Combination of cadmium and high cholesterol levels as a risk factor for heart fibrosis.

ToxSci Advance Access published March 13, 2015

Combination of Cadmium and High Cholesterol Levels as a Risk Factor for Heart Fibrosis Running Title: Cadmium, Cholesterol and the Heart

Türkcan A. *, Scharinger B.*, Grabmann G.†, Keppler B.†, Laufer G.*, Bernhard D.‡, Messner Downloaded from http://toxsci.oxfordjournals.org/ at Simon Fraser University on March 16, 2015

B*.

* Cardiac Surgery Research Laboratory, Surgical Research Laboratories, Department of Surgery, Medical University of Vienna, Vienna, Austria †

Institute of Inorganic Chemistry, University of Vienna, Währinger Str. 42, 1090, Vienna,

Austria ‡

Cardiac Surgery Research Laboratory Innsbruck, University Clinic for Cardiac Surgery,

Innsbruck Medical University, Innsbruck, Austria

Corresponding author: Messner Barbara Ph.D., Cardiac Surgery Research Laboratory, Department of Surgery, Medical University of Vienna, AKH, Ebene 8 G09/07, Währinger Gürtel 18-20. A-1090 Vienna. AUSTRIA. Tel.: 0043-(0)1-40400-69490; FAX: 0043-(0)1-40400-67820; e-mail: [email protected]

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ABSTRACT The deleterious effects of increased Cd serum levels on the cardiovascular system are proven by epidemiological and basic science studies. Cd exposure of animals and humans is known to impair myocardial function, possibly leading to heart failure. The present study aims at investigating the effect of Cd treatment on the cardiac system with emphasis on the combined effect of Cd and high serum cholesterol levels as an important cardiovascular risk factor. Detailed analyses of Cd induced effects on the heart of ApoE-/- mice fed a high fat diet, ApoE-/- mice fed a normal diet, and C57BL/6J mice fed a normal diet revealed proinflammatory and fibrotic changes in the presence of cellular hypertrophy but in the absence of organ hypertrophy. Hypercholesterolemia in ApoE-/- mice alone and in combination with

heart sections we conclude that severe hypercholesterolemia in combination with ApoE-/genotype as well as Cd treatment results in necrotic cardiomyocyte death. These data were supported by in vitro experiments showing a Cd induced depolarisation of the mitochondrial membrane and the permeabilization of the plasma membrane arguing for the occurrence of Cd induced necrotic cell death. In summary, we were able to show for the first time that the combination of high cholesterol and Cd levels increase the risk for heart failure through cardiac fibrosis. This observation could in part be explained by the dramatically increased deposition of Cd in the hearts of ApoE-/- mice fed a high fat diet.

Key words cell death, macrophages, cardiomyocyte hypertrophy, cholesterol, HL-1

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Downloaded from http://toxsci.oxfordjournals.org/ at Simon Fraser University on March 16, 2015

Cd treatment resulted in significant cardiomyocyte cell death. Based on further analyses of

INTRODUCTION As a naturally occurring heavy metal, cadmium (Cd) can be found throughout the environment at low levels but can accumulate significantly through human activities such as mining and iron smelting (ICdA, 2013). Human non-occupational exposure and uptake of Cd occurs mainly through ingestion of Cd contaminated food and to a lesser extent through ingestion of contaminated drinking water and inhalation of ambient air. Due to high amounts of Cd in tobacco leaves smoking is one of the most important sources for Cd intake in humans (doubling of the daily uptake) (EFSA, 2009; Friberg, 1986; Lewis et al., 1972). According to the WHO report on the Global Tobacco Epidemic from the year 2011, tobacco use causes more than 5 million deaths per year worldwide, rising up to 8 million deaths by

smoking induced deaths are in part attributable to Cd uptake, accumulation and toxicity. Absorption of Cd by the human body depends on the route of exposure: ingestion results in absorption rates of 1-10% whereas inhalation results in substantially higher absorption rates of 5-50%. In contrast, the excretion rate of Cd is very low (with a half-life of 10-30 years), leading to accumulation in the human body, particularly in the liver, kidneys and testis (ATSDR, 2012). As early as 1977 Voors and Shumann revealed a strong correlation between liver Cdconcentration and deaths from heart disease by analysing autopsy study data. In the same study the authors hypothesized that the cardiac conduction system is negatively influenced by Cd even at low concentrations (Voors and Shuman, 1977). In 2001, Abu-Hayyeh et al. detected Cd accumulation of up to 20µmol/l in the aortic wall of smokers and identified the vascular system as a new and important target of Cd deposition in the human body (AbuHayyeh et al., 2001). More recently, epidemiological studies linked increased CVD mortality to low level exposure to Cd (Menke et al., 2009; Tellez-Plaza et al., 2013; Tellez-Plaza et al., 2012), and data from the National Health and Nutrition Examination Survey (NHANES) led to the conclusion that increased Cd levels are associated with stroke and heart failure (Peters et al., 2010), peripheral arterial disease (Navas-Acien et al., 2004; Navas-Acien et al., 2005), myocardial infarction (Everett and Frithsen, 2008), and cerebrovascular diseases (Agarwal et al., 2011). Through extensive research using human samples, animal models, and cell culture approaches, Cd could also be identified as an independent risk factor for early atherosclerosis by our team (Messner et al., 2009; Messner et al., 2012). The cytotoxic effects of Cd on vascular endothelial cells (e.g. by inducing DNA damage, apoptotic-, necrotic-, and autophagic cell death) further demonstrated the pro-atherogenic and CVD causing potential of Cd (Dong et al., 2009; Jeong et al., 2004; Jung et al., 2008; Kolluru et al., 2006; Liu and Jan, 2000; Messner, Knoflach, Seubert, Ritsch, Pfaller, Henderson, Shen, Zeller, Willeit, Laufer, Wick, Kiechl and Bernhard, 2009;

