An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

Abstract

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain.

DOI: http://dx.doi.org/10.7554/eLife.19804.001

Introduction

As a constituent of hemoproteins, iron-sulphur proteins and other functional groups, iron is essential for cellular functions. Conversely, excess iron participates in cytotoxic Fenton-type chemical reactions. Thus, both iron deficiency and iron overload are detrimental to the cell. Therefore, the healthy functioning of tissues requires tight control of intracellular iron levels. These in turn are dependent both on cellular homeostatic pathways controlling iron uptake, usage, and storage, and on systemic pathways controlling iron levels in the plasma. In mammals, cellular iron homeostasis is controlled by the Iron Regulatory Proteins IRPs. Intracellular iron levels control the degradation of IRP2 and the conformational switch that confers the RNA-binding function of IRP1. IRPs in turn control the levels of iron uptake proteins such as transferrin receptor 1 (TfR1) and divalent metal transporter (DMT1), and the iron storage protein ferritin (Rouault and Klausner, 1997; Rouault, 2006). Systemic iron homeostasis is controlled by the hepcidin/ferroportin axis at the sites of iron entry into the circulation. Ferroportin (FPN), which is encoded by the Solute Carrier Family 40 Member 1 (Slc40a1), is the only known mammalian iron export protein and mediates iron release into the circulation from duodenal enterocytes, splenic reticuloendothelial macrophages and hepatocytes, the sites of iron absorption, recycling and storage respectively (Ganz, 2005; Donovan et al., 2005). FPN-mediated iron release is antagonized by the hormone hepcidin, also known as hepcidin antimicrobial peptide (HAMP). Produced primarily in the liver, hepcidin binds to and induces internalization of FPN, thereby limiting iron release into the circulation and its availability to peripheral tissues (Nemeth et al., 2004; Qiao et al., 2012). The importance of the HAMP/FPN axis is illustrated by diseases of systemic iron overload such as hemochromatosis and β-thalassaemia, where hepcidin production is impaired (Camaschella, 2005; Musallam et al., 2012), and in anaemia of chronic disease where hepcidin production is inappropriately elevated (De Falco et al., 2013; Nemeth and Ganz, 2014).

Other than the liver, hepcidin is also found in tissues with no recognized role in systemic iron homeostasis, including the heart (Merle et al., 2007), the brain (McCarthy and Kosman, 2014), the kidney (Kulaksiz, 2005) and the placenta (Evans et al., 2011). The function of this extra-hepatic hepcidin remains unknown, but one hypothesis is that it is involved in local iron control. Relevant to this hypothesis are our recent findings that FPN is also present in the heart, that it is essential for cardiomyocyte iron homeostasis and that its cardiomyocyte-specific deletion leads to fatal cardiac iron overload in mice (Lakhal-Littleton et al., 2015). Therefore, we hypothesised that cardiac HAMP regulates cardiac FPN, and that such regulation is important for local iron control and for cardiac function.

To test this hypothesis, we generated two novel mouse models; the first with a cardiomyocyte-specific deletion of the Hamp gene, and the second, with cardiomyocyte-specific knock-in of Slc40a1 C326Y, that encodes a FPN with intact iron export function but impaired HAMP binding (Schimanski et al., 2005; Drakesmith et al., 2005). We studied cardiac function and iron homeostasis longitudinally in both models and report that both develop fatal cardiac dysfunction and metabolic derangement as a consequence of cardiomyocyte iron deficiency. This occurs against a background of otherwise normal systemic iron homeostasis. Both cardiac dysfunction and metabolic derangement are prevented by intravenous iron supplementation.

Our findings demonstrate that, at least in the cardiomyocyte, endogenously-derived HAMP plays an essential role in cellular, rather than systemic iron homeostasis. It does this through the autocrine regulation of cardiomyocyte FPN. Disruption of this cardiac HAMP/FPN leads to fatal cardiac dysfunction.

Currently, there is considerable clinical interest in strategies that target the HAMP/FPN axis for the treatment of systemic iron overload and iron deficiency. Our findings suggest that these strategies may additionally alter cardiac iron homeostasis and function. Other than the heart, both HAMP and FPN are also found in the brain, kidney and placenta (McCarthy and Kosman, 2014; Kulaksiz, 2005; Evans et al., 2011; Rouault, 2013; Moos and Rosengren Nielsen, 2006; Bastin et al., 2006; Wolff et al., 2011). A pertinent question is the extent to which our findings in the heart extend to those tissues.

Results

Hamp expression and regulation in the heart

Hamp mRNA levels were approximately 30 fold lower in the adult mouse heart than in the liver (Figure 1A). Next, we examined the regulation of cardiac Hamp mRNA and HAMP protein in response to dietary iron manipulation, having first established that this dietary manipulation altered cardiac and liver iron levels (Figure 1—figure supplement 1). In both tissues, Hamp mRNA levels were decreased by the iron-deficient diet (Fe 2–5 ppm) and increased by the iron-loaded diet (Fe 5000 ppm) (Figure 1A). At the protein level, while changes in hepatic HAMP protein mirrored changes in its mRNA levels, cardiac HAMP protein was increased by the iron-deficient diet and unaffected by the iron-loaded diet (Figure 1B).10.7554/eLife.19804.003

Figure 1.

Hepcidin expression and regulation in the heart.

(A) Relative Hamp mRNA expression in heart and liver of adult C57BL/6 mice, under control conditions and after provision of low or high iron diets. *p=0.047, 0.001 respectively relative to control hearts, †p = 0.006, 0.019 respectively relative to control livers. (B) Corresponding immunohistochemical staining for HAMP in heart and liver. (C) Relative Hamp mRNA expression in primary adult mouse cardiomyocytes cultured under control conditions or in presence of FAC or DFO. *p=0.023, 0.001 and 0.014 respectively relative to control. †p = 0.024, 0.037, 0.016 and 0.037 respectively relative to control at the same timepoint. (D) Corresponding HAMP protein levels in supernatants of primary cardiomyocytes. DFO treatment was carried alone (DFO) or presence of Furin inhibitor (DFO+CMK). *p=0.002, 0.020, 0.028, 0.014, 0.015 respectively relative to control at the same timepoint. (E) Relative Hamp expression in heart and liver of 3 month old Hamp^fl/fl and Hamp^fl/fl;Myh6.Cre+ mice. *p=0.018 relative to cardiac Hamp in Hamp^fl/fl controls. (F) Corresponding immunohistochemical staining for HAMP in heart and liver. All values are plotted as mean ± SEM. Scale bar = 20 µm. n = 3 per group unless otherwise stated.

DOI: http://dx.doi.org/10.7554/eLife.19804.003

10.7554/eLife.19804.004

Figure 1—figure supplement 1.

Cardiac and liver iron following dietary iron manipulation.

Total elemental iron levels in heart and liver of C57BL/6 mice, under control conditions and after provision of low (Fe 5 ppm) or high iron (Fe 5000 ppm) diets from weaning for six weeks. *p=0.037 and 0.033 respectively relative to control heart, †p = 0.010 and 0.005 relative to control liver. n = 3. Data are represented as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.004

10.7554/eLife.19804.005

Figure 1—figure supplement 2.

