Elsevier Editorial System(tm) for Drug Discovery Today: Therapeutic Strategies Manuscript Draft Manuscript Number: Title: Biomarkers of oxidative stress in heart failure and identification of novel carbonylated proteins in plasma of failing rabbit hearts Article Type: Heart failure 2013 Corresponding Author: Prof. Rainer Schulz, PhD MD Corresponding Author's Institution: Justus-Liebig University First Author: Sara Menazza, PhD Order of Authors: Sara Menazza, PhD; Marcella Canton, PhD; Elisa Sorato, PhD; Kerstin Boengler, PhD; Rainer Schulz, PhD MD; Fabio Di Lisa, MD

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Dr. Raymond Baker and Eliot Ohlstein Editors-in-Chief Drug Discovery Today: Therapeutic Strategies Padova, August 30, 2013 Dear Editors, Please find attached our manuscript entitled "Old and new biomarkers of oxidative stress in heart failure" that we are submitting upon your kind invitation to contribute to Drug Discovery Today: Therapeutic Strategies. The submitted manuscript has not been published previously and is not under consideration for publication elsewhere. All Authors have approved the manuscript in its current form. If accepted, this work will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright-holder. Please note that as specified in the title page we propose to have both Prof. Rainer Schulz and Prof. Fabio Di Lisa as corresponding Authors. We hope that you will find our manuscript suitable for publication in Drug Discovery Today: Therapeutic Strategies. Best regards,

Prof. Fabio Di Lisa Department of Biomedical Sciences University of Padova Viale G. Colombo, 3 35131 Padova Italy

Cover Letter

Old and new biomarkers of oxidative stress in heart failure

Sara Menazzaa, Marcella Cantona, Elisa Soratoa, Kerstin Boenglerb, Rainer Schulzb*,

Fabio Di Lisaac*

a Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35131

Padova, Italy

b Institute of Physiology, Faculty of Medicine, Justus Liebig University, Aulweg 129, 35392

Giessen, Germany

c Institute of Neurosciences, CNR, University of Padova, Viale G. Colombo 3, 35131

Padova, Italy

* Corresponding Authors: Rainer Schulz and Fabio Di Lisa

Prof. Rainer Schulz

Institute of Physiology, Faculty of Medicine, Justus Liebig University, Aulweg 129,

35392 Giessen, Germany

Tel.: +496419947240

Email: Rainer.Schulz@physiologie.med.uni-giessen.de

Prof. Fabio Di Lisa

Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3,

35131 Padova, Italy

Tel.: +390498276132

Fax: +390498276140

Email: dilisa@bio.unipd.it

ManuscriptClick here to view linked References

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Highlights

Several molecular pathways and organs are involved in heart failure suggesting the

need for more objective measures for this disease.

Many oxidative stress biomarkers have been identified in plasma.

The relationship between oxidative stress within the failing heart and oxidative

event occurring in the plasma needs to be established.

Two novel targets of oxidative stress are identified in plasma and related to

contractile dysfunction in a heart failure model.

Abstract

Many cardiovascular diseases have been related to increased oxidative stress and

subsequent alterations in cardiomyocyte function and/or viability. As increased oxidative

stress might also modify non-cardiac proteins, quantitative relationships between plasma

proteins modified by reactive oxygen species and contractile abnormalities might be of

interest and become a diagnostic tool but have hardly been established yet. In the past

few decades several urine and serum biomarkers have been identified but the diagnostic

reproducibility of these tools as well as the scarcity of data evaluating their potential role in

heart failure development/progression is currently limited. Therefore, the different

biomarkers of oxidative stress and their relation to cardiac disease, especially heart failure,

are discussed and two novel plasma protein targets of oxidation – derived from

experimental studies - are identified.

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Introduction

The utilization of oxygen as the terminal acceptor of electrons in the mitochondrial

respiratory chain gives a very large advantage in the conversion of nutrients into energy

that can be utilized by cells, especially in the form of ATP synthesis. For instance, the

complete oxidation of glucose to CO2 and H2O yields 19 times more ATP than the

anaerobic conversion to lactate. For this tremendous increase in energy efficiency, living

organisms have to pay the price of coping with oxygen that, besides its transformation into

water, easily generates reactive and potentially toxic molecules. In fact, since oxygen

accepts only one electron at a time, its complete reduction into water requiring 4 electrons

inevitably goes through three steps that generate partially reduced forms of oxygen,

namely superoxide anion (O2·-), hydrogen peroxide and hydroxyl radical (OH·)(as covered

by countless reviews; for instance see [1-4]). These molecules are generally termed as

reactive oxygen species (ROS) that include also singlet oxygen (1O2), hypochlorous acid

(HOCl), as well as alkoxyl (RO·), peroxyl (ROO·) and hydroperoxyl (HOO·) radicals. In

addition, oxygen is utilized in the formation of nitric oxide and nitrogen-centered reactive

species (RNS) that can further react with ROS, as in the case of peroxynitrite (ONOO-)

resulting from the interaction between NO and superoxide.