Messner, Ploner, Laufer and

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the year 2030 (WHO, 2011). Based on this information it is reasonable to accept that

Bernhard, 2012; Woods et al., 2008). Additionally, it was observed that Cd altered the serum lipid and cholesterol profile to a more atherogenic state in mice (Messner, Knoflach, Seubert, Ritsch, Pfaller, Henderson, Shen, Zeller, Willeit, Laufer, Wick, Kiechl and Bernhard, 2009). After the discovery that the heart also accumulates Cd after low dose exposure of mammals (Kopp et al., 1978) animal studies followed, which could provide evidence that Cd treatment reduces myocardial contractility (Kopp et al., 1980), impairs mitochondrial respiration of cardiomyocytes (Kisling et al., 1987), induces damage to intercalated discs (Kolakowski et al., 1983), modifies the cardiac muscle ultrastructure (Kolakowski, Baranski and Opalska, 1983), and has a negative impact on the cardiac antioxidant system (Jamall and Smith, 1985). Further, in isolated cardiac mitochondria Cd results in the inhibition of the electron

Hypercholesterolemia alone is associated with cardiac failure as several animal studies have shown that hypercholesterolemia is not only associated with atherogenesis but also with cardiac hypertrophy and fibrosis. However, induction of cardiac hypertrophy and fibrosis develops over a much longer time – 26 weeks in LDLR-/- mice compared to 12 weeks in ApoE-/- mice – like in our setting (Kang et al., 2009). Reduction of the provisional tolerable weekly intake of Cd from 7µg/kg to 2.5µg/kg, by the European Food Safety Authority in 2009 shows that the adverse health effects of Cd were underestimated in the past (EFSA, 2011). Since a previous study suggested an increased cardiotoxic effect of Cd through long-term, low-dose Cd exposure (Kopp et al., 1982), and mechanistic data on the cardiotoxic properties of Cd are scarce, a significant increase in research in this area is absolutely crucial. This study is based on the previous finding of an increased heart weight/body weight ratio (indicative of a potential hypertrophy) in ApoE knock-out (ApoE-/-) mice under high fat diet (HFD) receiving Cd via drinking water compared to controls receiving normal drinking water (Messner, Knoflach, Seubert, Ritsch, Pfaller, Henderson, Shen, Zeller, Willeit, Laufer, Wick, Kiechl and Bernhard, 2009). The purpose of the present study is to reveal whether Cd alone or only in combination of high serum cholesterol levels is capable of inducing the increase in heart weight to body weight ratio. We also seek to define potential structural changes in mouse hearts under the indicated conditions (i.e. with and without Cd under high, moderate, and normal serum cholesterol levels), to study cardiotoxic, inflammatory, and fibrotic processes in mouse heart and to extend our analyses to in vitro work, which shall allow us to get a basic information of mechanistic principles of Cd-induced heart enlargement.

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transfer chain leading to reactive oxygen species (ROS) formation (Wang et al., 2004).

MATERIAL AND METHODS

Cell culture and treatment For in vitro experiments we used the HL-1 cardiomyocyte cell line provided by Prof. William C. Claycomb from the Louisiana State University (New Orleans, Louisiana). Handling of the cells was performed according to the instruction provided (Claycomb et al., 1998). For individual experiments cells were incubated with 2.5 or 5µmol/l CdCl2 (cadmium chloride, Sigma Aldrich; Vienna, Austria) for the indicated times. Quantification of Cd-induced cell death

procedure followed by FACS analyses (FACS Canto II; Becton & Dickinson; Vienna, Austria), as previously described (Bernhard et al., 2003).

Quantification of LDH release Cellular LDH release was measured using the LDH-Cytotoxicity Assay Kit II (Biovision; Mountain View, USA) as described in the user’s manual instructions. Measurement of Cd induced mitochondrial depolarization Measurement of the mitochondrial membrane potential was performed using the MitoProbe JC-1 assay kit (Molecular Probes; Vienna, Austria) according to the manufacturer’s instructions. For this purpose, HL-1 cells were stained and analysed using a FACS Canto II (Becton & Dickinson; Vienna, Austria). Quantification of intracellular DNA content After Cd treatment and following enzymatic detachment, cells were washed three times with PBS, permeabilised using saponin (1mg/ml), and subsequently stained with PI (50mg/ml). After an incubation time of 15min on ice cells were washed three times with PBS and analysed using a FACS Canto II (Becton & Dickinson; Vienna, Austria). Treatment of animals Ethics Statement: All animals received care in compliance with the ‘Principles of laboratory animal care’ formulated by the National Society for Medical Research and the ‘Guide for the care and use of laboratory animals’, prepared by the Institute of Laboratory Animal Resource and published by the NIH. This study was approved by the Austrian Ministry of Science and Research.

