Gordon S. Lynch

Force and power output of fast and slow skeletal muscles from mdx mice 6‐28 months old

1 Differences in the effect of age on structure‐function relationships of limb muscles of mdx (dystrophin null) and control mice have not been resolved. We tested the hypotheses that, compared with limb muscles from age‐matched control mice, limb muscles of 6‐ to 17‐month‐old mdx mice are larger but weaker, with lower normalised force and power, whereas those from 24‐ to 28‐month‐old mdx mice are smaller and weaker. 2 The maximum isometric tetanic force (Po) and power output of limb muscles from 6‐, 17‐, 24‐ and 28‐month‐old mdx and control mice were measured in vitro at 25 °C and normalised with respect to cross‐sectional area and muscle mass, respectively. 3 Body mass at 6 and 28 months was not signifcantly different in mdx and control mice, but that of control mice increased 16 % by 17 months and then declined 32 % by 28 months. The body masses of mdx mice declined linearly with age with a decrease of 25 % by 28 months. From 6 to 28 months of age, the range in the decline in the masses of EDL and soleus muscles of mdx and control mice was from 16 to 28 %. The muscle masses of mdx mice ranged from 9 % to 42 % greater than those of control mice at each of the four ages and, even at 28 months, the masses of EDL and soleus muscles of mdx mice were 17 % and 22 % greater than control values. 4 For mdx mice of all ages, muscle hypertrophy was highly effective in the maintenance of control values for absolute force for both EDL and soleus muscles and for absolute power of soleus muscles. Throughout their lifespan, muscles of mdx mice displayed significant weakness with values for specific Po and normalised power ≈20 % lower than values for control mice at each age. For muscles of both strains, normalised force and power decreased ≈28 % with age, and consequently weakness was more severe in muscles of old mdx than in those of old control mice.

Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy

This review discusses various issues to consider when developing standard operating procedures for pre-clinical studies in the mdx mouse model of Duchenne muscular dystrophy (DMD). The review describes and evaluates a wide range of techniques used to measure parameters of muscle pathology in mdx mice and identifies some basic techniques that might comprise standardised approaches for evaluation. While the central aim is to provide a basis for the development of standardised procedures to evaluate efficacy of a drug or a therapeutic strategy, a further aim is to gain insight into pathophysiological mechanisms in order to identify other therapeutic targets. The desired outcome is to enable easier and more rigorous comparison of pre-clinical data from different laboratories around the world, in order to accelerate identification of the best pre-clinical therapies in the mdx mouse that will fast-track translation into effective clinical treatments for DMD.

Cellular and molecular mechanisms underlying age-related skeletal muscle wasting and weakness

Some of the most serious consequences of ageing are its effects on skeletal muscle. The term ‘sarcopenia’ describes the slow but progressive loss of muscle mass with advancing age and is characterised by a deterioration of muscle quantity and quality leading to a gradual slowing of movement and a decline in strength. The loss of muscle mass and strength is thought to be attributed to the progressive atrophy and loss of individual muscle fibres associated with the loss of motor units, and a concomitant reduction in muscle ‘quality’ due to the infiltration of fat and other non-contractile material. These age-related changes in skeletal muscle can be largely attributed to the complex interaction of factors affecting neuromuscular transmission, muscle architecture, fibre composition, excitation–contraction coupling, and metabolism. Given the magnitude of the growing public health problems associated with sarcopenia, there is considerable interest in the development and evaluation of therapeutic strategies to attenuate, prevent, or ultimately reverse age-related muscle wasting and weakness. The aim is to review our current understanding of some of the cellular and molecular mechanisms responsible for age-related changes in skeletal muscle.

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca2+, which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca2+) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice

To identify mechanisms of anabolic androgen action in muscle, we generated male and female genomic androgen receptor (AR) knockout (ARKO) mice, and characterized muscle mass, contractile function, and gene expression. Muscle mass is decreased in ARKO males, but normal in ARKO females. The levator ani muscle, which fails to develop in normal females, is also absent in ARKO males. Force production is decreased from fast‐twitch ARKO male muscle, and slow‐twitch muscle has increased fatigue resistance. Microarray analysis shows up‐regulation of genes encoding slow‐twitch muscle contractile proteins. Realtime PCR confirms that expression of genes encoding polyamine biosynthetic enzymes, ornithine decarboxylase (Odc1), and S‐adenosylmethionine decarboxylase (Amd1), is reduced in ARKO muscle, suggesting androgens act through regulation of polyamine biosynthesis. Altered expression of regulators of myoblast progression from proliferation to terminal differentiation suggests androgens also promote muscle growth by maintaining myoblasts in the proliferate state and delaying differentiation (increased Cdkn1c and Igf2, decreased Itg1bp3). A similar pattern of gene expression is observed in orchidectomized male mice, during androgen withdrawal‐dependent muscle atrophy. In conclusion, androgens are not required for peak muscle mass in females. In males, androgens act through the AR to regulate multiple gene pathways that control muscle mass, strength, and fatigue resistance.—MacLean, H. E., Maria Chiu, W. S., Notini, A. J., Axell, A.‐M., Davey, R. A., McManus, J. F., Ma, C., Plant, D. R., Lynch, G. S., Zajac, J. D. Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. FASEB J. 22, 2676–2689 (2008)

Expression of the AMP-activated protein kinase β1 and β2 subunits in skeletal muscle