Cytochrome oxidase, the final reaction of the mitochondrial chain utilizing more than 90%

of oxygen supplied to cells, catalyzes the reduction into water proceeding through the

formation of potentially toxic intermediates that fortunately remain bound to the enzyme, so

that only the final product is released. Nevertheless, other physiological processes or

undesired reactions release ROS in every cell at any given time. A vast repertoire of

antioxidant defenses, both in the form of enzymatic activities and chemical scavengers,

abolishes ROS accumulation under physiological conditions. However, under pathological

conditions this delicate balance between ROS generation and removal is altered favoring

the increase in ROS levels as well as their deleterious attack to essential cellular

macromolecules, such as lipids, carbohydrates, proteins and nucleic acids. This imbalance

has been termed as oxidative stress that was originally introduced by H. Sies to describe

“a disturbance in the pro-oxidant–antioxidant balance in favour of the former, leading to

potential damage” [5].

Although ROS are involved in various physiological processes, the clinical interest related

to the development of diagnostic biomarkers is mostly, if not exclusively focused on the

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relationships between ROS and diseases. This article is not meant at covering the current

debate on how and to what extent oxidative stress contributes to a wide array of

pathologies. Here, we aim at reviewing the association that has been established between

oxidation of plasma components and cardiac pathologies, especially heart failure. In

addition, two novel plasma biomarkers are described that we have shown to be linked with

intracellular oxidative stress and contractile dysfunction in an experimental model of heart

failure.

Biomarkers of oxidative stress in body fluids

Although the participation of ROS in functional and structural derangements is commonly

accepted, discrepancies exist on causal links whereby pathological states would depend

on oxidative stress. Besides negative results in clinical trials evaluating the efficacy of

antioxidant interventions [6], the lack of conclusive evidence is mostly due to uncertainties

on the methodologies used to detect oxidative stress in clinical settings.

Techniques available for assessing directly ROS formation in tissues can hardly apply to

studies in humans that are mostly based upon the detection of metabolites in plasma or

urine. Since ROS are compounds with an extremely short half-life, their assessment in

extracellular fluids withdrawn from patients is not feasible. This limitation is circumvented

by measuring more stable results of ROS-induced changes of molecular targets that are

considered as biomarkers of oxidative stress. However and unfortunately, the reliability of

all of them is far from being optimal (as reviewed in [7-10]). The most relevant

shortcomings are that the available markers of oxidative stress are not specific for any

disease and do not allow the prediction of the evolution of any given pathology. Therefore,

data can be collected just as associations between a disease state and the increase of a

given biomarker without providing conclusive information on the severity of the disease, its

prognosis, the mechanisms causing the increase in modified plasma components and the

causal links between the disease and oxidative stress. These limitations obviously apply to

the validation and monitoring of antioxidant treatment as well. Notably, while oxidative

stress can be reflected reliably by an increased oxidized state within cells, this might not

be the case in the extracellular space. In fact, while the intracellular ratio between thiols

(reduced) and disulfides is > 100:1, as shown by the redox state of glutathione, in plasma

the cysteine/cystine ratio is 1:40 [10]. Therefore, not only it is unlikely that antioxidants can

5

increase the reduced state in plasma, but also this might not represent a desired goal. In

addition, biomarkers adopted for oxidative stress hardly satisfy several other criteria

required reliability. Most of them are not chemically stable, their levels are frequently

influenced by diet or pathological states independently of oxidative stress and standard

levels are difficult to be determined since large variations are present among not only

individuals, but also assay procedures. Furthermore, some assays are technically

demanding and require expensive instruments limiting their use in routinely clinical

evaluations.

Despite all these limitations, the wide and strong interest to ROS involvement in

pathological states prompted the development of a large number of biomarkers. A detailed

review listed 71 different indicators in 2009 [10], a number that is likely to be higher at

present. Products of lipid and protein oxidation represent the majority of circulating

biomarkers of oxidative stress that include also DNA oxidation, as reflected by 8-hydroxy-

2'-deoxyguanosine [8,10].

