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Cardiomyocyte hepcidin: From intracellular iron homeostasis to physiological function
CHAPTER NINE
Cardiomyocyte hepcidin: From intracellular iron homeostasis to physiological function S. Lakhal-Littleton* Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom *Corresponding author: e-mail address: [email protected]
Contents 1. Expression of cardiac hepcidin 1.1 Baseline expression relative to hepatic hepcidin 1.2 Baseline protein expression 1.3 Changes in cardiac hepcidin expression in pathophysiology 1.4 Known mechanisms of cardiac hepcidin regulation 2. The functions of cardiomyocyte hepcidin 3. Conclusions and future directions References
189 190 190 192 194 195 197 198
Abstract Cellular iron is required for the utilization of oxygen in the cell. Iron in iron-sulfur and heme groups is required for electron transfer and oxygen activation in oxidative phosphorylation, while labile free iron is required for oxygen activation by dioxygenases, and as a catalyst for redox signaling. At the same time, this reactivity with oxygen underpins the production of cell-damaging free radicals in the presence of excess iron. Because the cardiac cell is a major site of oxygen flux, it requires tight control of intracellular iron levels. Until recently, such control was thought to be mediated predominantly by the action of iron regulatory proteins. However, new evidence reveals that cardiomyocyte hepcidin is indispensable for the control of intracellular iron levels, normal metabolism and heart function. This new evidence highlights the need for better understanding of the regulation of cardiomyocyte hepcidin in health and disease.
1. Expression of cardiac hepcidin Since the discovery of hepcidin at the turn of the century, iron homeostasis research has focused on characterizing the expression, regulation and functions of hepatic hepcidin. The liver is the tissue with the most Vitamins and Hormones, Volume 110 ISSN 0083-6729 https://doi.org/10.1016/bs.vh.2019.01.009
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2019 Elsevier Inc. All rights reserved.
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abundant levels of hepcidin mRNA, with expression comparable to that of the house house-keeping gene GAPDH (Krause et al., 2000). In addition, the phenotype of animal models lacking hepatic hepcidin closely resembles the iron-loading seen in hemochromatosis patients, suggesting that hepatic hepcidin is the dominant regulator of systemic iron homeostasis (Zumerle et al., 2014). For these reasons, hepcidin expression in non-hepatic tissues has received little interest from researchers. However, recent evidence has uncovered the importance of locally produced hepcidin in the heart for cardiac iron homeostasis and function (Lakhal-Littleton et al., 2016). In light of this new evidence, the study of the regulation of cardiac hepcidin in health and disease becomes scientifically and clinically important.
1.1 Baseline expression relative to hepatic hepcidin In humans, rodents and fish, the heart is the second highest site of hepcidin mRNA expression. Table 1 summarizes the levels of cardiac hepcidin expression reported in various species. Within the heart, all the evidence points at the fact that cardiac hepcidin is primarily derived from cardiomyocytes and not from non-cardiomyocyte cell types such as fibroblasts. Indeed, in mice with a cardiomyocyte-specific deletion of the hepcidin gene, total cardiac mRNA analysis showed an almost complete ablation of hepcidin expression compared to control hearts (Lakhal-Littleton et al., 2016). This is consistent with an earlier finding in rat hearts, where the isolated cardiomyocyte fraction was shown to contain most of the hepcidin mRNA (Isoda et al., 2010).
1.2 Baseline protein expression Only a limited number of studies have reported on the detection or quantitation of cardiac hepcidin protein. Studies in guinea pig and rat hearts detected an immunoreactive band at 8 kDa, consistent with the size of pro-hepcidin (Schwarz et al., 2009; Simonis et al., 2010). Another study using an antibody for the N-terminal region of the hepcidin peptide in rat heart extracts detected an immunoreactive band at 9.5 kDa, while immunostaining of rat hearts detected strong signal in the intercalated disc region of the cardiomyocyte (Merle et al., 2007). The significance of this subcellular localization is not fully explained. Only two other studies included hepcidin immunostaining in the heart, reporting a more diffuse distribution of hepcidin staining within the cardiomyocytes (LakhalLittleton et al., 2016; Naito et al., 2014). Detection of the pro-hepcidin
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Table 1 Levels of cardiac hepcidin mRNA expression reported in different species. Expression relative Detection to liver Reference Species method
Human
q-RT-PCR 1/15
Krause et al. (2000)
Human
Northern Blot
Park, Valore, Waring, and Ganz (2001)
Mouse (Balb/cJ)
q-RT-PCR 1/100
Not quantified
Mouse (C57BL/6) q-RT-PCR 1/30
Ilyin et al. (2003) Lakhal-Littleton et al. (2016)
Male Sprague Dawley rats (200–250 g)
q-RT-PCR 1/40
Merle, Fein, Gehrke, Stremmel, and Kulaksiz (2007)
Zebrafish
q-RT-PCT 1/10
Shike, Shimizu, Lauth, and Burns (2004)
Turbot
RT-PCR
Chen et al. (2007)
Adult sheep
q-RT-PCT 2.9 104 Badial et al. (2011)
Ayu (Plecoglossus altivelis)
q-RT-PCR Not reported
Not quantified
Crocodylus siamensis q-RT-PCR 1/300 Convict cichlid
q-RT-PCR 1/500
Chen, Chen, Lu, and Shi (2010) Hao, Li, Xie, and Li (2012) Chi et al. (2015)
peptide in tissue sections and tissue lysates does not constitute definitive evidence that cardiac cells secrete the active mature hepcidin peptide. Such evidence was however demonstrated in primary cardiomyocytes isolated from mice. It was shown that the hepcidin peptide could be detected by ELISA in supernatants of primary cardiomyocytes from control mice, but not from mice with a cardiomyocyte-specific deletion of the hepcidin gene (Lakhal-Littleton et al., 2016). That study also demonstrated that the release of the hepcidin peptide is dependent on the pro-hormone convertase Furin, also known to mediate the cleavage of pro-hepcidin in hepatocytes (Valore & Ganz, 2008). In summary, in most species studied, the heart is the second most abundant site of hepcidin mRNA expression. Experiments with isolated primary cardiomyocytes have also demonstrated that cardiac hepcidin is primarily derived from cardiomyocytes, and that it is secreted in a manner dependent on the pro-hormone convertase Furin.
