Male Hjv^−/− (HJVKO) mice (kindly provided by Dr. Nancy C. Andrews, Duke University) were bred in-house at the University of Alberta Health Sciences Laboratory Animal Services facility. All experiments were performed in accordance with the University of Alberta institutional guidelines, which conformed to guidelines published by the Canadian Council on Animal Care and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (revised 2011). We performed advanced iron overload protocol by feeding 4 weeks old HJVKO mice with the high iron diet (28) (Prolab®RHM 3000 with iron 380 ppm) until they were 1 year old. At 9 months, the amlodipine group received amlodipine in drinking water (95 mg/L) for 3 months. The vehicle group comprised male HJVKO mice receiving a regular diet without treatment.

This patient was consented with written informed consent as part of the Heart Function and Cardiomyopathy Clinic registry, which is approved by the Ethics Committee at the University of Alberta (Pro00077124). Data for this patient's case was obtained through an electronic chart review.

The study was approved by the Ethics Committee at the University of Alberta, and all patients provided written informed consents in accordance with the Declaration of Helsinki (2008) of the World Medical Association. Left ventricular tissue was harvested from explanted human failing hearts and donor non-failing human hearts via HELP (Human Explanted Heart Program) at the Mazankowski Alberta Heart Institute and the HOPE (Human Organ Procurement and Exchange) program at the University of Alberta Hospital. The harvested hearts were preserved in cold cardioplegia solution, and collected samples were snap-frozen in liquid nitrogen within 15 min of explantation and stored at −80°C.

Samples (20 mg) of frozen tissue from LV were subjected to inductively coupled plasma resonance mass spectrometry to quantify iron levels at the Trace Metals Laboratory, London, Western Ontario. The samples were analyzed in triplicates, and the average values were used (15, 26).

Images were obtained on Hitachi S5500 SEM. Energy-dispersive X-ray spectra were acquired using Oxford INCA Energy in STEM mode with a 1.5 nm probe.

Transthoracic echocardiography was performed at 1 year of iron overload phenotype mice with the Vevo 2100 high-resolution imaging system equipped with a 30-MHz transducer using 1.5%–2% isoflurane (26, 29).

Mice were anesthetized with 1.5% isoflurane, and the right common carotid artery was cannulated. 1.4F Scisense catheter (Scisense Inc.) was advanced through the aortic valve and placed into the LV chamber. Pressure-volume loops were recorded via TCP-500 amplifier (Scisense Inc.) and analyzed offline using LabScribe 2 software (IWorks Inc.) as described previously (26, 29).

Myocytes were enzymatically isolated as described previously (30) without blebbistatin to preserve contractility. After isolation, myocytes were kept in the perfusion buffer solution (pH 7.4). An aliquot of isolated cardiomyocytes was transferred to a bath atop of an inverted microscope to measure excitability, contractility, or Ca²⁺ transients (loaded with FURA-2AM). The measurements were done in myocytes superfused with Tyrode's solution (1.2 mM Ca²⁺) at 35–36°C and paced with field stimulation at 1 Hz as previously described (26, 30, 31). Action potentials were recorded in the whole-cell ruptured-patch configuration with K⁺ pipette solution (31).

Mice were placed under isoflurane anesthesia (1.5%–2%) on a heated pad (body temperature maintained at 37°C, measured by the rectal probe). ECG leads were placed in Lead I configuration. The signal was digitized using acquisition interface ACQ-7700 (Data Science International, USA) with P3 Plus software (ver. 5.0, Data Science International, USA).

The excised hearts from anesthetized mice were arrested in diastole using saline with 15 mM KCl, fixed in 10% buffered formalin, and embedded in paraffin. Thin sections (5 µm) were stained with Prussian blue, picro-sirius red (PSR), Masson trichrome, or wheat germ agglutinin (WGA) stain as described previously (26, 32). Iron depositions were visualized as blue depositions using a bright field view. Myocardial fibrosis was evaluated by quantifying PSR stained sections, and myocyte cross-sectional area (MCSA) was assessed from WGA staining using an Olympus IX81 microscope. Image analysis was done using MetaMorph software (26, 32).

Immunofluorescence was performed on formalin-fixed paraffin-embedded heart sections (5 µm). Briefly, the sections were deparaffinized, followed by antigen retrieval and blocked with blocking buffer (1% BSA in 1X PBS) for 1 h. Similarly, OCT-embedded sections were fixed with 4% paraformaldehyde for 20 min and rehydrated in PBS for 30 min. Then, sections were incubated overnight in a humidified chamber at 4°C with primary antibody against rat anti-mouse neutrophil (Serotec), rat anti-mouse F4/80 (Serotec), mouse anti-nitrotyrosine (Santa Cruz), mouse anti-4-HNE (Abcam). Finally, sections were incubated with different fluorophore-conjugated secondary antibodies (Invitrogen USA), as described previously (26, 32).

