Iron deficiency impairs contractility of human cardiomyocytes through decreased mitochondrial function

Abstract

Aims: Iron deficiency is common in patients with heart failure and associated with a poor cardiac function and higher mortality. How iron deficiency impairs cardiac function on a cellular level in the human setting is unknown. This study aims to determine the direct effects of iron deficiency and iron repletion on human cardiomyocytes.

Methods and results: Human embryonic stem cell-derived cardiomyocytes were depleted of iron by incubation with the iron chelator deferoxamine (DFO). Mitochondrial respiration was determined by Seahorse Mito Stress test, and contractility was directly quantified using video analyses according to the BASiC method. The activity of the mitochondrial respiratory chain complexes was examined using spectrophotometric enzyme assays. Four days of iron depletion resulted in an 84% decrease in ferritin (P < 0.0001) and significantly increased gene expression of transferrin receptor 1 and divalent metal transporter 1 (both P < 0.001). Mitochondrial function was reduced in iron-deficient cardiomyocytes, in particular ATP-linked respiration and respiratory reserve were impaired (both P < 0.0001). Iron depletion affected mitochondrial function through reduced activity of the iron-sulfur cluster containing complexes I, II and III, but not complexes IV and V. Iron deficiency reduced cellular ATP levels by 74% (P < 0.0001) and reduced contractile force by 43% (P < 0.05). The maximum velocities during both systole and diastole were reduced by 64% and 85%, respectively (both P < 0.001). Supplementation of transferrin-bound iron recovered functional and morphological abnormalities within 3 days.

Conclusion: Iron deficiency directly affects human cardiomyocyte function, impairing mitochondrial respiration, and reducing contractility and relaxation. Restoration of intracellular iron levels can reverse these effects.

Keywords: Contractility; Heart failure; Human cardiomyocytes; Iron deficiency.

Figure 1

In vitro iron deficiency is obtained through deferoxamine (DFO) incubation. Ferritin levels decrease in a time‐dependent fashion during DFO incubation (A). Low iron levels lead to induced gene transcription levels of genes involved in iron uptake, transport, and storage (B). (C) Hypoxia‐inducible factor 1 alpha (HIF1α) protein levels in relation with α‐tubulin levels during DFO incubation. N.D., not determined; SLC11A2, solute carrier family 11 member 2; SLC39A14, solute carrier family 39 member 14; TfRC, transferrin receptor. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2

The effect of iron deficiency on mitochondrial function. Decreasing levels of intracellular iron correlate with ATP levels (A). Representative traces for control cardiomyocytes and cardiomyocytes treated with deferoxamine (DFO) for 2 and 4 days in a Mito Stress test (B). Effects of iron deficiency on ATP‐linked respiration and respiratory reserve are shown in (C). The enzymatic activity of each individual mitochondrial complex was analysed (D). OCR, oxygen consumption rate. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3

Effects of iron depletion are reversible by transferrin (Tf) administration. Following Tf‐bound iron supplementation, levels of ferritin (A), gene expression (B), mitochondrial respiration (C), of which ATP‐linked respiration and respiratory reserve shown in detail (D), hypoxia‐inducible factor 1 alpha (HIF1α) protein (E), and ATP (F) were mostly found to be restored. AntA, antimycin A; FCCP, carbonyl cyanide p‐tri‐fluoromethoxy‐phenyl‐hydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; Rot, rotenone. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Figure 4

Mitochondrial morphology is also affected by iron deficiency. Mitochondria in iron‐deficient cardiomyocytes appear swollen and contain electron dense inclusion bodies (A), which were found to contain nitrogen and sulfur based on energy dispersive X‐ray (EDX) analysis (B). Scale bar = 1 μm. DFO, deferoxamine.

Figure 5

Low iron levels resulted in reduced contractile force and impaired systolic and diastolic velocity. The fractional area change (FAC) of a single contraction for each condition (A) show that iron deficiency impairs contractile force (B). FAC, and maximum systolic and diastolic velocities (V_max) are affected by low iron levels, but are restored upon addition of transferrin (Tf)‐bound iron. *P < 0.05; **P < 0.01; ***P < 0.001. DFO, deferoxamine.

Figure 6

Reversible morphological aberrations during iron deficiency. Electron micrographs of (A) control, (B) iron‐deficient, and (C) transferrin‐treated cardiomyocytes. N, nucleus; G, glycogen; V, vacuole. Scale bar = 2 μm.