Markers of lipid oxidation

The oxidative degradation of polyunsaturated lipids, commonly refereed as

lipoperoxidation, generates a large number of end-products the detection of which allows

the assessment of oxidative stress in tissues and in extracellular fluids. Among them the

most utilized for plasma assays are malondialdehyde (MDA) and isoprostanes, especially

in cardiovascular studies.

The assessment of MDA is based upon its reaction with thiobarbituric acid (TBA)

generating a stable chromophore that can be detected either spectrophotometrically or by

means of HPLC [11]. The first approach is the easiest and thus the most utilized, yet it is

flawed by the TBA reaction with other aldehydes that are not related to lipoperoxidation.

The specificity, though also the procedure complexity, is increased by separating and then

detecting the MDA-TBA adduct by HPLC. In any case sources of errors should be

considered, such as MDA derived from dietary sources, the artifactual generation of MDA

during sample preparation, and the sensitivity to metals, as well as to metal chelating

agents used to prevent blood clotting [8,10].

Notably, MDA is not a specific marker of lipoperoxidation, since it can result also from the

ROS attack on sialic acid and deoxyribose. An absolute specificity for lipoperoxidation is

attributed to isoprostanes (isoPs) formed by non-enzymatic free-radical-induced

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peroxidation of arachidonic acid. These compounds have been termed F2-isoprostanes

(F2-IsoPs), since they are isomeric to prostaglandin (PG) F2 [8]. Although up to 64

isomers of F2-IsoPs can be formed, due to its abundance 15-F2-IsoP or 8-iso-PGF2 is the

product that is mostly used as a marker for oxidative stress [12].

IsoPs in plasma have a short half time, approximately 16 minutes [13], and are excreted

rapidly, which means that they must be formed constantly to maintain a steady-state

concentration. However, since IsoPs are structurally stable end-products, their excretion in

urine allows a cumulative detection of oxidative stress.

IsoPs can be assessed by either tandem mass-spectrometry (MS/MS) techniques (i.e.,

gas chromatograpy-MS/MS or liquid chromatography-MS/MS) or immunoassays, such as

EIA (enzyme immunoassay) or RIA (radioimmunoassay) [8,10,12]. MS-based assays are

more reliable and accurate, although they require a labor-intensive preparation of samples

along with expensive instruments.

IsoPs are likely to represent the best biomarker for lipid peroxidation. Nevertheless,

shortcomings have to be taken into account, such as elevation after fatty meal, plasma

concentrations close to detection limits, and direct excretion into urine of IsoPs generated

in the kidney.

Besides the lack of prognostic value, common concerns related to products of

lipoperoxidation are that they do not provide information on both the site of production and

the mechanisms linking oxidative stress in tissues with their elevation in body fluids. This

latter issue is complicated by the biological effects elicited by many of the molecules

generated by lipoperoxidation. Aldehydes are extremely reactive species that modify

covalently structures and functions of proteins and nucleic acids. In addition, by reacting

with glutathione aldehydes reduce its cellular content potentiating oxidative stress through

a decrease in antioxidant defenses [14,15]. IsoPs trigger signaling pathways upon their

binding to plasma membrane receptors [12,16]. Besides exerting vasoconstrictor and pro-

coagulant effects by means of effects on platelets and the endothelium [17,18], in liver

IsoPs have been shown to play a pro-fibrotic role by stimulating hepatic stellate cell

proliferation and collagen hyperproduction [16]. Therefore, the products of lipoperoxidation

generate a vicious cycle that is likely to amplify and exacerbate the initial injury. However,

causal relationships are ill defined, since it remains difficult to understand whether

molecules, such as MDA and IsoPs, are just biomarkers or contribute directly to causing

and maintaining pathological conditions.

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Markers of protein oxidation

Proteins undergo various forms of oxidation [19-22] the most abundant of which is protein

carbonylation. This covalent modification can result from the following types of reactions:

(i) metals- or H2O2-catalyzed formation of semialdehydes at the level of lysine, arginine

and proline residues; (ii) in a process termed as Michael addition, the side chain of lysine,

histidine or cysteine can react via nucleophilic attack on C3 of , unsaturated aldehydes

generated by lipoperoxidation, such as 4-hydroxynonenal or the above mentioned MDA.

