Carl Morris

Fast/Glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese mice.

In contrast to the well-established role of oxidative muscle fibers in regulating whole-body metabolism, little is known about the function of fast/glycolytic muscle fibers in these processes. Here, we generated a skeletal muscle-specific, conditional transgenic mouse expressing a constitutively active form of Akt1. Transgene activation led to muscle hypertrophy due to the growth of type IIb muscle fibers, which was accompanied by an increase in strength. Akt1 transgene induction in diet-induced obese mice led to reductions in body weight and fat mass, resolution of hepatic steatosis, and improved metabolic parameters. Akt1-mediated skeletal muscle growth opposed the effects of a high-fat/high-sucrose diet on transcript expression patterns in the liver and increased hepatic fatty acid oxidation and ketone body production. Our findings indicate that an increase in fast/glycolytic muscle mass can result in the regression of obesity and metabolic improvement through its ability to alter fatty acid oxidation in remote tissues.

The Role of GH and IGF-I in Mediating Anabolic Effects of Testosterone on Androgen-Responsive Muscle

Testosterone (T) supplementation increases skeletal muscle mass, circulating GH, IGF-I, and im IGF-I expression, but the role of GH and IGF-I in mediating Ts effects on the skeletal muscle remains poorly understood. Here, we show that T administration increased body weight and the mass of the androgen-dependent levator ani muscle in hypophysectomized as well as castrated plus hypophysectomized adult male rats. T stimulated the proliferation of primary human skeletal muscle cells (hSKMCs) in vitro, an effect blocked by transfecting hSKMCs with small interference RNA targeting human IGF-I receptor (IGF-IR). In differentiation conditions, T promoted the fusion of hSKMCs into larger myotubes, an effect attenuated by small interference RNA targeting human IGF-IR. Notably, MKR mice, which express a dominant negative form of the IGF-IR in skeletal muscle fibers, treated with a GnRH antagonist (acyline) to suppress endogenous T, responded to T administration by an attenuated increase in the levator ani muscle mass. In conclusion, circulating GH and IGF-I are not essential for mediating Ts effects on an androgen-responsive skeletal muscle. IGF-I signaling plays an important role in mediating Ts effects on skeletal muscle progenitor cell growth and differentiation in vitro. However, IGF-IR signaling in skeletal muscle fibers does not appear to be obligatory for mediating the anabolic effects of T on the mass of androgen-responsive skeletal muscles in mice.

Distinct protein degradation profiles are induced by different disuse models of skeletal muscle atrophy

Skeletal muscle atrophy can be a consequence of many diseases, environmental insults, inactivity, age, and injury. Atrophy is characterized by active degradation, removal of contractile proteins, and a reduction in muscle fiber size. Animal models have been extensively used to identify pathways that lead to atrophic conditions. We used genome-wide expression profiling analyses and quantitative PCR to identify the molecular changes that occur in two clinically relevant mouse models of muscle atrophy: hindlimb casting and Achilles tendon laceration (tenotomy). Gastrocnemius muscle samples were collected 2, 7, and 14 days after casting or injury. The total amount of muscle loss, as measured by wet weight and muscle fiber size, was equivalent between models on day 14, although tenotomy resulted in a more rapid induction of muscle atrophy. Furthermore, tenotomy resulted in the regulation of significantly more mRNA transcripts then did casting. Analysis of the regulated genes and pathways suggest that the mechanisms of atrophy are distinct between these models. The degradation following casting was ubiquitin-proteasome mediated, while degradation following tenotomy was lysosomal and matrix-metalloproteinase mediated, suggesting a possible role for autophagy. These data suggest that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat atrophy resulting from different conditions.

Muscle-Derived Proteins as Serum Biomarkers for Monitoring Disease Progression in Three Forms of Muscular Dystrophy

Abstract Background: Identifying translatable, non-invasive biomarkers of muscular dystrophy that better reflect the disease pathology than those currently available would aid the development of new therapies, the monitoring of disease progression and the response to therapy. Objective: The goal of this study was to evaluate a panel of serum protein biomarkers with the potential to specifically detect skeletal muscle injury. Method: Serum concentrations of skeletal troponin I (sTnI), myosin light chain 3 (Myl3), fatty acid binding protein 3 (FABP3) and muscle-type creatine kinase (CKM) proteins were measured in 74 Duchenne muscular dystrophy (DMD), 38 Becker muscular dystrophy (BMD) and 49 Limb-girdle muscular dystrophy type 2B (LGMD2B) patients and 32 healthy controls. Results: All four proteins were significantly elevated in the serum of these three muscular dystrophy patient populations when compared to healthy controls, but, interestingly, displayed different profiles depending on the type of muscular dystrophy. Additionally, the effects of patient age, ambulatory status, cardiac function and treatment status on the serum concentrations of the proteins were investigated. Statistical analysis revealed correlations between the serum concentrations and certain clinical endpoints including forced vital capacity in DMD patients and the time to walk ten meters in LGMD2B patients. Serum concentrations of these proteins were also elevated in two preclinical models of muscular dystrophy, the mdx mouse and the golden-retriever muscular dystrophy dog. Conclusions: These proteins, therefore, are potential muscular dystrophy biomarkers for monitoring disease progression and therapeutic response in both preclinical and clinical studies.