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1.3 Changes in cardiac hepcidin expression in pathophysiology A number of studies have reported changes in cardiac hepcidin mRNA and protein in the disease setting and in experimental models of heart disease. Broadly speaking, cardiac hepcidin appears to be responsive to hypoxia, inflammation, heart disease, and changes in systemic iron balance. The expression of hepcidin mRNA and protein in male Sprague Dawley rats was shown to be upregulated after 24 h and 5 day of exposure to 6% or 8% O2 (Merle et al., 2007). In mice, 1 week exposure to milder hypoxia (11% O2) was shown to downregulate hepcidin mRNA but to upregulate hepcidin protein. The divergence of mRNA and protein responses to hypoxia was further confirmed in isolated primary cardiomyocytes exposed to 5% O2 (Lakhal-Littleton et al., 2016). Cardiac hepcidin expression was also shown to be upregulated in turpentine experimental models of inflammation. Cardiac hepcidin mRNA was significantly augmented in the myocardium within 6 h of turpentine injection in one study and at 16 h post injection in another (Merle et al., 2007; Sheikh, Dudas, & Ramadori, 2007). In both studies, upregulation of cardiac hepcidin is described as an acute inflammatory response rather than a secondary response to change in systemic iron availability. The responsiveness of cardiac hepcidin to inflammation and hypoxia, singly or in concert, may underlie its upregulation in heart disease. In humans, hepcidin mRNA is markedly upregulated in hearts with myocarditis, an inflammatory condition of the myocardium. This observation was replicated in a rat model of experimental autoimmune myocarditis, where hepcidin mRNA was shown to increase after 9 days. In that setting, its levels correlated most closely with IL-6 expression, suggesting that IL-6 may be the cause of hepcidin upregulation in the inflamed heart (Isoda et al., 2010). In acute myocardial infarction in the rat, hepcidin mRNA increased dramatically in the infarct lesion within 1 day. In that setting, upregulation of hepcidin mRNA was largely attributed to upregulation of IL-6 in the heart (Isoda et al., 2010). The role of hypoxia-inducible factors (HIFs) in that study was not adequately assessed because the study only measured HIF-1α mRNA, while HIF responsiveness to hypoxia is known to be driven largely by post-translational modifications and changes in HIF protein stability. Another study of myocardial infarction in rats also reported marked upregulation of hepcidin mRNA in the ischemic and remote myocardium, and of pro-hepcidin peptide in the ischemic myocardium only 6 h post infarction. In that study, serum hepcidin levels were also elevated (Simonis et al., 2010). Of note, this finding mirrors
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observation in patients with myocardial infarction, in whom serum levels of the mature hepcidin peptide (hepcidin-25) and of a shorter derivative lacking the N-terminus (hepcidin-20) were both elevated 4 h post infarction, and remained high for 7 days thereafter (Suzuki et al., 2009). Because the cardiac and hepatic hepcidin proteins are not distinguishable in serum, it is not possible to attribute the observed elevation in serum hepcidin to increased cardiac secretion. Nonetheless, the acute nature of this serum hepcidin response is consistent with a direct and local response of cardiac hepcidin to myocardial infarction, rather than a secondary response of hepatic hepcidin to trans-acting factors. Of note, upregulation of serum hepcidin did not appear to correlate with inflammatory markers, suggesting that mechanisms other than inflammation underlie the increase in serum hepcidin. A different study looking at hepcidin levels 16 weeks post coronary ligation in rats also found upregulated levels of cardiac hepcidin mRNA, which were most closely correlated to levels of brain natriuretic peptide BNP, suggesting a role for tissue injury in modulating cardiac hepcidin levels in this setting (van Breda et al., 2016). Another physiological stress that appears to affect cardiac hepcidin expression is high salt diet. Specifically, in Dahl salt-sensitive rats, which develop cardiac hypertrophy, hepcidin mRNA and protein were markedly increased after 12 weeks of high salt diet, and in a manner that was not correlated to cardiac iron content (Naito et al., 2014). This study also found a modest attenuation of phenylephrine-induced cardiomyocyte hypertrophy by hepcidin siRNA, suggesting that increased hepcidin levels following provision of high salt diet may contribute to hypertrophy. Hepatic hepcidin is acutely sensitive to changes in dietary iron content, with its protein levels closely mirroring changes in mRNA levels. Cardiac hepcidin, on the other hand, appears to respond differently from hepatic hepcidin to changes in dietary iron content. Provision of an iron-deficient diet in mice markedly decreases cardiac hepcidin mRNA, but increases its protein. Provision of an iron-loaded diet markedly increases hepcidin mRNA levels, but does not affect its proteins levels. These results were largely replicated in primary cardiomyocytes treated with the iron chelator desferroxamine (DFO) or with iron in the form of ferric citrate, suggesting that such responses were dependent on autonomously acting pathways rather than mediated by changes in serum iron levels. The lack of concordance between RNA and protein responses is attributed to the manner in which the hepcidin pro-peptide is processed in the heart (Lakhal-Littleton et al., 2016). This is discussed further in Section 1.4.
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In summary, cardiac hepcidin mRNA appears to respond to cardiovascular stresses, both acute (myocardial infarction, ischemia, inflammation) and chronic (hypertrophy, hypoxia, dietary iron deficiency and loading). The mechanisms underlying such responses, and their importance or otherwise to cardiac physiology, remain largely unexplored.
