Molecular Responses to Oxidative Stress: From Extracellular Mitochondria to NRF2 Signaling and Translational Control

Abstract

Oxidative stress disrupts cardiovascular homeostasis by impairing mitochondrial integrity, depleting antioxidant defense, and reprogramming protein synthesis. The work presented here interrogates these responses across organelle, cellular, and organismal scales through integrative biochemical, omics, and in vivo approaches. An acute H2O2 exposure prompted AC16 cardiomyocytes to expel a distinct pool of fragmented extracellular mitochondria (EM). These organelles exhibited depolarized membrane potential, reduced bioenergetics, and altered cristae morphology. Untargeted lipidomics revealed ceramide enrichment and pharmacological inhibition of ceramide biosynthesis markedly suppressed mitochondrial extrusion. Functionally, EM served as damage-associated molecular patterns (DAMPs), driving inflammatory responses in macrophages and thereby linking mitochondria quality control to sterile inflammatory signaling. Cross-species analyses of human (GTEx), Rhesus monkey, Fischer rat, and C57BL/6J mouse myocardium uncovered an age-dependent decline in NRF2 expression and activity at RNA transcript or protein levels. Aged NRF2 knockout mice developed premature eccentric hypertrophy, diastolic dysfunction, and shortened lifespan. Our findings indicate an early onset of maladaptive cardiac remodeling with the loss of NRF2. Ribosome-bound mRNA sequencing of oxidatively stressed HeLa cells identified 21 selectively loaded transcripts, including ATF3 and MAFF. Increased ribosome-associated ATF3 mRNA coincided with higher ATF3 protein levels, whereas MAFF translation was delayed despite the polysome enrichment. Silencing the ribosomal quality control protein RACK1 eliminated this delay, indicating translational checkpoint on the ribosomes that modulates stress-associated protein synthesis. Together these findings delineate a coordinated triad of oxidative stress adaptations: (1) ceramide-dependent mitochondrial extrusion that drives inflammatory signaling, 2) an age-associated decline in NRF2 antioxidant response linked to cardiac remodeling, and 3) ribosome surveillance mechanisms that regulate protein synthesis. This work advances mechanistic insights into how redox imbalance culminates in cardiac pathology and identifies therapeutic entry points for mitigating oxidative damage.