Fig. 7. Effect of AAV-mediated expression of calpain 3 in DelMex5 mice.

(A) RT-PCR was performed using primers located in exons flanking each known C3 binding site on titin using extracts from heart of DelMex5 mice and skeletal muscle of DelMex5 and WT mice. (B) Survival curve of DelMex5 mice over a 30-day period after CAPN3 injection (n = 3 to 6). All mice were sacrificed at day 30. Control mice for this experiment are shown in Fig. 6B. (C) Expression of CAPN3 mRNA after systemic injection, quantified by RT-qPCR (copies per microgram of RNA), normalized to Rplp0 (rAAV-injected mice versus PBS). Statistical analyses were performed using nonparametric Kruskal-Wallis test and the post hoc multiple comparisons Dunn’s test (n = 3 to 4). *P < 0.05. (D) Western blot of calpain 3 in cardiac tissue. rAAV-injected mice versus PBS. (E) Heart sections were stained with Sirius red to highlight areas of fibrosis. Representative images of entire sections as well as ×2 magnification of one area are shown. rAAV-injected mice versus PBS. Scale bars, 100 μm. (F) Sirius red quantification. The amount of staining is expressed in percentage of the total tissue section. rAAV-injected mice versus PBS. Statistical analyses were performed using nonparametric Kruskal-Wallis test and post hoc multiple comparisons Dunn’s test (n = 3 to 6). *P < 0.05. (G) Fibronectin was quantified by RT- qPCR. Results were normalized to Rplp0 and compared to a PBS-injected WT condition. rAAV-injected mice versus PBS. Statistical analyses were performed using nonparametric Kruskal-Wallis test and post hoc multiple comparisons Dunn’s test (n = 3 to 7). (H) Western blot of calpain 3 in muscle tissue. Whole calpain 3 is expected at 94 kDa (black arrow), and cleavage products are expected at ~55 to 65 kDa (bracket). GAPDH was used to normalize the amount of protein loaded. rAAV-injected mice versus PBS. WT skeletal muscle (TA) was used as a positive control. (I) Stained HPS muscle sections. rAAV-injected mice versus PBS.