Furin regulation by iron.

(A) Relative Furin mRNA in primary adult mouse cardiomyocytes under control conditions and following treatment with DFO or FAC. *p=0.004, 0.001 and 0.001 respectively relative to control at the respective timepoint. (B) Relative Furin mRNA in hearts of mice provided control diet or iron-deficient diet (2–5 ppm) or iron-loaded diet (5000 ppm) from weaning for six weeks. *p=0.015 relative to control diet. n = 3. Data are plotted as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.005

10.7554/eLife.19804.006

Figure 1—figure supplement 3.

Relative Hamp mRNA expression in cardiomyocytes following treatment with Furin inhibitor CMK.

Relative Hamp mRNA expression in primary adult mouse cardiomyocytes under control conditions and following treatment with DFO or FAC, in presence or absence of Furin inhibitor CMK. n = 3. Data are plotted as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.006

10.7554/eLife.19804.007

Figure 1—figure supplement 4.

HAMP in supernatants of primary cardiomyocytes.

HAMP protein was measured by ELISA in supernatants of primary adult cardiomyocytes, derived from Hamp ^fl/fl or Hamp ^fl/fl;Myh6.Cre+ mice and cultured under control conditions or in presence of FAC or DFO. n = 3. Data are plotted as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.007

10.7554/eLife.19804.008

Figure 1—figure supplement 5.

Confirmation of HAMP antibody specificity.

(A) Staining with HAMP antibody (Abcam ab30760) in liver and heart of C57BL/6 mice is completely abrogated by co-incubation with hepcidin-25 blocking peptide (Abcam ab31875). Scale bar = 20 µm. (B) Loss of HAMP staining in Hamp^fl/fl;;Myh6.Cre+ hearts (Figure 1F) is consistent with the antibody detecting HAMP1 and not HAMP2, because Hamp2 mRNA expression is not altered in Hamp^fl/fl;Myh6.Cre+ mice relative to Hamp^fl/fl controls, either with control diet or iron-deficient diet (six weeks from weaning). *p=0.007 and 0.047 relative to Hamp^fl/fl control under respective diet. n = 3 per group. Values are plotted as mean ± SEM. N.S=not significant.

DOI: http://dx.doi.org/10.7554/eLife.19804.008

10.7554/eLife.19804.009

Figure 1—figure supplement 6.

HAMP detection by ELISA unaffected by FAC and DFO.

HAMP standard from mouse hepcidin ELISA (E91979Mu, Uscn) was diluted either in the kit’s own standard diluent, or in unconditioned growth medium alone, or containing 100 µmol/L DFO or 500 µmol/L FAC. ELISA was performed as per manufacturer’s instructions.

DOI: http://dx.doi.org/10.7554/eLife.19804.009

To explore further the regulation of cardiac hepcidin by iron, we isolated primary adult cardiomyocytes from mice, then carried out a timecourse treatment with the iron chelator desferroxamine DFO, or with ferric citrate FAC. Under control conditions, Hamp mRNA was upregulated following addition of fresh cardiomyocyte culture medium (cardiomyocytes are cultured for 2 hr in serum-free medium prior to this). Relative to control cardiomyocytes at the respective timepoint, Hamp mRNA was increased by FAC from 4 hr of treatment, and decreased by DFO at 4, 8 and 16 hr of treatment (Figure 1C). HAMP protein, measured by ELISA in supernatants was also increased following addition of fresh medium. Relative to control cardiomyocytes at the respective timepoint, HAMP protein in supernatants was increased by DFO as early as 2 hr, but remained unchanged by FAC at all timepoints (Figure 1D). Thus, the direction of response of the Hamp mRNA and HAMP protein to iron levels in vitro mirrored the responses seen in vivo.

Next, we aimed to understand the mechanisms underlying increased HAMP secretion in DFO-treated cardiomyocytes. To this end, we investigated the role of the prohormone convertase Furin, which in hepatocytes, is required for cleavage of the propeptide and the release of the mature HAMP peptide (Valore and Ganz, 2008). Furin expression has been reported in the heart (Beaubien et al., 1995; Ichiki et al., 2013), and we also found that its expression was upregulated in the hearts of mice provided an iron-deficient diet and in cardiomyocytes treated with DFO (Figure 1—figure supplement 2). When we measured HAMP in supernatants of cardiomyocytes treated with the Furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK), we found no increase in HAMP release following DFO treatment (Figure 1D). We confirmed that Hamp mRNA levels in cardiomyocytes were not altered by CMK treatment (Figure 1—figure supplement 3). Together, these results indicate that Furin upregulation mediates increased HAMP secretion from iron-deficient cardiomyocytes.

Having established that hepcidin is found in cardiomyocytes, we then aimed to define its function. To this end, we generated cardiomyocyte-specific Hamp knockout mice Hamp^fl/fl;Myh6.Cre+ by crossing in-house conditional Hamp floxed (fl) mice with mice transgenic for Cre recombinase under the control of cardiomyocyte-specific promoter Myosin Heavy Chain 6 (Myh6.Cre+). Hamp mRNA (Figure 1E) and HAMP protein (Figure 1F) in the hearts of Hamp^fl/fl;Myh6.Cre+ mice were significantly reduced compared to the hearts of Hamp^fl/fl controls. Furthermore, compared to Hamp^fl/fl cardiomyocytes, levels of HAMP protein in the supernatants of cardiomyocytes from Hamp^fl/fl;Myh6.Cre+ mice were either greatly reduced or undetectable, both under baseline conditions and following treatment with DFO or FAC (Figure 1—figure supplement 4). Near complete ablation of the cardiac Hamp mRNA and HAMP protein in Hamp^fl/fl;Myh6.Cre+ mice confirmed that cardiomyocytes were the primary site of hepcidin expression in the heart. Liver Hamp mRNA (Figure 1E) and HAMP protein (Figure 1F) were not different between Hamp^fl/fl;Myh6.Cre+ and Hamp^fl/fl controls, consistent with the cardiac-specific nature of Hamp gene deletion.

Also consistent with this cardiac-specific deletion, Hamp^fl/fl;Myh6.Cre+ mice had normal levels of liver iron stores and circulating markers of iron homeostasis when compared to Hamp^fl/fl controls, demonstrating that loss of cardiac hepcidin did not affect systemic iron homeostasis. In addition, circulating HAMP levels were not reduced in the serum of Hamp^fl/fl;Myh6.Cre+ mice, suggesting that cardiac hepcidin does not contribute significantly to circulating HAMP levels (Table 1).10.7554/eLife.19804.010

Table 1.

