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. 2008 Nov;22(11):3919–3924. doi: 10.1096/fj.08-113712

Nonshivering thermogenesis protects against defective calcium handling in muscle

Jan Aydin *, Irina G Shabalina †, Nicolas Place *, Steven Reiken ‡, Shi-Jin Zhang *, Andrew M Bellinger ‡, Jan Nedergaard †, Barbara Cannon †, Andrew R Marks ‡, Joseph D Bruton *, Håkan Westerblad *,1
PMCID: PMC3980658  PMID: 18687806

Abstract

When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein-1 (UCP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca2+ leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UCP1-deficient (UCP1-KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold-acclimated UCP1-KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca2+ release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca2+ and force were not decreased in FDB muscles of cold-acclimated UCP1-KO mice. These results indicate that activation of UCP1-mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles. Aydin, J., Shabalina, I. G., Place, N., Reiken, S., Zhang, S.-J., Bellinger, A. M., Nedergaard, J., Cannon, B., Marks, A. R., Bruton, J. D., Westerblad, H. Nonshivering thermogenesis protects against defective calcium handling in muscle.

Keywords: muscle contraction, ryanodine receptor, temperature control, increased β-adrenergic activity

When acutely exposed to a cold environment, mammals respond by repeated activation of skeletal muscles; that is, they shiver (1). The increased skeletal muscle activity generates heat, which makes it possible to maintain the body core temperature. During prolonged cold exposure, shivering can be replaced by adaptive nonshivering thermogenesis; that is, heat generated by increased metabolism in brown adipose tissue due to activation of uncoupling protein-1 (UCP1) (2, 3). Genetically engineered UCP1-deficient (UCP1-KO) mice cannot recruit UCP1-dependent adaptive nonshivering thermogenesis and therefore rely on shivering thermogenesis even during prolonged cold exposure (4, 5).

The stress induced by cold exposure increases the activity of the sympathetic nervous system (6,7,8). A prolonged increase in β-adrenergic activity during heart failure or a period of intensive exercise is associated with impaired contractile function due to adverse effects on the protein complex responsible for sarcoplasmic reticulum (SR) Ca2+ release, that is, the ryanodine receptor 1 (RyR1) channel complex (9,10,11). The mechanism underlying these latter effects involves increased SR Ca2+ leak due to protein kinase A (PKA) -induced hyperphosphorylation of RyR1 at Ser-2844 and dissociation of the channel-stabilizing subunit calstabin1 (also known as FKBP12) (12).

Here, we use wild-type (WT) and UCP1-KO mice kept in the cold (4°C; cold-acclimated) or at room temperature (24°C; control); for clarity, muscles and muscle fibers from these four groups of mice will be referred to as cold-acclimated WT, cold-acclimated UCP1-KO, control WT, and control UCP1-KO, respectively. We focus on possible PKA-induced changes in SR Ca2+ handling in soleus and flexor digitorum brevis (FDB) muscles. Soleus muscles participate in the shivering response, whereas shivering does not occur in superficial and distal muscles such as the FDB (1). We hypothesize that cold exposure has larger effects on soleus muscles of UCP1-KO than of WT mice, because shivering remains the major heat-generating system during prolonged cold exposure in UCP1-KO mice (4, 5). Conversely, cold exposure would induce similar changes in FDB muscles of UCP1-KO and WT mice, which then reflects the response to an increased β-adrenergic drive. In agreement with our hypotheses, cold exposure resulted in marked RyR1 PKA hyperphosphorylation and calstabin-1 dissociation, as well as decreased tetanic free myoplasmic [Ca2+] ([Ca2+]i) and force in soleus muscles of UCP1-KO but not of WT mice. Conversely, there was no significant difference in tetanic [Ca2+]i or force between FDB fibers of cold-acclimated WT and UCP1-KO mice.

