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The purpose of this study was to directly quantify the relative contribution of Ca2+ cycling to resting metabolic rate in mouse fast (extensor digitorum longus, EDL) and slow (soleus, SOL) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO2, μL/g wet weight/s) measured polarographically at 30°C was ~25% higher in SOL (0.61 ± .03) than EDL (0.46 ± .03). In order to quantify the specific contribution of Ca2+ cycling to resting metabolic rate, cyclopiazonic acid (CPA), a highly specific inhibitor of sarco(endo)plasmic Ca2+ ATPases (SERCAs), was added to the bath at different concentrations (1, 5, 10 and 15 μM). There was a concentration-dependent effect of CPA on oxygen consumption with increasing CPA concentrations up to 10 μM resulting in progressively greater reductions in muscle VO2. There were no differences between 10 and 15 μM CPA indicating that 10 μM CPA induces maximal inhibition of SERCAs in isolated muscle preparations. The relative (%) reduction in muscle VO2 in response to CPA was nearly identical in EDL (1 μM, 10.6 ± 3.0; 5 μM, 33.2 ± 3.4; 10 μM, 49.2 ± 2.9; 15 μM, 50.9 ± 2.1) and SOL (1 μM, 11.2 ± 1.5; 5 μM, 37.7 ± 2.4; 10 μM, 50.0 ± 1.3; 15 μM, 49.9 ± 1.6). The results indicate that ATP consumption by SERCAs is responsible for ~50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.
Skeletal muscle represents ~40% of body weight and accounts for ~20 to 30% of whole body basal metabolic rate (45,60). Like other cell types, a portion of basal metabolic rate in skeletal muscle can be attributed to the cycling of calcium ions across cell membranes but very few studies have attempted to quantify the relative contribution of Ca2+ cycling to resting muscle metabolism. Under basal conditions in skeletal muscle, sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) are responsible for maintaining a >104-fold Ca2+ concentration gradient across the sarcoplasmic reticulum (SR) membrane and for keeping the cytosolic free Ca2+ concentration ([Ca2+]f) at or below 100 nM (53). Under optimized states, SERCAs transport 2 mol of Ca2+ across the SR membrane upon the hydrolysis of 1 mol of ATP (15,31,51). Early estimates of the energy consumed by SERCAs in muscle under basal conditions were based on measurements of the rate of Ca2+ efflux from isolated SR vesicles and then the amount of ATP that would be required to reuptake that Ca2+ was calculated assuming an optimal Ca2+:ATP coupling ratio. This approach yielded values for the energetic cost of SR Ca2+ pumping of ~3.4 – 7% of resting muscle metabolism (13,27).
Although direct measurements are limited, there is evidence to suggest that these values largely underestimate the energetic cost of SR Ca2+ pumping in resting skeletal muscle. For example, another approach that has been employed to determine the relative contribution of SR Ca2+ cycling to resting energy expenditure in skeletal muscle is to measure the decrease in energy expenditure following exposure of the muscle to chemicals that indirectly inhibit SERCAs by inhibiting Ca2+ leakage from SR Ca2+ release channels (CRCs). Using this approach with direct calorimetry, Chinet et al. (1992) found that ~12–24% of resting energy expenditure in mouse soleus is related to Ca2+ cycling across the SR membrane. Similar experiments on mouse soleus and extensor digitorum longus (EDL) muscles showed that 18 – 22% of resting energy expenditure in both muscles is related to SR Ca2+ uptake (20). However, for several reasons these studies also likely underestimate the real contribution of SERCA activity to resting metabolic rate in skeletal muscle. First, there is no certainty that Ca2+ leakage through CRCs was completely blocked by the CRC inhibitors employed in those studies (2,3-butanedione monoxime, dantrolene sodium); second, SERCA pumps themselves are a very significant pathway for leakage of Ca2+ out of the SR (42); and third, SERCAs will be active in resting skeletal muscle as long as some Ca2+ is present in the cytoplasm, unless SERCA activity is directly inhibited.
