Skeletal muscle dysfunction in muscle-specific LKB1 knockout mice

J Appl Physiol 108: 1775–1785, 2010. First published April 1, 2010; doi:10.1152/japplphysiol.01293.2009.

Skeletal muscle dysfunction in muscle-specific LKB1 knockout mice David M. Thomson,1 Chad R. Hancock,2 Bradley G. Evanson,2 Steven G. Kenney,1 Brandon B. Malan,1 Anthony D. Mongillo,1 Jacob D. Brown,1 Squire Hepworth,2 Natasha Fillmore,1 Allen C. Parcell,3 David L. Kooyman,1 and William W. Winder1 Departments of 1Physiology and Developmental Biology, 2Nutrition, Dietetics and Food Science, and 3Exercise Sciences, Brigham Young University, Provo, Utah Submitted 17 November 2009; accepted in final form 31 March 2010

Thomson DM, Hancock CR, Evanson BG, Kenney SG, Malan BB, Mongillo AD, Brown JD, Hepworth S, Fillmore N, Parcell AC, Kooyman DL, Winder WW. Skeletal muscle dysfunction in musclespecific LKB1 knockout mice. J Appl Physiol 108: 1775–1785, 2010. First published April 1, 2010; doi:10.1152/japplphysiol.01293.2009.— Liver kinase B1 (LKB1) is a tumor-suppressing protein that is involved in the regulation of muscle metabolism and growth by phosphorylating and activating AMP-activated protein kinase (AMPK) family members. Here we report the development of a myopathic phenotype in skeletal and cardiac muscle-specific LKB1 knockout (mLKB1-KO) mice. The myopathic phenotype becomes overtly apparent at 30 –50 wk of age and is characterized by decreased body weight and a proportional reduction in fast-twitch skeletal muscle weight. The ability to ambulate is compromised with an often complete loss of hindlimb function. Skeletal muscle atrophy is associated with a 50 –75% reduction in mammalian target of rapamycin pathway phosphorylation, as well as lower peroxisome proliferator-activated receptor-␣ coactivator-1 content and cAMP response element binding protein phosphorylation (43 and 40% lower in mLKB1-KO mice, respectively). Maximum in situ specific force production is not affected, but fatigue is exaggerated, and relaxation kinetics are slowed in the myopathic mice. The increased fatigue is associated with a 30 –78% decrease in mitochondrial protein content, a shift away from type IIA/D toward type IIB muscle fibers, and a tendency (P ⫽ 0.07) for decreased capillarity in mLKB1-KO muscles. Hearts from myopathic mLKB1-KO mice exhibit grossly dilated atria, suggesting cardiac insufficiency and heart failure, which likely contributes to the phenotype. These findings indicate that LKB1 plays a critical role in the maintenance of both skeletal and cardiac function. adenosine 5=-monophosphate-activated protein kinase; mitochondria; cardiac myopathy; mammalian target of rapamycin; adenosine 3=,5=cyclic monophosphate response element binding protein LIVER KINASE B1 (LKB1) WAS first characterized as a tumorsuppressing protein, the mutation of which leads to PeutzJehgers syndrome, a condition characterized by intestinal polyps and an increased risk for contracting a variety of cancers (11). LKB1 phosphorylates members of the AMP-activated protein kinase (AMPK) family of protein kinases (16). Of the 14 AMPK family members, by far the best characterized are AMPK-␣1 and -␣2, which play important roles in cell metabolism and growth (35). Global LKB1 knock out results in embryonic lethality, showing the necessity of the gene during development (37). Tissue-specific knockouts, however, have proven valuable in understanding LKB1 function. Skeletal and cardiac musclespecific LKB1 knockout (mLKB1-KO) mice have been developed by us and other laboratories (14, 17, 23, 27, 30). Young

Address for reprint requests and other correspondence: D. M. Thomson, Dept. of Physiology and Developmental Biology, 589 WIDB, Brigham Young Univ., Provo, UT 84602 (e-mail: [email protected]). http://www.jap.org

mLKB1-KO mice appear to be healthy and without any obvious deficits in normal cage-based activity or behavior. However, when placed in a cage with an activity wheel, voluntary running is substantially lower in mLKB1-KO mice, as is forced treadmill running capacity (30). These deficits may be at least partly due to decreased contents of transcription factors and enzymes associated with mitochondrial biogenesis (14, 30), or fatty acid oxidation rates (27) in muscles from mLKB1-KO mice. This would be consistent with the known function of AMPK in stimulating mitochondrial biogenesis (33). We subsequently observed the overt development of a severe myopathic phenotype, particularly of the hindlimb musculature in mLKB1-KO mice, which becomes apparent after ⬃30 wk of age, on average. The purpose of this study was to characterize this myopathy and to determine potential factors involved in the dysfunctional phenotype. MATERIALS AND METHODS

Generation of mLKB1-KO mouse. As previously described (30), mLKB1-KO mice were generated on an albino FVB background strain by crossing mice expressing a floxed LKB1 gene with mice expressing Cre recombinase under the muscle creatine (Cr) kinase promoter. Genes driven by this promoter are only expressed in skeletal and cardiac muscle. Homozygous floxed LKB1-expressing mice (without Cre expression) were used as controls (CTRL). Genotype was determined by PCR using primers for floxed LKB1 and Cre and verified by Western blotting for LKB1. Before experimentation, all experimental procedures involving animals were approved by and performed in compliance with the Institutional Animal Care and Use Committee of Brigham Young University. In-cage activity measurement. Daily ambulatory cage activity of healthy 2- to 3-mo-old (n ⫽ 9) and healthy or myopathic 6- to 7-mo-old (n ⫽ 4 –11) mice was measured for 3 consecutive days with a computer attached to an infrared beam-based activity monitoring system (Columbus Instruments, Columbus, OH). Tissue collection. Female mLKB1-KO mice develop a dysfunctional hindlimb phenotype typically between 6 and 11 mo of age. Skeletal muscle and heart tissue was harvested from female mLKB1-KO and littermate CTRL mice in the fed state under 2.5% isoflurane anesthesia in supplemental oxygen ⬃2 wk after development of dysfunction, which ranged from 21 to 49 wk of age. Specific age ranges for each cohort of mice used for individual experiments are indicated in Figs. 1–7 legends. Tissue for protein analysis was snapfrozen between metal tongs at the temperature of liquid nitrogen. Tissue for histochemical analysis was embedded in OCT medium and then frozen in isopentane chilled to the temperature of liquid nitrogen. Tissues were then stored at ⫺95°C until analysis. In situ muscle contraction. To directly assess muscle function in the context of mLKB1 deficiency, measurement of in situ contractile function and fatigue of the gastrocnemius-plantaris-soleus (GPS) complex was made essentially as reported previously (9). Briefly, following anesthesia along with continuous supplemental oxygen, tetanic contractions were elicited in CTRL and mLKB1-KO mice

