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. 2010 Jun;108(6):1775-85.
doi: 10.1152/japplphysiol.01293.2009. Epub 2010 Apr 1.

Skeletal muscle dysfunction in muscle-specific LKB1 knockout mice

David M Thomson  1 , Chad R Hancock, Bradley G Evanson, Steven G Kenney, Brandon B Malan, Anthony D Mongillo, Jacob D Brown, Squire Hepworth, Natasha Fillmore, Allen C Parcell, David L Kooyman, William W Winder
Affiliations

Skeletal muscle dysfunction in muscle-specific LKB1 knockout mice

David M Thomson et al. J Appl Physiol (1985). 2010 Jun.

Abstract

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-alpha 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.

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Figures

Fig. 1.
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.
Fig. 2.
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.
Fig. 3.
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-3β (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.
Fig. 4.
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.
Fig. 5.
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 proliferator-activated receptor-α 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-1α, 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.
Fig. 6.
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.
Fig. 7.
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.
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