The sarcoplasmic reticulum (SR) is an intracellular membrane system dedicated to the active regulation of cytosolic calcium in muscle. The opening of Ca²⁺ channels in the SR results in a rapid increase in the myoplasmic Ca²⁺ concentration and the initiation of contraction. Closure of these channels allows the SR to re-accumulate the released Ca²⁺ which results in muscle relaxation. While it is known that a muscle fiber is stimulated to contract by the depolarization of the sarcolemma, it is not understood how this signal is communicated to the SR. The focus of this dissertation is twofold. The first objective is to gain an understanding of the mechanism of Ca²⁺ release from the SR. To this end, three studies have been performed which indicate that Ca²⁺ release is mediated by an oxidation reaction. The second goal is to gain insight into the function of the Ca²⁺ release channel. This is addressed by a fourth study which characterizes the effect of the plant alkaloid, ryanodine on channel operation. The anthraquinones mitoxantrone , doxorubicin, daunorubicin, and rubidazone are shown to be potent stimulators of Ca²⁺ release from SR vesicles. Anthraquinoneinduced Ca²⁺ release is shown to be via a specific interaction with the Ca²⁺ release system of the SR. In addition, a strong interaction between anthraquinone and caffeine binding sites on the Ca²⁺ release channel is observed when monitoring Ca²⁺ fluxes across the SR. It is shown that Ca²⁺ release stimulated by anthraquinones is inhibited by preincubating the quinone with dithionite, a strong reducing agent. Spectrophotometric measurements show that the dithionite treated quinone is in a reduced state. Previous work in this lab has shown that the photooxidizing xanthene dye rose bengal stimulates rapid Ca²⁺ release from skeletal muscle SR vesicles. In this thesis, it is shown that following fusion of vesicles to a bilayer lipid membrane (BLM), Ca²⁺ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. This stimulation is shown to be independent of the Ca²⁺ concentration but is inhibited by μM ruthenium red. The photooxidation of rose bengal is shown to not affect either the K+ or Cl- channels which are present in the SR. Exposure of the Ca²⁺ release channel to 500 nM rose bengal in the presence of light is shown to reverse the modification to the channel induced by μM ryanodine. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is directly observed upon measurement of [³H]ryanodine binding to TSR vesicles. Evidence is presented which suggests that Ca²⁺ release is mediated by singlet oxygen. Micromolar concentrations of the porphyrin meso-Tetra(4-N-methylpyridyl)porphine tetraiodide (TMPyP) is shown to induce the rapid release of Ca²⁺ from skeletal muscle SR vesicles. Porphyrin-induced Ca²⁺ release is stimulated by adenine nucleotides and μM Ca²⁺, and is inhibited by mM Mg²⁺ and μM ruthenium red. High-affinity [³H]ryanodine binding is also enhanced in the presence of the porphyrin. The presence of 1 mM Mg²⁺ in the assay medium sensitizes ryanodine binding to activation by ca²⁺. Porphyrin stimulated single channel activity is also sensitized to activation by Ca²⁺ in the presence of Mg²⁺. Reduction of the porphyrin by dithionite, a strong reducing agent, prior to exposure to the Ca²⁺ release channel inhibited the ability of TMPyP to stimulate Ca²⁺ release. These observations indicate that anthraquinones, rose bengal , and porphyrins induce a stimulation of the Ca²⁺ release protein from skeletal muscle SR by interacting with the ryanodine binding site. In addition, the mechanism of interaction for these compounds appears to be via an oxidation reaction. Nanomolar to micromolar concentrations of ryanodine are shown to alter the gating kinetics of the Ca²⁺ release channel from skeletal muscle SR fused with bilayer lipid membranes. In the presence of asymmetric CsCl, 5 to 40 nM concentrations of ryanodine are shown to activate the channel by increasing the open probability (P₀) without changing the conductance. Statistical analysis of gating kinetics reveal that the open and closed dwell times exhibit bi-exponential distributions that are significantly modified by nM ryanodine. The altered channel gating kinetics seen with low nM ryanodine is reversible and is shown to correlate with the binding kinetics of [³H]ryanodine with its highest affinity site under identical ionic conditions. Ryanodine concentrations between 20 and 50 nM are observed to induce occasional 1/2 conductance fluctuations while ryanodine concentrations greater than 50 nM stabilize the channel into a ½ conductance state which is not reversible. These results are shown to correlate with [³H]ryanodine binding to a second site having lower affinity than the first site. Ryanodine at concentrations greater than 70 μM from the 1/2 to a 1/4 conductance fluctuation , whereas ryanodine concentrations greater than 200 μM cause complete closure of the channel. The concentration of ryanodine required to stabilize either the 1/4 conductance transitions or channel closure do not directly correlate with the measured [³H]ryanodine equilibrium binding constants. However, these results can be explained by considering the association kinetics of ryanodine concentrations greater than 200 nM in the presence of 500 mM CsCl. These results indicate that ryanodine stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting ryanodine binding sites on the channel protein.