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MLKB1 KNOCKOUT INDUCES SKELETAL MUSCLE DYSFUNCTION

(n ⫽ 8 –9) via direct stimulation of the sciatic nerve (2– 4 V) for 10 min using a Grass S88X stimulator (15 trains/min, 100-ms train duration, 150-Hz pulse frequency). Twitch contractions were elicited in mice (n ⫽ 7–10) during separate experiments at a rate of 2 pulses/s. Tension was measured using a Grass force-displacement transducer FT03, and data were collected and analyzed with Labscribe2 software. Data were analyzed for peak force, fatigue, rate of force development, and relaxation times. Determination of high-energy phosphates. Metabolites relevant to high-energy phosphate metabolism [ATP, ADP, AMP, IMP, phosphocreatine (PCr), and Cr] were measured from contracted muscles and from the corresponding rested muscle on the contralateral leg, as done previously (9). Briefly, metabolites were extracted by homogenizing muscle in cold 3.5% perchloric acid, followed by centrifugation to remove protein and neutralization with tri-n-octylamine and 1,1,2-trichlorotrifluoroethane (4). Adenine nucleotides (ATP, ADP, and AMP) and IMP were quantified by reverse-phase HPLC, as described previously (31). PCr and Cr concentrations were quantified by ion-exchange HPLC (31, 34). Metabolites were expressed as micromoles per gram wet weight and corrected to the total Cr content of rested muscle (30 mmol/g wet wt) to account for fluid shifts that occur in response to muscle contractions. Myosin ATPase stain. Muscle fiber-type and cross-sectional area was determined using methods slightly modified from those of Brooke and Kaiser (3). Transverse medial gastrocnemius muscle sections (10 ␮m) from six to seven mice per group were cut on a cryostatmicrotome at ⫺20°C and stained for myosin ATPase following incubation at pH 4.4. Stained sections were viewed under a light microscope, and digital images were captured on a computer with imaging software. Muscle fiber cross-sectional areas of type I and II fibers were determined using computerized planimetry. At least 50 muscle fibers representing types I, IIA/D, and IIB for each sample were measured, and a mean cross-sectional area was calculated for each fiber type. Transmission electron microscopy. Plantaris muscles (n ⫽ 2–3) were excised, cut into 1mm ⫻ 2mm ⫻ 1mm blocks, and immersed in 2% gluteraldehyde for 2 hrs. Samples were then incubated in osmium tetroxide for two h. After removal of osmium tetroxide the samples were incubated in uranium acetate overnight. Dehydration of the samples was carried out with sequential 10-min washes in 10%, 30%, 50%, 70%, 95% acetone and then three 10-min washes in 100% acetone. The samples were then incubated in a 2:1 acetone/Spurrs epoxy mixture for 1 h, followed by a 1:2 acetone/Spurrs epoxy mixture for 1 h, and 100% Spurrs epoxy for 1 h. The samples were embedded in 100% Spurrs epoxy and allowed to harden. 100nm sections were cut with a microtome and fibers were visualized by transmission electron microscopy. Western blotting. Frozen muscles (n ⫽ 8 per group) were homogenized in 19 (gastrocnemius and heart) or 29 (soleus) volumes (wt/vol) of homogenization buffer (50 mM Tris·HCl, 250 mM mannitol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM dithiothreitol, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 ␮g/ml soybean trypsin inhibitor; pH 7.4) and clarified by centrifugation at 800 g and 4°C. Protein concentration of the homogenates was determined (DC Protein Assay, Biorad, Hercules, CA), and equal muscle protein was loaded on Tris·HCl gels (Bio-Rad Criterion System, Hercules, CA). Proteins were then transferred to polyvinylidene difluoride membranes, which were subsequently stained with Ponceau S to verify even transfer and protein loading across lanes. After blocking in 5% nonfat dry milk, membranes were incubated in primary antibody diluted in 1% BSA overnight at 4°C, washed again, and then incubated in secondary antibody diluted in 1% nonfat dry milk for 1 h at room temperature. Horseradish peroxidase activity was then detected using autoradiographic film (Classic Blue Sensitive; Midwest Scientific, St. Louis, MO). Relative band intensity was quantified using the Spot Denso function of AlphaEaseFC software (Alpha Innotech, San J Appl Physiol • VOL

