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Lipocalin-2 Induces Cardiomyocyte Apoptosis by Increasing Intracellular Iron Accumulation*

Open AccessPublished:November 22, 2011DOI:https://doi.org/10.1074/jbc.M111.275719
      Our objective was to determine whether lipocalin-2 (Lcn2) regulates cardiomyocyte apoptosis, the mechanisms involved, and the functional significance. Emerging evidence suggests that Lcn2 is a proinflammatory adipokine associated with insulin resistance and obesity-related complications, such as heart failure. Here, we used both primary neonatal rat cardiomyocytes and H9c2 cells and demonstrated for the first time that Lcn2 directly induced cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure. This was shown by detection of DNA fragmentation using TUNEL assay, phosphatidylserine exposure using flow cytometry to detect annexin V-positive cells, caspase-3 activity using enzymatic assay and immunofluorescence, and Western blotting for the detection of cleaved caspase-3. We also observed that Lcn2 caused translocation of the proapoptotic protein Bax to mitochondria and disruption of mitochondrial membrane potential. Using transient transfection of GFP-Bax, we confirmed that Lcn2 induced co-localization of Bax with MitoTracker® dye. Importantly, we used the fluorescent probe Phen Green SK to demonstrate an increase in intracellular iron in response to Lcn2, and depleting intracellular iron using an iron chelator prevented Lcn2-induced cardiomyocyte apoptosis. Administration of recombinant Lcn2 to mice for 14 days increased cardiomyocyte apoptosis as well as an acute inflammatory response with compensatory changes in cardiac functional parameters. In conclusion, Lcn2-induced cardiomyocyte apoptosis is of physiological significance and occurs via a mechanism involving elevated intracellular iron levels and Bax translocation.