Indices of systemic iron in six month old Hamp^fl/fl and Hamp^fl/fl;Myh6.Cre+ mice.

n = 6 per group. All values are shown as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.010

	Hampfl/fl	Hampfl/fl;Myh6.Cre+
liver total elemental iron (ng/mg tissue)	96.3 ± 12.2	88.7 ± 19.2
liver ferritin (µg/mg total protein)	0.65 ± 0.04	0.64 ± 0.05
serum iron (µmol/L)	28.60 ± 7.20	31.50 ± 8.40
serum ferritin (mg/L)	1.81 ± 0.04	1.88 ± 0.29
hemoglobin (g/L)	122.7 ± 11.5	116.0 ± 11.9
serum hepcidin (µg/L)	23.5 ± 7.6	23.9 ± 10.40

Fatal cardiac abnormalities in Hamp^fl/fl;Myh6.Cre+ mice

To determine the effects of loss of cardiac hepcidin, we first assessed the cumulative survival of Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls over a period of 52 weeks. Significantly greater mortality was observed amongst Hamp^fl/fl;Myh6.Cre+ mice, with only 29% of animals surviving to 52 weeks, compared with 90% of Hamp^fl/fl controls. The median survival of Hamp^fl/fl;Myh6.Cre+ mice was 28 weeks, whereas the majority of Hamp^fl/fl controls were still alive at 52 weeks (Figure 2A).10.7554/eLife.19804.011

Figure 2.

Fatal cardiac abnormalities in Hamp^fl/fl;Myh6.Cre+ mice.

(A) Cumulative survival of Hamp^fl/fl;Myh6.Cre+ mice (n = 50) and Hamp^fl/fl littermate controls (n = 47) over 52 weeks. (B) Representative H and E longitudinal heart sections from a six month old Hamp^fl/fl;Myh6.Cre+ mouse and Hamp^fl/fl littermate control. (C) Representative WGA cardiac staining from a six month old Hamp^fl/fl;Myh6.Cre+ mouse and Hamp^fl/fl littermate control. (D) Quantitation of cardiomyocyte size based on WGA staining in six month old Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. *p=0.001 relative to Hamp^fl/fl littermate controls. (E) Relative expression of the hypertrophic gene markers Myh7 and Nppb in hearts of 6 month old Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. *p=0.001, 0.047 for the respective gene relative to Hamp^fl/fl littermate controls. (F) Representative cardiac in-situ TUNEL staining from a six month old Hamp^fl/fl;Myh6.Cre+ mouse and Hamp^fl/fl littermate control. (G) Quantitation of percentage of apoptotic cardiomyocytes based on in-situ TUNEL staining in six month old Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls *p=0.001 relative to Hamp^fl/fl littermate controls. (H) Representative midventricular Cine MR images of hearts from Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl controls at 3, 6 and 9 months of age. (I–K) Cine MRI measurements of LV lumen, at end-systole (LVES), end-diastole (LVED), and of ejection fraction (LVEF) in Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls at three months (n = 8 per group), six months (n = 11 per group, *p=0.043 for LVES, 0.047 for LVED and 0.020 for LVEF) and nine months (n = 5 per group, *p=0.044 for LVES, 0.042 for LVED and 0.034 for LVEF). p values are relative to Hamp^fl/fl controls of the respective age. All Values are plotted as mean ± SEM. n = 3 per group unless otherwise stated. Scale bar = 50 µm.

DOI: http://dx.doi.org/10.7554/eLife.19804.011

Six-month old mice were sacrificed for assessment of cardiac morphology, which showed gross enlargement of the left ventricle (LV) in Hamp^fl/fl;Myh6.Cre+ hearts compared to Hamp^fl/fl controls (Figure 2B). Assessment of cardiomyocyte size by wheat germ agglutinin (WGA) staining confirmed that Hamp^fl/fl;Myh6.Cre+ cardiomyocytes were hypertrophied (Figure 2C–D). This was accompanied by upregulation of expression of hypertrophic gene markers myosin heavy chain (Myh7) and natriuretic peptide precursor (Nppb) (Figure 2E). TUNEL staining for in-situ detection of cell death also showed significantly greater apoptosis in the hearts of Hamp^fl/fl;Myh6.Cre+ mice than in Hamp^fl/fl controls (Figure 2F–G).

To characterise further the phenotype caused by loss of cardiac hepcidin, we used cine MRI in anaesthetised Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls at 3, 6 and 9 months of age. Mid-ventricular cine MR images showed no differences between the two genotypes at 3 months of age. At 6 and 9 months of age, cine MR images showed marked enlargement of the LV in Hamp^fl/fl;Myh6.Cre+ mice compared to Hamp^fl/fl controls (Figure 2H). Formal quantitation of cardiac parameters by cine MRI confirmed enlargement of the LV lumen in Hamp^fl/fl;Myh6.Cre+ mice, both at end-systole (LVES) (Figure 2I) and at end-diastole (LVED) (Figure 2J), accompanied by a decrease in LV ejection fraction (LVEF) from 62% to 42% (Figure 2K). Other parameters of cardiac performance were not significantly altered between mice from the two genotypes (Table 2). Taken together, histological examination of the hearts and cine MRI studies indicated that Hamp^fl/fl;Myh6.Cre+ mice developed fatal LV dysfunction with reduced LVEF.10.7554/eLife.19804.013

Table 2.

Non-LV parameters of cardiac function are not altered between Hamp^fl/fl and Hamp^fl/fl;;Myh6.Cre+ mice.

Cine MRI measurements of cardiac function in Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl controls at three months (n = 8 per group), six months (n = 11 per group) and nine months (n = 5 per group) of age. Values are shown as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.013

	3 months		6 months		9 months
	Hamp fl/fl	Hamp fl/fl; Myh6.Cre+	Hamp fl/fl	Hamp fl/fl; Myh6.Cre+	Hamp fl/fl	Hamp fl/fl; Myh6.Cre+
Average mass (mg)	70.51 ± 7.47	70.38 ± 5.33	72.59 ± 5.38	82.54 ± 11.32	78.30 ± 4.92	83.07 ± 5.44
RVED lumen (µl)	31.37 ± 2.55	26.81 ± 2.30	33.12 ± 3.19	30.63 ± 2.35	38.39 ± 3.88	39.95 ± 3.00
RVES lumen (µl)	7.43 ± 0.79	5.23 ± 0.67	8.70 ± 1.22	8.34 ± 1.06	13.36 ± 2.21	15.51 ± 2.47
RVEF (%)	76.42 ± 1.32	79.88 ± 2.80	73.66 ± 2.50	73.42 ± 2.22	65.20 ± 4.36	61.95 ± 3.53
Stroke volume (µl)	25.47 ± 1.99	22.73 ± 2.11	25.70 ± 2.70	23.76 ± 1.85	29.79 ± 2.30	29.84 ± 1.59
Cardiac output (ml/min)	10.34 ± 1.06	9.62 ± 0.90	10.72 ± 1.07	10.53 ± 1.05	11.46 ± 1.15	11.99 ± 1.38
Heart Rate (bpm)	404.07 ± 18.69	426.90 ± 17.94	419.64 ± 19.05	436.14 ± 14.30	384.84 ± 28.55	400.97 ± 41.20
Heart/body weight ratio x1000	2.80 ± 0.18	3.09 ± 0.26	2.51 ± 0.23	2.98 ± 0.37	2.36 ± 0.28	2.88 ± 0.27

Such changes in cardiac performance could not be attributed to Cre recombinase toxicity in the heart as we have previously shown that Myh6.Cre+ mice have normal cardiac function compared to wild type littermate controls (Lakhal-Littleton et al., 2015).