Leandro, CA). All primary antibodies were from Cell Signaling Technology (Beverly, MA), unless indicated otherwise. Flag-tagged recombinant mouse peroxisome proliferator-activated receptor-␣ coactivator-1 (PGC-1␣) was used to verify the efficacy of the PGC-1␣ antibody (courtesy of Dong-Ho Han from Dr. John Holloszy’s laboratory, Washington University). Primary antibodies used were as follows: LKB1 (no. 07– 694; Upstate, Lake Placid, NY), PGC-1 (no. 516557; Calbiochem, La Jolla, CA), cytochrome c (no. 13156; Santa Cruz, Santa Cruz, CA), phospho-mammalian target of rapamycin (mTOR) (Ser2448; no. 2971), total mTOR (no. 2983), phospho-S6 kinase (S6k) (Thr389; no. 9234), total S6k (no. 2708), phospho-S6 (Ser235/236; no. 2211), total S6 (no. 2217), phospho-eukaryotic initiation factor 4E-binding protein (4EBP1) (Thr37/46; no. 2855), total 4EBP1 (no. 9452), phospho-eukaryotic elongation factor 2 (eEF2) (Thr56; no. 2331), total eEF2 (no. 2332), phospho-Akt (Ser473; no. 9271), total Akt (no. 9272), phospho-cAMP response element binding protein (CREB) (Ser131; no. 9191), phospho-proline-rich Akt substrate of 40 kDa (PRAS40) (Thr246; no. 2997), total PRAS40 (no. 2691), phospho-glycogen synthase kinase-3␤ (GSK3) (Ser21/9; no. 9331). Determination of capillarity. Eight-micrometer medial gastrocnemius muscle cross sections were cut onto glass slides, air dried, and fixed in acetone at 4°C for 10 min. Sections were permeabilized with 0.3% Triton X-100 in PBS for 10 min at 4°C, blocked in 5% normal goat serum for 30 min at room temperature, then incubated overnight in a 1:50 dilution of CD31 antibody (Serotech no. MCA2388GA) in 5% normal goat serum. After washing 3 ⫻ 5 min in PBS, sections were then incubated in the dark in a 1:100 dilution of cy3-conjugated goat anti-rat IgG secondary antibody (Jackson Immunoresearch) for 30 min at room temperature. Slides were washed again in PBS, then mounted with coverslips and visualized with fluorescence microscopy. The total number of capillaries and muscle fibers per field were counted, and capillary-to-fiber ratio was calculated. Citrate synthase activity assay. Citrate synthase activity (n ⫽ 8/group) was determined on diluted raw homogenates using the method described by Srere (26). Statistics. The data are expressed as means ⫾ SE. Comparisons between mLKB1-KO and CTRL mice were made using Student’s t-test. Results were considered significant at P ⬍ 0.05. RESULTS

Muscle-specific LKB1 deletion leads to weight loss and muscle myopathy in female mice. Young mLKB1-KO mice are indistinguishable from their CTRL littermates. However, while attempting to raise mice for use in metabolic studies in old age, we observed the development of a crippling dysfunction of the hindlimbs in female mLKB1-KO mice that occurs as early as 21 wk of age, but at 30 wk of age on average. We have subsequently noted the development of a similar phenotype in male mice. However, all observations reported herein are on female mice, unless otherwise noted. On average, the body weight of mLKB1-KO mice plateaus at ⬃25–26 wk of age and thereafter begins to decline, while the body weight of littermate CTRL mice continues to increase (Fig. 1A). By 29 wk of age, the difference in body weight between CTRL and mLKB1-KO mice is statistically significant. At approximately the same time as the onset of weight loss, the mLKB1-KO mice develop the dysfunctional-hindlimb phenotype. The onset of overt myopathy is most common at ⬃30 wk of age, and, by 36 wk of age, approximately twothirds of the mice are affected. By 1 yr, ⬃90% of all female mice develop the myopathic phenotype. The severity of dysfunction is variable, but, in subjective terms, with hindlimb extension the legs appear stiff and rigid, as if relaxation is

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Fig. 1. Muscle-specific deletion of liver kinase B1 knockout (mLKB1-KO) leads to weight loss and atrophy of type II skeletal muscle in mLKB1-KO. A: body weight over time for female mLKB1-KO mice and littermate controls (CTRL); n ⫽ 17–23 mice per time point. B: body weight and whole muscle weight for gastrocnemius (gastroc), tibialis anterior (TA), and soleus muscles in mLKB1-KO vs. littermate CTRL mice; n ⫽ 9/group at 30 – 45 wk of age (after onset of myopathy in mLKB1-KO mice). C: type I, IIA/D, and IIB muscle fiber areas in mLKB1-KO vs. CTRL medial gastrocnemius muscles; n ⫽ 6 –7/group. Mice were killed at 30 – 45 wk of age (after development of myopathy in mLKB1-KO mice). Values are averages ⫾ SE. *Significant (P ⬍ 0.05) difference between mLKB1-KO and CTRL.

impaired. Thus ambulation is very difficult for the animal. Commonly, at later stages, the mouse is only able to ambulate using its forelimbs (see videos 1 and 2 in supplemental materials; the online version of this article contains supplemental data.). Fast-twitch skeletal muscle atrophy occurs with mLKB1-KO. Muscle mass decreases proportionally with body weight, but primarily in fast-twitch, weight-bearing muscles (Fig. 1B). At the time of death (1–2 wk after development of overt myopathy), gastrocnemius muscle weight is decreased significantly by ⬃18% (93.4 ⫾ 2.3 vs. 77.1 ⫾ 4.1 mg) in mLKB1-KO mice. The non-weight-bearing tibialis anterior (TA) muscle is 9% smaller in mLKB1-KO mice, but this difference was not significant. The slow-twitch soleus muscle weight was likewise not significantly different between genotypes. In agreement with this, the cross-sectional area of type I muscle fibers from the medial gastrocnemius is not affected by genotype, while type II fibers are significantly smaller in mLKB1-KO medial gastrocnemius muscle (Fig. 1C). Weights of some internal J Appl Physiol • VOL

organs (liver and spleen) are also reduced in myopathic mLKB1-KO mice (data not shown). In-cage activity progressively decreases in mLKB1-KO mice. To determine whether a decline in activity precedes the development of overt myopathy, we measured activity levels of 2- to 3-mo-old and 6- to 7-mo-old mLKB1-KO and CTRL mice using an infrared beam-based monitoring system. Young mLKB1-KO mice are normally active (Fig. 2A). At 6 mo of age, however, activity levels of apparently healthy mLKB1-KO mice, with no overt myopathy, were ⬃54% lower than those of CTRL littermates, and activity of myopathic mLKB1-KO mice was predictably approaching zero (Fig. 2B). Skeletal muscle atrophy is associated with decreased mTOR pathway activation. The importance of the mTOR signaling pathway in the regulation of muscle size is well established. Activation of AMPK is known to inhibit activation of the mTOR signaling pathway in skeletal muscle (2, 28), and it might be expected that activation of mTOR in mLKB1-KO muscles would be enhanced. However, with the pronounced

