ABSTRACT
Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.
Subject(s)
Muscle, Skeletal , Ryanodine Receptor Calcium Release Channel , Animals , Humans , Calcium/metabolism , Muscle, Skeletal/metabolism , Protein Isoforms/metabolism , Ryanodine/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/metabolism , Thermogenesis , AmphibiansABSTRACT
Store-operated Ca2+ entry (SOCE) is critical to cell function. In skeletal muscle, SOCE has evolved alongside excitation-contraction coupling (EC coupling); as a result, it displays unique properties compared to SOCE in other cells. The plasma membrane of skeletal muscle is mostly internalized as the tubular system, with the tubules meeting the sarcoplasmic reticulum (SR) terminal cisternae, forming junctions where the proteins that regulate EC coupling and SOCE are positioned. In this review, we describe the properties and roles of SOCE based on direct measurements of Ca2+ influx during SR Ca2+ release and leak. SOCE is activated immediately and locally as the [Ca2+ ] of the junctional SR terminal cisternae ([Ca2+ ]jSR ) depletes. [Ca2+ ]jSR changes rapidly and steeply with increasing activity of the SR ryanodine receptor isoform 1 (RyR1). The high fidelity of [Ca2+ ]jSR with RyR1 activity probably depends on the SR Ca2+ -buffer calsequestrin that is located immediately behind RyR1 inside the SR. This arrangement provides in-phase activation and deactivation of SOCE with a large dynamic range, allowing precise grading of SOCE flux. The in-phase activation of SOCE as the SR partially depletes traps Ca2+ in the cytoplasm, preventing net Ca2+ loss. Mild presentation of RyR1 leak can occur under physiological conditions, providing fibre Ca2+ redistribution without changing fibre Ca2+ content. This condition preserves normal contractile function at the same time as increasing basal metabolic rate. However, higher RyR1 leak drives excess cytoplasmic and mitochondrial Ca2+ load, setting a deleterious intracellular environment that compromises the function of the skeletal muscle.
Subject(s)
Muscle, Skeletal , Ryanodine Receptor Calcium Release Channel , Ryanodine Receptor Calcium Release Channel/metabolism , Muscle, Skeletal/physiology , Calcium Signaling , Sarcoplasmic Reticulum/metabolism , Cytoplasm/metabolism , Calcium/metabolismABSTRACT
Ca2+ is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca2+ is reported to modulate the rate of ATP resynthesis, regulate the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1α) following exercise, and drive the generation of reactive oxygen species (ROS). Due to the latter, mitochondrial Ca2+ overload is recognized as a pathophysiological event but the former events represent important physiological functions in need of tight regulation. Recently, we described the relationship between [Ca2+]mito and resting [Ca2+]cyto and other mitochondrial Ca2+-handling properties of skeletal muscle. An important next step is to understand the triggers for Ca2+ redistribution between intracellular compartments, which determine the mitochondrial Ca2+ load. These triggers in both physiological and pathophysiological scenarios can be traced to the coupled activity of the ryanodine receptor 1 (RyR1) and store-operated Ca2+ entry (SOCE) in the resting muscle. In this piece, we will discuss some issues regarding Ca2+ measurements relevant to mitochondrial Ca2+-handling, the steady-state relationship between cytoplasmic and mitochondrial Ca2+, and the potential implications for Ca2+ handling by muscle mitochondria and cellular function.
Subject(s)
Calcium , Ryanodine Receptor Calcium Release Channel , Adenosine Triphosphate/metabolism , Calcium/metabolism , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Peroxisome Proliferator-Activated Receptors/metabolism , Reactive Oxygen Species/metabolism , Ryanodine Receptor Calcium Release Channel/metabolismABSTRACT
Muscle contraction depends on tightly regulated Ca2+ release. Aberrant Ca2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca2+ leak initiates a cascade of events that cause precise redistribution of Ca2+ among the SR, cytoplasm, and mitochondria through altering the Ca2+ permeability of the transverse tubular system membrane. This redistribution of Ca2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.
