Determination of inflammatory and Ca2+ profile by flow cytometry in PBMC of non-STEMI versus STEMI patients

Abstract

Myocardial infarction affects more than 120,000 people every year in France. Despite the increasing effectiveness of patient management, the morbi-mortality remains high. Peripheral blood mononuclear cells (PBMC) have been recently shown to present a phenotype characteristic of a pathology, which could provide prognostic and/or diagnostic information. We aim to characterize the inflammatory and Ca2+ profile of PBMC from non-STEMI versus STEMI patients. Regarding cell phenotyping, lymphocytes were distinguished from monocytes according to their morphological criteria. Using the CD14, CD16, CCR2, and CX3CR1 markers, pro-inflammatory monocytes were further differentiated from anti-inflammatory monocytes. To analyze Ca2+ fluxes, different cell compartments were marked with chemical probes: FuraRed-AM (cytosol), Rhod2-AM (mitochondria), and MagFluo4-AM (reticulum). Using a pharmacological approach, the optimal conditions for measuring Ca2+ exchange between the different cell compartments were applied to a cohort of STEMI and non-STEMI patients. Decreased lymphocyte proportion towards an increase in the monocyte proportion, specifically classical monocytes, was measured in STEMI patients. Caffeine stimulation led to a non-significant decrease in Ca2+ transfer to the cytosol and to mitochondria and in the reticular Ca2+ release, in the STEMI PBMC. A significant reduced IP3R-induced Ca2+ response in the cytosol and in the mitochondrial compartment was also reported in the STEMI PBMC. Our study led to the setup of a multiparametric analysis by flow cytometry of Ca2+ and inflammation in PBMC. We demonstrated an altered Ca2+ homeostasis in STEMI PBMC. A higher heterogeneity in the inflammatory profile and calcium response in STEMI patients suggests a potential molecular signature of PBMC according to the clinical outcome. Therefore, we now plan to analyze PBMC from an existing and still ongoing cohort (HIBISCUS) of post-myocardial infarction patients with a 3-year clinical follow-up to identify new circulating prognosis biomarkers.