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Fig. 2. In-cage ambulatory activity progressively declines in mLKB1-KO mice. A: in-cage ambulatory activity at 2–3 mo of age for mLKB1-KO and littermate CTRL mice; n ⫽ 9/group. B: in-cage activity at 6 –7 mo of age for healthy mLKB1-KO, healthy CTRL, and myopathic mLKB1-KO mice; n ⫽ 11 CTRL, 7 healthy mLKB1-KO, 4 myopathic mLKB1-KO. Values are averages ⫾ SE.

atrophy in these muscles, we hypothesized that mTOR activity would be reduced. Despite the lack of LKB1 and essentially absent AMPK phosphorylation/activation (data not shown), phosphorylation of mTOR, S6k, and rpS6 (downstream components of the mTOR pathway) were all greatly decreased (34, 25, and 48% of CTRL values, respectively) in the gastrocnemius muscle of mLKB1-KO mice compared with CTRLs, while phosphorylation of 4EBP1 at Thr37/46 and that of eEF2 were unchanged (Fig. 3A). Total protein levels for mTOR and S6 were also reduced significantly in mLKB1-KO muscles (by 45 and 59%, respectively). Total 4EBP1 content was elevated by 42% (Fig. 3B). Phosphorylation of 4EBP1 prevents its ability to downregulate translational initiation by sequestering eIF4E. Since 4EBP1 phosphorylation was unaffected by genotype, the observed increase in total 4EBP1 indicates that the level of unphosphorylated, active 4EBP1 was elevated, which is also consistent with the atrophic phenotype. S6k and eEF2 protein contents were unaffected by genotype (Fig. 3B). Since activation of mTOR in response to growth factor stimulation and insulin is dependent on Akt, we hypothesized that its phosphorylation would, like mTOR, be decreased. Contrary to our hypothesis, phosphorylation of Akt at Ser473 J Appl Physiol • VOL

tended to be elevated (61% greater than CTRL; P ⫽ 0.09) in mLKB1-KO muscles, as did phosphorylation of the Akt target GSK-3␤ (67% greater than CTRL; P ⫽ 0.06). Surprisingly, phosphorylation of PRAS40 at Thr246, another potential Akt target protein, was significantly decreased by 39% in mLKB1-KO muscles (Fig. 3C). Total Akt and PRAS40 content was not different between genotypes. Skeletal muscle fatigability and relaxation time are increased in muscle from myopathic mLKB1-KO mice. To determine whether skeletal muscle performance was affected in mLKB1-KO mice, we measured in situ force production and fatigue of the GPS muscles from myopathic mLKB1-KO mice and littermate CTRL mice. The absolute maximum tetanic tension and peak twitch tension was significantly lower in mLKB1-KO mice (Table 1). However, when tension was normalized to the weight of the GPS, no difference in force production was observed between genotypes. The rate of force production for tetanic contractions was not different between groups (data not shown). In response to a fatiguing protocol of 15 tetanic contractions/min, mLKB1-KO mice exhibited significantly greater fatigue than the CTRL mice (Fig. 4A). Consistent with a compromised energetic state, mLKB1-KO mus-

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Fig. 3. Phosphorylation of mammalian target of rapamycin (mTOR) pathway proteins is decreased in myopathic mLKB1-KO gastrocnemius muscles. A: phosphorylation (p) of the mTOR, p70 ribosomal protein S6 kinase (S6K), ribosomal protein S6 (S6), eukaryotic initiation factor 4E binding protein (4EBP1), and eukaryotic elongation factor 2 (eEF2) in gastrocnemius muscles from mLKB1-KO (KO) and littermate CTRL (C) mice. Mice were killed at 40 – 49 wk of age (after development of myopathy in mLKB1-KO mice). B: total protein content for mTOR (t-mTOR), S6k (t-S6k), S6 (t-S6), 4EBP1 (t-4EBP1), and eEF2 (t-eEF2) in gastrocnemius muscles from KO and C mice. Mice were killed at 40 – 49 wk of age (after development of myopathy in mLKB1-KO mice). C: phosphorylation of Akt, glycogen synthase kinase-3␤ (GSK3), and proline-rich Akt substrate of 40 kDa (PRAS40) in gastrocnemius muscles from KO and C mice; n ⫽ 8/group. Mice were killed at 21–36 wk of age (after development of myopathy in mLKB1-KO mice). Values are averages ⫾ SE. *Significant (P ⬍ 0.05) difference between mLKB1-KO and CTRL.

cles also displayed slower relaxation rates compared with the CTRL mice during fatiguing contractions (Fig. 4B). High-energy phosphate metabolites were measured in GPS muscles at rest and following a 10-min series of tetanic contractions (15/min). Resting concentrations of PCr and Cr were not different between genotypes (PCr 14.1 ⫾ 0.58 and 14.2 ⫾ 0.48; Cr 14.1 ⫾ 0.97 and 16.7 ⫾ 0.77 in CTRL compared with mLKB1-KO muscles, respectively). Furthermore, resting concentrations of each of the metabolites measured were not different between the two genotypes (Table 2). Following 10 min of tetanic contractions (15/min) a significantly greater reduction in ATP and PCr was observed in mLKB1-KO muscle (Table 2). This corresponded with a greater accumulation of IMP in mLKB1-KO muscle as well (Table 2). Thus the increased fatigue and slowed relaxation Table 1. Peak tension during in situ muscle contraction Body weight, g Peak tetanic tension, g Peak tetanic tension, g/mg muscle Peak twitch tension, g Peak twitch tension, g/mg muscle

