Background <p>Carbon monoxide poisoning (COP) induces systemic hypoxia and oxidative stress–related injury, leading to myocardial injury and persistent cardiac dysfunction. However, reliable biomarkers for monitoring long-term cardiac sequelae and therapeutic responses remain lacking. Extracellular vesicles (EVs), which reflect the molecular status of their cells of origin, may serve as candidate biomarkers for organ-specific injury. This study investigated whether cardiac EV proteins capture COP-induced myocardial and mitochondrial dysfunction and reflect the therapeutic effects of hyperbaric oxygen (HBO) therapy.</p> Methods <p>A rat model of COP was established with or without HBO treatment. Cardiac function was assessed by echocardiography, and myocardial injury was evaluated using histological, ultrastructural, and biochemical analyses. Cardiac-enriched EVs isolated from ex vivo whole-heart perfusate were used for global proteomic profiling. Candidate differentially abundant proteins were analyzed with emphasis on pathways related to mitochondrial dynamics, mitochondrial energy metabolism, calcium handling, and myocardial contractility. Key EV-associated and tissue proteins were further validated, and selected candidates were examined in serum-derived EVs as preliminary targeted circulating EV validation.</p> Results <p>COP induced significant cardiac dysfunction, as evidenced by reduced ejection fraction and fractional shortening, together with histological myocardial injury, all of which were attenuated by HBO treatment. Proteomic analysis demonstrated that COP reshaped the cardiac EV proteome in a manner consistent with mitochondrial abnormalities, altered calcium-handling protein profiles, and impaired myocardial contractile function. These EV proteomic alterations were enriched in pathways related to mitochondrial dynamics, calcium signaling, and cardiac contractile regulation. Specifically, COP was associated with dysregulation of mitochondrial dynamics regulators, including optic atrophy type 1 (Opa1) and mitochondrial fission protein 1 (FIS1), as well as calcium-handling proteins such as ryanodine receptor 2 (Ryr2) and phospholamban (Pln). Ultrastructural and biochemical analyses showed mitochondrial cristae disruption, altered mitochondrial fusion–fission protein profiles, mitophagy-related protein changes, and pyroptosis-associated signaling in cardiac tissue following COP, whereas HBO mitigated these abnormalities. Notably, EV-associated Opa1 and FIS1 were associated with COP-related alterations in mitochondrial dynamic balance, whereas EV-associated Ryr2 and Pln were associated with impaired myocardial contractile parameters. Additional analysis of EV proteins related to mitochondrial function and ATP energy production further supported COP-associated mitochondrial energy metabolism–related protein remodeling. Targeted analysis of serum-derived EVs further showed that selected calcium-handling proteins, including Ryr2 and Pln, were detectable in circulating EVs and exhibited COP-associated changes consistent with cardiac tissue alterations. These findings support selected cardiac-enriched EV proteins as candidate molecular readouts of COP-associated myocardial, mitochondrial, and contractile abnormalities.</p> Conclusions <p>Cardiac EV proteomic remodeling reflects COP-associated mitochondrial and contractile abnormalities and captures the therapeutic effects of HBO. These findings identify cardiac-enriched EV proteins as candidate molecular readouts of myocardial injury and treatment response, providing a cardiac-enriched EV discovery framework for future blood-based biomarker development. The observed alterations in mitochondrial dynamics–, mitochondrial energy metabolism–, and calcium-handling–related proteins provide hypothesis-generating insight into molecular pathways associated with COP-induced cardiac dysfunction. Further validation using circulating EV proteomics, biomarker classifier analyses, and dedicated redox proteomics will be required to establish clinical utility and redox-regulated mechanistic relevance.</p>