Figure 5. Endogenous cardiac glycosides regulate Beclin 1/Na⁺,K⁺-ATPase binding and autosis during exercise.

(A and B) Representative Western blots (A) and quantitation (B) of coimmunoprecipitation of Beclin 1 with the α subunit of Na⁺,K⁺-ATPase in hearts from mice treated with either intraperitoneal vehicle or cardiac glycoside–blocking antibody DigiFab (10 mg/kg) before 80 minutes of resting or exercise. For A, the same issue sample of the resting group without DigiFab (lane 1) was used as a control for IgG immunoprecipitation. In B, bars represent mean ± SD (n = 10 mice per group). (C and D) Representative images (C) and quantitation (D) of PLAs of Beclin 1 and the α subunit of Na⁺,K⁺-ATPase in hearts from mice treated with either intraperitoneal vehicle or cardiac glycoside–blocking antibody DigiFab (10 mg/kg) before 80 minutes of resting or exercise. In D, bars represent mean ± SEM (n = 6–8 mice per group, 10 randomly selected fields analyzed per mouse). (E and F) Representative micrographs (E) and quantitation (F) of autotic noncardiomyocyte cells in hearts from mice treated with either intraperitoneal vehicle or cardiac glycoside blocking antibody DigiFab (10 mg/kg) before resting or running until exhaustion. In F, bars represent mean ± SEM (n = 6 randomly selected areas from 3 mice per condition, more than 1,000 analyzed cells per area). Asterisk, nonspecific band. Scale bars: 10 μm (B), 1 μm (C). *P < 0.05, **P < 0.01, and ***P < 0.001; 2-way ANOVA test.