Increased fatigue resistance linked to Ca 2+ -stimulated mitochondrial biogenesis in muscle fibres of cold-acclimated mice

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1 J Physiol (2010) pp Increased fatigue resistance linked to Ca 2+ -stimulated mitochondrial biogenesis in muscle fibres of cold-acclimated mice Joseph D. Bruton 1, Jan Aydin 1, Takashi Yamada 1, Irina G. Shabalina 2, Niklas Ivarsson 1, Shi-Jin Zhang 1, Masanobu Wada 3, Pasi Tavi 4, Jan Nedergaard 2,AbramKatz 1 and Håkan Westerblad 1 1 Department of Physiology and Pharmacology, Karolinska Institutet, SE Stockholm, Sweden 2 The Wenner-Gren Institute, the Arrhenius Laboratories F3, Stockholm University, SE Stockholm, Sweden 3 Graduate School of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan 4 Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, FI Kuopio, Finland Mammals exposed to a cold environment initially generate heat by repetitive muscle activity (shivering). Shivering is successively replaced by the recruitment of uncoupling protein-1 (UCP1)-dependent heat production in brown adipose tissue. Interestingly, adaptations observed in skeletal muscles of cold-exposed animals are similar to those observed with endurance training. We hypothesized that increased myoplasmic free [Ca 2+ ] ([Ca 2+ ] i ) is important for these adaptations. To test this hypothesis, experiments were performed on flexor digitorum brevis (FDB) muscles, which do not participate in the shivering response, of adult wild-type (WT) and UCP1-ablated (UCP1-KO) mice kept either at room temperature (24 C) or cold-acclimated (4 C) for 4 5 weeks. [Ca 2+ ] i (measured with indo-1) and force were measured under control conditions and during fatigue induced by repeated tetanic stimulation in intact single fibres. The results show no differences between fibres from WT and UCP1-KO mice. However, muscle fibres from cold-acclimated mice showed significant increases in basal [Ca 2+ ] i ( 50%), tetanic [Ca 2+ ] i ( 40%), and sarcoplasmic reticulum (SR) Ca 2+ leak ( fourfold) as compared to fibres from room-temperature mice. Muscles of cold-acclimated mice showed increased expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and increased citrate synthase activity (reflecting increased mitochondrial content). Fibres of cold-acclimated mice were more fatigue resistant with higher tetanic [Ca 2+ ] i and less force loss during fatiguing stimulation. In conclusion, cold exposure induces changes in FDB muscles similar to those observed with endurance training and we propose that increased [Ca 2+ ] i is a key factor underlying these adaptations. (Resubmitted 26 August 2010; accepted 7 September 2010; first published online 13 September 2010) Corresponding author H. Westerblad: Department of Physiology and Pharmacology, Karolinska Institutet, SE Stockholm, Sweden. hakan.westerblad@ki.se Abbreviations AMPK, AMP-activated protein kinase; β-had, β-hydroxyacyl-coa dehydrogenase; BSA, bovine serum albumin; [Ca 2+ ] i, myoplasmic free [Ca 2+ ]; CaMK, Ca 2+ /calmodulin-dependent protein kinase; CS, citrate synthase; DHPR, dihydropyridine receptor; FDB, flexor digitorum brevis; FKBP12, 12 kda FK506 binding protein; KO, knock-out; MyHC, myosin heavy chain; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; RT-PCR, real-time PCR; RyR1, ryanodine receptor 1; SR, sarcoplasmic reticulum; SERCA, SR Ca 2+ ATPase; TBS-T, Tris-buffered saline containing 0.05% Tween 20; UCP, uncoupling protein; WT, wild-type; ww, wet weight. Introduction Skeletal muscles of animals exposed to a cold environment show increases in mitochondrial biogenesis and oxidative capacity, which are also hallmarks of endurance training (Holloszy & Coyle, 1984; Bourhim et al. 1990; Booth & Thomason, 1991; Puigserver et al. 1998; Schaeffer et al. 2003; Oliveira et al. 2004; Arruda et al. 2008). The mechanisms underlying the stimulation of mitochondrial biogenesis induced either by cold exposure or by endurance training are not fully understood. However, it has become increasingly clear that the peroxisome DOI: /jphysiol

2 4276 J. D. Bruton and others J Physiol proliferator-activated receptor-γ coactivator-1 (PGC-1) family of transcriptional coactivators plays a key role in both situations (Puigserver et al. 1998; Oliveira et al. 2004; Handschin & Spiegelman, 2006; Arany, 2008). Endurance exercise increases PGC-1α expression in muscle both at the mrna and protein level, whereas PGC-1β expression is less affected (Baar et al. 2002; Norrbom et al. 2004; Arany, 2008). Cold exposure and exercise may promote PGC-1α expression, and hence mitochondrial biogenesis, via multiple signalling pathways triggered by, for instance, changes in the concentration of energy metabolites, reactive oxygen species and Ca 2+, as well as altered neuro-endocrine signalling (Arany, 2008). While it is well established that the increased mitochondrial content in skeletal muscle induced by physical exercise results in increased fatigue resistance, the situation after cold exposure is less clear. In this case, the increased mitochondrial content in skeletal muscle might be counteracted by decreased mitochondrial efficiency in using fuel oxidation to convert ADP to ATP (Wu et al. 1999; Zaninovich et al. 2003; Oliveira et al. 2004; Mollica et al. 2005) and/or by increased ATP consumption due to e.g. ineffective SR Ca 2+ pumping (Arruda et al. 2008). When placed in a cold environment, mammals initially shiver to maintain body temperature (Hemingway, 1963). During prolonged cold exposure, shivering is successively replaced by the recruitment of uncoupling protein (UCP) 1-dependent heat production in brown adipose tissue (Heaton et al. 1978; Cannon & Nedergaard, 2004). Mice lacking UCP1 (UCP1-KO mice) continue to depend on heat generated by shivering during prolonged cold exposure (Enerbäck et al. 1997; Golozoubova et al. 2001, 2006). The myoplasmic