Surprisingly, no study has used specific inhibitors of SERCAs to directly quantify the relative contribution of SR Ca2+ pumping to resting metabolic rate in intact skeletal muscle. Chinet et al. (1992) attempted this approach but found it problematic to separate the effects of blocking SR Ca2+ uptake from the resulting rise in [Ca2+]f and development of contracture on muscle energy expenditure. In order to accurately quantify the contribution of Ca2+ cycling to resting metabolic rate in skeletal muscle, SERCA activity must be inhibited directly in conjunction with myosin ATPase activity. This can be accomplished pharmacologically using the myosin-II inhibitor N-benzyl-p-toluene sulfonamide (BTS), which appears to selectively inhibit cross-bridge cycling but only in fast-twitch muscles (11,49,58). An alternative method that should be equally effective in fast-and slow-twitch muscles, is to simply pre-stretch the muscle to eliminate filament overlap prior to inhibiting SERCA activity (6,43,56). Here, we used cyclopiazonic acid (CPA), a highly specific inhibitor of SERCA activity (23,48), in conjunction with the pre- stretch technique, in order to more accurately quantify the contribution of SR Ca2+ pumping to resting metabolic rate in mouse fast- and slow-twitch skeletal muscle. For comparison, some experiments with fast-twitch muscles were also performed with BTS.
A total of 93 sexually mature (8–12 weeks) male C57BL/6 mice weighing an average of 23.8 ± 0.4 g were used in this study. Animals were housed 2–4 per cage in an environmentally controlled room with a standard 12:12 light/dark cycle and allowed access to food (Tekland 22/5 Rodent Diet, Harland- Tekland, Madison, WI) and water ad libitum. Animals were euthanized and the EDL or soleus muscles were carefully removed from both hindlimbs with tendons intact. Due to the time required for each experiment, only 1 muscle (EDL or soleus) could be assessed from each mouse. In total, 40 EDL muscles and 40 soleus muscles were randomly assigned to 1 of 5 experimental conditions consisting of control (ie. no CPA) and four CPA conditions (i.e. 1, 5, 10, and 15 μM). The control trials were conducted with the CPA solvent, dimethyl sulfoxide (DMSO). An additional 5 mice were used for EDL experiments with BTS and an additional 8 mice were used for experiments to assess the effects of time on muscle VO2 in pre-stretched resting soleus and EDL. The study was approved by the Animal Care Committee at the University of Waterloo and all procedures were performed in accordance with the Canadian Council on Animal Care.
The isolated EDL and soleus muscles were mounted in the TIOX tissue bath system (Hugo Sachs Electronik-Harvard Apparatus, Germany) for the measurement of resting muscle oxygen consumption (VO2). The TIOX tissue bath system consists of a moveable platform which supports the muscle and a force transducer (F30 type 372) for measuring contractile force. A movable jacketed tissue chamber that is fitted with a temperature probe and a Clarke type PO2 electrode (model 1302) are also mounted onto the platform. The jacketed reservoir is connected to a thermocirculator to maintain constant chamber temperatures. Two parallel platinum plate electrodes are located on either side of the muscle for stimulation enabling simultaneous measurement of force production and oxygen consumption. All components of the TIOX system are connected and controlled by individual PLUGSYS-modules and the data acquired from the individual modules are compiled, filtered, displayed and stored by the HSE-HA ACAD data acquisition software (Hugo Sachs Electronik-Harvard Apparatus, Germany).
Using 4/0 surgical silk, the isolated muscles were mounted onto a fixed lower hook attached to the platform and a top hook which passes through a hole in the lid of the platform and connects to the external force transducer. After mounting the muscle, the tissue chamber was closed to the atmosphere by gently raising it to the platform and tightly sealing it with screws and wing nuts to fasten the chamber in place. The system was made air tight by closing off the hole in the lid of the platform using grease (baysilone paste, GE Bayer Silicones). The chamber was then filled with Ringer solution (121 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 24 mM NaHCO3, 5.5 mM glucose and 0.1 mM EDTA, pH 7.3), which was pre-heated to 30°C and aerated with 95% oxygen and 5% carbon dioxide, through a port on the bottom of the bath until it overflowed out of the ventilation cap in the lid of the platform which was then sealed. The contents of the chamber were constantly stirred with a magnetic bar and stirrer located directly under the oxygen electrode to ensure consistent oxygen concentration throughout the solution. The muscle length was adjusted to achieve optimal length (Lo) for twitch force production and then the muscle was given 10 minutes to equilibrate inside the chamber prior to initiating data collection.