CTRL

mLKB1-KO

27.2 ⫾ 0.3 392 ⫾ 30 3.32 ⫾ 0.11 136 ⫾ 9.2 1.11 ⫾ 0.08

22.3 ⫾ 0.6* 292 ⫾ 12* 3.26 ⫾ 0.08 87 ⫾ 3.4* 0.96 ⫾ 0.04

Values are means ⫾ SE; n ⫽ 6 –9 animals/group at 26 –53 wk of age (after onset of overt myopathy). CTRL, littermate control mice; mLKB1-KO, muscle-specific LKB1-knockout mice. *P ⬍ 0.05 compared with CTRL. J Appl Physiol • VOL

kinetics are consistent with compromised capacity for ATP production in the mLKB1-KO muscle. Increased fatigability is associated with decreased mitochondrial content in muscle from myopathic mLKB1-KO mice. To determine contributing factors in the fatigability of mLKB1-KO muscle, we measured mitochondrial protein content in the muscles of mLKB1-KO mice and hypothesized that, like young, healthy mLKB1-KO mice (30), they would be lower in muscle from myopathic mLKB1-KO mice. Indeed, content of many mitochondrial enzymes was decreased in mLKB1-KO gastrocnemius muscle (Fig. 5A), including components of complexes 1 (decreased 34%) and 2 (decreased 31%), Core2 (complex 3; decreased 78%), Cox1 (complex 4; decreased 57%), and cytochrome c (decreased 40%). Citrate synthase activity was 40% lower in mLKB1-KO gastrocnemius muscles and 63% lower in the heart in mLKB1-KO vs. CTRL mice (Fig. 5B). PGC-1␣ is an important cofactor in the transcription of mitochondrial genes. We observed a decrease of ⬃40% in PGC-1␣ levels in the mLKB1-KO muscles. Some concern exists concerning the specificity of the commercially available antibodies against PGC-1␣. The PG-1␣ antibody from Calbiochem detected a band of ⬃109 kDa in our gastrocnemius muscle samples. As positive controls we ran brown adipose tissue and recombinant PGC-1 protein alongside the muscle samples (see supplemental data). As expected, the putative

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Fig. 4. In situ fatigue and relaxation time are increased in mLKB1-KO gastrocnemius-plantaris-soleus (GPS) complex muscles. A: in situ skeletal muscle force production from 15 tetanic GPS contractions/min, expressed as %initial force in mLKB1-KO and littermate CTRL mice. Contractions were elicited by sciatic nerve stimulation. B: 50 –75% relaxation time for in mLKB1-KO and CTRL GPS muscles during contraction bout described in A above; n ⫽ 8 –9 mice/group. Mice were killed at 26 –53 wk of age (after development of myopathy in mLKB1-KO mice). Values are averages ⫾ SE. *Significant (P ⬍ 0.05) difference between mLKB1-KO and CTRL.

PGC-1␣ band for brown adipose tissue was very strong and ran at the same molecular weight as in the muscle samples. The antibody also strongly detected recombinant PGC-1␣, which ran slightly slower than the muscle and adipose tissue bands due to the associated flag-tag. Therefore, our antibody detects PGC-1␣, although we cannot say with certainty that a confounding protein of the same molecular weight as PGC-1␣ is not also detected by this antibody. However, since we also

observed decreased expression of mitochondrial proteins whose expression is regulated by PGC-1␣, it is likely that the observed decrease in band intensity in the mLKB1-KO muscles is due at least in part to decreased PGC-1␣ protein content, which was 40% lower in mLKB1-KO vs. CTRL gastrocnemius muscles. (Fig. 5C). The transcription factor CREB is also involved in mitochondrial gene expression in muscle (8). Although CREB protein content was unchanged with genotype (data not shown), phosphorylation of CREB, which corresponds to increased activity, was decreased by 40% in both gastrocnemius and heart muscle from mLKB1-KO vs. CTRL mice (Fig. 5D). As observed by electron microscopy, mitochondrial content and thickness of the subsarcolemmal layer were clearly reduced (Fig. 5E). Muscle fiber-type distribution and capillarity is altered in myopathic mLKB1-KO mice. The percentage of type I fibers, as assessed by myosin ATPase staining, is unaltered by genotype (CTRL ⫽ 14.1 ⫾ 0.03%, mLKB1-KO ⫽ 12.7 ⫾ 0.04%), but we observed a shift from type IIA/D (CTRL ⫽ 17.3 ⫾ 3.0%, mLKB1-KO ⫽ 7.0 ⫾ 1.4%) toward the less oxidative type IIB fibers (CTRL ⫽ 68.6 ⫾ 5.8%, mLKB1-KO ⫽ 80.4 ⫾ 4.0%) in myopathic mLKB1-KO medial gastrocnemius muscles (Fig. 6A). Although not significantly different, the number of capillaries per muscle fiber tended (P ⫽ 0.07) to be lower in mLKB1-KO gastrocnemius muscle (Fig. 6B). Gross atrial dilation occurs in mLKB1-KO mice. Skeletal muscle atrophy and dysfunction is common in cardiac failure, suggesting that heart failure might play a role in the skeletal muscle dysfunction in mLKB1-KO mice. Consistent with this hypothesis, gross atrial dilation and discoloration were observed in all myopathic mLKB1-KO mice (Fig. 7A), suggesting cardiac insufficiency, which confirms a recent report of congestive heart failure in cardiac muscle-specific mLKB1-KO mice (12). While absolute heart weight (Fig. 7B) is not increased (111 ⫾ 3.2 in CTRL and 115 ⫾ 4.4 mg in mLKB1-KO mice), heart-to-body weight ratio (Fig. 7C) is significantly increased in female mLKB1-KO mice (4.5 ⫾ 0.1 vs. 5.7 ⫾ 0.2 mg/g). DISCUSSION

Here we have described the development of a dysfunctional, myopathic phenotype in mice lacking LKB1 in skeletal and cardiac muscle. This condition is characterized by decreased ambulatory activity, generalized atrophy and weight loss, and crippling skeletal muscle dysfunction, particularly in the hindlimbs. The dysfunction is also associated with a specific decrease in type II muscle fiber area, a shift from type IIA/D to