1.4 Known mechanisms of cardiac hepcidin regulation Other than correlative studies of cardiac hepcidin expression, there is little definitive evidence supporting specific mechanisms of cardiac hepcidin regulation. One mechanism that has been demonstrated is the Furin-dependent increase in cardiac hepcidin peptide secretion in response to hypoxia and iron deficiency (Lakhal-Littleton & Robbins, 2017; Lakhal-Littleton et al., 2016). It was found that increased secretion of hepcidin peptide was dependent on the activity of the pro-hormone convertase Furin. Importantly, the expression of Furin in the heart and in primary cardiomyocytes was also increased by hypoxia and iron deficiency. These conditions would both stabilize HIF protein by limiting the activity of the prolyl hydroxylases that target HIF protein for degradation in an oxygen- and iron-dependent manner. Consistent with this, Furin is a known HIF-target gene (Silvestri, Pagani, & Camaschella, 2008). Interestingly, as well as the processing of the hepcidin peptide, Furin in hepatocytes is also known to regulate hepcidin RNA expression, by cleaving the bone morphogenetic protein co-receptor, hemojuvelin. In this setting, hypoxia decreases hepcidin mRNA through Furin-mediated increase in hemojuvelin cleavage (Silvestri et al., 2008). That cardiac hepcidin secretion is upregulated rather than downregulated by Furin and hypoxia suggests that the hemojuvelin pathway may not operate to regulate hepcidin expression in cardiomyocytes, in the same manner that it does in hepatocytes. These observations pose broader questions over the differences between hepatic and cardiac hepcidins, in terms of transcriptional, translational and post-translational processing. In the liver, the consensus has been that hepcidin regulation is primarily achieved at the transcriptional level, meaning that protein measurements are not always conducted to support any observed changes in hepatic hepcidin mRNA levels. The work presented by Lakhal-Littleton et al. is the first example of opposing responses of the hepcidin transcript and protein to a given stimulus. Furthermore, the need for differential regulation between hepatic and cardiac hepcidins may reflect their distinct functions in relation to iron homeostasis. The function of cardiomyocyte hepcidin is discussed in Section 2.
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2. The functions of cardiomyocyte hepcidin The role of hepcidin as master regulator of systemic iron homeostasis was established over a decade ago. A number of subsequent studies established that it operated primarily through antagonism of the iron export protein ferroportin, at three important sites: duodenal enterocytes (the site of iron absorption), reticuloendothelial macrophages (the site of iron recycling) and hepatocytes (the site of iron storage) (Donovan et al., 2005; Nemeth et al., 2004). The use of liver-specific hepcidin knockout mice established that such function was primarily mediated by hepatic hepcidin, because the phenotype of such animals appeared to closely resemble that of ubiquitous hepcidin knockouts (Zumerle et al., 2014). Thus, the importance, if any, of non-hepatic hepcidins remained unexplored. Furthermore, such studies were not possible in the ubiquitous knockout mouse models that existed at the time, because of the confounding effects of altered systemic iron homeostasis, which would mask any effects of hepcidin in non-hepatic tissues. As hepcidin is a hormone that affects iron homeostasis through its target ferroportin, there was additionally the need to establish the exact site of action of non-hepatic hepcidins. In the context of the heart, two studies performed by Lakhal-Littleton et al. provided the first evidence of the importance of cardiomyocyte hepcidin and further identified the site of its action. In the first study, mice carrying a cardiomyocyte-specific deletion of the ferroportin gene were found to develop fatal left ventricular dysfunction by 3 months of age. The dysfunction was caused by a threefold increase in iron levels within cardiomyocytes. Importantly, downregulation of Transferrin receptor (TfR1) was not sufficient to prevent iron overload in ferroportin-deficient hearts, suggesting that ferroportin-mediated iron release is an essential component of iron homeostasis in the heart (LakhalLittleton et al., 2015). In the second study, mice carrying a cardiomyocytespecific deletion of hepcidin developed fatal left ventricular dysfunction between 3 and 6 months of age, despite the maintenance of normal systemic iron levels. A similar result was obtained in animals with cardiomyocytespecific knock-in of the ferroportin isoform C326Y, which retains its iron export function but loses its hepcidin binding. In both settings, the cardiomyocytes were found to be iron deficient due to increased iron export. Intravenous iron supplementation from 3 months of age prevented the development of cardiac dysfunction in cardiac-hepcidin knockouts, demonstrating the importance of cardiomyocyte iron deficiency in the cardiac dysfunction seen in these models (Lakhal-Littleton et al., 2016). Another
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important observation is that mice lacking cardiac hepcidin did not have reduced serum hepcidin levels, nor did they manifest any changes in hemoglobin, serum iron indices or liver iron stores. This observation is consistent with the notion that cardiomyocyte hepcidin, at least in the normal healthy heart, does not act in a paracrine manner. It also reflects the fact that, at such low levels of expression (relative to hepatic hepcidin), cardiomyocyte hepcidin is indeed only able to affect ferroportin locally within the heart. Taken together, these two studies demonstrate that the cardiac hepcidin/ ferroportin axis is essential for the cell autonomous control of the intracellular iron pool upon which normal cardiac function depends. Cardiomyocyte hepcidin also appears to protect the heart from the effects of systemic iron deficiency. Indeed, it was found that hepcidin-deficient hearts developed a greater hypertrophic response to sustained dietary iron restriction than their littermate controls. This finding, together with the observation that cardiac hepcidin protein is upregulated rather than downregulated by dietary iron restriction, indicates that cardiomyocyte hepcidin is part of cardioprotective mechanism that mitigates against reduced systemic iron availability by increasing retention of intracellular iron. Fig. 1 summarizes the current thinking around the function of cardiomyocyte hepcidin.
Fig. 1 The role of cardiomyocyte hepcidin in maintenance of intracellular iron levels.
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There is some evidence that cardiomyocyte hepcidin dysregulation may impinge on severity and progression of certain types of heart disease. In a mouse model of dilated cardiomyopathy, transgenic for the R141W mutation of the Troponin gene in cardiac cells, cardiac hepcidin levels were found to be less than half of those measured in normal control hearts. Furthermore, transgenic overexpression of hepcidin in the hearts of these mice reduced cardiac ferroportin levels, restored cardiac iron content to that of controls, and improved left ventricle morphology and ejection fraction. Interestingly, the effects of transgenic hepcidin overexpression were also associated with improvement of mitochondrial morphology (Zhang et al., 2012). The use of overexpression of the hepcidin transgene in the heart may not fully recapitulate the true physiological function of hepcidin. Nonetheless, the findings of this study are consistent with the idea that hepcidin acts in an autonomous manner to regulate cardiac ferroportin, and that such regulation is important for normal cardiac metabolism and function. Under normal conditions, cardiomyocyte hepcidin appears not to contribute to serum hepcidin levels or the control of systemic iron homeostasis. However, the finding that it is increased dramatically in the infarcted heart, and acutely in the serum of patients following myocardial infarction, suggests that it may have an effect on systemic iron homeostasis in this particular setting (Simonis et al., 2010; Suzuki et al., 2009). Clinical studies have shown that iron deficiency in acute heart disease correlates with poor prognosis ( Jankowska et al., 2014). It would be important to assess the extent to which cardiac hepcidin contributes to iron deficiency in acute heart failure. Iron deficiency and high serum hepcidin are also correlated with poor prognosis in chronic heart failure (Comin-Colet et al., 2013). In this setting it is assumed that the liver is the source of hepcidin, and that IL-6-driven inflammation is the cause. Experimental models have shown that cardiomyocyte hepcidin can be dramatically increased in the inflammatory setting (Merle et al., 2007; Sheikh et al., 2007). Therefore, it would be important to establish to what extent cardiomyocyte hepcidin accounts for iron deficiency in chronic heart failure.