The role of cardiomyocyte FPN in the phenotype of Hamp^fl/fl;Myh6.Cre+ mice

We examined FPN protein in the hearts of Hamp^fl/fl;Myh6.Cre+ mice, and found that FPN protein was markedly upregulated compared to Hamp^fl/fl controls (Figure 3A), consistent with the idea that loss of cardiac HAMP was acting through upregulation of cardiomyocyte FPN. In order to test whether cardiac dysfunction arose from upregulation of cardiomyocyte FPN, we engineered mice where the Slc40a1 gene harbours a conditional cardiac-specific C326Y point mutation, which confers HAMP-resistance while conserving the iron export function of FPN (Schimanski et al., 2005; Drakesmith et al., 2005). We confirmed that the Slc40a1 C326Y fl allele produced the C326Y transcript specifically in the heart (Figure 3—figure supplement 1) and that Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice did not exhibit changes in systemic iron indices (Table 3). As seen in Hamp^fl/fl;Myh6.Cre+ mice, cardiomyocyte FPN was indeed upregulated in Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice (Figure 3B).10.7554/eLife.19804.014

Figure 3.

The role of cardiomyocyte FPN.

(A–B) Immunohistochemical staining for FPN in the hearts of three month old Hamp^fl/fl;Myh6.Cre+, Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice and respective controls. (C) Cumulative survival of Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice (n = 36) and Slc40a1 C326Y^fl/fl littermate controls (n = 31) over 52 weeks. (D) Representative H and E-stained longitudinal heart sections from a six month old Slc40a1 C326Y^fl/fl;Myh6.Cre+ mouse and a Slc40a1 C326Y^fl/fl control. (E) Representative WGA cardiac staining from a six month old Slc40a1 C326Y^fl/fl;Myh6.Cre+ mouse and Slc40a1 C326Y^fl/fl control. (F) Quantitation of cardiomyocyte size based on WGA staining. n = 3 per group. *p=0.001 relative to Slc40a1 C326Y^fl/fl controls. (G) Relative expression of Myh7 and Nppb in hearts of 6 month old Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice and Slc40a1 C326Y^fl/fl controls. n = 3 per group. *p=0.032, 0.044 for the respective gene relative to Slc40a1 C326Y^fl/fl controls. (H) Representative cardiac TUNEL staining from a six month old Slc40a1 C326Y^fl/fl;Myh6.Cre+ mouse and Slc40a1 C326Y^fl/fl control. (I) Quantitation of percentage of apoptotic cardiomyocytes based on TUNEL staining, n = 3 per group. *p=0.0003 relative to Slc40a1 C326Y^fl/fl controls. (J–L) Cine MRI measurements of LVES, LVED and LVEF in Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice and Slc40a1 C326Y^fl/fl controls at three months (n = 6 per group), six months (n = 6 per group, *p=0.003 for LVES, 0.043 for LVED and 0.001 for LVEF) and nine months (n = 5 per group, *p=0.033 for LVES, 0.047 for LVED and 0.023 for LVEF). P values are relative to Slc40a1 C326Y^fl/fl controls of the same age. (M) Percentage Fe55 efflux in cardiomyocytes from Hamp^fl/fl;Myh6.Cre+ mice, Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice and respective controls, in presence or absence of HAMP peptide. *p=0.018, 0.006 and 0.007 respectively (N) Elemental iron levels in cardiomyocyte fractions (CF) from the hearts of Hamp^fl/fl;Myh6.Cre+ mice, Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice and their respective controls. n = 4 per group. *p=0.032, 0.044, 0.047 and 0.031 respectively. (O–P) Relative TfR1 (*p=0.038, 0.001) and Fpn (*p=0.039, 0.047) expression in hearts of 3 month old Hamp^fl/fl;Myh6.Cre+ mice, Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice and their respective controls. All values are plotted as mean ± SEM. Scale bar = 50 µm.

DOI: http://dx.doi.org/10.7554/eLife.19804.014

10.7554/eLife.19804.016

Figure 3—figure supplement 1.

Characterisation of Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice.

Total mRNA was extracted from the liver and heart of an adult a Slc40a1 C326Y^fl/+;Myh6.Cre+ mouse, reverse transcribed using primers for exon 7 of the Slc40a1 mRNA transcript. Products were sequenced to confirm successful heterozygous expression of the C326Y transcript in the heart but not in the liver.

DOI: http://dx.doi.org/10.7554/eLife.19804.016

10.7554/eLife.19804.017

Figure 3—figure supplement 2.

Elemental iron levels in total hearts of Hamp^fl/fl;Myh6.

Cre+ mice, Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice and their respective controls at three months and 6 months of age. n = 4 per group. Data are plotted as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.017

10.7554/eLife.19804.018

Table 3.

Characterisation of Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice.

Indices of iron status in Slc40a1 C326Y^fl/fl and Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice at six months of age (n = 4 per group). Values are shown as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.018

	Slc40a1 C326Yfl/fl	Slc40a1 C326Y fl/fl; Myh6.Cre+
liver total elemental iron (ng/mg tissue)	92.77 ± 21.30	84.00 ± 26.00
liver ferritin (µg/mg total protein)	0.87 ± 0.06	0.92 ± 0.05
serum iron (µmol/L)	27.30 ± 5.20	29.60 ± 7.20
serum ferritin (mg/L)	2.10 ± 0.04	2.20 ± 0.15
hemoglobin (g/L)	125.70 ± 8.80	126.00 ± 12.30
serum hepcidin (µg/L)	25.90 ± 11.60	27.50 ± 8.40

Like Hamp^fl/fl;Myh6.Cre+ mice, Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice also had increased mortality relative to their littermate controls (Figure 3C). We then determined whether Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice developed the same phenotype of cardiac dysfunction as Hamp^fl/fl;Myh6.Cre+ mice. Histologically, Slc40a1 C326Y^fl/fl;Myh6.Cre+ hearts from six month old mice also showed LV enlargement (Figure 3D), hypertrophied cardiomyocytes (Figure 3E–F), upregulation of hypertrophy markers Myh7 and Nppb (Figure 3G) and a greater degree of apoptosis compared to Slc40a1 C326Y^fl/fl controls (Figure 3H–I). When we measured cardiac performance by cine MRI, we found that Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice also developed LV dysfunction by 6 months of age, with a reduction in LVEF from 73% to 55% (Figure 3J–L).

