JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 ª 2021 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER THE CC BY-NC-ND LICENSE (http://creativecommons.org/licenses/by-nc-nd/4.0/). CLINICAL RESEARCH Implications of Iron Deficiency in STEMI Patients and in a Murine Model of Myocardial Infarction Javier Inserte, PHD,a,b,c José A. Barrabés, MD, PHD,a,b,c David Aluja, BSC,a,b,c Imanol Otaegui, MD,a,b,c Jordi Bañeras, MD, PHD,a,b,c Laura Castellote, MD,d Ana Sánchez, BSC,a,b,c José F. Rodríguez-Palomares, MD, PHD,a,b,c Víctor Pineda, MD, PHD,e Elisabet Miró-Casas, BSC,a,b,c Laia Milà, MD,a,b,c Rosa-Maria Lidón, MD,a,b,c Antonia Sambola, MD, PHD,a,b,c Filipa Valente, MD,a,b,c Agnès Rafecas, MD,a,b,c Marisol Ruiz-Meana, PHD,a,b,c Antonio Rodríguez-Sinovas, PHD,a,b,c Begoña Benito, MD, PHD,a,b,c Irene Buera, MD,a,b,c Sara Delgado-Tomás, BSC,a,b,c David Beneítez, MD,f Ignacio Ferreira-González, MD, PHDa,b,c VISUAL ABSTRACT HIGHLIGHTS  In patients with STEMI treated with primary percutaneous coronary intervention, iron deficiency is associated with larger infarcts, more extensive microvascular obstruction, and a higher frequency of adverse left ventricular remodeling.  An iron-deficient diet reduces the toler- ance to ischemia/reperfusion in mice at least in part by interfering with the car- dioprotective pathway eNOS/soluble guanylate cyclase/protein kinase G.  An iron-deficient diet reduces eNOS ac- tivity by increasing oxidative/nitrosative stress and its proteasome-dependent degradation.  Not only iron excess but also iron Inserte, J. et al. J Am Coll Cardiol Basic Trans Science. 2021;6(7):567–80. deficiency may have deleterious effects in the context of acute myocardial ischemia. From the aDepartment of Cardiology, Vall d’Hebron Hospital Universitari and Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain; bUniversitat Autònoma de Barcelona, Bellaterra, Spain; cCentro de Investigación en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain; dDepartment of Biochemistry, Vall d’Hebron Hospital Universitari, Barcelona, Spain; eInstitut de Diagnòstic per la Imatge, Barcelona, Spain; and the fDepartment of Hematology, Vall d’Hebron Hospital Universitari and Vall d’Hebron Institut d’Oncologia (VHIO), Barcelona, Spain. The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the Author Center. Manuscript received March 9, 2021; revised manuscript received May 10, 2021, accepted May 10, 2021. ISSN 2452-302X https://doi.org/10.1016/j.jacbts.2021.05.004 568 Inserte et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Impact of Iron Deficiency in Myocardial Infarction JULY 2021:567–580 ABBREVIATIONS SUMMARY AND ACRONYMS CK-MB = creatine kinase- In patients with a first anterior ST-segment elevation myocardial infarction treated with primary percutaneous myocardial band coronary intervention, iron deficiency (ID) was associated with larger infarcts, more extensive microvascular CMR = cardiac magnetic obstruction, and higher frequency of adverse left ventricular remodeling as assessed by cardiac magnetic resonance resonance imaging. In mice, an ID diet reduced the activity of the endothelial nitric oxide synthase/ eNOS = endothelial nitric soluble guanylate cyclase/protein kinase G pathway in association with oxidative/nitrosative stress oxide synthase and increased infarct size after transient coronary occlusion. Iron supplementation or administration HSP90 = heat-shock protein 90 of an sGC activator before ischemia prevented the effects of the ID diet in mice. Not only iron excess, but also ID, may have deleterious effects in the setting of ischemia and reperfusion. ID = iron deficiency (J Am Coll Cardiol Basic Trans Science 2021;6:567–80) © 2021 The Authors. Published by Elsevier on behalf iNOS = inducible nitric oxide synthase of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND LV = left ventricular license (http://creativecommons.org/licenses/by-nc-nd/4.0/). MVO = microvascular I obstruction PKG = protein kinase G ron is essential for normal cardiovascu- and post-infarction left ventricular (LV) remodeling, sGC = soluble guanylyl cyclase lar function. In patients with chronic because of its effect on infarct size (21,22), directly STEMI = ST-segment elevation heart failure, iron deficiency (ID) is com- through the aforementioned mechanisms (23) or by acute myocardial infarction mon irrespective of anemia, it portends a enhancing inflammation (24,25). However, the STIR = short tau inversion worse prognosis, and its correction improves implications of ID in acute myocardial infarction recovery symptoms and quality of life (1,2). The signif- remain unclear, and the involvement of the eNOS/ VASP = vasodilator-stimulated icance of ID in acute coronary syndromes has sGC/PKG pathway in this setting is unknown. phosphoprotein been less investigated. Recent studies re- Accordingly, we aimed to assess whether ID is ported a high prevalence of ID in this setting but associated with infarct size and with adverse LV were inconsistent regarding its association with out- remodeling in patients with ST-segment elevation comes (3–6). acute myocardial infarction (STEMI), whether it Both iron excess and ID may negatively influence modifies myocardial tolerance to ischemia/reperfu- cardiomyocyte survival after transient ischemia. sion injury by altering the activity of the eNOS/sGC/ Because of its ability to redox cycle, iron excess pro- PKG pathway in mice, and whether in this latter motes oxidative stress via Fenton-type reactions (7), model its effects are antagonized by iron supple- and iron chelation has been shown to reduce experi- mentation or sGC activation. mental ischemia/reperfusion injury (8,9). Conversely, METHODS ID may affect the activity of essential iron-based en- zymes, leading to reactive oxygen species production The protocol was approved by the Ethics Committees (10). Enhanced myocardial oxidative/nitrosative on Clinical Research (JAB-HIE-2017-01) and