Table 2. Metabolites from rested and stimulated gastrocnemius, plantaris, and soleus muscles CTRL

mLKB1-KO

Metabolite

Rest

15 Tetani/min

Rest

15 Tetani/min

ATP ADP AMP IMP Total AdN ⫹ IMP DPCr with stim

4.85 ⫾ 0.16 0.81 ⫾ 0.03 0.088 ⫾ 0.008 0.020 ⫾ 0.007 5.77 ⫾ 0.19

3.27 ⫾ 0.27 0.71 ⫾ 0.02 0.076 ⫾ 0.013 0.78 ⫾ 0.26 4.83 ⫾ 0.11 8.69 ⫾ 0.80

5.02 ⫾ 0.23 0.86 ⫾ 0.04 0.068 ⫾ 0.008 0.019 ⫾ 0.003 5.97 ⫾ 0.27

2.39 ⫾ 0.18* 0.74 ⫾ 0.03 0.20 ⫾ 0.019* 1.55 ⫾ 0.17* 5.09 ⫾ 0.25 11.82 ⫾ 0.52*

Values are means ⫾ SE. All metabolites are expressed as ␮mol/g muscle wet weight. For rested values, n ⫽ 13–14, and for stimulated values, n ⫽ 4 –7, at 26 –53 wk of age (after onset of overt myopathy). AdN, ATP ⫹ ADP ⫹ AMP; ⌬PCr with stim, the calculated within-animal difference of the measured PCr in the rested muscle and the stimulated muscle. *P ⬍ 0.05 compared with CTRL animals with the same treatment. J Appl Physiol • VOL

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Fig. 5. Mitochondrial content is decreased in mLKB1-KO skeletal muscle. A: skeletal muscle content of complex 1 (Cplx1), complex 2 (Cplx2), complex 5 (Cplx5), cytochrome-c oxidase (Cox1), core 2 of complex 3 (Core2), and cytochrome c (Cyto C) of the electron transport chain in mLKB1-KO and littermate CTRL mice; n ⫽ 8/group. B: citrate synthase activity in gastrocnemius from mLKB1-KO and CTRL mice; n ⫽ 8/group. C: peroxisome proliferatoractivated receptor-␣ coactivator-1 (PGC-1) content in the heart from mLKB1-KO and CTRL mice; n ⫽ 8/group. D: phosphorylation of cAMP response element binding protein (CREB) at Ser133 in gastrocnemius from mLKB1-KO and CTRL mice. Mice were killed at 40–49 wk of age (after development of myopathy in mLKB1-KO mice), except for PGC-1␣, which were at 21–36 wk of age. Values are averages ⫾ SE. *Significant (P ⬍ 0.05) difference between mLKB1-KO and CTRL. E: representative transmission electron micrographs showing decreased thickness of subsarcolemmal mitochondrial layer (marked by arrows) in mLKB1-KO vs. CTRL plantaris muscle; n ⫽ 3/group.

type IIB muscle fibers, increased skeletal muscle fatigability, decreased mitochondrial content, and prolonged relaxation time in situ. These changes in the mLKB1-KO mice are associated with decreased mTOR pathway phosphorylation, CREB phosphorylation, and PGC-1 content, as well as decreased mitochondrial enzyme contents. Loss of mTOR, CREB, or PGC-1 activity through skeletal muscle-specific transgenic mouse models all lead to a myopathic phenotype (1, 10, 22), suggesting that they may play a role in the dysfunctional phenotype observed in our mLKB1-KO mice. Since AMPK is known to inhibit mTOR activity, the loss of LKB1 would be expected to lead to increased mTOR activity (2, 28). However, since mTOR is a major regulator of muscle size, we hypothesized that its activation, as assessed by levels of phosphorylated pathway proteins, would be decreased in the atrophying skeletal muscle from the mLKB1-KO mice. Indeed, we found that phosphorylation of mTOR and downstream mTOR targets S6k and S6 were greatly reduced in mLKB1-KO J Appl Physiol • VOL

gastrocnemius muscles, suggestive of decreased protein synthesis. Regulation of mTOR activity is complex, but the classic upstream mTOR activator is Akt. However, phosphorylation of Akt was not altered significantly by genotype, but instead tended to be elevated, suggesting that the reduction in mTOR activity was not due to decreased Akt activity. The tendency for elevated Akt phosphorylation in our fed mice is consistent with increased insulin-stimulated Akt activity in mLKB1-KO mice observed by Koh et al. (14), potentially due to decreased TRB3 levels. Akt regulates mTOR, in part, through phosphorylation and subsequent sequestration of the mTOR inhibitor PRAS40 (32). However, PRAS40 phosphorylation at the Akt site (Thr246) was significantly decreased in the mLKB1-KO mice, indicating that signaling through a pathway other than Akt is likely regulating this protein. PRAS40 phosphorylation at Thr246 under leucine stimulation is controlled by something other than Akt (24), suggesting that amino-acid signaling to the

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Fig. 6. Skeletal muscle fiber-type shifts away from type IIA/D in mLKB1-KO gastrocnemius muscle. A: fiber-type percentage for mLKB1-KO and littermate CTRL medial gastrocnemius muscles, as determined by myosin ATPase staining. B: capillary-to-muscle fiber ratio for CTRL and mLKB1-KO medial gastrocnemius muscles as determined by CD31 immunofluorescence; n ⫽ 6 –7/group. Mice were killed at 30 – 45 wk of age (after development of myopathy in mLKB1-KO mice). Values are averages ⫾ SE. *Significant (P ⬍ 0.05) difference between mLKB1-KO and CTRL.