free [Ca 2+ ] ([Ca 2+ ] i ) is considered to be a key factor in the control of PGC-1α and mitochondrial biogenesis in skeletal muscle (Handschin et al. 2003; Wright et al. 2007; Arany, 2008). Prolongedcoldexposure can affect the intracellular Ca 2+ handling of skeletal muscle cells both regarding the SR Ca 2+ release via the ryanodine receptor 1 (RyR1) and the active re-uptake via the SR Ca 2+ -ATPase (SERCA) (Arruda et al. 2008; Aydin et al. 2008). Here we studied the effect of prolonged cold exposure on the interplay between intracellular Ca 2+ handling, mitochondrial biogenesis and fatigue resistance. Experiments were performed on the distal, superficial flexor digitorum brevis (FDB) muscles, which are unlikely to participate in the shivering response that mainly involves postural limb and trunk muscles (Hemingway, 1963). We used UCP1-KO and wild-type (WT) mice which allowed us, in a non-shivering muscle, to detect possible additional (systemic) effects caused by sustained shivering of postural limb and trunk muscles in the UCP1-KO mice. We hypothesized that cold exposure triggers the following sequence of events: altered SR Ca 2+ handling leads to increased [Ca 2+ ] i, which induces PGC-1α expression and mitochondrial biogenesis resulting in increased fatigue resistance. Methods Animal description and ethical approval UCP1-ablated mice (Enerbäck et al. 1997) were backcrossed to C57Bl/6 mice for 10 generations and after intercrossing maintained as UCP1 / (UCP1-KO) and UCP1 +/+ (WT) strains on C57Bl/6 background. Western blotting confirmed that UCP1 was not expressed in littermates to the UCP1-KO mice used here (Shabalina et al. 2010b).Themicewerehousedatroomtemperature (24 C) with a 12:12 h light dark cycle and free access to water and fed ad libitum (R70 Standard Diet, Lactamin; Vadstena, Sweden). Adult female mice were divided into age- (7 8 weeks old) and body weight- (17 18 g) matched groups and kept at 24 C (room temperature), or successively acclimated to cold by first placing them at 18 C for 4 weeks and then at 4 C for 4 5 weeks (cold-acclimated) (Golozoubova et al. 2001). Animals were killed by rapid neck disarticulation and the FDB muscles removed. A total of 20 mice were used. The studies were approved by the Stockholm North local ethical committee. [Ca 2+ ] i, force and fatigue properties Intact, single muscle fibres were dissected from FDB muscles as describedelsewhere (Lännergren & Westerblad, 1987). The isolated fibre was mounted in a stimulation chamber at optimum length and superfused with Tyrode solution (mm): NaCl, 121; KCl, 5.0; CaCl 2, 1.8; MgCl 2, 0.5; NaH 2 PO 4,0.4;NaHCO 3, 24.0; EDTA, 0.1; glucose, 5.5. Fetal calf serum (0.2%) was added to the solution to improve muscle fibre survival. The solution was bubbled with 5% CO 2 95% O 2, which gives an extracellular ph of 7.4. Experiments were performed at room temperature ( 25 C). Tetanic stimulation was achieved by supramaximum current pulses (duration 0.5 ms) delivered via platinum plate electrodes lying parallel to the muscle fibre. The fluorescent Ca 2+ indicator indo-1 (Invitrogen/Molecular Probes) was microinjected into the isolated fibre. The fluorescence of indo-1 was converted to [Ca 2+ ] i using an intracellularly established calibration curve (Andrade et al. 1998). Tetanic [Ca 2+ ] i was measured as the mean indo-1 fluorescence during tetanic stimulation trains, and basal [Ca 2+ ] i as the mean over 200 ms in the absence of stimulation. Possible changes in SR Ca 2+ pumping and/or passive SR Ca 2+ leak were assessed by measuring the tails of elevated [Ca 2+ ] i after 100 Hz tetani (Klein et al. 1991; Westerblad & Allen, 1994). Tetanic force

3 J Physiol Increased fatigue resistance in cold-acclimated skeletal muscle fibres 4277 was measured as the mean over 100 ms where force was maximal. The rate of force relaxation was assessed by measuring the half-relaxation time, i.e. the time from the end of stimulation until force had decreased by 50%. The fibre was allowed to rest for at least 30 min after being injected with indo-1. It was then stimulated by individual 500 ms stimulation trains at 10 to 100 Hz given at 1 min intervals. Force [Ca 2+ ] i curves were constructed from these contractions by fitting data points to the following equation: P = P max [Ca 2+ ] N i /(Ca N 50 + [Ca2+ ] N i ) (1) where P is the force, P max is the force at saturating [Ca 2+ ] i, Ca 50 is the [Ca 2+ ] i giving 50% of P max,andn is a Hill coefficient that describes the steepness of the function. Fatigue was induced by 50 tetanic stimulations (70 Hz, 300 ms duration) given at 2 s intervals. Citrate synthase and β-hydroxyacyl-coa dehydrogenase activities FDB muscles were homogenized with a ground glass homogenizer in ice-cold buffer (50 μl(mgwetweight) 1 ) consisting of (mm): Tris, 50; sodium citrate, 5; MnCl 2, 0.6; cysteine, 1; 0.05% (v/v) Triton X-100 (ph 7.4). The homogenate was centrifuged for 1 min at 1400 g (4 C) and aliquots of the supernatant were frozen for citrate synthase and β-hydroxyacyl-coa dehydrogenase (β-had) analyses; the pellets were stored for fibre typing (see below). Citrate synthase and β-had activities were analysed with standard spectrophotometric techniques (Bass et al. 1969). The activities were measured at room temperature under conditions that yielded linearity with respect to extract volume and time. The supernatant protein content was determined using the Bradford assay (BioRad, UK) and activities were adjusted for protein content. Fibre typing Myosin heavy chain (MyHC) isoforms were separated with electrophoresis using a 7% polyacrylamide slab gel (Yamada et al. 2006). The myofibrillar fraction was extracted from the pellet of centrifuged homogenates (see above). The pellet was homogenized in 20 volumes of a solution containing 5 M urea, 2 M thiourea, 0.17% (v/v) 2-mercaptoethanol and 10mM sodium pyrophosphate. Aliquots