What’s Good for the Marrow Is Bad for the Heart: Improving Anemia in a Cardiac Iron Overload Model of Hbbth1/th1 Mice.
Authors: Ginzburg Yelena Z. , Suzuka Sandra M. , Fabry Mary E. , Nagel Ronald L. 
Published: October 2019

β-thalassemia is a β-globin gene mutation that leads to ineffective erythropoiesis, increased apoptosis, shortened red blood cell (RBC) survival, and anemia. The severity of anemia is variable, ranging from β-thalassemia major patients requiring transfusion therapy to β-thalassemia intermedia patients who are transfusion independent but absorb more iron from the gastrointestinal tract. Both types develop iron overload, with cardiac complications accounting for the majority of deaths. We hypothesized that cardiac iron overload in β-thalassemia intermedia occurs as a consequence of increased iron demand in the hematopoietic organs which, when not met, unexpectedly leads to relative iron deficiency and increased iron absorption. Iron is not present in the hearts of Hbbth1/th1 mice at baseline. However, abnormal echocardiogram results were observed in our mice relative to wild type, with lower ejection fractions (P=0.004, n=6) and a proportionally larger left ventricular mass (P=0.005, n=6) at baseline. We also developed a mouse model of cardiac iron overload in Hbbth1/th1 mice using iron dextran injections. After iron dextran, we demonstrated a significant decrease in left ventricular mass (P=0.008, n=4) compared to baseline and in a pilot experiment, observed a 150-fold increase in non-heme iron in heart as measured by spectrophotometry,. Furthermore, 130 mg of iron dextran injected over four weeks increases the reticulocyte count (P=0.03, n=5) as well as the hematocrit (P=0.01, n=5). Transferrin saturation with this method of iron loading increased from 35% to 100% (P=6x10−9 relative to baseline, n=6). Extramedullary hematopoiesis in the liver increased proportionally to the amount of iron administered and could be visualized near iron-laden Kupffer cells. Due to the increased iron demand in β-thalassemia and improved reticulocyte count and hemoglobin with the administration of iron, we postulated that Hbbth1/th1 mice are relatively iron deficient and set out to analyze the physiological mechanism of improved hematopoiesis after iron. With ter-119 and CD-71 to identify RBC precursors, and Annexin-V to identify apoptotic cells, we used flow cytometry to show that iron dextran administration decreases the degree of apoptosis in RBC precursors (from 21% to 8.5%) in Hbbth1/th1 mice to levels measured in wildtype mice at baseline. Preliminary results on RBC lifespan show that there is a shortened RBC survival in Hbbth1/th1 mice, with 50% of Hbbth1/th1 RBCs destroyed at 10.1 days relative to 21.8 days in C57 controls (P<0.0001, n=5). This analysis confirms the utility of this method for measuring the effects of iron on RBC survival in Hbbth1/th1 mice. In conclusion, the administration of iron dextran leads to clear improvement in erythropoiesis in Hbbth1/th1 mice and is in part due to increased extramedullary hematopoiesis, decreased apoptosis of RBC precursors, and possibly improved RBC survival. The loss of left ventricular mass after iron dextran administration implies myocyte death and supports our use of Hbbth1/th1 mice as a model of cardiac iron overload in β-thalassemia intermedia.