ABSTRACT
In vitro studies have shown that alterations in redox state can cause a range of opposing effects on the properties of the contractile apparatus in skeletal muscle fibers. To test whether and how redox changes occurring in vivo affect the contractile properties, vastus lateralis muscle fibers from seven healthy young adults were examined at rest (PRE) and following (POST) high-intensity intermittent cycling exercise. Individual mechanically skinned muscle fibers were exposed to heavily buffered solutions at progressively higher free [Ca2+] to determine their force-Ca2+ relationship. Following acute exercise, Ca2+ sensitivity was significantly decreased in type I fibers (by 0.06 pCa unit) but not in type II fibers (0.01 pCa unit). Specific force decreased after the exercise in type II fibers (-18%) but was unchanged in type I fibers. Treatment with the reducing agent dithiothreitol (DTT) caused a small decrease in Ca2+-sensitivity in type II fibers at PRE (by â¼0.014 pCa units) and a significantly larger decrease at POST (â¼0.035 pCa units), indicating that the exercise had increased S-glutathionylation of fast troponin I. DTT treatment also increased specific force (by â¼4%), but only at POST. In contrast, DTT treatment had no effect on either parameter in type I fibers at either PRE or POST. In type I fibers, the decreased Ca2+ sensitivity was not due to reversible oxidative changes and may have contributed to a decrease in power production during vigorous exercises. In type II fibers, exercise-induced redox changes help counter the decline in Ca2+-sensitivity while causing a small decline in maximum force.NEW & NOTEWORTHY This study identified important cellular changes occurring in human skeletal muscle fibers following high-intensity intermittent exercise: 1) a decrease in contractile apparatus Ca2+ sensitivity in type I but not type II fibers, 2) a decrease in specific force only in type II muscle fibers, and 3) a redox-dependent increase in Ca2+ sensitivity occurring only in type II fibers, which would help maintain muscle performance by countering the normal metabolite-induced decline in Ca2+ sensitivity.
Subject(s)
High-Intensity Interval Training , Calcium , Humans , Muscle Contraction , Muscle Fibers, Fast-Twitch , Muscle Fibers, Skeletal , Muscle, Skeletal , Young AdultABSTRACT
This study investigated the effect of S-glutathionylation on passive force in skeletal muscle fibres, to determine whether activity-related redox reactions could modulate the passive force properties of muscle. Mechanically-skinned fibres were freshly obtained from human and rat muscle, setting sarcomere length (SL) by laser diffraction. Larger stretches were required to produce passive force in human fibres compared to rat fibres, but there were no fibre-type differences in either species. When fibres were exposed to glutathione disulfide (GSSG; 20 mM, 15 min) whilst stretched (at a SL where passive force reached ~ 20% of maximal Ca2+-activated force, denoted as SL20 % max), passive force was subsequently decreased at all SLs in both type I and type II fibres of rat and human (e.g., passive force at SL20 % max decreased by 12 to 25%). This decrease was fully reversed by subsequent reducing treatment with dithiothreitol (DTT; 10 mM for 10 min). If freshly skinned fibres were initially treated with DTT, there was an increase in passive force in type II fibres (by 10 ± 3% and 9 ± 2% in rat and human fibres, respectively), but not in type I fibres. These results indicate that (i) S-glutathionylation, presumably in titin, causes a decrease in passive force in skeletal muscle fibres, but the reduction is relatively smaller than that reported in cardiac muscle, (ii) in rested muscle in vivo, there appears to be some level of reversible oxidative modification, probably involving S-glutathionylation of titin, in type II fibres, but not in type I fibres.
Subject(s)
Muscle Fibers, Skeletal/drug effects , Protein S/metabolism , Animals , Humans , RatsABSTRACT
This study investigated the effects of fibre swelling on force production in rat and human skinned muscle fibres, using osmotic compression to reverse the fibre swelling. In mechanically-skinned fibres, the sarcolemma is removed but normal excitation-contraction coupling remains functional. Force responses in mechanically-skinned fibres were examined with and without osmotic compression by polyvinylpyrrolidone 40 kDa (PVP-40) or Dextran 500 kDa (dextran). Fibre diameter increased to 116 ± 2% (mean ± SEM) when rat skinned type II fibres were immersed in the standard intracellular solution, but remained close to the in situ size when 3% (mass/volume) PVP-40 or 4% Dextran were present. Myofibrillar Ca2+ sensitivity, as indicated by pCa50 (- log10[Ca2+] at half-maximal force), was increased in 4% Dextran (0.072 ± 0.007 pCa50 shift), but was not significantly changed in 3% PVP-40. Maximum Ca2+-activated force increased slightly to 103 ± 1% and 104 ± 1% in PVP-40 and Dextran, respectively. Both tetanic and depolarization-induced force responses in rat skinned type II fibres, elicited by electrical stimulation and ion substitution respectively, were increased by ~ 10 to 15% when the fibres were returned to their normal in situ diameter by addition of PVP-40 or Dextran. Interestingly, the potentiation of these force responses in PVP-40 was appreciably greater than could be explained by potentiation of myofibrillar function alone. These results indicate that muscle fibre swelling, as can occur with intense exercise, decreases evoked force responses by reducing both the Ca2+-sensitivity of the contractile apparatus properties and Ca2+ release from the sarcoplasmic reticulum.