of the extracts containing 0.5 μg myofibrillar protein were applied to the gel. Electrophoresis was run at 4 C for 24 h at 300 V. Gels were silver-stained and gel images were acquired using the GelDoc imaging system. The relative distribution of MyHC isoforms was densitometrically evaluated with PeakFit (SeaSolve Software Inc., Framingham, MA, USA). Western blotting SERCA1, AMP-activated protein kinase (AMPK), phospho-thr 172 -AMPK, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), phospho-thr 286 -CaMKII and dihydropyridine receptor (DHPR). FDB muscles were homogenized in ice-cold homogenization buffer (ph 7.4) (10 μl per mg wet weight) consisting of (mm): Tris HCl, 50; NaCl, 150; EDTA, 1; sodium fluoride, 50; sodium pyrophosphate, 5; sodium orthovanadate, 2; phenylmethylsulfonyl fluoride, 1; plus glycerophosphate 2mgml 1 ; 1μgml 1 of the following: aprotinin, pepstatin, leupeptin; sodium deoxycholate 0.25% (v/v) and Triton X 1% (v/v). The homogenate was centrifuged at 700 g for 15 min at 4 C. Twenty micrograms of protein for SERCA1, 30 μg for AMPK and 60 μg for CaMKII were loaded into each well and separated by electrophoresis using NuPAGE Novex 4 12% Bis-Tris Gels (Invitrogen) and transferred onto polyvinylidine fluoride membranes (Immobilon FL, Millipore). Membranes were blocked for 1 h at room temperature in 3% (w/v) bovine serum albumin (BSA) in Tris-buffered saline containing 0.05% Tween 20 (TBS-T), followed by incubation overnight at 4 C with the following antibodies diluted in blocking buffer: mouse anti-serca1 ATPase (1:2500, no. ab2818, Abcam), rabbit anti-ampk (1:1000, no. 2532, Cell Signaling Technology), rabbit anti-phospho-thr 172 -AMPK (1:1000, no. 2535, Cell Signaling Technology), mouse anti-camkii (0.1 μgml 1, no , BD Biosciences), rabbit anti-phospho-thr 286 -CaMKII (1:1000, no. 3361, Cell Signaling Technology), and mouse anti-dihydropyridine receptor (DHPR; 1:500, no. ab2864, Abcam). Membranes were then washed in TBS-T and incubated for 1 h at room temperature with IRDye 680-conjugated goat anti-mouse IgG and IRDye 800-conjugated goat anti-mouse IgG (1:15,000, LI-COR) diluted in TBS-T with 3% (w/v) BSA and 0.01% SDS. Membranes were then washed in TBS-T. Immunoreactive bands were visualized using infrared fluorescence (IR-Odyssey scanner, LI-COR Biosciences). PGC-1α and DHPR. FDB muscles were homogenized in ice-cold homogenization buffer (20 μl per mg wet weight) consisting of (mm): Tris, 50; dithiothreitol, 20; 2-mercaptoethanol, 127; EDTA, 5; potassium fluoride, 20; plus 1% (v/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 0.01% (v/v) bromophenol blue, and one tablet of protease inhibitor cocktail (Roche) per 50 ml of buffer. The homogenate was heated to 100 C for 5 min and then centrifuged at 23,000 g for 5 min at room temperature. Fifteen micrograms of protein were loaded onto each well and separated by electrophoresis using NuPAGE Novex 12% Bis-Tris Gels (Invitrogen) and transferred onto polyvinylidine fluoride membranes (Bio-Rad). Membranes were blocked in 5% (w/v) non-fat milk

4 4278 J. D. Bruton and others J Physiol Tris-buffered saline containing 0.05% Tween 20 followed by incubation overnight at 4 C with mouse anti-pgc-1α antibody (1:1000 dilution, no. ST1202, Calbiochem) in 5% (w/v) bovine serum albumin. Membranes were then washed and incubated for 1.5 h at room temperature with horseradish peroxidase-conjugated antibody (goat-anti-mouse IgG, 1:10,000 dilution, Bio-Rad). Immunoreactive bands were visualized using enhanced chemiluminiscence (Super Signal; Pierce, Rockford, IL, USA). Membranes were thereafter re-blotted against the mouse anti-dihydropyridine receptor (DHPR) antibody (1:500 dilution, no. ab2864, Abcam) followed by incubation with horseradish peroxidase-conjugated antibody (anti-mouse IgG, 1:1000 dilution, Pierce). Control experiments with the anti-pgc1α antibody showed the expected markedly higher band intensities in samples from mouse heart and slow-twitch soleus muscles, which have higher mitochondrial content than fast-twitch extensor digitorum longus and FDB muscles (Supplemental Fig. S1A). Control experiments were also performed on skeletal muscle samples from transgenic mice overexpressing PGC-1α in their skeletal muscle and these showed markedly higher band intensities than controls (Supplemental Fig. S1B). Band densities were analysed with Image J (NIH, USA; The DHPR band intensity showed no difference between the different groups of muscles and was used as loading controls. Equal protein loading was also verified with protein staining of membranes (SimplyBlue Safe Stain, Invitrogen). Data for protein expression are presented relative to DHPR expression and the mean value in muscles from room-temperature mice was set to 100%. RNA isolation and quantitative PCR Total RNA from FDB muscles was isolated using the RNeasy Mini Kit (Qiagen). cdna was synthesized using the RevertAid First Strand cdna Synthesis Kit (MBI Fermentas), and quantitative PCR reactions were performed with the ABI 7700 Sequence Detection System (Applied Biosystems, USA) using TaqMan chemistry. The forward and reverse primer sequences were as follows: Tfam 5 -TTCGTTACGACAATGAAATGAAGTC-3 and 5 - TCGACGGATGAGATCACTTCG-3 (NM ); SERCA2A 5 -CAGCCATGGAGAACGCTCA-3 and 5 -TCGTTGACCCCGAAGTGG-3 (NM ); MyHC type IIa 5 -CCGAAGCGAGGCACAAA-3 and 5 -TTGGGCTTTTTATTTCCTTACAACA-3 (NM ); MyHC type IIb (Myh4) 5 -GAAGA- GCCGAGAGGTTCACAC-3 and 5 -CAGGACAGTGA- CAAAGAACGTC-3 (NM ); MyHC type IIx (Myh1, MyhIId, MyhIIx, MyhIIx/d) 5 -GAAGAGTGATTGATCCAAGTG-3 and 5 -TAT- CTCCCAAAGTTATGAGTACA-3 (NM ); MyHC type Ib (MHC-Ib, Myo1b) 5 -AATTCA- CAGACCAGCAGAAACTTATTT-3 and 5 -TGCCCAA- CACTAGAAGGATATAAAGC-3 (NM ). The fluorogenic probes were: Tfam, 5 -Fam-TGGGAAGA- GCAGATGGCTGAAGTTGG-Tamra-3 ; SERCA2A, 5 - Fam-ACAAAGACCGTGGAGGAGGTGCTGG-Tamra-3 ; MyHC type IIa, 5 -Fam-TCATGCGCCTGTGTGATTCT- ATTCCATC-Tamra-3 ; MyHC type IIb, 5 -Fam-AT- CCATCTTTCTGTTGAGAGGTGAC-Tamra-3 ; MyHC typeiix,5 -Fam-TGACCAAAGAGATGAGCAAAATGTG- Tamra-3 ;MyHC-Ib,5 -Fam-AGCTCGAGGCCAGCGAA- CTCTTCAA-Tamra-3. The results were normalized to 18S rrna quantified from the same samples using the forward and reverse primers 5 -TGGTT- GCAAAGCTGAAACTTAAAG-3 and 5 -AGTC- AAATTAAGCCGCAGGC-3, and the fluorogenic probe 5 -Vic-CCTGGTGGTGCCCTTCCGTCA-Tamra-3. Statistics Data are presented as mean ± S.E.M. Two-wayrepeated measures ANOVA (SigmaStat 3.11, Systat Software) was used when comparing repeated measurements (frequency or time and temperature) and when this showed a significant difference, the Holm Sidak post hoc test was performed. Student s unpaired t test was used to detect differences between single measurements. P < 0.05 was considered statistically significant. Results Tetanic and basal [Ca 2+ ] i is higher in muscle fibres of cold-acclimated mice [Ca 2+ ] i and force were measured during contractions at different frequencies. The difference in tetanic [Ca 2+ ] i between FDB fibres of room-temperature (kept at 24 C) WT and UCP1-KO mice was insignificant (less than 10%) at all frequencies and this was also the case for cold-acclimated (kept at 4 C) WT and UCP1-KO mice (Fig. 1A). As there was no effect of genotype on this parameter, we have pooled the data and these show higher [Ca 2+ ] i at 70 and 100 Hz stimulation in fibres of cold-acclimated than of room-temperature mice (Fig. 1B). Tetanic forces at all stimulation frequencies were similar in the four groups of fibres, and hence pooled data from fibres of cold-acclimated and room-temperature mice show similar forces at all stimulation frequencies (Fig. 1C). Note that the higher [Ca 2+ ] i in cold-acclimated fibres at 70 and 100 Hz did not result in higher forces because force was close to maximal at these frequencies. The mean force [Ca 2+ ] i relationships of fibres from

5 J Physiol Increased fatigue resistance in cold-acclimated skeletal muscle fibres 4279 cold-acclimated and room-temperature mice were similar (Fig.1D) and hence analyses (using eqn (1)) in individual fibres show no differences between the groups regarding the maximum force (P max ; 399 ± 23 vs. 391 ± 28 kpa), the [Ca 2+ ] i required to obtain half-maximum tetanic force (Ca 50 ; 0.61 ± 0.05 vs ± 0.05 μm) and the steepness of the relationship (N;4.0± 0.4 vs.4.7± 0.7). Basal [Ca 2+ ] i was similar in WT and UCP1-KO fibres (difference < 5%) obtained from cold-acclimated mice, and the situation was the same for room-temperature mice. Pooled data from WT and UCP1-KO fibres show an 50% (P < 0.01) higher basal [Ca 2+ ] i in fibres of cold-acclimated compared to room-temperature mice (Fig. 2A). Two possible mechanisms behind the increased basal [Ca 2+ ] i in fibres of cold-acclimated mice are increased SR Ca 2+ leak and impaired SR Ca 2+ pumping. To differentiate between these two possibilities, we analysed the tails of increased [Ca 2+ ] i after tetanic contraction using a model originally described by Klein and co-workers (1991). Fig. 2B shows averaged tails of [Ca 2+ ] i after 100 Hz tetani in fibres of cold-acclimated and room-temperature mice. The tails were fitted to a double exponential function (dashed lines). These fits were used to calculate d[ca 2+ ] i /dt vs. [Ca 2+ ] i at regular intervals, and the resulting data points are plotted in Fig. 2C. The following equation was fitted to these points: d[ca 2+ ] i /dt = A[Ca 2+ ] n i L (2) where A reflects the rate of SR Ca 2+ uptake, L is the SR Ca 2+ leak, and n is a power function (Klein et al. 1991; Westerblad & Allen, 1994). To enable comparisons of A and L between groups, n wassetto4andthisgaveagood fit (Klein et al. 1991). The [Ca 2+ ] i tail analysis showed a fourfold higher SR Ca 2+ leak (L) in cells of cold-acclimated mice (29.4 nm s 1 ) as compared to cells of room-temperature mice (6.8 nm s 1 ). The value of A, which reflects the pumping rate, was lower in fibres of cold-acclimated mice ( vs nm 3 s 1 ). Thus, the increase in basal [Ca 2+ ] i can be explained by a markedly increased SR Ca 2+ leak combined with a slowed SR Ca 2+ pumping. To assess the relative importance of these two changes, we entered the individual changes in L and A into eqn (2) and calculated the change in basal [Ca 2+ ] i (i.e. when d[ca 2+ ] i /dt = 0). This simulation indicated that about 70% of the increase in basal [Ca 2+ ] i was due to increased SR Ca 2+ leak and the remaining 30% to slowed SR Ca 2+ pumping. Western blotting of SERCA1 (the predominant isoform in fast-twitch skeletal muscle) was used to study whether decreased expression of these pumps can explain the slowed SR Ca 2+ pumping. The results, however, did not show any difference in SERCA1 expression between muscles from room-temperature and cold-acclimated mice (Fig. 2D ande). The mitochondrial content is increased in muscles of cold-acclimated mice According to our hypotheses, the increased [Ca 2+ ] i observed in FDB muscle fibres of cold-acclimated mice would lead to enhanced mitochondrial biogenesis mediated via increased PGC-1α expression and activity (Ojuka et al. 2003; Wright et al. 2007; Arany, 2008). We therefore measured PGC-1α protein expression as well as citrate synthase and β-had actitivities, which reflect Figure 1. Tetanic [Ca 2+ ] i is increased in FDB fibres of cold-acclimated mice A, mean data (± S.E.M.) of tetanic [Ca 2+ ] i vs. the stimulation frequency. Fibres of cold-acclimated (4 C) WT (, n = 4) and UCP1-KO (, n = 6) mice and room-temperature (24 C) WT (, n = 6) and UCP1-KO (, n = 5) mice. Pooled data of tetanic [Ca 2+ ] i (B) andforce(c) vs. stimulation frequency from fibres of cold-acclimated ( ) and room-temperature ( ) mice.d, pooled data of force vs. [Ca 2+ ] i obtained at different stimulation frequencies in cold-acclimated ( ) and room-temperature ( ) mice; data from B and C. P < 0.01.