The similarity between Hamp^fl/fl;Myh6.Cre+ and Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice in terms of the nature and time course of cardiac dysfunction suggests a common mechanism of cardiac dysfunction involving upregulation of cardiomyocyte FPN. Therefore, we tested whether this upregulation of FPN resulted in increased iron efflux from cardiomyocytes. Iron Fe55 efflux was indeed significantly greater in cardiomyocytes isolated from Hamp^fl/fl;Myh6.Cre+ and Slc40a1 C326Y^fl/fl;Myh6.Cre+ hearts than in cardiomyocytes isolated from their respective controls (Figure 3M). Addition of exogenous mouse HAMP in the efflux medium inhibited the increase in Fe55 efflux from Hamp^fl/fl;Myh6.Cre+ cardiomyocytes but not from Slc40a1 C326Y^fl/fl;Myh6.Cre+ cardiomyocytes, consistent with the HAMP-resistant mutation in Slc40a1 C326Y^fl/fl;Myh6.Cre+ cardiomyocytes. We hypothesised that upregulation of cardiac FPN and iron export in Hamp^fl/fl;Myh6.Cre+ and Slc40a1 C326Y^fl/fl;Myh6.Cre+ hearts caused cardiomyocyte iron depletion. To test this hypothesis, we quantified iron levels both in total hearts and in the isolated cardiomyocyte fractions at 3 months and 6 months of age. While iron levels in total hearts were not significantly different between any of the genotypes (Figure 3—figure supplement 2), the iron content of the cardiomyocyte fraction was significantly lower in Hamp^fl/fl;Myh6.Cre+ and in Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice than in their respective controls (Figure 3N). Furthermore, expression of TfR1 mRNA was upregulated (Figure 3O) and Slc40a1 mRNA was downregulated (Figure 3P) in Hamp^fl/fl;Myh6.Cre+ and Slc40a1 C326Y^fl/fl;Myh6.Cre+ hearts relative to their respective controls, consistent with a transcriptional response to intracellular iron deficiency (Rouault and Klausner, 1997; Rouault, 2006; Ward and Kaplan, 1823). Together these results demonstrate that loss of either cardiac hepcidin or hepcidin responsiveness in the heart results in upregulation of cardiomyocyte FPN, and that cardiomyocytes of Hamp^fl/fl;Myh6.Cre+ and Slc40a1 C326Y^fl/fl;Myh6.Cre+ hearts are iron deficient as a result of upregulation of FPN-mediated iron export.

The role of cardiomyocyte iron deficiency and metabolic derangement in cardiac dysfunction

As cardiomyocyte iron deficiency preceded the development of cardiac dysfunction, we hypothesised it is the cause of the cardiac phenotype in Hamp^fl/fl;Myh6.Cre+ mice. To test this hypothesis, we treated Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl controls with fortnightly intravenous injections of ferric carboxymaltose solution containing 0.5 mg iron from three months of age, and confirmed the effects of this treatment on cardiac and systemic iron indices at 6 months of age (Table 4). At this timepoint, we performed cine MRI and found that the LV enlargement and the reduced LVEF, seen in untreated Hamp^fl/fl;Myh6.Cre+ mice, were prevented in iron-treated Hamp^fl/fl;Myh6.Cre+ mice (Figure 4A–C). The transcriptional response to intracellular iron deficiency in untreated Hamp^fl/fl;Myh6.Cre+ mice (upregulation of TfR1 mRNA and downregulation of Slc40a1 mRNA relative to Hamp^fl/fl controls), was absent in iron-treated Hamp^fl/fl;Myh6.Cre+ mice, consistent with correction of cardiomyocyte iron deficiency (Figure 4D–E). Prevention of cardiac dysfunction in Hamp^fl/fl;Myh6.Cre+ mice by intravenous iron treatment confirms the causal relationship between cardiomyocyte iron deficiency and cardiac dysfunction in this setting.10.7554/eLife.19804.019

Figure 4.

The role of cardiomyocyte iron deficiency and metabolic derangement in cardiac dysfunction.

(A–C) Cine MRI measurements of LV lumen, at end-systole (LVES, *p=0.048), end-diastole (LVED, *p=0.031), and of ejection fraction (LVEF, *p=0.004) in 6-month old untreated (-iron) and I.V iron-treated (+iron) Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. n = 5 per group. (D–E) Relative TfR1 (*p=0.001) and Fpn (*p=0.002) expression in hearts of 6-month old untreated (-iron) and I.V iron-treated (+iron) Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. n = 4 per group. (F–H) Enzymatic activities of Aconitase I (*p=0.035, 0.041), Complex I (*p=0.004, 0.030) and Complex IV (*p=0.003, 0.026) in untreated (-iron) 3-month and 6-month old and in I.V iron-treated (+iron) 6-month old Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. n = 4 per group. (I) Representative EM micrographs of hearts from untreated (-iron) 3-month and 6-month old and I.V iron-treated (+iron) 6-month old Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. Scale bar = 2 µm. (J–L) Relative Hk2 (*p=0.010, 0.002), Eno (*p=0.014, 0.021) and Ldha (*p=0.003, 0.001) expression levels in hearts of untreated (-iron) 3-month and 6-month old and in I.V iron-treated (+iron) 6-month old Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. n = 4 per group. NS=not significant. All values are plotted as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.019

10.7554/eLife.19804.021

Table 4.

Effect of intravenous iron treatment on iron indices.

Total cardiac and liver elemental iron, serum iron and circulating HAMP in 6-month old untreated and I.V iron-treated Hamp^fl/fl;Myh6.Cre+ mice and Hamp^fl/fl littermate controls. Treated mice were injected with 0.5 mg iron fortnighly from the age of 3 months. Tissues and serum were harvested 12 hr after the final injection. n = 5 per group. *p<0.05 relative to untreated Hamp^fl/fl mice. †p < 0.05 relative to untreated Hamp^fl/fl;Myh6.Cre+. Values are shown as mean ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.19804.021

	Hampfl/fl		Hamp fl/fl;Myh6.Cre+
	untreated	treated with I.V iron	untreated	treated with I.V iron
cardiac total elemental iron (ng/mg tissue)	82.2 ± 16.9	331.3 ± 21.5*	74.9 ± 7	399.8 ± 68.5†
liver total elemental iron (ng/mg tissue)	100.4 ± 11	2527.6 ± 27.63*	96.3 ± 14	2258.2 ± 239.9†
serum iron (µmol/L)	30.09 ± 6.37	74.48 ± 17.96*	31.5 ± 6.9	80.12 ± 24.9†
serum hepcidin (µg/L)	27.41 ± 6.7	237.3 ± 16.7*	28.9 ± 9.4	209.8 ± 38.8†

Having confirmed a causal relationship between cardiomyocyte iron deficiency and cardiac dysfunction, we aimed to understand the mechanisms linking the two. Iron is a cofactor for several enzymes involved in metabolism (Meyer, 2008; Sono et al., 1996; Solomon et al., 2003), and metabolic derangement is a well-recognized precursor to cardiac dysfunction (Belke et al., 2000; Kakinuma et al., 2000; Ashrafian et al., 2007). Iron deficiency has been reported to reduce the levels and/or activities of key metabolic iron-containing enzymes, in cell lines, in the hearts of mice with impaired cardiac iron uptake (cardiac-specific TfR1 knockouts) and in the hearts of mice fed an iron-deficient diet (Dallman, 1986; Dhur et al., 1989; Xu et al., 2015; Oexle et al., 1999). Based on those studies, we postulated that iron deficiency in the cardiomyocytes of Hamp^fl/fl;Myh6.Cre+ mice would also result in reduction in the activities of key metabolic iron-containing enzymes. To test this hypothesis, we measured the activities of the iron-sulphur containing enzyme Aconitase I as well as electron transport chain (ETC) complexes in cardiac lysates from Hamp^fl/fl;Myh6.Cre+ hearts at 3 months and 6 months of age. We found that Aconitase I, Complex I and Complex IV activities were significantly reduced in Hamp^fl/fl;Myh6.Cre+ hearts compared to Hamp^fl/fl controls at 3 months and 6 months of age, and that this reduction in activity was prevented in iron-treated 6-month old mice (Figure 4F–H). As ETC activity is essential to mitochondrial function, we examined whether Hamp^fl/fl;Myh6.Cre+ hearts had signs of mitochondrial failure, and whether such mitochondrial failure was prevented by intravenous iron supplementation. By electron microscopy (EM), dilation of mitochondrial cristae was seen in Hamp^fl/fl;Myh6.Cre+ hearts as early as 3 months of age and progressed further at 6 months of age. However, this was prevented in iron-treated Hamp^fl/fl;Myh6.Cre+ mice, which had healthy-looking mitochondria at 6 months of age (Figure 4I). Similar changes in mitochondrial morphology were seen in the hearts of 3 month old Slc40a1 C326Y^fl/fl;Myh6.Cre+ mice (data not shown).

