Tajes et al. Cell Biosci (2021) 11:89 https://doi.org/10.1186/s13578-021-00605-5 Cell & Bioscience RESEARCH Open Access Neurohormonal activation induces intracellular iron deficiency and mitochondrial dysfunction in cardiac cells M. Tajes1,2, C. Díez‑López1,2,3,7, C. Enjuanes1,2,4, P. Moliner1,2,4, J. L. Ferreiro1,2, A. Garay1,2,4, S. Jiménez‑Marrero1,2,4, S. Yun1,4,9, S. G. Sosa1,2, L. Alcoberro1,2,4, J. González‑Costello1,2,3,7, E. García‑Romero1,2,3, L. Yañez‑Bisbe5, B. Benito5,6,8 and J. Comín‑Colet1,2,4,7* Abstract Background: Iron deficiency (ID) is common in patients with heart failure (HF) and is associated with poor outcomes, yet its role in the pathophysiology of HF is not well-defined. We sought to determine the consequences of HF neuro‑ hormonal activation in iron homeostasis and mitochondrial function in cardiac cells. Methods: HF was induced in C57BL/6 mice by using isoproterenol osmotic pumps and embryonic rat heart-derived H9c2 cells were subsequently challenged with Angiotensin II and/or Norepinephrine. The expression of several genes and proteins related to intracellular iron metabolism were assessed by Real time-PCR and immunoblotting, respec‑ tively. The intracellular iron levels were also determined. Mitochondrial function was analyzed by studying the mito‑ chondrial membrane potential, the accumulation of radical oxygen species (ROS) and the adenosine triphosphate (ATP) production. Results: Hearts from isoproterenol-stimulated mice showed a decreased in both mRNA and protein levels of iron regulatory proteins, transferrin receptor 1, ferroportin 1 and hepcidin compared to control mice. Furthermore, mitofer‑ rin 2 and mitochondrial ferritin were also downregulated in the hearts from HF mice. Similar data regarding these key iron regulatory molecules were found in the H9c2 cells challenged with neurohormonal stimuli. Accordingly, a deple‑ tion of intracellular iron levels was found in the stimulated cells compared to non-stimulated cells, as well as in the hearts from the isoproterenol-induced HF mice. Finally, neurohormonal activation impaired mitochondrial function as indicated by the accumulation of ROS, the impaired mitochondrial membrane potential and the decrease in the ATP levels in the cardiac cells. Conclusions: HF characteristic neurohormonal activation induced changes in the regulation of key molecules involved in iron homeostasis, reduced intracellular iron levels and impaired mitochondrial function. The current results suggest that iron could be involved in the pathophysiology of HF. Keywords: Neurohormonal activation, Heart failure, Iron deficiency, Cardiac cell, Mitochondria function Background Heart failure (HF) is a devastating condition and repre- sents a challenge for public healthcare systems [1]. The *Correspondence: josepcomin@gmail.com; jcomin@bellvitgehospital.cat current understanding of its pathophysiology is based on 1 Bio‑Heart Cardiovascular Diseases Research Group, Bellvitge Biomedical the “neurohormonal hypothesis”, which states that HF Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain progression is promoted by the long-term maladaptive Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​mmons.​org/​publi​cdoma​in/​ zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tajes et al. Cell Biosci (2021) 11:89 Page 2 of 18 and deleterious effects of sustained neurohormonal In spite of all the accumulating data, the interplay of activation in the heart and in rest of the cardiovascular iron in the pathophysiology of HF is not well-defined. We system [2, 3]. Many of classical neurohormones such as hypothesized that neurohormonal activation could be norepinephrine (Nor) and angiotensin II (Ang II) are involved in the generation of ID in cardiac cells, directly known to be synthesized directly within the myocardium impacting on the mitochondria function. Therefore, the and, therefore, act in an autocrine or paracrine manner aim of this study was to analyze the relationship between [3, 4]. In fact, the sustained activation of these pathways neurohormonal stimuli and iron homeostasis, as well as lead to an increased plasma Ang II, epinephrine and Nor their role regulating the mitochondrial function in car- levels [5, 6]. The inhibition of these neurohormonal sys- diac cells. tems has demonstrated a consistent reduction in morbid- ity and mortality in patients with systolic HF and is the Results basis of modern pharmacological treatment [7, 8]. We designed a two-step experimental approach. First, the However, these effective therapies have failed to pro- effect of HF over several molecules involved in iron regu- mote a complete remission of symptoms and restore lation was evaluated in hearts from a well-established life expectancy in many patients. This has motivated an isoproterenol-induced HF mice [24]. Second, the mecha- increasing interest in emerging therapeutic targets. In nisms linking iron regulation, energy metabolism and this regard, iron deficiency (ID) is