Excitation–contraction coupling represents a coordinated sequence of events that results in skeletal muscle contraction. Ultimately, three key enzymes hydrolyze ATP during this process to enable repeated muscle contraction to occur, including the Na⁺/K⁺ ATPase (NKA), the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA), and myosin ATPase (¹). Although myosin ATPase consumes the greatest amount of ATP during muscle contraction, and is directly responsible for generating muscle force, repeated contractions cannot occur without NKA and SERCA activity (^2,3).

In the past several decades, it has been well established that SERCA has an essential role in processes linked to muscle contraction, fatigue, and energy metabolism (^3–5). SERCA is a transmembrane protein localized in the sarcoplasmic reticulum (SR) membrane that uses the energy derived from the hydrolysis of ATP to transport 2 Ca²⁺ ions across the SR membrane (⁶). Because the binding of Ca²⁺ to troponin induces cross-bridge cycling, SERCA-mediated sequestering of Ca²⁺ is required for muscle relaxation, while at the same time reestablishes SR Ca²⁺ stores necessary for a subsequent muscle contraction (⁶). More recently, SERCA flux has also been linked to energy expenditure and the susceptibility for the development of obesity through sarcolipin-mediated “uncoupling” of SERCA (^4,7). These data highlight a fundamental role for SERCA in controlling metabolic homeostasis across a range of cellular stresses.

The NKA is a transmembrane protein localized in the sarcolemma and transverse tubules (t-tubules) where it contributes substantially to the regeneration of resting membrane potential after depolarization-induced muscle contraction (^8,9). In this respect, despite the small contribution to total energy expenditure (~5% during contraction), NKA plays a key role in skeletal muscle metabolism (^8,10). In contrast to SERCA, which has been extensively studied, and despite the necessity of NKA for proper skeletal muscle function, very little information exists regarding the function/regulation of this enzyme. The paucity of information likely stems from methodological constraints, as small amounts of contamination from other ATPases (e.g., myosin ATPase and SERCA) prevent the assessment of NKA activity in muscle homogenates (¹¹). Consequently, contemporary approaches to ascertain NKA enzymatic function require the purification of muscle samples, which requires substantial amounts of muscle tissue (^12,13). This approach results in very low recovery of total protein and has likely contributed to the inadequate examination of NKA function in human skeletal muscle.

Therefore, we sought to determine whether pharmacological inhibitors of myosin ATPase and SERCA could be used to establish NKA activity assays in muscle homogenates. Importantly, we established the accuracy and feasibility of determining NKA activity in small rodent muscle samples (~10 mg) and in human skeletal muscle.

METHODS

Animals

Sedentary C57BL/6 wild-type male mice (n = 6) were housed in a temperature- and light-controlled room and given free access to food and water at the University of Guelph (Guelph, ON, Canada). Soleus and extensor digitorum longus (EDL) muscles were obtained from animals anesthetized with sodium pentobarbital (60 mg·kg⁻¹). All procedures in this study were approved by the University of Guelph Animal Care Committee.

Human subjects and muscle biopsy

Three healthy older adult females (65.6 ± 2.3 yr, 1.61 ± 0.03 m, 62.1 ± 4.1 kg) participated in the study. Written informed consent was received from each subject after a detailed explanation of the experimental protocol and any associated risks. On the day of the experiment, subjects reported to the laboratory for a resting skeletal muscle biopsy under local anesthesia (2% lidocaine without epinephrine) from the vastus lateralis muscle, using the percutaneous needle biopsy technique described by Bergström (¹⁴). The experiments were approved by the University of Guelph and McMaster University Research Ethics Boards according to the principles expressed in the Declaration of Helsinki.

ATPase activity assays

Muscle samples from mice (5–10 mg) and humans (30–50 mg) were washed (two to three times with homogenizing buffer on a sterilized gauze), weighed, diluted (101:1 and 11:1 [vol/wt], respectively) in ice-cold buffer containing (in mM) 0.2 PMSF, 250 sucrose, 5 HEPES, and 0.2% NaN₃ (pH 7.5), and homogenized using a handheld glass pestle and glass mortar. The homogenate was separated into two to five aliquots, flash-frozen in liquid nitrogen, and stored at −80°C for future analyses. The bicinchoninic acid assay was performed to determine protein content of homogenate.