Impairment of electron transport is known to drive glycolysis, as an alternative route of ATP production (Schönekess et al., 1997; Kato et al., 2010). Therefore, we examined the expression of a number of genes encoding glycolytic enzymes. We found that expression of genes encoding Hexokinase 2 (Hk2) which catalyses the first step of glycolysis, Enolase (Eno), which catalyses the penultimate step of glycolysis and of Lactate dehydrogenase A (Ldha) which catalyses the ultimate step of glycolysis were all significantly increased at 3 months and 6 months of age in hearts of untreated Hamp^fl/fl;Myh6.Cre+ mice. This upregulation of glycolytic genes was not seen in iron-treated Hamp^fl/fl;Myh6.Cre+ mice (Figure 4J–L). These results demonstrate that reduction in the activities of key iron-containing metabolic enzymes, mitochondrial failure and upregulated glycolysis precede the development of cardiac dysfunction in Hamp^fl/fl;Myh6.Cre+ hearts, and are prevented by intravenous iron treatment.

Discussion

The major finding of this study is that cardiomyocyte hepcidin is required for autonomous cellular iron homeostasis. Loss of hepcidin responsiveness specifically in cardiomyocytes engendered the same effects as loss of cardiac hepcidin, demonstrating that cardiac hepcidin operates in an autocrine fashion by regulating cardiomyocyte FPN. A role in cellular iron homeostasis, and an autocrine mode of action for the HAMP/FPN axis have not been described previously in any other tissue. Indeed, hepcidin and FPN are better known to interact in an endocrine fashion, at the level of the gut, spleen and liver, to regulate systemic iron homeostasis.

A second important finding is that this cardiac HAMP/FPN axis is essential for normal heart function, and that its disruption leads to ultimately fatal cardiac metabolic and contractile dysfunction, even against a background of intact systemic iron homeostasis. Metabolic and contractile dysfunction are preceded by cardiomyocyte iron deficiency and prevented by intravenous iron supplementation, indicating a causal relationship between cardiomyocyte iron deficiency and cardiac dysfunction. This causal relationship has previously been demonstrated in studies using mice lacking cardiac TfR1, in which cardiac iron deficiency also affects the heart against a background of otherwise intact systemic iron homeostasis (Xu et al., 2015). Furthermore, the importance of metabolic derangement seen in our mouse models is also consistent with the findings in mice lacking the cardiacTfR1, and in mice and rats fed iron deficient diets (Dhur et al., 1989; Xu et al., 2015; Tanne et al., 1994; Walter et al., 2002). Correction of metabolic and contractile dysfunction by intravenous iron treatment likely involves not only increased iron availability for uptake into cardiomyocytes, but also the effects of increased circulating HAMP on cardiomyocyte FPN.

Surprisingly, we found that, both in vitro and in vivo, cardiac HAMP protein responded differently from its transcript to changes in iron levels. The current understanding of hepcidin regulation is based on studies of hepatic hepcidin. In that setting, release of the active mature HAMP peptide is dependent on cleavage of the propeptide by Furin (Valore and Ganz, 2008). We found that Furin inhibition increased iron export from Hamp^fl/fl but not from Hamp^fl/fl;Myh6.Cre+ cardiomyocytes (Appendix 1—figure 1), demonstrating that cardiomyocytes secrete an active HAMP peptide in a Furin-dependent manner. Furthermore, we found that increased HAMP release from iron-deficient cardiomyocytes depended on Furin, and that cardiac Furin itself is upregulated by iron deficiency both in vitro and in vivo. The latter finding is consistent with the reported regulation of Furin by iron deficiency through Hypoxia-Inducible Factors HIFs (Silvestri et al., 2008). These data suggest that differential regulation of Furin by iron may explain the divergent effects of iron on Hamp transcript and HAMP protein. Comprehensive studies using cardiac-specific knockouts of putative regulators will be required to explore formally the regulation or otherwise, of cardiac hepcidin by pathways known to regulate hepatic hepcidin.

The upregulation of cardiac HAMP in mice fed an iron-deficient diet raises the possibility that it may be involved in protecting the heart in the setting of systemic iron deficiency. This hypothesis is supported by the finding that, when provided an iron-deficient diet, Hamp^fl/fl;Myh6.Cre+ mice exhibited a greater cardiac hypertrophic response than their Hamp^fl/fl littermate controls (Appendix 1—figure 2).

Intracellular iron levels are dependent both on cellular homeostatic pathways and on systemic iron availability in plasma. Therefore, the interplay between the cardiac and the systemic HAMP/FPN axes is important in determining cardiomyocyte iron levels. Some insight into this interplay is gained from comparing systemic and cardiac mouse models of disrupted iron homeostasis. It is interesting that ubiquitous Hamp knockout (Lakhal-Littleton et al., 2015) and ubiquitous Slc40a1 C326Y knock-in mice (Appendix 1—figure 3), both models of systemic iron overload, do not develop the cardiac dysfunction seen in cardiomyocyte-specific Hamp knockout and cardiomyocyte-specific Slc40a1 C326Y knock-in mice. This suggests that, while upregulation of cardiomyocyte FPN under conditions of normal iron availability (cardiac-specific models described in this study) is detrimental to cardiac function, it is protective under conditions of increased systemic iron availability (systemic models). Previously, we also showed that deletion of cardiomyocyte FPN resulted in fatal cardiomyocyte iron overload, preventable by dietary iron restriction (Lakhal-Littleton et al., 2015). Together, our studies demonstrate that iron levels within cardiomyocytes are a balance between cellular iron efflux which is regulated by the cardiac HAMP/FPN axis, and systemic iron availability which is regulated by the systemic HAMP/FPN axis (Figure 5).10.7554/eLife.19804.022

Figure 5.

Interplay between systemic and cardiac iron HAMP/FPN axes.

Cardiomyocyte iron content is determined by both systemic iron availability, which is regulated by liver HAMP, and by the cardiac HAMP/FPN axis, which regulates cardiomyocyte iron efflux. In the wild type heart, cardiac HAMP regulates the levels of cardiac FPN and iron release from cardiomyocytes. In this study, we have demonstrated that loss of cardiac HAMP (cardiac Hamp KO) or loss of cardiac HAMP responsiveness (cardiac Slc40a1 C326Y KI) result in cardiomyocyte iron deficiency due to increased cardiomyocyte FPN and iron release. Previously, we also demonstrated that loss of cardiomyocyte FPN caused cardiomyocyte iron overload. In these two sets of conditions, cardiomyocyte iron deficiency and cardiomyocyte iron overload cause cardiac dysfunction. We have also shown that upregulation of cardiac FPN occurs as a result of loss of either systemic HAMP or systemic HAMP responsiveness, and is protective against the otherwise detrimental effects of systemic iron overload.

