Methionine redox regulation of actin-interacting proteins primarily governs antioxidative signaling and response to the salvianolic acid B treatment in EA.hy926 cells.

Actin-interacting proteins are important molecules for filament assembly and cytoskeletal signaling within vascular endothelium. Disruption in their interactions causes endothelial pathogenesis through redox imbalance. Actin filament redox regulation remains largely unexplored, in the context of pharmacological treatment. This work focused on the peptidyl methionine (M) redox regulation of actin-interacting proteins, aiming at elucidating its role on governing antioxidative signaling and response. Endothelial EA.hy926 cells were subjected to treatment with salvianolic acid B (Sal B) and tert-butyl-hydroperoxide (tBHP) stimulation. Mass spectrometry was employed to characterize redox status of proteins, including actin, myosin-9, kelch-like erythroid-derived cap-n-collar homology-associated protein 1 (Keap1), plastin-3, prelamin-A/C and vimentin. The protein redox landscape revealed distinct stoichiometric ratios or reaction site transitions mediated by M sulfoxide reductase and reactive oxygen species. In comparison with effects of tBHP stimulation, Sal B treatment prevented oxidation at actin M325, myosin-9 M1489/1565, Keap1 M120, plastin-3 M592, prelamin-A/C M187/371/540 and vimentin M344. For Keap1, reaction site was transitioned within its scaffolding region to the actin ring. These protein M oxidation regulations contributed to the Sal B cytoprotective effects on actin filament. Additionally, regarding the Keap1 homo-dimerization region, Sal B preventive roles against M120 oxidation acted as a primary signal driver to activate nuclear factor erythroid 2-related factor 2 (Nrf2). Transcriptional splicing of non-POU domain-containing octamer-binding protein was validated during the Sal B-mediated overexpression of NAD(P)H dehydrogenase [quinone] 1. This molecular redox regulation of actin-interacting proteins provided valuable insights into the phenolic structures of Sal B analogs, showing potential antioxidative effects on vascular endothelium.

PGC-1α/NRF1-dependent cardiac mitochondrial biogenesis: A druggable pathway of calycosin against triptolide cardiotoxicity.

Mitochondrion-related cardiotoxicity due to cardiotoxin stimuli is closely linked to abnormal activities of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), followed by co-inactivation of nuclear respiratory factor-1(NRF1). Pharmacological interventions targeting mitochondria may be effective for developing agents against cardiotoxicity. Herein, in triptolide-treated H9C2 cardiomyocytes, we observed defective mitochondrial biogenesis and respiration, characterized by depletion of mitochondrial mass and mitochondrial DNA copy number, downregulation of mitochondrial respiratory chain complexes subunits, and disorders of mitochondrial membrane potential and mitochondrial oxidative phosphorylation. Dysregulation of mitochondria led to cardiac pathological features, such as myocardial fiber fracture, intercellular space enlargement, and elevation of serum aspartate aminotransferase, creatine kinase isoenzyme, lactate dehydrogenase, and cardiac troponin I. However, following calycosin treatment, an active compound from Astragali Radix, the mitochondrion-related disorders at both cell and tissue levels were significantly ameliorated, which was facilitated by the activation of PGC-1α via deacetylation, followed by NRF1 co-activation. Calycosin-enhanced PGC-1α deacetylation is impelled by increasing sirtuin-1 expression and NAD+/NADH ratio. PGC-1α/NRF1 signaling in calycosin-mediated mitochondrial biogenesis protection was further confirmed by NRF1 knockdown and PGC-1α inhibition with SR18292. We conclude that calycosin ameliorated triptolide-induced cardiotoxicity by protecting PGC-1α/NRF1-dependent cardiac mitochondrial biogenesis and respiration, which is the druggable pathway for cardiotoxicity mitigation.