Demembranated skeletal and cardiac fibers produce less force with altered cross-bridge kinetics in a mouse model for limb-girdle muscular dystrophy 2i.

Limb-girdle muscular dystrophy 2i (LGMD2i) is a dystroglycanopathy that compromises myofiber integrity and primarily reduces power output in limb muscles but can influence cardiac muscle as well. Previous studies of LGMD2i made use of a transgenic mouse model in which a proline-to-leucine (P448L) mutation in fukutin-related protein severely reduces glycosylation of α-dystroglycan. Muscle function is compromised in P448L mice in a manner similar to human patients with LGMD2i. In situ studies reported lower maximal twitch force and depressed force-velocity curves in medial gastrocnemius (MG) muscles from male P448L mice. Here, we measured Ca2+-activated force generation and cross-bridge kinetics in both demembranated MG fibers and papillary muscle strips from P448L mice. Maximal activated tension was 37% lower in MG fibers and 18% lower in papillary strips from P448L mice than controls. We also found slightly faster rates of cross-bridge recruitment and detachment in MG fibers from P448L than control mice. These increases in skeletal cross-bridge cycling could reduce the unitary force output from individual cross bridges by lowering the ratio of time spent in a force-bearing state to total cycle time. This suggests that the decreased force production in LGMD2i may be due (at least in part) to altered cross-bridge kinetics. This finding is notable, as the majority of studies germane to muscular dystrophies have focused on sarcolemma or whole muscle properties, whereas our findings suggest that the disease pathology is also influenced by potential downstream effects on cross-bridge behavior.

Myostatin regulates pituitary development and hepatic IGF1.

Circulating myostatin-attenuating agents are being developed to treat muscle-wasting disease despite their potential to produce serious off-target effects, as myostatin/activin receptors are widely distributed among many nonmuscle tissues. Our studies suggest that the myokine not only inhibits striated muscle growth but also regulates pituitary development and growth hormone (GH) action in the liver. Using a novel myostatin-null label-retaining model (Jekyll mice), we determined that the heterogeneous pool of pituitary stem, transit-amplifying, and progenitor cells in Jekyll mice depletes more rapidly after birth than the pool in wild-type mice. This correlated with increased levels of GH, prolactin, and the cells that secrete these hormones, somatotropes and lactotropes, respectively, in Jekyll pituitaries. Recombinant myostatin also stimulated GH release and gene expression in pituitary cell cultures although inhibiting prolactin release. In primary hepatocytes, recombinant myostatin blocked GH-stimulated expression of two key mediators of growth, insulin-like growth factor (IGF)1 and the acid labile subunit and increased expression of an inhibitor, IGF-binding protein-1. The significance of these findings was demonstrated by smaller muscle fiber size in a model lacking myostatin and liver IGF1 expression (LID-o-Mighty mice) compared with that in myostatin-null (Mighty) mice. These data together suggest that myostatin may regulate pituitary development and function and that its inhibitory actions in muscle may be partly mediated by attenuating GH action in the liver. They also suggest that circulating pharmacological inhibitors of myostatin could produce unintended consequences in these and possibly other tissues.

Reduced Circulating GDF11 Is Unlikely Responsible for Age-Dependent Changes in Mouse Heart, Muscle, and Brain.

Recent high-profile studies report conflicting data on the age-related change in circulating growth/differentiation factor 11 (GDF11) and myostatin as well as the former's influence on muscle regeneration. Both ligands bind and activate ActRIIB receptors with similar affinities and should therefore have similar actions, yet these studies suggest that GDF11 activates muscle regeneration whereas myostatin is well known to inhibit it. They also suggest that circulating GDF11 levels, but not those of myostatin, decline with age. We performed a careful assessment of the ELISA used to quantify circulating myostatin in these studies and determined that assay reagents significantly cross react with each protein, each of which is highly homologous. Circulating myostatin levels decreased with age and estimates of GDF11 levels using myostatin null mice indicate that they were almost 500 times lower than those for myostatin. This suggests that circulating GDF11 has little physiological relevance as it could not outcompete myostatin for ActRIIB binding sites. Together, these results further suggest that the previously reported aging muscle, heart, and brain phenotypes attributed to reduced circulating GDF11 should be reconsidered.