The levels of malondialdehyde (MDA), an indicator of lipid peroxidation, were measured in myocardial tissue (100–150 mg) by using a commercially available kit (Bioxytech, MDA-586TM assay, Oxis International Inc., Foster City, CA). Myocardial reduced (GSH) and oxidized glutathione (GSSG) levels were measured as described previously (26, 33).

mRNA expression levels were evaluated using TaqMan real-time PCR. Total RNA was extracted from flash-frozen LV tissue using the TRIzol RNA extraction method. RNA (1 µg) was subjected to reverse transcription to synthesize cDNA. Samples were loaded in triplicate, and the data were analyzed by Roche's Light cycler® 480 system.

Transcriptome sequencing, including sample preparation, library construction, and Illumina sequencing, were carried out by Novogene Corporation Inc. (California, USA). The reported methods below were modified based on the standard procedures provided by Novogene. Total RNAs from left ventricles (4 hearts/group) were extracted. The pathway analysis was performed using iDEP.94 http://bioinformatics.sdstate.edu/idep94/ (34).

Western blot analysis was performed on flash-frozen LV tissue samples as previously described (26, 32). Briefly, we extracted protein from LV tissues and performed immunoblotting for various proteins using the following primary antibodies: SERCA2a, NCX1 (Thermo Fisher Scientific), PLN-P^Ser16, total PLN (Badrilla Ltd), Akt-P^Ser473, Akt-P^Thr308, total Akt (Cell Signaling), AMPK-P^Thr172 and total AMPK (Cell Signaling) and subsequently incubated with HRP conjugated secondary antibodies respectively.

All data are presented as mean ± SEM. The explanted heart was assessed using the one-sample t-test. In the case of multiple group comparisons, differences were evaluated by one-way ANOVA followed by Tukey's post-test. Statistical analysis was performed using Origin 2020 software (OriginLab).

HJVKO mice were fed a high-iron diet to achieve iron overload as mice aged to 12 months. In the rescue group, amlodipine was introduced at 9 months (Figure 1A). Prussian blue histological staining showed a marked increase in iron deposition in groups receiving the high-iron diet (Figure 1B). Direct measurement of iron levels with inductively-coupled plasma mass spectrometry showed that the iron-fed group had markedly elevated iron levels and the amlodipine group had significantly reduced iron levels (Figure 1C). At the cellular level, iron deposition occurred as sub-micrometer particles (Figure 1D). The composition of the deposits was confirmed using energy-dispersive X-ray spectra, which showed a characteristic Fe peak at 6.4 keV (Figure 1D). In contrast to the heart, liver iron levels were increased at 12 months in both iron-fed groups (placebo and amlodipine; see Figures 1E, F). These results demonstrate that amlodipine reduces the iron load in the heart but not in the liver.

Iron accumulation led to elevated expression levels of heart failure markers (natriuretic peptides, ANF and BNP, and myosin heavy chain β, β-MHC) in the iron group. In contrast, the amlodipine-treated group exhibited overall normalization of expression of heart failure markers (Figure 3A). In addition, the morphometric index of hypertrophy, LV mass adjusted by tibia length (LV/TL), was increased in the iron group but normalized in the amlodipine-treated group treated with amlodipine (Figure 3B). At the cellular level, measurement of length (L) and width (W) of isolated cardiomyocytes demonstrated that myocytes were elongated (increased L:W ratio) in the iron group and had an L:W ratio close to normal in the amlodipine-treated group (Figure 3C). Assessment of hypertrophy at the tissue level using WGA staining (Figure 3D) revealed increased MCSA in the iron group and substantially reduced myocyte area in the amlodipine-treated group. The data suggest that iron overload leads to eccentric-hypertrophy remodeling, and amlodipine treatment reversed a significant part of hypertrophic remodeling.

Cardiac fibrosis was assessed using Mason's trichrome (Figure 3E) and PSR (Figure 3F) staining. The quantification of PSR staining (Figure 3G) showed a marked increase in interstitial fibrosis in iron-overloaded hearts and a considerable reduction of fibrosis in the hearts from the amlodipine-treated group. Expression levels of pro-fibrotic markers (pro-collagen Iα1 and IIIα1) followed a similar pattern: upregulation in the iron overload group and normalization in the amlodipine-treated group (Figure 3H).

