Abstract Body (Do not enter title and authors here):
Background: Myocardial infarction (MI) is a leading cause of heart failure. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show therapeutic potential for MI, largely attributed to paracrine effects. However, the role of microvesicles (MVs), a major class of extracellular vesicles, n this effect remains unclear. Therefore, it is imperative to ascertain whether hiPSC-CM derived MVs exhibit cardioprotective effects and to elucidate the underlying mechanisms in both murine and porcine models of MI.

Methods: hiPSC-CM-MVs were isolated by differential ultracentrifugation and characterized via nanoparticle tracking, electron microscopy, flow cytometry, and western blot. Their therapeutic effects were evaluated in oxygen–glucose deprivation (OGD)-injured hiPSC-CMs and murine MI models. Proteomic profiling identified candidate effectors, which were further validated using cardiac-specific gene overexpression or knockout transgenic mouse models. A porcine MI model was also used to assess the safety and efficacy of MV through MDCT, hemodynamic measurements, and histological analysis.

Results: In vitro, we initially isolated high-purity MVs from hiPSC-CMs, exhibiting a distinct enrichment of mitochondrial constituents.These MVs conferred robust cytoprotection against OGD-induced injury by suppressing cardiomyocyte apoptosis, augmenting mitochondrial membrane potential and calcium uptake capacity, and thereby sustaining calcium homeostasis. In vivo, the trentment of MV alleviated progressive cardiac dysfunction following MI in mice and concurrently suppressed a spectrum of pathological remodeling events, including myocardial hypertrophy, fibrosis, apoptosis, and calcium overload. The cardioprotective effects observed were likely the result of enhanced mitochondrial calcium uptake, which contributed to restore calcium homeostasis. To explore the underlying mechanism, proteomic profiling of MVs identified DExH-box helicase 9 (DHX9) as a key regulatory cargo. Cardiac-specific overexpression of DHX9 partially reproduced while knockout DHX9 abolished the effects of MVs. Finally, in a porcine MI model, MVs exhibited robust therapeutic efficacy and safety without systemic toxicity.

Conclusion: This study identifies a novel mechanism by which hiPSC-CM-MVs restore calcium homeostasis and improve cardiac function post-MI via DHX9. These findings provide preclinical evidence supporting MVs as a safe and effective therapeutic strategy for MI.