PO2 measurements were recorded every 4 seconds during each of 3 separate experimental trials at 30°C designed to quantify resting muscle VO2 and SERCA pump energetics. The PO2 in the bath (~620 mmHg) should enable adequate diffusive oxygen supply to support resting muscle metabolism of mouse soleus and EDL (8); however, to prevent the formation of hypoxia in the core of the muscle, all experiments were terminated before the PO2 of the Ringer solution fell below 580 mmHg. First, the decrease in PO2 of the Ringer solution was recorded in the presence of a resting muscle at Lo for 30 minutes. Secondly, the muscle was lengthened to ~1.42 Lo to eliminate sarcomere overlap and prevent activation of myosin ATPase activity during CPA trials. The bath was then emptied and refilled with fresh Ringer solution and the PO2 was recorded for 30 minutes. Lastly, with the muscle still pre-stretched, the bath was emptied and refilled with fresh Ringer solution and either CPA (1, 5, 10 or 15 μM) or vehicle (DMSO) was added with a Hamilton syringe through the vent in the lid of the platform and PO2 was recorded for 30 minutes. Following this final 30 minute period, the muscle was returned to its optimal length and a single twitch was applied to ensure continued viability following CPA exposure. In order to further demonstrate the viability of each muscle preparation and the adequacy of O2 diffusion, after the final twitch, muscles were removed and frozen immediately in liquid nitrogen for later assessment of high energy phosphates (ATP, PCr) and muscle metabolites (IMP, CR). Since the CPA trial was always performed last, it was also important to demonstrate that muscle VO2 in resting soleus and EDL is constant over the full duration of the experiment (i.e. 90 min). Therefore, in a subset of experiments (n=8), muscle VO2 in pre-stretched resting soleus and EDL was recorded for 90 min in the absence of CPA (3 × 30 min trials). Finally, in a subset of experiments with EDL (n=5), BTS was used to inhibit myosin ATPase activity as opposed to pre-stretch. For these experiments, following the measurement of resting VO2 for 30 min, EDL muscles were incubated with 25 μM BTS (in DMSO) for 45 min to inhibit myosin ATPase activity, followed by incubation with both 25 μM BTS and 10 μM CPA for an additional 30 min. The muscles were maintained at Lo for all of these incubations.
The muscle VO2 was calculated by multiplying the measured drop in partial pressure of oxygen (PO2) with time by the solubility of oxygen in Ringer’s solution at 30°C and the chamber volume (12.7 mL). The solubility of oxygen at 30°C was calculated to be 0.001203 M/atm using the following equation:
where kH is solubility constant of O2 at experimental temperature, kHo is the solubility constant of O2 in pure water at standard temperature of 25°C (0.0013 M/atm), H is the enthalpy to dissolve O2 (gas) in water (11.7 kJ/mol), R is the gas constant (0.008312 kJ/mol/K), T is the experimental temperature (30°C or 303.15 K) and To is standard temperature (25°C or 298.15 K).
In reality, the TIOX system is not completely closed to the atmosphere resulting in leak of oxygen out of the solution even with no muscle mounted inside the chamber. Therefore, in order to account for the oxygen leak, blank trials were done at the beginning and at the end of daily data collection. A blank trial measures the rate of oxygen loss in an empty chamber (i.e. no muscle). The average daily rate of oxygen leak is then subtracted from the oxygen loss in the presence of the muscle to give the muscle VO2. A sample PO2 tracing of a leak, rest, and CPA trial is depicted in Fig. 1. The average rate of oxygen leak (μL/s) over the course of the experiment was found to be 0.010 ± .00002 with a coefficient of variation of 5.6%. Muscle VO2 is reported at 23°C and 1 atm and expressed relative to muscle wet weight (μL/g wet weight/s).
