Red Blood Cell-Mediated Cardioprotection Is Impaired in ST-Segment Elevation Myocardial Infarction Patients With Anemia

Abstract

Red blood cells (RBCs) contribute to the regulation of nitric oxide (NO) bioactivity and cardiovascular function. In experimental and clinical studies, it has been shown that RBC-derived endothelial nitric oxide synthase (eNOS) provides cardioprotection in acute myocardial infarction (AMI).1, 2, 3 This cardioprotective role of RBCs appears to be mediated at least in part by activation of purinergic signaling via the P2Y13 receptor and the NO–soluble guanylyl cyclase pathway within the RBCs and protein kinase G in the heart. 1 Anemia is a frequent comorbidity in AMI and leads to a worse prognosis with increased morbidity and mortality. 4 , 5 Severe complications, such as bleeding, thromboembolic events, stroke, hypertension, arrhythmias, and inflammation, frequently coincide with anemia in AMI. These deleterious effects are observed even under moderate anemia with maintained oxygen carrying capacity of circulating blood at least under resting conditions. In an experimental ischemia/reperfusion model in isolated hearts, we have shown that RBCs from anemic mice lose their cardioprotective properties. Simultaneous application of the reactive oxygen species scavenger N-Acetyl-L-Cystein rescued RBC-mediated cardioprotection, indicating a potential target for therapy. 2 Experimental models and studies of anemia showed that remaining circulating RBCs become dysfunctional with signs of increased turnover, eryptosis, exaggerated membrane fragility, disturbed redox balance with increased production of reactive oxygen species, and uncoupled RBC-derived eNOS with reduced NO bioactivity.1, 2, 3 In this study, we hypothesized that the circulating NO pool in patients with ST-segment elevation myocardial infarction (STEMI) and anemia on admission is changed and that cardioprotective capacity of RBCs is impaired. Anemia was defined according to the World Health Organization as hemoglobin (Hb) levels <13.0 g/dL in adult men and <12.0 g/dL in adult nonpregnant women. Whole blood from STEMI patients without anemia (n = 42; mean Hb: 14.7 ± 1.3 g/dL) and without anemia (n = 25; mean Hb: 11.6 ± 1.3 g/dL; P < 0.001) was drawn within 24 hours after admission, and RBCs were immediately prepared for experiments (Figure 1A). STEMI patients with anemia were characterized as having mild normocytic, hypochromic anemia. STEMI patients with anemia were older and more likely hypertensive, but other baseline demographics and cardiovascular risk profiles were not statistically different. Mean RBC distribution width was significantly increased in STEMI patients with anemia (14.0 ± 1.6) compared with STEMI patients without anemia (13.3 ± 0.8; P = 0.025) indicating an enhanced turnover of RBCs. The content of oxidative metabolites (NOx: nitrate and nitrite), were measured in RBCs and plasma of STEMI patients using a gas phase chemiluminescence detector and high-pressure liquid chromatography nitrite/nitrate analyzer (ENO30, Eicom). The RBC content of nitrate and nitrite were significantly decreased (Figure 1B), whereas these metabolites were not altered in plasma (data not shown). The decrease in absolute numbers of RBCs in anemic patients together with a reduction of intracellular NOx of the remaining RBCs led to an altered circulating NO pool. Figure 1 Work Flow and Results of Sample Phenotyping (A) Overview of patient characterization and red blood cell (RBC) sample preparation from ST-segment elevation myocardial infarction (STEMI) patients with anemia (n = 25) and without anemia (n = 42). Blood was collected from STEMI patients with and without anemia undergoing primary percutaneous coronary intervention (pPCI). RBCs were processed according to our established laboratory protocols before being subjected to the appropriate experiment. (B) Analysis of oxidative NO products (NO2 - and NO3 -) in the deproteinized and N-ethylmaleimide-treated RBCs from STEMI patients with anemia (red circles) and without anemia (black circles). (C) RBC suspension (40% hematocrit) from STEMI patients with anemia (red circles) and without anemia (black circles) loaded on mouse (C57BL/6J) hearts in a Langendorff system. After a brief ischemia, the coronary flow, the left ventricular developed pressure (LVDP), maximal rate of raise of pressure increase (dP/dtmax), and minimal rate of raise of pressure increase (dP/dtmin) were recorded. Data represent the percentage recovery of left ventricular function after 120 minutes of reperfusion time from baseline. All values are mean ± SD. ∗P ≤ 0.05; ∗∗P ≤ 0.01. Statistical differences were analyzed with 2-way analysis of variance including all time points, and Bonferroni‘s post hoc test was used to compare groups. To evaluate the cardioprotective properties of RBCs, isolated and resuspended RBCs from STEMI patients undergoing primary percutaneous coronary intervention were transferred to isolated mouse hearts subjected to 40 minutes of global ischemia and 120 minutes of reperfusion. RBCs from nonanemic STEMI patients demonstrated improved left ventricular (LV) function during reperfusion compared with RBCs from anemic STEMI patients, indicated by an increased LV developed pressure and maximal and minimal rate of raise of LV pressure. These cardioprotective properties were severely impaired in RBCs from STEMI patients with anemia (Figure 1C). Coronary flow was maintained in both groups. These effects were specific for RBCs, as preloading with plasma from either anemic or nonanemic STEMI patients did not affect any parameter of LV function after ischemia/reperfusion. Our study unequivocally demonstrates that in STEMI patients with anemia, RBCs are dysfunctional and display a reduced circulating NO pool. In ex vivo transfer experiments, dysfunctional RBCs lose their cardioprotective properties in isolated mouse hearts subjected to ischemia/reperfusion. Impairment of the circulating NO pool and noncanonical, cardioprotective RBC function might contribute to the deleterious effects of anemia in AMI. P values were not adjusted for multiple testing; thus, future large-scale clinical studies are needed to confirm these findings and to prove that markers of RBC dysfunction may facilitate risk stratification in high-risk STEMI patients with anemia independent of hemoglobin levels. Additional trials may test whether RBC-specific antioxidant therapies can restore cardioprotection. In addition, new clinical trials are needed to address the controversial issue of hemoglobin thresholds for RBC transfusion in AMI in patients with anemia. New transfusion strategies, such as those tested in the MINT (Myocardial Ischemia and Transfusion; NCT02981407) trial, could help to optimize the individual treatment management of high-risk anemic STEMI patients and thereby improve clinical outcomes.