Subject(s)
Depression/etiology , Muscle Fatigue/physiology , Muscle Fibers, Skeletal/pathology , Adult , Animals , Humans , Male , Rats , Rats, Sprague-Dawley , Young AdultABSTRACT
The contractile properties of vastus lateralis muscle fibres were examined in prostate cancer (PrCa) patients undergoing androgen deprivation therapy (ADT) and in age- and activity-matched healthy male subjects (Control). Mechanically-skinned muscle fibres were exposed to a sequence of heavily Ca2+ -buffered solutions at progressively higher free [Ca2+ ] to determine their force-Ca2+ relationship. Ca2+ -sensitivity was decreased in both type I and type II muscle fibres of ADT subjects relative to Controls (by -0.05 and -0.04 pCa units, respectively, P < .02), and specific force was around 13% lower in type I fibres of ADT subjects than in Controls (P = .02), whereas there was no significant difference in type II fibres. Treatment with the reducing agent dithiothreitol slightly increased specific force in type I and type II fibres of ADT subjects (by ~2%-3%, P < .05) but not in Controls. Pure type IIx fibres were found frequently in muscle from ADT subjects but not in Controls, and the overall percentage of myosin heavy chain IIx in muscle samples was 2.5 times higher in ADT subjects (P < .01). The findings suggest that testosterone suppression can negatively impact the contractile properties by (i) reducing Ca2+ -sensitivity in both type I and type II fibres and (ii) reducing maximum specific force in type I fibres.
Subject(s)
Goserelin/therapeutic use , Muscle Contraction/drug effects , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/physiology , Prostatic Neoplasms/drug therapy , Aged , Androgen Antagonists , Antineoplastic Agents, Hormonal/therapeutic use , Humans , MaleABSTRACT
Na(+), K(+)-ATPase (NKA) isoforms (α1,α2,α3,ß1,ß2,ß3) are involved in the maintenance of membrane potential and hence are important regulators of cellular homeostasis. Given the age-related decline in skeletal muscle function, we investigated whether the natural physiological process of aging is associated with altered abundance of NKA isoforms (α1,α2,α3,ß1,ß2,ß3) or of the commonly used control protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Importantly, measurements were made in both whole muscle or specific fiber types obtained from skeletal muscle biopsies. Seventeen healthy older (AGED, 69.4 ± 3.5 years, mean ± SD) and 14 younger (YOUNG, 25.5 ± 2.8 years) adults underwent a muscle biopsy for biochemical analyses. Comparing homogenates from AGED and YOUNG individuals revealed higher ß3 isoform (p<0.05) and lower GAPDH (p<0.05). Analysis of individual fibers in muscle from YOUNG individuals, showed greater α3 and ß2 isoforms, and more GAPDH in Type II compared with Type I fibers (p<0.05). In the AGED, GAPDH was higher in Type II compared with Type I fibers (p<0.05), there were no fiber type differences in the NKA isoforms (p>0.05). Compared with the same fiber type in YOUNG, α1 was greater (Type I) and α3 lower (Type II), while in both fiber types, ß2 was lower, ß3 greater and GAPDH lower, in muscle from AGED individuals (all p<0.05). Overall, we demonstrate that (i) GAPDH is an inappropriate choice of protein for normalization in all skeletal muscle research and (ii) full understanding of the role of NKA isoforms in human skeletal muscle requires consideration of age and muscle fiber type.
