Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

The Myopathic Protein Myotilin in Developing Mouse and in Muscle Function

Skeletal muscle cells are highly specialised in order to accomplish their function. During development, the fusion of hundreds of immature myoblasts creates large syncytial myofibres with a highly ordered cytoplasm filled with packed myofibrils. The assembly and organisation of contractile myofibrils must be tightly controlled. Indeed, the number of proteins involved in sarcomere building is impressive, and the role of many of them has only recently begun to be elucidated. Myotilin was originally identified as a high affinity a-actinin binding protein in yeast twohybrid screen. It was then found to interact also with filamin C, actin, ZASP and FATZ-1. Human myotilin is mainly expressed in striated muscle and induces efficient actin bundling in vitro and in cells. Moreover, mutations in myotilin cause different forms of muscle disease, now collectively known as myotilinopathies. In this thesis, consisting of three publications, the work on the mouse orthologue is presented. First, the cloning and molecular characterisation of the mouse myotilin gene showed that human and mouse myotilin share high sequence homology and a similar expression pattern and gene regulation. Functional analysis of the mouse promoter revealed the myogenic factor-binding elements that are required for myotilin gene transcription. Secondly, expression of myotilin was studied during mouse embryogenesis. Surprisingly, myotilin was expressed in a wide array of tissues at some stages of development; its expression pattern became more restricted at perinatal stages and in adult life. Immunostaining of human embryos confirmed broader myotilin expression compared to the sarcomeric marker titin. Finally, in the third article, targeted deletion of myotilin gene in mice revealed that it is not essential for muscle development and function. These data altogether indicate that the mouse can be used as a model for human myotilinopathy and that loss of myotilin does not alter significantly muscle structure and function. Therefore, disease-associated mutant myotilin may act as a dominant myopathic factor.

The Role of Membrane Lipid Composition on Skeletal Muscle Damage in the Rodent Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a X-linked muscle disease, which leads to alterations in membrane phospholipid fatty acid (FA) composition and skeletal muscle damage. Increased membrane saturated FA in muscular dystrophy may suggest its association with increased susceptibility (as being the cause or consequence) to muscle damage. It was hypothesised that increased saturation is positively correlated to increased muscle damage. Correlations were hypothesized to be greater in extensor digitorum longus (EDL) at 20 weeks compared to soleus (SOL) at 10 weeks in dystrophin deficient (mdx) mice. Increased saturation was correlated to damage in EDL at both 10 and 20 weeks, with stronger correlations at 10 weeks. The results suggest that membrane PL FA composition may be associated with damage through two possible means. Increased saturation may be a cause or consequence of membrane damage. Association of membrane composition with eccentric induced damage has underscored the importance of saturated PL FA compositions in damage to dystrophic myofibres.

Muscular Dystrophy in Dogs: Does the Crossing of Breeds Influence Disease Phenotype?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Exercise-induced muscle damage and running economy in humans

Running economy (RE), defined as the energy demand for a given velocity of submaximal running, has been identified as a critical factor of overall distance running performance. Plyometric and resistance trainings, performed during a relatively short period of time (15-30 days), have been successfully used to improve RE in trained athletes. However, these exercise types, particularly when they are unaccustomed activities for the individuals, may cause delayed onset muscle soreness, swelling, and reduced muscle strength. Some studies have demonstrated that exercise-induced muscle damage has a negative impact on endurance running performance. Specifically, the muscular damage induced by an acute bout of downhill running has been shown to reduce RE during subsequent moderate and high-intensity exercise (>65% VOax). However, strength exercise (i.e., jumps, isoinertial and isokinetic eccentric exercises) seems to impair RE only for subsequent high-intensity exercise (90% VOax). Finally, a single session of resistance exercise or downhill running (i.e., repeated bout effect) attenuates changes in indirect markers of muscle damage and blunts changes in RE. © 2013 Cláudio de Oliveira Assumpção et al.