Skeletal muscle contains an internal membrane system called the sarcoplasmic reticulum (SR) whose function is to regulate the Ca2+ concentration of the myoplasm. Ca2+ is transported into the SR from the myoplasm via a Ca2+ dependent ATPase thus lowering the myoplasmic Ca2+ concentration. Ca2+ exits from the SR via a Ca2+ releqse pathway resultingin the increase of myoplasmic Ca2+. Muscles contract when the myoplasmic Ca2+ concentration is > 5 uM and relax when the Ca2+ concentration is lowered below 1 uM. The Ca2+ dependent ATPase has been extensively studied but the Ca2+ release system is less well understood. SR vesicles release their internal Ca2+ when a reactive thiol group is oxidized (oxidation-induced Ca2+ release). It is shown in this dissertation that oxidation-induced Ca2+ release is stimulated by adenine nucleotides with an order of effectiveness of: ATP > AMP-PCP > cAMP > AMP > adenine. The stimulatory effect is not dependent upon phosphorylation of a protein because AMP-PCP, a nonhydrolyzable analogue of ATP, is almost as effective as ATP in stimulating oxidation-induced Ca2+ release. It is also shown in this dissertation that photooxidation of histidyl residues results in an increase Ca2+ permeability of the SR. Unlike oxidation-induced Ca2+ release, photooxidation-induced Ca2+ release is Mg2+ independent, not inhibited by ruthenium red and inhibited by adenine nucleotides. Covalent modification of histidyl residues with ethoxyformic anhydride results in the increased permeability of SR vesicles. Similar to photooxidation-induced Ca2+ efflux, EFA-induced Ca2+ efflux is Mg2+ independent and is inhibited by ATP. The AMP-PCP protection of SR proteins from modification with EFA is similar to non-competitive inhibition with a KI = 50 uM. The photooxidation effect is not on membrane lipids but on a protein component which may be an ion transport system, other than the Ca2+ release protein, altered in such a way that it now transports Ca2+.

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. / 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.

Skeletal muscle fatigue can be defined as the inability to produce a desired amount of force. Fatigue can not only limit athletic performance and rehabilitation, but it can affect one's ability to perform every day activity as well. Despite extensive investigation of muscle fatigue, little is known about the exact mechanisms that result in decreased muscle performance. It likely involves several factors that are themselves dependent upon activation patterns and intensity. The process of excitation-contraction (EC) coupling is of particular importance with respect to regulation of force production. The release of calcium (Ca²⁺) from the sarcoplasmic reticulum (SR), which is stimulated by the depolarization of the sarcolemma, causes muscle contraction. The SR Ca²⁺-adenosine triphosphatase (ATPase) drives the translocation of two Ca²⁺ ions into the SR, utilizing the energy derived from the hydrolysis of one adenosine triphosphate (ATP) molecule. The process of SR Ca²⁺ uptake causes muscle relaxation. It has been proposed that both glycogen and glycolytic enzymes are associated with the SR membrane (SR-glycogenolytic complex). Interestingly, glycogen phosphorylase, an enzyme involved in glycogen breakdown, seems to be associated with the SR-glycogenolytic complex through its binding to glycogen. The presence of the SR-glycogenolytic system may serve to locally regenerate ATP utilized by the SR Ca²⁺-ATPase. The purpose of the present study was to investigate the effects of prolonged muscle contraction on glycogen concentration, glycogen phosphorylase content and activity, and maximum Ca²⁺ uptake rate associated with the SR. Tetanic contractions, elicited once per second for 15 minutes, significantly reduced glycogen associated with SR to 5.1% of control from 401.17 ± 79.81 to 20.46 ± 2.16 mg/mg SR protein (£ 0.05). The optical density of glycogen phosphorylase from SDS-PAGE was significantly reduced to 21.2% of control (£ 0.05). Activity of glycogen phosphorylase, in the direction of glycogen breakdown, was significantly reduced to 4.1% of control (£ 0.05). Pyridoxal 5'-phosphate (PLP) concentration, a quantitative indicator of glycogen phosphorylase content, was significantly reduced to 3.3% of control (£ 0.05). Maximum SR Ca²⁺ uptake rates were significantly reduced to 80.8% of control (£ 0.05). These data suggest reduced glycogen and glycogen phosphorylase may be involved, either directly or indirectly, in a mechanism that causes decreased SR Ca²⁺ uptake normally found in fatigue. / Master of Science

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xiii, 134 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 119-128).

