Induced degeneration and regeneration in aged muscle reduce tubular aggregates but not muscle function.

Introduction: Tubular aggregates (TA) are skeletal muscle structures that arise from the progressive accumulation of sarcoplasmic reticulum proteins. Cytoplasmic aggregates in muscle fibers have already been observed in mice and humans, mainly during aging and muscle disease processes. However, the effects of muscle regeneration on TA formation have not yet been reported. This study aimed to investigate the relationship between degeneration/regeneration and TA in aged murine models. We investigated the presence and quantity of TA in old males from two murine models with intense muscle degeneration and regeneration. Methods: One murine lineage was a Dmdmdx model of Duchenne muscular dystrophy (n = 6). In the other model, muscle damage was induced by electroporation in C57BL/6J wild-type mice, and analyzed after 5, 15, and 30 days post-electroporation (dpe; n = 15). Regeneration was evaluated based on the quantity of developmental myosin heavy chain (dMyHC)-positive fibers. Results: The frequency of fibers containing TA was higher in aged C57BL/6J (26 ± 8.3%) than in old dystrophic Dmdmdx mice (2.4 ± 2%). Comparing the data from induced degeneration/regeneration in normal mice revealed a reduced proportion of TA-containing fibers after 5 and 30 dpe. Normal aged muscle was able to regenerate and form dMyHC+ fibers, mainly at 5 dpe (0.1 ± 0.1 vs. 16.5 ± 2.6%). However, there was no difference in force or resistance between normal and 30 dpe animals, except for the measurements by the Actimeter device, which showed the worst parameters in the second group. Discussion: Our results suggest that TA also forms in the Dmdmdx muscle but in smaller amounts. The intense degeneration and regeneration of the old dystrophic model resulted in the generation of new muscle fibers with a lower quantity of TA. Data from electroporated wild-type mice support the idea that muscle regeneration leads to a reduction in the amount of TA. We suggest that TA accumulates in muscle fibers throughout physiological aging and that regeneration leads to the formation of new fibers without these structures. In addition, these new fibers do not confer functional benefits to the muscle.

Pathogenic DPAGT1 variants in limb-girdle congenital myasthenic syndrome (LG-CMS) associated with tubular aggregates and ORAI1 hypoglycosylation.

AIMS: Limb-girdle congenital myasthenic syndrome (LG-CMS) is a genetically heterogeneous disorder characterized by muscle weakness and fatigability. The LG-CMS gene DPAGT1 codes for an essential enzyme of the glycosylation pathway, a posttranslational modification mechanism shaping the structure and function of proteins. In DPAGT1-related LG-CMS, reduced glycosylation of the acetylcholine receptor (AChR) reduces its localization at the neuromuscular junction (NMJ), and results in diminished neuromuscular transmission. LG-CMS patients also show tubular aggregates on muscle biopsy, but the origin and potential contribution of the aggregates to disease development are not understood. Here, we describe two LG-CMS patients with the aim of providing a molecular diagnosis and to shed light on the pathways implicated in tubular aggregate formation. METHODS: Following clinical examination of the patients, we performed next-generation sequencing (NGS) to identify the genetic causes, analysed the biopsies at the histological and ultrastructural levels, investigated the composition of the tubular aggregates, and performed experiments on protein glycosylation. RESULTS: We identified novel pathogenic DPAGT1 variants in both patients, and pyridostigmine treatment quantitatively improved muscle force and function. The tubular aggregates contained proteins of the sarcoplasmic reticulum (SR) and structurally conformed to the aggregates observed in tubular aggregate myopathy (TAM). TAM arises from overactivation of the plasma membrane calcium channel ORAI1, and functional studies on muscle extracts from our LG-CMS patients evidenced abnormal ORAI1 glycosylation. CONCLUSIONS: We expand the genetic variant spectrum of LG-CMS and provide a genotype/phenotype correlation for pathogenic DPAGT1 variants. The discovery of ORAI1 hypoglycosylation in our patients highlights a physiopathological link between LG-CMS and TAM.

Tubular Aggregate Myopathy: A Case Report

We report a first Korean case of presumably dominantly inherited primary tubular aggregate myopathy in a 19-yr-old man, who presented with slowly progressive proximal muscle stiffness and weakness. In hematoxylin and eosin stain, it showed subsarcolemmal, or central pale basophilic granular vacuoles, which stained red with modified Gomori's trichrome and intensive blue with nicotinamide adenonine dinucleotide-tetrazolium reductase, respectively. Ultrastructurally, aggregates of 60 nm-sized hexagonal tubules were found in both type 1 and type 2 fibers. We briefly review the pathologic findings of the previously reported cases of tubular aggregate myopathy and discuss the possible pathogenesis of this disease. We briefly discuss the possible pathogenesis of sarcoplasmic reticulum and review the ultrastructural characteristics.