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Cell death was detected and quantified using the AnnexinV/propidium iodide (PI) staining

Female C57/BL6 mice and female ApoE-/- mice were purchased from Charles River Laboratories (USA). All mice were kept at the Institute of Biomedical Research of the Medical University of Vienna at 24°C and a 12h light/dark cycle. After 8 weeks the mice were randomly divided into three groups: (i) ApoE-/- mice receiving a Western type high fat diet (ApoE-/- HFD, n=6 control and n=7 Cd-treated; crude fat 21.2%; Ssniff, Soest, Germany), (ii) ApoE-/- mice receiving a normal diet (ApoE-/- ND, n=3 control and n=3 Cd-treated; R/M-H feed, Sniff, Germany) and C57BL6/J mice (n=3 control and n=3 Cd-treated) receiving a normal diet. The three Cd-treated groups received 100mg/l CdCl2 via drinking water. After 12 weeks of treatment animals were anesthetized with xylasol/ketamine, weighted, and blood samples were drawn from the inferior vena cava after opening of the abdomen.

samples were stored at -80°C). Thereafter, spleen, liver, kidneys, lung and heart were harvested, divided into two parts of equal size, and stored in liquid nitrogen or in formalin for dehydration and histological processing. Quantification of Cd concentrations in organ samples Digestion of kidneys and hearts of treated and untreated mice was performed in duplicates by microwave irradiation (MLS, Milestone, Germany). Wet sample tissue (approx. 15-30mg) was digested with 32.5% HNO3 (1ml). The digested samples and the microwave blanks were diluted with water to a total weight of ca. 10g. Limit of detection (3pg/g) and quantification (10pg/g) of the microwave digested samples were calculated from the microwave blanks in solution. The determination of Cd in mouse organs was performed on an ICP-MS 7500ce (Agilent, Waldbronn, Germany) measuring

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Cd (analyte) and 115In (internal standard). The

instrument was equipped with a concentric nebulizer, a Scott spray chamber and Ni cones. The measurement parameters were as follows: RF power was set to 1500W, a carrier gas flow of 0.98L/min and a makeup gas flow of 0.14L/min were applied. Detection of the peak maximum was chosen as mode of acquisition with a dwell time of 0.3s and a replication rate of 10 per mass. Standard stock solutions were purchased from CPI International (USA). Each element was diluted with 3.2% HNO3 (sub-boiled) yielding a final concentration of 0.01– 5ng/g Cd and 5ng/g. Measurement of TGF-β concentrations in the serum of Cd-treated mice After blood collection and serum preparation, TGF-β concentrations were measured using the TGF-β 1 Ready-Set-Go ELISA according to the manufacturer’s instructions (eBioscience, Austria).

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Subsequently, blood samples were subjected to serum preparations (until further use, serum

Histological analyses After fixation in 4.5% formaldehyde and dehydration mouse hearts were embedded in paraffin and cross sections of 5µm thickness were prepared. After deparaffinization using a decreasing ethanol series, heart tissue was stained with Masson-Trichrome-Goldner (MTG) (MTG-staining kit from Merck, Gemany) according to manufacturer’s instructions (extended incubation period of 30min in the Azophloxin staining solution). Image acquisition was performed with an Axio Cam (Zeiss, Germany) and for the quantification of blue fibrotic tissue area (compared to whole organ area) Axio Software (Zeiss, Germany) was used. The MTG stained heart sections were further used for the measurement of the left ventricular (LV) wall thickness (as percent of the total heart diameter). Using Picro-Sirius stained

purpose 5µm sections were deparaffinized with a decreasing ethanol series, incubated with Weigert’s Hämatoxylin solution for 8min, rinsed with tap water for 10min, and stained PicroSirius Red Solution (Sigma Aldrich, Austria). After staining, the slides were rinsed in 0.5% acetic acid, rehydrated in an increasing alcohol series, and fixed with Histokitt (Firma Assistant, Germany). Image acquisition was performed with the Mosaix Software of Axio Cam to obtain whole pictures of mice hearts (Zeiss, Germany). TUNEL assay on histological sections The presence of DNA strand breaks on heart sections of control and Cd-treated mice was analysed using the In Situ Cell Death Detection Kit (POD, Roche, Germany) according to the manufacturer’s instructions. Image acquisition was conducted using a LSM510 Meta attached to an Axioplan 2 imaging MOT using ZEN software (Zeiss, Austria). Blinded image analysis was performed by two independent researchers by counting the number of TUNELpositive cells per total cell count (identified by staining of the nuclei with PI). Immunofluorescence analyses of heart sections Immunofluorescence staining was performed on 3µm heart sections. Heart tissue sections were subjected to standard deparaffinization technique followed by heat-mediated antigen retrieval. Blocking of unspecific binding sites was performed using 2% bovine serum albumin (BSA). Visualization of nuclei was performed either by staining with PI for 1.5 min or TOPRO3 for 10min. Heart muscle structure was depicted by staining with Alexa 488 conjugated Phalloidin (Life Technologies; Vienna, Austria). Primary antibodies used were anti-vimentin antibody (sc-5565, Santa Cruz, USA), anti-CD68 antibody (MAB6564, Abnova; Germany), anti-monocyte chemoattractant protein 1 (MCP-1) antibody (ab9669, Abcam; UK), anticleaved caspase-3 antibody (#9661 Cell Signaling Technology; USA) at overnight incubation. Thereafter, slides were incubated with secondary antibodies for 1h in the dark, using either