DOI: http://dx.doi.org/10.7554/eLife.19804.022

Iron overload is detrimental to cardiac health, as demonstrated by iron overload cardiomyopathy in hemochromatosis and thalassemia major patients (Gulati et al., 2014). Our model of cardiac iron homeostasis implies that the cardiac HAMP/FPN axis may have a modifying effect on the severity of iron-overload cardiomyopathy. Thus, it would be interesting to explore whether differences in the levels of cardiac FPN and HAMP, possibly due to different local stimulatory and suppressive signals (e.g local inflammation, local ischemia), explain the reported lack of concordance between the degrees of cardiac iron overload and liver iron overload in a significant proportion of hemochromatosis and thalassemia major patients (Anderson et al., 2001; Noetzli et al., 2008).

Iron deficiency is also detrimental to cardiac health. Indeed, systemic iron deficiency correlates with functional and molecular markers of disease severity in patients with chronic heart failure (CHF) (Klip et al., 2013; Comín-Colet et al., 2013),and also appears to contribute to the risk of death after an episode of acute heart failure (Jankowska et al., 2014). Given the high prevalence of iron deficiency in patients with CHF, ranging between 30–50% (Klip et al., 2013; Nanas et al., 2006; Ezekowitz et al., 2003), the European Society of Cardiology recently recommended the assessment of iron deficiency as a comorbidity in CHF. Furthermore, several clinical trials have now established the benefits of intravenous iron supplementation in CHF patients, with or without anaemia (Ponikowski et al., 2015; Anker et al., 2009; Avni et al., 2012). The mechanisms underlying the anaemia-independent effects of iron deficiency and the benefits of intravenous iron in CHF patients are not fully understood. In light of the direct effect of cardiomyocyte iron deficiency on heart function, demonstrated in this and other studies, it would interesting to explore whether systemic iron deficiency in CHF patients is accompanied by cardiomyocyte iron deficiency, and whether correction of the later underlies the benefits of intravenous iron supplementation in non-anaemic patients.

Another open question is whether disruption of the cardiac HAMP/FPN axis contributes to the pathophysiology of heart disease. It has been shown, in the rat model of myocardial infarction (MI), that Hamp mRNA and HAMP protein are elevated in the ischemic myocardium during the acute phase (Simonis et al., 2010). In humans, circulating HAMP was shown to be elevated in the serum within 4 hr of MI, although the tissue source of this hepcidin was not identified (Suzuki et al., 2009). In addition, decreased cardiac HAMP expression has been reported in a transgenic mouse model of dilated cardiomyopathy, where the phenotype was ameliorated following transgenic overexpression of cardiac hepcidin (Zhang et al., 2012). Thus, further studies are warranted in humans to explore formally the role of the cardiac HAMP/FPN axis in the aetiology of heart disease.

Currently, there is considerable interest in targeting the HAMP/FPN axis for the treatment of iron overload and iron deficiency. Our studies suggest that such strategies may also impinge on cardiac iron homeostasis and function. Other than the heart, both FPN and hepcidin are also found in the brain, kidney and placenta (McCarthy and Kosman, 2014; Kulaksiz, 2005; Evans et al., 2011; Rouault, 2013; Moos and Rosengren Nielsen, 2006; Bastin et al., 2006; Wolff et al., 2011). It would be important to establish whether our findings in the heart extend to these tissues.

Materials and methods

Mice

All animal procedures were compliant with and approved under the UK Home Office Animals (Scientific Procedures) Act 1986. Both males and females were used in experiments, with the respective littermate control being of a matching sex.

The strategy for generating cardiac Hamp knockout mice is outlined in Appendix 1—figure 4. Briefly, a targeting vector was designed to introduce a floxed Hamp allele into C57BL/6N mouse ES cells (JM8F6) with exons 2 and 3, which encode the majority of the peptide, flanked by LoxP sites and a line of floxed mice was generated by blastocyst injection of targeted ES cells, as previous described (Lakhal-Littleton et al., 2015). Further breeding with a C57BL/6 Flp recombinase deleter mouse allowed removal of the Neomycin resistance cassette. Cardiac Hamp knockouts were then generated by by crossing Hamp^fl/fl animals with mice transgenic for Cre recombinase, under the control of cardiomyocyte-specific Alpha Myosin Heavy Chain (Myh6) promoter (B6.FVB-Tg(Myh6-cre)2182Mds/J).The subsequent breeding strategy was designed to produce cardiac Hamp knockouts and homozygous floxed controls (Hamp^fl/fl;Myh6.Cre+ and Hamp^fl/fl respectively) in the same litter.

The strategy for generating Cardiac Slc40a1 C326Y knock-in mice is outlined in Appendix 1—figure 5, and further details are provided in the Appendix.

Cine MRI

Mice were anaesthetized with 2% isofluorane in O₂ and positioned supine in a purpose-built cradle. ECG electrodes were inserted into the forepaws, a respiration loop was taped across the chest and heart and respiration signals were monitored using a custom-built physiological motion gating device. The cradle was lowered into a vertical-bore, 11.7 T MR system with a 40 mm birdcage coil (Rapid Biomedical, Würzburg, Germany) and visualised using a Bruker console running Paravision 2.1.1. A stack of contiguous 1 mm thick true short-axis ECG and respiration-gated cine-FLASH images were acquired. The entire in vivo imaging protocol was performed in approximately 60 min. Image analysis was performed using ImageJ (NIH Image, Bethesda, MD). Left ventricular volumes and ejection fractions were calculated from the stack of cine images as described (Lakhal-Littleton et al., 2015).

Dietary iron content

Unless otherwise stated, animals were provided with a standard rodent chow diet containing 200 ppm iron. In iron manipulation experiments, mice were given an iron-deficient diet (2–5 ppm iron; Teklad TD.99397; Harlan Laboratories), or an iron-loaded diet (5000 ppm iron; Teklad TD.140464) or a matched control diet (200 ppm iron; Teklad TD.08713) from weaning for six weeks.

Isolation of primary adult mouse cardiomyocytes and in vitro treatment

Adult primary cardiomyocytes were isolated from eight week old C57BL/6 mice. Hearts were cannulated and mounted on a langendorff apparatus, then perfused using a liberase solution for 10 min. After filtration through a 400 µm gauze, cells were cultured in MEM medium containing Hanks salts, L-glutamine and antibiotics. Within 2 hr of cardiomyocyte culture, supernatants were replaced with fresh medium containing 10% Fetal calf serum, with 0.5 mmol/L ferric citrate (FAC) (F3388, Sigma Aldrich) or 100 µmol/L desferroxamine (D9533, Sigma Aldrich) for 8 hr. The Furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) (N1505, Bachem) was added at a concentration of 50 µmol/L for the duration of DFO and FAC treatment.

