Critical evaluation of the use of cell cultures for inclusion in clinical trials of patients affected by collagen VI myopathies.

Collagen VI myopathies (Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), and myosclerosis myopathy) share a common pathogenesis, that is, mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). This effect was first identified in the Col6a1(-/-) mouse model and then in muscle cell cultures from UCMD and BM patients; the normalizing effect of cyclosporin A (CsA) confirmed the pathogenic role of PTP opening. In order to determine whether mitochondrial performance can be used as a criterion for inclusion in clinical trials and as an outcome measure of the patient response to therapy, it is mandatory to establish whether mitochondrial dysfunction is conserved in primary cell cultures from UCMD and BM patients. In this study we report evidence that mitochondrial dysfunction and the consequent increase of apoptotic rate can be detected not only, as previously reported, in muscle, but also in fibroblast cell cultures established from muscle biopsies of collagen VI-related myopathic patients. However, the mitochondrial phenotype is no longer maintained after nine passages in culture. These data demonstrate that the dire consequences of mitochondrial dysfunction are not limited to myogenic cells, and that this parameter can be used as a suitable diagnostic criterion, provided that the cell culture conditions are carefully established.

The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1-/- myopathic mice.

BACKGROUND AND PURPOSE: We have investigated the therapeutic effects of the selective cyclophilin inhibitor D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025) in myopathic Col6a1(-/-) mice, a model of muscular dystrophies due to defects of collagen VI. EXPERIMENTAL APPROACH: We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca(2+) and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1(-/-) mice before after treatment with Debio 025. KEY RESULTS: Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1(-/-) mice. CONCLUSIONS AND IMPLICATIONS: Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1(-/-) myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.