A NRF2/β3-adrenoreceptor axis drives a sustained antioxidant and metabolic rewiring through the pentose-phosphate pathway to alleviate cardiac stress

Background Cardiac β3-adrenergic receptors (β3AR) are upregulated in diseased hearts and mediate antithetic effects to those of β1AR and β2AR. β3AR agonists were recently shown to protect from myocardial remodeling in preclinical studies and to improve systolic function in patients with severe heart failure. The underlying mechanisms, however, remain elusive. Methods To dissect functional, transcriptional and metabolic effects, hearts and isolated ventricular myocytes from mice harboring a moderate, cardiac-specific expression of a human ADRB3 transgene (β3AR-Tg) and subjected to transverse aortic constriction (TAC) were assessed using echocardiography, RNAseq, PET scan, metabolomics, seahorse and metabolic flux analysis. Subsequently, signaling and metabolic pathways were investigated further in vivo in β3AR-Tg and in vitro in neonatal rat ventricular myocytes adenovirally infected to express β3AR and subjected to neurohormonal stress. These results were completed with an analysis of single nucleus RNAseq data from human cardiac myocytes from heart failure patients. Results Compared with WT littermate, β3AR-Tg mice were protected from hypertrophy after transaortic constriction (TAC), while systolic function was preserved. β3AR-expressing hearts displayed enhanced myocardial glucose uptake under stress in absence of increased lactate levels. Instead, metabolomic and metabolic flux analyses in stressed hearts revealed an increase in intermediates of the Pentose-Phosphate Pathway (PPP) in β3AR-Tg, an alternative route of glucose utilization, paralleled with increased transcript levels of NADPH-producing and rate-limiting enzymes of the PPP, without fueling the hexosamine metabolism. The ensuing increased content of NADPH and of reduced glutathione decreased myocyte oxidant stress, while downstream oxidative metabolism assessed by oxygen consumption was preserved with higher glucose oxidation in β3AR-Tg post-TAC compared to WT, together with increased mitochondrial biogenesis. Unbiased transcriptomics and pathway analysis identified NRF2 (NFE2L2) as upstream transcription factor which was functionally verified in β3AR-expressing cardiac myocytes where its translocation and nuclear activity was dependent on β3AR activation of nitric-oxide synthase (NOS) NO production. Conclusion Moderate expression of cardiac β3AR, at levels observed in human cardiac myocardium, exerts antioxidant effects through activation of the PPP and NRF2 pathway, thereby preserving myocardial oxidative metabolism, function and integrity under pathophysiological stress. ### Competing Interest Statement The authors have declared no competing interest.