Muscle metabolites were extracted from freeze dried tissue (25,26) and both adenosine triphosphate (ATP) and inosine monophosphate (IMP) were measured using ion-pair reversed-phase high-performance liquid chromatography (HPLC) procedures originally developed by Ingebretson et al. (32) and subsequently modified by Green et al. (1989). Measurements of phosphocreatine (PCr) and creatine (Cr) were accomplished using fluorometric procedures (37) with modifications (24).
For Western blotting, homogenates were prepared with whole soleus and EDL muscles diluted 19:1 (vol/wt) in an ice-cold buffer containing 250 mM sucrose, 5 mM HEPES, 10 mM NaN3, and 0.2 mM PMSF (pH 7.5) by using a handheld glass homogenizer (Duall 20, Kontes). Total protein concentration of the homogenates was measured by the method of Lowry, as modified by Schacterle and Pollock (46).
SR membrane fractions were isolated from white portions of gastrocnemius muscles and whole heart from 3–4 month old Sprague-Dawley rats as described in earlier publications (47,54). Subsequently, SERCA1a and SERCA2a were immunoprecipitated from the gastrocnemius and heart fractions, respectively (22). The purified protein (SERCA1a or SERCA2a) was confirmed through polyacrylamide gel electrophoresis and Ponceau (Sigma) staining. The purified protein was also differentiated with different amounts of BSA standard through SDS-PAGE, transferred to PVDF membrane, stained with Ponceau, and quantified using a bio-imaging system and densitometric analysis with the GeneTools software (Syngene).
Western blotting was performed to determine the content and relative proportion of SERCA1a and SERCA2a in EDL and soleus muscles. Homogenates (SERCA1a, 0.075 and 0.25 μg total protein for EDL and soleus, respectively; SERCA2a, 4 and 1 μg total protein for EDL and soleus, respectively) were applied to 7% polyacrylamide gels, and proteins were separated using standard SDS-PAGE protocols (34) and then transferred to polyvinlyidene difluoride (PVDF) membranes (BIO-RAD). After blocking with a 5% skim milk suspension, the membranes were incubated for 1 hr with either anti-SERCA1a monoclonal antibody A52 (59) or anti-SERCA2a antibody 2A7-A1 (Affinity Bioreagents Inc.). Then, after washing in Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20 (TBS-T), the membranes were treated with horseradish peroxidase-conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology). Membranes were washed in TBS-T, and the signals were detected with an enhanced chemiluminescence kit (GE Healthcare) using a bio-imaging system and densitometric analysis was performed using the GeneTools software (Syngene). For quantification of SERCA1a and SERCA2a, standard curves were plotted (density versus known amounts of pure protein [SERCA1a or SERCA2a]) and the amounts of SERCA1a or SERCA2a in a given sample (molecules/pg total protein) were determined from standard curves on the same Western blot (42).
A two-way ANOVA was employed to analyze the muscle type specific (soleus vs. EDL) changes in VO2 in response to different concentrations of CPA (DMSO only vs. 1 μM vs. 5 μM vs. 10 μM vs. 15 μM). Student’s t test was utilized for all other comparisons. The significance level was set at 0.05, and when appropriate, a Newman-Keuls post hoc test was used to compare specific means. Values are means ± SE.
The twitch and passive force responses recorded for an EDL muscle throughout a single experiment are shown in Fig. 2. The force responses for soleus were nearly identical to the response shown in Fig. 2 (data not shown). Twitch force was measured at the start of each experiment at Lo (A) and then at progressively longer muscle lengths up to ~1.42 Lo (B) where minimum twitch force was achieved (~7% of twitch force measured at Lo). There was a slow but steady decline in passive tension during pre-stretch trials (without CPA) probably due to relaxation of series and parallel elastic elements within the muscle. Although active twitch force could not be completely eliminated by pre-stretch, importantly passive muscle tension remained fairly constant during CPA trials (D) indicating minimal activation of myosin ATPase activity. Twitch force measured at Lo at the end of each experiment following the CPA trial (E) was ~76% of initial twitch force measured at Lo. These force responses are similar to what has been reported previously for mouse EDL under similar conditions (8).
