Figure 4. Temporal knockout of Pik3c2b at both early and late stages of disease rescues the Mtm1 disease phenotype.

(A) RT-PCR showing excision of floxed Pik3c2b. (B) Western blot reveals undetectable PIK3C2B protein in cKO muscle. (C) Early-TAM-cKO (induced by tamoxifen treatment starting at 21 days) or late-TAM-cKO (starting at 30 days) of Pik3c2b confers a survival benefit to Mtm1 KO mice. Mtm1 KO alone plus TAM median survival = 42 days, while both early-TAM-cKO (n = 3) and late-TAM-cKO (n = 7) animals live at least 100 days, with the oldest survivors more than 160 days old. (D) Body weights of early-TAM-cKO (n = 3), late-TAM-cKO (n = 7), and Mtm1 KO mice (n = 4) are initially similar to those of Mtm1 KOs, but from 51 days of age onward they have weights similar to WT. (E) Late-TAM-cKOs appear similar (thought slightly smaller) than WTs (age = 160 days). (F) Grip strength measured at 28 and 100 days of age. At 28 days of age, Mtm1 KOs have a significant decrease in mean grip strength (from Figure 1D). After induction of cKO at 30 days, however, grip strength of late-TAM-cKO (n = 4), as measured at 100 days of age, equals that of WT (94% ± 3.1% vs. WT 100% ± 7.5% [P = 0.38]). (G) Late-TAM-cKOs have normalized treadmill behavior. As demonstrated in Figure 1E, Mtm1 KOs have reduced average propulsion time at 28 days (77± 7.2 ms). However, conditional KO of Pik3c2b in Mtm1 KOs starting at 30 days results in normal propulsion at 100 days of age (119 ± 8 ms, n = 4, vs. 125 ± 0.7 ms for WT, n = 3, P = 0.27). (H) Early-TAM-cKOs have normal myofiber size and structure and an absence of central nuclei. Late-TAM-cKO muscle structure is also relatively restored, with slight reductions in myofiber size and small increases in central nuclei (arrows). Scale bars: 20 μm. Comparisons were done using ANOVA followed by Dunnett’s multicomparison test.