6 4280 J. D. Bruton and others J Physiol the cellular mitochondrial content and the mitochondrial capacity for fatty acid β-oxidation, respectively (Holloszy & Coyle, 1984; Reisch & Elpeleg, 2007). Neither of these measurements showed any significant difference between muscles obtained from WT and UCP1-KO mice kept either in the cold or at room temperature (differences generally <10% with P values ranging from 0.18 to 0.96). Therefore, we used pooled data to compare muscles from cold-acclimated and room-temperature mice. The protein expression of PGC-1α and the citrate synthase activity were 50% higher in muscles of cold-acclimated than of room-temperature mice (Fig. 3A and B). The activity of β-had was also higher in muscles of cold-acclimated than of room-temperature mice (3.05 ± 0.13 vs ± 0.12 μmol (g ww) 1 min 1, n = 10 in both groups, P < 0.05). The PGC-1α-mediated stimulation of mitochondrial biogenesis involves increased expression of the mitochondrial transcription factor A (Tfam), which translocates from the nucleus into mitochondria where it initiates transcription and replication of the mitochondrial genome (Wu et al. 1999). We used real-time PCR (RT-PCR) to quantify the expression of transcripts encoding for Tfam and observed the expected increase in muscles of cold-acclimated as compared to room-temperature mice (Fig. 3C). Activation of the Ca 2+ /calmodulin-dependent protein phosphatase calcineurin is one tentative mechanism linking elevated [Ca 2+ ] i to increased mitochondrial biogenesis (Arany, 2008). Activation of calcineurin promotes the conversion of fast-twitch type II fibres to slow-twitch type I fibres, at least in developing or regenerating muscle (Chin et al. 1998; Naya et al. 2000; McCullagh et al. 2004; Schiaffino, 2010). We therefore assessed the fibre type distribution in FDB muscles of cold-acclimated and room-temperature mice by measuring MyHC isoforms but the results showed no difference between the two groups (Fig. 4A). We also measured the expression of transcripts encoding for the different MyHC isoforms. In this case, the results showed about a doubling of the mrna expression of MyHC type 1, whereas the expression of the type II isoforms were not changed (Fig. 4B). This indicates a drive towards more MyHC type I, which was not translated into a detectable change at the protein level. Another cellular Ca 2+ sensor that may translate an increased [Ca 2+ ] i into increased mitochondrial biogenesis is CaMKII (Wright et al. 2007). The expression and Figure 2. FDB fibres of cold-acclimated mice show altered SR Ca 2+ handling A, mean data (± S.E.M.) of basal [Ca 2+ ] i in fibres of room-temperature (n = 12; filled bar) and cold-acclimated (n = 10; open bar) mice. P < B, average records of tails of elevated [Ca 2+ ] i after the end of 100 Hz tetanic stimulation in fibres from cold-acclimated (n = 10) and room-temperature (n = 11) mice as indicated. Time axis starts at the end of tetanic stimulation. The tails were fitted to a double exponential function (dashed lines). C, data points of the rate of [Ca 2+ ] i change ( d[ca 2+ ] i dt 1 ) vs. [Ca 2+ ] i obtained from the curve fits in A (room temperature, ; cold acclimated, ). The lines represent curve fitting of these data points to eqn (2). D, representative Western blots for SERCA1 and DHPR (loading control) in FDB muscles of WT and UCP1-KO mice kept at room temperature or in the cold as indicated. E, pooled mean data (± S.E.M.) of SERCA1 protein expression relative to DHPR in muscles from room-temperature (n = 5; mean value set to 100%) and cold-acclimated (n = 6) mice.

7 J Physiol Increased fatigue resistance in cold-acclimated skeletal muscle fibres 4281 phosphorylation of CaMKII have been shown to increase with endurance training in human muscle (Rose et al. 2007). We measured the protein expression of CaMKII but did not observe any difference between FDB muscles from room-temperature and cold-acclimated mice (Supplemental Fig. S1B). We also attempted to measure the extent of phosphorylation of CaMKII but were unable to get reliable data from these experiments. Increased [Ca 2+ ] i may induce changes in the cellular energy status that may be sensed by AMPK. Activation (phosphorylation) of AMPK has been shown to increase the expression of PGC-1α and mitochondrial biogenesis in muscle (Jäger et al. 2007). Total AMPK protein abundance did not differ between FDB muscles of room-temperature and cold-acclimated mice (100 ± 2 vs. 104 ± 2%, n = 6 in both groups). Moreover, the extent of AMPK phosphorylation did not differ between muscles from room-temperature and cold-acclimated mice (Fig. 4C). On the other hand, in control experiments where FDB muscles were subjected to repeated tetanic stimulation there was about a threefold increase in the phosphorylation of AMPK. (i.e. decreased P max ), followed by a force decrease along a curve that is shifted towards higher [Ca 2+ ] i as compared to the un-fatigued state (Fig. 6B). Fibres of cold-acclimated mice showed the initial reduction in force due to decreased P max, but thereafter tetanic [Ca 2+ ] i never decreased to the extent required for force to start to decline along the steep part of the force [Ca 2+ ] i relationship. The fact that fibres from cold-acclimated mice never entered the steep part of the force [Ca 2+ ] i relationship was not simply a consequence of their higher initial tetanic [Ca 2+ ] i, because tetanic [Ca 2+ ] i was, if anything, better maintained in fibres of cold-acclimated than in fibres of room-temperature mice; tetanic [Ca 2+ ] i in the last tetanus was 116 ± 8and 89± 10% of the initial, respectively (see Fig. 6A). Slowing of force relaxation is a common characteristic of skeletal muscle fatigue (Allen et al. 2008b). We measured the half-relaxation time in the first, tenth and last tetanus of fatigue runs (Fig. 6C). The half-relaxation time in fibres of room-temperature mice was increased by Muscle fibres of cold-acclimated mice are more fatigue resistant In a final set of experiments we tested whether the increased mitochondrial content in FDB muscles of cold-acclimated mice resulted in increased fatigue resistance. Isolated fibres were fatigued by 50 repeated contractions. Fibres of cold-acclimated mice were generally more fatigue-resistant than fibres of room-temperature mice, and representative force and [Ca 2+ ] i recordsareshowninfig.5.meandatashowhigher tetanic [Ca 2+ ] i throughout fatiguing stimulation in fibres of cold-acclimated as compared to room-temperature mice (Fig. 6A, upper panel). Force was decreased by 10% during the first ten tetani in both groups. Thereafter force remained almost constant throughout fatiguing stimulation in fibres of cold-acclimated mice, whereas it started to