are not simply due to changes in myocardial function. In fact, recent evidence has indicated that reductions in skeletal muscle strength and endurance during CHF significantly impact exercise capacity of the CHF patient. Currently, it is believed that alterations in skeletal muscle phenotype, or more specifically a slow to fast transformation in phenotypic protein isoforms contribute to the reductions in muscle function. However, currently there are few data which directly document this slow to fast transformation of the skeletal muscle. Interestingly, it is well established that exercise training can cause changes in skeletal muscle phenotype, more specifically in the fast to slow direction. This is in direct contrast to what is known to occur during CHF. However, it is unclear if similar adaptations will result from training in a CHF patient. Also, it is not clear if the adaptations are due to alterations in the myocardium or changes directly imposed upon the muscle by the exercise training. Therefore, the purpose of this study was two-fold: 1) to clarify the changes in skeletal muscle myosin heavy chain (MHC) during CHF and 2) to determine if skeletal muscle can adapt to increased activity levels, utilizing functional overload (FO) without significantly improving cardiac function. In the first study the mixed plantaris muscles from rats afflicted with severe CHF demonstrated a significant (p<0.05) increase in fast MHC (e.g. IIb expression at the expense of IIx expression) compared to the control animal (SHAM). The mixed red gastrocnemius, regardless of the severity of CHF, exhibited significant (p<0.05) changes in all of the MHC isoforms. The slow soleus and fast white gastrocnemius did not display any significant changes in MHC expression. The changes in MHC isoform significantly correlated with indicators of disease severity, suggesting there may be an existing relationship between skeletal muscle MHC expression and alterations in myocardial function. In the second study, there were no differences exhibited between CHF and SHAM absolute or specific plantaris mass. There was a significant (p<0.05) 30% increase in both absolute and specific mass of the plantaris in the CHF-FO and SHAM-FO groups compared to the CHF and SHAM groups. There was a significant (p<0.05) 3.5% increase in slow MHC I expression and a significant (p<0.05) 6.5% decrease in fast MHC IIb expression in the CHF-FO group compared to the CHF group. In the SHAM-FO group, there was a significant (p<0.05) 4% increase in MHC I expression and a subsequent 8% decrease in fast MHC IIx+IIb in the SHAM-FO compared to the SHAM groups. There were no differences detected in the rates of Ca²⁺ uptake between the CHF-FO, SHAM, and SHAM-FO. However, Ca²⁺ uptake rates were significantly (p<0.05) elevated by 44% in the CHF group when compared to the other three groups. There were very few changes in plantaris SERCA 1 or 2 protein expression between the four groups. These data suggest that during CHF there are alterations in skeletal muscle isoform expression. However, at least some of the data suggest that changes in function are not always associated with changes in phenotype. Instead, it seems that the changes in Ca²⁺ handling may be due to an alteration in a regulatory mechanism. Also, the data indicate that skeletal muscle is adaptable to increases in activity levels without significantly altering myocardial morphology. / Ph. D.