Calpainopathy, or limb–girdle muscular dystrophy type R1/2A (LGMDR1/2A), is the most prevalent form of LGMD, comprising about 32% of all cases. The disease is caused by mutations in the CAPN3 gene, leading to dysfunction of the corresponding protein–an enzyme critical for muscle fiber cytoskeleton remodeling and protein signaling regulation through selective proteolysis. Clinical manifestations demonstrate significant phenotypic polymorphisms, ranging from oligosymptomatic forms to severe early-onset cases, with the loss of ambulation occurring 10–25 years after disease onset. A characteristic feature is predominantly symmetrical involvement of limb and trunk muscles, leading to early mobility loss, disability, and reduced work capacity. Noninvasive imaging can suggest dystrophic muscle disease but requires differentiation from other myopathies. Confirming the diagnosis involves histological, immunological, and molecular genetic studies to identify calpain-3 activity or CAPN3 gene expression alterations. Currently, no targeted or etiological therapies are available for calpainopathy. Treatment focuses on symptom management, complication prevention, and slowing disease progression. Preclinical research demands the development of an appropriate animal model that displays disease phenotypes mirroring those observed in humans. Preclinical and clinical research are also investigating therapeutic options, including the use of drugs that have proven effective in other myopathies and genome editing via transgenic CAPN3 delivery to restore protein activity. Gene therapy has shown promise in murine models, but safety concerns, particularly systemic toxicity affecting the heart and other organs, remain significant. This review comprehensively analyzes the clinical features, diagnostic approaches, and advancements in modeling and therapeutic development for calpainopathy.

Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.

BACKGROUND: With the sharp increase in mine-explosive injuries, one of the key tasks of modern histology is to assess the morphogenesis of high-energy limb tissue damage. AIM: This study aimed to characterize striated skeletal muscle tissue in the zone of mine-explosive limb segment avulsion during the early post-traumatic period (days 1–4). METHODS: Histological methods (hematoxylin and eosin staining and Martius Scarlet Blue [MSB] staining for fibrin age determination), immunohistochemical and immunofluorescence techniques (antibodies to CD3, CD20, CD31 [Platelet Endothelial Cell Adhesion Molecule-1], CD68, and NETs [Neutrophil Extracellular Traps]), transmission electron microscopy, and morphometric analyses were applied. RESULTS: Post-traumatic pathological changes affected both muscle tissue (necrosis, fiber fragmentation, traumatic edema) and endomysial and perimysial connective tissue (traumatic edema, fibrin infiltration). Disruption of the cytoskeletal structure (sarcomeres), loss of myosatellite cells, and endothelial cell death in capillaries were observed. Local hemodynamic disturbances manifested as mosaic thrombosis, embolism, and hemorrhage. Within the first 24 hours, arterial and arteriolar spasms led to reduced tissue perfusion. Partial resolution of the spasms by day 4 likely corresponded to the second stage of traumatic shock (the torpid phase). Among leukocytes, CD68+ macrophages were the first to respond, with extravasation beginning on day 2 after injury. A similar trend was observed for CD3+ T cells. CD20+ B cells showed no morphological signs of reactivity during this period. NET formation was observed both intravascularly and in the extravascular compartment. CONCLUSION: Structural and ultrastructural changes in skeletal muscle tissue within the first 4 days after mine-explosive injury have been characterized.

The aim of the work was behavioral and pathomorphological phenotyping of the mice knockout for the DYSF gene, which plays an important role in the development and progression of dysferlinopathy.Materials and methods. A B6.A-Dysf^prmd/GeneJ (Bla/J) mice subline was used in the work. During the study, a muscle activity was determined basing on the following tests: “Inverted grid”, “Grip strength”, “Wire Hanging”, “Weight-loaded swimming”, Vertical Pole”. Histological and immunofluorescent examinations of skeletal muscles (m. gastrocnemius, m. tibialis) were performed. The presence and distribution of the dysferlin protein was assessed, and general histological changes in the skeletal muscle characteristics of mice at the age of 12 and 24 weeks, were described. A morphometric analysis with the determination of the following parameters was performed: the proportion of necrotic muscle fibers; the proportion of fibers with centrally located nuclei; the mean muscle fiber diameter.Results. The “Grip strength” test and the “Weight-loaded swimming” test revealed a decrease in the strength of the forelimbs and endurance in the studied mice of the Bla/J subline compared to the control line. The safety of physical performance was checked using the “Wire Hanging” test and the “Vertical Pole” test, which showed a statistically significant difference between the studied mice and control. The coordination of movements and muscle strength of the limbs examined in the “Inverted Grid” test did not change in these age marks. Decreased grip strength of the forelimbs, decreased physical endurance with age, reflects the progression of the underlying muscular disease. Histological methods in the skeletal muscles revealed signs of a myopathic damage pattern: necrotic muscle fibers, moderate lympho-macrophage infiltration, an increase in the proportion of fibers with centrally located nuclei, and an increase in the average fiber diameter compared to the control. The dysferlin protein was not found out in the muscle tissues.Conclusion. Taking into account the results of the tests performed, it was shown that the absence of Dysf^-/^- gene expressionin Bla/J subline mice led to muscular dystrophy with the onset of the development of phenotypic disease manifestations at the age of 12 weeks and their peak at 24 weeks. Histopathological phenotypic manifestations of the disease are generally nonspecific and corresponded to the data of intravital pathoanatomical examination in diferlinopathy patients. The mice of the studied subline Bla/J are a representative model of dysferlinopathy and can be used to evaluate new therapeutic agents for the treatment of this disease.

