CAPN6 is expressed in regenerating skeletal muscles and suppresses regeneration.

<p>(A) RT-PCR with total RNAs extracted from skeletal muscles of 7-wk-old wild-type mice 2 d, 4 d, and 7 d after cardiotoxin or PBS (control) injection. The PCR bands of <i>Capn6</i> were amplified with the primers that detect full-length <i>Capn6</i> mRNA. <i>Myh3</i> and <i>Myh8</i> mRNA was amplified to confirm that skeletal muscles successfully regenerated after their cardiotoxin-induced degeneration; <i>Capn1</i> mRNA was detected as an internal control. (B) Western blot analysis using antibodies against CAPN6, of skeletal muscle lysates of 7-wk-old <i>Capn6^lacZ/+</i> and <i>Capn6^lacZ/lacZ</i> mice, 5 days after cardiotoxin or PBS (control) injection. The specific CAPN6 band, indicated by an arrow, was detected around 70 kDa, and was not detected in <i>Capn6^lacZ/lacZ</i> mice. (C–E) Histological analysis of the TA muscles of 7-wk-old <i>Capn6^lacZ/+</i> and <i>Capn6^lacZ/lacZ</i> mice after a cardiotoxin or PBS (control) injection. Regenerating skeletal muscles with central nuclei were observed 4 d after the cardiotoxin injection. Remarkable degeneration was not observed in TA muscles at 4d after a PBS injection (shown as control) (C). Scale bar: 50 µm. Seven days after the injection, the average number of nuclei per cell was significantly greater in <i>Capn6^lacZ/lacZ</i> (1.43 [mean]±0.06 [s.e.m.]) than in <i>Capn6^lacZ/+</i> (1.21±0.03) (D), and the mean CSA of the regenerating muscle myofibers was also significantly greater in <i>Capn6^lacZ/lacZ</i> (1608.0 [mean]±78.5 [s.e.m.] µm) than in <i>Capn6^lacZ/+</i> (938.0±67.2 µm) (E). *, <i>P</i><0.05; **, <i>P</i><0.01 by Student's t-test. Each value is the mean from five experiments, and 50 cells were counted in each experiment. (F) Western blot analysis with skeletal muscle lysates of 7-wk-old <i>Capn6^lacZ/Y</i> and <i>Capn6^+/Y</i> mice 2d and 7 d after cardiotoxin or PBS (control) injection using antibodies against MYH3, MYH8 and CAPN1. CBB staining was used as an internal control. Wt, <i>Capn6^+/Y</i> (♂); Hemi, <i>Capn6^lacZ/Y</i> (♂); Het, <i>Capn6^lacZ/+</i> (♀); Ho, <i>Capn6^lacZ/lacZ</i> (♀).