NHE9 regulates exosomal stress response to hypoxia in cardiomyocytes

Hypoxia, a major stressor in conditions like ischemia, significantly impacts the function and communication of cardiomyocytes. Cells adapt to hypoxia through various mechanisms, including the release of exosomes. Exosomes are nanoscale vesicles that facilitate intercellular signaling by transferring specific biomolecular cargos. Despite this understanding, the molecules driving these stress responses and the release of exosomes remain unclear. Recent research efforts are focused on identifying the key molecular players involved in exosome release during hypoxic conditions to gain a clearer understanding of these adaptive mechanisms. The endosomal sodium-proton exchanger NHE9 was recently identified as a crucial regulator of exosome biogenesis. In this study, we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and H9c2, a mouse cardiomyocyte cell line as models to show that hypoxia leads to significant upregulation of NHE9. To investigate the role of NHE9 upregulation during hypoxia, we generated genetically engineered cell lines with altered NHE9 expression. We then examined exosome dynamics under hypoxic conditions in cell lines where NHE9 was either overexpressed or knocked down. Exosomes were isolated and characterized using nanoparticle tracking analysis and Western blotting. Our results demonstrate that NHE9 upregulation leads to a significant increase in exosome secretion and enriches these exosomes with stress response proteins, specifically hypoxia-inducible factor 1-alpha (HIF1α) and heat shock protein 70 (Hsp70). Therefore, this study reveals a novel role for NHE9 as a critical regulator of stress signaling in hypoxic environments, offering new insights into cardiomyocyte adaptation and potential therapeutic interventions.