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sections measurements of cross-sectional myocyte diameter were performed. For this

anti-rabbit or anti-mouse-Alexa 488 antibodies (Life technologies, Austria). After washing in PBS slides were mounted with ProLong Gold antifade reagent (Life technologies, Austria). Image acquisition was conducted using a LSM 510 Meta attached to an Axioplan 2 imaging MOT using ZEN software 2008 (Zeiss, Germany). Statistical analyses All data were analysed for Gaussian distribution and consequently subjected either to parametric tests (one-way ANOVA or two-sided t-test) or non-parametric tests (MannWhitney U Test), using SPSS software.

The combination of Cd treatment and high serum cholesterol levels induces a significant increase in heart weight/body weight ratio and causes cardiac fibrosis. The initial observation after subjecting mice to Cd in drinking water was, depending on the mouse strain and food cholesterol load, that Cd treatment induces an increase in mouse heart weight to body weight ratio only in ApoE-/- HFD mice. Additionally, ApoE-/- HFD mice showed a lower heart to body weight ratio compared to ApoE-/- ND and C57BL/6J ND mice (Figure 1A). The treatment of mice with Cd contained drinking water also induced detectable structural changes in the myocardium. Histological analyses of MTG and Sirius Red stained heart sections identified the presence of fibrotic areas within the myocardium (Figure 1D & Supplemental Figure 1). Areal quantification revealed that of all groups, only the ApoE-/HFD mice developed an increase in cardiac fibrosis (Figure 1B). Investigation of the localisation of fibrotic deposition demonstrated a Cd dependent increase in intracardial collagen deposition in the ApoE-/- HFD group and a Cd induced perivascular collagen deposition in all three groups (Supplemental Figure 1). To explore a possible connection between the vascular system and the morphological changes of the heart, the influence of Cd on the development on atherosclerotic lesions in mouse aortas was investigated. Only ApoE-/- HFD mice, where a rise in atherosclerotic area was measured showed a pro-atherogenic of Cd. In ApoE -/- ND mice atherosclerotic plaque area did not differ between control and Cd treated mice (Supplemental Table 1). Further, the effect of Cd administration and high serum cholesterol levels on the structure of the heart’s left ventricular (LV) wall thickness was examined. Measurements of the LV wall thickness in relation to total heart diameter resulted in no significant differences among the different animal and treatment groups (Figure 1C). Although neither Cd treatment nor hypercholesterolemia triggered structural changes of the LV, treatment of ApoE-/- HFD and

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RESULTS

ApoE-/- ND mice with Cd increased the cross-sectional diameter of cardiomyocytes in vivo (Figure 2A and 2B). Cd and HFD induce DNA damage, mitochondrial depolarisation and necrotic cell death in cardiomyocytes. DNA strand breaks are a known feature preceding cell death. In vivo quantification of DNA strand break positive nuclei was performed using the TUNEL assay (Figure 3B). Cd uptake through drinking water led to an increased number of DNA strand break positive cell nuclei in C57BL/6J ND and ApoE-/- ND mice, whereas this effect was not observed in ApoE-/- HFD mice when compared to the corresponding control (Figure 3A). Among the control groups it

damage in ApoE-/- mice when compared to C57BL/6J ND indicated my a statistically nonsignificant trend (p=0.068) (Figure 3A).To study whether this increase in DNA strand breaks in vivo led to an activation of apoptotic cell death we analysed section for the occurrence of active caspase-3, a downstream executioner enzyme in apoptotic signalling. The presence of active caspase-3 could only be detected in ApoE-/- HFD mice (Figure 3C and D). Following the observation of Cd-induced cardiomyocyte death in vivo we aimed at elucidating the mechanism of Cd induced cell death by subjecting cardiomyocytes (HL-1 cells) to physiologically relevant Cd concentrations (i.e. 2.5 and 5µmol/l) in vitro. FACS based analyses of AnnexinV/PI stained cardiomyocytes suggested that Cd induces mainly apoptotic cell death after 96h (Figure 4A). An involvement of mitochondrial signalling in Cd-induced cell death was confirmed using JC-1 staining of mitochondrial membrane potential and FACS analyses. In these studies we could show that Cd treatment leads to a significant increase in cells exhibiting depolarized mitochondria (Figure 4B). As we could previously show that Cd treatment of endothelial cells leads to a form of cell death that is characterized by DNA degradation which mimics apoptotic cell death (absence of PI positive cells in AnnexinV/PI staining) but is in fact a necrotic cell death, we measured cellular DNA content in Cd treated HL-1 cells. Over the period of 96h where cell death occurs it was also observed that DNA content is substantially decreased in HL-1 cells. Additional measurements of LDH release by cardiomyocytes following Cd treatment showed that Cd-induced cell death in HL-1 cells is characterised by cell permeabilization and the release of cellular contents, i.e. pure necrosis. Cd and HFD diet induce MCP-1 and CD68 expression in cardiac tissue. One of the best studied chemokines involved in the development of cardiac fibrosis is MCP1. Immunofluorescence analysis of heart sections stained with anti-MCP-1 antibody (Figure 5B) revealed that a high fat diet in ApoE-/- mice without Cd treatment is sufficient to increase cardiac MCP-1 expression (Figure 5A). Apo -/- mice fed a normal diet in combination with Cd