MATERIALS AND METHODS

Animals

UCP1-ablated mice (progeny of those described previously; ref. 4) were backcrossed to C57BL/6 mice for 10 generations and after intercrossing maintained as UCP1(−/−) (UCP1-KO) and UCP1(+/+) (WT) strains on a C57BL/6 background. The mice were fed ad libitum (R70 Standard Diet; Lactamin, Vadstena, Sweden), had free access to water, and were kept on a 12:12-h light-dark cycle at 24°C. For experiments on cold-acclimated animals, adult female mice were divided into age- (7 to 8 wk old) and body weight (17–18 g) -matched groups and kept at 24°C (control) or successively acclimated to cold by first placing them at 18°C for 4 wk and then at 4°C for 4–5 wk (cold-acclimated); the intermediate 18°C step is required to allow for survival of the UCP1-KO animals at 4°C (5). Animals were killed by rapid neck disarticulation. The experiments were approved by the Animal Ethics Committee of the North Stockholm region and by the Institutional Animal Care and Use Committee of Columbia University.

RyR1 immunoprecipitation and Western blot analysis

Soleus and FDB muscles were isolated and immediately frozen in liquid nitrogen. Muscle homogenate was prepared by isotonic lysis of 5 mg of muscle samples. RyR1 was immunoprecipitated from 100 μg of homogenate with anti-RyR antibody (2 μl 5029 Ab) in 0.5 ml of a modified RIPA buffer (50 mM Tris-HCl, pH 7.4; 0.9% NaCl; 5.0 mM NaF; 1.0 mM Na3VO4; 0.5% Triton-X100; and protease inhibitors) for 1 h at 4°C. The samples were incubated with protein A Sepharose beads (Amersham Pharmacia, Piscataway, NJ, USA) at 4°C for 1 h, and the beads were washed 3 times with buffer. Control samples were treated with either protein phosphatase 1 (PP1, 5 U/reaction; Calbiochem, San Diego, CA, USA) or PKA + 100 μM mg-ATP (5 U/reaction; Sigma, St. Louis, MO, USA). The product was size-fractionated on SDS-PAGE gels (4–20% gradient) and transferred onto nitrocellulose membranes for 2 h at 200 mA (SemiDry transfer blot; Bio-Rad, Hercules, CA, USA). After incubation with blocking solution (Li-COR Biosciences, Lincoln, NE, USA) to prevent nonspecific antibody binding and a wash in Tris-buffered saline with 0.1% Tween-20, membranes were incubated for 1–2 h at room temperature with the primary antibodies anti-calstabin (1:2500 in blocking buffer), anti-RyR1 (5029, 1:5000), or anti-phospho-RyR2-pSer2809 (1:5000). After 3 washes, membranes were incubated with infrared-labeled secondary antibodies (1:10,000 dilution, Li-COR Biosystems). Band densities were quantified using the Odyssey Infrared Imaging System (Li-COR Biosciences).

[Ca2+]i and force measurements in soleus muscle

Small bundles of muscle fibers (2–4 cells) were dissected from soleus muscles. The fiber bundle was mounted in a stimulation chamber at optimum length and superfused with Tyrode solution (mM): 121 NaCl, 5.0 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24.0 NaHCO3, 0.1 EDTA, and 5.5 glucose. Fetal calf serum (0.2%) was added to the solution to improve muscle fiber survival. The solution was bubbled with 5% CO2/95% O2, which gives an extracellular pH of 7.4. Experiments were performed at 25°C. Tetanic stimulation was achieved by supramaximum current pulses (duration 0.5 ms) delivered via platinum plate electrodes lying parallel to the fibers. [Ca2+]i was measured with the fluorescent Ca2+ indicator indo-1 (Invitrogen/Molecular Probes, Carlsbad, CA, USA), which was microinjected into one fiber in the bundle. The mean fluorescence of indo-1 at rest or during tetanic contractions was measured and converted to [Ca2+]i using an intracellular Ca2+ calibration curve (13). After injection, the fiber bundle was allowed to rest for at least 30 min. It was then tested at regular intervals to ascertain that tetanic [Ca2+]i was stable. Cells where tetanic [Ca2+]i was unstable or showed marked irregularities during contractions were discarded. The fiber bundle was then stimulated with 600-ms pulse trains at 10 to 100 Hz at 1-min intervals, and tetanic [Ca2+]i was measured. Force was recorded to ascertain normal function of the preparation in this respect, that is, force increased and became more fused as the stimulation frequency was increased; bundles that either showed an abnormal force response to an increase in the stimulation frequency or a markedly decreased (>10%) tetanic force after indo-1 injection were excluded.

