Higher calcium levels exacerbate sickling of RBC-retaining mitochondria in pediatric SCD patients

● SCD RBCs with mitochondria exhibit high calcium, increased sickling, reduced lifespan, obstruction in microfluidic channels, and reduced Hb. ● Ex vivo calcium chelation reduces RBC sickling in pediatric SCD patients and mouse models Sickle cell disease (SCD) is a genetic disorder characterized by hemoglobin (Hb) polymerization leading to red blood cell (RBC) sickling, oxidative stress, and vaso-occlusive episodes. Advancing our understanding of disease mechanisms is crucial for developing effective, disease-modifying therapies. This work demonstrates that mitochondrial retention in RBCs is associated with increased intracellular calcium, increased sickling, lower Hb levels, and reduced RBC survival. Our work investigated the impact of RBC mitochondrial retention and calcium dysregulation on sickling propensity in pediatric SCD patients across diverse genotypes (HbSS, HbSβ 0 , HbSC, HbSβ + ) and Townes mice. Patients with high mitochondrial retention had lower Hb levels and higher reticulocytosis than patients with low mitochondrial retention. Higher intracellular calcium levels were observed in the RBCs of pediatric SCD patients, and these levels were further elevated in mitochondria-positive RBCs. Reactive oxygen species (ROS) production levels were also elevated in mitochondria-positive RBCs in pediatric SCD patients compared to mitochondria-negative RBCs. Using the ImageStream flow cytometer, we demonstrated that, in both patient samples and Townes mice, mitochondrial retention and higher intracellular calcium levels were associated with a greater propensity for sickling. We observed reduced RBC lifespan in mitochondria-retaining RBCs and RBCs with higher calcium. This was done via labeling studies in the Townes mice. In parallel, calcium chelation reduced sickling in both patient-derived RBCs and those from Townes mice, with corresponding decrease in mitochondrial and cytosolic ROS in Townes mice. This work demonstrates the suitability of the ImageStream flow cytometer in analyzing and developing disease-modifying therapies for SCD mitochondrial-associated complications.