Animal stress has been found in animals fed with low-iron Experimentation (CEEA-13.17) of Hospital Vall d’He- diets, an effect attributed to increased superoxide bron. All patients gave written informed consent to production caused by mitochondrial dysfunction and participate. The experimental procedures conformed inducible nitric oxide synthase (iNOS) expression (11) to the EU directive 2010/63EU. or to reduced antioxidant activity (12). ID-induced oxidative stress may affect the activity PATIENTS. The study cohort consisted of 141 patients of signaling pathways involved in cardiomyocyte admitted to our acute cardiac care unit between survival after reperfusion (13,14). The endothelial October 2013 and May 2019 with a first anterior STEMI nitric oxide synthase (eNOS)/soluble guanylyl cyclase treated with primary percutaneous coronary inter- (sGC)/protein kinase G (PKG) axis influences cell fate vention. Anterior STEMI was diagnosed by the pres- after restoration of flow, and increasing cGMP ence of prolonged anginal symptoms associated with synthesis or reducing its degradation attenuates car- ST-segment elevation $0.2 mV in $2 anterior leads diomyocyte death in this setting (15,16). However, and with a significant elevation of cardiac bio- oxidative stress dramatically affects cGMP production markers. Patients were not included if they had a (17–19). Because eNOS and sGC are iron-based history of previous infarction, significant valvular or enzymes, ID might also directly affect their activity myocardial disease, persistent clinical instability, (20). Finally, ID could influence myocardial healing persistent arrhythmia, or contraindications for JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Inserte et al. 569 JULY 2021:567–580 Impact of Iron Deficiency in Myocardial Infarction cardiac magnetic resonance (CMR). Sixteen patients F I G U R E 1 Patient Flowchart were excluded for different reasons, and 125 were finally included, of whom 109 underwent a second CMR at 6 months (Figure 1). Patients’ management was in accordance with practice guidelines. CLINICAL AND LABORATORY VARIABLES. Demographic data, cardiovascular risk factors, comorbidities, hos- pitalization data, and pharmacologic therapy were prospectively collected. Anterograde flow in the culprit vessel was characterized using the TIMI (Thrombolysis In Myocardial Infarction) scale. The extent of coronary artery disease was assessed as the number of vessels with lesions $50%. Revasculari- zation of significant distant lesions was often per- formed in a staged procedure. Blood levels of creatine kinase-myocardial band (CK-MB) were determined upon admission, at 4- to 6-hour intervals during the first 24 hours, and at 12-hour intervals between 24-48 hours after admission. Troponin values were CMR ¼ cardiac magnetic resonance. disregarded because different assays were used throughout the study period. The morning after admission, a fasting blood starting 10-15 min after contrast administration (ma- sample was obtained for standard hematological and trix 256  197; voxel size 2.0  1.6  6 mm; TE 4.91 ms; biochemical determinations. An additional sample of TR 700 ms; flip angle 30 ; bandwidth 140 Hz/pixel). 10 mL of blood was collected, and serum was ob- Studies were analyzed by an experienced operator tained by centrifugation and stored at 80  C. Free unaware of laboratory results. LV end-diastolic and iron, ferritin, transferrin, and transferrin saturation end-systolic volumes and ejection fraction were were measured in the stored samples. ID was defined calculated. In the initial study, myocardial edema was as ferritin level <100 m g/L or 100-299 mg/L if trans- quantified in STIR sequences delineating the areas of ferrin saturation was <20% (1,2). hyperintensity with 2 SD above average, obtained from CMR IMAGING. Studies were performed with 1.5-T the remote healthy myocardium and normalized by LV equipment (Siemens Sonata or Avanto), as described mass. Intramyocardial hemorrhage was identified as (22,26). Electrocardiogram-gated breath-hold short- the hypointense core within the hyperintense regions axis cine views were performed to quantify volumes in STIR images, and its mass was calculated as pub- and ejection fraction (steady-state free procession se- lished elsewhere (21). Infarct size was quantified by quences; slice thickness 6 mm; space between slices delineating the enhanced areas in the late gadolinium 67%; matrix 256  256; field of view 300 mm-370 mm; enhancement sequences with 5 SD above average, temporal resolution <50 ms). Additional 2-chamber, 3- obtained from the remote healthy myocardium and chamber, and 4-chamber views were obtained. Short normalized by LV mass. Microvascular obstruction tau inversion recovery (STIR) sequences were used in (MVO) was defined by the presence of hypoenhanced the same view as the cine sequences, in mid-diastole, zones inside hyperenhanced areas. Adverse LV to evaluate edema and hemorrhage (slice thickness remodeling was defined as an increase in LV end- 8 mm; space between slices 20%; matrix 256  256; diastolic volume $20% at 6 months with respect to field of view 300-370 mm; temporal resolution <50 ms; baseline (26). repetition time 2 R-R intervals; echo time 100 ms; MOUSE MODEL OF ID AND MONITORING. Male  inversion time 170 ms; flip angle 160 ; bandwidth C57BL/6N mice (Charles River Laboratories) aged 781 Hz/pixel). Late gadolinium enhancement images 6 weeks were kept on a control diet (control group; were acquired at identical slice positions to the cine D10012M, Research Diets Inc), or after 250- to 300-m L images after administration of 0.2 mmol/kg blood withdrawal, on an ID diet (ID group; 3 mg/kg gadolinium-DTPA (Berlex). A segmented inversion- Fe; D03062702, Research Diets Inc) for 4 weeks, as recovery gradient-echo sequence was acquired previously described (27). To determine the effect of 570 Inserte et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Impact