mTOR pathway might be defective in mLKB1-KO mice. However, the most obvious defect in the pathway that could explain the decreased mTOR signaling is simply that mTOR levels were lower in the mLKB1-KO mice, suggesting that the defect may not be due to upstream signaling at all, but instead to a lack of mTOR available for activation. Since muscle inactivity through denervation can lead to decreased mTOR levels and phosphorylation (7), a likely mechanism for the decrease in mTOR levels is the progressive decline in in-cage activity that we observed in the mLKB1-KO mice. Regardless of mechanism, the decreased mTOR activity likely plays a role in the myopathic phenotype, since muscle-specific mTOR knockout leads to a similar dystrophic phenotype, albeit at an earlier age (22). Levels of PGC-1␣ were also decreased substantially in mLKB1-KO gastrocnemius muscles, as were levels for many mitochondrial proteins. Skeletal muscle-specific knockout of PGC-1␣ leads to a similar myopathic phenotype, as observed in our mLKB1-KO mice (10). Although PGC-1␣ levels are regulated by skeletal muscle activity (25), our laboratory previously reported decreased PGC-1␣ levels, along with decreased mitochondrial protein levels, in young, apparently healthy mice (30) at an age when basal in-cage activity is not different between genotypes, indicating that inactivity is not the primary mechanism for the decreased PGC-1␣ levels. Since AMPK is also known to regulate PGC-1␣ expression J Appl Physiol • VOL

(13), the lack of AMPK activity in the mLKB1-KO muscles likely plays an important role in this defect. It is possible that other AMPK family members that are activated by LKB1 may also play a role. Similar to the knockout of mTOR and PGC-1␣, skeletal muscle-specific expression of a dominant-negative CREB construct also leads to skeletal muscle atrophy and dysfunction (1). It was determined that this myopathic phenotype was due in part to decreased CREB-dependent expression of salt-inducible kinase (SIK) and a resultant suppression of myocyte enhancer factor-2 activity through hyperactivity of histone deacytylase (HDAC) activity. We observed greatly reduced CREB phosphorylation in mLKB1-KO mice, suggesting that a similar mechanism may play a role in these mice as well. Additionally, SIK is an AMPK family member that is phosphorylated and activated by LKB1, and class II HDACs are also phosphorylated by AMPK (18). Thus the lack of LKB1 may affect SIK1/HDAC signaling to MEF-2, both by CREB-dependent SIK expression, decreased LKB1-dependent SIK phosphorylation, and decreased LKB1-dependent AMPK phosphorylation. Further study will be required to determine whether the SIK/HDAC/ MEF-2 pathway is affected in mLKB1-KO mice. In addition to the roles that decreased mTOR, PGC-1, and CREB activities may play in the development of the myopathic phenotype in the mLKB1-KO mice, heart failure, which independently leads to skeletal muscle atrophy and weakness (5, 6, 21), is very likely an important contributing factor. Although we do not present any functional data characterizing heart performance in our mice, the atria are grossly dilated in mLKB1-KO mice, strongly suggesting the presence of cardiac insufficiency and failure. This observation is in agreement with a recent report indicating that cardiac-specific LKB1 knockout results in heart dysfunction and greatly increased rate of mortality associated with cardiac hypertrophy in male cardiac muscle-specific LKB1-KO mice (12). The loss of body weight in our mLKB1-KO mouse is associated with atrophy of the skeletal muscle, as well as some internal organs (in which LKB1 levels are normal), suggesting that it is mediated, in part, by systemic factors, which may be derived from the failing heart. Although we are unaware of any evidence that heart failure alone can produce the extreme crippling phenotype observed in our mice (see Supplemental Videos 1 and 2), many of the observed differences between mLKB1-KO and CTRL skeletal muscle are consistent with changes observed in heart failure, such as atrophy, decreased capillarity, fiber-type shifting, and decreased PGC-1␣ and mitochondrial content (19). Although the extent of these changes in heart failure is variable, it is likely that the heart is playing a role, to some extent, in our mice. However, as mentioned previously, the changes we observed in PGC-1␣ content, mitochondrial protein levels, and CREB phosphorylation all are present in young, apparently healthy mice, suggesting that these defects are likely primary to the LKB1 knockout and not secondary to heart failure. Thus we think it is likely that the myopathic phenotype in skeletal muscle may be either triggered by a failing heart and exacerbated by the lack of LKB1, or vice versa. This will be the topic of continuing research. Despite the systemic nature of the atrophy that we observed in the mLKB1-KO mice, soleus muscle mass was not decreased, nor was the area for type I fibers lower in the medial

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Fig. 7. Atrial dilation and increased cardiac weight relative to body weight in mLKB1-KO mice. A: gross atrial dilation in female mLKB1-KO vs. CTRL heart. B: absolute heart weight in female mKLB1-KO and CTRL mice; n ⫽ 9/group. C: heart weight relative to body weight (BW) for female mLKB1-KO and CTRL mice; n ⫽ 9/group. All mice were killed at 30 – 45 wk of age (after development of myopathy in mLKB1-KO mice). *Significant (P ⬍ 0.05) difference between mLKB1-KO and CTRL.

gastrocnemius muscle of mLKB1-KO mice, despite a great reduction in gastrocnemius mass and fiber area for type II fibers. These findings are in general agreement with observations that oxidative muscle fibers are resistant against atrophy in a model of chronic heart failure (15). We do not know, however, whether muscle function is altered in the soleus of mLKB1-KO mice. It has been reported that muscle Cr kinasedriven expression of transgenic genes is ineffective in slowvs. fast-twitch muscle fibers (36). However, LKB1 expression was knocked down to a similar extent in soleus and gastrocnemius muscles, indicating that this does not explain the lack of atrophy in the soleus. Interestingly, the TA muscle, despite being fast twitch, was not significantly smaller in mLKB1-KO vs. CTRL mice. Unlike the gastrocnemius muscle, the TA, being a dorsiflexor, is not a weightbearing muscle. Therefore, it appears that skeletal muscle myopathy in this model may be exacerbated by increased loading and/or energetic demand. Although not significant, mLKB1-KO medial gastrocnemius muscles tended (P ⫽ 0.07) to have a decreased capillary-tofiber ratio. If real, this effect is likely due, in part, to the lack of AMPK activity, which is thought to control the basal level of capillarity in skeletal muscle (38), presumably by controlling the expression of vascular endothelial growth factor (20). Predictably, due to decreased muscle mass, absolute force production by the GPS complex in mLKB1-KO mice is much lower than by that of CTRL muscles. However, when normalized to muscle mass, peak force measures are similar between genotypes, suggesting that general muscle mechanics are not impaired in the context of chronic LKB1 deficiency. In contrast, in situ GPS fatigue was greater in mLKB1-KO muscles. This is consistent with the decreased voluntary running ability that we and others have previously observed in young mLKB1-KO mice (14, 30). The increased fatigue is likely due, at least in part, to a reduction in mitochondrial content and capillarity of the mLKB1-KO muscle. We also noted a shift J Appl Physiol • VOL