A novel protocol for assessing exercise performance and dystropathophysiology in the mdx mouse.

INTRODUCTION: Dystrophinopathy in the young mdx mouse model of Duchenne muscular dystrophy is comparatively mild, requires induction, and is rarely assessed with tests of systemic muscle function in whole animals. METHODS: A modified TREAT-NMD induction protocol was used to evaluate respiratory and exercise performance, starting and ending with maximum oxygen consumption (VO2max ) tests. RESULTS: The initial and/or final VO2max , time to exhaustion, speed at exhaustion, and total expended calories were significantly lower in mdx mice. Episodic VO2 and VCO2 fluctuations occurred during training and resulted in dissociated patterns of VO2 and respiratory exchange ratio (RER). These fluctuations further resulted in significantly greater VO2 coefficient of variation and RER values and lower minimal VO2 values. CONCLUSIONS: Quantifying respiratory performance during exercise is a potentially useful means for studying pathophysiology in mdx mice, as it assesses intact animals over time, is more sensitive than some histological markers, and assesses systemic muscle function.

Evidence for expression of both classical and non-classical major histocompatibility complex class I genes in bovine trophoblast cells.

PROBLEM: During the third trimester of pregnancy bovine trophoblast cells in the interplacentomal and arcade regions of the placenta express major histocompatibility complex class I (MHC-I) antigens. At parturition immunological recognition of MHC-I antigens appears to contribute to normal placental release. Therefore, we hypothesized that during late pregnancy bovine trophoblast cells express polymorphic, classical MHC-I antigens. METHOD OF STUDY: Cloning, microarray screening and sequencing of cDNA were used to study transcription of MHC-I genes in peripheral blood mononuclear cells (PBMC) and interplacentomal, trophoblast cells. Real-time reverse transcription, polymerase chain reaction (RT-PCR) was used to compare the abundance of MHC-I transcripts in PBMC and trophoblast cells. RESULTS: Screening of cloned MHC-I cDNA on MHC-I microarrays indicated that in PBMC 90-98% of MHC-I transcripts were encoded by classical MHC-I genes with the remainder encoded by non-classical MHC-I genes. In contrast, 21-66% of MHC-I transcripts from interplacentomal trophoblast cells were from classical genes and 34-79% were from non-classical genes. Transcripts from four non-classical MHC-I loci were identified by sequence analysis. Real-time RT-PCR indicated that the overall levels of MHC-I gene expression in PBMC and trophoblast were similar. CONCLUSION: Bovine interplacentomal trophoblast cells express both classical and non-classical MHC-I genes, but the relative level of expression varies considerably.

Characterization of Bison bison major histocompatibility complex class IIa haplotypes.

American bison (Bison bison) and domestic cattle (Bos taurus and Bos indicus) evolved from a common ancestor 1-1.4 million years ago. Nevertheless, they show dramatic differences in their susceptibility to infectious diseases, including malignant catarrhal fever (MCF). Although bison are highly susceptible to ovine herpesvirus-2 (OvHV-2) associated MCF, about 20% of healthy domesticated and wild bison are positive for OvHV-2 antibody. We are interested in testing the hypothesis that, within the bison population, the polymorphism of major histocompatibility complex (MHC) class II genes influences resistance to MCF. However, since little was known about the MHC class II genes of bison, it was necessary to first characterize class II haplotypes present in Bi. bison (Bibi). Thus, the MHC class II haplotypes carried by 14 bison were characterized by the PCR-based cloning and sequencing of their DRB3, DQA, and DQB alleles. Twelve MHC class II haplotypes were identified in the 14 bison. These haplotypes comprised six previously reported and six new Bibi-DRB3 alleles, along with 11 Bibi-DQA and 10 Bibi-DQB alleles. For each bison class II allele, it was possible to identify closely related cattle sequences. The closest bison and bovine DQA, DQB, and DRB3 alleles, on average, differed by only 1.3, 3.5, and 5.8 amino acids, respectively. Furthermore, bison MHC haplotypes with both nonduplicated and duplicated DQ genes were identified; these haplotypes appear to have originated from the same ancestral haplotypes as orthologous cattle haplotypes.