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xv, 181 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / The Ca2+ pump or Ca2+-ATPase of cardiac sarcoplasmic reticulum, SERCA2a, is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the apparent Ca2+ affinity of the enzyme. We propose that PLB decreases Ca2+ affinity by stabilizing the Ca2+-free, E2·ATP state of the enzyme, thus blocking the transition to E1, the high Ca2+ affinity state required for Ca2+ binding and ATP hydrolysis. The purpose of this dissertation research is to critically evaluate this idea using series of cross-linkable PLB mutants of increasing inhibitory strength (N30C-PLB < PLB3 < PLB4). Three hypotheses were tested; each specifically designed to address a fundamental point in the mechanism of PLB action. Hypothesis 1: SERCA2a with PLB bound is catalytically inactive. The catalytic activity of SERCA2a irreversibly cross-linked to PLB (PLB/SER) was assessed. Ca2+-ATPase activity, and formation of the phosphorylated intermediates were all completely inhibited. Thus, PLB/SER is entirely catalytically inactive. Hypothesis 2: PLB decreases the Ca2+ affinity of SERCA2a by competing with Ca2+ for binding to SERCA2a. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca2+-ATPase activity and phosphoenzyme formation were measured, and correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca2+ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca2+ for binding to SERCA2a. This was confirmed with the Ca2+ pump mutant, D351A, which is catalytically inactive but retains strong Ca2+ binding. Increasingly higher Ca2+ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB competes with Ca2+ for binding to the Ca2+ pump. Hypothesis 3: PLB binds exclusively to the Ca2+-free E2 state with bound nucleotide (E2·ATP). Thapsigargin, vanadate, and nucleotide effects on PLB cross-linking to SERCA2a were determined. All three PLB mutants bound preferentially to E2 state with bound nucleotide (E2·ATP), and not at all to the thapsigargin or vanadate bound states. We conclude that PLB inhibits SERCA2a activity by stabilizing a unique E2·ATP conformation that cannot bind Ca2+.

PURPOSE: Muscle overuse is characterized by inflammation, reduced strength and muscle damage. It has been proposed that calcium (Ca2+) accumulation during muscle contraction, is responsible for muscle damage. Muscle contractile properties are regulated by calcium regulatory excitation contraction coupling mechanisms. Therefore, the main aim of the present study was to investigate the effects of voluntary repetitive tasks during six weeks on the rate of sarcoplasmic reticulum (SR) Ca2+-uptake, and Ca2+-release, in young female Sprague-Dawley rats. Secondly, this study aims to evaluate the effect of the training on muscular strength and the relationship between SR Ca2+-kinetics and grip strength test performance. METHODS: Six rats were trained (EXP), using a well-established model of reaching and handle pulling with the upper extremities (2 hr/day, 3days/week, 6 weeks), six control rats (KON) were included that were not exposed to the task. Grip strength were evaluated using a grip strength meter for rodents, two weeks prior the training was initiated, and two days after the training period was concluded. Tissue samples were obtained from the supraspinatus and trapezius muscle, and the rate of SR Ca2+-uptake and SR Ca2+-release were analysed using the fluorescent Ca2+ indicator indo 1. RESULTS: The analysis revealed that EXP had a significant higher rate of SR Ca2+-uptake, in both supraspinatus (33%, P &lt; 0,05) and trapezius (14%, P &lt; 0,05), compared to KON. However, no significant differences in SR Ca2+-release rate were found between groups, in neither of the muscles. A decline in grip strength were found in both EXP and KON, with no significant differences between groups. No significant correlation between grip strength and the Ca2+ release uptake variables could be found. CONCLUSION: The present results suggests that repetitive voluntary reaching and handle pulling with the upper extremities during six weeks, induce extant changes in SR Ca2+-uptake rate in rats.

One advantage of higher life-forms over less developed organisms is their ability to respond to signals from their environment with motion. This requires highly specialized contractile cells and a whole locomotion apparatus. In vertebrates, the cells responsible for movement are the skeletal muscle cells. They receive signals from the autonomic nervous system in the form of an action potential, and they contract in an appropriate manner. Calcium is a vital intracellular passenger whose role in muscular function is to initiate contraction. It is released via specific channel proteins from an internal Ca++ store, the sarcoplasmic reticulum, and triggers muscular contraction, the actual interplay of actin and myosin filaments. The step that is still not fully understood is the coupling process between arrival of an action potential and the subsequent contraction, called excitation-contraction coupling. Several theories have been proposed to explain this process. Some years ago, our laboratory introduced the hypothesis that an oxidation-reduction reaction of critical sulfhydryls associated with the Ca+t channel protein are involved in the regulation of channel gating. In an effort to understand more about the Ca++ channel gating mechanism at the molecular level, this thesis focuses on the interaction between o-phthalaldehyde, a reagent which specifically forms an isoindole derivative with the amino acids cysteine and lysine, and the Ca++ release channel complex. In this thesis, the planar lipid bilayer technique was used to study the Ca++ release channel protein from skeletal muscle sarcoplasmic reticulum at the single channel level. Utilizing this experimental technique, the direct interaction between OP A and the channel was investigated. In this study, it was shown that the interaction of o-phthalaldehyde with the channel increases the channel's open probability as well as its mean open time. Furthermore, the covalent nature of o-phthalaldehyde binding to the calcium release channel complex is shown and its inhibiting effects on chloride channels are demonstrated.