The rates of oxygen consumption of the soleus and EDL muscles were measured at optimal length for 30 minutes at 30°C (n=40 each). The average resting VO2 (μL/g wet weight/s) at 30°C was ~25% higher (P<0.05) in soleus (0.61 ± .03) compared with EDL (0.46 ± .03) (Fig. 3, open bars). Pre-stretching the muscles had no effect (P>0.1) on muscle VO2 for either soleus or EDL muscles (Fig. 3). Resting VO2 of both soleus and EDL was stable with time as the rates measured from 0–30 min of incubation were not different (P>0.05) than measurements taken from 60–90 min of incubation (Fig. 4).
The percent contribution of Ca2+ pumping by SERCAs to resting VO2 was calculated by dividing the difference between the pre-stretch trial and the CPA trial by resting VO2 (at Lo). A range of CPA concentrations (1, 5, 10, and 15 μM) was used to determine the optimal concentration that would elicit maximal inhibition of SERCA activity without compromising mitochondrial function, which was assessed indirectly by measuring muscle high energy phosphates (ATP, PCr) and metabolites (IMP, Cr). Control trials were conducted with the CPA solvent DMSO which had no effect on muscle VO2 (P>0.1) in either EDL or soleus muscles (Fig. 5). In both EDL and soleus, there was a concentration-dependent effect of CPA on oxygen consumption with increasing CPA concentrations up to 10 μM, which resulted in progressively greater reductions in muscle VO2 (Fig. 5). There were no differences (P>0.1) between 10 and 15 μM CPA indicating that 10 μM CPA induces maximal inhibition of SERCAs in isolated muscle preparations. The relative (%) reduction in muscle VO2 in response to CPA was nearly identical in EDL (1 μM, 10.6 ± 3.0; 5 μM, 33.2 ± 3.4; 10 μM, 49.2 ± 2.9; 15 μM, 50.9 ± 2.1) and soleus (1 μM, 11.2 ± 1.5; 5 μM, 37.7 ± 2.4; 10 μM, 50.0 ± 1.3; 15 μM, 49.9 ± 1.6). Importantly, when BTS was used to inhibit myosin ATPase activity in EDL muscles instead of pre-stretch, similar results were obtained where 10 μM CPA caused a 50.9 ± 7.4% reduction in muscle VO2 (Fig. 6).
High energy phosphates (ATP, PCr) and metabolites (IMP, Cr) were measured in both EDL and soleus muscles which were freeze clamped following the CPA trial in the TIOX. Metabolite measurements were made on the freeze-dried tissue. Fig. 7 shows the high energy phosphate and metabolite levels for the DMSO and 10 μM CPA conditions. There were no differences (p>0.1) between the DMSO and 10 μM CPA condition for any of the metabolites measured in either EDL or soleus.
The content and relative distribution of SERCA pump isoforms (SERCA1a + SERCA2a) were compared in EDL and soleus by quantitative Western blotting as described under “Experimental Procedures” (e.g. Fig. 8). The amount of SERCA1a (mg/g total muscle protein) was ~5.2-fold higher (P<0.05) in EDL (46 ± 4.3) compared with soleus (8.9 ± 0.5) and the amount of SERCA2a (mg/g total muscle protein) was not significantly different (p=0.08) between EDL (0.51 ± 0.07) and soleus (0.65 ± 0.18). The number of SERCA1a and SERCA2a pumps per fg total muscle protein was 254 ± 23 and 2.8 ± 0.4, respectively, in EDL and 49 ± 3 and 3.6 ± 1, respectively, in soleus. Therefore, the ratio of SERCA1a:SERCA2a was ~91:1 in EDL compared with ~14:1 in soleus and in total there were ~4.9-fold more SERCA pumps (SERCA1a + SERCA2a) in EDL than soleus.