Velocity recovery cycles of human muscle action potentials and their sensitivity to ischemia

This study was undertaken to test whether recovery cycle measurements can provide useful information about the membrane potential of human muscle fibers. Multifiber responses to direct muscle stimulation through needle electrodes were recorded from the brachioradialis of healthy volunteers, and the latency changes measured as conditioning stimuli were applied at interstimulus intervals of 2-1000 ms. In all subjects, the relative refractory period (RRP), which lasted 3.27 +/- 0.45 ms (mean +/- SD, n = 12), was followed by a phase of supernormality, in which the velocity increased by 9.3 +/- 3.4% at 6.1 +/- 1.3 ms, and recovered over 1 s. A broad hump of additional supernormality was seen at around 100 ms. Extra conditioning stimuli had little effect on the early supernormality but increased the later component. The two phases of supernormality resembled early and late afterpotentials, attributable respectively to the passive decay of membrane charge and potassium accumulation in the t-tubules. Five minutes of ischemia progressively prolonged the RRP and reduced supernormality, confirming that these parameters are sensitive to membrane depolarization. Velocity recovery cycles may provide useful information about altered muscle membrane potential and t-tubule function in muscle disease. Muscle Nerve, 2008.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

AIMS:Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. METHODS AND RESULTS:Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. CONCLUSION:Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

Protein adduct species in muscle and liver of rats following acute ethanol administration

Aims: Previous immunohistochemical studies have shown that the post-translational formation of aldehyde-protein adducts may be an important process in the aetiology of alcohol-induced muscle disease. However, other studies have shown that in a variety of tissues, alcohol induces the formation of various other adduct species, including hybrid acetaldehyde-malondialdehyde-protein adducts and adducts with free radicals themselves, e.g. hydroxyethyl radical (HER)-protein adducts. Furthermore, acetaldehyde-protein adducts may be formed in reducing or non-reducing environments resulting in distinct molecular entities, each with unique features of stability and immunogenicity. Some in vitro studies have also suggested that unreduced adducts may be converted to reduced adducts in situ. Our objective was to test the hypothesis that in muscle a variety of different adduct species are formed after acute alcohol exposure and that unreduced adducts predominate. Methods: Rabbit polyclonal antibodies were raised against unreduced and reduced aldehydes and the HER-protein adducts. These were used to assay different adduct species in soleus (type I fibre-predominant) and plantaris (type II fibre-predominant) muscles and liver in four groups of rats administered acutely with either [A] saline (control); [B] cyanamide (an aldehyde dehydrogenase inhibitor); [C] ethanol; [D] cyanamide+ethanol. Results: Amounts of unreduced acetaldehyde and malondialdehyde adducts were increased in both muscles of alcohol-dosed rats. However there was no increase in the amounts of reduced acetaldehyde adducts, as detected by both the rabbit polyclonal antibody and the RT1.1 mouse monoclonal antibody. Furthermore, there was no detectable increase in malondialdehyde-acetaldehyde and HER-protein adducts. Similar results were obtained in the liver. Conclusions: Adducts formed in skeletal muscle and liver of rats exposed acutely to ethanol are mainly unreduced acetaldehyde and malondialdehyde species.

NDRG2 overexpression effects on skeletal muscle cell growth and stress-response

NDRG2 is a gene in skeletal muscle. My PhD thesis determined that increased levels of NDRG2 promoted the growth of muscle cells and also protected against cell death. This research contributed to the understanding of NDRG2 in the hope that in future this knowledge can help combat muscle diseases.

Investigation of physiological properties of nerves and muscles using electromyography.

The measurement and representation of the electrical activity of muscles [electromyography (EMG)] have a long history from the Victorian Era until today. Currently, EMG has uses both as a research tool, in noninvasively recording muscle activation, and clinically in the diagnosis and assessment of nerve and muscle disease and injury as well as in assessing the recovery of neuromuscular function after nerve damage. In the present report, we describe the use of a basic EMG setup in our teaching laboratories to demonstrate some of these current applications. Our practical also illustrates some fundamental physiological and structural properties of nerves and muscles. Learning activities include 1) displaying the recruitment of muscle fibers with increasing force development; 2) the measurement of conduction velocity of motor nerves; 3) the assessment of reflex delay and demonstration of Jendrassik's maneuver; and 4) a Hoffman reflex experiment that illustrates the composition of mixed nerves and the differential excitability thresholds of fibers within the same nerve, thus aiding an understanding of the reflex nature of muscle control. We can set up the classes at various levels of inquiry depending on the needs/professional requirements of the class. The results can then provide an ideal platform for a discovery learning session/tutorial on how the central nervous system controls muscles, giving insights on how supraspinal control interacts with reflexes to give smooth, precise muscular activation.

