The Abnormal Presence of Mitochondria in Circulating Red Blood Cells Cause an Increased Oxygen Consumption Rate, ROS Generation and Hemolysis in Patients with Sickle Cell Disease

Sickle Cell Disease (SCD) is an inherited blood disorder that affects millions of people worldwide, and it is caused by a mutation of the β-globin gene which results in the polymerization of HbS when deoxygenated. Patients with sickle cell disease experience severe pain, multi organ damage, and shortened life span. Changes arising from sickle hemoglobin (HbS) inside the red blood cell (RBC) leads to an imbalance of the oxidative reactions which increases reactive oxygen species (ROS) generation. ROS causes hemolysis and damages blood vessels. Previously, our lab demonstrated that SCD patients have a significant fraction of mitochondria retaining-RBCs which were associated with excessive levels of ROS. In addition immature mitochondria containing reticulocytes, R1 reticulocytes, are increased in the peripheral blood of SCD patients compared to control subjects. Here we report that the percentage of mitochondria retaining-RBCs directly correlated with the absolute number of reticulocytes (n=18,r=0.69, P< 0.001). As would be expected we were able to link mitochondria-retention with hemolysis. In addition, when we investigated energy metabolism we made the novel finding that SCD RBCs have an increased oxygen consumption rate (OCR). Mitochondrial-RBC retention and markers of hemolysis (serum bilirubin, and LDH) were compared by using Pearson's correlation coefficient analysis. The total serum bilirubin was directly associated with the percentage of mitochondria-containing RBCs in SCD patients' blood samples (r=0.31, n=41, p<0.04). A subgroup analysis showed a strong association between bilirubin and mitochondria-retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.55, n=17, P<0.02). Lactate dehydrogenase (LDH) is a known biomarker for hemolysis-and associated disease complications and early mortality in patients with SCD. There was a less significant association of percentage of mitochondria- retaining RBCs in SCD with serum LDH (r=0.02, n=40, P<0.2) when all patients were analyzed, however, a strong association was seen with serum LDH and mitochondria retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.67, n=17, P<0.002). We further investigated the mitochondrial RBCs energy metabolism in SCD as compared to controls in both human and mice. RBCs obtained from Townes SCD or control mice were seeded in an Agilent Seahorse XF 24-well plate using the Seahorse XF Base Medium with supplements, and we monitored the OCR and extracellular acidification rate (ECAR), in real time by Seahorse XFe-extracellular flux analyzer. OCR(pmol/min) standardized for protein was significantly higher in sickle cell RBCs of both human and mice origin compared to control. [((SCD patients(Hb SS): 6.92± 1.23, n=2 and control subjects (Hb AA)2.55±0.17, n=2, p<0.006)). ((Townes SCD (HbSS): 169.3± 30.8, n=3 and Transgenic control (HbAA):5.0±0.8, n=2, p<0.001))]. OCR was reduced to normal levels with oligomycin which is a known blocker of mitochondrial respiration. ECAR, a measure of glycolysis, was significantly higher levels in SCD patient and mouse samples compared to control. These findings support our premise that mitochondria retaining RBCs play a role in intravascular hemolysis. In addition, they indicate that RBC from SCD patients have increased oxygen consumption. Mitochondrial retention in RBCs have two possible mechanism of damage to the RBC, increased ROS formation and increased deoxygenated HbS polymer formation due to mitochondrial oxygen consumption. These results support further investigation into the role of mitochondrial energy metabolism in the pathogenesis of sickle cell disease.