New Insights into the Hepcidin-Ferroportin Axis and Iron Homeostasis in iPSC-Derived Cardiomyocytes from Friedreich’s Ataxia Patient

Hindawi Oxidative Medicine and Cellular Longevity Volume 2019, Article ID 7623023, 11 pages https://doi.org/10.1155/2019/7623023 Research Article New Insights into the Hepcidin-Ferroportin Axis and Iron Homeostasis in iPSC-Derived Cardiomyocytes from Friedreich’s Ataxia Patient Alessandra Bolotta ,1,2 Provvidenza Maria Abruzzo ,1,2 Vito Antonio Baldassarro ,3 Alessandro Ghezzo ,1 Katia Scotlandi,4 Marina Marini ,1,2 and Cinzia Zucchini 1 1 Department of Experimental, Diagnostic and Specialty Medicine, Bologna University, 40126 Bologna, Italy IRCCS Fondazione Don Carlo Gnocchi, 20148 Milan, Italy 3 Interdepartmental Centre for Industrial Research in Health Sciences and Technologies (ICIR-HST), University of Bologna, 40064 Ozzano, Bologna, Italy 4 CRS Development of Biomolecular Therapies, Experimental Oncology Laboratory, Orthopedic Rizzoli Institute, 40136 Bologna, Italy 2 Correspondence should be addressed to Provvidenza Maria Abruzzo; Received 11 September 2018; Accepted 4 December 2018; Published 27 March 2019 Guest Editor: Giorgos Sakkas Copyright © 2019 Alessandra Bolotta et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Iron homeostasis in the cardiac tissue as well as the involvement of the hepcidin-ferroportin (HAMP-FPN) axis in this process and in cardiac functionality are not fully understood. Imbalance of iron homeostasis occurs in several cardiac diseases, including iron-overload cardiomyopathies such as Friedreich’s ataxia (FRDA, OMIM no. 229300), a hereditary neurodegenerative disorder. Exploiting the induced pluripotent stem cells (iPSCs) technology and the iPSC capacity to differentiate into specific cell types, we derived cardiomyocytes of a FRDA patient and of a healthy control subject in order to study the cardiac iron homeostasis and the HAMP-FPN axis. Both CTR and FRDA iPSCs-derived cardiomyocytes express cardiac differentiation markers; in addition, FRDA cardiomyocytes maintain the FRDA-like phenotype. We found that FRDA cardiomyocytes show an increase in the protein expression of HAMP and FPN. Moreover, immunofluorescence analysis revealed for the first time an unexpected nuclear localization of FPN in both CTR and FRDA cardiomyocytes. However, the amount of the nuclear FPN was less in FRDA cardiomyocytes than in controls. These and other data suggest that iron handling and the HAMP-FPN axis regulation in FRDA cardiac cells are hampered and that FPN may have new, still not fully understood, functions. These findings underline the complexity of the cardiac iron homeostasis. 1. Introduction Iron is a trace metal essential for numerous biological processes. Its homeostasis is finely regulated, since both iron excess and deficiency are potential detrimental. In fact, iron excess favors the formation of oxygen radicals, while iron deficiency impairs enzyme functionality affecting oxygen metabolism. It has been demonstrated that the dysregulation of iron homeostasis is involved in different pathological conditions, including cancer, anemia, neurodegenerative disorders, and cardiac diseases [1]. Iron deficiency was found to occur in heart failure patients, independently of normal systemic iron concentration, causing morphological and functional mitochondrial alterations and consequently ATP depletion [2]. These dysfunctions, in turn, impair cardiac contractility and relaxation. Ironically, cardiomyopathy can be induced also by systemic iron overload, as in hereditary hemochromatosis (HH) and β-thalassemia, and by iron misdistribution in the cellular organelles, as in Friedreich’s ataxia (FRDA) [3]. Iron excess causes an alteration of systolic and diastolic functions through the decrease of L-type channel activity, essential for the heart contraction. In addition, at 2 the cellular level, iron misdistribution in cellular organelles, such as the mitochondria, can damage the cells through oxygen radical production. Cardiomyocytes, being endowed of poor antioxidant defenses, are more susceptible to reactive species of oxygen (ROS) damage via Fenton and Heiber-Weiss-typereactions [3, 4]. Iron homeostasis is regulated by several proteins involved in the iron uptake, transport, storage, and export. These proteins cooperate with ferrireductases, ferroxidases, and chaperones to regulate the cellular iron trafficking and to limit the unbound labile iron pool (LIP), potential source of ROS. Iron exists within heme molecules such as hemoglobin and cytochromes or in iron-sulfur cluster- (ISC-) containing proteins such as succinate dehydrogenase; moreover, nonheme/non-ISC iron-containing proteins are present in the cells [5]. Nonheme iron is transported into the cells by iron-binding proteins, such as transferrin. Cellular uptake of iron from transferrin is initiated by the binding of transferrin to transferrin receptor 1 (TFRC). TFRC is a transmembrane protein that assists iron uptake through receptor-mediated endocytosis of iron-loaded transferrin [5]. In addition, iron chaperones such as frataxin, a nuclear-encoded protein localized into the mitochondrial matrix, act as iron sensor and storage proteins as well as iron chaperons during cellular Fe-S cluster biosynthesis [6]. In iron homeostasis, a central regulatory mechanism is the binding of the hormone hepcidin (HAMP) to the iron exporter ferroportin (FPN). FPN is the only iron-exporting protein localized in the cell membrane; it was independently discovered by three different groups [7–9]. The FPN structure has not been completely defined; it is characterized by 9-12 transmembrane domains (TMs), organized into two six-helix halves, which are connected by a large cytoplasmic loop between the 6th and the 7th domain [10, 11]. Furthermore, whether the functional form of FPN is monomeric or dimeric remains an open question. Genetic and biochemical evidences support the dimeric form [12]. However, different groups reported that FPN is a monomer, and that, in this form, it is able to bind HAMP [13, 14]. Regulation of FPN occurs at multiple levels, transcriptional, posttranscriptional, and posttranslational. FPN expression is regulated at the transcriptional level by hypoxia inducible factor-2alpha (HIF2α) in response to hypoxia and inflammation; moreover, it is induced by iron heme and other metals. Posttranscriptionally, FPN synthesis is regulated by iron regulatory proteins (IRPs), which bind to an iron responsive element (IRE) located in its 5′UTR. In addition, posttranslational regulation of FPN is mediated by HAMP. HAMP binds FPN and triggers its internalization, ubiquitination, and subsequent lysosomal degradation [10, 11]. At systemic level, circulating HAMP is synthesized by the liver, where it is induced in iron overloading conditions and is inhibited by iron deficiency due to anemia, hypoxia, ineffective erythropoiesis, and inflammation [10, 11]. HAM (...truncated)