Abstract Body: Introduction:
Mitochondrial dysfunction and Inflammation are hallmarks of heart failure (HF). We previously found mitochondrial function of the circulating immune cells are linked to its inflammatory state. Here we test the hypothesis that mitochondria-containing extracellular vesicles (EV-Mito) released from monocytes act as immunogenic mediators of sterile inflammation in HF and are regulated by mitochondrial quality-control pathways.

Methods and Results:
Plasma from stage-D HFrEF patients and healthy controls was analyzed using volumetric flow cytometry with mitochondrial- and cytoplasmic-specific markers. HF plasma demonstrated a 2.5-fold increase in mitochondrial–cytoplasmic double-positive microparticles (EV-Mito), representing ~60% of circulating microparticles. EV-Mito isolated from HF plasma induced greater cytokine production in macrophages than soluble plasma fractions, identifying EV-Mito as a major source of plasma immunogenicity. Surface marker profiling of EV-mito implicated CD14-positive monocytes as a primary source.

Mechanistically, mtDNA–mediated TLR9 activation induced mitochondrial injury and suppressed mitophagy by destabilizing PI3K-III complex II, triggering EV-Mito release. To identify regulators, we performed an siRNA screen targeting 140 membrane trafficking genes and found RhoA was a top hit for EV-mito release.

CRISPR-mediated RhoA deletion reduced mitochondria–ER contact, suggesting disruption of mitochondria-associated membranes (MAMs), a key site of mitophagy initiation. RhoA-null cells also exhibited significantly increased release of LC3-negative EV-Mito, indicating functional impairment of mitophagy. Further, RhoA deletion increased expression of SNX9, a regulator of mitochondrial-derived vesicle formation. Lastly, EV-Mito isolated from RhoA-deficient cells efficiently induced IL-1β and IL-6 production in macrophages.

Conclusion:
Monocyte-derived EV-Mito are markedly increased in advanced HF and act as potent mediators of sterile inflammation. Mechanistically, RhoA preserves mitochondrial quality control by maintaining MAM-dependent mitophagy, thereby limiting SNX9-associated mitochondrial vesicle formation and the release of pro-inflammatory EV-Mito.