3. Conclusions and future directions In most species, the heart is the second highest site of hepcidin expression. Within the heart, hepcidin appears to be expressed primarily in cardiomyocytes, at levels that are orders of magnitude lower that those seen
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in the liver. In the normal healthy heart, cardiomyocyte hepcidin is indispensable for control of intracellular iron. It does this through cell autonomous control of ferroportin-mediated iron release. This function is essential for maintenance of normal cardiac metabolism and contractile function. Cardiomyocyte hepcidin is regulated post-translationally by hypoxia and iron deficiency in a manner that is distinct from that of hepatic hepcidin. This distinct regulation is likely a cardioprotective mechanism, particularly in the setting of reduced systemic iron availability. Finally, cardiomyocyte hepcidin appears to be responsive to inflammation and local ischemia in the heart, raising the possibility that it may contribute to changes in local and systemic iron levels in that setting. Future studies are needed to address a number of pertinent questions relating to how the cardiac hepcidin gene is regulated, and how post-translational modifications fine-tune the amount of hepcidin secreted from the heart. Additionally, comparative structural studies need to be carried out to compare cardiac and hepatic hepcidin proteins, and predict any further differences relating to stability and potency. Finally, the possibility that newly developed hepcidin mimics (for the treatment of iron-overload) may alter cardiac iron levels directly by acting on cardiomyocyte ferroportin should not be overlooked.
References Badial, P. R., Oliveira Filho, J. P., Cunha, P. H., Cagnini, D. Q., Araujo, J. P., Jr., Winand, N. J., et al. (2011). Identification, characterization and expression analysis of hepcidin gene in sheep. Research in Veterinary Science, 90(3), 443–450. Chen, M. Z., Chen, J., Lu, X. J., & Shi, Y. H. (2010). Molecular cloning, sequence analysis and expression pattern of hepcidin gene in ayu (Plecoglossus altivelis). Dong wu xue yan jiu ¼ Zoological Research, 31(6), 595–600. Chen, S. L., Li, W., Meng, L., Sha, Z. X., Wang, Z. J., & Ren, G. C. (2007). Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot (Scophthalmus maximus). Fish & Shellfish Immunology, 22(3), 172–181. Chi, J. R., Liao, L. S., Wang, R. G., Jhu, C. S., Wu, J. L., & Hu, S. Y. (2015). Molecular cloning and functional characterization of the hepcidin gene from the convict cichlid (Amatitlania nigrofasciata) and its expression pattern in response to lipopolysaccharide challenge. Fish Physiology and Biochemistry, 41(2), 449–461. Comin-Colet, J., Enjuanes, C., Gonzalez, G., Torrens, A., Cladellas, M., Merono, O., et al. (2013). Iron deficiency is a key determinant of health-related quality of life in patients with chronic heart failure regardless of anaemia status. European Journal of Heart Failure, 15(10), 1164–1172. Donovan, A., Lima, C. A., Pinkus, J. L., Pinkus, G. S., Zon, L. I., Robine, S., et al. (2005). The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metabolism, 1(3), 191–200. Hao, J., Li, Y. W., Xie, M. Q., & Li, A. X. (2012). Molecular cloning, recombinant expression and antibacterial activity analysis of hepcidin from Simensis crocodile (Crocodylus siamensis). Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 163(3–4), 309–315.
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Ilyin, G., Courselaud, B., Troadec, M. B., Pigeon, C., Alizadeh, M., Leroyer, P., et al. (2003). Comparative analysis of mouse hepcidin 1 and 2 genes: Evidence for different patterns of expression and co-inducibility during iron overload. FEBS Letters, 542(1–3), 22–26. Isoda, M., Hanawa, H., Watanabe, R., Yoshida, T., Toba, K., Yoshida, K., et al. (2010). Expression of the peptide hormone hepcidin increases in cardiomyocytes under myocarditis and myocardial infarction. The Journal of Nutritional Biochemistry, 21(8), 749–756. Jankowska, E. A., Kasztura, M., Sokolski, M., Bronisz, M., Nawrocka, S., OleskowskaFlorek, W., et al. (2014). Iron deficiency defined as depleted iron stores accompanied by unmet cellular iron requirements identifies patients at the highest risk of death after an episode of acute heart failure. European Heart Journal, 35(36), 2468–2476. Krause, A., Neitz, S., Magert, H. J., Schulz, A., Forssmann, W. G., Schulz-Knappe, P., et al. (2000). LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Letters, 480(2–3), 147–150. Lakhal-Littleton, S., & Robbins, P. A. (2017). The interplay between iron and oxygen homeostasis with a particular focus on the heart. Journal of Applied Physiology (Bethesda, MD: 1985), 123(4), 967–973. Lakhal-Littleton, S., Wolna, M., Carr, C. A., Miller, J. J., Christian, H. C., Ball, V., et al. (2015). Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function. Proceedings of the National Academy of Sciences of the United States of America, 112(10), 3164–3169. Lakhal-Littleton, S., Wolna, M., Chung, Y. J., Christian, H. C., Heather, L. C., Brescia, M., et al. (2016). An essential cell-autonomous role for hepcidin in cardiac iron homeostasis. eLife, 5, e19804. Merle, U., Fein, E., Gehrke, S. G., Stremmel, W., & Kulaksiz, H. (2007). The iron regulatory peptide hepcidin is expressed in the heart and regulated by hypoxia and inflammation. Endocrinology, 148(6), 2663–2668. Naito, Y., Hosokawa, M., Sawada, H., Oboshi, M., Iwasaku, T., Okuhara, Y., et al. (2014). Hepcidin is increased in the hypertrophied heart of Dahl salt-sensitive rats. International Journal of Cardiology, 172(1), e45–e47. Nemeth, E., Tuttle, M. S., Powelson, J., Vaughn, M. B., Donovan, A., Ward, D. M., et al. (2004). Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 306(5704), 2090–2093. Park, C. H., Valore, E. V., Waring, A. J., & Ganz, T. (2001). Hepcidin, a urinary antimicrobial peptide synthesized in the liver. The Journal of Biological Chemistry, 276(11), 7806–7810. Schwarz, P., Strnad, P., Singer, N., Oswald, F., Ehehalt, R., Adler, G., et al. (2009). Identification, sequencing, and cellular localization of hepcidin in guinea pig (Cavia porcellus). The Journal of Endocrinology, 202(3), 389–396. Sheikh, N., Dudas, J., & Ramadori, G. (2007). Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat. Laboratory Investigation, 87(7), 713–725. Shike, H., Shimizu, C., Lauth, X., & Burns, J. C. (2004). Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates. Developmental and Comparative Immunology, 28(7–8), 747–754. Silvestri, L., Pagani, A., & Camaschella, C. (2008). Furin-mediated release of soluble hemojuvelin: A new link between hypoxia and iron homeostasis. Blood, 111(2), 924–931. Simonis, G., Mueller, K., Schwarz, P., Wiedemann, S., Adler, G., Strasser, R. H., et al. (2010). The iron-regulatory peptide hepcidin is upregulated in the ischemic and in the remote myocardium after myocardial infarction. Peptides, 31(9), 1786–1790.
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Suzuki, H., Toba, K., Kato, K., Ozawa, T., Tomosugi, N., Higuchi, M., et al. (2009). Serum hepcidin-20 is elevated during the acute phase of myocardial infarction. The Tohoku Journal of Experimental Medicine, 218(2), 93–98. Valore, E. V., & Ganz, T. (2008). Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin. Blood Cells, Molecules & Diseases, 40(1), 132–138. van Breda, G. F., Bongartz, L. G., Zhuang, W., van Swelm, R. P., Pertijs, J., Braam, B., et al. (2016). Cardiac hepcidin expression associates with injury independent of iron. American Journal of Nephrology, 44(5), 368–378. Zhang, L., Lu, D., Zhang, W., Quan, X., Dong, W., Xu, Y., et al. (2012). Cardioprotection by Hepc1 in cTnT(R141W) transgenic mice. Transgenic Research, 21(4), 867–878. Zumerle, S., Mathieu, J. R., Delga, S., Heinis, M., Viatte, L., Vaulont, S., et al. (2014). Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype. Blood, 123(23), 3646–3650.
Cardiomyocyte hepcidin: From intracellular iron homeostasis to physiological function S. Lakhal-Littleton* Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom *Corresponding author: e-mail address: [email protected]
Contents 1. Expression of cardiac hepcidin 1.1 Baseline expression relative to hepatic hepcidin 1.2 Baseline protein expression 1.3 Changes in cardiac hepcidin expression in pathophysiology 1.4 Known mechanisms of cardiac hepcidin regulation 2. The functions of cardiomyocyte hepcidin 3. Conclusions and future directions References
189 190 190 192 194 195 197 198
Abstract Cellular iron is required for the utilization of oxygen in the cell. Iron in iron-sulfur and heme groups is required for electron transfer and oxygen activation in oxidative phosphorylation, while labile free iron is required for oxygen activation by dioxygenases, and as a catalyst for redox signaling. At the same time, this reactivity with oxygen underpins the production of cell-damaging free radicals in the presence of excess iron. Because the cardiac cell is a major site of oxygen flux, it requires tight control of intracellular iron levels. Until recently, such control was thought to be mediated predominantly by the action of iron regulatory proteins. However, new evidence reveals that cardiomyocyte hepcidin is indispensable for the control of intracellular iron levels, normal metabolism and heart function. This new evidence highlights the need for better understanding of the regulation of cardiomyocyte hepcidin in health and disease.
1. Expression of cardiac hepcidin Since the discovery of hepcidin at the turn of the century, iron homeostasis research has focused on characterizing the expression, regulation and functions of hepatic hepcidin. The liver is the tissue with the most Vitamins and Hormones, Volume 110 ISSN 0083-6729 https://doi.org/10.1016/bs.vh.2019.01.009
#
2019 Elsevier Inc. All rights reserved.
189
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abundant levels of hepcidin mRNA, with expression comparable to that of the house house-keeping gene GAPDH (Krause et al., 2000). In addition, the phenotype of animal models lacking hepatic hepcidin closely resembles the iron-loading seen in hemochromatosis patients, suggesting that hepatic hepcidin is the dominant regulator of systemic iron homeostasis (Zumerle et al., 2014). For these reasons, hepcidin expression in non-hepatic tissues has received little interest from researchers. However, recent evidence has uncovered the importance of locally produced hepcidin in the heart for cardiac iron homeostasis and function (Lakhal-Littleton et al., 2016). In light of this new evidence, the study of the regulation of cardiac hepcidin in health and disease becomes scientifically and clinically important.
1.1 Baseline expression relative to hepatic hepcidin In humans, rodents and fish, the heart is the second highest site of hepcidin mRNA expression. Table 1 summarizes the levels of cardiac hepcidin expression reported in various species. Within the heart, all the evidence points at the fact that cardiac hepcidin is primarily derived from cardiomyocytes and not from non-cardiomyocyte cell types such as fibroblasts. Indeed, in mice with a cardiomyocyte-specific deletion of the hepcidin gene, total cardiac mRNA analysis showed an almost complete ablation of hepcidin expression compared to control hearts (Lakhal-Littleton et al., 2016). This is consistent with an earlier finding in rat hearts, where the isolated cardiomyocyte fraction was shown to contain most of the hepcidin mRNA (Isoda et al., 2010).