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was observed and quantified that a high fat diet resulted in marginally increased DNA

treatment also showed increased cardiac MCP-1 expression (Figure 5A; p=0.003). Comparing Cd treated groups at different feedings we were able to show that ApoE-/genotype and a high fat diet aggravate the Cd induced effects of MCP-1 expression in the mouse heart (Figure 5A and 5B). Detection of macrophages within the LV was performed by immunofluorescent staining with an anti-CD68 antibody (Figure 5D). Quantification of cardiac CD68 expression in the control groups at different feeding revealed an increase in CD68 positive macrophages in the ApoE-/- ND control mice (Figure 5C). Additionally, Cd treatment enhances CD68 expression in ApoE-/- ND mice (also compared to Cd treated ApoE-/- HFD mice; Figure 5C). No increase in CD68 signal could be detected in the other two groups (ApoE -/- HFD and C57BL/6J; Figure 5C and 5D).

One of the most prominent cytokines known to be involved in fibrosis development is transforming growth factor-beta (TGF-β). To elucidate the role of TGF-β in Cd and high fat diet induced cardiac changes, we measured serum TGF-β concentrations in controls and Cd treated mice in the three feeding groups. ELISA based TGF- β quantification revealed no difference between the control and the Cd treated animals, both in the high fat diet and in the two normal diet groups (Figure 6A). Serum measurements have shown that TGF-β concentrations are increased in the Cd-treated ApoE-/- ND group compared to the Cdtreated C57BL6/J ND group (Figure 6A). After having analysed systemic TGF-β concentrations, immunofluorescent staining of heart sections with anti-TGF-β antibody revealed no change in tissue expression of this cytokine (Supplemental Figure 4). We also performed immunofluorescence triple staining to visualize heart muscle (Phalloidin staining – green signal), nuclei (TOP-RO-3 – blue signal) and fibroblasts (Vimentin – red signal) in control and Cd treated mice of the three feeding groups. Images shown in Figure 6B suggest that fibroblast accumulate both in and around fibrotic areas in Cd treated ApoE-/- mice. Additionally, it appears that close to the fibrotic areas, some cells are double positive for vimentin and phalloidin (cardiomyocytes), suggesting the presence of myofibroblasts. Although no visible fibrotic areas were detectable in ApoE-/- ND, small areas with fibroblast clustering are observable which are non-existent in control ApoE-/- ND mice (Figure 6B, second row). Interestingly, neither Cd treated wild type mice, nor the controls with different feeding showed fibroblast accumulation. Deposition of Cd in cardiac and kidney tissue Since the kidneys are one of the major target organs for Cd deposition we analysed the concentration of this metal in the different treatment and feeding groups. Cd treatment of mice, regardless of feeding, increases renal Cd. ICP-MS based analyses of kidney tissue

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Influence of the pro-fibrotic cytokine TGF-β in Cd and HFD induced fibrosis.

revealed that Cd deposition in ApoE-/- HFD mice is massively increased compared to ApoE/- ND and C57BL/6J ND mice (Figure 7A). Comparable results are provided by the analyses of Cd concentration in the heart of the three feeding groups. Measurements of Cd content in the heart showed an increase in Cd concentration between the control and treated groups of ApoE-/- HFD and ApoE-/- ND mice. The difference in Cd concentration between the control and the Cd treated animals of the C57BL/6J group did not reach statistical significance. Similar to renal Cd deposition, hearts of the ApoE-/- HFD mice showed massively increased Cd accumulation when compared to the corresponding control (Figure 7B). Interestingly, Cd concentration in hearts of ApoE-/- ND mice was also increased compared to untreated controls whereas C57BL6J mice did not show Cd accumulation in the heart even following Downloaded from http://toxsci.oxfordjournals.org/ at Simon Fraser University on March 16, 2015

Cd treatment (Figure 7B).

