Unraveling the Role of PIEZO1 in Stressed Erythropoiesis: Implications for Dyserythropoiesis and Potential Therapeutic Targets in Dehydrated Hereditary Stomatocytosis

PIEZO1 is a mechanosensitive cation channel that plays a crucial role in various physiological processes as a mechanical force sensor. In erythrocytes, it is responsible for regulating cellular volume and hydration. Gain-of-function (GoF) mutations in PIEZO1 lead to dehydrated hereditary stomatocytosis (DHS) by slowing down the channel's inactivation kinetics. DHS patients often exhibit a range of clinical presentations, including mild to severe hemolytic anemia, and iron overload. In addition, studies on erythroid progenitor cells from DHS patients have indicated a mutation-dependent delay in erythroid differentiation. Furthermore, studies on constitutive GoF Piezo1 mice have revealed stressed erythropoiesis characterized by increased erythrocyte turnover. The primary aim of this study is to investigate the role of PIEZO1 during stressed erythropoiesis in DHS and identify potential druggable targets. A total of 80 DHS patients were analysed, and their BMRI (reticulocyte count × patient's Hb/normal Hb), EPO, and ERFE levels were measured. The findings revealed mutation-dependent dyserythropoietic features similar to those observed in CDAII patients (characterized by dyserythropoiesis). The engineered erythroid model of DHS, Hudep2-PIEZO1-KI (Hudep2-KI), was subjected to erythroid differentiation for 12 days and compared to Hudep2-WT. A notable reduction in CD235a expression was detected on the last day of differentiation, indicating that PIEZO1-mediated alteration occurs during the late stages of differentiation. Morphological analysis of differentiating cells indicated that KI cells experienced a significant reduction in proliferation rate (1.5-fold increase vs. day 0) compared to Hudep2-WT (3.1-fold increase vs. day 0) starting from day 7. By day 12 of differentiation, KI cells displayed only half the cell count of WT cells, with a substantial decrease in the percentage of orthochromatic normoblasts (13.7% vs. WT 54.5%). This was accompanied by a relative increase in reticulocytes (Hudep2-WT: 45.5% vs. Hudep2-KI: 86.3%). To identify the altered signalling pathways involved in differentiation deregulation, RNAseq was performed at various time points during differentiation. The gene ontology analysis indicated that the most enriched biological processes were related to apoptosis, cellular response to hypoxia, and glycolysis. During the differentiation process, various apoptosis-related processes were found significantly enriched on different days. Upon con