Measurement of NKA activity

The measurement of NKA activity was performed by an enzyme-coupled method using an ATP regenerating assay, which uses the pyruvate kinase (PK) and the lactate dehydrogenase (LDH) reactions to link ATP hydrolysis with the degradation of NADH (¹⁵):

After freeze–thawing muscle homogenates five times with liquid nitrogen, an aliquot (15 μL of 60–191 μg of protein) was incubated for ~10 min at 37°C in a buffer containing (mM) 50 Tris-HCl, 15 KCl, 1 EGTA, 4 MgCl₂, 2.5 PEP, 4 MgATP, and 5 KN₃ (pH 7.4). Before starting the reaction, 18 U·mL⁻¹ (3.5 μL) of PK (Sigma-Aldrich, St. Louis, MO) and LDH (Sigma-Aldrich) and 0.3 mM NADH (Sigma-Aldrich) were added to a cuvette containing ~1.5 mL of reaction buffer. Assays were performed using a fluorometer (Lumina; Thermo Scientific, Fisher, Hampton, NH) set at an excitation wavelength of 340 nm and an emission wavelength of 460 nm. The reaction was started by the addition of 80 mM NaCl in the presence of 25 μM blebbistatin (BLEB), a specific inhibitor of myosin ATPase (¹⁶). NKA activity was calculated from the negative slope (fluorescence per second) of NADH from a standard curve established with the same reaction conditions (Fig. 1). NKA maximal activity was obtained by subtracting ATPase activity in the presence and absence of 2 mM ouabain (ouab) (^13,17), using the following equation:

where Y_[Na] is the change in fluorescence in the presence of 80 mM Na, Y_[ouab] is the change in fluorescence after the addition of 2 mM ouab, and m is the slope obtained from standard curve.

Measurement of SERCA activity

Total SERCA activity was measured using a fluorometric assay based on the method developed by Simonides and van Hardeveld (¹⁶). Specifically, the reaction buffer contained (mM) 200 KCl, 20 HEPES, 15 MgCl₂, 1 EGTA, 10 NaN₃, 5 ATP, and 10 PEP (pH 7.0 at 37°C). Before starting the reaction, 18 U·mL⁻¹ LDH, 18 U·mL⁻¹ PK, 5 μL homogenate, and 0.2 mM NADH were added to a cuvette containing ~1.5 mL of reaction buffer in the presence or absence of 25 μM BLEB (B0560; Sigma, St Louis, MO). Assays were performed at 37°C using a fluorometer (Lumina; Thermo Scientific, Fisher, Hampton, NH) set at an excitation wavelength of 340 nm and an emission wavelength of 460 nm. SERCA activity was assessed by adding 15 μL of 10 mM CaCl₂ every 2 min. SERCA activity increases with free Ca²⁺ concentrations ([Ca²⁺]f) until a plateau occurs, indicating maximal activity is reached. The [Ca²⁺]f corresponding to each CaCl₂ addition was determined using an online calculator (¹⁸) with the following constants for input fields: ionic strength 0.28, pH 7.0, temperature 37°C, 1 mM EGTA, 5 mM ATP, and 15 mM Mg²⁺. The rate of ATPase activity was calculated from the negative slope (fluorescence per second) of NADH from a standard curve established with the same reaction conditions. SERCA activity was obtained by subtracting ATPase activity in the presence and absence of 40 μM cyclopiazonic acid (CPA), a specific inhibitor of SERCA (^19,20), from total ATPase activity at different [Ca²⁺]f.

To obtain the kinetic properties of the enzyme, SERCA activity was plotted against the negative logarithm of [Ca²⁺]f (pCa). V_max (μmol⋅min⁻¹⋅mg⁻¹ protein) represented the peak value, whereas Ca50 represented the measurement obtained from a sigmoid fit of the data, which yielded 50% of V_max. The Hill coefficient was determined through a nonlinear regression (variable slope) with Prism software (GraphPad Software, Inc., La Jolla, CA) by using a portion of the curve that corresponded to between 10% and 90% of maximal activity, using the following sigmoidal dose–response equation:

where Y is the plateau, Y_bot is the value at the bottom of the plateau, Y_top is the value at the top, log Ca50 is the concentration that gives a response halfway between Y_bot and Y_top, x is the [Ca²⁺]f corresponding to each CaCl₂ addition, and n_H is the Hill coefficient.

Statistics

Data are presented as means ± SEM. A Student’s t-test (two-way) was used to detect differences with/without pharmacological inhibitors and between SOL and EDL muscles for SERCA and NKA activities. Coefficient of variation (CV) from duplicate measurements was calculated as [(SD/mean) × 100]. Significance was determined at P < 0.05. GraphPad Prism program, version 7.0 (GraphPad Software, Inc.), was used for all statistical analyses.