present in up to 50% mitochondrial function, were further explored in depth of HF patients [9, 10] and is associated with a higher risk in heart-derived cell cultures H9c2 exposed to the neuro- of mortality and hospitalization [10, 11], reduced func- hormonal activation typically found in HF. tional capacity, and impaired health-related quality of life [12], independent of the presence of anemia [10, 12, 13]. Changes in the gene expression and protein levels of iron Interestingly, intravenous iron supplementation has been regulatory molecules in the heart of isoproterenol‑induced recommended by European Society of Cardiology (ESC) HF mice HF guidelines as a potential therapeutic approach, since First, we analyzed the cardiomyocyte area on histological the correction of ID with intravenous iron alleviates HF sections from mice hearts (Fig. 1a). As it is shown in the symptoms, reduces risk of hospitalization and improves Fig. 1, isoproterenol induced cellular hypertrophy, com- quality of life [14–16]. pared to control animals (Fig. 1a, b). Although ID has been mainly considered as an extra- Both, the mRNA and protein levels of the main mol- cardiac co-morbidity complicating the course of HF, ecules related to cellular iron uptake, release and stor- recent data suggest that it may actually interact directly age, were analyzed in HF and control mice. First, it was with the mechanisms involved in HF initiation and pro- determined the two iron regulatory proteins (Irp1 and gression [17–20]. Previous studies from our group sug- gested an association between HF neurohormonal activation and systemic ID [17]. It has been shown that in chronic HF patients, the increase in the sympathetic activity (measured by Nor systemic levels) was associ- ated with ID, independently of anemia [17]. In addition, Maeder et al. suggested that the sympathetic activation found in HF patients may contribute to iron depletion, specifically in heart, since Nor stimulation may reduce the intracellular iron levels in cardiac cells by downreg- ulating the transferrin receptor 1 (Tfr1) expression [18]. Other studies have shown that iron content in myocardial was reduced by 20–30% in patients with advanced HF [21]. Apart from its role in oxygen transport, iron plays an important role in cellular metabolism, impacting on the regulation of oxidative stress and ATP synthesis, as an enzymatic cofactor in the mitochondria [19, 22]. Radi- cal oxygen species (ROS) directly impacts on the mito- Fig. 1 Isoproterenol induces cardiac hypertrophy. a Masson’s chondrial ­Ca2+ homeostasis contributing to an altered Trichrome staining of sections from ventricular myocardial of control contractibility and increasing the cardiac wall stiffness (Ct) and isoproterenol (Iso) stimulated mice. b Quantification of [23]. Hence, iron homeostasis is crucial for cells with cardiomyocytes area. Data were expressed as mean ± SEM (n = 8 per high energy demand, such as cardiomyocytes [10, 19]. group) (***p < 0.001 vs control) Tajes et al. Cell Biosci (2021) 11:89 Page 3 of 18 2), which regulate the translation of several proteins both Ang II and Nor induced an increase in the mRNA involved in iron metabolism [21]. Irp1 (Fig. 2a) and 2 levels of Bnp and Myh7 (Fig. 6a). Similar results were (Fig. 2b) were down-regulated in mice stimulated with found when the MYH7 protein levels were analyzed isoproterenol compared to control animals. Next, the lev- (Fig. 6b). els of the main iron transporters into the cell, Tfr1 and divalent metal transporter 1 (Dmt1), were also deter- Neurohormonal activation induced changes mined. Whereas Dmt1 was unaltered (Fig. 2c), Tfr1 was in the intracellular iron metabolism molecules in H9c2 cells reduced in the hearts of HF mice (Fig. 2d). The same molecules studied in mice were subsequently In order to look deeper into the role of the molecules assessed in the in vitro neurohormonal activation model. related to the cellular iron release, ferroportin 1 (Fpn1) Irp1 and 2 were significantly reduced when cells were (Fig. 3a), and its inhibitor, hepcidin antimicrobial pep- exposed to all the experimental conditions (Fig. 7a, b). In tide (Hamp) (Fig. 3b) were analyzed. In the isoprotere- addition, Tfr1 and Dmt1 were down-regulated in the set- nol-stimulated mice, the levels of both molecules were ting of any of the neurohormonal challenges (Fig. 8a, b). down-regulated when compared to control animals. With respect to cellular iron release, whereas Fpn1 Finally, the cytoplasmic iron storage molecule ferri- mRNA levels were reduced under Ang II or Nor treat- tin, encoded by ferritin heavy chain 1 (Fth1) and ferri- ment alone, there were no changes in its expression tin light chain 1 (Ftl1), was analyzed. No changes were when cells were simultaneously treated with both stimuli found between the experimental groups in the Fth1 and (Fig. 9a, left). Fpn1 protein levels did not change in any Ftl1 mRNA (Fig. 3c), neither in the Ferritin protein lev- experimental