of Iron Deficiency in Myocardial Infarction JULY 2021:567–580 subsequent iron repletion, an additional group fed Changes in total and dimeric eNOS protein levels with ID diet received 2 mg/kg intravenous iron hy- were also evaluated in isolated cardiomyocytes droxide in sucrose (Vifor Pharma) once a week (IDþFe obtained as previously described (29) and incubated group). On week 4, plasma hemoglobin concentration with the iron chelator deferasirox (Selleckchem) at was determined, and ferritin, transferrin, and iron 200 m mol/L for 4 hours in Hanks’ balanced salt solu- concentrations were measured using an immuno- tion (Gibco). turbidimetric and colorimetric assay (AU5800 Beck- Polyubiquitination levels of eNOS were evaluated man Coulter analyzer). Myocardial iron content was by using agarose-tandem ubiquitin-binding entities determined in digested tissue samples using induc- (LifeSensors) following the manufacturer’s protocol. tively coupled plasma-mass spectrometry (Nexion Pulled-down myocardial samples were analyzed by 350x, PerkinElmer) and was normalized to the dry immunoblotting using an eNOS antibody (BD Trans- weight of myocardium. Transthoracic echocardiogra- duction Laboratories) and a polyubiquitin FK2 phy was performed at baseline and at week 4 using a monoclonal antibody (Enzo Life Sciences). Vivid-Q portable ultrasound system equipped with a IN SITU MYOCARDIAL INFARCTION. Mice were i12L-RS 13 MHz transducer (GE Healthcare) as anesthetized (40 mg/kg pentobarbital–40 mg/kg described earlier (28). ketamine) and subjected to 45-min left anterior QUANTIFICATION OF OXIDATIVE AND NITROSATIVE descendent coronary artery ligation followed by 24-h STRESS. Production of superoxide was detected in reperfusion. Hearts were removed and retrogradely myocardial cryosections by oxidation of dihy- perfused with Krebs-Henseleit buffer for 1 min. The droethidium (Thermo Fisher Scientific) as previously coronary artery was relegated at the same point, and described (13). Myocardial extracts were used to the heart was perfused with 5% Evans blue and sliced measure protein carbonyl, 8-isoprostane, and in 6 short-axis sections (Zivic Instruments heart slicer 3-nitrotyrosine as markers of oxidative and nitro- matrix) that were incubated with 1% triphenylte- sative stress (Cayman, items 10005020 and 516351, trazolium chloride (Sigma Chemical) for 10 min at and Elabscience, respectively), and total antioxidant 37  C. The slices were weighed and photographed from capacity (Cayman, item 709001) and superoxide both sides. The area at risk (Evans blue–negative) dismutase-1 activity (Cayman, item 706002). Pro- and the area of necrosis (Evans blue–negative and duction of H 2O 2 in isolated fresh mitochondria was triphenyltetrazolium chloride–negative) were semi- monitored in a spectrofluorimeter (SpectraMax Gem- automatically determined (Image ProPlus software iniXS, Molecular Devices) using malate, glutamate or version 4.5, Media Cybernetics) in the digital images succinate substrates as previously described (28). and expressed as average percent area from both sides of each slice and corrected for slice weight: area at risk ANALYSIS OF THE eNOS/sGC/PKG PATHWAY. Total weight ¼ (weight slice 1  % area at risk slice 1) þ myocardial nitrate/nitrite and cGMP were measured . þ (weight slice N  % area at risk slice N). Area of in myocardial homogenates by using, respectively, a necrosis weight was calculated in the same manner. nitrate/nitrite colorimetric assay and a competitive Finally, infarct size was calculated as percentage of the enzyme-linked immunosorbent assay (Cayman, items area at risk (28). To explore the effect of sGC activation 780001 and 581021, respectively) following the man- on myocardial infarction, a subgroup of animals from ufacturer’s instructions. the control and ID groups received 10 m g/kg ataciguat Proteins were separated by Western blot as previ- (HMR1766, Sigma-Aldrich) intraperitoneally 10 min ously described (13). Primary antibodies used were before ischemia. raised against eNOS and pS1177-eNOS (BD Trans- duction Laboratories), iNOS (Abcam), heat-shock STATISTICAL ANALYSIS. Data analysis was per- protein 90 (HSP90, Abcam), PKG (Enzo Life Sciences), formed using SPSS software, version 20.0.0. (IBM). ubiquitin (Abcam), vasodilator-stimulated phospho- Continuous variables are described as mean  SEM or protein (VASP) and pS237-VASP (Cell Signaling as median with 25th and 75th percentiles (Q1-Q3) if Technology), and GAPDH (Genetex). Dimeric and not normally distributed. Categorical variables are monomeric forms of eNOS were separated by described as counts (percentages). Comparisons be- low-temperature electrophoresis as previously tween continuous variables were performed by described (13). Protein bands were detected by chem- means of Student’s t-tests, Mann-Whitney U-test or iluminescence (ECL, Amersham) and quantified using by 1-way analysis of variance with Tukey’s post hoc a charge-coupled device system (Image Reader test applied for multiple pairwise comparisons, when LAS-3000, Fujifilm) and image analysis software appropriate. Changes in CMR endpoints from baseline (Image Gauge, version 4.0 Fujifilm). to 6 months were assessed by paired Student’s t-tests. