An NRF2/ß3-Adrenoreceptor Axis Drives a Sustained Antioxidant and Metabolic Rewiring Through the Pentose-Phosphate Pathway to Alleviate Cardiac Stress

BACKGROUND: Cardiac ß3-adrenergic receptors (ARs) are upregulated in diseased hearts and mediate antithetic effects to those of ß1AR and ß2AR. ß3AR agonists were recently shown to protect against myocardial remodeling in preclinical studies and to improve systolic function in patients with severe heart failure. However, the underlying mechanisms 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 transgene (ß3AR-Tg) and subjected to transverse aortic constriction were assessed with echocardiography, RNA sequencing, positron emission tomography scan, metabolomics, and metabolic flux analysis. Subsequently, signaling and metabolic pathways were further investigated in vivo in ß3AR-Tg and ex vivo in neonatal rat ventricular myocytes adenovirally infected to express ß3AR and subjected to neurohormonal stress. These results were complemented with an analysis of single-nucleus RNA-sequencing data from human cardiac myocytes from patients with heart failure. RESULTS: Compared with wild-type littermates, ß3AR-Tg mice were protected from hypertrophy after transaortic constriction, and systolic function was preserved. ß3AR-expressing hearts displayed enhanced myocardial glucose uptake under stress in the absence of increased lactate levels. Instead, metabolomic and metabolic flux analyses in stressed hearts revealed an increase in intermediates of the pentose-phosphate pathway in ß3AR-Tg, an alternative route of glucose utilization, paralleled with increased transcript levels of NADPH-producing and rate-limiting enzymes of the pentose-phosphate pathway, without fueling the hexosamine metabolism. The ensuing increased content of NADPH and of reduced glutathione decreased myocyte oxidant stress, whereas downstream oxidative metabolism assessed by oxygen consumption was preserved with higher glucose oxidation in ß3AR-Tg mice after transaortic constriction compared with wild type, together with increased mitochondrial biogenesis. Unbiased transcriptomics and pathway analysis identified NRF2 (NFE2L2) as an upstream transcription factor that was functionally verified in vivo and in ß3AR-expressing cardiac myocytes, where its translocation and nuclear activity were dependent on ß3AR activation of nitric oxide synthase and nitric oxide production through S-nitrosation of the NRF2-negative regulator Keap1. CONCLUSIONS: Moderate expression of cardiac ß3AR, at levels observed in human cardiac myocardium, exerts metabolic and antioxidant effects through activation of the pentose-phosphate pathway and NRF2 pathway through S-nitrosation of Keap1, thereby preserving myocardial oxidative metabolism, function, and integrity under pathophysiological stress.