decrease rather rapidly after 20 tetani in fibres of room-temperature mice (Fig. 6A, lower panel). At the end of fatiguing stimulation, force was higher in fibres of cold-acclimated (decreased by 20%) than of room-temperature (decreased by 60%) mice. The force decrease during fatigue induced by repeated tetanic stimulation is generally due to the combined effect of decreases in P max, myofibrillar Ca 2+ sensitivity and tetanic [Ca 2+ ] i, where the decrease in P max is the dominating factor in early fatigue and the other two factors become increasingly important as fatigue progresses (Allen et al. 2008a). Force [Ca 2+ ] i curves of fibres from room-temperature mice illustrate this pattern with an initial reduction in force despite increasing tetanic [Ca 2+ ] i Figure 3. Increased mitochondrial biogenesis in FDB muscles of cold-acclimated mice A, upper part shows representative Western blots for PGC-1α and DHPR in muscles of room-temperature and cold-acclimated mice as indicated. Lower part shows pooled mean data (± S.E.M.) of protein expression of PGC-1α relative to DHPR in muscles from room-temperature (n = 10; mean value set to 100%) and cold-acclimated (n = 10) mice. B, mean data of citrate synthase (CS) activity in muscles of room-temperature (n = 12) and cold-acclimated (n = 12) mice. C, mean data of Tfam mrna expression relative to 18 s expression in muscles of room-temperature (n = 9) and cold-acclimated (n = 8) mice. Filled bars, room-temperature mice; open bars, cold-acclimated mice. P < 0.05; P <

8 4282 J. D. Bruton and others J Physiol % in the tenth tetanus and by 45% in the last tetanus as compared to the starting value (P < 0.01). On the other hand, fibres of cold-acclimated mice showed no change of the half-relaxation time during fatiguing stimulation (P > 0.05). Consequently, at the end of fatiguing stimulation the half-relaxation time was significantly shorter in fibres of cold-acclimated as compared to room-temperature mice. Another frequent feature of fatigue in fast-twitch fibres is an increase in basal [Ca 2+ ] i (Westerblad & Allen, 1991). At the end of fatiguing stimulation, basal [Ca 2+ ] i was increased by 159 ± 28 and 80 ± 18 nm in fibres of room-temperature and cold-acclimated mice, respectively; thus, the increase was about twice as large in fibres of room-temperature mice (P < 0.05). Discussion We studied skeletal muscle function in mice exposed to a cold environment with special focus on the interaction between intracellular Ca 2+ handling, mitochondrial biogenesis and fatigue resistance. FDB fibres of cold-acclimated mice showed higher basal and tetanic [Ca 2+ ] i and increased mitochondrial content compared to fibres of room-temperature mice. Furthermore, during fatiguing stimulation, tetanic force was better maintained, relaxation was less slowed and the increase in basal [Ca 2+ ] i was smaller in fibres of cold-acclimated as compared to room-temperature mice. Thus, FDB fibres of cold-acclimated mice show increased mitochondrial content and hence increased fatigue resistance and we propose that these changes are mediated by the observed increase in [Ca 2+ ] i. The absence of UCP1 did not affect the response of FDB muscles to cold exposure In this study we used WT and UCP1-KO mice because their response to prolonged cold exposure differs. In WT mice shivering is successively replaced by the recruitment of UCP1-dependent heat generation in brown adipose tissue, whereas UCP1-KO mice continue to depend on heat generated by shivering (Enerbäck et al. 1997; Golozoubova et al. 2001, 2006). Shivering limb muscles of Figure 4. No change in the composition MyHC protein isoforms or AMPK phosphorylation in FDB muscles of cold-acclimated mice A, relative expression (mean ± S.E.M.) of myosin heavy chain (MyHC) isoforms in FDB muscles of room-temperature (n = 3) and cold-acclimated (n = 3) mice. The gel shows representative examples as indicated. The Marker lane was obtained from a mixture of fast-twitch extensor digitorum longus and slow-twitch soleus muscles. Note that the IIa and IIx bands overlap and they are analysed as one band. The IIb band was not detected in the FDB muscles. B, mean data of the mrna expression (relative to 18 s expression) of the different MyHC isoforms in muscles of room-temperature (n = 9) and cold-acclimated (n = 8) mice. C, upper part shows representative Western blots for phosphorylated and total AMPK in muscles of room-temperature and cold-acclimated mice as indicated. Lanes to the right obtained from control experiment where one muscle was frozen after being kept at rest and the other after being stimulated with repeated tetani (100 ms, 50 Hz contractions given every 500 ms for 20 min); note the marked increase in phosphorylated AMPK after stimulation. Lower part shows mean data of phosphorylated to total AMPK of muscles from room-temperature (n = 6; mean value set to 100%) and cold-acclimated (n = 6) mice. Grey bars show measurements from the control experiment. Black bars, room-temperature mice; white bars, cold-acclimated mice. P < 0.01.

9 J Physiol Increased fatigue resistance in cold-acclimated skeletal muscle fibres 4283 Figure 5. FDB fibres of cold-acclimated mice show increased fatigue resistance Representative continuous force records from fatiguing stimulation of a fibre from a room-temperature (A) and a cold-acclimated (B) mouse, respectively. C and D show records of [Ca 2+ ] i (upper part) and force (lower part) from the first, tenth and last (50th) tetani of the fatigue runs depicted above. cold-acclimated UCP1-KO mice display severe contractile dysfunction, altered mitochondrial substrate utilization and increased UCP3 expression (Aydin et al. 2008; Shabalina et al. 2010a). The present study was performed on the distal, superficial FDB muscles, which are unlikely to participate in the shivering response (Hemingway, 1963). This experimental design enabled us to examine possible additional systemic effects caused by the sustained Figure 6. Tetanic [Ca 2+ ] i and force are higher in fatigued FDB fibres of cold-acclimated than of room-temperature mice A, mean data (± S.E.M.) tetanic [Ca 2+ ] i (upper part) and force (lower part) during fatiguing stimulation of FDB fibres from room-temperature (, n = 12) and cold-acclimated (, n = 9) mice. Tetanic force at the start of each fatigue run was set to 100%. P < 0.05; P < B, datafroma were used to plot the relation between force and [Ca 2+ ] i during fatiguing stimulation of fibres from room-temperature (upper part) and cold-acclimated (lower part) mice. Curves (without symbols) show the mean respective force [Ca 2+ ] i relationship under control conditions. C, the half-relaxation time was measured in the first, tenth and 50th tetanus of fatigue runs.

