Exploring the mechanisms of metabolic adaptations to cardiotoxic doxorubicin
Authors: Guerra G , Russo M , Priolo R , Riganti C , Reano S , Filigheddu N , Hirsch E , Ghigo A 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): Leducq Foundation

Background
Doxorubicin (DOX) is an anthracycline chemotherapeutic agent whose clinical application is hindered by the emergence of early and late cardiotoxic effects. Recent findings demonstrated that DOX damages cardiac mitochondria, with subsequent metabolic perturbation and energetic imbalance. We previously observed that phosphoinositide 3-kinase γ (PI3Kγ) contributes to DOX-induced cardiotoxicity, mediating mitophagy inhibition and accumulation of damaged mitochondria into cardiac cells.

Objective
Here we intend to describe the cardiotoxic metabolic phenotype of DOX-treated hearts, unravelling the contribution of PI3Kγ signalling to this process.

Methods
Wild-type (WT) and knock-in mice expressing a kinase-inactive PI3Kγ (kinase-dead; KD) were treated with DOX at day 0, 7 and 14 (cumulative dose 12 mg/kg). To explore the contribution of PI3Kγ on the early cardiac metabolic rewiring, metabolomic analysis, mitochondrial respiration capacity and glycolytic enzymes activity were evaluated at day 3. To investigate the role of autophagy, mice were infected with an adeno-associated virus 9 (AAV9) carrying a vector expressing a short-hairpin RNA against ATG7 (ATG7sh). To test the role of PI3Kγ in glycolysis regulation, glucose uptake and plasma membrane exposure of GLUT-4 were measured in neonatal mouse cardiomyocytes with DOX acute treatment (1µM for 3h). To evaluate the contribution of different substrates to oxygen consumption, OROBOROS analysis were performed on myocardial slices from mice sacrificed at day 3 and 42.

Results
DOX-treated cardiomyocytes exhibited a metabolic phenotype characterized by glucose as the primary energy substrate, with increased uptake and augmented glycolytic enzymes activity. Metabolomic analysis of DOX-treated hearts indicated diminished pyruvate levels, the glycolysis end-product, unaltered lactate but increased Acetyl-CoA quantity, suggesting enhanced glucose processing for the TCA cycle. Accordingly, oxygen consumption after pyruvate supplementation significantly increased in DOX-treated condition, resulting in the generation of cytotoxic ROS rather than energy production. This was mainly due to DOX-induced mitochondrial damage, with impaired fatty acid oxidation and electron transport chain activity, resulting in TCA cycle slow-down, with accumulation of cardiotoxic glucose-derived intermediates. These metabolic alterations were completely prevented in KD hearts. In vitro experiments demonstrated that inhibiting PI3Kγ reduced the activity of pyruvate dehydrogenase (PDH), the key enzyme of Randle cycle, regulating the switch from fatty acids to glucose usage, while decreasing DOX-induced mobilization of GLUT-4-carrying vesicles to the plasma membrane, limiting subsequent glucose uptake.

Conclusion
These results demonstrate that DOX promotes an early metabolic rewiring with PI3Kγ-dependent increased glucose uptake and modulation of the Randle cycle towards augmented glucose utilization.