Sarcolemmal neuronal nitric oxide synthase defect in limb-girdle muscular dystrophy: an adverse modulating factor in the disease course?

Reduction of neuronal nitric oxide synthase (nNOS) has been associated with the pathogenesis and clinical expression of inherited myopathies. To determine whether a defect in nNOS might be an adverse modulating factor in the course of limb-girdle muscular dystrophy, we investigated cytosolic and sarcolemmal nNOS expression in muscle biopsies from 32 patients with 7 forms of limb-girdle muscular dystrophy. Primary calpainopathy, dysferlinopathy, and caveolinopathy biopsies showed normal levels of cytosolic nNOS and preserved sarcolemmal nNOS immunoreactivity. By contrast, the cytosolic nNOS levels in sarcoglycanopathy muscles were variably reduced. Sarcolemmal nNOS immunoreactivity varied from absent to reduced, depending on the integrity of the sarcoglycan complex. In muscles with loss of the entire sarcoglycan complex, sarcolemmal nNOS was absent; it otherwise depended on the specific sarcoglycan gene and type of mutation. The integrity of the entire sarcoglycan complex is, therefore, essential for the stabilization of nNOS to the sarcolemma. Absence of sarcolemmal nNOS in sarcoglycanopathy muscle was always associated with severe muscular dystrophy and sometimes with dilated cardiomyopathy, supporting the hypothesis that nNOS defect might contribute to skeletal and cardiac muscle disease progression. These results emphasize the value of nNOS immunohistochemical analysis in limb-girdle muscular dystrophy and provide additional insights for future therapeutic interventions in these disorders.

Cytoplasmic gamma-actin expression in diverse animal models of muscular dystrophy.

We recently showed that cytoplasmic gamma-actin (gamma(cyto)-actin) is dramatically elevated in striated muscle of dystrophin-deficient mdx mice. Here, we demonstrate that gamma(cyto)-actin is markedly increased in golden retriever muscular dystrophy (GRMD), which better recapitulates the dystrophinopathy phenotype in humans. Gamma(cyto)-Actin was also elevated in muscle from alpha-sarcoglycan null mice, but not in several other dystrophic animal models, including mice deficient in beta-sarcoglycan, alpha-dystrobrevin, laminin-2, or alpha7 integrin. Muscle from mice lacking dystrophin and utrophin also expressed elevated gamma(cyto)-actin, which was not restored to normal by transgenic overexpression of alpha7 integrin. However, gamma(cyto)-actin was further elevated in skeletal muscle from GRMD animals treated with the glucocorticoid prednisone at doses shown to improve the dystrophic phenotype and muscle function. These data suggest that elevated gamma(cyto)-actin is part of a compensatory cytoskeletal remodeling program that may partially stabilize dystrophic muscle in some cases where the dystrophin-glycoprotein complex is compromised.

A novel paradigm for therapeutic basis of advanced heart failure--assessment by gene therapy.

The precise mechanism(s) of the progression of advanced heart failure (HF) should be determined to establish strategies for its treatment or prevention. Based on pathological, molecular, and physiological findings in 3 animal models and human cases, we propose a novel scheme that a vicious cycle formed by increased sarcolemma (SL) permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin (Dys) commonly lead to advanced HF. The aim of this article was to assess our recent paradigm that disruption of myocardial Dys is a final common pathway to advanced HF, irrespective of its hereditary or acquired origin, but not intended to provide a comprehensive overview of the various factors that may be involved in the course of HF in different clinical settings. In addition, each component of Dys-associated proteins (DAP) was heterogeneously degraded in vivo and in vitro, i.e. Dys and alpha-sarcoglycan (SG) were markedly destroyed using isolated calpain 2, while delta-SG was not degraded at all. The up-regulation of calpain 2 was confirmed through previously published data that remain insufficient for precise evaluation, supporting our new scheme that the activation of calpain(s) is involved in the steady process of Dys cleavage. In addition, somatic gene therapy is discussed as a potential option to ameliorate the physiological/metabolic indices and to improve the prognosis.

Muscular dystrophy with reduced beta-sarcoglycan in a cat.

A partial beta-sarcoglycan (SG) deficiency with retention of other components of the SG complex (SGC) is described in 6-month-old, intact male domestic shorthaired kitten that was referred for evaluation of weakness, reluctance to move and dyspnoea. Neurological deficits were restricted to the neuromuscular system. Muscle biopsy revealed moderate variability in myofibre size, with numerous atrophic rounded fibres, rare myofibre necrosis, regeneration and moderate perimysial and endomysial fibrosis. Immunohistochemistry revealed decreased expression of beta- and gamma-SG and western blotting revealed markedly decreased beta-SG with normal expression of alpha-, gamma- and delta-SG, caveolin-3 and calpain-3. Sarcoglycanopathy has not previously been described in cats. In human and canine sarcoglycanopathies the deficiency in any one of the SGs leads to secondary deficiency of the entire SGC. Such spontaneously arising muscular disease in animals can provide valuable models for equivalent human disorders.

Zeta-sarcoglycan is a functional homologue of gamma-sarcoglycan in the formation of the sarcoglycan complex.

The sarcoglycans (SGs), transmembrane components of the dystrophin-associated glycoprotein complex, are stable and functional only when they assemble into a tetrameric complex in muscle cells. A defect in any one of the four SG members disrupts the entire SG complex (SGC) and causes limb-girdle muscular dystrophy. zeta-SG has been recently found as a transmembrane protein homologous to gamma-SG and delta-SG. To characterize zeta-SG in complex formation, we co-transfected expression vectors encoding all six SGs (alpha-, beta-, gamma-, delta-, epsilon- and zeta-SG) and dystroglycan into Chinese hamster ovary cells. Immunoprecipitation analysis showed that zeta-SG or gamma-SG formed a SGC with beta-SG and delta-SG plus alpha-SG or epsilon-SG, revealing that zeta-SG can form two types of SGCs (alpha-beta-zeta-delta or epsilon-beta-zeta-delta). This result indicates the functional resemblance of zeta-SG to gamma-SG rather than delta-SG, although phylogenetic analysis suggests that zeta-SG is evolutionally closer to delta-SG than to gamma-SG. Reverse transcription (RT)-PCR showed that the expression pattern of the transcript was almost the reciprocal of that of gamma-SG in various mouse tissues and that the zeta-SG transcript was especially abundant in the brain, suggesting that zeta-SG might play a particular role in the central nervous system.