Few studies have attempted to quantify the specific contribution of SR Ca2+ pumping to resting metabolic rate in muscle. This is the first study to use a direct inhibitor of SERCA (i.e. CPA) to quantify the contribution of SERCA activity to resting energy expenditure in mouse skeletal muscle. The approach used in previous studies involved the use of CRC blockers (i.e. BDM and dantrolene) to inhibit SR Ca2+ release thus lowering [Ca2+]f and indirectly inhibiting SERCA activity (12,20). We hypothesized that this approach likely underestimates the ATP turnover by SERCAs in resting muscle because even if BDM and dantrolene completely inhibit Ca2+ release through CRCs, SERCAs would remain active and contribute to energy consumption at rest (4,42). Indeed, our results indicate that ATP consumption by SERCAs is responsible for ~50% of resting metabolic rate in both EDL and soleus, a quantity that is at least 2-fold higher than previously reported (12,20). However, consistent with our results, Dulloo et al. (1994) found that the relative (%) contribution of Ca2+ dependent ATP turnover to resting metabolic rate was equal between EDL and soleus.
In this study, we measured rates of oxygen consumption at 30°C in resting mouse EDL and soleus using the TIOX tissue bath system. With the exception of at least one other study (14), our finding that resting VO2 was ~25% higher in soleus compared with EDL is in agreement with most previous studies (18,20,56). The differences in muscle VO2 we observed between EDL and soleus correspond to an absolute ATP turnover rate by SERCAs at the whole muscle level that is ~24% lower in EDL (159 ± 8.8 nmol/g/s) than SOL (209 ± 11.5 nmol/g/s). Assuming that [Ca2+]f is constant in resting skeletal muscle, basically 2 main factors determine the ATP turnover rate by SERCAs in resting muscle: 1) the rate of Ca2+ leakage out of the SR and 2) the stoichiometry of Ca2+ uptake by SERCAs (i.e. Ca2+/ATP ratio). Measurements in mechanically skinned fibres from rat EDL and soleus indicate that under normal resting conditions, Ca2+ leaks out of the SR at approximately the same absolute rate in both muscles (38,39). CRCs and SERCAs represent the major pathways for Ca2+ leakage from the SR, although SERCAs appear to be of primary importance in this regard (38,39,42). It is well known that the density of CRCs is ~2–3-fold higher in EDL than soleus (3,19) and the SERCA density is ~3–8-fold higher in EDL than soleus (42,55,57). In this study, we found that mouse EDL contained ~4.9-fold more SERCA pumps than soleus. Therefore, it might seem surprising that the rate of Ca2+ leakage from the SR is the same in EDL and soleus; however, Murphy et al. (2009) recently showed that the high content of calsequestrin in EDL muscle fibres helps to keep the free [Ca2+] in the SR at sufficiently low levels to prevent high rates of Ca2+ leakage through the high density of SERCA pumps. Assuming that rat and mouse fibres are comparable and the SR Ca2+ leak rates are the same in mouse EDL and soleus, the differences we observed between EDL and soleus in ATP turnover by SERCAs would therefore suggest that Ca2+ uptake by SERCAs is less efficient in soleus than EDL, at least under resting conditions.