      Introduction

      Cardiovascular disease is the leading cause of death worldwide, and individuals with obesity and type 2 diabetes have an increase in prevalence for cardiovascular morbidity and mortality (
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      Cardiovascular disease risk in type 2 diabetes mellitus: insights from mechanistic studies.
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      ,
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      Adipokines, myokines and cardiovascular disease.
      ). Heart failure is one example, but mechanisms of obesity- and diabetes-induced heart disease are multifaceted and remain to be defined clearly (
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      Impact of obesity on the risk of heart failure and its prognosis.
      ). A potential mechanism may be changes in cardiac remodeling as a result of altered circulating adipokine and cytokine profiles (
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      Cardiac remodeling in obesity.
      ). Lipocalin-2 (Lcn2,
      The abbreviations used are: Lcn2
      lipocalin-2
      AV
      annexin V
      2′-DPD
      2,2′-dipyridyl
      HBSS
      Hanks' buffered salt solution
      PG-SK
      Phen Green SK
      PI
      propidium iodide
      TMRE
      tetramethylrhodamine ethylester.
      also often termed neutrophil gelatinase-associated lipocalin (NGAL) or oncogene 24p3), is a small, secreted adipokine and belongs to a diverse family of lipocalins that has the characteristics of binding and transporting small molecules such as retinoid, fatty acid, steroid, and iron (
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      ). Recent studies show that Lcn2 is a proinflammatory marker associated with insulin resistance and obesity-related metabolic disorders (
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      Emerging clinical and experimental evidence for the role of lipocalin-2 in metabolic syndrome.
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      ). An increase of Lcn2 expression in adipose tissue is observed in various experimental models of obesity and in obese humans (
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      The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance.
      ,
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      • Hu F.B.
      Lipocalins and insulin resistance: etiological role of retinol-binding protein 4 and lipocalin-2.
      ). It was suggested that Lcn2 may mediate the innate immune responses in the pathogenesis of heart failure (
      • Damman K.
      • van Veldhuisen D.J.
      • Navis G.
      • Voors A.A.
      • Hillege H.L.
      Urinary neutrophil gelatinase-associated lipocalin (NGAL), a marker of tubular damage, is increased in patients with chronic heart failure.
      ,
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      Increased plasma neutrophil gelatinase-associated lipocalin levels predict mortality in elderly patients with chronic heart failure.
      ,
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      • Malyszko J.
      • Bachorzewska-Gajewska H.
      • Malyszko J.S.
      • Dobrzycki S.
      Serum neutrophil gelatinase-associated lipocalin as a marker of renal function in patients with chronic heart failure and coronary artery disease.
      ,
      • Yndestad A.
      • Landrø L.
      • Ueland T.
      • Dahl C.P.
      • Flo T.H.
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      • Espevik T.
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      • Husberg C.
      • Christensen G.
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      • Øie E.
      • Gullestad L.
      • Aukrust P.
      Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure.
      ). Lcn2 expression is significantly augmented in patients with coronary heart disease and myocardial infarction (
      • Choi K.M.
      • Lee J.S.
      • Kim E.J.
      • Baik S.H.
      • Seo H.S.
      • Choi D.S.
      • Oh D.J.
      • Park C.G.
      Implication of lipocalin-2 and visfatin levels in patients with coronary heart disease.
      ,
      • Hemdahl A.L.
      • Gabrielsen A.
      • Zhu C.
      • Eriksson P.
      • Hedin U.
      • Kastrup J.
      • Thorén P.
      • Hansson G.K.
      Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction.
      ). Plasma Lcn2 was increased after carotid artery injury in rats (
      • Bu D.X.
      • Hemdahl A.L.
      • Gabrielsen A.
      • Fuxe J.
      • Zhu C.
      • Eriksson P.
      • Yan Z.Q.
      Induction of neutrophil gelatinase-associated lipocalin in vascular injury via activation of nuclear factor-κB.
      ) and in a heterotopic mouse transplanted heart after ischemia/reperfusion (
      • Aigner F.
      • Maier H.T.
      • Schwelberger H.G.
      • Wallnöfer E.A.
      • Amberger A.
      • Obrist P.
      • Berger T.
      • Mak T.W.
      • Maglione M.
      • Margreiter R.
      • Schneeberger S.
      • Troppmair J.
      Lipocalin-2 regulates the inflammatory response during ischemia and reperfusion of the transplanted heart.
      ).