10 4284 J. D. Bruton and others J Physiol shivering of postural limb and trunk muscles in UCP1-KO mice. However, neither of the measured parameters showed any difference between FDB muscles from WT and UCP1-KO mice. Thus, the changes in FDB muscle properties observed in cold-acclimated mice were caused by effects common to both WT and UCP1-KO mice and no additional effects related to the sustained shivering in UCP1-KO mice could be detected. Mechanisms behind increased basal and tetanic [Ca 2+ ] i in fibres of cold-acclimated mice The basal [Ca 2+ ] i was 50% higher in FDB fibres of cold-acclimated as compared to room-temperature mice. To assess the mechanism behind this difference we analysed the tail of elevated [Ca 2+ ] i after the end of tetanic stimulation (see Fig. 2). Changes in [Ca 2+ ] i during this short time frame (<2s)aredominatedbyCa 2+ pumping into the SR and Ca 2+ leakage out of the SR (Klein et al. 1991). Previous analyses with this method in mouse FDB fibres showed the expected results. For instance, a decrease in the SR Ca 2+ uptake (reduced A in eqn (2)) with little effect on the leak (L in eqn (2)) was observed when the SR Ca 2+ pumps were pharmacologically inhibited by 2,5-di(tert-butyl)-1,4-benzohydroquinone (Westerblad & Allen, 1994). The present analysis of the tetanic [Ca 2+ ] i decline showed an about fourfold increase in the SR Ca 2+ leak and a slowed SR Ca 2+ uptake in fibres of cold-acclimated mice. While both these changes would increase basal [Ca 2+ ] i, the increased leak appeared to be the dominating factor. The present results also show 50% higher tetanic [Ca 2+ ] i at 70 and 100 Hz stimulation in FDB fibres of cold-acclimated mice than in fibres of room-temperature mice. The higher basal and tetanic [Ca 2+ ] i in FDB fibres of cold-acclimated mice may involve changes in the SR Ca 2+ release mechanism and the RyR1 channel complex. We recently observed an increase in RyR1 phosphorylation and a slight decrease in the amount of 12 kda FK506 binding protein (FKBP12, also called calstabin1) bound to the RyR1 channel complex in FDB fibres of cold-acclimated mice (Aydin et al. 2008). Such modifications have been associated with prolonged β-adrenergic stimulation resulting in increased SR Ca 2+ leak (Zalk et al. 2007) and cold acclimation is associated with a chronically increased sympathetic activity in skeletal muscle (Dulloo et al. 1988). Moreover, intense physical exercise has been shown to induce major modifications in the RyR1 channel complex (Bellinger et al. 2008). There are differences in the motor behaviour of room-temperature and cold-acclimated mice, which might include the FDB muscles. Therefore we cannot exclude that a shivering-independent increase in contractile activity in FDB muscles of cold-exposed mice contributes to the increased SR Ca 2+ leak. The active SR Ca 2+ uptake via SERCA is another site that might be involved in the increased basal and tetanic [Ca 2+ ] i in FDB fibres of cold-acclimated mice. Obviously the effectiveness by which Ca 2+ is pumped into the SR can affect basal [Ca 2+ ] i and the rate of [Ca 2+ ] i decline after tetanic stimulation (Westerblad & Allen, 1994). Furthermore, SR Ca 2+ leakage may occur via SERCA (Inesi & de Meis, 1989), and recent results indicate that this is the dominating leak pathway in rat fast-twitch muscle fibres at basal [Ca 2+ ] i (Murphy et al. 2009). The protein expression of the predominant isoform of SERCA in fast-twitch muscle, SERCA1, did not differ between room-temperature and cold-acclimated mice (see Fig.2D and E). Furthermore, the mrna expression of SERCA2 (the dominating isoform in slow-twitch muscle) was similar in the two groups (data not shown). Thus, SERCA-related alterations in Ca 2+ handling in muscle fibres of cold-acclimated mice would be due to a change in SERCA function rather than altered expression. The increase with cold acclimatization appeared larger for tetanic [Ca 2+ ] i (from 2 to3μm in 100 Hz tetani; see Fig. 1A and B) than for basal [Ca 2+ ] i (from 57 to 86 nm; seefig.2a). A previous study on mouse FDB fibres has shown that application of a sub-contracture concentration of caffeine, which mainly facilitates SR Ca 2+ release, results in more than a doubling of tetanic [Ca 2+ ] i, whereas basal [Ca 2+ ] i was only increased by 20% (Allen & Westerblad, 1995). Furthermore, inhibition of SERCA with 2,5-di(tert-butyl)-1,4-benzohydroquinone (100 nm) gave 80% increase in tetanic and 30% increase in resting [Ca 2+ ] i (Westerblad & Allen, 1994). Thus, a larger effect on tetanic than on basal [Ca 2+ ] i is in agreement with previous results from mouse FDB fibres both regarding increased SR Ca 2+ release and slowed SR Ca 2+ pumping. Mechanisms underlying the increased cellular mitochondrial content in fibres of cold-acclimated mice Previous studies, as well as the present results (see Fig. 3A), show that cold exposure induces PGC-1α expression in skeletal muscle (Puigserver et al. 1998; Oliveira et al. 2004). PGC-1α has been identified as a powerful mediator of skeletal muscle adaptations, including mitochondrial biogenesis (Arany, 2008). Accordingly, increased mitochondrial enzyme activities and O 2 consumption have been observed in skeletal muscle of cold-acclimated animals (Bourhim et al. 1990; Arruda et al. 2008), which is consistent with the present finding of increased citrate synthase and β-had activities and Tfam mrna expression in FDB muscles of cold-acclimated mice (see Fig. 3B and C).

11 J Physiol Increased fatigue resistance in cold-acclimated skeletal muscle fibres 4285 We show an increased [Ca 2+ ] i in FDB fibres of cold-acclimated mice. [Ca 2+ ] i is considered a key regulator of PGC-1α and mitochondrial biogenesis in skeletal muscle (Handschin et al. 2003; Wright et al. 2007; Arany, 2008). [Ca 2+ ] i may regulate PGC-1α expression via two Ca 2+ /calmodulin-dependent enzymes, calcineurin and CaMKII (Chin, 2004; Lira et al. 2010). Activation of calcineurin promotes a gene programme that acts towards a switch from fast-twitch type II to slow-twitch type I muscle fibres (Chin et al. 1998; Naya et al. 2000). While increased calcineurin activity results in a switch towards moreslow-twitchmyhctypeiproteinindevelopingand regenerating muscle, no or little change in the relation between MyHC type I and type II protein is observed in adult muscle (McCullagh et al. 2004; Schiaffino, 2010). Moreover, endurance training does not induce any major change towards more MyHC type I protein in either human or rodent muscles (Okumoto et al. 1996; Harridge, 2007; Glaser et al. 2010) and a relatively large increase in MyHC type I mrna abundance can be accompanied by only a minor change at the protein level (Short et al. 2005). Our results show a more than doubling of the MyHC type I mrna expression in fibres of cold-acclimated as compared to room-temperature mice, whereas no difference between the two groups were detected at the MyHC protein level. Thus, the present results are consistent with increased calcineurin activation in fibres of cold-acclimated mice. Previous studies support a central role of CaMKII in the signalling cascade relaying increased [Ca 2+ ] i to increased mitochondrial biogenesis. For instance, caffeine has been used to increase [Ca 2+ ] i below the contraction threshold in fast-twitch rat epitrochlearis muscles and this resulted in increased PGC-1α expression and increased mitochondrial biogenesis (Wright et al. 2007). The effects were mediated via p38 mitogen-activated protein kinase and were blocked by pharmacological inhibition of CaMKII (Wright et al. 2007). CaMKII inhibition also completely blocked the mitochondrial biogenesis induced by increasing [Ca 2+ ] i in L6 myotubes (Ojuka et al. 2003). The protein expression of CaMKII has been shown to increase with endurance training (Rose et al. 2007). In the present study, we measured CaMKII protein expression but did not find an increased expression in