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DISCUSSION The development of heart failure is based on fundamental structural and functional changes within the heart muscle and is characterized by cardiomyocyte hypertrophy, death, and the development of fibrosis resulting in loss of function and organ hypertrophy (Berk et al., 2007). The present study demonstrates for the first time that Cd exposure induces cardiac fibrosis however only in combination with a high fat diet in ApoE-/- mice. Based on our data we hypothesize

that

continuous

Cd

intoxication

in

combination

with

moderate

hypercholesterolemia or normal cholesterol levels induces pro-fibrotic processes possibly leading to fibrosis development at later time point. Initially, it is important to state that the Cd concentrations applied in vivo are likely to be of

administrated via the drinking water leads to concentrations in the blood comparable to physiological concentrations in humans (Thijssen et al., 2007). Our previous observation that Cd treatment of ApoE-/- mice results in an increased heart weight to body weight ratio could be confirmed by the present study and we could also extend the knowledge in this field by demonstrating that this effect is tightly associated with the ApoE-/- genotype and HFD. Detailed morphological evaluation of the hearts revealed the presence of cardiac fibrosis only in ApoE-/- HFD. However, an increase in perivascular fibrosis could be observed in Cd treated mice with moderate and normal cholesterol levels suggesting the occurrence of earlystage cardiac fibrosis. Although the increased heart weight to body weight ratio and the presence of cardiac fibrosis are typical signs for the occurrence of hypertrophic changes in LV diameter were not induced by Cd. Notably, hypercholesterolemia alone is known to induce hypertrophy albeit only after longer treatment times of 26 weeks (Kang, Wang, Palade, Sharma and Mehta, 2009). Although cardiac fibrosis and hypertrophy are known to be induced mainly by hypertension in humans (Drazner, 2011), Kang et al, reported a form of hypertension-independent hypertrophy in mice (Kang, Wang, Palade, Sharma and Mehta, 2009). Additionally, it has been established that a high fat diet alone does not trigger hypertension in ApoE-/- mice within 12 weeks and is therefore not a trigger for increased heart weight and cardiac remodelling (Hartley et al., 2000). Although we could show that Cd did not increase blood pressure in female C57BL/6J mice over 6 weeks it is important to consider that an association of Cd and hypertension has been controversially discussed: some studies show increased systolic blood pressure especially in male ICR mice and male Wistar rats over a period of 8 weeks of Cd treatment (Almenara et al., 2013; Donpunha et al., 2011), other studies could not detect a correlation between Cd and hypertension in rats (Dudley et al., 1982; Kotsonis and Klaassen, 1977). Similarly, in humans the connection between Cd and hypertension (and gender) is inconclusive especially since patients with itaiitai disease did not develop hypertension (Gallagher and Meliker, 2010; Nakagawa and

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physiological relevance as Thijssen et al. reported that a concentration of 100mg/l Cd

Nishijo, 1996; Whittemore et al., 1991). Although neither Cd treatment nor high cholesterol levels induced changes of LV diameter we were able to show that Cd in combination with high and moderate cholesterol levels triggers cardiomyocyte hypertrophy on a cellular level in vivo. Based on this observation we hypothesize that the combination of high serum cholesterol levels and Cd provokes cellular hypertrophy, which may precede organ hypertrophy. Cardiac fibrosis development is known to be triggered by myocyte cell death (Gandhi et al., 2011). As we could observe pronounced Cd induced DNA damage and as a result cardiomyocyte death, particularly in the ApoE-/- HFD mice showing significant increase in fibrotic area, it seems likely that Cd-induced myocyte death is responsible for consequent shown in rats and rabbits (Obradovic et al., 2013; Zarzoso et al., 2013) this cardiotoxic effect is not enough to trigger fibrosis. For the results of the present study we suggest that high cholesterol levels may mainly serve as a Cd deposition accelerating factor and that Cd is the central element that defines the amount of cell death. The absence of a difference in the amount of cells with DNA strand breaks between control and Cd-treated animals in the ApoE-/- HFD mice group could be explained by the fact that within or near the fibrotic areas cell death may already have occurred at an earlier time point. As cell death mode is essential for further processes in the tissue, e.g. inflammation, in vitro and in vivo studies were performed. Our analyses of the active subunit of caspase-3 in vivo have shown that apoptotic cardiomyocyte death occurs only in a minor fraction of cardiomyocytes and that the predominant type of cell death induced by Cd and high hypercholesterolemia is necrosis. Also the in vitro data proved that 5µmol/l Cd (treatment of mice with 100mg/l Cd leads to concentrations of 5-8µmol/l in cardiac tissue) induces necrotic cell death with an involvement of mitochondrial depolarisation arguing for programmed necrosis of cardiomyocytes as already observed by Gandhi et al. (Gandhi, Kamalov, Shahbaz, Bhattacharya, Ahokas, Sun, Gerling and Weber, 2011). Additionally, live cell imaging analyses on cardiomyocyte contractility showed that Cd treatment significantly reduces cardiomyocyte function in vitro (Video 1 and Video 2). As we observed an interference of Cd with cardiomyocyte contractility in cell cultures it is evident that Cd also reduced heart function on the cellular level and may thereby also contribute to cardiomyocyte hypertrophy. Further evidence for the cell death – inflammation – fibrosis axis being active in mice with high and moderate hypercholesterolemia and Cd treatment stem from our studies on the proinflammatory cytokine MCP-1 known to be involved in ischemic cardiomyopathy (Frangogiannis et al., 2007). A significant increase in MCP-1 expression could be observed in Cd treated mice with moderate hypercholesterolemia arguing for our hypothesis that under this treatment the fibrotic process is currently in progress. In contrast, in ApoE-/- mice with