RESULTS

Myosin ATPase, a significant contributor to background activity in the NKA assay

We first aimed to determine the feasibility of measuring NKA activity in muscle homogenates. It has been suggested that the high background observed for NKA assays performed in muscle homogenates is due to SERCA and myosin ATPase activities (^11,21). On this basis, we sought to examine if the inhibition of these enzymes would decrease the background previously observed for NKA assays performed in muscle homogenates. First, we performed a titration curve with different concentrations of BLEB and observed that the addition of ≥25 μM of BLEB reduced background noise similarly (Fig. 2A). Thus, we used 25 μM BLEB for the following experiments, which importantly reduced background NKA activity approximately threefold (Fig. 2A, B, C), but the addition of 40 μM CPA had no effect (Fig. 2C). We next determined if our preparation was sensitive enough to detect a change in NKA activity after the addition of saturating concentrations of NKA substrate NaCl (80 mM), and whether these changes were specific to NKA enzyme. First, we observed a substantial increase in the slope after the addition of 80 mM NaCl (from 19.1 to 26.5 RFU/min/100), and the subsequent addition of 2 mM ouab, a specific inhibitor of NKA (^13,17), decreased the slope to basal levels (20.0) (Fig. 1B). Finally, to validate the responsiveness of the system with our preparation, we performed NKA maximal activity assays in soleus and EDL muscles. The maximal NKA activity of soleus (31 ± 6 μmol⋅min⁻¹⋅g⁻¹ protein) was significantly higher than EDL (15 ± 5 μmol⋅min⁻¹⋅g⁻¹ protein) (P < 0.01; Fig. 2D). The CV for the NKA assays was 6.2% ± 2.2%. Altogether, these results suggest that myosin ATPase is the major contributor to background activity of NKA assay in skeletal muscle homogenates, and the presence of BLEB, which inhibits myosin ATPase, enables the determination of NKA activity in muscle homogenates.

Inhibition of myosin ATPase does not affect kinetic properties of SERCA

To get a specific reading of SERCA kinetic function from skeletal muscle homogenates, the inhibition of Ca²⁺-dependent and Ca²⁺-independent ATPases is essential. During the measurement of SERCA activity, the major contributor to background activity is thought to be myosin ATPase (²²), as the contribution of mitochondrial ATP synthase is minimized by the addition of azide within the buffer solutions (²³). Although the Ca²⁺-dependent portion of myosin ATPase can be inhibited by increasing the ionic strength of the solution (I > 0.2) (²²), the presence of Mg²⁺ also stimulates this enzyme (^22,24), possibly affecting validity of the assay. On this basis, we determined whether there was a significant presence of myosin ATPase activity under high ionic strength conditions (I = 0.28). The addition of 25 μM BLEB had minimal effects on background SERCA activity in baseline Ca²⁺-stimulated conditions (Fig. 3A, B). Similarly, the addition of BLEB did not affect SERCA V_max, Ca50, or Hill slope (Fig. 3C, D). We therefore then performed experiments with our preparation to determine potential differences between the kinetic properties of SERCA in soleus (red) and EDL (white) muscles. Similar to previous studies (^18,24), we observed approximately fivefold decreased SERCA maximal activity in soleus (304 ± 32 μmol⋅min⁻¹⋅mg⁻¹ protein) compared with EDL (1488 ± 133 μmol⋅min⁻¹⋅mg⁻¹ protein) (Fig. 4A; P < 0.01), with no differences in Ca50 (soleus, 5.8 ± 0.1; EDL, 5.8 ± 0.1) or Hill slopes (soleus, 1.9 ± 0.1; EDL, 2.2 ± 0.2) (Fig. 4B, C). The CV for SERCA assays was 4.4% ± 1.0%. These experiments demonstrated that unlike NKA, the addition of BLEB is not required to accurately determine the kinetic properties of SERCA, and verified the use of a fluorometric approach to study NKA and SERCA activity in rodent homogenates.

SERCA and NKA activity assays in older adults

We finally applied our technique to detect ATPase activities in human skeletal muscle. As expected, we observed typical kinetic properties of SERCA activity with the titration of different Ca²⁺ concentrations (Fig. 5A). Furthermore, the values of Ca50 (5.4 ± 0.1) and Hill slope (1.5 ± 0.3) were within the range of published studies in humans (Fig. 5B) (^26,27). Lastly, with our preparation, NKA maximal activity was 18 ± 2 μmol⋅min⁻¹⋅g⁻¹ protein, approximately three times higher than previously published results obtained from young human skeletal muscle (²⁸).

In human skeletal muscle, the theoretical maximal activity of NKA can be calculated by multiplying the content of NKA by the ATP turnover rate of the pump. The NKA content measured in human skeletal muscle using [3H]ouabain binding is ~360 pmol⋅g⁻¹ wet weight (¹⁰). Thus, 1 g of skeletal muscle has ~360 pmol NKA pumps. The NKA pump turnover rate is ~8000 molecules of ATP per NKA molecule per min (¹¹). Thus, 360 pmol × 8000 ATP/min = 2.9 μmol ATP⋅min⁻¹⋅g⁻¹ muscle. Assuming that 1 g of muscle has ~150 mg of protein, NKA max activity would be 2.9 × 6.7 = 19.4 μmol⋅min⁻¹⋅g⁻¹ prot, similar to the values generated in the present study (¹¹) (Fig. 5C). Therefore, these results demonstrated that our preparation was suitable to measure NKA and SERCA activities in human skeletal muscle.