condition (Fig. 9b, left). On the other hand, els (Fig. 3d). while Hamp was induced by Ang II treatment, its levels were reduced under the influence of Nor and simulta- neous treatment with Nor and Ang II (Fig. 9a, right and Regulation of mitochondrial iron metabolism molecules Fig. 9b right). in the heart of HF mice Finally, although the stimulation with Nor produced a Due to the role of iron in mitochondrial function, the slight reduction in the Fth1 expression, there were no sig- mitochondrial iron uptake transporters Mitoferrin 1 nificant changes when exposing cardiac cells to Ang II or (Mfrn1) (Fig. 4a) and 2 (Mfrn2) (Fig. 4b), and the mito- both stimuli at the same time (Fig. 10a, right). Also, no chondrial iron storage molecule Mitochondrial ferri- significant changes in Ftl1 expression were found in any tin (Ftmt) (Fig. 4c) were analyzed. Whereas the Mfnr1 experimental condition (Fig. 10a, left). Accordingly, no was not modified, the Mfrn2 was down-regulated in the significant changes were found in the cytoplasmic Ferri- hearts from the HF animals. In addition, Ftmt was also tin protein levels (Fig. 10b). down-regulated in hearts from isoproterenol-stimulated mice. Neurohormonal activation depleted intracellular iron in H9c2 cells Intracellular iron depletion in the heart Beyond the effects of neurohormonal activation on the of isoproterenol‑induced HF mice molecules involved in iron metabolism, its effect on the Finally, the total heart iron (Fig. 5a), as well as the iron intracellular iron levels was assessed in H9c2 cells. As it ions ­Fe2+ (Fig. 5b) and ­Fe3+ (Fig. 5c), were analyzed in is shown in Fig. 11, the total iron (Fig. 11a), as well as the mice. As it is shown in Fig. 5, the iron ions were reduced ­ e2+ (Fig. 11b) and F iron ions F ­ e3+ (Fig. 11c), were reduced in the hearts from the animals stimulated with isoproter- in the cells challenged with Nor, Ang II, or their combi- enol compared to control ones. nation, when compared to control cells. Neurohormonal stimulation induced hypertrophy in H9c2 Neurohormonal activation promoted down‑regulation cells of Ftmt levels in H9c2 cells To further explore the role of neurohormonal activation The exposure to Nor and/or Ang II did not induce in iron metabolism at cellular level, an in vitro cardiac changes in the levels of the mitochondrial iron trans- cell model was studied. Since myocardial hypertrophy is porters Mfrn1 and Mfrn2 (Fig. 12a, b). Interestingly, the a hallmark of HF [3], some of the classical markers of mRNA and protein levels of the Ftmt were reduced in all cardiac hypertrophy (Bnp and Myh7) [25] were assessed the experimental conditions (Fig. 12c, d). in the H9c2 cells stimulated with the neurohormonal stimuli (Ang II and/or Nor). As it is shown in Fig. 6, Tajes et al. Cell Biosci (2021) 11:89 Page 4 of 18 Fig. 2 Isoproterenol-induced HF mice heart presents changes in iron regulatory molecules. Analysis of (a) Irp1 mRNA and protein levels; b Irp2 mRNA and protein levels; c Dmt1 mRNA and protein levels; and d Tfr1 mRNA and protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA or protein levels and expressed as mean ± SEM (n = 7 per group for mRNA; n = 5 per group for protein); (*p < 0.05 and ** p < 0.01 vs control) Tajes et al. Cell Biosci (2021) 11:89 Page 5 of 18 Fig. 3 Isoproterenol-induced HF mice heart presents changes in release and storage iron regulatory molecules. Analysis of (a) Fpn1 mRNA and protein levels, b Hamp mRNA and protein levels; c Fth1 and Ftl1 mRNA levels; and d Ferritin protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA or protein levels and expressed as mean ± SEM (n = 7 per group for mRNA; n = 5 per group for protein); (*p < 0.05 and **p < 0.01 vs control) Tajes et al. Cell Biosci (2021) 11:89 Page 6 of 18 Fig. 4 Isoproterenol-induced HF mice myocardial tissue presents changes in mitochondrial iron regulatory molecules. Analysis of (a) Mfrn1 mRNA and protein levels, b Mfrn2 mRNA and protein levels, c Ftmt mRNA levels and protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA protein levels and expressed as mean ± SEM (n = 7 per group for mRNA; n = 5 per group for protein); (*p < 0.05 and *** p < 0.001 vs control) Neurohormonal activation impaired mitochondrial (Fig. 13b), along with a decrease in the ATP levels function in H9c2 cells (Fig. 13c), regardless of the type of stimuli. Several parameters were assessed to characterize mito- chondrial function in the neurohormonal activation in vitro model. In the stimulated cells, the mitochon- Discussion drial membrane potential was reduced (Fig. 13a). Addi- ID has been associated with adverse outcomes in HF tionally, there was an increase in the ROS production [26, 27]. Despite the clinical association between HF and ID, it is currently unknown whether intracellular Tajes et al. Cell Biosci (2021) 11:89 Page 7 of 18 iron levels and impaired mitochondrial function in car- diac cells. Although the unequivocal benefits of intravenous iron may be related