Fig. 1

Generation of transgenic mice with inducible cardiomyocyte-specific expression of mitochondria-targeting dominant negative AKT1. A The mito-dnAkt1 construct contains a dominant negative AKT1 with a mitochondria localization sequence and His-tag. B Scheme of model to overexpress cardiac-specific mitochondria-targeting dominant negative AKT1 (CAMDAKT). Transgenic mice harboring mito-dnAkt1 were crossed with Myh6-Cre mice (Cre recombinase expressed in cardiomyocytes) to generate CAMDAKT bi-genic mice. The Neo cassette, containing Neo cDNA followed by an SV40 PolyA signal to terminate transcription, was removed by Cre-mediated recombination upon injection with tamoxifen (TAM). C Mitochondria specific expression of mito-dnAKT1 in CAMDAKT heart. Eight-week-old CAMDAKT mice were injected with TAM or vehicle-corn oil (CO). 10 h after injection, the mitochondrial fraction was isolated and expression of mito-dnAKT1 was analyzed by western blotting. The mito-dnAKT1 protein, detected by 6x-His antibodies, was expressed in cardiomyocyte mitochondria isolated from TAM-CAMDAKT mice but not in CO-CAMDAKT mice, TAM-Myh6-Cre mice or TAM-wildtype (WT) mice. VDAC1 was used as a loading control and mitochondria marker. D Cardiac specific expression in CAMDAKT mice after TAM induction. Total protein was isolated from the specified organs of CAMDAKT mice 10 h after TAM injection. Tamoxifen-induced mito-dnAKT1 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. E Mitochondria specific localization of mito-dnAKT1. Co-localization of mito-dnAKT1 and mitochondria was visualized by immunostaining. 24 h after injection with TAM, hearts were fixed and embedded with paraffin. 4-micron thin sections were immunostained with 6x-His antibodies (red) followed by staining with MitoTracker™ Green FM (green) and DAPI (blue). Mito-dnAKT1was detected in TAM-CAMDAKT cardiomyocytes and colocalized with mitochondria. Scale bar = 50 μm (upper), 20 μm (lower). F Inactivation of AKT activity in CAMDAKT cardiac mitochondria. AKT activity in the mitochondrial protein fractions was analyzed by an in vitro kinase assay using recombinant GSK3α as substrate, 24 h after TAM injection