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Inserte et al. 571 JULY 2021:567–580 Impact of Iron Deficiency in Myocardial Infarction Values of P < 0.05 were considered statisti- T A B L E 1 Demographic and Baseline Clinical Characteristics of Patients With cally significant. and Without ID RESULTS No ID (n ¼71) ID (n ¼ 54) P Value Age, y 58.8  1.4 60.8  1.7 0.359 CLINICAL AND LABORATORY DATA. Mean age of the Male 64 (90.1) 40 (74.1) 0.017 125 patients included was 59.7  1.1 years and 104 Body mass index, kg/m2 27.6  0.5 27.5  0.6 0.908 Active smoking 38 (53.5) 22 (40.7) 0.157 (89.2%) were male. ID was present in 54 (43.2%). Hypertension 29 (40.8) 30 (55.6) 0.103 Demographic characteristics and frequency of car- Diabetes mellitus 11 (15.5) 12 (22.2) 0.336 diovascular risk factors and comorbidities were Dyslipidemia 41 (57.7) 27 (50.0) 0.389 comparable in patients with and without ID but for a Chronic angina or previous PCI 3 (4.2) 1 (1.9) 0.419 higher frequency of female sex in the former (Table 1). Previous heart failure 0 (0) 0 (0) — Clinical features on presentation, angiographic data, Previous stroke 0 (0) 0 (0) — complications, and in-hospital management were Peripheral artery disease 0 (0.0) 3 (5.6) 0.078 also similar in both groups but for a significantly Severe chronic renal failure 1 (1.4) 0 (0.0) 0.568 Pulmonary disease 4 (5.6) 3 (5.6) 1.000 higher frequency of glycoprotein IIb/IIIa inhibitor use Previous antiplatelet agents 5 (7.0) 7 (13.0) 0.266 in patients with ID (Table 2). No patient received iron Previous beta blockers 7 (9.9) 3 (5.6) 0.585 supplements during hospitalization or at discharge. Previous ACE inhibitors/ARB 19 (26.8) 21 (38.9) 0.150 Table 3 summarizes the main laboratory data. Previous aldosterone inhibitors 0 (0) 0 (0) — Hemoglobin levels were comparable in patients with Previous diuretics 9 (12.7) 12 (22.2) 0.157 and without ID. Twelve patients (9.6%) had mild Previous oral antidiabetic drugs 7 (9.9) 7 (13.0) 0.586 anemia (lowest hemoglobin value, 11.4 g/dL). Blood Previous insulin 3 (4.2) 2 (3.7) 0.628 cell counts, glucose levels, and renal function were Previous statins 16 (22.5) 14 (25.9) 0.660 comparable among groups. Serum levels of iron and Values are means  SEM or n (%). ferritin and transferrin saturation were lower in ACE ¼ angiotensin-converting enzyme; ARB ¼ angiotensin receptor blockers; ID ¼ iron defi- ciency; PCI ¼ percutaneous coronary intervention. patients with ID than in the rest. Remarkably, CK-MB peak was significantly higher in patients with ID than in those without ID (median [Q1-Q3]: 335 ng/mL [249-477 ng/mL] vs 246 ng/mL [139-335 ng/mL], respectively [P ¼ 0.003]). T A B L E 2 Clinical and Angiographic Characteristics and Hospital Management CMR IMAGING. In the initial exam (median [Q1-Q3]: of Patients With and Without ID 5 days [3 days-8 days] after admission), LV mass No ID (n ¼ 71) ID (n ¼ 54) P Value averaged 126.0  3.1 g (64.9  1.4 g/m 2), the area at Time symptom onset-balloon, min 169 (138-257) 189 (134-273) 0.942 risk (n ¼ 90) averaged 41.9  16.8 g (34.1%  12.0% of Initial systolic blood pressure, mm Hg 146  3 146  4 0.932 LV mass), infarct size averaged 24.7  1.6 g (19.4%  Initial heart rate, beats/min 75 (65-86) 76 (62-87) 0.822 1.0% of LV mass, 59.6%  24.7% of the area at risk), Killip class $2 10 (14.1) 8 (14.8) 0.908 and 64 patients (54.7%) had MVO, which involved GRACE score 107  4 112  4 0.328 Initial TIMI flow grade 0-1 51(85.0) 47 (95.9) 0.118 a median (Q1-Q3) of 1 segments (0 segments-3 Final TIMI flow grade 3 68 (97.1) 54 (100) 0.317 segments). Nineteen patients (17.3%) had intra- Multivessel disease 24 (34.3) 22 (42.3) 0.366 myocardial hemorrhage, which when present, aver- Reinfarction 1 (1.4) 2 (3.7) 0.398 aged 2.7  0.5 g (2.2%  0.4% of LV mass). Among Worse Killip class $3 1 (1.4) 3 (5.6) 0.214 patients with a follow-up CMR, LV end-diastolic vol- Ventricular fibrillation 7 (9.9) 10 (18.5) 0.162 ume increased at a median (Q1-Q3) of 6 months (5 Paroxysmal atrial fibrillation 3 (4.2) 2 (3.7) 0.628 months-6 months) by 8.7%  2.3% (from 77.5  Advanced auriculoventricular block 0 (0.0) 2 (3.7) 0.185 2 2 1.6 mL/m to 82.9  1.9 mL/m ; P ¼ 0.001), end- Glycoprotein IIb/IIIa inhibitors 24 (34.3) 29 (53.7) 0.030 Dual antiplatelet therapy at discharge 71 (100) 52 (96.3) 0.185 systolic volume did not change significantly (from 2 2 Oral anticoagulants at discharge 5 (7.0) 4 (7.4) 1.000 42.0  1.2 mL/m to 41.6 mL/m ; P ¼ 0.817), and Beta-blockers at discharge 68 (95.8) 48 (88.9) 0.260 ejection fraction increased by 5.4%  0.7% (from ACE inhibitors/ARB at discharge 70 (98.6) 52 (96.3) 0.398 46.0%  0.9% to 51.4%  1.0%; P < 0.001). Twenty-six Aldosterone inhibitors at discharge 18 (25.4) 12 (22.2) 0.685 patients (23.9%) had adverse LV remodeling as Statins at discharge 70 (98.6) 51 (94.4) 0.214 defined before. Values are median (25th-75th percentiles), mean  SEM, or n (%). Patients with ID had significantly larger mean GRACE ¼ Global Registry of Acute Coronary Events; TIMI ¼ Thrombolysis In Myocardial infarct size (22.8%  1.4% vs 16.8%  1.2% of LV mass; Infarction; other abbreviations in Table 1. P ¼ 0.002, 68.0%  3.6% vs 52.8%  3.5% of the area 572 Inserte et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Impact of Iron Deficiency in Myocardial Infarction JULY 2021:567–580 percentage (38.1% vs 12.8%; P ¼ 0.019) and extent T A B L E 3 Laboratory Data in Patients With and Without ID (1.0%  0.3% vs 0.2%  0.1% of LV mass; P ¼ 0.042) of No ID (n ¼ 71) ID (n ¼ 54) P Value intramyocardial hemorrhage. Hemoglobin levels, g/dL 14.4  0.1 14.2  0.2 0.381 BASELINE CHARACTERISTICS AND ECHOCARDIO- Hematocrit value, % 43.1  0.4 42.8  0.5 0.665 GRAPHIC DATA IN MICE. Hematological parameters, Mean corpuscular volume, fL 91.7  0.6 91.2  0.8 0.646 Leukocyte count/mL 10.7 (9.4-13.4) 12.3 (9.2-14.9) 0.224 myocardial iron content, and hemodynamic and Platelet count/mL 226.7  7.5 230.2  8.6 0.759 echocardiographic data in the experimental groups Serum iron, mg/dL 70.9  3.2 55.9  4.1 0.004 are summarized in Table 5. Plasma hemoglobin was Serum ferritin, mg/dL 233 (168-383) 96 (62-171) <0.001 mildly reduced by ID diet with respect to control Serum transferrin, mg/dL 224.3  3.4 234.6  5.0 0.081 subjects, although values remained within the normal Transferrin saturation, % 31.9  1.5 24.0  1.8 0.001 range. Serum ferritin, serum iron content, and Glucose levels, mg/dL 130 (113-159) 138 (111-170) 0.360 transferrin saturation were reduced by ID diet, Creatinine levels, mg/dL 0.88  0.03 0.89  0.03 0.768 eGFR, mL/min/1.73 m 2 99.7  3.2 91.4  3.2 0.071 whereas serum transferrin was increased. Most Peak