Alteration of Sarcoplasmic Reticulum Ca²⁺ Release in Skeletal Muscle from Calpain 3-Deficient Mice

Figure 1

Morphological and molecular features of skeletal muscle cells used throughout this study. (a) Phase contrast and fluorescence micrographs of murine skeletal muscle cells in primary culture. Typical morphology of the living cells (left upper panel: myoblasts; right upper panel: myotubes) used for calcium measurements observed by phase contrast microscopy. Circles indicate the region of drug application and monitoring of [Ca²⁺]_i. Immunological staining of myogenin on myoblasts (left middle panel) and myotubes (right middle panel) was visualized by confocal microscopy using a FITC-labelled secondary antibody (green fluorescence). Myosine Heavy Chain (MHC) was similarly observed in myoblasts (left lower panel) and myotubes (right lower panel) using a TRITC-labelled secondary antibody (red fluorescence). Myoblasts and myotubes were obtained after 6 or 11 days in culture, respectively. (b) Detection of calpain 3-mRNA in wild type (+/+) myoblasts and myotubes by RT-PCR. Gel electrophoresis of the RT-PCR reactions obtained using the primer pairs p94sys3, p94sys5 and p94sys6 (see Section 2) on murine myoblast (MB) or myotube (MT) mRNA. (c) Detection of the ryanodine receptor in skeletal muscle from normal and capn3-deficient mice. Muscle from normal (Lane 3) and capn3-deficient mice (Lane 2) were extracted and left 30 min at room temperature to allow the cleavage of RyR and were then subjected to SDS-PAGE. Human muscle was used as control (Lane1). No difference in the cleavage pattern was observed, indicating that the partial cleavage of RyR also occurs in the absence of calpain 3 in this biochemical assay. (d) Measurement of caspase 3 activity in wild type and capn3-deficient myoblasts. The graph displays the levels of substrate cleavage expressed as means ± S.D. in arbitrary units. The results are based on 4 different experiments. The differences in the median values among the two groups are greater than would be expected by chance; there is a statistically significant difference (𝑃=0.029), as indicated by a Mann-Whitney Rank Sum test.