to a peripheral effect [14, 28], recent stud- ies support a cardiac-centered hypothesis. First, ferric carboxymaltose administration was associated with car- diac iron repletion in cardiac magnetic resonance T2* and T1 mapping sequences [29]. Second, Doppler and strain rate echocardiography parameters were signifi- cantly improved after intravenous iron administration in patients with stable systolic HF and ID without ane- mia [30]. Third, recent data in patients with HF have correlated ID and reduced peak exercise capacity with impaired myocardial contractile reserve [31]. We set up a study with both in vivo and in vitro approaches. First, we used a well-established HF mice model induced by isoproterenol (β-adrenoceptor ago- nist) osmotic pumps [24, 32]. Interestingly, these animals showed an increase of Ang II in blood and heart [33], thereby being a good model to study the effect of neuro- hormonal activation. Moreover, the results found in the in vivo model were validated in H9c2 cardiac cells chal- lenged with AngII, Nor or both, in order to mimic the neurohormonal activation with more than just on stimuli [3, 6]. To validate the neurohormonal activation cell model, we analyzed the expression of cardiac hypertrophy mol- ecules (Bnp and Myh7) as hallmarks of HF [3, 34]. Our neurohormonal activation in vitro model induced both mRNA and protein levels of cardiac hypertrophy markers in all the experimental conditions. Nevertheless, no syn- ergistic effect was observed in cells stimulated with both stimuli simultaneously, possibly because the stimulation with each of them separately already achieved the maxi- mum effect. Similarly, isoproterenol also induced cardio- myocyte hypertrophy in mice. Our data showed that the neurohormonal activation was related to a reduction of the intracellular iron lev- els (both F­ e2+ and F ­ e3+), thereby suggesting that neuro- hormonal stimuli may contribute to cardiac alteration Fig. 5 Iron content depletion in the myocardial tissue of through intracellular ID. Interestingly, previous stud- isoproterenol-induced HF mice. Analysis of intracellular iron levels in hearts: (a) total iron content, b ­Fe2+ ion content and c ­Fe3+ ion ies reported iron depletion in myocardial tissue of HF content. Data were normalized by protein levels and expressed as patients [18, 22] and highlighted the relevance of neuro- mean ± SEM (n = 5 per group); (**p < 0.01 vs control) hormonal activation leading to iron depletion. At a cardiac cell level, ID may be due to several mecha- nisms involving iron uptake, release and storage. Interest- ID in HF-cardiac cells is a consequence of the disease ingly, Irps are able to regulate the translation of different or, otherwise, directly takes part in the cellular altera- molecules related to these processes depending on iron tions leading the pathology. In the present study we levels [35]. As a matter of fact, Haddad et al. showed that demonstrated that neurohormonal activation related Irps are crucial to maintain the iron supply in cardiomyo- to HF promoted significant abnormalities in the iron cytes and to prevent HF in mice [21]. Furthermore, the metabolism regulatory systems, reduced intracellular same study showed a decrease in the Irp activity from HF Tajes et al. Cell Biosci (2021) 11:89 Page 8 of 18 Fig. 6 Neurohormonal activation induces hypertrophy in H9c2 cells. Analysis of (a) Myh7 and Bnp mRNA levels. b Western blot showing MYH7 protein levels. Data were normalized by the Gapdh mRNA (a) or protein (b) levels and expressed as mean ± SEM (n = 7 per group); (*p < 0.05, **p < 0.01 and *** p < 0.001 vs control) patients, related to a depletion of iron in the heart [21]. in hearts from isoproterenol-induced HF mice. As Kobak Accordingly, our study evidenced a down-regulation of et al. recently reviewed, Dmt1 uptake could act as a com- the Irps in both of our experimental models. Next, we pensatory mechanism of the Tfr1-mediated iron import assessed the levels of Tfr1, which is involved in the tran- [39]. ferrin-bound iron intake [36]. Xu et al. showed that the Neurohormonal stimuli also regulate the molecules lack of Tfr1 in knock-out mice produced a lethal cardio- involved in the cellular iron release, Fpn1 and Hamp. In myopathy by diminishing the intracellular iron content the heart, Hamp participates in the local iron homeo- and impairing mitochondrial function [37]. Besides, the stasis regulation by inhibiting Fpn1 function [40]. Fpn1 levels of Tfr1 were found reduced in the myocardial tis- exports intracellular iron outside the cell [41]. Deregu- sue of HF patients [18, 21]. Interestingly, Maeder et al. lations that affect the homeostasis of the Fpn1/Hepci- demonstrated that cardiomyocytes treated with aldoster- din axis are known to produce iron overload or anemia, one or Nor showed a decrease in the Tfr1 levels [18]. In depending on the direction of the functional changes line with these studies, we found that Tfr1 was decreased [42]. In the