creatine kinase-MB, ng/mL 246 (139-335) 335 (249-477) 0.003 importantly, ID diet induced a significant decrease in myocardial iron content. All these parameters were Values are mean  SEM or median (25th-75th percentiles). normalized after weekly administration of iron- eGFR ¼ estimated glomerular filtration rate; ID ¼ iron deficiency; MB ¼ myocardial band. sucrose. ID diet lacked any effects on hemodynamic or transthoracic echocardiography measurements. at risk; P ¼ 0.004 [n ¼ 90]) and more frequent and ACTIVITY OF THE eNOS/sGC/PKG PATHWAY. West- extensive MVO than those without ID, though a ern blot analysis of eNOS demonstrated a significant similar extent of myocardial hemorrhage (Table 4). decrease in total protein without changes in the They also showed significantly greater increases in extent of its phosphorylation in ID mice (P ¼ 0.023) ventricular volumes at 6 months than patients (Figure 2A). The stability of eNOS, calculated as the without ID, with similar changes in ejection fraction. ratio between its dimeric and monomeric forms, was Adverse LV remodeling occurred in 37.8% of patients reduced in myocardial homogenates from ID mice with ID and in 14.1% of those without ID (P ¼ 0.004). (P ¼ 0.020) (Figure 2B). The effect of ID on total and Compared with the remaining patients, those devel- dimeric eNOS protein levels was reproduced in iso- oping adverse remodeling had larger mean infarct lated cardiomyocytes incubated with the iron size (24.8%  1.9% vs 17.0%  1.1% of LV mass; chelator deferasirox (P ¼ 0.005 and P < 0.001, P ¼ 0.001), more extensive MVO (median [Q1-Q3]: 3 respectively) (Figure 2C). Measurement of myocardial segments [1 segments-4 segments] vs 0 segment [0 nitrate/nitrite concentration confirmed the decreased segment-2 segments]; P < 0.001) and a higher eNOS activity in iron-depleted hearts (P ¼ 0.005) (Figure 2D). Attenuation of eNOS activity correlated with the inhibition of the sGC/PKG pathway as indi- T A B L E 4 Main Results of CMR Imaging in Patients With and Without ID cated by the reduction in the myocardial cGMP con- No ID (n ¼ 71) ID (n ¼ 54) P Value tent (P ¼ 0.005) (Figure 2D) and by the reduced Area at risk, ga 43.7  2.7 39.6  2.2 0.266 phosphorylation of the PKG substrate VASP Area at risk, % of LV massa 35.3  1.9 32.6  1.7 0.296 (P ¼ 0.023) (Figure 2E). No significant differences in Infarct size, g 20.7  1.6 30.1  2.9 0.003 the myocardial expression of iNOS were observed Infarct size, % of LV mass 16.8  1.2 22.8  1.4 0.002 Infarct size, % of the area at risk a 52.8  3.5 68.0  3.6 0.004 (Figure 2F). No changes in total PKG levels were No. segments with MVO 0 (0-2) 2 (0-3) 0.002 observed (data not shown). Iron administration $3 segments with MVO, % 12 (17.9) 20 (40.0) 0.008 effectively restored the activity of the eNOS/sGC/PKG Any hemorrhage, % 9 (13.6) 10 (22.7) 0.217 pathway (Figure 2). Hemorrhage, % of LV mass 0.3  0.1 0.5  0.2 0.238 Relative increase in LVEDV, %b 3.7  2.5 15.9  4.1 0.012 CYTOSOLIC OXIDATIVE AND NITROSATIVE Relative increase in LVESV, %b -7.2  3.0 9.3  4.7 0.002 STRESS. Mice fed with the ID diet showed increased Increase in LVEF, %b 6.4  0.9 3.9  1.1 0.080 oxidative and nitrosative stress as indicated by the Adverse LV remodeling, %b 9 (14.1) 17 (37.8) 0.004 enhanced production of isoprostane (P ¼ 0.008), Values are mean  SEM or median (25th-75th percentiles). aValues from 90 patients with protein carbonyl (P ¼ 0.011), and tyrosine nitration adequate measurements. bValues from the 109 patients (45 with ID and 64 without ID) with (P ¼ 0.043) in myocardial extracts (Figure 3A). cardiac magnetic resonance imaging repeated at 6 months. CMR ¼ cardiac magnetic resonance; ID ¼ iron deficiency; LV ¼ left ventricular; LVEDV ¼ left Increased levels of cytosolic O 2 in ID mice was ventricular end-diastolic volume; LVEF ¼ left ventricular ejection fraction; LVESV ¼ left ven- confirmed by measuring the fluorescence produced tricular end-systolic volume. by dihydroethidium in heart sections (P ¼ 0.002) JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Inserte et al. 573 JULY 2021:567–580 Impact of Iron Deficiency in Myocardial Infarction (Figure 3B). Oxidative stress was associated with a T A B L E 5 Hematological Parameters, Cardiac Iron Content, and significant reduction in the total myocardial antioxi- Hemodynamic and Echocardiographic Data in Mice dant capacity (P ¼ 0.008) and in the activity of the Control Diet ID Diet ID DietþFe antioxidant enzyme superoxide dismutase (n ¼ 8) (n ¼ 8) (n ¼ 8) P Value (P ¼ 0.032) (Figure 3C). The effect of ID on mito- Body weight, g 34.6  1.8 35.4  1.1 36.0  1.5 0.710 chondrial H2O 2 production was analyzed in isolated Hemoglobin, g/dL 13.8  0.2 12.6  0.4* 13.7  0.4 0.024 mitochondria incubated with complex-I substrates Serum ferritin, ng/mL 527  37 343  14‡ 581  36 <0.001 (malate and glutamate) or succinate. ID diet did not Serum iron, mmol/L 36.7  3.0 24.4  2.5‡ 45.5  4.2 0.001 affect H2O 2 production driven by malate plus gluta- Cardiac iron, mg/g d.w.k 124.4  3.1 84.0  8.6† 143.9  11.2 0.010 Serum transferrin, g/L 0.66  0.08 1.24  0.10§ 0.80  0.09 <0.001 mate. Hydrogen peroxide increased linearly with the Transferrin saturation, % 55.7  5.2 19.4  1.9§ 52.3  3.4 <0.001 addition of succinate, but no significant differences Heart rate, beats/min 421  12 427  16 412  15 0.654 associated with diet were observed after 5 min of in- MBP, mm Hg 84  3 89  5 81  4 0.547 cubation (Figure 3D). Iron supplementation prevented LVEDD, mm 4.23  0.17 4.32  0.15 4.18  0.13 0.712 both oxidative/nitrosative stress and reduced anti- LVESD, mm 3.04  0.17 3.06  0.10 2.99  0.15 0.550 oxidant capacity (Figure 3). IVS, mm 0.74  0.05 0.78  0.09 0.70  0.08 0.274 LVPW, mm 0.73  0.04 0.74  0.07 0.72  0.08 0.651 eNOS UBIQUITINATION. Because total eNOS protein LVEF, % 66.3  3.2 63.6  4.5 67.1  7.6 0.198 levels were reduced in ID mice, we next examined whether ID downregulates eNOS by increasing its Values