Collagen VI is an extracellular matrix protein forming a microfilamentous network in skeletal muscle and other organs. Mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, whose pathogenesis is not known. A study has been performed in collaboration with the University of Padova (Prof. P. Bonaldo) on previously generated collagen VI-deficient (Col6a1-/-) mice. The published results (Irwin et al. Nature Genet 2003;35:367-371) have been cited among the "Neurogenetics top research advances from 2003" by Neurology Today (2004 4: 5-6).
The results show that Col6a1-/- muscle has a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis.
Electron microscopic analysis identified ultrastructural defects in Col6a1-/- muscle. About 30% of the fibers had structural alterations of mitochondria (abnormal cristae with tubular shape and altered matrix density associated with the presence of dense bodies) and SR (marked dilations, especially at the level of triadic system, but sarcomeres, sarcolemma and basal lamina appeared normal). We detected myonuclei with the typical hallmarks of apoptosis in Col6a1-/- fibers with organelle alterations. None of these defects were present in wild-type muscle. Taken together with the presence of Evans blue-positive fibers, this finding suggests that collagen VI-deficient muscle contain differently affected fibers that might represent various steps of the dystrophic process. The mitochondrial function was also investigated in cultured FDB myofibers. Mitochondrial transmembrane potential was monitored by tetramethylrhodamine methyl ester, a fluorescent probe that accumulates in polarized mitochondria and is released when transmembrane potential decreases. A latent mitochondrial dysfunction was found in myofibers of Col6a1-/- mice on incubation with the selective F1FO-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo.
These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis. Lack of collagen VI might cause mitochondrial dysfunction and increased PTP opening through an abnormal engagement of integrins, in keeping with the recent finding that integrin-mediated signaling regulates mitochondrial function. Increased PTP opening would alter Ca2+ handling by SR, cause Ca2+ deregulation with further increase of the PTP open time and thus set in motion a self-amplifying loop eventually leading to structural defects of both mitochondria and SR. Our finding that the structural alterations and apoptotic defects of Col6a1-/- muscles in vivo can be rescued by CsA through a mitochondrial mechanism may open the way to a pharmacological treatment for Bethlem myopathy and Ullrich congenital muscular dystrophy.
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