The state of the intrinsic antioxidant system was assessed by measuring the concentration of GSH and GSSG. Consistent with a heightened degree of oxidative stress, the iron group had low GSH and high GSSG concentrations (Figure 6A); on the other hand, the amlodipine-treated group had a close-to-normal pattern of GSH-GSSG distribution (high GSH with low GSSG) (Figure 6A). MDA was elevated in the iron group but largely normalized levels in the amlodipine-treated group, corroborating GSH-GSSG results that oxidative stress was elevated in the iron group and was at near-normal levels in the amlodipine-treated hearts (Figure 6A). Assessment of metabolic signaling showed that iron overload was associated with (i) decreased Akt phosphorylation at both Thr308 (Akt-T308) and Ser473 (Akt-S473) and (ii) increased AMPK phosphorylation (Thr172) (Figure 6B). In contrast, the amlodipine-treated group had near-normal phosphorylation levels at all three sites (Figure 6B), suggesting that the amlodipine treatment normalizes pathological metabolic signaling in cardiac tissue.

Iron overload alters the expression of multiple genes involved in cardiac muscle contraction and the citrate cycle. We used bulk RNA sequencing to assess broad changes in expression levels (Data Supplement and Supplementary Figure S1) and found two important pathways being affected: cardiac muscle contraction and citrate cycle. In both pathways, iron overload led to an overall reduction in the expression levels (cardiac muscle contraction pathway Figure 7A and citrate cycle Figure 7B). Amlodipine therapy partially normalized the changes in myocardial gene expression in response to iron overload (Figures 7A, B).

A 16-year-old girl with β-thalassemia major was admitted with refractory heart failure (HF). Echocardiogram showed dilation with bi-ventricular systolic and diastolic dysfunction with LVEF of 25%. The pre-transplant iron panel showed elevated serum iron (31 µmol/L; normal: 9.0–30 µmol/L), severely high transferrin iron saturation (94%; normal: 25%–35%) and low hemoglobin (94 g/L; normal: 120–160 g/L). Pre-transplant cardiac MRI indicated severe iron overload. With proper consent, samples were collected from the explanted heart for further comparison to non-failing control heart samples obtained from HOPE.

Prussian blue staining showed widespread myocardial iron deposition in the IOC heart but no detectable staining in the non-failing control hearts (Figure 8A). The IOC heart had marked fibrosis, as evident from trichrome staining (Figure 8B) and PSR staining (Figure 8C), in association with marked hypertrophy judging by the increase in MCSA (Figure 8D). Scanning electron microscopy revealed micron-sized iron deposits in the IOC heart but not in non-failing control (Figure 8E), with spectral analysis showing a marked increase of characteristic Fe peak at 6.4 keV (Figure 8F). Assessment of key proteins involved in Ca²⁺ cycling showed a reduction in the levels of SERCA2 and a compensatory increase in the levels of NCX1 (Figure 8G). Iron overload was also associated with metabolic remodeling, as evidenced by increased phosphorylation ratios of Akt^Thr308 and AMPK (Figure 8H). Assessment of oxidative stress status showed a marked increase in MDA (Figure 8I), reduction in GSH, increase in GSSG, and lower GSH/GSSG ratio (Figure 8J). In this clinical case, IOC exhibited iron deposition (including intracellular micrometer-size particles), fibrosis, hypertrophy, remodeling of Ca²⁺ cycling, metabolic remodeling, and a heightened degree of oxidative stress compared to non-failing control hearts. One-year post-cardiac transplant, this patient is on chelators (deferiprone and deferoxamine) and L-type Ca²⁺ channel blocker (amlodipine), has no signs of heart failure, and is followed up regularly. Cardiac MRI showed no evidence of myocardial (T2* = 45.5 ms) or hepatic (T2* = 17.5 ms) iron overload and normal bi-ventricular size and function, with LVEF of 66% and normal right ventricular ejection fraction of 63%.