This second type is the most abundant; (iii) Schiff base formation between a lipid aldehyde

and the amino group of lysine residues. Notably, this type of lipid-protein modification, do

not generate a detectable free carbonyl group. Lysine residues can also react with

carbonyl derivatives formed by carbohydrate oxidation [20]. These glycation reactions

result in the formation of the so called advanced glycation end-products (AGEs) that are

suggested to play a relevant role in mechanisms of injury related to diabetes, whereas the

products of lipid-protein reactions, termed as advanced lipoxidation end products (ALEs),

are especially involved in atherosclerosis [10]. For instance, circulating MDA is not free,

but mostly bound to oxidized LDL (oxLDL) generated by macrophages within

atherosclerotic lesions and then released into the blood stream. Conceivably, the

assessment of MDA-oxLDL represents a valuable tool for investigating pathologies related

to atherosclerosis, including coronary artery disease [8,10,23].

Among the various amino acids in proteins, cysteinyl residues in the highly nucleophilic

thiolate form represent the major site for ROS attack. The initial oxidation to sulfenic acids

(R-SOH) can proceed to potentially reversible steps forming disulfides (R-SS-R),

sulfonamides and sulfinic acids that can further be oxidized irreversibly into sulfonic acids.

Additional reversible modifications of sulfenic acid are given by reacting with thiol

compounds, such as glutathione or cysteine, or with reactive nitrogen species generating

nitrosothiols in a process termed as S-nitrosation or S-nitrosylation. These reactions add to

cysteine carbonylation described above. The other sulfur-containing amino acid,

methionine, also is prone to oxidation generating methionine sulfoxide which can be

catalytically reduced by methionine sulfoxidases and methionine sulfoxide reductases

repairing oxidative damage to methionine in native proteins or further oxidized irreversibly

to methionine sulfone [22].

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The general outcome of protein oxidation is inactivation. However, notable exceptions,

especially in signaling pathways, have been described in which carbonyl formation as well

as cysteine and methionine oxidation result in gain of function [20,22].

Products of protein oxidation can be detected by means of dedicated proteomic analyses,

or in some cases by immunoblotting [24]. Of note, protein carbonyls are assessed by

means of their reaction with 2,4 dinitrophenylhydrazine (DPNH) [25]. Western blot stained

with anti-DPNH antibodies (also termed as OxyBlot) is used to separate and identify the

oxidized proteins as also performed in the present study for the identification of novel

biomarkers.

Being the most abundant protein in plasma albumin is relevant as a circulating biomarker

of oxidative stress [26]. Albumin ability to bind and transport metals favors carbonylation.

On the other hand the redox properties of human serum albumin (HSA) are mostly related

to Cys34 whose low pKa (<6.7) favors the thiolate form. Cys34 in HSA represents the great

majority ( 80%) of all free thiols in plasma. Therefore, data on circulating free thiols by

which a decrease is a marker of oxidative stress can be considered a rough estimate of

HSA redox state that can be considered a significant fraction of plasma antioxidant

capacity. Various oxidized forms of albumin have been demonstrated in humans,

especially in kidney failure, but the relationships among them have not been investigated

yet.

Albumin exemplifies unsolved issues and open questions in the field of biomarkers for

oxidative stress and more in general in the clinical evaluation of ROS contribution to

pathophysiology, as well as in the diagnosis and therapy of a wide array of diseases.

Major questions appear to be as follows:

(i) Sensitivity and specificity. Is albumin oxidation more or less specific and sensitive than

that of other plasma proteins? Due to its exchange with the extravascular/interstitial space,

albumin could sense tissue oxidative stress before proteins confined to the intravascular

space. Its abundance could make it as a general probe of oxidative stress with little

specificity for a subset of diseases. A high sensitivity with a narrower broad of specificity

could pertain to proteins that are more directly involved in metal transport. By the way,

albumin oxidation affects its metal binding ability resulting in increases in free metal levels

that are likely to exacerbate oxidative stress;

(ii) Kinetics. How much oxidation affects plasma protein turnover/removal? And in the case

of albumin how much is HSA exchange among compartments impaired? For instance,

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nitroalbumin crosses the blood-brain barrier 4 times faster than albumin, yet no information

is available for other modified forms;

(iii) Causal relationships. Do specific links exist between the various oxidized forms and

diseases?