Transcriptional regulation differs in affected facioscapulohumeral muscular dystrophy patients compared to asymptomatic related carriers

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder that has been associated with a contraction of 3.3-kb repeats on chromosome 4q35. FSHD is characterized by a wide clinical inter- and intrafamilial variability, ranging from wheelchair-bound patients to asymptomatic carriers. Our study is unique in comparing the gene expression profiles from related affected, asymptomatic carrier, and control individuals. Our results suggest that the expression of genes on chromosome 4q is altered in affected and asymptomatic individuals. Remarkably, the changes seen in asymptomatic samples are largely in products of genes encoding several chemokines, whereas the changes seen in affected samples are largely in genes governing the synthesis of GPI-linked proteins and histone acetylation. Besides this, the affected patient and related asymptomatic carrier share the 4qA161 haplotype. Thus, these polymorphisms by themselves do not explain the pathogenicity of the contracted allele. Interestingly, our results also suggest that the miRNAs might mediate the regulatory network in FSHD. Together, our results support the previous evidence that FSHD may be caused by transcriptional dysregulation of multiple genes, in cis and in trans, and suggest some factors potentially important for FSHD pathogenesis. The study of the gene expression profiles from asymptomatic carriers and related affected patients is a unique approach to try to enhance our understanding of the missing link between the contraction in D4Z4 repeats and muscle disease, while minimizing the effects of differences resulting from genetic background.

Aspectos morfológicos de biópsias musculares em equinos com miopatia sob forma de surto

As miopatias em equinos são classificadas de acordo com aspectos clínicos, morfológicos e moleculares, em três grandes grupos: não associadas ao exercício, associadas ao exercício e devido alteração da condução elétrica do sarcolema. Apesar dos avanços no diagnóstico, a literatura ainda relata surtos de miopatia em equinos sem etiologia esclarecida em diversos países. O objetivo desse estudo foi descrever as alterações histológicas e histoquímicas de biópsias musculares de equinos acometidos por miopatia. Sete equinos da raça Quarto de Milha, com 18-24 meses de idade, apresentaram sinais clínicos de miopatia. Dentre esses animais, cinco apresentaram sinais subagudos leves a moderados e dois apresentaram sinais hiperagudos severos e decúbito lateral. Foram realizadas biópsias musculares utilizando a técnica percutânea, por agulha tipo Bergström, no músculo glúteo médio em todos os animais acometidos. As amostras foram processadas por meio de técnicas histológicas (HE, Tricrômio de Gomori modificado) e histoquímicas (PAS, NADH, ATPase). Nos quadros clínicos mais leves, a principal alteração encontrada foi a presença de fibras vermelhas rajadas do tipo I e IIA, que estão associadas às alterações do metabolismo oxidativo e das funções mitocondriais, como ocorrem nas miopatias mitocondriais. Também foram observadas fibras atróficas do tipo I e IIA, além da presença de agregados subsarcolemais. Nos quadros mais severos o tecido muscular apresentou infiltrado inflamatório, aumento de colágeno, fagocitose, necrose, calcificação e regeneração muscular. Diante dos achados morfológicos, da resposta à terapia com vitamina E e Se e da baixa mortalidade quando comparado aos relatos de miopatia atípica, conclui-se que esse surto foi desencadeado por lesões mitocondriais, caracterizadas pelas fibras musculares vermelhas rajadas, possivelmente devido uma quebra da homeostase de vitamina E e Se, sendo compatível com o diagnóstico de miopatia nutricional.