Oxidative damage to urinary proteins from the GRMD dog and mdx mouse as biomarkers of dystropathology in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, X-chromosome linked muscle-wasting disease affecting about 1 in 3500-6000 boys worldwide. Myofibre necrosis and subsequent loss of muscle mass are due to several molecular sequelae, such as inflammation and oxidative stress. We have recently shown increased neutrophils, highly reactive oxidant hypochlorous acid (HOCl) generation by myeloperoxidase (MPO), and associated oxidative stress in muscle from the GRMD dog and mdx mouse models for DMD. These findings have led us to hypothesise that generation of HOCl by myeloperoxidase released from neutrophils has a significant role in dystropathology. Since access to muscle from DMD patients is limited, the aim of this study was to develop methods to study this pathway in urine. Using immunoblotting to measure markers of protein oxidation, we show increased labelling of proteins with antibodies to dinitrophenylhydrazine (DNP, oxidative damage) and DiBrY (halogenation by reactive oxidants from myeloperoxidase) in GRMD and mdx urine. A strong positive correlation was observed between DiBrY labelling in dog urine and muscle. A strong positive correlation was also observed when comparing DNP and DiBrY labelling (in muscle and urine) to markers of dystropathology (plasma creatine kinase) and neutrophil presence (muscle MPO). Our results indicate the presence of neutrophil mediated oxidative stress in both models, and suggest that urine is a suitable bio-fluid for the measurement of such biomarkers. These methods could be employed in future studies into the role of neutrophil mediated oxidative stress in DMD and other inflammatory pathologies.

Nebulin nemaline myopathy recapitulated in a compound heterozygous mouse model with both a missense and a nonsense mutation in Neb.

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, NebY2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, NebY2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.

Doublet stimulation increases Ca2+ binding to troponin C to ensure rapid force development in skeletal muscle.

Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca2+ during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca2+ release, binding of Ca2+ to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca2+ spikes, at doublet frequencies up to 1 KHz. A 200-Hz doublet at the start of a tetanic stimulation train (70 Hz) decreases the drop in free Ca2+ between the first three Ca2+ spikes of the transient, maintaining a higher overall free Ca2+ level during first 20-30 ms of the response. Doublet stimulation also increased the rate of force development in isolated fast-twitch muscles. We also modeled SR Ca2+ release rates during doublet stimulation and showed that Ca2+-dependent inactivation of ryanodine receptor activity is rapid, occurring ≤1ms after initial release. Furthermore, we modeled Ca2+ binding to the main intracellular Ca2+ buffers of troponin C (TnC), parvalbumin, and the SR Ca2+ pump during Ca2+ release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca2+-binding site on TnC (TnC2), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca2+ levels for longer in the early phase of the Ca2+ response, resulting in faster saturation of TnC2 with Ca2+, faster initiation of cross-bridge cycling, and more rapid force development.