A 69-year-old man with a history of primary hemochromatosis (PH) (C282Y/H63D compound heterozygote) and myelodysplastic syndrome (MDS) presented with IOC leading to HF and conduction abnormalities. He reported fatigue, shortness of breath, paroxysmal nocturnal dyspnea, and bilateral leg swelling [New York Heart Association (NYHA) Class III]. Blood work demonstrated anemia (hemoglobin = 87 g/L), elevated serum ferritin (1,230 µg/L), and elevated B-type natriuretic peptide (1,224 ng/L). Electrocardiogram showed abnormal conduction delays with underlying atrial flutter (Figure 9A). Cardiac magnetic resonance imaging (CMR) demonstrated dilated left ventricle with moderately reduced systolic function (LVEF = 40%). Cardiac T2 mapping showed evidence of moderate myocardial iron loading (T2* 12–13 ms) and hepatic iron overload (T2* 2 ms) (Figure 9B). Medical therapies were initiated to manage this patient's IOC and subsequent HF. Given this patient's concurrent MDS and PH, therapeutic phlebotomy was not feasible. Oral iron chelation therapy was undertaken with increasing doses of deferasirox; however, iron saturation remained elevated at 70%. Despite improvements, this patient still expressed marked limitations in physical activities (NYHA class III). He was started on amlodipine 2.5 mg orally daily in addition to iron chelation and was titrated to the maximum dose of 10 mg daily. During follow-up 2 years after adding amlodipine, the patient reported marked improvement in HF symptoms (NYHA class I). Comparing initial CMR imaging to 24 months demonstrated improvement in LVEF and RVEF from 40% to 66% and 41% to 64%, respectively, with normalized ventricular volumes and mass. Myocardial T2* improved from 12.5 ms to 30 ms, indicating a marked reduction in myocardial iron loading (Figure 9C).

Hemojuvelin-deficient mice on an iron-rich diet are an established IOC model that exhibits both systolic and diastolic dysfunction (26, 27). Iron levels obtained in the hearts of the animal model (2.7 mg/g dry weight) were approaching myocardial iron levels (3.5–9.2 mg/g dry weight) reported in patients with IOC and associated heart failure. The presented animal model replicated many crucial changes that develop during IOC in patients. We observed deterioration of cardiac function and micron-sized iron particles in cardiac tissue both in the explanted human heart with IOC and in the mouse model. In the case of the explanted heart, the heart failure was very severe (LVEF = 25%), whereas mice on the iron-rich diet had moderate cardiomyopathy (LVEF = 48%). Despite these few differences, both the explanted heart and the mouse model had fibrosis, hypertrophy, remodeling of Ca²⁺ cycling (decrease in SERCa2 levels accompanied by a compensatory increase in NCX levels), metabolic remodeling (inhibition of Akt activity coupled with upregulation of AMPK activity), and oxidative stress (increased MDA levels and low GSH/GSSG ratio). Accumulation of Fe²⁺ inside the cardiac cells is known to lead to the excessive production of ROS and trigger cell death due to ferroptosis (42), resulting in heart failure (35). In this animal model of IOC, we observed no changes in cardiac excitability but impaired cardiac cellular contractility and relaxation. The diminished contractility was associated with the reduction in Ca²⁺ release suggesting that changes in Ca²⁺ cycling underly reduced cardiac contractility.

As an L-type Ca²⁺ channel blocker, amlodipine can reduce the entry of non-transferrin-bound iron (Fe²⁺) via L-type Ca²⁺ channels, resulting in lower levels of cardiac iron and reduced oxidative stress in cardiac tissue. Mitigated damage from oxidative stress will contribute to the normalization of expression of Ca²⁺ cycling proteins and collagens, leading to the reversion of fibrosis and normalization of Ca²⁺ cycling, which translates to the normalization of systolic and diastolic dysfunctions at the whole-heart level. Besides being a channel blocker, amlodipine has been shown to have highly efficient antioxidant properties due to high lipophilicity and the ability to quench free radical formation (21). Antioxidants have been shown to mitigate damage associated with iron toxicity. For example, resveratrol (26, 27), thrombopoetin (43), and ebselen (44) have been shown to protect the heart in the settings of iron overload in mouse models. In our study, amlodipine demonstrated properties that can also be attributed to antioxidant activity (normalization of GSH, GSSG, and MDA levels).

In the reported case of primary hemochromatosis and IOC, the myelodysplastic syndrome prevented therapeutic phlebotomy. Iron chelation alone (deferasirox) was insufficient to reduce the burden of cardiac iron overload. However, adjunctive amlodipine therapy provided objective improvement in cardiac iron overload on imaging and symptomatic improvement in functional status for this patient. Despite randomized clinical trials demonstrating that amlodipine did not exhibit favorable clinical effects for the treatment of chronic heart failure (45, 46), amlodipine therapy could be used as adjunctive therapy in cases of iron overload, as seen in our patient's case and recent randomized clinical trials of amlodipine in patients with iron overload (17, 19). Therefore, any beneficial action from reducing iron deposition in the heart may also be reinforced by the antioxidant properties of amlodipine, suggesting that amlodipine is a good option for an adjuvant agent in chelation therapy.