in fiber type away from type IIA/D muscle fibers, presumably toward type IIB fibers, which would be expected to confer less fatigue resistance on the muscle as a whole. Mitochondrial insufficiency in and of itself is capable of inducing skeletal muscle myopathy and may, therefore, contribute to other aspects of the dysfunctional phenotype observed besides fatigue. Decreased mitochondria are likely due, in part, to a reduction in PGC-1␣ content and CREB phosphorylation, which were both greatly reduced in the gastrocnemius. CREB is known to regulate basal mitochondrial biogenesis during muscle development (8). Its phosphorylation is mediated by a number of kinases, including protein kinase A, mitogen-activated kinases, Ca 2⫹ / calmodulin-dependent protein kinase, GSK3, and, notably in the context of this study, AMPK (29). In addition to decreased AMPK activity in mLKB1-KO mice, we also observed increased a strong, but insignificant, tendency for increased GSK3 phosphorylation. GSK3 is inhibited by phosphorylation at the measured site. Therefore, GSK3 and AMPK might both play a role in the decreased CREB phosphorylation that we observed in the myopathic mice. We did not measure any indicators of activity for any other CREB kinases. In situ relaxation time was also increased in the mLKB1-KO muscles. Whether this is due to alterations in calcium handling by the cell, or is secondary to fatigue is not known, but it is consistent with the subjective analysis of the ambulatory movements of the myopathic mLKB1-KO mice in which the mice appear to have difficulty relaxing the hindlimbs after contraction. In conclusion, we have characterized a myopathic phenotype in mLKB1-KO mice. The root causes of the skeletal muscle dysfunction observed in these mice are not clear, but likely involve mitochondrial insufficiency and other effects of decreased PGC-1␣ content, as well as decreased phosphorylation of CREB and mTOR, and/or cardiac cachexia. Our findings are

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indicative of an important role for LKB1 in skeletal muscle function. ACKNOWLEDGMENTS We thank Steven Ellsworth and Ryan DiGiovanni for assistance with data collection , Michael Standing for technical assistance with electron microscopy, Jiping Zou for assistance with HPLC, Dong-Ho Han for provision of recombinant PGC-1␣, and Hoon Kim and Troy Tenney for assistance with animal husbandry and genotyping. GRANTS This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-51928. DISCLOSURES No conflicts of interest (financial or otherwise) are declared by the author(s). REFERENCES 1. Berdeaux R, Goebel N, Banaszynski L, Takemori H, Wandless T, Shelton GD, Montminy M. SIK1 is a class II HDAC kinase that promotes survival of skeletal myocytes. Nat Med 13: 597–603, 2007. 2. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 277: 23977–23980, 2002. 3. Brooke MH, Kaiser KK. Three “myosin adenosine triphosphatase” systems: the nature of their pH lability and sulfhydryl dependence. J Histochem Cytochem 18: 670 –672, 1970. 4. Chen SC, Brown PR, Rosie DM. Extraction procedures for use prior to HPLC nucleotide analysis using microparticle chemically bonded packings. J Chromatogr Sci 15: 218 –221, 1977. 5. De Sousa E, Veksler V, Bigard X, Mateo P, Ventura-Clapier R. Heart failure affects mitochondrial but not myofibrillar intrinsic properties of skeletal muscle. Circulation 102: 1847–1853, 2000. 6. Degens H, Alway SE. Control of muscle size during disuse, disease, and aging. Int J Sports Med 27: 94 –99, 2006. 7. Dreyer HC, Glynn EL, Lujan HL, Fry CS, DiCarlo SE, Rasmussen BB. Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway. J Appl Physiol 104: 27–33, 2008. 8. Franko A, Mayer S, Thiel G, Mercy L, Arnould T, Hornig-Do HT, Wiesner RJ, Goffart S. CREB-1 alpha is recruited to and mediates upregulation of the cytochrome c promoter during enhanced mitochondrial biogenesis accompanying skeletal muscle differentiation. Mol Cell Biol 28: 2446 –2459, 2008. 9. Hancock CR, Janssen E, Terjung RL. Skeletal muscle contractile performance and ADP accumulation in adenylate kinase-deficient mice. Am J Physiol Cell Physiol 288: C1287–C1297, 2005. 10. Handschin C, Chin S, Li P, Liu F, Maratos-Flier E, Lebrasseur NK, Yan Z, Spiegelman BM. Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1 alpha muscle-specific knock-out animals. J Biol Chem 282: 30014 –30021, 2007. 11. Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, Bignell G, Warren W, Aminoff M, Hoglund P, Jarvinen H, Kristo P, Pelin K, Ridanpaa M, Salovaara R, Toro T, Bodmer W, Olschwang S, Olsen AS, Stratton MR, de la Chapelle A, Aaltonen LA. A serine/ threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 391: 184 –187, 1998. 12. Ikeda Y, Sato K, Pimentel DR, Sam F, Shaw RJ, Dyck JR, Walsh K. Cardiac-specific deletion of LKB1 leads to hypertrophy and dysfunction. J Biol Chem 284: 35839 –35849, 2009. 13. Irrcher I, Adhihetty PJ, Sheehan T, Joseph AM, Hood DA. PPARgamma coactivator-1 alpha expression during thyroid hormone- and contractile activity-induced mitochondrial adaptations. Am J Physiol Cell Physiol 284: C1669 –C1677, 2003. 14. Koh HJ, Arnolds DE, Fujii N, Tran TT, Rogers MJ, Jessen N, Li Y, Liew CW, Ho RC, Hirshman MF, Kulkarni RN, Kahn CR, Goodyear LJ. Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3. Mol Cell Biol 26: 8217–8227, 2006. J Appl Physiol • VOL

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