DISCUSSION

In the present study, we observed that myosin ATPase activity was the major interfering factor for determining NKA activity in skeletal muscle homogenates. As a result, the addition of BLEB, a specific myosin ATPase inhibitor, minimized background “noise” and enabled the determination of NKA activity in skeletal muscle homogenates. Consequently, the present study established a methodology to determine NKA activity from a common muscle homogenate, using extremely small muscle samples in rodent and human skeletal muscle (5–10 mg). This methodology may help remove a technical barrier to studying NKA in diverse metabolic situations.

A major aim of the present study was to determine whether the pharmacological inhibition of myosin ATPase and SERCA enabled the assessment of NKA activity in skeletal muscle homogenates (^11,21). Although the CPA-mediated inhibition of SERCA had no effect on NKA activity, the addition of BLEB decreased basal background activity threefold, suggesting that myosin ATPase was a major contributor to the high background that previously prevented NKA detection in muscle homogenates. In support of this, and similar to previous reports using isolated plasma membranes (^12,17), NKA maximal activity determined in the presence of BLEB was higher in the soleus compared with the EDL. However, although the basic fiber-type pattern appeared similar to previous reports, we were able to generate reliable measurements of NKA maximal activities in rodent and age human muscle which were approximately three times higher than previously published results (^17,28) and closer to the maximal theoretical value proposed by Clausen (¹¹). These results can be attributed to the larger population of NKA enzyme (i.e., t-tubules, sarcolemma, and subcellular compartments) that our assay appeared to detect, and to the preservation of NKA content in age human skeletal muscle (¹⁰).

Methodological aspects

In the present study, we also aimed to determine the feasibility of measuring NKA and SERCA in a common homogenate. This was necessary as the homogenate used for assessing rodent tissue was 10-fold more dilute than typically reported for SERCA kinetic analysis. The kinetic data generated in the present study (maximal activity, Ca50, and Hill slope) was comparable with published reports in rodents (^19,25) and humans (^26,27), and although BLEB did not statistically reduce SERCA V_max, it was slightly lower (~8%) in the presence of BLEB. These data may suggest that BLEB could marginally impair SERCA activity. However, this is unlikely because as the high ionic strength (I > 0.2) of the assay solution used in the present study inhibits a significant portion of myosin ATPase (^22,24), BLEB did not dramatically reduce activity after CPA, and the kinetic properties of SERCA were similar between conditions. Most probably, the variability in V_max in the presence of BLEB likely reflects the small sample size used in the present study.

The significant background contamination from other ATPases has prevented the use of a reliable approach to measure NKA activity in muscle homogenates. Previous methods have attempted to circumvent this issue by purifying muscle samples using ultracentrifugation procedures (²¹), or by using the artificial substrate 3-O-methylfluorescein phosphate (3-O-MFP) to measure NKA activity (^12,13). The disadvantages of these approaches are that the former results in very low recovery of total protein and NKA maximal activity, and the latter measures NKA activity under Na⁺ free conditions, although Na⁺ is one of the major regulators of NKA pump (^8,9). This has likely contributed to the contradicting results regarding NKA activity in human skeletal muscle. With the present approach, NKA activity was calculated indirectly by measuring oxidation of NADH in the presence of glycolytic auxiliary enzymes. A major advantage of this method is that a steady level of ATP is maintained by constant conversion of ADP, removing the effects of ADP on NKA enzyme (⁸). Furthermore, this method appears to detect a significant population of NKA enzyme, leading to values near the theoretical maximum while using low quantities of muscle tissue.

Perspective and significance

The present study establishes a cost-effective approach to determine NKA activity from a single homogenate using significantly less muscle tissue (5–10 mg) than historically required. The dramatic reduction in tissue requirements should enable a broader examination of NKA activity in diverse metabolic situations, especially for situations with compromised muscle mass/quality where tissue is a limiting factor for analysis (e.g., aging and inactivity). In addition, the use of muscle homogenates to determine the total cellular activity of NKA enzyme (i.e., t-tubules and sarcolemma) negates the need for sarcolemmal purification, which may contribute to the observation that the present approach resulted in NKA values similar to the theoretical maximal value for muscle. In addition, removing the requirement for expensive centrifuges should make the assessment of NKA activity more accessible. As a result, the establishment of the present methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations and clearly highlights the importance of inhibiting myosin ATPase for the determination of NKA function in skeletal muscle homogenates.