The force output of the fiber bundles was not further analyzed because it emerged from more cells than those in which [Ca2+]i was measured. We instead performed additional experiments on isolated whole soleus muscles, which were mounted in a stimulation chamber filled with Tyrode solution (see above). The muscle length was adjusted to that giving the maximum tetanic force response. Muscles were then allowed to rest for 30 min. The force-frequency relation was studied by sequentially stimulating the muscle to produce a tetanic contraction at 10–100 Hz at 1-min intervals. At the end of the experiment, the muscle length was measured, and muscles were then taken out of the chamber, tendons were removed, and the muscle was weighed. The cross-sectional area was calculated as muscle weight/(muscle length×1.056). Forces were measured as the peak force.

Measurements of [Ca2+]i and force in FDB muscle fibers

Intact, single muscle fibers were dissected from FDB muscles of the hind limb as described elsewhere (14). The isolated fiber was mounted in a stimulation chamber and stimulated with current pulses while [Ca2+]i and force were measured as described above. The fiber was allowed to rest for at least 30 min after being injected with indo-1; fibers that showed a marked force decrease (>10%) after injection were discarded. It was then stimulated by individual 500-ms stimulation trains at 10 to 100 Hz, given at 1-min intervals.

Statistics

Data are presented as means ± se. SigmaStat (Systat Software, Chicago, IL, USA) was used to test for significant differences between groups, and the significance level was set to P < 0.05. Differences between single measurements in two groups were determined with Student′s unpaired t test. For repeated measurements in the same preparation, we used 2-way repeated-measures ANOVA. When the ANOVA analysis showed significance, the Holm-Sidak post hoc test was performed.

RESULTS

Compared to their controls, cold-acclimated UCP1-KO soleus muscles displayed marked PKA hyperphosphorylation of RyR1 and calstabin1 depletion from the RyR1 channel complex (Fig. 1A–C). Conversely, in WT soleus muscles, cold exposure only caused a minor decrease in RyR1 calstabin1 binding, whereas RyR1 PKA phosphorylation was not significantly affected. FDB muscles of both WT and UCP1-KO mice showed a pattern similar to that observed in WT soleus muscles; that is, cold exposure induced only minor changes, with slight increases in RyR1 PKA phosphorylation and decreases in RyR1 calstabin1 binding (Fig. 1D–F). Thus, RyR1 PKA hyperphosphorylation and marked calstabin1 depletion from the RyR1 channel complex occurred only in cold-acclimated UCP1-KO soleus muscles.

Figure 1.

Figure 1

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Soleus muscles of cold-acclimated UCP1-KO mice display RyR1 PKA hyperphosphorylation and calstabin1 depletion. RyR1 immunoprecipitated from soleus (A) and FDB (D) muscle of WT and UCP1-KO mice housed at 4°C (indicated by + below images) and 24°C and assessed for total RyR1, PKA phosphorylation of RyR1 (RyR1-pS2844), and calstabin1 bound to RyR. PP1- or PKA-treated immunoprecipitates are shown to indicate minimum and maximum phosphorylation levels, respectively. Soleus (B) and FDB (E) muscle data (mean±se, n=4 or 5) of relative RyR1 PKA phosphorylation (normalized to total RyR1 and compared to PKA-treated sample). Soleus (C) and FDB (F) muscle binding of calstabin1 (normalized to total RyR1). White bars, control muscles; black bars, cold-acclimated muscles. **P < 0.01, ***P < 0.001 for cold-acclimated mice vs. their respective controls.

