Ana Domazetovska

An αtropomyosin mutation alters dimer preference in nemaline myopathy

Nemaline myopathy is a human neuromuscular disorder associated with muscle weakness, Z‐line accumulations (rods), and myofibrillar disorganization. Disease‐causing mutations have been identified in genes encoding muscle thin filament proteins: actin, nebulin, slow troponin T, βTropomyosin, and αTropomyosinslow. Skeletal muscle expresses three tropomyosin (Tm) isoforms from separate genes: αTmfast(αTm, TPM1), βTm (TPM2), and αTmslow (γTm, TPM3). In this article, we show that the level of βTm, but not αTmfast protein, is reduced in human patients with mutations in αTmslow and in a transgenic mouse model of αTmslow(Met9Arg) nemaline myopathy. A postnatal time course of Tm expression in muscles of the mice indicated that the onset of αTmslow(Met9Arg) expression coincides with the decline of βTm. Reduction of βTm levels is independent of the degree of pathology (rods) within a muscle and is detected before the onset of muscle weakness. Thus, reduction in the level of βTm represents an early clinical diagnostic marker for αTmslow‐based mutations. Examinations of tropomyosin dimer formation using either recombinant proteins or sarcomeric extracts show that the mutation reduces the formation of the preferred α/β heterodimer. We suggest this perturbation of tropomyosin isoform levels and dimer preference alters sarcomeric thin filament dynamics and contributes to muscle weakness in nemaline myopathy. Ann Neurol 2004

Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy

Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary L-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that L-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.

Intranuclear Rod Myopathy : Molecular Pathogenesis and Mechanisms of Weakness

Mutations in the α‐skeletal actin gene (ACTA1) result in a variety of inherited muscle disorders characterized by different pathologies and variable clinical phenotypes. Mutations at Val163 in ACTA1 result in pure intranuclear rod myopathy; however, the molecular mechanisms by which mutations at Val163 lead to intranuclear rod formation and muscle weakness are unknown.

Disease Severity and Thin Filament Regulation in M9R TPM3 Nemaline Myopathy

The mechanism of muscle weakness was investigated in an Australian family with an M9R mutation in TPM3 (&agr;-tropomyosinslow). Detailed protein analyses of 5 muscle samples from 2 patients showed that nemaline bodies are restricted to atrophied Type 1 (slow) fibers in which the TPM3 gene is expressed. Developmental expression studies showed that &agr;-tropomyosinslow is not expressed at significant levels until after birth, thereby likely explaining the childhood (rather than congenital) disease onset in TPM3 nemaline myopathy. Isoelectric focusing demonstrated that &agr;-tropomyosinslow dimers, composed of equal ratios of wild-type and M9R-&agr;-tropomyosinslow, are the dominant tropomyosin species in 3 separate muscle groups from an affected patient. These findings suggest that myopathy-related slow fiber predominance likely contributes to the severity of weakness in TPM3 nemaline myopathy because of increased proportions of fibers that express the mutant protein. Using recombinant proteins and far Western blot, we demonstrated a higher affinity of tropomodulin for &agr;-tropomyosinslow compared with &bgr;-tropomyosin; the M9R substitution within &agr;-tropomyosinslow greatly reduced this interaction. Finally, transfection of the M9R mutated and wild-type &agr;-tropomyosinslow into myoblasts revealed reduced incorporation into stress fibers and disruption of the filamentous actin network by the mutant protein. Collectively, these results provide insights into the clinical features and pathogenesis of M9R-TPM3 nemaline myopathy.

In Vitro Analysis of Rod Composition and Actin Dynamics in Inherited Myopathies

Rods are the pathological hallmark of nemaline myopathy, but they can also occur as a secondary phenomenon in other disorders, including mitochondrial myopathies such as complex I deficiency. The mechanisms of rod formation are not well understood, particularly when rods occur in diverse disorders with very different structural and metabolic defects. We compared the characteristics of rods associated with abnormalities in structural components of skeletal muscle thin filament (3 mutations in the skeletal actin gene ACTA1) with those of rods induced by the metabolic cell stress of adenosine triphosphate depletion. C2C12 and NIH/3T3 cell culture models and immunocytochemistry were used to study rod composition and conformation. Fluorescent recovery after photobleaching was used to measure actin dynamics inside the rods. We demonstrate that not all rods are the same. Rods formed under different conditions contain a unique fingerprint of actin-binding proteins (cofilin and &agr;-actinin) and display differences in actin dynamics that are specific to the mutation, to the cellular location of the rods (intranuclear vs cytoplasmic), and/or to the underlying pathological process (i.e. mutant actin or adenosine triphosphate depletion). Thus, rods likely represent a common morphological end point of a variety of different pathological processes, either structural or metabolic.

Mosaic caveolin-3 expression in acquired rippling muscle disease without evidence of myasthenia gravis or acetylcholine receptor autoantibodies

Inherited rippling muscle disease is an autosomal dominant disorder usually associated with caveolin-3 mutations. Rare cases of acquired rippling muscle disease with abnormal caveolin-3 localisation have been reported, without primary caveolin-3 mutations and in association with myasthenia gravis and acetylcholine receptor autoantibodies, or thymoma. We present three new patients with electrically-silent muscle rippling and abnormal caveolin-3 localisation, but without acetylcholine receptor autoantibodies, or clinical or electrophysiological evidence of myasthenia gravis. An autoimmune basis for rippling muscle disease is supported by spontaneous recovery and normalisation of caveolin-3 staining in one patient and alleviation of symptoms in response to plasmapheresis and immunosuppression in another. These patients expand the autoimmune rippling muscle disease phenotype, and suggest that autoantibodies to additional unidentified muscle proteins result in autoimmune rippling muscle disease.