Dysferlinopathy represents an orphan disease within the spectrum of progressive muscular dystrophies, occurring at a frequency of 1 to 9 cases per 1.000.000 individuals (Orphanet, 2024). It arises from mutations in the DYSF gene (OMIM 603009, 2p13, NM_003494.4), which is responsible for coding the transmembrane protein dysferlin. Dysferlin plays a critical role in the repair of muscle fiber membranes and the cellular processes of skeletal muscle regeneration. Although the molecular mechanisms of dysferlin-mediated repair are under active investigation, reports on the ultrastructural alterations in human skeletal muscles due to dysferlin deficiency are sparse. Objective. To identify the ultrastructural pathomorphological features of skeletal muscles in 6 patients with dysferlinopathy. Material and methods. This study presents pathomorphological, immunohistochemical, and ultrastructural data from skeletal muscle biopsies of 6 patients with molecularly confirmed dysferlinopathy. Results. Examination of paraffin-embedded sections of the anterior tibialis and vastus lateralis muscles, stained with hematoxylin and eosin, identified a primarily myopathic pattern of skeletal muscle injury. Immunohistochemical staining with dysferlin antibodies revealed the absence of the protein in muscle tissue compared to the positive control. Transmission electron microscopy has revealed ultrastructural alterations characteristic of dysferlinopathy, although not specific, including thickening and fragmentation of the basal membrane, thinning and lysis of myofibrils, folding and disruptions of the sarcolemma, destruction of mitochondria, and, newly described in this disease, necrosis of myosatellite cells and telocytes in skeletal muscles. Conclusion. Despite the non-specificity of the identified ultrastructural alterations, electron microscopy of skeletal muscle biopsies in dysferlinopathy can provide additional information about the mechanisms underlying the disease development. The finding of myosatellite cell and telocyte necrosis indicates the impairment of skeletal muscle regenerative capacity, which may be a novel link in the pathogenesis of dysferlinopathy.

BACKGROUND: Striated cardiac muscle tissue in dysferlinopathy, a rare hereditary muscular dystrophy, has been the subject of limited research. Dysferlinopathy is traditionally considered a disease that predominantly affects skeletal muscles, as clinically significant heart failure is rare in affected individuals. However, myocardial involvement due to hereditary dysferlin deficiency has been described in only a few studies. The development of heart failure in these patients may result from both circulatory remodeling due to hypodynamia and direct myocardial damage. Structural changes observed in Bla/J mice with dysferlinopathy provide evidence for direct myocardial damage. However, submicroscopic alterations in cardiomyocytes and stromal myocardial cells (fibroblasts, endothelial cells, telocytes), as well as their role in the pathomorphogenesis of dysferlinopathy, remain insufficiently studied. AIM: To characterize the ultrastructure of cardiomyocytes and stromal myocardial cells in the left ventricle of dysferlin-deficient Bla/J mice. METHODS: Myocardial left ventricle fragments from Bla/J and C57BL/6 (control group) mice at 3, 6, 9, and 12 months of age were fixed and embedded in Araldite resin. Ultrathin sections (50–100 nm) were prepared, stained using Reynolds’ method, and examined via transmission electron microscopy. RESULTS: Ultrastructural changes in the myocardium of Bla/J dysferlin-deficient mice included: destruction of the sarcolemma and intercalated discs; expansion and vacuolization of the sarcoplasmic reticulum; mitochondrial polymorphism. Additionally, myelin-like structures were detected in subsarcolemmal spaces and sarcoplasmic reticulum cisterns. In dysferlin-deficient mice, telocytes exhibited signs of degeneration. In contrast, the control group (C57BL/6 mice) showed no significant ultrastructural changes. CONCLUSION: Ultrastructural evidence of myocardial damage in dysferlin-deficient Bla/J mice suggests a potential role of dysferlin in maintaining the structural integrity of cardiomyocytes and stromal cells.