Tetanic [Ca2+]i was measured in intact single soleus fibers, and tetanic force was measured in isolated whole soleus muscles. Both tetanic [Ca2+]i and force were markedly lower in cold-acclimated UCP1-KO as compared to cold-acclimated WT soleus muscle (Fig. 2). The lower force in cold-acclimated UCP1-KO than in cold-acclimated WT soleus muscles was not due to decreased muscle size, because there was no significant difference in either muscle length or weight between the two groups, and hence, the cross-sectional areas were similar (0.68±0.04 vs. 0.74±0.04 mm2; P=0.26).

Figure 2.

Figure 2

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Tetanic [Ca2+]i and force are decreased in cold-acclimated soleus muscle of UCP1-KO mice. A) Representative [Ca2+]i records from 100-Hz tetanic contractions produced in a WT (left) and a UCP1-KO (right) soleus fiber, respectively. B) Representative force records from 100-Hz tetanic contractions in soleus muscles. C) Mean ± se [Ca2+]i at 10- to 100-Hz stimulation in WT (○, n=5) and UCP1-KO (•, n=3) single soleus fibers, respectively. D) Mean forces in WT (○, n=7) and UCP1-KO (•, n=6) soleus muscles, respectively. *P < 0.05; **P < 0.01; ***P < 0.001.

In contrast to the situation in cold-acclimated soleus muscles, there was no significant difference in tetanic [Ca2+]i or force between control UCP1-KO and control WT soleus muscle at any stimulation frequency. Pooled data of control soleus fibers show tetanic [Ca2+]i values that lie between those observed in soleus fibers from cold-acclimated mice; for instance, tetanic [Ca2+]i with 100-Hz stimulation was 1.02 ± 0.21 μM (n=6) in control fibers, which compares to mean values of 0.51 and 1.43 μM in cold-acclimated UCP1-KO and cold-acclimated WT fibers, respectively.

Contrary to the situation in soleus muscle, there was no significant difference in tetanic [Ca2+]i or force between cold-acclimated WT and cold-acclimated UCP1-KO FDB fibers at any stimulation frequency (Fig. 3). There was also no difference in tetanic [Ca2+]i or force between control WT and control UCP1-KO FDB fibers at any stimulation frequency. Intriguingly, tetanic [Ca2+]i was generally higher in cold-acclimated as compared to control FDB fibers; for instance, [Ca2+]i during 100 Hz tetani was 3.10 ± 0.34 μM (n=10) in cold-adapted and 2.03 ± 0.30 μM (n=11) in control FDB fibers (P<0.05).

Figure 3.

Figure 3

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Tetanic [Ca2+]i and force do not differ in cold-acclimated WT and UCP1-KO FDB fibers. A, B) Representative [Ca2+]i (A) and force (B) records from 100-Hz tetani produced in cold-acclimated WT (left) and UCP1-KO (right) FDB fibers. C, D) Mean ± se [Ca2+]i (C) and force (D) at 10- to 100-Hz stimulation in WT (○, n=4) and UCP1-KO (•, n=6) FDB fibers, respectively.

DISCUSSION

We studied cold-induced adaptation of skeletal muscle with a special focus on intracellular Ca2+ handling in WT mice, which can induce adaptive nonshivering thermogenesis, and UCP1-KO mice, which depend on shivering to generate heat (4, 5). We used soleus muscles, which would contribute to the shivering response during cold exposure, and the distally and superficially located FDB muscles, which would not shiver (1). The major novel result is that prolonged shivering during cold exposure can result in severely decreased contractile force due to impaired SR Ca2+ release associated with RyR1 PKA hyperphosphorylation and calstabin1 depletion from the RyR1 channel complex. These cold-induced dysfunctions only occurred in soleus muscles of UCP1-KO mice, which continued to shiver in the cold environment.

