Proteomic and β-hydroxybutyrylation Landscapes Reveal AMPKα2-Associated Modulation in HUCMSC-Derived Exosome-Mediated Amelioration of Cardiac Aging.

Abstract

Cardiac aging is characterized by progressive functional decline and metabolic remodeling. We previously found that lysine β-hydroxybutyrylation (Kbhb) is altered in wild-type (WT) aged and AMP-activated protein kinase alpha 2 (AMPKα2)-knockout (AK) young hearts, but whether this landscape can be reversed therapeutically remains unknown. We evaluated the therapeutic potential of human umbilical cord mesenchymal stromal cell-derived exosomes (hUCMSC-exos) in WT and AK aged mice, using young and aged untreated cohorts as controls. Quantitative proteomics across six experimental groups identified 1,755 Kbhb sites on 652 proteins, mapping the dynamic cardiac Kbhb landscape under aging, AMPKα2 deficiency, and exosome intervention. In wild-type aged (WTA) mice, hUCMSC-exos improved systolic and diastolic function, enhanced grip strength, reduced fibrosis, and restored ATP levels, accompanied by predominant downregulation of Kbhb on enzymes of glycolysis/gluconeogenesis, fatty acid oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. In AMPKα2-knockout aged (AKA) mice, exosomes partially improved mitochondrial ultrastructure and diastolic indices but did not rescue systolic function or fibrosis. Site-level analysis revealed that exosomes selectively normalized Kbhb sites elevated during aging, whereas AMPKα2 deficiency caused nonselective global depletion. The candidate AMPKα2-SIRT3 pathway, supported by SIRT5, may underlie this Kbhb reprogramming. We validated carnitine acetyltransferase (CRAT) K141bhb as an age-accumulated modification reversed by exosomes in an AMPKα2- associated manner. Molecular modeling indicated that K141bhb neutralizes the positive charge of K141, disrupting the local salt bridge between K141 and E144 and destabilizing the α-helical structure, suggesting that K141bhb may serve as a metabolic brake on CRAT activity. These findings map the cardiac Kbhb landscape and show that hUCMSC-exos ameliorate cardiac aging through AMPKα2- associated Kbhb modulation.