1.2 Baseline protein expression Only a limited number of studies have reported on the detection or quantitation of cardiac hepcidin protein. Studies in guinea pig and rat hearts detected an immunoreactive band at 8 kDa, consistent with the size of pro-hepcidin (Schwarz et al., 2009; Simonis et al., 2010). Another study using an antibody for the N-terminal region of the hepcidin peptide in rat heart extracts detected an immunoreactive band at 9.5 kDa, while immunostaining of rat hearts detected strong signal in the intercalated disc region of the cardiomyocyte (Merle et al., 2007). The significance of this subcellular localization is not fully explained. Only two other studies included hepcidin immunostaining in the heart, reporting a more diffuse distribution of hepcidin staining within the cardiomyocytes (LakhalLittleton et al., 2016; Naito et al., 2014). Detection of the pro-hepcidin
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Table 1 Levels of cardiac hepcidin mRNA expression reported in different species. Expression relative Detection to liver Reference Species method
Human
q-RT-PCR 1/15
Krause et al. (2000)
Human
Northern Blot
Park, Valore, Waring, and Ganz (2001)
Mouse (Balb/cJ)
q-RT-PCR 1/100
Not quantified
Mouse (C57BL/6) q-RT-PCR 1/30
Ilyin et al. (2003) Lakhal-Littleton et al. (2016)
Male Sprague Dawley rats (200–250 g)
q-RT-PCR 1/40
Merle, Fein, Gehrke, Stremmel, and Kulaksiz (2007)
Zebrafish
q-RT-PCT 1/10
Shike, Shimizu, Lauth, and Burns (2004)
Turbot
RT-PCR
Chen et al. (2007)
Adult sheep
q-RT-PCT 2.9 104 Badial et al. (2011)
Ayu (Plecoglossus altivelis)
q-RT-PCR Not reported
Not quantified
Crocodylus siamensis q-RT-PCR 1/300 Convict cichlid
q-RT-PCR 1/500
Chen, Chen, Lu, and Shi (2010) Hao, Li, Xie, and Li (2012) Chi et al. (2015)
peptide in tissue sections and tissue lysates does not constitute definitive evidence that cardiac cells secrete the active mature hepcidin peptide. Such evidence was however demonstrated in primary cardiomyocytes isolated from mice. It was shown that the hepcidin peptide could be detected by ELISA in supernatants of primary cardiomyocytes from control mice, but not from mice with a cardiomyocyte-specific deletion of the hepcidin gene (Lakhal-Littleton et al., 2016). That study also demonstrated that the release of the hepcidin peptide is dependent on the pro-hormone convertase Furin, also known to mediate the cleavage of pro-hepcidin in hepatocytes (Valore & Ganz, 2008). In summary, in most species studied, the heart is the second most abundant site of hepcidin mRNA expression. Experiments with isolated primary cardiomyocytes have also demonstrated that cardiac hepcidin is primarily derived from cardiomyocytes, and that it is secreted in a manner dependent on the pro-hormone convertase Furin.
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1.3 Changes in cardiac hepcidin expression in pathophysiology A number of studies have reported changes in cardiac hepcidin mRNA and protein in the disease setting and in experimental models of heart disease. Broadly speaking, cardiac hepcidin appears to be responsive to hypoxia, inflammation, heart disease, and changes in systemic iron balance. The expression of hepcidin mRNA and protein in male Sprague Dawley rats was shown to be upregulated after 24 h and 5 day of exposure to 6% or 8% O2 (Merle et al., 2007). In mice, 1 week exposure to milder hypoxia (11% O2) was shown to downregulate hepcidin mRNA but to upregulate hepcidin protein. The divergence of mRNA and protein responses to hypoxia was further confirmed in isolated primary cardiomyocytes exposed to 5% O2 (Lakhal-Littleton et al., 2016). Cardiac hepcidin expression was also shown to be upregulated in turpentine experimental models of inflammation. Cardiac hepcidin mRNA was significantly augmented in the myocardium within 6 h of turpentine injection in one study and at 16 h post injection in another (Merle et al., 2007; Sheikh, Dudas, & Ramadori, 2007). In both studies, upregulation of cardiac hepcidin is described as an acute inflammatory response rather than a secondary response to change in systemic iron availability. The responsiveness of cardiac hepcidin to inflammation and hypoxia, singly or in concert, may underlie its upregulation in heart disease. In humans, hepcidin mRNA is markedly upregulated in hearts with myocarditis, an inflammatory condition of the myocardium. This observation was replicated in a rat model of experimental autoimmune myocarditis, where hepcidin mRNA was shown to increase after 9 days. In that setting, its levels correlated most closely with IL-6 expression, suggesting that IL-6 may be the cause of hepcidin upregulation in the inflamed heart (Isoda et al., 2010). In acute myocardial infarction in the rat, hepcidin mRNA increased dramatically in the infarct lesion within 1 day. In that setting, upregulation of hepcidin mRNA was largely attributed to upregulation of IL-6 in the heart (Isoda et al., 2010). The role of hypoxia-inducible factors (HIFs) in that study was not adequately assessed because the study only measured HIF-1α mRNA, while HIF responsiveness to hypoxia is known to be driven largely by post-translational modifications and changes in HIF protein stability. Another study of myocardial infarction in rats also reported marked upregulation of hepcidin mRNA in the ischemic and remote myocardium, and of pro-hepcidin peptide in the ischemic myocardium only 6 h post infarction. In that study, serum hepcidin levels were also elevated (Simonis et al., 2010). Of note, this finding mirrors
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observation in patients with myocardial infarction, in whom serum levels of the mature hepcidin peptide (hepcidin-25) and of a shorter derivative lacking the N-terminus (hepcidin-20) were both elevated 4 h post infarction, and remained high for 7 days thereafter (Suzuki et al., 2009). Because the cardiac and hepatic hepcidin proteins are not distinguishable in serum, it is not possible to attribute the observed elevation in serum hepcidin to increased cardiac secretion. Nonetheless, the acute nature of this serum hepcidin response is consistent with a direct and local response of cardiac hepcidin to myocardial infarction, rather than a secondary response of hepatic hepcidin to trans-acting factors. Of note, upregulation of serum hepcidin did not appear to correlate with inflammatory markers, suggesting that mechanisms other than inflammation underlie the increase in serum hepcidin. A different study looking at hepcidin levels 16 weeks post coronary ligation in rats also found upregulated levels of cardiac hepcidin mRNA, which were most closely correlated to levels of brain natriuretic peptide BNP, suggesting a role for tissue injury in modulating cardiac hepcidin levels in this setting (van Breda et al., 2016). Another physiological stress that appears to affect cardiac hepcidin expression is high salt diet. Specifically, in Dahl salt-sensitive rats, which develop cardiac hypertrophy, hepcidin mRNA and protein were markedly increased after 12 weeks of high salt diet, and in a manner that was not correlated to cardiac iron content (Naito et al., 2014). This study also found a modest attenuation of phenylephrine-induced cardiomyocyte hypertrophy by hepcidin siRNA, suggesting that increased hepcidin levels following provision of high salt diet may contribute to hypertrophy. Hepatic hepcidin is acutely sensitive to changes in dietary iron content, with its protein levels closely mirroring changes in mRNA levels. Cardiac hepcidin, on the other hand, appears to respond differently from hepatic hepcidin to changes in dietary iron content. Provision of an iron-deficient diet in mice markedly decreases cardiac hepcidin mRNA, but increases its protein. Provision of an iron-loaded diet markedly increases hepcidin mRNA levels, but does not affect its proteins levels. These results were largely replicated in primary cardiomyocytes treated with the iron chelator desferroxamine (DFO) or with iron in the form of ferric citrate, suggesting that such responses were dependent on autonomously acting pathways rather than mediated by changes in serum iron levels. The lack of concordance between RNA and protein responses is attributed to the manner in which the hepcidin pro-peptide is processed in the heart (Lakhal-Littleton et al., 2016). This is discussed further in Section 1.4.