      Cardiomyocyte apoptosis can play a critical role in the progression of heart failure. For example, in patients with end-stage cardiomyopathy in dilated and ischemic heart disease, hypertrophic heart disease and arrhythmogenic right ventricular dysplasia, loss of cardiomyocytes due to apoptosis can be observed that leads to the progression of cardiac dysfunction, ultimately heart failure (
      • Olivetti G.
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      • Beltrami C.A.
      • Krajewski S.
      • Reed J.C.
      • Anversa P.
      Apoptosis in the failing human heart.
      ,
      • Takemura G.
      • Fujiwara H.
      Role of apoptosis in remodeling after myocardial infarction.
      ,
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      • Kolodgie F.D.
      • Hajjar R.J.
      • Schmidt U.
      • Semigran M.J.
      • Dec G.W.
      • Khaw B.A.
      Apoptosis in myocytes in end-stage heart failure.
      ,
      • Narula J.
      • Kolodgie F.D.
      • Virmani R.
      Apoptosis and cardiomyopathy.
      ). Lcn2 has been implicated in the regulation of cell death in various cell types (
      • Devireddy L.R.
      • Teodoro J.G.
      • Richard F.A.
      • Green M.R.
      Induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by IL-3 deprivation.
      ,
      • Lee S.
      • Park J.Y.
      • Lee W.H.
      • Kim H.
      • Park H.C.
      • Mori K.
      • Suk K.
      Lipocalin-2 is an autocrine mediator of reactive astrocytosis.
      ,
      • Bong J.J.
      • Seol M.B.
      • Kim H.H.
      • Han O.
      • Back K.
      • Baik M.
      The 24p3 gene is induced during involution of the mammary gland and induces apoptosis of mammary epithelial cells.
      ,
      • Zheng L.T.
      • Lee S.
      • Yin G.N.
      • Mori K.
      • Suk K.
      Down-regulation of lipocalin 2 contributes to chemoresistance in glioblastoma cells.
      ), although whether Lcn2 directly regulates cardiomyocyte apoptosis remains unknown. It has been shown that changes in intracellular iron content can mediate apoptosis in a variety of cell types (
      • Kooncumchoo P.
      • Sharma S.
      • Porter J.
      • Govitrapong P.
      • Ebadi M.
      Coenzyme Q(10) provides neuroprotection in iron-induced apoptosis in dopaminergic neurons.
      ,
      • Velez-Pardo C.
      • Jimenez Del Rio M.
      • Verschueren H.
      • Ebinger G.
      • Vauquelin G.
      Dopamine and iron induce apoptosis in PC12 cells.
      ,
      • Devireddy L.R.
      • Gazin C.
      • Zhu X.
      • Green M.R.
      A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake.
      ,
      • Oudit G.Y.
      • Trivieri M.G.
      • Khaper N.
      • Husain T.
      • Wilson G.J.
      • Liu P.
      • Sole M.J.
      • Backx P.H.
      Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model.
      ). Furthermore, iron overload is associated with cardiomyopathy involving apoptosis and fibrosis leading to heart failure (
      • Whittaker P.
      • Hines F.A.
      • Robl M.G.
      • Dunkel V.C.
      Histopathological evaluation of liver, pancreas, spleen, and heart from iron-overloaded Sprague-Dawley rats.
      ,
      • Arvapalli R.K.
      • Paturi S.
      • Laurino J.P.
      • Katta A.
      • Kakarla S.K.
      • Gadde M.K.
      • Wu M.
      • Rice K.M.
      • Walker E.M.
      • Wehner P.
      • Blough E.R.
      Deferasirox decreases age-associated iron accumulation in the aging F344XBN rat heart and liver.
      ,
      • Wang Y.
      • Wu M.
      • Al-Rousan R.
      • Liu H.
      • Fannin J.
      • Paturi S.
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      • Katta A.
      • Kakarla S.K.
      • Rice K.M.
      • Triest W.E.
      • Blough E.R.
      Iron-induced cardiac damage: role of apoptosis and deferasirox intervention.
      ,
      • Kohgo Y.
      • Ikuta K.
      • Ohtake T.
      • Torimoto Y.
      • Kato J.
      Body iron metabolism and pathophysiology of iron overload.
      ). Because Lcn2 has the capability to bind iron (
      • Bachman M.A.
      • Miller V.L.
      • Weiser J.N.
      Mucosal lipocalin 2 has proinflammatory and iron-sequestering effects in response to bacterial enterobactin.
      ,
      • Schmidt-Ott K.M.
      • Mori K.
      • Kalandadze A.
      • Li J.Y.
      • Paragas N.
      • Nicholas T.
      • Devarajan P.
      • Barasch J.
      Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia.
      ,
      • Goetz D.H.
      • Holmes M.A.
      • Borregaard N.
      • Bluhm M.E.
      • Raymond K.N.
      • Strong R.K.
      The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.
      ,
      • Yang J.
      • Goetz D.
      • Li J.Y.
      • Wang W.
      • Mori K.
      • Setlik D.
      • Du T.
      • Erdjument-Bromage H.
      • Tempst P.
      • Strong R.
      • Barasch J.
      An iron delivery pathway mediated by a lipocalin.
      ), we hypothesized that the alteration of intracellular iron levels is an important mechanism in Lcn2-induced apoptosis. This study therefore set out to analyze the role of Lcn2 on apoptosis at cellular and molecular levels, which may lead to better understanding of the pathophysiological role of Lcn2 in cardiomyopathy.