A heterotrimeric member of the AMP‐activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an α2 catalytic and two non‐catalytic subunits, β2 and γ1. The AMPK β2 cDNA (271 amino acids (aa), molecular weight (MW)=30 307, pI 6.3) was cloned from skeletal muscle and found to share an overall identity of 70% with β1 (270 aa, MW=30 475, pI 6.0). In the liver AMPK β1 subunit, Ser‐182 is constitutively phosphorylated whereas in skeletal muscle β2 isoform, we find that Ser‐182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser‐24, Ser‐25 found in the β1 are replaced by Ala‐Glu in the β2 isoform. β2 contains seven more Ser and one less Thr residues than β1, raising the possibility of differential post‐translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively β1 associated with α2, whereas extensor digitorum longus muscle α2 (EDL, fast twitch) associates with β2 as well as β1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co‐immunoprecipitated with the AMPK α2β2γ1 complex.

Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.

Adipose triacylglycerol lipase deletion alters whole body energy metabolism and impairs exercise performance in mice.

Adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) are essential for efficient lipolysis in adipose tissue and skeletal muscle. Herein, we utilized whole body knockout mice to address the importance of ATGL and HSL for metabolic function and exercise performance. ATGL deletion severely disrupts whole-body substrate partitioning at rest; reducing plasma free fatty acid (FFA) availability (WT: 0.49 +/- 0.06 vs. ATGL(-/-) 0.34 +/- 0.03 mM), which in turn enhances carbohydrate oxidation during fasting (mean RER, WT: 0.86 +/- 0.02, ATGL(-/-) 0.90 +/- 0.01) and is associated with depleted muscle and liver glycogen stores. While plasma FFA was modestly reduced (23%) and whole body carbohydrate metabolism increased in HSL(-/-) mice, resting glycogen storage was not compromised. Studies in isolated muscles revealed that the capacity of ATGL and HSL(-/-) muscle to transport exogenous fatty acids is not compromised and the capacity to oxidize fatty acids is actually increased (3.7- and 1.3-fold above WT for ATGL and HSL). The exercise-induced increase in plasma FFA and glycerol was blunted with ATGL or HSL deletion, demonstrating an impaired capacity for exercise-induced lipolysis in these mice. Carbohydrate oxidation was increased concomitantly during exercise in ATGL(-/-) and HSL(-/-) mice, resulting in more muscle and liver glycogen depletion. Maximal running velocity and endurance capacity were reduced by 42% and 46% in ATGL(-/-) mice, but not in HSL(-/-) mice. The reduction in performance in ATGL(-/-) mice was not due to defective muscle contractile performance. These results demonstrate an essential role for both ATGL and HSL in maintaining adequate FFA supply to sustain normal substrate metabolism at rest and during exercise.

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation.

The activation of AMP‐activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl‐CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser‐221 phosphorylation does not always correlate with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole‐4‐carboxymide‐1‐β‐d‐ribofuranoside (AICAR) and contraction‐stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle‐specific kinase dead (KD) AMPK α2. In wild‐type (WT) mice, AICAR and contraction increased AMPK α2 and α1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl‐CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained, malonyl‐CoA levels were reduced and rates of fatty acid oxidation were comparable between genotypes. During treadmill exercise both KD and WT mice had similar values of respiratory exchange ratio. These studies suggested the presence of an alternative ACC2 kinase(s). Using a phosphoproteomics‐based approach we identified 18 Ser/Thr protein kinases whose phosphorylation was increased by greater than 25% in contracted KD relative to WT muscle. Utilizing bioinformatics we predicted that extracellular regulated protein‐serine kinase (ERK1/2), inhibitor of nuclear factor (NF)‐κB protein‐serine kinase β (IKKβ) and protein kinase D (PKD) may phosphorylate ACC2 at Ser‐221 but during in vitro phosphorylation assays only AMPK phosphorylated ACC2. These data demonstrate that AMPK is not essential for the regulation of fatty acid oxidation by AICAR or muscle contraction.

Improved contractile function of the mdx dystrophic mouse diaphragm muscle after insulin-like growth factor-I administration

Limited knowledge exists regarding the efficacy of insulin-like growth factor I (IGF-I) administration as a therapeutic intervention for muscular dystrophies, although findings from other muscle pathology models suggest clinical potential. The diaphragm muscles of mdx mice (a model for Duchenne muscular dystrophy) were examined after 8 weeks of IGF-I administration (1 mg/kg s.c.) to test the hypothesis that IGF-I would improve the functional properties of dystrophic skeletal muscles. Force per cross-sectional area was approximately 49% greater in the muscles of treated mdx mice (149.6 +/- 9.6 kN/m(2)) compared with untreated mice (100.1 +/- 4.6 kN/m(2), P < 0.05), and maintenance of force over repeated maximal contraction was enhanced approximately 30% in muscles of treated mice (P < 0.05). Diaphragm muscles from treated mice comprised fibers with approximately 36% elevated activity of the oxidative enzyme succinate dehydrogenase, and approximately 23% reduction in the proportion of fast IId/x muscle fibers with concomitant increase in the proportion of type IIa fibers compared with untreated mice (P < 0.05). The data demonstrate that IGF-I administration can enhance the fatigue resistance of respiratory muscles in an animal model of dystrophin deficiency, in conjunction with enhancing energenic enzyme activity. As respiratory function is a mortality predictor in Duchenne muscular dystrophy patients, further evaluation of IGF-I intervention is recommended.