are mean  SEM. *P < 0.05 vs control diet. †P < 0.05. ‡P < 0.01. §P < 0.001 versus control diet and ID dietþFe. kCardiac iron was measured in 6 additional mice. Statistical signifi- degradation through the ubiquitin/proteasome cance tested with the use of analysis of variance with Tukey’s post hoc test applied for multiple pathway. We observed a significant reduction in the pairwise comparisons. d.w. ¼ dry weight; ID ¼ iron deficiency; IVS ¼ interventricular septum. LVEDD ¼ left ventricular levels of the chaperon protein HSP90 in myocardial end-diastolic diameter. LVEF; left ventricular ejection fraction. LVESD ¼ left ventricular end- samples from ID-mice (P ¼ 0.031) (Figure 4A). systolic diameter. LVPW ¼ left ventricular posterior wall; MBP ¼ mean blood pressure. Because HSP90 has been proposed to modulate eNOS stability in a ubiquitin-dependent manner (30), polyubiquitinated proteins were pulled down from DISCUSSION cardiac extracts using the agarose-tandem ubiquitin- binding entities molecular tool and probed with In this study, 43% of patients with a first anterior eNOS antibody. The results showed that ID diet in- STEMI undergoing primary percutaneous coronary creases myocardial eNOS ubiquitination (P ¼ 0.008) intervention had ID. Patients with ID had larger in- (Figure 4B) and reduces eNOS protein levels farcts and more severe MVO acutely and a higher (P ¼ 0.007) (data not shown). Iron supplementation frequency of adverse LV remodeling at 6 months than prevented the decrease in HSP90 protein content and the remaining patients. Mice fed with the ID diet had the enhanced eNOS ubiquitination and degrada- larger infarcts after transient coronary occlusion than tion (Figure 4). control mice and iron supplementation prevented MYOCARDIAL TOLERANCE TO ISCHEMIA/REPERFUSION. this effect. ID diet inhibited the activity of the eNOS/ In mice subjected to 45-min coronary ligation, mor- sGC/PKG pathway, which was associated with tality rate during the procedure was 15% (2 animals increased oxidative/nitrosative stress linked to from the IDþFe group and 1 from each of the altered antioxidant defense and to eNOS degradation remaining groups). The mass of myocardium at risk via ubiquitin/proteasome system. sGC activators was 32.8%  2.8% of LV in the control group and was reverted the detrimental effects of ID in mice. similar among groups (Figure 5). By contrast, infarct Few studies have assessed the prevalence and size measured after 24 h of reperfusion was signifi- significance of ID in patients with acute coronary cantly larger in mice fed with ID diet than in controls syndromes. ID was found in 29% to 60% of patients, (58.1%  3.0% vs 40.4%  3.7% of the area at risk; but its clinical implications have been inconsistent P ¼ 0.004), and iron supplementation prevented this between studies (3–6). ID was related to a worse effect (36.3%  4.0%; P ¼ 0.407 vs control group). The functional recovery (3) or to a higher long-term sGC activator ataciguat induced a trend toward occurrence of myocardial infarction/cardiovascular reduced infarct size in mice fed with control diet death or total mortality in unselected acute coronary (27.5%  2.8%; P ¼ 0.061 vs control group) and syndrome patients (4,5). By contrast, in a study in prevented the increase in infarct size observed in STEMI patients undergoing mechanical reperfusion, ID-mice (31.0%  2.5%; P ¼ 0.287 vs con- those with ID had higher admission troponin levels trolþataciguat group). but a more benign in-hospital clinical course, and 574 Inserte et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Impact of Iron Deficiency in Myocardial Infarction JULY 2021:567–580 F I G U R E 2 ID Inhibits the eNOS/cGMP/PKG Pathway Representative Western blot and quantification of (A) total eNOS and its phosphorylated form and (B) the sodium dodecyl sulfate–resistant eNOS dimer in cardiac lysates obtained from the different experimental groups, n ¼ 5 per group. (C) Representative Western blot and quantification of total eNOS and its dimeric form in isolated cardiomyocytes treated with deferasirox (DFX), n ¼ 4 per group. (D) Quantification of myocardial content of nitrate/nitrite (NOx) and cGMP, n ¼ 5 per group. (E) protein kinase G (PKG) activity evaluated by Western blot as PKG-dependent phosphorylation of its substrate vasodilator-stimulated phosphoprotein (VASP), n ¼ 5 per group. (F) Representative Western blot of inducible nitric oxide synthase (iNOS). LPS corresponds to a mouse spleen sample obtained 6 h after lipopolysaccharide injection (10 mg/kg intraperitoneally). *P < 0.05, **P < 0.01 vs IDD group. CD ¼ control diet; eNOS ¼ endothelial nitric oxide synthase; IDD ¼ iron-deficient diet; IDDþFe ¼ IDD plus iron supplementation. Data are mean  SEM. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Inserte et al. 575 JULY 2021:567–580 Impact of Iron Deficiency in Myocardial Infarction F I G U R E 3 ID Increases Oxidative/Nitrosative Stress by Reducing Myocardial Antioxidant Capacity (A) Quantification of markers of oxidative (8-isoprostane and protein carbonyl) and nitrosative (nitrotyrosine) stress, n ¼ 5 per group. (B) Representative ventricular cross-sections incubated with dihydroethidium (DHE). DHE fluorescence is expressed as arbitrary units, n ¼ 4 per group. (C) Quantification of total antioxidant capacity and superoxide dismutase-1 (SOD1) activity, n ¼5 per group. (D) Time course of H2O2 production from isolated mitochondria after the addition of malate plus glutamate or succinate. F/F0 corresponds to arbitrary fluorescence values normalized with respect to baseline (excitation at 320 nm, emission at 440 nm), n ¼ 3 per group. Data are mean  SEM. *P < 0.05, **P < 0.01 vs IDD groups. Abbreviations as in Figure 2. among those with a CMR performed, similar infarct aimed to account in part for the nonspecific L-ferritin size, higher myocardial salvage index, and less MVO increase occurring in acute disorders (1,2). In our se- as compared