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fibrosis. Although a high fat diet alone induces cardiomyocyte death, which has already been

high cholesterol levels the proinflammatory signalling has already taken place and cardiac fibrosis is apparently present. The observation of raised MCP-1 signalling within the myocardium of ApoE-/- HFD mice is of particular interest since it is not seen in ApoE-/- ND and C57BL/6J mice and to our knowledge the first description of this phenomenon in mice. In our histological studies we were also able to show macrophage infiltrates in the hearts of Cd treated animals. Similar to the results of MCP-1 expression pronounced macrophage infiltration

was

only

detectable

in

Cd

treated

ApoE-/-

mice

with

moderate

hypercholesterolemia. An association between cell death, macrophage infiltration and the development of fibrosis are typically known to be linked by TGF-β (secreted by e.g. macrophages) thereby activating fibroblasts and inducing the differentiation of precursor cells

through the enhancement of TIMP activity (tissue inhibitor of metalloproteinases). Importantly, it has been demonstrated that macrophages and other cell types can cause fibrosis also independent from TGF-β stimulation (Wynn and Barron, 2010). Although TGF-β is one prominent and extensively investigated cardiac fibrosis inducing factor our results suggest that Cd induced fibrosis in ApoE-/- HFD mice is not associated with or dependent on TGF-β signalling as it was neither increased in blood nor in tissue. Nevertheless, we hypothesize that Cd treatment of hypercholesterolemic ApoE-/- mice induces the recruitment of myofibroblasts to the proximity of fibrotic areas, which are known to reinforce ECM production as well as the release of further proinflammatory cytokines (Creemers and Pinto, 2011). As already mentioned above, high and moderate serum cholesterol levels increases Cd deposition in target organs based on the heavy metal’s tendency to accumulate in adipose tissue thus explaining the advanced toxic and fibrotic effect of Cd in ApoE-/- HFD and ApoE/- ND mice. Especially Cd deposition in mouse hearts depends on high blood cholesterol levels since Cd treated C57BL/6J mice did not accumulate significantly higher Cd concentrations compared to the control. Important to note is that at very low Cd concentrations, even below the detection limit as seen in C57BL/6J mice, significant cardiomyocyte cell death is still occurring. In summary, we were able to show that Cd has cell death inducing- and inflammatoryproperties in cardiac tissue but disease progression leading to the development of cardiac fibrosis depends on lipid concentrations, as a high fat diet accelerates Cd deposition in organs immensely, especially in the heart. Based on the obtained results we hypothesize that the combination of the two cardiovascular risk factors hypercholesterolemia and increased cardiac Cd concentrations, as it can be seen in e.g. smokers, possibly accelerate and aggravate the pathological processes otherwise induced individually.

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to myofibroblasts. All these processes are leading to an accumulation of extracellular matrix

SUPPLEMENTARY DATA DESCRIPTION The supplementary data section illustrates additional findings of Cd interactions with the cardiovascular system in mice namely its impact on blood pressure and aortic plaque development in the investigated mice. Sirius Red staining of collagen deposition and immunofluorescence staining of the profibrotic signalling molecule TGF-β give insights into fibrotic remodelling in cardiac tissue after Cd incubation. In a HL-1 cell culture, Cd causes decreased cell viability and contractility as seen by live cell imaging.

This work was supported by the Austrian National Bank Project 14745 to D. Bernhard and Austrian National Bank Project 14590 to B. Messner.

ACKNOWLEDGMENTS The authors would like to thank Anneliese Steinacher-Nigisch, Birgitta Winter, and Eva Eichmair for their excellent technical assistance.

CONFLICT OF INTEREST The authors declare no conflict of interest.

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FUNDING SOURCE

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FIGURES AND FIGURE LEGENDS Figure 1: Cd and HFD induced changes in myocardial structure. Figure 1A shows the heart weight to body weight ratio in percent of control and Cd treated mice. Quantification of myocardial fibrosis of the three groups is shown in Figure 1B. Figure 1C shows the results of the LV wall thickness measurements of control and treated hearts. Representative images of MTG stained heart sections are depicted in Figure 1D (magnification 20x) including a zoom image of a highly fibrotic area in ApoE-/- HFD (magnification 63x). Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. Figure 2: Cd induced cardiomyocyte hypertrophy. Quantification of cardiomyocyte