A 2:1 ratio of Ca2+ transport to ATP hydrolysis by SERCAs is considered to be optimal as it corresponds to the stoichiometry of two Ca2+ binding sites and one ATP binding site on each SERCA pump (40,52). However, our values for ATP turnover by SERCAs strongly suggest that the Ca2+:ATP coupling ratio is actually much lower than 2:1 under physiological conditions. If the coupling ratio was 2 Ca2+:1ATP, the rate of Ca2+ uptake (which is equal to the rate of SR Ca2+ leakage in resting muscle) would be ~318 nmol/g/s in EDL and ~418 nmol/g/s in soleus which is ~22–26-fold higher than what has been measured in mechanically skinned EDL and soleus fibers from the rat (35,38,39). Taking into account the ATP turnover rates we measured in this study and the SR Ca2+ leak rate reported by Lamb and Cellini (1999) for rat EDL (which would be equal in soleus), we can calculate coupling ratios of ~0.09 Ca2+/1ATP in EDL and ~0.07 Ca2+/1ATP in soleus. Interestingly, the calculated coupling ratios for mouse EDL and SOL are similar to measurements made in SR preparations isolated from canine cardiac muscle (10,21), human skeletal muscle (28) and various rat skeletal muscles (29). However, we are likely underestimating the true SR Ca2+ leak rates in EDL and soleus under the experimental conditions employed in this study; therefore, these calculations also likely underestimate the coupling ratios in EDL and soleus. The amount of slippage/leakage of SERCA pumps increases with increasing temperature (16) and [ADP] (38). Compared with the skinned fiber study by Lamb and Cellini (1999), both the experimental temperature (23 vs. 30°C) and the [ADP] concentration in the fibers (~0.1 μM vs. ~10 μM) were higher in the present study. Higher coupling ratios have been reported for rabbit skeletal muscle preparations and, in contrast with our calculations, the Ca2+/ATP ratio was higher in slow-twitch muscle SR vesicles (i.e. 0.98 Ca2+/1ATP) compared with fast-twitch skeletal muscle SR vesicles (i.e. 0.37 Ca2+/1ATP) (44). The coupling ratio differences found between slow and fast muscle were attributed to differences in SERCA isoform expression since SR vesicles from rabbit fast-twitch skeletal muscle only expressed SERCA1a whereas vesicles from rabbit slow-twitch skeletal muscle expressed preferentially SERCA2a (44). In contrast, we found that over 90% of the SERCA molecules found in both mouse EDL and soleus were SERCA1a which could explain why the calculated coupling ratios for each muscle are closer to the ratios that were reported for rabbit fast-twitch skeletal muscle vesicles in the study by Reis et al. (2002).
One possible explanation for reduced Ca2+ transport efficiency of SERCAs in mouse soleus compared with EDL is related to differences in the expression of both phospholamban (PLN) and sarcolipin (SLN), two homologous SERCA binding proteins that regulate SERCA activity by lowering the apparent Ca2+ affinity of the pump. Comparisons between wild type and PLN-null mice have shown that PLN also decreases the Ca2+:ATP coupling ratio in cardiac SR preparations, specifically at low [Ca2+]f (21). Likewise, reconstitution experiments have shown that SLN uncouples ATP hydrolysis from Ca2+ transport by SERCAs (51) and increases the amount of heat released per mol of ATP hydrolyzed (41). These results could be explained by SLN causing an increased rate of slippage on SERCAs (41,51) which would decrease the fraction of energy released during ATP hydrolysis that is converted into work and increase the amount of heat released (16,17). Both PLN and SLN are expressed abundantly in mouse soleus (5,50) but not EDL. This could explain why the absolute ATP turnover by SERCAs is higher in mouse soleus than EDL under resting conditions but since resting VO2 is higher in soleus, the relative contribution of SERCA activity to muscle VO2 is the same in EDL and soleus.