Treatment with rhBMP12 or rhBMP13 increase the rate and the quality of rat Achilles tendon repair

Tendon injuries that result in partial or complete tears often come from chronic, repetitive use, or from sudden trauma. In some cases, torn tendons can be repaired, but such repairs often fail to completely restore tendon function. We used global gene expression profiling and histological examination to study tendon repair to elucidate key molecular processes that regulate the rate and quality of tissue restoration. Using a rat Achilles tendon transection model, tissue was collected at 3, 7, 10, and 15 days postinjury. The pattern of gene expression in the repairing tissue paralleled the healing phases of inflammation, matrix formation, and matrix reorganization. Newly formed repaired tissue is characterized by cells expressing many genes associated with tendon formation, thereby potentially distinguishing this repair tissue from other types of repair or scar tissue. Addition of recombinant human bone morphogenic protein (rhBMP)12 or rhBMP13, also known as growth and differentiation factors (GDFs) 6 and 7, 1 day after injury yielded increases in tissue volume, rate of cellular infiltration, and in changes in levels of key mRNAs involved in tendon repair. Altogether, our results indicate that rhBMP12 or rhBMP13 enhance the rate of tendon repair. A better understanding of the key molecular regulators of tendon repair could lead to the development of new therapies for tendon injuries and the identification of diagnostic markers that indicate the status of tendon repair after injury.

Dystrophic muscle improvement in zebrafish via increased heme oxygenase signaling

Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx(5cv) mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications.

A proteasome inhibitor fails to attenuate dystrophic pathology in mdx mice.

Dystrophin deficiency leads to increased proteasome activity in skeletal muscle. Previous observations suggest short-term inhibition of the proteasome restores dystrophin expression. Contrary to our hypothesis, eight days of MG-132 administration to mdx mice increased susceptibility to contraction induced injury and Evans blue dye penetration compared to controls. Following six weeks of MG-132 administration muscle function was similar to control animals. These data suggest that proteasome inhibition does not reduce the severity of muscle dysfunction caused by dystrophin-deficiency.

Reduced serum myostatin concentrations associated with genetic muscle disease progression

Myostatin is a highly conserved protein secreted primarily from skeletal muscle that can potently suppress muscle growth. This ability to regulate skeletal muscle mass has sparked intense interest in the development of anti-myostatin therapies for a wide array of muscle disorders including sarcopenia, cachexia and genetic neuromuscular diseases. While a number of studies have examined the circulating myostatin concentrations in healthy and sarcopenic populations, very little data are available from inherited muscle disease patients. Here, we have measured the myostatin concentration in serum from seven genetic neuromuscular disorder patient populations using immunoaffinity LC–MS/MS. Average serum concentrations of myostatin in all seven muscle disease patient groups were significantly less than those measured in healthy controls. Furthermore, circulating myostatin concentrations correlated with clinical measures of disease progression for five of the muscle disease patient populations. These findings greatly expand the understanding of myostatin in neuromuscular disease and suggest its potential utility as a biomarker of disease progression.

Quantitative measurements of GDF-8 using immunoaffinity LC-MS/MS

Growth and differentiation factor 8 (GDF‐8) is a negative regulator of skeletal muscle mass and targeted by inhibitors to treat diseases associated with muscle loss. In order to enable clinical and translational investigations of GDF‐8 inhibitors, specific and sensitive measurements of GDF‐8 are necessary.

1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)indoline-4-carbonitrile Derivatives as Potent and Tissue Selective Androgen Receptor Modulators

We present a novel series of selective androgen receptor modulators (SARMs) which shows excellent biological activity and physical properties. 1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)-indoline-4-carbonitriles showed potent binding to the androgen receptor (AR) and activated AR-mediated transcription in vitro. Representative compounds demonstrated diminished activity in promoting the intramolecular interaction between the AR carboxyl (C) and amino (N) termini. This N/C-termini interaction is a biomarker assay for the undesired androgenic responses in vivo. In orchidectomized rats, daily administration of a lead compound from this series showed anabolic activity by increasing levator ani muscle weight. Importantly, minimal androgenic effects (increased tissue weights) were observed in the prostate and seminal vesicles, along with minimal repression of circulating luteinizing hormone (LH) levels and no change in the lipid and triglyceride levels. This lead compound completed a two week rat toxicology study, and was well tolerated at doses up to 100 mg/kg/day, the highest dose tested, for 14 consecutive days.