Subject(s)
Aging/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/analysis , Muscle, Skeletal/enzymology , Sodium-Potassium-Exchanging ATPase/analysis , Adult , Aged , Aging/pathology , Female , Humans , Isoenzymes/analysis , Male , Muscle Fibers, Fast-Twitch/enzymology , Muscle Fibers, Slow-Twitch/enzymology , Muscle Proteins/analysis , Muscle, Skeletal/cytologyABSTRACT
We describe a new method for determining the concentration of total Ca in whole skeletal muscle samples ([CaT]WM in units of mmoles/kg wet weight) using the Ca-dependent UV absorbance spectra of the Ca chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). Muscle tissue was homogenized in a solution containing 0.15 mM BAPTA and 0.5% sodium dodecyl sulfate (to permeabilize membranes and denature proteins) and then centrifuged. The solution volume was adjusted so that BAPTA captured essentially all of the Ca. [CaT]WM was obtained with Beer's law from the absorbance change produced by adding 1 mM EGTA to capture Ca from BAPTA. Results from mouse, rat, and frog muscles were reasonably consistent with results obtained using other methods for estimating total [Ca] in whole muscles and in single muscle fibers. Results with external Ca removed before determining [CaT]WM indicate that most of the Ca was intracellular, indicative of a lack of bound Ca in the extracellular space. In both fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from mice, [CaT]WM increased approximately linearly with decreasing muscle weight, increasing approximately twofold with a twofold decrease in muscle weight. This suggests that the Ca concentration of smaller muscles might be increased relative to that in larger muscles, thereby increasing the specific force to compensate for the smaller mass. Knocking out the high capacity Ca-binding protein calsequestrin (CSQ) did not significantly reduce [CaT]WM in mouse EDL or soleus muscle. However, in EDL muscles lacking CSQ, muscle weights were significantly lower than in wild-type (WT) muscles and the values of [CaT]WM were, on average, about half the expected WT values, taking into account the above [CaT]WM versus muscle weight relationship. Because greater reductions in [CaT]WM would be predicted in both muscle types, we hypothesize that there is a substantial increase in Ca bound to other sites in the CSQ knockout muscles.
Subject(s)
Calcium/metabolism , Calsequestrin/metabolism , Muscle, Skeletal/metabolism , Animals , Anura , Calcium Chelating Agents/chemistry , Egtazic Acid/analogs & derivatives , Egtazic Acid/chemistry , Mice , Rats , Spectrometry, Fluorescence/methodsABSTRACT
Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits release when [Ca(T)](SR) declines to a low level.
Subject(s)
Calcium Signaling , Calcium/metabolism , Muscle Fibers, Fast-Twitch/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Buffers , Calsequestrin/metabolism , Hydrogen-Ion Concentration , Membrane Potentials , Muscle Fibers, Fast-Twitch/physiology , Rana temporariaABSTRACT
One aim of this article was to determine the resting concentration of free Ca(2+) in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca(2+)](SR,R)) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca(2+)](SR,R) to TMX's apparent dissociation constant for Ca(2+) (K(app)) in order to establish the capability of monitoring [Ca(2+)](SR)(t) during SR Ca(2+) release - a signal needed to determine the Ca(2+) permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K(app) as well as a dependence of K(app) on TMX concentration, the results indicate an average [Ca(2+)](SR,R) ranging from 0.43 to 1.70mM. The ratio [Ca(2+)](SR,R)/K(app) averaged 0.256, a relatively low value which should not depend on factors influencing K(app). As a result, the time course of [Ca(2+)](SR)(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.
Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Coloring Agents/analysis , Murexide/analogs & derivatives , Muscle Fibers, Skeletal/metabolism , Sarcoplasmic Reticulum/metabolism , Action Potentials/physiology , Animals , Hydrogen-Ion Concentration , Models, Biological , Murexide/analysis , Muscle Fibers, Skeletal/physiology , Proton Ionophores , Rana catesbeiana , Saponins , Time FactorsABSTRACT
Whether the use of pre-exercise hyperhydration could improve the performance of athletes who do not hydrate sufficiently during prolonged exercise is still unknown. We therefore compared the effects of pre-exercise hyperhydration and pre-exercise euhydration on endurance capacity, peak power output and selected components of the cardiovascular and thermoregulatory systems during prolonged cycling. Using a randomized, crossover experimental design, 6 endurance-trained subjects underwent a pre-exercise hyperhydration (26 ml of water x kg body mass(-1) with 1.2 g glycerol x kg body mass(-1)) or pre-exercise euhydration period of 80 min, followed by 2 h of cycling at 65% maximal oxygen consumption (VO(.)2max) (26-27 degrees C) that were interspersed by 5, 2-min intervals performed at 80% V(.)O2max. Following the 2 h cycling exercise, subjects underwent an incremental cycling test to exhaustion. Pre-exercise hyperhydration increased body water by 16.1+/-2.2 ml.kg body mass(-1). During exercise, subjects received 12.5 ml of sports drink x kg body mass(-1). With pre-exercise hyperhydration and pre-exercise euhydration, respectively, fluid ingestion during exercise replaced 31.0+/-2.9% and 37.1+/-6.8% of sweat losses (p>0.05). Body mass loss at the end of exercise reached 1.7+/-0.3% with pre-exercise hyperhydration and 3.3+/-0.4% with pre-exercise euhydration (p<0.05). During the 2 h of cycling, pre-exercise hyperhydration significantly decreased heart rate and perceived thirst, but rectal temperature, sweat rate, perceived exertion and perceived heat-stress did not differ between conditions. Pre-exercise hyperhydration significantly increased time to exhaustion and peak power output, compared with pre-exercise euhydration. We conclude that pre-exercise hyperhydration improves endurance capacity and peak power output and decreases heart rate and thirst sensation, but does not reduce rectal temperature during 2 h of moderate to intense cycling in a moderate environment when fluid consumption is 33% of sweat losses.
