Introduction: Protein kinase B (Akt) is involved in the regulation of multiple cellular processes including metabolism, proliferation and cell death. Combined cardiomyocyte specific deletion of Akt-isoforms Akt1 and Akt2 results in a severe heart failure phenotype that is characterized by cardiac atrophy and energy depletion. Ingenuity pathway analysis of RNA microarray data at day 21 after induction of Akt knock out showed a strong downregulation of pathways related to different levels of cellular metabolism including ‘mitochondrial dysfunction’, ‘TCA cycle’, or ‘fatty acid β-oxidation’. Therefore, we aimed to investigate the functional consequences of these transcriptional alterations on cardiac substrate metabolism.

Methods: Experiments were performed using male mice with inducible, cardiomyocyte-restricted Akt 1 and Akt2 knock out (KO), or wild type animals (WT) at day 21 (d21) after 4-hydroxytamoxifen induced knock out induction. In a first experimental series, cardiac substrate metabolism was investigated in intact cardiac tissue slices using the Seahorse XFe24 extracellular flux analyser. Either glucose/pyruvate/glutamine or palmitate/glucose/glutamine were used as substrate combinations. Oxygen consumption rates (OCR) were measured at baseline and after mitochondrial uncoupling with FCCP (uncoupled), and coupling ratios were calculated as uncoupled/basal OCR. Long chain fatty acid (LCFA) metabolism was inhibited by etomoxir. In a second series of experiments, cardiac mitochondria were isolated by differential centrifugation, and ADP stimulated (state 3) and resting respiration (state 4) were measured using complex I substrate pyruvate/glutamate/malate or palmitoyl-l-carnitine. Data are mean±SD; unpaired two-sample t-test.

Results: In experimental series 1, the coupling ratio using glucose/pyruvate/glutamine as substrates did not show a difference between WT and KO (2.5±0.6 vs 2.3±1.0; p=0.554; n=7 per group) whereas the coupling ratio in KO was 31% lower compared to WT (p=0.003; n=4-5) with palmitate/glucose/glutamine as substrate indicating substrate specific metabolic alterations potentially caused by defects in LCFA oxidation. Inhibition of CPT1 in WT reduced uncoupled OCR by 46±16%, and in KO by 28±8% (p=0.06) using palmitate/glucose/glutamine as substrate further supporting defects in LCFA oxidation. Western blot analysis demonstrated a downregulation of CPT2 by 40% in d21 KO hearts. In experimental series 2, analysis of state 4 respiration showed no difference between WT and KO, neither using complex I substrate nor palmitoyl-l-carnitine. In contrast, stimulated state 3 respiration using complex I substrate was comparable between WT and KO (325±83 vs. 316±56 nmol O₂/mg/min; p=0.767; n=11 per group), whilst state 3 was reduced in KO (283±39 vs 238±30 nmol O₂/mg/min; p=0.008; n=10-11) using palmitoyl-l-carnitine as substrate.

Conclusion: The cardiomyocyte restricted depletion of Akt isoforms causes severe defects in cardiac metabolism. Our data points towards substrate specific defects with a major contribution of LCFA oxidation, especially mitochondrial LCFA uptake and/or β-oxidation.

(Funding: DFG IRTG 1902, Project 8)