of cardiomyocyte diameter measurements. Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. Figure 3: Effect of Cd treatment and high cholesterol levels on the viability of cardiomyocytes in vivo. Figure 3A shows the quantification of TUNEL staining performed on heart sections of the three groups. Figure 3B shows representative pictures of cells with DNA strand breaks (shown in green) and PI stained cell nuclei shown in red (magnification 63x). Figure 3C shows the quantification of caspase-3 positive cells on heart sections of the three groups. Figure 3D shows representative pictures of caspase-3 positive cells (shown in green) and PI stained cell nuclei shown in red (magnification 63x). Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. Figure 4: Effect of Cd on cardiomyocyte viability, DNA content, mitochondrial membrane potential and plasma membrane integrity. Figure 4A shows the FACS based quantification of AnnexinV/PI stainings of Cd treated cardiomyocytes. Figure 4B shows the amount of cells with depolarised mitochondria after Cd treatment for 72h. Quantification of LDH release into the cell culture supernatant of Cd treated cardiomyocytes is depicted in Figure 4C. Figure 4D shows the results of intracellular DNA quantification in Cd treated HL-1 compared to control. All experiments were performed in triplicates and were repeated at least three times. Results depict the mean ± standard deviation. Figure 5: Detection of proinflammatory signals within the heart of animals treated with Cd and different feedings. Figure 5A shows the quantification of MCP-1 positive signal within the hearts of control and Cd treated animals of different feeding groups. Representative images of immunofluorescent staining of MCP-1 are shown in Figure 5B (magnification 40x). Quantitative analyses displaying the presence of macrophages are shown in Figure 5C and representative images are depicted in Figure 5D (magnification 63x).

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diameter is depicted in Figure 2A expressed in µm. Figure 2B shows representative images

Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. Figure 6: Influence of Cd treatment and high cholesterol levels on the serum concentration of TGF-β and the myocardial fibroblast distribution. Figure 6A depicts the results of the ELISA based quantifications of TGF-β expressed as pg/ml. Figure 6B depicts images of triple-stained heart sections to visualize heart muscle (Phalloidin-staining), nuclei (TO-PRO3) and fibroblasts (Vimentin-staining). Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. Figure 7: Quantification of organ Cd concentration. After twelve weeks of treatment,

(Figure 7B) of control versus Cd treated animals. Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation.

24


animals were sacrificed to analyse Cd concentrations within kidney (Figure 7A) and heart


Figure 1: Cd and HFD induced changes in myocardial structure. Figure 1A shows the heart weight to body weight ratio in percent of control and Cd treated mice. Quantification of myocardial fibrosis of the three groups is shown in Figure 1B. Figure 1C shows the results of the LV wall thickness measurements of control and treated hearts. Representative images of MTG stained heart sections are depicted in Figure 1D (magnification 20x) including a zoom image of a highly fibrotic area in ApoE-/- HFD (magnification 63x). Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. 498x370mm (300 x 300 DPI)


Figure 2: Cd induced cardiomyocyte hypertrophy. Quantification of cardiomyocyte diameter is depicted in Figure 2A expressed in µm. Figure 2B shows representative images of cardiomyocyte diameter measurements. Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. 207x338mm (300 x 300 DPI)


Figure 3: Effect of Cd treatment and high cholesterol levels on the viability of cardiomyocytes in vivo. Figure 3A shows the quantification of TUNEL staining performed on heart sections of the three groups. Figure 3B shows representative pictures of cells with DNA strand breaks (shown in green) and PI stained cell nuclei shown in red (magnification 63x). Figure 3C shows the quantification of caspase-3 positive cells on heart sections of the three groups. Figure 3D shows representative pictures of caspase-3 positive cells (shown in green) and PI stained cell nuclei shown in red (magnification 63x). Number of animals per group: ApoE-/HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. 415x309mm (300 x 300 DPI)


Figure 4: Effect of Cd on cardiomyocyte viability, DNA content, mitochondrial membrane potential and plasma membrane integrity. Figure 4A shows the FACS based quantification of AnnexinV/PI stainings of Cd treated cardiomyocytes. Figure 4B shows the amount of cells with depolarised mitochondria after Cd treatment for 72h. Quantification of LDH release into the cell culture supernatant of Cd treated cardiomyocytes is depicted in Figure 4C. Figure 4D shows the results of intracellular DNA quantification in Cd treated HL-1 compared to control. All experiments were performed in triplicates and were repeated at least three times. Results depict the mean ± standard deviation. 304x250mm (300 x 300 DPI)


Figure 5: Detection of proinflammatory signals within the heart of animals treated with Cd and different feedings. Figure 5A shows the quantification of MCP-1 positive signal within the hearts of control and Cd treated animals of different feeding groups. Representative images of immunofluorescent staining of MCP-1 are shown in Figure 5B (magnification 40x). Quantitative analyses displaying the presence of macrophages are shown in Figure 5C and representative images are depicted in Figure 5D (magnification 63x). Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. 376x402mm (300 x 300 DPI)


Figure 6: Influence of Cd treatment and high cholesterol levels on the serum concentration of TGF-β and the myocardial fibroblast distribution. Figure 6A depicts the results of the ELISA based quantifications of TGF-β expressed as pg/ml. Figure 6B depicts images of triple-stained heart sections to visualize heart muscle (Phalloidin-staining), nuclei (TOPRO3) and fibroblasts (Vimentin-staining). Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. 198x305mm (300 x 300 DPI)


Figure 7: Quantification of organ Cd concentration. After twelve weeks of treatment, animals were sacrificed to analyse Cd concentrations within kidney (Figure 7A) and heart (Figure 7B) of control versus Cd treated animals. Number of animals per group: ApoE-/- HFD = 13, ApoE-/- ND = 6, and C57BL/6J ND = 6. Results depict the mean ± standard deviation. 154x201mm (300 x 300 DPI)

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