Quantitative PCR

Total RNA extraction and cDNA synthesis were carried out as previously described (Lakhal-Littleton et al., 2015). Gene expression was measured using Applied Biosystems Taqman gene expression assay probes for Slc40a1, Hamp, TfR1, Myh7, Nppb, Ldha, Hk2, Eno and house-keeping gene β-Actin (Life Technologies, Carlsbad, CA). The CT value for the gene of interest was first normalised by deducting CT value for β-Actin to obtain a delta CT value. Delta CT values of test samples were further normalised to the average of the delta CT values for control samples to obtain delta delta CT values. Relative gene expression levels were then calculated as 2^{-delta deltaCT}.

Immunohistochemistry

Tissues were prepared as described previously (Lakhal-Littleton et al., 2015) and stained with rabbit polyclonal anti-mouse HAMP antibody (ab30760, Abcam, RRID:AB_2115844) at 1/40 dilution, or rabbit polyclonal anti-mouse FPN antibody (MTP11-A, Alpha Diagnostics, RRID:AB_1619475) at 1/200 dilution. Results of control experiments confirming the specificity of the HAMP antibody are shown in Figure 1—figure supplement 5.

HAMP enzyme-linked immunosorbent assay (ELISA)

HAMP was measured in mouse sera and in cardiomyocyte supernatants using a HAMP ELISA kit (E91979Mu, USCN) according to the manufacturer’s instructions. Results of control experiments confirming that in vitro treatments did not affect HAMP peptide detection by this ELISA kit are shown in Figure 1—figure supplement 6.

DAB-enhanced perls stain

Formalin-fixed paraffin-embedded tissue sections were deparaffinised using Xylene, then rehydrated in ethanol. Slides were then stained for 1 hr with 1% potassium ferricyanide in 0.1 mol/L HCl buffer. Endogenous peroxidase activity was quenched, then slides were stained with DAB chromogen substrate and counterstained with haematoxylin. They were visualised using a standard brightfield microscope.

Electron microscopy (EM)

Hearts were dissected and 0.5–1 mm³ slices were fixed by immersion for 2 hr in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer and prepared for electron microscopy by standard methods. Briefly, cells were post-fixed in osmium tetroxide (1% w/v in 0.1 mol/L phosphate buffer), stained with uranyl acetate (2% w/v in distilled water), dehydrated through increasing concentrations of ethanol (70–100%) and acetone and embedded in TAAB resin (TAAB, Aldermaston, UK). Ultrathin sections (50–80 nm) were prepared using a Reichert ultracut S microtome and mounted on 200 mesh nickel grids. Sections were lightly counterstained with lead citrate and uranyl acetate and examined with a Jeol transmission electron microscope (JEM-1010, JEOL, Peabody MA).

Fe55 efflux in primary adult cardiomyocytes

Adult cardiomyocytes were isolated from mice of the desired genotype at 9 weeks of age as described above. Cardiomyocytes were then cultured in 24-well plates at equal densities for 16 hr before the efflux experiment was performed as described (McKie et al., 2000). Briefly, after washing with three times PBS, cells were incubated for 30 min in 200 µl uptake solution (98 mmol/L NaCl, 2.0 mmol/L KCl, 0.6 mmol/L CaCl2, 1.0 mmol/L MgCl2, 1.0 mmol/L ascorbic acid, 10 mmol/L HEPES [pH 6.0] with Tris base, 50 μmol/L Fe55 [NEN, Boston, MA]), then washed three times with PBS and incubated for 30 min with efflux solution (98 mmol/L NaCl, 2.0 mmol/L KCl, 0.6 mmol/L CaCl2, 1.0 mmol/L MgCl2, 10 mmol/L HEPES [pH 7.4] with Tris base, 300 U/ml bovine ceruloplasmin (cp) (Sigma) and 40 μg/ml human apotransferrin (tf) [Sigma]),in the absence or presence of 0.5 µmol/L mouse HAMP peptide (Peptides International). The efflux medium was then removed, the cells washed three times in ice-cold PBS and disrupted by incubation in 100 µl of 10% SDS solution for 10 min.The efflux solution and cell lysates were then transferred into scintillation vials for Fe55 counting. Where Furin inhibition was carried out, CMK was added to the culture medium at 50 µmol/L 2 hr before the efflux experiment was carried out.

Iron quantitation

Ferritin concentration in serum and in liver lysates was determined using the ferritin ELISA kit (ICL, Inc. Portland). Serum iron levels were determined using the ABX-Pentra system (Horiba Medical, CA). Determination of total elemental iron in the heart was carried out by inductively coupled plasma mass spectrometry (ICP-MS) as described previously (Lakhal-Littleton et al., 2015). Calibration was achieved using the process of standard additions, where spikes of 0 ng/g, 0,5 ng/g, 1 ng/g, 10 ng/g, 20 ng/g and 100 ng/g iron were added to replicates of a selected sample. An external iron standard (High Purity Standards ICP-MS-68-A solution) was diluted and measured to confirm the validity of the calibration. Rhodium was also spiked onto each blank, standard and sample as an internal standard at a concentration of 1 ng/g. Concentrations from ICP-MS were normalised to starting tissue weight.

Isolation of cardiomyocyte and non-cardiomyocyte fractions for iron quantitation

Following cardiac perfusion, hearts were dissected into small pieces in ice-cold Hanks buffer, and subject to collagenase P digestion at 37C for 1 hr (11213857001, Roche Diagnostics). Following lysis of red blood cells, cell suspensions were passed through a 70 µm sieve, before being labelled using cardiomyocyte isolation kit (130-100-825, Miltenyi Biotec). Separation of cardiomyocyte and non-cardiomyocyte fractions was carried out using MACS magnetic separation system according to the manufacturer’s instructions. Cardiac fractions were lysed immediately for ICP-MS analysis.

Activity assays for aconitase I and electron transport chain (ETC) complexes

Approximately 10 mg of frozen, crushed tissues were suspended in 200 µl of ice-cold KME buffer (100 mmol/L KCl, 50 mmol/L Mops, 0.5 mmol/L EGTA, pH 7.4), then homogenized by rupturing with a TissueRuptor (Qiagen, UK) over ice. In a plastic cuvette, the cardiac lysate is mixed with assay buffer and slotted into a spectrophotometer. Details of assay buffers and of reaction procedure for each enzyme are detailed in the Appendix.

Statistics

Values are shown as mean ± standard error of the mean (SEM). Comparison of iron indices, enzyme activities and parameters of cardiac function between groups was performed using Student’s T test. p values < 0.05 were deemed as indicating significant differences between groups. Where significant, exact p values for a figure panel are stated in the corresponding figure legend. No explicit power analysis was performed prior to the experiments to determine sample size, since we had no means to reliably estimate the size and variability of the effects of deleting hepcidin on parameters of cardiac function. For Cine MRI assessment of cardiac function, typically 5–11 animals of each genotype were used, with a matching number of littermate controls. For gene expression, iron quantitation and enzyme activity assays from mouse tissues, typically, 3–6 independent biological replicates and matching littermate controls were analysed. Since significant results were obtained from these set of experiments, no further animals were sacrificed. All 'n' values reported refer to independent biological replicates.