muscles of cold-acclimated as compared to room-temperature mice. Ca 2+ -induced activation of CaMKII involves autophosphorylation of the enzyme (Hudmon & Schulman, 2002) and we attempted to measure the extent of CaMKII phosphorylation but were unable to get reliable data on this point. Thus, increased CaMKII activity would be expected in FDB muscles of cold-acclimated as compared to room-temperature mice but we are unable to present direct evidence in favour of this notion. Changes in [Ca 2+ ] i can induce changes in the cellular energy status that might be sensed by AMPK, and activation (phosphorylation) of this enzyme increases both the activity and expression of PGC-1α (Jäger et al. 2007). Activation of AMPK has been observed in gastrocnemius muscles of rats kept at 4 C for 4 days (Oliveira et al. 2004). Shivering is still important for maintaining body temperature after 4 days in a cold environment (Cannon & Nedergaard, 2004). The activation of AMPK in the study of Oliveira et al. (2004) might then be associated with metabolic changes induced by the contractile activity during shivering. On the other hand, in the present study we used 4 5 weeks cold exposure and FDB muscles that do not participate in the shivering response and did not observe any difference in the extent of AMPK phosphorylation between muscles of cold-acclimated and room-temperature mice. To sum up, calcineurin and CaMKII are likely to sense the increased [Ca 2+ ] i and induce mitochondrial biogenesis in FDB fibres of cold-acclimated mice, but a contribution from other signalling pathways cannot be excluded. Mechanisms underlying the increased fatigue resistance in fibres of cold-acclimated mice The development of fatigue induced by repeated short tetani in fast-twitch muscle fibres usually follows a characteristic pattern: an initial force decrease of 10 15% over ten tetani accompanied by increased tetanic [Ca 2+ ] i (Phase 1), which is followed by a period of slow force decline (Phase 2), and finally tetanic force and [Ca 2+ ] i decrease more rapidly (Phase 3) (Allen et al. 2008b). The force decrease during Phase 1 was not different in muscle fibres of cold-acclimated and room-temperature mice. This initial force decrease is not determined by the mitochondrial function, because it is little affected by cyanide or decreased oxygen pressure (Lännergren & Westerblad, 1991; Westerblad & Allen, 1991; Stary & Hogan, 2000). Instead it is considered to be caused by breakdown of phosphocreatine, resulting in increased myoplasmic concentration of inorganic phosphate, which acts on cross-bridge function to decrease P max and myofibrillar Ca 2+ sensitivity (increased Ca 50 )(Dahlstedtet al. 2003; Allen et al. 2008b). The duration of Phase 2 was increased in fibres from cold-acclimated mice so that these fibres never entered Phase 3; in other words, the tetanic [Ca 2+ ] i did not decrease to the steep part of the force [Ca 2+ ] i relationship in fibres from cold-acclimated mice (see Fig. 6A). The duration of Phase 2 depends on mitochondrial function because it is markedly shortened by mitochondrial inhibition with cyanide or exposure to decreased oxygen pressure (Lännergren & Westerblad, 1991; Westerblad & Allen, 1991; Stary & Hogan, 2000). In addition to the better maintained force during fatigue in fibres of cold-acclimated mice compared to fibres

12 4286 J. D. Bruton and others J Physiol of room-temperature mice, the former also displayed no slowing of force relaxation and a smaller rise of basal [Ca 2+ ] i, which further emphasizes that they were metabolically less affected by fatiguing stimulation. Thus, our results are consistent with the increased mitochondrial content in FDB fibres of cold-acclimated mice resulting in an increased ability to use oxidative metabolism to generate ATP and hence increased fatigue resistance. An increased cellular mitochondrial content is generally accompanied by an increased fatigue resistance (Burke et al. 1973; Holloszy & Coyle, 1984; Booth & Thomason, 1991; Allen et al. 2008b). The present finding of an increased fatigue resistance in muscle fibres of cold-acclimated mice would then be expected. However, there are several reasons as to why the situation may be more complex. For instance, there might be a decreased mitochondrial efficiency in using fuel oxidation and to convert ADP to ATP in skeletal muscle of cold-exposed mammals (Wu et al. 1999; Zaninovich et al. 2003; Oliveira et al. 2004; Mollica et al. 2005). Moreover, the increase in SR Ca 2+ leak in muscle fibres of cold-acclimated mice will increase the ATP consumption and this additional energy utilization will generate heat rather than being relayed into force production. This thermogenic process would not contribute to the overall temperature control in the cold, because the changes in basal and tetanic [Ca 2+ ] i induced by cold exposure did not differ between FDB fibres from WT and UCP1-KO mice. Nevertheless, we suggest that the heat generated is important for keeping the temperature of superficially and distally located muscles at a level where contractile performance can be maintained. In this context it should be noted that the in vivo temperature in FDB musclesofmicekeptatroomtemperatureisonlyabout 30 C,hencewellbelowthecorebodytemperature(Bruton et al. 1998). To conclude, the increased fatigue resistance in FDB fibres of cold-acclimated mice shows that even if mitochondrial efficiency was reduced and Ca 2+ -related ATP consumption increased, the magnitude of these changes was too small to counteract the increase in mitochondrial biogenesis and oxidative capacity. Conclusions and general implications In the present study we show that a physiological intervention, prolonged cold exposure, can enhance the fatigue resistance of skeletal muscle cells. We show that the increased stress induced by cold exposure results in altered SR Ca 2+ handling and increased [Ca 2+ ] I,andwepropose that this, via activation of PGC-1α, triggers mitochondrial biogenesis. Similar Ca 2+ -dependent signalling has been proposed to underlie the increased aerobic capacity and fatigue resistance induced by endurance training (see Wright et al. (2007) and Rose et al. (2007) and references therein). However, we are not aware of any in vivo study where training-induced changes in basal and tetanic [Ca 2+ ] i have been directly measured. Furthermore, the repeated contractile activity during endurance training will cause marked changes in, for instance, muscle energy metabolism and mechanical load, which may also trigger signalling leading to increased mitochondrial biogenesis. Such changes will not occur in FDB muscles of cold-acclimated mice and these can therefore serve as a more specific model to test the effects of increased [Ca 2+ ] i on mitochondrial biogenesis in vivo. There appears to be a fine balance between beneficial and deleterious changes of Ca 2+ handling in muscle cells. For instance, soleus fibres of cold-acclimated UCP1-KO mice, which are exposed to both the general stress induced by cold exposure and prolonged shivering, display impaired cellular Ca 2+ handling and contractile dysfunction (Aydin et al. 2008). Moreover, very intensive exercise may result in overtraining with markedly increased Ca 2+ leak through the RyR1 channel complex and impaired contractile performance (Bellinger et al. 2008). In conclusion, interventions that induce a modest increase in [Ca 2+ ] i in skeletal muscle cells can have positive functional consequences, e.g. cause an increase in mitochondrial biogenesis and fatigue resistance, whereas more dramatic changes in cellular Ca 2+ handling can be deleterious. References Allen DG, Lamb GD & Westerblad H (2008a). Impaired calcium release during fatigue. JApplPhysiol104, Allen DG, Lamb GD & Westerblad H (2008b). Skeletal muscle fatigue: cellular mechanisms. Physiol Rev 88, Allen DG & Westerblad H (1995). 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