studied models we found different behav- in both hearts of HF mice and in the cardiac cells, in the iors when analyzing these components. Whereas Fpn1 setting of any of the neurohormonal challenges. Finally, mRNA levels were reduced under AngII and Nor stim- although the Dmt1, related to the non-transferrin bound ulation, the combination of both stimuli not induces iron uptake [38], was slightly down-regulated in the neu- changes in the expression of this gene in cardiac cells. rohormonal-stimulated cells, we found no modifications In contrast, in the isoproterenol treated mice, we Tajes et al. Cell Biosci (2021) 11:89 Page 9 of 18 Fig. 7 Neurohormonal activation modifies iron metabolism related molecules in H9c2 cells. Analysis of (a) Irp1 (left) and Irp2 (right) mRNA levels and (b) IRP1 (left) and IRP2 (right) protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA (a) or protein (b) levels and expressed as mean ± SEM (n = 7 per group); (*p < 0.05, **p < 0.01 and *** p < 0.001 vs control) observed a decreased of Fpn1 compared to the control iron by Fpn1. Nevertheless, Hamp was upregulated in mice. On the other hand, Hamp was downregulated the cells challenged only with Ang II. Interestingly, the in isoproterenol-stimulated mice, as well as in cells expression of Hamp is up-regulated in iron-deprived challenged with Nor and both stimuli together. There- environments to secure the intracellular iron content fore, although the Fpn1 levels decrease in the HF mice by exerting a negative feed-back on Fpn1 in the car- model, probably as an attempt to stop the iron release, diomyocytes [40]. Our data suggest that while Ang II- the reduced Hamp levels could favor the release of stimulated cells would try to retain the intracellular Tajes et al. Cell Biosci (2021) 11:89 Page 10 of 18 Fig. 8 Neurohormonal activation modifies iron uptake molecules in H9c2 cells. Analysis of (a) Tfr1 (left) and Dmt1 (right) mRNA levels; b TFR1 (left) and DMT1 (right) protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA (a) or protein (b) levels and expressed as mean ± SEM (n = 7 per group); (*p < 0.05, **p < 0.01 and ***p < 0.001 vs control) iron by increasing the levels of Hamp, the exposure to On the other hand, our study did not evidence a more intense neurohormonal insult would counteract changes in the cytoplasmic iron storage molecule fer- this compensatory mechanism, and could favored the ritin by the neurohormonal activation stimuli (in any iron release. To note, the Hamp knock-out mice induce experimental condition). It is worth to note that ferritin myocardial ID and dilated cardiomyopathy due to an may be further regulated by mechanisms others than increase of iron release [42]. Irps, such as oxidative stress [43]. Tajes et al. Cell Biosci (2021) 11:89 Page 11 of 18 Fig. 9 Neurohormonal activation modifies iron released molecules in H9c2 cells. Analysis of (a) Fpn1 (left) and Hamp (right) mRNA levels; (b) FPN1 (left) and HAMP (right) protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA (a) or protein (b) levels and expressed as mean ± SEM (n = 7 per group); (*p < 0.05, **p < 0.01 and ***p < 0.001 vs control) Altogether, our results suggest that neurohormonal intracellular iron deprivation. However, further stud- activation may contribute to intracellular iron depletion ies should be conducted to show the exact mechanisms by both increasing intracellular iron release and reduc- underlying the neurohormonal regulation of these com- ing extracellular iron uptake in cardiac cells. It means ponents in the failing heart. that the neurohormonal activation observed in HF The role of iron in cardiac function is closely related patients could participate in disease progression through to the mitochondria because of its role as an enzymatic Tajes et al. Cell Biosci (2021) 11:89 Page 12 of 18 Fig. 10 Neurohormonal activation does not modify ferritin in H9c2 cells. Analysis of (a) Ftl1 (left) and Fth1 (right) mRNA levels; (b) Ferritin protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA (a) or protein (b) levels and expressed as mean ± SEM (n = 7 per group); (*p < 0.05 vs control) cofactor, and the role of mitochondrial dysfunction in cardiac cells more sensitive to oxidative stress. This HF is being increasingly recognized [44, 45]. Due to hypothesis is further supported by other studies showing the importance of mitochondria in cardiomyocytes, the decrease of antioxidant enzymes in hearts from HF we assessed the levels of the mitochondrial iron uptake patients [22]. transporter Mfrn and the iron storage molecule Ftmt. Our data revealed that neurohormonal activation Mfrn1 and Mfrn2 were not altered in the neurohormo- increased ROS production and reduced mitochondrial nal-stimulated cells, although in hearts from isopro- membrane potential in cardiac cells, showing impairment terenol-induced mice the Mfrn2 was down-regulated. in the mitochondria. Mitochondria are crucial to main- Paradkar et al. showed that the reduction in