Changes of mitochondrial TMRM fluorescence induced by oligomycin in muscle cells isolated from healthy donors and from UCMD and Bethlem myopathy patients before and after treatment with CsA. Muscle cells obtained from biopsies of two healthy donors (a and b), four patients with UCMD (c–f; patients 1–4, respectively), and one with Bethlem myopathy (g; patient 5) were seeded onto glass coverslips precoated with laminin/poly-l-lysine, loaded with TMRM and studied as described in Experimental Procedures. The procedure was repeated after 1 month of oral treatment with 5 mg/kg CsA per day (c′–g′; patients 1–5, respectively). Where indicated, 6 μM oligomycin (arrows) or 4 μM carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (arrowheads) was added. Each line represents one individual cell.

Changes of mitochondrial TMRM fluorescence induced by oligomycin in muscle fibers isolated from Col6a1^−/− mice. Flexor digitorum brevis muscle fibers were isolated from Col6a1^−/− mice treated with vehicle (a) or with 10 mg/kg CsA in two divided doses per day for 4 days and assayed 7 h (b), 17 h (c), or 27 h (d) after the last treatment with CsA. Fibers from two individuals per time point were loaded with TMRM and studied as described in Experimental Procedures, and the results for each time point were pooled. Where indicated, 6 μM oligomycin (arrows) or 4 μM carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (arrowheads) was added. Each line represents one individual fiber.

Apoptosis and regeneration in muscle biopsies from UCMD and Bethlem myopathy patients before and after treatment with CsA. (A) (Bottom) Biopsies from four patients with UCMD (P1–P4) and one patient with Bethlem myopathy (P5) were scored for the presence of apoptotic nuclei with the TUNEL reaction as described in Experimental Procedures before (open bars) and after 1 month of oral treatment with 5 mg/kg CsA per day (filled bars). Data are expressed as mean ± SEM of three independent experiments. *, P < 0.05. The incidence of apoptotic nuclei in healthy donor (control) biopsies was 0.39 ± 0.3 per square millimeter. (Top and Middle) Representative fields of sections derived from the biopsy of one healthy donor (control) and of patient 1 (P1) before and after CsA treatment, stained with TUNEL and Hoechst. Arrows indicate TUNEL-positive nuclei. (Scale bar: 50 μm.) (B) Representative cross-sections of muscle biopsies of UCMD patient 3 before (basal) and after (CsA) 1 month of oral treatment with 5 mg/kg CsA per day. The sections were stained with DAPI to identify nuclei (blue) and with antibodies against developmental myosin heavy chain (green) and desmin (red). (Left) Overlay between DAPI and developmental myosin heavy chain. (Right) Overlay between developmental myosin heavy chains and desmin. (C) Occurrence of regenerating fibers before (open bars) and after treatment with CsA (filled bars) identified as illustrated for B. Data represent mean ± SD. *, P < 0.05.