Subject(s)
Bicycling/physiology , Dehydration/prevention & control , Drinking/physiology , Exercise/physiology , Physical Endurance/physiology , Body Temperature , Climate , Cross-Over Studies , Female , Heart Rate , Humans , MaleABSTRACT
The aim of this study was to determine the effects of oral beta-hydroxy-beta-methylbutyrate (HMB) supplementation (3 g/d) on selected components of aerobic performance and body composition of active college students. Subjects were randomly assigned to either an HMB (n=8) or a placebo (PLA) group (n=8) for a 5-wk supplementation period during which they underwent interval training 3 times a week on a treadmill. Aerobic-performance components were measured using a respiratory-gas analyzer. Body composition was determined using dual-energy X-ray absorptiometry. After the intervention, there were significant differences (P<0.05) between the 2 groups in gains in maximal oxygen consumption (+8.4% for PLA and +13.4% for HMB). Regarding body composition, there were no significant differences. The authors concluded that HMB supplementation positively affects selected components of aerobic performance in active college students.
Subject(s)
Body Composition/drug effects , Exercise/physiology , Oxygen Consumption/drug effects , Valerates/pharmacology , Absorptiometry, Photon , Adult , Body Composition/physiology , Dietary Supplements , Energy Metabolism/drug effects , Energy Metabolism/physiology , Exercise Test , Female , Humans , Male , Oxygen Consumption/physiology , Respiration/drug effects , Valerates/administration & dosageABSTRACT
Calsequestrin is a large-capacity Ca-binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ([Ca2+](SR)) serving to maintain the driving force for SR Ca2+ release. Essentially all of the functional studies on calsequestrin to date have been carried out on purified calsequestrin or on disrupted muscle preparations such as terminal cisternae vesicles. To obtain information about calsequestrin's properties during physiological SR Ca2+ release, experiments were carried out on frog cut skeletal muscle fibres using two optical methods. One - the EGTA-phenol red method - monitored the content of total Ca in the SR ([Ca(T)](SR)) and the other used the low affinity Ca indicator tetramethylmurexide (TMX) to monitor the concentration of free Ca in the SR. Both methods relied on a large concentration of the Ca buffer EGTA (20 mM), in the latter case to greatly reduce the increase in myoplasmic [Ca2+] caused by SR Ca2+ release thereby almost eliminating the myoplasmic component of the TMX signal. By releasing almost all of the SR Ca, these optical signals provided information about [Ca(T)](SR) versus [Ca2+](SR) as [Ca2+](SR) varied from its resting level ([Ca2+](SR,R)) to near zero. Since almost all of the Ca in the SR is bound to calsequestrin, this information closely resembles the binding curve of the Ca-calsequestrin reaction. Calcium binding to calsequestrin was found to be cooperative (estimated Hill coefficient = 2.95) and to have a very high capacity (at the start of Ca2+ release, 23 times more Ca was estimated to initiate from calsequestrin as opposed to the pool of free Ca in the SR). The latter result contrasts with an earlier report that only approximately 25% of released Ca2+ comes from calsequestrin and approximately 75% comes from the free pool. The value of [Ca2+](SR,R) was close to the K(D) for calsequestrin, which has a value near 1 mm in in vitro studies. Other evidence indicates that [Ca2+](SR,R) is near 1 mM in cut fibres. These results along with the known rapid kinetics of the Ca-calsequestrin binding reaction indicate that calsequestrin's properties are optimized to buffer [Ca2+](SR) during rapid, physiological SR Ca2+ release. Although the results do not entirely rule out a more active role in the excitation-contraction coupling process, they do indicate that passive buffering of [Ca2+](SR) is a very important function of calsequestrin.