CFTR corrector C17 rescues defective SERCA1 in bovine pseudomyotonia: a potential therapy for Brody myopathy

Figures

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  Figure 1. CFTR correctors promoted the rescue of R164H and G211V mutant SERCA1 in cells. (A) HEK293 cells transfected with WT and R164H mutated SERCA1 cDNAs were treated with the proteasome inhibitor MG132 (10 μM), the vehicle DMSO (0.1%), CFTR correctors C2 (5 μM), C3 (10 μM), C4 (10 μM), C5 (15 μM), C6 (10 μM), C9 (10 μM), C13 (5 μM), C14 (5 μM), C15 (15 μM), C17 (2 μM), VX-809 (10 μM), VX-661 (10 μM) and the molecule glafenine (10 μM). All molecules were dissolved in the DMSO. An equal quantity of protein from total cell lysates was separated by SDS-PAGE and subjected to immunoblot analysis with antibodies specific to SERCA1 and 43 kDa beta actin, used as loading control. The graph shows the average values (± SD) of SERCA1 expression determined by densitometric analyses of four independent experiments (n = 4). Values are expressed as percentage of the SERCA protein content present in cells expressing the wild type form. Statistical analysis was performed by one-way ANOVA test, multiple comparisons Dunnett test; ****P ≤ 0.0001; **P ≤ 0.01; *P ≤ 0.05. (B) HeLa cells were transfected with WT or with G211V mutated SERCA1 cDNAs, as indicated. Twenty-four hours after transfection, C17 (5 μM)…

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  Figure 2. Expression level of mutated SERCA1 in microsomes isolated from skeletal muscle after treatment with corrector C17. Microsomal fractions enriched in content of SR membranes were obtained from semimembranosus muscle biopsies of case 1 and case 2 PMT affected calves. Biopsies were taken from two injection sites treated with C17, the contralateral treated with the vehicle or untreated sites for case 1 (A) or from two injection sites treated with C17 and the contralateral untreated site, for case 2 (B). Microsomal fractions were separated by SDS-PAGE and blotted onto nitrocellulose. The blots were incubated with antibodies to SERCA1 (protein loading was 3 μg/lane and 5 μg/lane in a and B, respectively). Staining with Ponceau red or incubation with the indicated antibodies (calreticulin and the non-junctional sarcalumenin and its splice variant 53 kDa glycoprotein), were used as loading control. A representative western blot is shown. Quantification by densitometric analysis of western blots from case 1 (C) and case 2 (D). The average amount of SERCA1 is expressed as fold increase (± SD) of the protein content compared with the contralateral untreated site. Data are from four technical…

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  Figure 3. Distribution of SERCA1 protein within muscle subfractions. Muscle homogenates from the two injection sites treated with C17 and the contralateral untreated semimembranosus muscles of PMT-affected case 2 calf, were sub-fractionated as described in materials and methods to obtain a crude microsomal fraction enriched in content of SR membranes (lanes 1, 4, 7), a soluble supernatant (lanes 2, 5, 8), and a myofibrillar fraction (lanes 3, 6, 9). Proteins were separated by SDS-PAGE and blotted onto nitrocellulose. The blots were incubated with antibodies to SERCA1. The lower part of the panel shows Ponceau red staining, used as loading control.

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  Figure 4. Ca2+-ATPase activity in SR microsomes isolated from semimembranosus skeletal muscle biopsies after treatment with corrector C17. Ca2+-ATPase activity assay at pCa5 was performed on SR microsomal fractions obtained from two injection sites treated with C17, the contralateral treated with the vehicle or untreated sites for case 1 (A), or from the two injection sites treated with C17 and the contralateral untreated site, for case 2 (B). Data (± SD) were obtained from four technical replicates in which each sample was tested in duplicate. Statistical analysis was performed by one-way ANOVA test—Multiple comparisons Dunnett test; ****P ≤ 0.0001; ***P ≤ 0.001.

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  Figure 5. Histological staining on semimembranosus muscle sections from PMT-affected subjects after treatment with C17 CFTR corrector. Transversal sections from semimembranosus muscle biopsies taken at the end of muscle treatment protocol with C17 molecule. Biopsies were taken from the injection sites (A, B, C) and contralateral untreated site (G) for case 1; from injection sites (D, E) and contralateral untreated site (F) for case 2. Sections were stained with Hematoxylin and eosin (H&E) (A, B, D, E) or azan–Mallory (C, F, G) methods. Scale bars 50 μm.

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  Figure 6. Ex vivo treatment with C17 CFTR corrector of semimembranosus muscle fibers from case 1 PMT affected calf. Bundles of fibers from muscle biopsy taken from contralateral untreated muscle of PMT-affected case 1, were maintained under tissue culture conditions (untreated) or were incubated with MG132 (5 μM), CFTR correctors C17 (5 and 10 μM) and C4 (5, 10 and 15 μM) at different concentrations or with their vehicle, DMSO (0,1%), as indicated. After treatment, fibers were homogenized and sub-fractionated as described in materials and methods. An equal quantity of proteins from the crude microsomal fraction enriched in content of SR was separated by SDS-PAGE and blotted into nitrocellulose (protein loading 3 μg/lane). The SR membrane fraction from healthy Romagnola calf was used as control. Blots were incubated with antibody to SERCA1. The lower panel is Ponceau red staining of the membrane fractions, used as loading control.

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  Figure 7. Ultrastructural studies of skeletal muscles from bovine PMT- affected case 1. Electron microscopy of semimembranosus (A) semitendinosus (B) and quadriceps (C, D) muscle biopsies from PMT-affected subject. Samples were collected after a severe contractural crisis. Scale bars, 2 μm (a, B, C) and 1 μm (D).

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  Figure 8. Cell viability assays in bovine cell lines treated with C17 corrector at different concentrations. Dose–response curves of C17 in BFH12 (A) and MDBK (B) cells after 24 and 48 hrs of incubation. Data are expressed as the mean viability rate (%) (± SD) of three biological replicates (n = 3 independent experiments), each performed in sextuplicate.