We are confident in the validity of our results and our conclusion that SERCA pumps account for ~50% of resting muscle metabolic rate, for several reasons. First, we used a range of CPA concentrations to ensure maximal inhibition of SERCA activity was achieved in the isolated muscle preparations. The reduction in muscle VO2 due to CPA exposure reached a plateau at 10 μM CPA in both EDL and soleus suggesting that SERCAs were completely inhibited at this concentration. If SERCA inhibition was incomplete, even at 15 μM CPA, this would mean that if anything we could be underestimating the contribution of SERCA activity to resting muscle VO2. Secondly, to prevent the expected contracture of the muscle following the addition of CPA, the pre-stretch method was used to inhibit cross-bridge formation. Importantly, pre-stretch had no effects on muscle VO2 in either EDL or soleus suggesting that SERCA activity was not altered by stretch. As further support for this view, we have found that 10 μM CPA inhibits resting VO2 in mouse EDL by ~50% regardless of whether pre-stretch or BTS was used to inhibit myosin ATPase activity. It is also important to point out that even if pre-stretch and/or BTS did not completely prevent activation of myosin ATPase activity as a result of elevated [Ca2+]f during CPA trials, this would mean that we are in fact underestimating the contribution of SERCAs to resting muscle metabolic rate. Similarly, we could be underestimating the contribution of SERCAs to resting muscle metabolism if [Ca2+]f was increased during CPA exposure and if Ca2+ directly increased mitochondrial respiration (9). Third, 10 μM CPA should have highly specific effects on SERCAs in skeletal muscle preparations (30,48). Nevertheless, we assessed muscle metabolite levels in EDL and soleus as a marker of mitochondrial function in order to rule out the possibility that the effects of CPA on muscle VO2 were due to impaired oxidative phosphorylation. CPA had no effects on muscle high energy phosphates (ATP, PCr) or related by-products (IMP, Cr) and the metabolite concentrations found in both EDL and soleus are within the normal range for resting mouse muscle (33). These results indicate that diffusive O2 supply in these experiments was adequate to support resting metabolism and importantly we can rule out the possibility that the effects of CPA on muscle VO2 involved impairment of mitochondrial O2 consumption. Finally, since resting VO2 in both EDL and soleus was stable for at least 90 min, we can conclude that the effects of CPA on muscle VO2 were specifically due to inhibition of SERCAs and not a result of a coinciding progressive decrement in muscle metabolic rate with time.
In adult C57BL/6 mice, the EDL contains >90% type II fibers (mostly IIB) and <10% type I fibers whereas the soleus contains ~50% type I and type II fibers (mostly IIA) (1,2). Our results do not identify the contribution of the different fiber types to muscle VO2 or the relative contribution of SERCA activity to the metabolic rate of the different fiber types. It is worth noting that Murphy et al. (2009) reported that virtually all of the SERCA1a found in rat soleus muscles were present in the fast-twitch fibers. Interestingly, the relative abundance of SERCA1a and SERCA2a mRNA levels in mouse (strain 129sV/Swiss 50/50) soleus also appears to match closely the fiber type distribution (i.e. 58% SERCA2 and 42% SERCA1) (55). Importantly, the protein contents of the different SERCA isoforms in mouse soleus have not been quantified previously. In this study, SERCA1a and SERCA2a were quantified by comparing the Western blot band intensities of mouse muscle samples relative to purified proteins obtained from rat muscles. Although it is quite possible that the SERCA antibodies were differentially sensitive to the mouse and rat SERCA proteins, which would have caused an unknown level of error in the quantification, based on our results showing that over 90% of the SERCA molecules found in mouse soleus were SERCA1a, it is likely that SERCA1a is present in at least some type I mouse soleus fibers. Future studies should examine SERCA isoform protein expression in individual fiber types from mouse muscles to test this postulate.
It is well known that SERCAs play an important role in thermogenesis (9,16) but SERCAs may also play an important role in the regulation of whole body energy balance. Assuming that skeletal muscle accounts for 30% of basal metabolic rate in mice (45) and that the results of our current study can be extrapolated to all mouse muscles at physiological temperature, SERCA activity can explain ~15% of whole animal resting VO2. It is also well known that SERCAs contribute ~30–40% to the energy cost associated with muscle contraction (for review see (7)). Therefore, given that basal metabolic rate and physical activity account for ~60 and 30% of total daily energy expenditure, respectively, (36,45) and that skeletal muscle accounts for ~90% of the increased energy requirements associated with physical activity (45), SERCAs in skeletal muscle may explain ~17–20% of whole body total daily energy expenditure.
In summary, the results from this study support the conclusion that ATP consumption by SERCAs is responsible for ~50% of resting metabolic rate in both mouse EDL and soleus muscles at 30°C. These results have important implications for the basic understanding of muscle energetics and suggest that the SERCA pump in skeletal muscle represents an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity and other metabolic disorders.
This work was supported by Grant MOP-86618 from the Canadian Institutes of Health Research (to A. R. T.). S. M. Norris was supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship M award. E. Bombardier, I. C. Smith and C. Vigna are all supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship D award. We thank Dr. Richard Hughson and Dr. James Rush, for helpful comments on the manuscript.