Residual Erythropoiesis Protects Against Cardiac Iron Loading in Transfusion Dependent Thalassaemia (TDT) By Lowering Labile Plasma Iron (LPI) through Transient Apotransferrin Generation

Introduction

Myocardial iron loading frequently complicates TDT but factors affecting the highly variable risk are not fully understood and current available predictors do not explain this variability. Chronic blood transfusion in TDT, by correcting anemia, is life saving but also aims to reduce ineffective erythropoiesis (IE) and its complications. Such transfusion will also decrease utilisation of transferrin (Tf) iron by the erythron and increase Tf saturation (TfSat) (Porter et al, Brit J Haematol 2014). Here we examine for the first time, how variable levels of residual erythropoiesis in TDT (marked by soluble transferrin receptors, sTfR), and the consequent presence of ApoTf, affect myocardial iron loading. We then examine how the difference between the transfusional iron loading rate (ILR) and Tf iron utilisation (calculated from sTfR levels) impact on myocardial iron loading. We finally test the hypotheses generated by these findings, using in vitro models of cellular uptake of NTBI and its redox active component LPI, the main conduits of myocardial iron uptake, in the presence and absence of ApoTf.

Methods

A cohort of 73 TDT patients on deferasirox (DFX) had cardiac MRI and blood biomarkers measured at baseline and at 1.5 years later, over which time the ILR was calculated. Patients were divided into those with cardiac iron (24 cases, cardiac T2*<20ms, R2* >50s^-1) and those without (59 controls, T2*>20ms, R2* <50s^-1): biomarkers of iron metabolism and routine biochemical variables were compared in both groups with data shown for the 1.5 year measurements. 48h washout from DFX was ensured prior to blood sampling for which patients gave written informed consent. NTBI, LPI, sTfR, TfSat were performed as in Porter et al 2014, mouse HL-1 cardiomyocytes grown as in Claycomb et al, PNAS 1998, Ferric citrate (Fe-cit) prepared as in Evans et al, J Biol Inorg Chem 2008, and total cellular iron was assayed by ferrozine as in Riemer et al Anal Biochem 2004.

Results and Discussion

Tf iron utilisation and myocardial iron in TDT. Of all biomarkers analysed, sTfR was most significantly associated with heart iron (p<0.001) being >3x higher in controls than cases. NTBI, LPI, TfSat and ILR were not significantly different in cases and controls, but LIC and ferritin were higher in cases (p=0.003 and 0.002). With sTfR expressed as Tf iron utilisation rate (or erythroid Tf uptake rate, ETUR) after Beguin et al, Blood 1993 and compared to ILR, myocardial iron was present only when the net ILR (ILR-ETUR) exceeded 0.21 mg/kg/d (p<0.001). Above and below this threshold LIC, TfSat and NTBI were similar (9.7 vs 10.1 mg/g dry weight, 96 vs 98%, 3 vs 2.6µM, p=ns), while LPI was 3x higher (0.35 vs 0.1 µM p=0.01). Thus LPI was high only in patients where the net ILR exceeded 0.21 whereas LIC, TfSat, NTBI showed no difference between patients with and without myocardial siderosis, even when adjusted for the net ILR (Figure 1).

Effect of ApoTf and Fe:cit ratio on NTBI uptake into cultured cardiomyocytes.  ApoTf or TfSat <100% inhibited NTBI uptake into HL-1 cells as well as LPI but did not increase iron release from cells preloaded with iron. Fe-cit, the main NTBI species, is less visible as LPI than NTA iron. Increasing excess of cit:Fe increases the proportion of iron visible to the LPI assay, increases myocardial iron uptake and iron binding to Tf. Previous work (Evans et al J Biol Inorg Chem 2008) showed that Fe:cit ratio does not affect the NTBI assay, by contrast we show it affects the LPI assay. The ratio affects uptake and because uptake may be redox dependent (Oudit et al, Nat Med 2003), it is very likely that a redox facile NTBI, i.e. LPI, is the key species taken up by the heart, further supported by Fe:cit ratios <1:100 (more citrate) accounting for the majority of uptake (EC₉₀=1.09µM,  Figure 2)

Conclusion

Taken together these data suggest that the risk of myocardial siderosis in TDT is critically dependent on the degree of residual erythropoiesis and the net balance between the transfusional iron delivery (ILR) and the iron utilisation rate by the erythron (ETUR). Myocardial siderosis occurs when the net ILR-ETUR exceeds 0.21 mg/kg/day, the same threshold at which LPI is increased. The likely mechanism is that higher ILR-ETUR values decrease TfSat<100% hence allowing transiently formed ApoTf to decrease LPI. This mechanism is supported by the finding that as little as 1µM ApoTf inhibits NTBI uptake into cardiomyocytes in culture while also decreasing LPI.