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In summary, cardiac hepcidin mRNA appears to respond to cardiovascular stresses, both acute (myocardial infarction, ischemia, inflammation) and chronic (hypertrophy, hypoxia, dietary iron deficiency and loading). The mechanisms underlying such responses, and their importance or otherwise to cardiac physiology, remain largely unexplored.
1.4 Known mechanisms of cardiac hepcidin regulation Other than correlative studies of cardiac hepcidin expression, there is little definitive evidence supporting specific mechanisms of cardiac hepcidin regulation. One mechanism that has been demonstrated is the Furin-dependent increase in cardiac hepcidin peptide secretion in response to hypoxia and iron deficiency (Lakhal-Littleton & Robbins, 2017; Lakhal-Littleton et al., 2016). It was found that increased secretion of hepcidin peptide was dependent on the activity of the pro-hormone convertase Furin. Importantly, the expression of Furin in the heart and in primary cardiomyocytes was also increased by hypoxia and iron deficiency. These conditions would both stabilize HIF protein by limiting the activity of the prolyl hydroxylases that target HIF protein for degradation in an oxygen- and iron-dependent manner. Consistent with this, Furin is a known HIF-target gene (Silvestri, Pagani, & Camaschella, 2008). Interestingly, as well as the processing of the hepcidin peptide, Furin in hepatocytes is also known to regulate hepcidin RNA expression, by cleaving the bone morphogenetic protein co-receptor, hemojuvelin. In this setting, hypoxia decreases hepcidin mRNA through Furin-mediated increase in hemojuvelin cleavage (Silvestri et al., 2008). That cardiac hepcidin secretion is upregulated rather than downregulated by Furin and hypoxia suggests that the hemojuvelin pathway may not operate to regulate hepcidin expression in cardiomyocytes, in the same manner that it does in hepatocytes. These observations pose broader questions over the differences between hepatic and cardiac hepcidins, in terms of transcriptional, translational and post-translational processing. In the liver, the consensus has been that hepcidin regulation is primarily achieved at the transcriptional level, meaning that protein measurements are not always conducted to support any observed changes in hepatic hepcidin mRNA levels. The work presented by Lakhal-Littleton et al. is the first example of opposing responses of the hepcidin transcript and protein to a given stimulus. Furthermore, the need for differential regulation between hepatic and cardiac hepcidins may reflect their distinct functions in relation to iron homeostasis. The function of cardiomyocyte hepcidin is discussed in Section 2.
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2. The functions of cardiomyocyte hepcidin The role of hepcidin as master regulator of systemic iron homeostasis was established over a decade ago. A number of subsequent studies established that it operated primarily through antagonism of the iron export protein ferroportin, at three important sites: duodenal enterocytes (the site of iron absorption), reticuloendothelial macrophages (the site of iron recycling) and hepatocytes (the site of iron storage) (Donovan et al., 2005; Nemeth et al., 2004). The use of liver-specific hepcidin knockout mice established that such function was primarily mediated by hepatic hepcidin, because the phenotype of such animals appeared to closely resemble that of ubiquitous hepcidin knockouts (Zumerle et al., 2014). Thus, the importance, if any, of non-hepatic hepcidins remained unexplored. Furthermore, such studies were not possible in the ubiquitous knockout mouse models that existed at the time, because of the confounding effects of altered systemic iron homeostasis, which would mask any effects of hepcidin in non-hepatic tissues. As hepcidin is a hormone that affects iron homeostasis through its target ferroportin, there was additionally the need to establish the exact site of action of non-hepatic hepcidins. In the context of the heart, two studies performed by Lakhal-Littleton et al. provided the first evidence of the importance of cardiomyocyte hepcidin and further identified the site of its action. In the first study, mice carrying a cardiomyocyte-specific deletion of the ferroportin gene were found to develop fatal left ventricular dysfunction by 3 months of age. The dysfunction was caused by a threefold increase in iron levels within cardiomyocytes. Importantly, downregulation of Transferrin receptor (TfR1) was not sufficient to prevent iron overload in ferroportin-deficient hearts, suggesting that ferroportin-mediated iron release is an essential component of iron homeostasis in the heart (LakhalLittleton et al., 2015). In the second study, mice carrying a cardiomyocytespecific deletion of hepcidin developed fatal left ventricular dysfunction between 3 and 6 months of age, despite the maintenance of normal systemic iron levels. A similar result was obtained in animals with cardiomyocytespecific knock-in of the ferroportin isoform C326Y, which retains its iron export function but loses its hepcidin binding. In both settings, the cardiomyocytes were found to be iron deficient due to increased iron export. Intravenous iron supplementation from 3 months of age prevented the development of cardiac dysfunction in cardiac-hepcidin knockouts, demonstrating the importance of cardiomyocyte iron deficiency in the cardiac dysfunction seen in these models (Lakhal-Littleton et al., 2016). Another
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important observation is that mice lacking cardiac hepcidin did not have reduced serum hepcidin levels, nor did they manifest any changes in hemoglobin, serum iron indices or liver iron stores. This observation is consistent with the notion that cardiomyocyte hepcidin, at least in the normal healthy heart, does not act in a paracrine manner. It also reflects the fact that, at such low levels of expression (relative to hepatic hepcidin), cardiomyocyte hepcidin is indeed only able to affect ferroportin locally within the heart. Taken together, these two studies demonstrate that the cardiac hepcidin/ ferroportin axis is essential for the cell autonomous control of the intracellular iron pool upon which normal cardiac function depends. Cardiomyocyte hepcidin also appears to protect the heart from the effects of systemic iron deficiency. Indeed, it was found that hepcidin-deficient hearts developed a greater hypertrophic response to sustained dietary iron restriction than their littermate controls. This finding, together with the observation that cardiac hepcidin protein is upregulated rather than downregulated by dietary iron restriction, indicates that cardiomyocyte hepcidin is part of cardioprotective mechanism that mitigates against reduced systemic iron availability by increasing retention of intracellular iron. Fig. 1 summarizes the current thinking around the function of cardiomyocyte hepcidin.