with those without ID (6). The reasons ries, ID was present in 43% of cases and was signifi- for these discrepancies are unclear but might be cantly associated with larger infarcts—either related in part to the diverse patient populations and estimated by CK-MB peak or by CMR—and with a the different definitions of ID used. more extensive MVO. Of note, this association would To facilitate interpretation and comparability of still be present if ID were defined as a transferrin our results, we defined ID using the criteria that have saturation <20% irrespective of ferritin levels (data become standard in the setting of acute heart failure, not shown). Because patients with and without ID 576 Inserte et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Impact of Iron Deficiency in Myocardial Infarction JULY 2021:567–580 F I G U R E 4 ID Reduces HSP90 Levels and Increases the Extent of eNOS Ubiquitination (A) Representative Western blot and quantification of HSP90. (B) Polyubiquitination levels of eNOS in cardiac lysates analyzed by using agarose-tandem ubiquitin-binding entities pull-downs. eNOS bands were normalized to their corresponding ubiquitin smears and ratios were normalized to ubiquitinated eNOS level in control samples. Representative Western blots are shown in all panels. Data are mean  SEM. *P < 0.05, **P < 0.01 vs IDD group. Abbreviations as in Figure 2. had comparable baseline characteristics and were Rineau et al (27) study, ours showed a slight decline in treated similarly, the results suggest that ID enhanced hemoglobin levels, although values remained within myocardial injury. The higher use of glycoprotein IIb/ the physiological range. Blood analyses and myocar- IIIa inhibitors in patients with ID is in line with this dial iron quantification confirmed significant ID that interpretation. On the one hand, it might reflect a was fully prevented by iron supplementation. ID did higher thrombotic burden in these patients, and on not induce ventricular dilatation or dysfunction, in the other hand, these drugs are potentially protective agreement with the Rineau et al (27) study and in against ischemia/reperfusion injury (31). contrast with the myocardial remodeling described in To the best of our knowledge, no previous experi- models of long-term ID anemia (11). mental studies have analyzed the effect of dietary ID The larger infarct size observed in ID mice with on myocardial tolerance to transient ischemia. In our respect to control mice concurs with our findings in experiments in mice, we used a model of ID without STEMI patients. As expected (10–12), this effect anemia recently described by Rineau et al (27) to coincided with increased myocardial oxidative/ isolate the effects of ID from those of anemia, usually nitrosative stress. Our data indicated that ID in- present in dietary models of ID or in models with creases cytosolic O 2 levels in association with repeated blood withdrawal (11,32). In contrast to the reduced total myocardial antioxidant capacity JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Inserte et al. 577 JULY 2021:567–580 Impact of Iron Deficiency in Myocardial Infarction F I G U R E 5 ID Increases Myocardial Susceptibility to Ischemia/Reperfusion Injury Area at risk, expressed as a percentage of left ventricular mass, and infarct size, measured by triphenyltetrazolium chloride staining and expressed as a percentage of the area at risk, in mouse hearts after 24-h of reperfusion in the different experimental groups. Representative examples are shown. Data are mean  SEM. n ¼ 6-7 per group. *P < 0.05 vs the CD group. Ata ¼ ataciguat; other abbreviations as in Figure 2. caused, at least partially, by the altered activity of study, ID increased myocardial nitrotyrosine, a antioxidant enzymes with no evidence of changes in fingerprint of the in vivo presence of ONOO , and the mitochondrial contribution to oxidative stress. reduced the dimeric form of eNOS, reflecting its These alterations were previously documented in uncoupling, and myocardial nitrates and nitrites blood from patients with ID anemia and in the levels, used as surrogate markers for NO. As a result, myocardium from animals with dietary ID and were myocardial cGMP content and the phosphorylation of reverted by iron therapy (12,33). Similarly, our results VASP, a protein substrate of PKG, but not total PKG showed reduced oxidative stress coupled with levels, were reduced. Further supporting the inhibi- enhanced antioxidant capacity in mice receiving ID tion of the eNOS/sGC/PKG pathway and its contribu- diet plus iron supplementation. tion to the effects of ID, administration of the sGC Abundant evidence demonstrates that preserva- activator ataciguat prevented the increased suscep- tion of the eNOS/sGC/PKG pathway attenuates tibility to ischemia in ID mice. myocardial cell death after ischemia/reperfusion Our data show that ID, in addition to its oxidation, (15,16). This pathway is highly susceptible to oxida- decreased myocardial eNOS protein content and did tion (13,17–19) and can be affected by ID at different not induce iNOS expression. These results differ from levels. First, O 2 combines with NO to generate other studies describing eNOS and iNOS up- ONOO, which apart from reducing NO availability, regulation and increased nitro-oxidative stress in induces nitro-oxidative stress toxicity (34). Second, the heart of anemic rats in parallel with the devel- O 2 and ONOO  can oxidize tetrahydrobiopterin, a opment of ventricular dilatation (11). Recently, a cofactor essential for proper eNOS function, leading study described reduced eNOS function and to eNOS uncoupling (19). Third, O2 oxidizes the increased reactive oxygen species formation in red heme group of sGC, decreasing its responsiveness to blood cells from anemic mice, which was compen- NO and reducing cGMP production and activation of sated by enhanced expression of eNOS in aorta, PKG, the final