Mfrn1 and tain the high-energy demand of the heart [10, 22]. Actu- Mfrn2 by RNA interference resulted in a decreased mito- ally, mitochondrial function has been found impaired chondrial iron accumulation, heme synthesis and iron- in myocardial tissue of advanced HF patients [10]. The sulfur cluster synthesis [46]. Interestingly, Mfrn2 is more role of myocardial and cardiomyocyte iron depletion ubiquitously expressed than Mfrn1. Besides, we observed leading to mitochondrial malfunction has been high- a down-regulation of the Ftmt levels in all the experimen- lighted as one of the drivers impairing contractile func- tal conditions. Our data are in line with those reported tion [19]. Accordingly, mitochondrial dysfunction found in hearts from ­Ftmt−/− mice, since these animals showed in the cells challenged with the neurohormonal stimuli mitochondrial damage and fibril disorganization [40]. was related to a decrease in the ATP levels. Altogether, Moreover, given the antioxidant role of Ftmt [47], neu- our results are in line with the data reviewed by Brown rohormonal activation may directly contribute to make Tajes et al. Cell Biosci (2021) 11:89 Page 13 of 18 intracellular iron deficiency and the mitochondrial dys- function at cardiac cell level (Fig. 14). Conclusions In conclusion, our data provide evidences that the neu- rohormonal activation observed in the setting of HF pro- motes a reduction in intracellular iron levels within cardiac cells and hampers mitochondrial function. The molecu- lar mechanisms mediating this relationship may involve a transcriptional regulation of several iron metabolism genes in the cell. The current results suggest that ID could be a key element in the pathophysiological sequence that leads to the progression of HF. Nevertheless, further research is necessary to fully characterize the role of iron in the patho- physiology of HF. Materials and methods Animal model Sixteen 10-week-old male C57BL/6 mice received a con- tinuous infusion of isoproterenol (ISO group; n = 8) or vehicle (saline) (Control group; n = 8) at a rate of 30 mg/kg/ day for 28 days using a subcutaneously implanted osmotic mini-pump (Alzet, model 1004), to generate a validated model of experimental HF in mice [24]. The animals were anesthetized by isoflurane inhalation during the implan- tation pump. Buprenorphine (0.3 mg/kg, i.p.) was admin- istered 10 min before surgery and after 24 h. Mice were euthanized by an i.p. injection of pentobarbital sodium (100 mg/kg). The experimental protocol was approved by the local Institutional Ethics Committee of the Institut Municipal d’Investigacions Mèdiques-Universitat Pompeu Fabra (CEEA-PRBB 16-1814I) and all animal procedures performed according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Heart samples were fixed in buffered 4% paraformalde- hyde, embedded in paraffin and cut into 4 µm-thick slices. Heart sections were deparaffinized and rehydrated with xylene, etanol (100%, 90%, 70%) and water, and stained with Masson’s Trichrome Stain Kit (Polyscience) to assess cardiomyocyte hypertrophy. Representative ventricular photomicrographs (5–10 per animal) were acquired at 400 Fig. 11 Neurohormonal activation depletes intracellular iron in × magnification with a light microscope (BX61, Olympus) H9c2 cells. Analysis of intracellular iron levels: (a) total iron content, b ­Fe2+ ion content and (c) ­Fe3+ ion content. Data were normalized and a mounted digital camera (DP72, Olympus). LV cardi- by protein levels and expressed as mean ± SEM (n = 7 per group); omyocyte area was measured in at least 30 random cardio- (*p < 0.05 and **p < 0.01 vs Control) myocytes by outlining round to cuboidal-shaped nucleated cardiomyocites using the ImageJ software. et al. suggesting that mitochondrial impairment could be Cell culture model linked to cardiomyocyte injury and HF progression [45]. Embryonic rat heart-derived cells (H9c2 cells) were main- Finally, Fig. 14 summarized the key results obtained tained in a high-glucose Dulbecco’s modified Eagle’s in this work, showing the effect of neurohormonal acti- medium (4.5 g/l glucose) (DMEM; supplemented with vation over the main iron metabolism molecules, the 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ Tajes et al. Cell Biosci (2021) 11:89 Page 14 of 18 ml streptomycin) at 37 °C and 5% ­CO2. Cells were seeded phosphatase and protease inhibitors (Roche Diagnos- in multi-well plates and serum deprived in DMEM sup- tics; Basel, Switzerland) and western blot analyses were plemented with 1% fetal bovine serum, 100 U/ml penicillin performed using antibodies against MYH7, TFR1, FT, and 100 μg/ml streptomycin for 16 h, and then stimulated HAMP, FTMT and GAPDH from Abcam (USA), and with 1 µM of Ang II (Sigma Aldrich, Spain) or/and 2 µM FPN1, IRP1, IRP2, MFRN1, MFRN2 and DMT1 from of Nor (Sigma Aldrich, Spain) for 48 (n = 7 in each experi- Thermo Scientific (Spain). Detection was performed mental group). using the appropriate horseradish