Fig. 1 The role of cardiomyocyte hepcidin in maintenance of intracellular iron levels.
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There is some evidence that cardiomyocyte hepcidin dysregulation may impinge on severity and progression of certain types of heart disease. In a mouse model of dilated cardiomyopathy, transgenic for the R141W mutation of the Troponin gene in cardiac cells, cardiac hepcidin levels were found to be less than half of those measured in normal control hearts. Furthermore, transgenic overexpression of hepcidin in the hearts of these mice reduced cardiac ferroportin levels, restored cardiac iron content to that of controls, and improved left ventricle morphology and ejection fraction. Interestingly, the effects of transgenic hepcidin overexpression were also associated with improvement of mitochondrial morphology (Zhang et al., 2012). The use of overexpression of the hepcidin transgene in the heart may not fully recapitulate the true physiological function of hepcidin. Nonetheless, the findings of this study are consistent with the idea that hepcidin acts in an autonomous manner to regulate cardiac ferroportin, and that such regulation is important for normal cardiac metabolism and function. Under normal conditions, cardiomyocyte hepcidin appears not to contribute to serum hepcidin levels or the control of systemic iron homeostasis. However, the finding that it is increased dramatically in the infarcted heart, and acutely in the serum of patients following myocardial infarction, suggests that it may have an effect on systemic iron homeostasis in this particular setting (Simonis et al., 2010; Suzuki et al., 2009). Clinical studies have shown that iron deficiency in acute heart disease correlates with poor prognosis ( Jankowska et al., 2014). It would be important to assess the extent to which cardiac hepcidin contributes to iron deficiency in acute heart failure. Iron deficiency and high serum hepcidin are also correlated with poor prognosis in chronic heart failure (Comin-Colet et al., 2013). In this setting it is assumed that the liver is the source of hepcidin, and that IL-6-driven inflammation is the cause. Experimental models have shown that cardiomyocyte hepcidin can be dramatically increased in the inflammatory setting (Merle et al., 2007; Sheikh et al., 2007). Therefore, it would be important to establish to what extent cardiomyocyte hepcidin accounts for iron deficiency in chronic heart failure.
3. Conclusions and future directions In most species, the heart is the second highest site of hepcidin expression. Within the heart, hepcidin appears to be expressed primarily in cardiomyocytes, at levels that are orders of magnitude lower that those seen
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in the liver. In the normal healthy heart, cardiomyocyte hepcidin is indispensable for control of intracellular iron. It does this through cell autonomous control of ferroportin-mediated iron release. This function is essential for maintenance of normal cardiac metabolism and contractile function. Cardiomyocyte hepcidin is regulated post-translationally by hypoxia and iron deficiency in a manner that is distinct from that of hepatic hepcidin. This distinct regulation is likely a cardioprotective mechanism, particularly in the setting of reduced systemic iron availability. Finally, cardiomyocyte hepcidin appears to be responsive to inflammation and local ischemia in the heart, raising the possibility that it may contribute to changes in local and systemic iron levels in that setting. Future studies are needed to address a number of pertinent questions relating to how the cardiac hepcidin gene is regulated, and how post-translational modifications fine-tune the amount of hepcidin secreted from the heart. Additionally, comparative structural studies need to be carried out to compare cardiac and hepatic hepcidin proteins, and predict any further differences relating to stability and potency. Finally, the possibility that newly developed hepcidin mimics (for the treatment of iron-overload) may alter cardiac iron levels directly by acting on cardiomyocyte ferroportin should not be overlooked.
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Suzuki, H., Toba, K., Kato, K., Ozawa, T., Tomosugi, N., Higuchi, M., et al. (2009). Serum hepcidin-20 is elevated during the acute phase of myocardial infarction. The Tohoku Journal of Experimental Medicine, 218(2), 93–98. Valore, E. V., & Ganz, T. (2008). Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin. Blood Cells, Molecules & Diseases, 40(1), 132–138. van Breda, G. F., Bongartz, L. G., Zhuang, W., van Swelm, R. P., Pertijs, J., Braam, B., et al. (2016). Cardiac hepcidin expression associates with injury independent of iron. American Journal of Nephrology, 44(5), 368–378. Zhang, L., Lu, D., Zhang, W., Quan, X., Dong, W., Xu, Y., et al. (2012). Cardioprotection by Hepc1 in cTnT(R141W) transgenic mice. Transgenic Research, 21(4), 867–878. Zumerle, S., Mathieu, J. R., Delga, S., Heinis, M., Viatte, L., Vaulont, S., et al. (2014). Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype. Blood, 123(23), 3646–3650.