      DISCUSSION

      Emerging data indicate that circulating levels of Lcn2 correlate positively with various aspects of the metabolic syndrome (
      • Jang Y.
      • Lee J.
      • Wang Y.
      • Sweeney G.
      Emerging clinical and experimental evidence for the role of lipocalin-2 in metabolic syndrome.
      ). Work to date has focused principally on the proinflammatory and metabolic effects of Lcn2 (
      • Wang Y.
      • Lam K.S.
      • Kraegen E.W.
      • Sweeney G.
      • Zhang J.
      • Tso A.W.
      • Chow W.S.
      • Wat N.M.
      • Xu J.Y.
      • Hoo R.L.
      • Xu A.
      Lipocalin-2 is an inflammatory marker closely associated with obesity, insulin resistance, and hyperglycemia in humans.
      ,
      • Law I.K.
      • Xu A.
      • Lam K.S.
      • Berger T.
      • Mak T.W.
      • Vanhoutte P.M.
      • Liu J.T.
      • Sweeney G.
      • Zhou M.
      • Yang B.
      • Wang Y.
      Lipocalin-2 deficiency attenuates insulin resistance associated with aging and obesity.
      ,
      • Mishra J.
      • Dent C.
      • Tarabishi R.
      • Mitsnefes M.M.
      • Ma Q.
      • Kelly C.
      • Ruff S.M.
      • Zahedi K.
      • Shao M.
      • Bean J.
      • Mori K.
      • Barasch J.
      • Devarajan P.
      Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery.
      ,
      • Flo T.H.
      • Smith K.D.
      • Sato S.
      • Rodriguez D.J.
      • Holmes M.A.
      • Strong R.K.
      • Akira S.
      • Aderem A.
      Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.
      ,
      • Chan Y.R.
      • Liu J.S.
      • Pociask D.A.
      • Zheng M.
      • Mietzner T.A.
      • Berger T.
      • Mak T.W.
      • Clifton M.C.
      • Strong R.K.
      • Ray P.
      • Kolls J.K.
      Lipocalin 2 is required for pulmonary host defense against Klebsiella infection.
      ), yet less is known regarding cardiovascular effects. Several studies have now shown that Lcn2 was elevated during ischemia-reperfusion processes and in coronary heart disease and myocardial infarction (
      • Choi K.M.
      • Lee J.S.
      • Kim E.J.
      • Baik S.H.
      • Seo H.S.
      • Choi D.S.
      • Oh D.J.
      • Park C.G.
      Implication of lipocalin-2 and visfatin levels in patients with coronary heart disease.
      ,
      • Yndestad A.
      • Landrø L.
      • Ueland T.
      • Dahl C.P.
      • Flo T.H.
      • Vinge L.E.
      • Espevik T.
      • Frøland S.S.
      • Husberg C.
      • Christensen G.
      • Dickstein K.
      • Kjekshus J.
      • Øie E.
      • Gullestad L.
      • Aukrust P.
      Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure.
      ,
      • Hemdahl A.L.
      • Gabrielsen A.
      • Zhu C.
      • Eriksson P.
      • Hedin U.
      • Kastrup J.
      • Thorén P.
      • Hansson G.K.
      Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction.
      ,
      • Aigner F.
      • Maier H.T.
      • Schwelberger H.G.
      • Wallnöfer E.A.
      • Amberger A.
      • Obrist P.
      • Berger T.
      • Mak T.W.
      • Maglione M.
      • Margreiter R.
      • Schneeberger S.
      • Troppmair J.
      Lipocalin-2 regulates the inflammatory response during ischemia and reperfusion of the transplanted heart.
      ). Here, we analyzed the effect of Lcn2 on cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure (
      • Olivetti G.
      • Abbi R.
      • Quaini F.
      • Kajstura J.
      • Cheng W.
      • Nitahara J.A.
      • Quaini E.
      • Di Loreto C.
      • Beltrami C.A.
      • Krajewski S.
      • Reed J.C.
      • Anversa P.
      Apoptosis in the failing human heart.
      ,
      • Takemura G.
      • Fujiwara H.
      Role of apoptosis in remodeling after myocardial infarction.
      ).
      Our data using FACS analysis of annexin V binding to phosphatidylserine, TUNEL staining, and various measures of caspase-3 activation clearly demonstrate that recombinant Lcn2 induced apoptosis in primary neonatal cardiomyocytes and H9c2 cells. Because this is a novel observation and little is known about Lcn2 signaling we next investigated potential mechanisms responsible for Lcn2-induced apoptosis. Our studies uncovered an initially unexpected and novel mechanism that integrates the ability of Lcn2 to regulate intracellular iron levels with known proapoptotic effects of iron. Although playing a vital role in cellular functions, such as enzyme activity, intracellular iron levels must be carefully regulated because iron, especially ferrous (Fe2+) form, has high potential to cause toxicity. The Haber-Weiss (Fenton) reaction, catalyzed by ferrous iron, generates hydroxyl radicals. Generation of reactive oxygen species is thus one way in which increased intracellular ferrous iron has been proposed to induce apoptosis (
      • Munoz J.P.
      • Chiong M.
      • García L.
      • Troncoso R.
      • Toro B.
      • Pedrozo Z.
      • Diaz-Elizondo J.
      • Salas D.
      • Parra V.
      • Núñez M.T.
      • Hidalgo C.
      • Lavandero S.
      Iron induces protection and necrosis in cultured cardiomyocytes: role of reactive oxygen species and nitric oxide.
      ). It was also shown recently that iron enhanced doxorubicin-induced cardiomyocyte apoptosis which was prevented by iron chelation using dexrazoxane, likely via preventing iron-mediated degradation of hypoxia-inducible factors (
      • Spagnuolo R.D.
      • Recalcati S.
      • Tacchini L.
      • Cairo G.
      Role of hypoxia-inducible factors in the dexrazoxane-mediated protection of cardiomyocytes from doxorubicin-induced toxicity.
      ). Iron nanoparticles have also been shown to enhance the apoptotic effects of 7-ketocholesterol in cardiomyocytes (
      • Kahn E.
      • Baarine M.
      • Pelloux S.
      • Riedinger J.M.
      • Frouin F.
      • Tourneur Y.
      • Lizard G.
      Iron nanoparticles increase 7-ketocholesterol-induced cell death, inflammation, and oxidation on murine cardiac HL1-NB cells.
      ). In general, iron distribution must be tightly regulated due to the high potential of iron for inducing biological toxicity (
      • Goswami T.
      • Rolfs A.
      • Hediger M.A.
      Iron transport: emerging roles in health and disease.
      ,

