Beyond H 2 O 2 : Effects of NH 4 + and catecholaldehydes on mitochondrial metabolism following MAO-mediated norepinephrine metabolism in the heart

Rachel Crawford; Ethan Anderson

doi:10.1152/physiol.2026.41.S1.2299579

Abstract only Background: Monoamine oxidase (MAO) highly is expressed in nearly all cell types, and outside the central nervous system, is the primary enzyme responsible for metabolism of catecholamines and other neurotransmitters. Oxidative deamination of the norepinephrine (NE) by MAO generates the reactive metabolites hydrogen peroxide (H 2 O 2 ), ammonia (NH 4 + ), and the catecholaldehyde 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL). Studies have associated MAO and catecholamine metabolites with a variety of neurodegenerative, cardiovascular, and inflammatory diseases. Mechanisms underlying MAO’s pathogenicity are not entirely understood, in part because studies have focused exclusively on oxidative stress generated by MAO-mediated H 2 O 2 production while ignoring NH 4 + and catecholaldehydes. Catecholaldehydes are particularly difficult to detect in biological systems owing to their high reactivity. Our lab has shown that the dipeptide carnosine readily forms stable conjugates with DOPEGAL, illustrating a potential new method to track DOPEGAL formation in cells. We also hypothesized that NH 4 + generated via MAO-mediated NE metabolism may participate in mitochondrial substrate shuttling and directly impact respiration. Purpose: In this study we went ‘beyond H 2 O 2 ’ to investigate MAO-mediated DOPEGAL-carnosine formation in primary cardiomyocytes, and examined the energetic effects of MAO-mediated NH 4 + in cardiac mitochondria. Methods: Mitochondrial respiration (JO 2 ) and ATP synthesis (JATP) were measured in mitochondria isolated from left ventricle/septum of healthy, young adult (8-15 weeks) wild-type (WT) and cardiomyocyte-specific MAO-A deficient (cMAO-Adef) mice, using a customized oxifluorimeter system (Oroboros O2K and Horiba, Inc.). Respiration was supported by a range of substrates (pyruvate, palmitoyl carnitine, glutamate) while clamped in phosphorylating state (0.1mM ADP) followed by NE or NH 4 + administration. ATP was indirectly measured via enzyme coupled generation of NADPH, as we and others have previously shown. DOPEGAL formation in primary adult cardiomyocytes was detected by measuring DOPEGAL-carnosine conjugates using LC-MS/MS. Cells were incubated with NE (100μM), in presence and absence of carnosine (1mM) and MAO inhibitors (MAOIs) for up to 24 hours. Results: NE (5, 10µM) had no effect on JO 2 supported by pyruvate or palmitoyl-carnitine, but NE increased glutamate-supported JO 2 by 15%. This effect was abrogated in cMAO-Adef mice and with MAOIs. Importantly, the effect of NE was phenocopied with NH 4 + and attenuated with an inhibitor of glutamic oxaloacetic transaminase-1 (GOT1), a key enzyme in the malate-aspartate shuttle and glutamine synthesis pathway. Mitochondrial ATP synthesis rates were not affected by NE at any concentration, for any substrate tested. MS analysis of conditioned media following cardiomyocyte incubation showed a peak at 1.64 retention time with same m/z and fragmentation pattern seen with the DOPEGAL-carnosine standard, and MAOIs completely blunted this peak formation. Conclusion: Our data indicates that the NH 4 + formed during MAO-mediated NE metabolism is linked to mitochondrial glutamate uptake and oxidation via GOT1, revealing a potential new pathway by which MAO impacts mitochondrial OxPHOS. Finally, our detection of DOPEGAL-carnosine adducts in conditioned media via LC-MS/MS validates the use of this biomarker as an index of MAO activity in cardiomyocytes, which can be exploited to develop novel pharmacotherapies which mitigate pathogenic effects of MAO. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Beyond H 2 O 2 : Effects of NH 4 + and catecholaldehydes on mitochondrial metabolism following MAO-mediated norepinephrine metabolism in the heart

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