Transcriptional and histopathological profiling of skeletal muscle in Bla/J mice at the stage of dysferlinopathy manifestation.

Abstract

Dysferlinopathy is a rare muscular dystrophy characterized by chronic muscle damage and ineffective regeneration. While late-stage morphological changes, such as fibroadipose replacement, are well described, the early molecular mechanisms driving muscle fiber loss and regenerative failure at the onset of the disease remain largely uncharacterized. To address this gap, we investigated the skeletal muscles of dysferlin-deficient Bla/J mice during the early manifestation stage (3 months of age). This exploratory study aimed to identify primary pathomorphogenetic events by correlating the transcriptomic profile of the tissue with its specific histopathological and ultrastructural alterations. We performed a comparative analysis of the m. gastrocnemius in 3-month-old Bla/J mice versus wild-type controls using RNA sequencing, RT-qPCR, histomorphometry and transmission electron microscopy. The results revealed atrophy and muscle fiber necrosis without the expected induction of Fbxo32 and Trim63 ubiquitin ligases, suggesting ubiquitin-proteasome system-independent muscle mass loss. Furthermore, the absence of Casp3, Bak1, and Bad induction, confirmed by the lack of active caspase-3, excluded apoptosis as the primary death mechanism. A differentiation block in satellite cells was confirmed by the lack of Myf5, Myod1, and Myog induction and a trend toward Tead4 suppression, pointing to an early failure of the reparative program. Exploratory RNA sequencing also identified a suppression of Prkn expression accompanied by LC3B-II-positive autophagosome accumulation. Immunohistochemical and immunofluorescent evaluation of the mitochondrial network (TOMM20) revealed abnormal accumulations and dense clumping, indicating impaired organelle clearance. Furthermore, ultrastructural analysis demonstrated internal organelle damage and the presence of myelin-like structures, consistent with a state of stalled mitophagy. Collectively, this exploratory study demonstrates that early muscle atrophy and myofiber necrosis in dysferlinopathy occur independently of canonical ubiquitin-proteasome and apoptotic pathways. Instead, the disease manifestation stage is structurally characterized by stalled mitochondrial clearance and a delayed regenerative response.