Cold exposure activates the sympathetic nervous system (6,7,8), and the increased adrenergic activity is essential for the activation of UCP1 in brown adipose tissue and, hence, the adaptive nonshivering thermogenesis (3). Also, in skeletal muscle, cold acclimation is associated with a chronically increased sympathetic activity (15). β-Adrenergic stimulation increases the second messenger cAMP via a G-protein-mediated activation of adenylyl cyclase. cAMP activates PKA, which catalyzes phosphorylation of numerous skeletal muscle proteins (16). Acute β-adrenergic stimulation of skeletal muscle leads to improved contractile function (16,17,18). The mechanism behind this force increase has been studied by exposing mouse FDB fibers to the β-adrenergic agonist terbutaline, and the results showed increased tetanic [Ca2+]i, which was suggested to be caused by cAMP-dependent phosphorylation of RyR1 (18). Conversely, chronically increased β-adrenergic activity may cause a hyperphosphorylation of RyR1 and calstabin1 depletion from the RyR1 complex (9, 10). Normally, calstabin1 stabilizes RyR1 channels in the closed state, and this inhibition of RyR1 channel opening is relieved when calstabin1 dissociates, resulting in increased SR Ca2+ leak and impaired muscle function (9). Thus, while acute β-adrenergic stimulation of skeletal muscle leads to improved contractile function (16,17,18), prolonged stimulation may impair contractile function due to adverse effects on the RyR1 channel complex, as has been observed in association with heart failure (9, 10) and a period of intense exercise (11).

We observed only minor increases in RyR1 PKA phosphorylation and decreases in calstabin1 binding to the RyR1 channel complex in nonshivering cold-acclimated muscles (i.e., WT soleus, UCP1-KO FDB, and WT FDB; see Fig. 1) and fibers of these muscles displayed higher tetanic [Ca2+]i than the control fibers. Thus, the changes in nonshivering cold-acclimated muscles may reflect the response to an increased β-adrenergic stimulation on its own.

Conversely, cold-acclimated UCP1-KO soleus muscles showed markedly decreased tetanic [Ca2+]i and force accompanied by RyR PKA hyperphosphorylation and severe calstabin1 depletion. These cold-acclimated UCP1-KO soleus muscles were exposed to both an increased β-adrenergic drive and the stress induced by shivering. Thus, it appears that the combination of β-adrenergic stress and continuous contractile activity during cold exposure is required to drive muscle cells into a defective state with RyR1 PKA hyperphosphorylation and severe calstabin1 depletion from the RyR1 channel complex. However, from the present experiments, we cannot exclude that other factors may also contribute to the severe changes observed in cold-acclimated UCP1-KO soleus but not FDB muscles. For instance, β-adrenergic stimulation results in PKA-induced phosphorylation of phospholamban, which leads to an increased rate of SR Ca2+ pumping, and phospholamban is expressed in slow-twitch soleus muscles but not in fast-twitch FDB muscles (19).

The defective muscle function in cold-acclimated UCP1-KO soleus muscles does not prevent them from shivering, because these mice can maintain their body temperature in the cold where shivering remains the mechanism of heat generation (5). However, although UCP1-KO mice can survive in the cold, they have a much shorter life span in the cold than WT mice with a median survival of only 13 wk (5). It might be speculated that the premature death in cold exposed UCP1-KO mice occurs at a time point when the functional impairment of shivering muscles has become so severe that effective shivering no longer occurs and hence the body temperature cannot be defended.

In conclusion, activation of UCP1-mediated adaptive nonshivering thermogenesis in brown adipose tissue reduces the necessity for shivering, which can be considered as a protective mechanism against the development of shivering-induced skeletal muscle dysfunction.

Acknowledgments

This study was supported by the Swedish Research Council, the Swedish National Center for Sports Research, funds from the Karolinska Institute, and the U.S. Defense Advanced Research Projects Agency (DARPA). N.P. was supported by the Swedish Institute.

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