calpain and Distal-Myopathies

Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively.

Topics: Amino Acid Motifs; Animals; Calpain; Connectin; Distal Myopathies; Female; Humans; Male; Mice; Mice, Inbred C57BL; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Protein Binding; Protein Kinases; Protein Processing, Post-Translational; Proteolysis

The dominant tibial muscular dystrophy (TMD) and recessive limb-girdle muscular dystrophy 2J are allelic disorders caused by mutations in the C-terminus of titin, a giant sarcomeric protein. Both clinical presentations were initially identified in a large Finnish family and linked to a founder mutation (FINmaj). To further understand the physiopathology of these two diseases, we generated a mouse model carrying the FINmaj mutation. In heterozygous mice, dystrophic myopathology appears late at 9 months of age in few distal muscles. In homozygous (HO) mice, the first signs appear in the Soleus at 1 month of age and extend to most muscles at 6 months of age. Interestingly, the heart is also severely affected in HO mice. The mutation leads to the loss of the very C-terminal end of titin and to a secondary deficiency of calpain 3, a partner of titin. By crossing the FINmaj model with a calpain 3-deficient model, the TMD phenotype was corrected, demonstrating a participation of calpain 3 in the pathogenesis of this disease.

Topics: Animals; Blotting, Western; Calpain; Connectin; Disease Models, Animal; Distal Myopathies; DNA Mutational Analysis; Echocardiography; Genetic Linkage; Genetic Predisposition to Disease; Heterozygote; Mice; Microscopy, Electron; Muscle Proteins; Muscular Dystrophies, Limb-Girdle; Mutation; Polymerase Chain Reaction; Protein Kinases; Sarcomeres

We reported a 23-year-old woman with distal myopathy and highly elevated serum creatine kinase (CK) caused by calpainopathy. Although muscle weakness was not evident, a muscle CT scan revealed replacement by adipose tissue in the medial head of the gastrocnemius. The gluteus maximus and biceps femoris were also affected to a lesser degree, but the lateral head of the gastrocnemius was preserved. A histological study of a biopsied specimen of the biceps brachii revealed obvious variation in fiber size and a few necrotic or regenerating fibers. Rimmed vacuoles or lobulated fibers were absent in vacuoles. Although the clinical features suggested Miyoshi's distal myopathy, gene analysis of calpain 3 revealed a c.802-9G > A mutation in intron 5 and a c.1319G > A (p.Arg440Gln) in exon 10. Mini-multiplex Western Blotting (MMW) of the patient's muscle showed no band in calpain 3 (p94) and calpain 3 30 kDa fragments and immunoblotting did not reveal any dysferlin abnormalities. Calpainopathy should be also considered in patients with clinical manifestations of Miyoshi distal myopathy.

Topics: Calpain; Creatine Kinase; Diagnosis, Differential; Distal Myopathies; Female; Humans; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Mutation; Tomography, X-Ray Computed; Young Adult