Characterization of the reticulum-mitochondria coupling in human immortalized B lymphocytes

Abstract

During myocardial infarction (MI), dying cardiomyocytes induce an acute inflammatory response that, if prolonged, will weaken the heart even beyond the initially injured area and promote the occurrence of secondary cardiac events. This inflammatory response is partially mediated by circulating immune cells, including B lymphocytes which have emerged as a new therapeutic target of interest to prevent damages related to this post-MI inflammation. B cell activation is a Ca2± dependent process, which promotes the production of pro- and anti-inflammatory cytokines. Ca2+ homeostasis is therefore essential to ensure their proper functioning. Whether an alteration of Ca2+ homeostasis in B lymphocytes occurs during MI remains unknown. Ca2+ signaling relies notably on the reticulum-mitochondria coupling (MAM); however, the role of MAM in B cell activation is not yet elucidated. Our aim is to characterize the role of the reticulum-mitochondria coupling in B lymphocyte function in order to further establish their potential alteration during MI. B cells from three healthy patients were immortalized with the Epstein–Barr virus, as an alternative to native B cells since their short lifespan limits analyses. MAM composition and structure were respectively analyzed by Western blot and electron microscopy (EM). Intracellular Ca2+ measurements in the different cell compartments (cytosol, endoplasmic reticulum and mitochondria) were performed using fluorescent chemical or genetic Ca2+ probes followed by wield-field microscopy. We confirmed the expression of proteins involved in Ca2+ transfer at MAM interface (IP3R, GRP75, VDAC1, MCU, MICU1) in immortalized B lymphocytes of the three patients. EM preliminary results revealed a reticulum-mitochondria median width of ∼31.15 nm compatible with Ca2+ transfer, and a median of ∼10.9% of the mitochondrial surface involved in MAM formation, with no significant difference between patients. Ca2+ imaging is still ongoing. Immortalized B lymphocytes display MAM structural and functional characteristics. Therefore, immortalized B cells may represent a relevant model to study the role of MAM during MI.