      Deleted in proof

      ).
      Lcn2 has the capability to bind iron, and the complex can internalize through binding to two potential Lcn2 receptors, 24p3R and/or megalin (
      • Devireddy L.R.
      • Gazin C.
      • Zhu X.
      • Green M.R.
      A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake.
      ,
      • Hvidberg V.
      • Jacobsen C.
      • Strong R.K.
      • Cowland J.B.
      • Moestrup S.K.
      • Borregaard N.
      The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake.
      ). We used PG-SK dye which is quenched by binding iron, especially ferrous form, and showed that Lcn2 treatment of cardiomyocytes resulted in a decrease of PG-SK fluorescence, indicating an increase of intracellular iron pool. We also confirmed that increasing intracellular iron with FeSO4 induced cardiomyocyte apoptosis and that chemical chelation of free intracellular iron attenuated Lcn2-induced caspase-3 activity. This is in keeping with the literature cited above showing an important role for iron in cardiomyocyte apoptosis (
      • Munoz J.P.
      • Chiong M.
      • García L.
      • Troncoso R.
      • Toro B.
      • Pedrozo Z.
      • Diaz-Elizondo J.
      • Salas D.
      • Parra V.
      • Núñez M.T.
      • Hidalgo C.
      • Lavandero S.
      Iron induces protection and necrosis in cultured cardiomyocytes: role of reactive oxygen species and nitric oxide.
      ,
      • Spagnuolo R.D.
      • Recalcati S.
      • Tacchini L.
      • Cairo G.
      Role of hypoxia-inducible factors in the dexrazoxane-mediated protection of cardiomyocytes from doxorubicin-induced toxicity.
      ,
      • Kahn E.
      • Baarine M.
      • Pelloux S.
      • Riedinger J.M.
      • Frouin F.
      • Tourneur Y.
      • Lizard G.
      Iron nanoparticles increase 7-ketocholesterol-induced cell death, inflammation, and oxidation on murine cardiac HL1-NB cells.
      ). One study suggested that treatment of the atrial derived HL-1 cell line with recombinant enterobactin-Fe3+ loaded or iron-free Lcn2 protein showed no apoptosis induction (
      • Aigner F.
      • Maier H.T.
      • Schwelberger H.G.
      • Wallnöfer E.A.
      • Amberger A.
      • Obrist P.
      • Berger T.
      • Mak T.W.
      • Maglione M.
      • Margreiter R.
      • Schneeberger S.
      • Troppmair J.
      Lipocalin-2 regulates the inflammatory response during ischemia and reperfusion of the transplanted heart.
      ). However, it may be that ferrous iron is important in mediating the proapoptotic effects of Lcn2. Interestingly, although the average intracellular iron level increased in the cell population treated by Lcn2, cell by cell image analysis indicated that the effect was more pronounced in a subpopulation of cells. We therefore examined whether individual cells showing the most pronounced increase in intracellular iron showed an increase in cell death. Indeed, decreased PG-SK fluorescence correlated well with cleaved caspase-3 immunoreactivity in individual cells. Because iron has multiple roles in health and disease (
      • Goswami T.
      • Rolfs A.
      • Hediger M.A.
      Iron transport: emerging roles in health and disease.
      ,

