Fig. 4. Exercise or β-adrenergic stimulation increases truncation of myocardial AIF in Dsg2^mut/mut myocytes.

(A) Representative immunoblots of AIF with the mature AIF (mAIF) indicated at 62 kDa and truncated AIF (tAIF) at 57 kDa in hearts of mice of the indicated genotype under sedentary (rest) or exercised (swim) conditions. GADPH and cytC served as loading controls. (B and C) Quantification of mAIF and tAIF in hearts from the indicated mice. Data are presented as mean ± SEM [n = 6 per cohort per condition; *P < 0.05 compared to WT (rest); †P < 0.05 compared to WT (swim); and ǂP < 0.05 compared to Dsg2^mut/mut (rest)] using one-way ANOVA in (B) and two-tailed t test in (C). (D) Representative immunoblots of AIF in subcellular fractions of ventricular lysates from exercised mice. Cyto., cytosolic extracts; Mito. Bound, mitochondrial fraction; Nucl., nuclear fraction; Chrom. Bound, chromatin-bound fraction. (E to H) Quantification of AIF in the indicated subcellular fraction. mAIF was quantified for cytosolic and mitochondrial fractions (E and F); tAIF was quantified for nuclear and chromatin-bound fractions (G and H). Data are presented as means ± SEM [n ≥ 6 per cohort per compartment; *P < 0.05 compared to WT (swim)] using two-tailed t test. (I and J) Representative immunoblots from WT and Dsg2^mut/mut ES-CMs treated for 1 day or 7 days with either isoproterenol (ISO; 50 μM), calcium (Ca²⁺ 1 μM), or both (ISO/Ca²⁺). Data are representative of one of six experiments. (K) Representative immunoblots from WT and Dsg2^mut/mut ES-CMs treated for 7 days with ISO/Ca²⁺, with or without calpeptin (50 μM) pretreatment. (L to N) Quantification of mAIF (62 kDa), tAIF (57 kDa), and active CAPN1 in ES-CMs of the indicated genotypes subjected to conditions as in (K). Data are presented as means ± SEM (n = 6 per genotype per parameter; *P < 0.05 untreated Dsg2^mut/mut ES-CMs compared to untreated WT ES-CMs; †P < 0.05 calpeptin-pretreated Dsg2^mut/mut ES-CMs compared to untreated Dsg2^mut/mut ES-CMs using one-way ANOVA).