A NRF2/beta3-adrenoreceptor axis drives a sustained antioxidant and metabolic rewiring through the pentose-phosphate pathway to alleviate cardiac stress

Background: Cardiac beta3-adrenergic receptors (beta3AR) are upregulated in diseased hearts and mediate antithetic effects to those of beta1AR and beta2AR. Beta3AR 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 (beta3AR-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 beta3AR-Tg and in vitro in neonatal rat ventricular myocytes adenovirally infected to express beta3AR 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, beta3AR-Tg mice were protected from hypertrophy after transaortic constriction (TAC), while systolic function was preserved. Beta3AR-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 beta3AR-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 beta3AR-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 beta3AR- expressing cardiac myocytes where its translocation and nuclear activity was dependent on beta3AR activation of nitric-oxide synthase (NOS) NO production. Conclusion: Moderate expression of cardiac beta3AR, 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.