Hypoxic Human Induced Pluripotent Stem Cell‐Derived Cardiomyocytes Release Large Extracellular Vesicles That Promote a Pro‐Inflammatory Phenotype in Macrophages

Directly post‐infarction, myocardial tissue release signals that trigger an acute inflammatory response to clear debris and initiate cardiac repair. However, prolonged inflammation may drive adverse remodelling and heart failure in the long‐term. Therefore, understanding the mechanisms of acute and chronic inflammation after myocardial infarction (MI) is crucial for developing targeted treatments. Extracellular vesicles (EVs) mediate intercellular communication by carrying diverse biomolecules, such as proteins, that influence recipient cell behaviour. Cardiomyocyte‐derived EVs (CM‐EVs) are known to play a role in both cardiac repair and the progression of heart failure following MI. However, little is known about CM‐EV subpopulations and how they modulate post‐infarction inflammation. Here, we demonstrate that human CM‐EV subpopulations can modulate macrophage phenotype. We identified three EV subpopulations, defined by their sedimentation coefficients, with distinct density and proteome profiles released by human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs). The 2K, 10K and 100K pellets, collected by differential ultracentrifugation of the conditioned medium, are enriched, respectively, in sarcomeric components, metabolic proteins and proteins involved in transport to the plasma membrane. Proteomic analysis revealed that hypoxia changes the proteome of EV‐containing pellets, enriching for proteins involved in cellular stress responses, such as chaperones, ribosomal, proteasomal and metabolic proteins. Next, we observed that both normoxic and hypoxic CM‐EVs are internalized by macrophages, and the hypoxic 10K pellet induces a pro‐inflammatory phenotype in these cells. Future research should elucidate whether CM‐EVs contribute to excessive inflammation and adverse cardiac remodelling post‐MI in vivo and if this can be modulated by blocking the release of specific CM‐EV subpopulations.