Additional file 1: Figure S1. Cardiac-specific induction of Cre recombinase after TAM Induction in CAMDAKT Mice. A. CAMDAKT mice were administered TAM or CO once, as indicated at 2 months of age. 10 h after tamoxifen injection, the presence of Cre recombinase in the nuclei was examined by immunohistochemistry analysis. Figure S2. Development of Myocardial Fibrosis and Cardiomyopathy Caused by Impaired Mitochondrial AKT1 Signaling. A. Representative images of trichrome stained mid-heart cross sections. TAM or CO was administered once, as indicated. 7 days after injection, cardiac fibrosis was quantified by trichrome staining. Scale bar = 500 μm. B. Hearts were weighed and reported as a ratio of heart mass to body mass. (n = 6 (TAM-Myh6-Cre), n = 6 (TAM-MDNAKT), n = 5 (CO-CAMDAKT), n = 13 (TAM-CAMDAKT); p < 0.05). C. Left, representative images of trichrome stained myocardial sections. TAM or CO was administered once, as indicated. 7 days after injection, cardiac fibrosis was quantified by trichrome staining. Scale bar = 100 μm. Right, quantification of cardiac fibrosis. (n = 10 (TAM-Myh6-Cre), n = 9 (TAM-MDNAKT), n = 9 (CO-CAMDAKT), n = 9 (TAM-CAMDAKT); p < 0.01). Figure S3. AKT1 Interacted with the ATP Synthase Complex. A. Scheme of sucrose gradient used to separate mitochondrial protein complexes. Figure S4. Generation of Transgenic Mice with Inducible Cardiomyocyte-Specific Expression of Mitochondria-Targeting Constitutively Active AKT1. A. Scheme of model to overexpress cardiac-specific mitochondria-targeting constitutively active AKT1 (CAMCAKT). Transgenic mice harboring mito-caAkt1 were crossed with Myh6-Cre mice (Cre recombinase expressed in cardiomyocytes) to generate CAMCAKT bi-genic mice for this series of experiments. The Neo cassette, containing Neo cDNA followed by an SV40 PolyA signal to terminate transcription, is removed by Cre-mediated recombination upon injection with tamoxifen (TAM). B. Cardiac specific expression in CAMCAKT mice after TAM induction. Total protein was isolated from the specified organs of CAMCAKT mice 10 h after TAM injection. Tamoxifen-induced mito-caAKT1 protein expression was detected by 6x-His antibodies in the heart specifically and not detected in other organs examined. Actinin was used as a loading control. C. Mitochondria specific localization of mito-caAKT1. Co-localization of mito-caAKT1 and mitochondria was visualized by immunostaining. 24 h after injection with TAM, hearts were fixed and embedded with...