ATP consumption by sarcoplasmic reticulum Ca²⁺ pumps accounts for 40-50% of resting metabolic rate in mouse fast and slow twitch skeletal muscle

Abstract

The main 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) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO₂, µL/g wet weight/s) measured polarographically at 30°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca²⁺ cycling to resting metabolic rate, the concentration of MgCl₂ in the bath was increased to 10 mM to block Ca²⁺ release through the ryanodine receptor, thus eliminating a major source of Ca²⁺ leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca²⁺-ATPases (SERCAs). The relative (%) reduction in muscle VO₂ in response to 10 mM MgCl₂ was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid (CPA, up to 160 µM), but were unsuccessful in removing the energetic cost of Ca²⁺ cycling in resting isolated muscles. The results of the MgCl₂ experiments indicate that ATP consumption by SERCAs is responsible for 40-50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C, or 12-15% of whole body resting VO₂. 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.

Figure 1. Effects of 10 mM Mg²⁺ on force and the indo-1 fluorescence ratio during caffeine-induced contracture.

(A) The force response of an intact mouse lumbrical muscle loaded with AM-indo-1 in a circulating oxygenated buffer. The source of the buffer was alternated between a large reservoir (used during the initial 5 min incubation and during 2-3 min washout periods) and a small reservoir (used as site of Mg²⁺ or Na⁺ salt addition, and either contained or did not contain 15 mM caffeine as indicated). Switches between the two reservoirs are marked by the dashed arrows. Fluorescent measures (B) were collected immediately prior to the introduction of each solution originating from the small reservoir (labeled Pre and 60 s), and 60 s after the introduction of the Mg²⁺ or Na⁺ salt to the muscle (labeled 120 s). Bars correspond to the 1.0 s average of the 405/495 nm indo-1 emission ratio normalized to the average of the Pre ratios and are relative indicators of what is happening to the cytosolic Ca²⁺ concentration. *Significantly different than the preceding pre-switch value (P<0.05). †significantly different than all other values (P<0.05). Fluorescent responses of muscles to MgCl₂ and MgSO₄ in absence of caffeine are shown in panels C and D, respectively. Bars are analogous to 60 s and 120 s from Panel B *Significantly different than control value (P<0.05).

Figure 2. Representative raw tracing of PO₂ decline over 30 minutes at 30^°C for an O₂ leak trial (No Muscle, Δ), a resting muscle VO₂ trial (Rest, ○) and a high MgCl₂ trial (10 mM MgCl₂, □) for a soleus muscle.

Figure 3. Representative tracing of muscle force for one EDL muscle experiment.

(A) Twitch force at L_o (no CPA); (B) twitch force at lengths >L_o (no CPA); (C) passive force throughout the period of pre-stretch (no CPA); (D) passive force throughout the CPA trial; and (E) twitch force at L_o following the CPA trial. Thin black arrows represent muscle stimulation (pulse duration, 0.2 ms; voltage, 10 V). Thick grey arrows indicate when fresh buffer and CPA were added. ↑S indicates where the incremental stretches were applied to the muscle.

Figure 4. Resting oxygen consumption of EDL and soleus muscles at optimal length (L_o) compared to oxygen consumption during stretch trial (no sarcomere overlap) at 30^°C for 30 minutes.

VO₂ is expressed relative to average resting VO₂ at L_o. Values are mean ± SE (n=6).

Figure 5. Effects of 10 mM MgCl₂ on resting VO₂ of isolated soleus and EDL muscles.

(A) Resting VO₂ of isolated soleus (n=14) and EDL (n=13) muscles during serial 30 minute incubations in solutions containing 0.5 (Basal and Washout) or 10 (High MgCl₂) mM MgCl₂. Incubation with 10 mM MgCl₂ reversibly reduced resting VO₂ in both soleus and EDL muscles (Main effect, High MgCl₂ < Basal, Washout; P<0.001). (B) Percent reduction in resting VO₂ of isolated soleus and EDL muscles induced by incubation in high (10 mM) MgCl₂.

Figure 6. Effects of CPA on resting VO₂ and baseline tension of isolated soleus and EDL muscles.

(A) Representative raw tracings of PO₂ decline over 30 minutes at 30^°C for an O₂ leak trial (No Muscle, Δ), a resting muscle VO₂ trial (Rest, ○) and a 160 µM CPA trial (□) for a soleus muscle and muscle force for two different EDL (B) and soleus (C) muscles showing changes in resting tension at L_o following a single addition of 160 µM CPA (thick lines) and serial dilutions of CPA (thin lines) with cumulative CPA concentrations enumerated.

Figure 7. Quantification of SERCA1a and SERCA2a in mouse EDL and soleus muscle homogenates.

Protein from mouse EDL and soleus whole muscle homogenates (0.05-15.0 µg total protein, as indicated) and 1.0-8.0 ng purified SERCA1a (prepared from rat skeletal muscle) or SERCA2a (prepared from rat heart) were separated on a 7% SDS-PAGE gel and SERCA1a (A) or SERCA2a (C) were detected by Western blotting using the A52 and 2A7-A1 antibodies, respectively. Density of band for purified SERCA1a (B) and SERCA2a (D) was plotted against amount of protein. The amount of SERCA1a and SERCA2a in EDL and soleus homogenates was determined from intensity of bands that fell within the linear range for samples of purified SERCA1a and SERCA2a on the same gel.