peroxidase (HRP)-con- jugated secondary antibody (Dako; Glostrup, Denmark). RNA preparation and quantitative real time reverse The bands were visualized using ClarityTM Western transcription‑polymerase chain reaction (RT‑PCR) analysis ECL Substrate (BioRad, Spain) with the Quantity One Briefly, levels of mRNA were assessed by RT-PCR. Total software (BioRad, Spain). Differences in the protein lev- RNA was isolated from H9c2 cells and mice heart using els were expressed as arbitrary units (A.U.) percentage Nucleospin RNA II kit (Macherey–Nagel, Spain). RNA induction over control. was quantified by a NanoDrop 1000 Spectrophotom- eter (Thermo Scientific, Spain). TaqMan gene expression Intracellular iron determination assays-on-demand (Thermo Scientific, Spain) were used The intracellular iron ions content was determined in for rat myosin heavy chain 7 (Myh7) (Rn00691731_m1), hearts and H9c2 cells using the colorimetric Iron Assay rat natriuretic peptide B (Bnp) (Rn00580641_m1), rat iron kit (Abcam, Spain), following the manufacturer’s instruc- regulatory protein 1 (Irp1) (Rn00569045_m1) and mouse tions. All assays were performed in duplicate, and Irp1 (Mm01223514_m1), rat iron regulatory protein 2 measured on a Tecan Infinite F200 microplate reader. (Irp2) (Rn00575852_m1) and mouse Irp2 (Mm01179595_ ­ e2+ and F Levels of total iron, as well as the ions F ­ e3+, m1), rat ferroportin (Fpn1) (Rn00591187_m1) and mouse were expressed relative to untreated controls. Fpn1 (Mm01254822_m1), rat hepcidin antimicrobial peptide (Hamp) (Rn00584987_m1) and mouse Hamp Mitochondrial membrane potential (Mm00519025_m1), rat transferrin receptor 1(Tfr1) The mitochondrial membrane potential was determined (Rn01474701_m1) and mouse Tfr1 (Mm01344485_m1), using the reagent TMRE-Mitochondrial Membrane rat divalent metal transporter 1 (Dmt1) (Rn01533109_ Potential Assay Kit (Abcam; Spain). All assays were per- m1) and mouse Dmt1 (Mm01308330_s1), rat ferri- formed in duplicate, and measured on a Tecan Infinite tin heavy chain 1 (Fth1) (Rn00820640_g1) and mouse F200 microplate reader. Levels of fluorescence were Fth1 (Mm00850707_g1), rat ferritin light chain 1 (Ftl1) expressed relative to untreated controls. (Rn04341729_g1) and mouse Ftl1 (Mm03030144_g1), rat mitochondrial ferritin (Ftmt) (Rn01492073_s1) and mouse Ftmt (Mm01268428_s1), rat mitoferrin 1 (Mfrn1) Intracellular radical oxygen species (ROS) determination (Rn01753423_m1) and mouse Mfrn1 (Mm00471133_ Intracellular ROS levels were determined using Cellular m1), rat mitoferrin 2 (Mfrn2) (Rn01411393_m1) and Reactive Oxygen Species Detection Assay Kit (Abcam; mouse Mfrn2 (Mm01199497_m1). Glyceraldehyde- Spain). All assays were performed in duplicate, and meas- 3-phosphate dehydrogenase (Gapdh) was used as the ured on a Tecan Infinite F200 microplate reader. Levels of endogenous control, rat (Rn01775763_g1) and mouse ROS were expressed relative to untreated controls. (Mm99999915_g1). The results were normalized to Gadph, and relative quantification was performed using Detection of adenosine triphosphate (ATP) content the comparative Ct (2-DDCt) method. mRNA levels were Intracellular ATP content was determined by the ATP expressed as fold induction over control. Assay kit (Abcam; Spain). All assays were performed in duplicate, and measured on a Tecan Infinite F200 micro- Immunoblotting plate reader. Levels of ATP were expressed relative to Whole protein extracts were collected from H9c2 cells untreated controls. in RIPA buffer (0.1% SDS, 150 mM NaCl, 1% Nonidet P40, 50 mM Tris–HCl, 0.5% deoxycholate) containing (See figure on next page.) Fig. 12 Neurohormonal activation modifies mitochondrial iron storage related molecules in H9c2 cells. Analysis of (a) Mfrn1 and Mfrn2 mRNA levels, (b) MFRN1 and MFRN2 protein levels, (c) Ftmt mRNA levels and (d) FTMT protein levels. Representative Western Blot image of all the analyzed proteins. Data were normalized by the Gapdh mRNA (a, c) or protein (b, d) levels and expressed as mean ± SEM (n = 7 per group); (*p < 0.05 and **p < 0.01 vs Control) Tajes et al. Cell Biosci (2021) 11:89 Page 15 of 18 Tajes et al. Cell Biosci (2021) 11:89 Page 16 of 18 Fig. 14 Changes in iron metabolism due to neurohormonal activation in cardiac cells. This illustration describes the key iron molecules modified under neurohormonal activation in cardiac cells. It is also defined the iron deficiency and the mitochondrial dysfunction present under the neurohormonal stimuli. ATP adenosine triphosphate, Dmt1 divalent metal transporter 1, Fe iron, Fpn1 ferroportin 1, Ftmt mitochondrial ferritin, Hamp hepcidin, Irp1/2 iron regulatory proteins ½, ROS reactive oxygen species, Tf-Fe transferrin-bound iron, Tfr1 transferrin receptor 1 (ANOVA), followed by Bonferroni’s post hoc test, as appropriate. Data were analysed by using the GraphPad Instat programme (GraphPad Prism 6.01 Software Inc., USA). Differences were considered statistically signifi- cant at P-value < 0.05. Abbreviations HF: Heart failure; Bnp: Brain natriuretic peptide; Irps: Iron regulatory proteins; Fth1: Ferritin heavy chain 1; Ftl1: Ferritin light chain 1; Hamp: Hepcidin anti‑ microbial peptide; Tfr1: Transferrin receptor 1; Myh7: Myosin heavy chain 7; Dmt1: Divalent metal transporter 1; AngII: Angiotensin II; Nor: Norepinephrine; ID: Iron deficiency; RNA: Ribonucleic acid; ROS: Radical oxygen species; Fpn1: Ferroportin 1; Ftmt: Mitochondrial ferritin; Mfrn: Mitoferrin; ATP: Adenosin triphosphate. Acknowledgements We thank CERCA Programme/Generalitat de Catalunya for institutional support. Authors’ contributions Fig. 13 Neurohormonal activation impaires mitochondrial function. Conceptualization, MT, CDL, CE, PM and JCC; methodology, MT, CDL, LY, SGS, Quantification of different parameters: (a) Mitochondrial membrane JLF, and JCC; validation, MT, CE, PM, SGS, LY, BB and JCC; formal analysis, MT, CDL, CE, PM and JCC; investigation, MT, CDL, CE, PM, JGC and JCC; resources, potential, b intracellular ROS production, and (c) ATP levels. Data MT, CE and JCC; writing—original draft preparation, MT, CDL and JCC; writ‑ were normalized by the cellular protein content and expressed ing—review and editing, MT, CDL, CE, PM, AG, SJM, SY, LA, JGC, EGR, BB and as mean ± SEM (n = 7 per group); (**p < 0.01 and *** p < 0.001 vs JCC; supervision, MT, CE and JCC; project administration, MT, CE and JCC; control) funding acquisition, MT, CE, PM and JCC. All authors read and approved the final manuscript. Funding This work was funded by the following Grants: unrestricted grant from Vifor Statistical analysis Pharma and Basic Research Competitive Grant in Cardiology from the Spanish Data are expressed as the mean ± standard error of the Society of Cardiology 2015. mean (SEM). Significant differences were established Availability of data and materials using Student’s t-test or one-way analysis of variance All relevant data are included in this published article. Tajes et al. Cell Biosci (2021) 11:89 Page 17 of 18 Declarations and hospitalizations: insights from an observational cohort study. BMC Cardiovasc Disord. 2018;18:206. Ethics approval and consent to participate 12. Enjuanes C, Bruguera J, Grau M, et al. Iron status in chronic heart failure: The experimental protocol was approved by the local Institutional Ethics impact on symptoms, functional class and submaximal exercise capacity. Committee of the Institut Municipal d’Investigacions Mèdiques-Universitat Rev Esp Cardiol (Engl Ed). 2016;69:247–55. Pompeu Fabra (CEEA-PRBB 16-1814I) and all animal procedures performed 13. Enjuanes C, Klip IT, Bruguera J, et al. Iron deficiency and health-related according to the guidelines from Directive 2010/63/EU of the European Parlia‑ quality of life in chronic heart failure: results from a multicenter European ment on the protection of animals used for scientific purposes. study. Int J Cardiol. 2014;174:268–75. 14. Anker SD, Comin Colet J, Filippatos G, et al. Ferric carboxymaltose Consent for publication in patients with heart failure and iron deficiency. N Engl J Med. Not applicable. 2009;361:2436–48. 15. Comin-Colet J, Lainscak M, Dickstein K, et al. The effect of intravenous Competing interests ferric carboxymaltose on health-related quality of life in patients with Dr. Josep Comín-Colet has received honoraria for speaking for Vifor Pharma chronic heart failure and iron deficiency: a subanalysis of the FAIR-HF and consultancy fees from Vifor Pharma. study. Eur Heart J. 2013;34:30–8. 16. Anker SD, Kirwan BA, van Veldhuisen DJ, et al. Effects of ferric carboxy‑ Author details maltose on hospitalisations and mortality rates in iron-deficient heart 1 Bio‑Heart Cardiovascular Diseases Research Group, Bellvitge Biomedical failure patients: an individual patient data meta-analysis. Eur J Heart Fail. Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain. 2 Cardi‑ 2018;20:125–33. ology Department, Bellvitge University Hospital, L’Hospitalet de Llobregat, Bar‑ 17. Moliner P, Enjuanes C, Tajes M, et al. Association between norepineph‑ celona, Spain. 3 Advanced Heart Failure and Heart Transplant Unit, Cardiology rine levels and abnormal iron status in patients with chronic heart Department, Bellvitge University Hospital, L’Hospitalet de Llobregat, Barcelona, failure: is iron deficiency more than a comorbidity? J Am Heart Assoc. Spain. 4 Community Heart Failure Program, Cardiology Department, Bellvitge 2019;8:e010887. University Hospital, L’Hospitalet de Llobregat, Barcelona, Spain. 5 Vascular 18. Maeder MT, Khammy O, dos Remedios C, Kaye DM. Myocardial and sys‑ Biology and Metabolism Program, Vall d’Hebron Research Institute (VHIR), temic iron depletion in heart failure implications for anemia accompany‑ Barcelona, Spain. 6 Cardiology Department, Hospital Vall d’Hebron Hospital, ing heart failure. J Am Coll Cardiol. 2011;58:474–80. Barcelona, Spain. 7 Department of Clinical Sciences, School of Medicine, Uni‑ 19. 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