effector of this pathway (17,18). Finally, although no significant differences in myocardial both eNOS and sGC contain a prosthetic heme group eNOS expression were observed (32). Altogether, and their enzymatic activity may be inhibited inde- these results suggest that eNOS response to ID may pendently of oxidative stress by severe ID (20). In our vary depending on the presence of anemia or tissue 578 Inserte et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Impact of Iron Deficiency in Myocardial Infarction JULY 2021:567–580 hypoxia. The reduction in myocardial eNOS content relationship between ID and the increased post- in ID mice correlated with lower HSP90 protein levels ischemic damage in STEMI patients. However, this and increased eNOS ubiquitination. In endothelial interpretation concurred with the hypothesis of the cells, HSP90 associates with eNOS, maintains its study and was strongly supported by the results of functional dimeric structure, and regulates its our experiments in mice. Although only male mice degradation by ubiquitination (30). Iron chelators were used, increased oxidative stress in response to downregulate HSP90 expression in vitro (35) and ox- ID (12) and the cardioprotective role of eNOS/sGC ygen radicals cleave HSP90, disrupting its chaper- pathway (43) have been demonstrated in both sexes, oning function (36). Based on our results and this and no sex differences in infarct size were observed in previous evidence, it is tempting to speculate that ID- previous studies from our group (28), suggesting that induced oxidative stress may result in a defective the proposed effect of ID on acute myocardial toler- proteasomal degradation of eNOS by reducing HSP90 ance to ischemia is independent of sex. Despite that protein content. our data obtained in isolated cardiomyocytes support In agreement with previous studies (21,22), infarct a direct effect of ID on these cells, our study does not size, MVO, and intramyocardial hemorrhage predicted rule out the contribution of endothelial cells to the adverse remodeling. It is therefore possible that the myocardial effects of ID. Finally, although we provide association between ID and adverse LV remodeling in a plausible mechanism for the detrimental effect of ID our STEMI patients was mainly driven by their more in STEMI patients, we cannot exclude that others severe damage acutely, and a direct effect of ID on are involved. ventricular healing can be neither supported nor excluded based on our data. Previous evidence in this CONCLUSIONS respect is contradictory, as some observational studies in STEMI patients suggested that ID could be detri- This study shows that ID is associated with larger mental to the remodeling process (25,37), whereas infarcts and more frequent adverse LV remodeling in other studies reported an attenuated LV dilatation in patients with a reperfused first anterior STEMI and experimental animals treated with post-infarction di- that dietary ID without anemia reduces the myocar- etary iron restriction (38) or iron chelation (39). dial tolerance to ischemia/reperfusion in mice. This Erythropoietin administration has not improved clin- effect is produced at least in part by inhibiting the ical outcomes or LV performance after STEMI (40), eNOS/sGC/PKG pathway and involves increased indicating that increasing erythropoiesis does not oxidative/nitrosative stress and the proteasome- ameliorate post-infarction remodeling, but these re- dependent degradation of eNOS. Our findings high- sults do not exclude that ID is detrimental to this pro- light that not only iron excess but also ID may have cess. In fact, erythropoietin mobilizes iron from deleterious effects on myocardial salvage and on storage sites for hemoglobin synthesis, potentially fa- ventricular remodeling in patients with acute voring absolute ID, and lowers percent transferrin myocardial infarction and justify a word of caution on saturation in the presence of adequate iron stores, fa- the use of iron-lowering therapies in this setting. voring functional ID (41). FUNDING SUPPORT AND AUTHOR DISCLOSURES STUDY LIMITATIONS. Our relatively low-risk patients may not be representative of the overall STEMI pop- This study was funded by Instituto de Salud Carlos III, Spain, through ulation, but the inclusion criteria were selected to the project PI16/00232 and the research network CIBERCV (CB16/11/ 00479), both cofunded by European Regional Development Fund, ensure homogeneity and to avoid any confounding and by the Sociedad Española de Cardiología (Proyecto de Inves- factors for CMR analysis. Nine percent had mild tigación Traslacional en Cardiología 2016). The authors have reported anemia, but none of the main results would have that they have no relationships relevant to the contents of this paper differed had these patients been excluded (data not to disclose. shown). Measuring the area at risk based on edema quantification in STIR images is subject to limitations ADDRESS FOR CORRESPONDENCE: Dr Javier Inserte (42). No adjustment for multiple comparisons was or Dr. José A. Barrabés, Department of Cardiology, applied in the statistical differences depicted in the Vall d’Hebron Hospital Universitari, Passeig Vall tables. The observational nature of our clinical study d’Hebron 119-129, 08035 Barcelona, Spain. E-mail: prevents us from establishing a cause-effect javier.inserte@vhir.org OR jabarrabes@vhebron.net. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 6, NO. 7, 2021 Inserte et al. 579 JULY 2021:567–580 Impact of Iron Deficiency in Myocardial Infarction PERSPECTIVES COMPETENCY IN MEDICAL KNOWLEDGE: In pa- TRANSLATIONAL OUTLOOK: In patients with acute tients with STEMI, ID is associated with larger infarcts and myocardial infarction, the definition of ID needs to be with a higher frequency of LV adverse remodeling. ID improved and the optimal iron status clarified. 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