Dynamic distribution of muscle-specific calpain in mice has a key role in physical-stress adaptation and is impaired in muscular dystrophy
Koichi Ojima, … , Atsu Aiba, Hiroyuki Sorimachi
Koichi Ojima, … , Atsu Aiba, Hiroyuki Sorimachi
Published July 1, 2010
Citation Information: J Clin Invest. 2010;120(8):2672-2683. https://doi.org/10.1172/JCI40658.
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Research Article Muscle biology

Dynamic distribution of muscle-specific calpain in mice has a key role in physical-stress adaptation and is impaired in muscular dystrophy

Abstract

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle–specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretch-induced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.

Authors

Koichi Ojima, Yukiko Kawabata, Harumi Nakao, Kazuki Nakao, Naoko Doi, Fujiko Kitamura, Yasuko Ono, Shoji Hata, Hidenori Suzuki, Hiroyuki Kawahara, Julius Bogomolovas, Christian Witt, Coen Ottenheijm, Siegfried Labeit, Henk Granzier, Noriko Toyama-Sorimachi, Michiko Sorimachi, Koichi Suzuki, Tatsuya Maeda, Keiko Abe, Atsu Aiba, Hiroyuki Sorimachi

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Figure 3

Translocation of p94 in response to sarcomeric length.

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Translocation of p94 in response to sarcomeric length. (A–C) Direct obse...
(A–C) Direct observation of rapid GFP-p94 exchange in cultured myotubes by FRAP. GFP-p94WT and GFP-p94CS were exogenously expressed in cultured myotubes from WT (A) and p94KI (B) mice and were predominantly localized to the M-lines of connectin/titin. Reference images were acquired before photobleaching (Pre). Rectangles indicate the photobleached areas. The signals in the M-line were calculated as the ratio of GFP intensity at the indicated time points, and exponential curves were fitted (C). The mobile fraction (M), rate constant (k), and recovery half-time (t1/2) were calculated from the FRAP curve. Note that a single exponential fit provided the best result, and k (i.e., t1/2) was significantly different between WT and p94KI (see Supplemental Figures 5 and 6). (D–P) Confocal micrographs of p94 localization in longitudinal sections of EDL from WT (D–F and J–L) and p94KI (G–I and M–O) mice. p94, the Z-bands, and connectin/titin N2A region were detected with anti-pNS (E, H, K, and N), anti–s-α-actinin (D and G; arrows), and anti-connectin/titin-N2A (J and M; arrows) Abs, respectively. p94 was localized to the M-lines (open arrowheads), N2A regions, and/or Z-bands (brackets) of both mice, but predominantly to the N2A regions in extended sarcomeres (K and N). Ratio of the p94 signal in the M-line to that in the N2A region was plotted against the sarcomeric length (P). The 2 estimated lines were statistically shown to be significantly different (see Supplemental Figure 6 legend). Scale bars: 10 μm.