      Deleted in proof

      ) our observations on a mechanistic role for iron in proapoptotic effects of Lcn2 may have more widespread, and thus far unappreciated, implications in understanding additional physiological effects of Lcn2 (
      • Jang Y.
      • Lee J.
      • Wang Y.
      • Sweeney G.
      Emerging clinical and experimental evidence for the role of lipocalin-2 in metabolic syndrome.
      ).
      Further investigation of the intracellular mechanisms of Lcn2-induced cardiomyocyte apoptosis demonstrated activation of the intrinsic apoptotic pathway. In particular, we used several approaches to demonstrate Bax translocation to the mitochondrial membrane, a subsequent disruption of mitochondrial membrane potential, and caspase-3 activation. This is in keeping with published work showing that FeSO4 induced mitochondrial damage leading to nuclear DNA condensation and fragmentation (
      • Kooncumchoo P.
      • Sharma S.
      • Porter J.
      • Govitrapong P.
      • Ebadi M.
      Coenzyme Q(10) provides neuroprotection in iron-induced apoptosis in dopaminergic neurons.
      ). It has also been shown that iron induced mitochondrial damage and caused caspase-3 activation, NF-κB induction, and decreased Bcl-2 expression (
      • Kooncumchoo P.
      • Sharma S.
      • Porter J.
      • Govitrapong P.
      • Ebadi M.
      Coenzyme Q(10) provides neuroprotection in iron-induced apoptosis in dopaminergic neurons.
      ). Here, we also confirmed that increasing intracellular iron with FeSO4 induced cardiomyocyte apoptosis and that chemical chelation of free intracellular iron attenuated Lcn2-induced Bax translocation to mitochondria.
      To investigate the physiological significance of our observations we then conducted in vivo studies in mice administered Lcn2 for 14 days. Good rationale for these studies also comes from the fact that iron accumulation has been identified as an important mediator of age-associated cardiac apoptosis and proposed as an effective pharmacologic target for therapeutic intervention (
      • Arvapalli R.K.
      • Paturi S.
      • Laurino J.P.
      • Katta A.
      • Kakarla S.K.
      • Gadde M.K.
      • Wu M.
      • Rice K.M.
      • Walker E.M.
      • Wehner P.
      • Blough E.R.
      Deferasirox decreases age-associated iron accumulation in the aging F344XBN rat heart and liver.
      ). Analysis of TUNEL-positive cells showed a characteristic low value in wild-type mice of <0.5%, but this was significantly increased in animals treated with Lcn2, thus confirming translation of our cell-based studies to an animal model. We analyzed functional parameters in the heart and found that Lcn2 administration tended to induce an unexpected improvement in cardiac function. However, it is important to note that Lcn2 also promotes an acute inflammatory response, as we observed here via CD68 immunofluorescence to monitor macrophage infiltration. It is known that inflammation can induce intracardiac volume depletion, inducing more rapid heart rate and reduced size of cardiac chambers. Additional effects of Lcn2, such as changes in cardiac metabolism, which are still unknown, may also contribute to functional parameters we measured here using echocardiography. Thus, our study indicated an early compensatory stage of cardiac remodeling in the face of elevated apoptosis and inflammation. The temporal nature of cardiac remodeling events is highly significant (
      • Abel E.D.
      • Litwin S.E.
      • Sweeney G.
      Cardiac remodeling in obesity.
      ), and this is also likely to be the case for Lcn2-mediated changes. It will be of interest to perform more chronic studies and assess long term changes in cardiac remodeling induced by Lcn2 and whether cardiac iron content is increased in such animals. Indeed, iron overload is known to cause elevated oxidative stress, myocardial apoptosis, systolic and diastolic dysfunction, and increased morbidity (
      • Oudit G.Y.
      • Trivieri M.G.
      • Khaper N.
      • Husain T.
      • Wilson G.J.
      • Liu P.
      • Sole M.J.
      • Backx P.H.
      Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model.
      ,
      • Wang Y.
      • Wu M.
      • Al-Rousan R.
      • Liu H.
      • Fannin J.
      • Paturi S.
      • Arvapalli R.K.
      • Katta A.
      • Kakarla S.K.
      • Rice K.M.
      • Triest W.E.
      • Blough E.R.
      Iron-induced cardiac damage: role of apoptosis and deferasirox intervention.
      ,
      • Borgna-Pignatti C.
      • Cappellini M.D.
      • De Stefano P.
      • Del Vecchio G.C.
      • Forni G.L.
      • Gamberini M.R.
      • Ghilardi R.
      • Piga A.
      • Romeo M.A.
      • Zhao H.
      • Cnaan A.
      Cardiac morbidity and mortality in deferoxamine- or deferiprone-treated patients with thalassemia major.
      ).
      In conclusion, Lcn2-induced cardiomyocyte apoptosis is at least in part mediated via an increase in intracellular iron levels and induction of the intrinsic mitochondrial pathway via translocation and activation of Bax. Further experiments to understand fully the regulation of various components of cardiac remodeling by Lcn2 and the long term consequences of such changes in vivo will now be of great interest.

      Acknowledgments

      We thank Helen K. W. Law (Institut Pasteur Paris) for discussion and analysis of flow cytometry data.

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