Biomarkers of oxidative stress and heart failure

Oxidative stress is associated with most, if not all cardiovascular disorders and contributes

largely to their development and worsening. This applies to large ROS formation, since a

mild oxidative stress elicits protective mechanisms and appear to be necessary for

physiological responses to various forms of stress. The link between cardiovascular

diseases and abnormal ROS formation has been established by countless experimental

and clinical studies and covered by numerous excellent reviews (for instance, see [6,27-

33]). Initial evidence has been obtained in ischemia/reperfusion (I/R) injury during the

eighties [29]. Then, besides its role in inflammation and atherosclerosis, the contribution of

oxidative stress to HF, even independently of I/R, became evident. Indeed, ROS are

involved in every step leading to HF, such as development of hypertrophy, contractile

dysfunction, interstitial cardiac fibrosis, adverse remodelling after myocardial infarction,

loss of viability and endothelial dysfunction [32]. It is worth pointing out that, although ROS

contribution has been suggested for many cardiac diseases, including diabetic

cardiomyopathy [34] and aging [35], a non-ambiguous causal relationship can be

described only in a limited number of cases, such as Keshan syndrome due to selenium

deficiency [36] and anthracycline cardiotoxicity [37,38].

In HF, oxidative stress appears to be triggered and/or exacerbated by hormones and

cytokines released from the diseased heart creating a vicious cycle that amplifies the initial

injury. In this respect, catecholamines, angiotensin II, endothelin 1, aldosterone and TNF

appear to be the main agonists [39]. Their contribution is likely to be reinforced by

lipotoxicity in the case of diabetes and obesity [40-43]. All these factors trigger signaling

pathways that cause an increase in ROS formation within cardiomyocytes and vascular

cells. Among the many sources the most relevant are NADPH oxidase [44] and

mitochondrial dysfunction [45-48] that can be contributed by the increased activity of

specific enzymes, such as MAO [49,50] or p66Shc [51]. Eventually, the oxidative attack on

cellular components makes contractile derangements hardly surprising. In fact, ROS

10

hamper mitochondrial function and energy metabolism [52], channel activities and ion

homeostasis [53,54], and structure-function relationships in myofibrillar proteins [55-58].

These concepts have been established by experimental studies allowing to obtain direct

evidence of ROS formation and causal relationships with biochemical and functional

alterations occurring within cardiomyocytes. Nevertheless, many of the above mentioned

biomarkers have been utilized to provide clinical support to the association between

oxidative stress and HF [23,59-75] (reviewed in [6] and [76]). Most of the markers utilized

belongs to products of lipoperoxidation. Initial evidence was obtained by assessing MDA,

especially in patients suffering from ischemic heart disease [59-61]. In this respect, the

assessment of MDA-oxLDL has been suggested to provide reliable information of the

severity of vascular lesions along with having a significant prognostic value [23]. As

discussed above, MDA and oxLDL have an intrinsic link with atherosclerotic lesions and

coronary artery disease. Evidence of oxidative stress in HF independent of myocardial

ischemia has been obtained by measuring IsoPs in various body fluids and especially in

urine of HF patients [65,67,69,70,72-74]. IsoPs were found elevated also in patients

suffering for coronary artery disease [59,76], indicating that independently of

etiopathogenesis HF is associated with an abnormal generation of ROS that is reflected by

plasma and urinary biomarkers of oxidative stress. Indeed, MDA and IsoPS were

significantly higher in severe heart failure (NYHA class III and IV) and their levels

correlated with the severity of contractile dysfunction and/or left ventricle dilation

[64,65,67,69,70,72,73].

Recently, the involvement of oxidative stress in human I/R injury has been argued by

showing the absence of changes in arterio-venous differences of several circulating

biomarkers of oxidative stress in patients undergoing I/R protocols due to surgical

interventions on heart or kidney [77]. The argument is based upon measurements during

only the first hour of reperfusion. Interestingly, at least in kidney evidence of tissue

oxidative stress was documented by an increased expression of Nrf2, a transcription factor

stimulating the expression of antioxidant defenses. Therefore, this study does not appear

to challenge the involvement of oxidative stress in human I/R injury, as also demonstrated

previously by other means [78]. Rather, evidence is provided indicating that a prolonged

and/or more severe oxidative stress in tissues is required for detecting changes in

circulating biomarkers, and also that in acute phases oxidation events follows ROS-

induced changes within tissues. This might not be the case in chronic diseases, especially

when inflammatory responses involving circulating cells become relevant.

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The increase in MDA and IsoPs was also associated with a decrease of circulating

antioxidants [61,63,75] along with an increase in SOD [61,75] and a decrease in

glutathione peroxidase [64,75]. However, since these enzymes are released by tissues,

the interpretation of their changes in plasma is not straightforward, also because the

activity assessment was not followed by structural studies documenting their oxidation.

This is a general caveat in the available studies that monitored lipoperoxidation products

with scarce attention to oxidation-induced changes in plasma proteins. For instance, it is

rather surprising that, at least to our knowledge, albumin oxidation was not assessed

directly, so that the evidence of its occurrence is left to indirect measurements. Few

studies reported the decrease in plasma free thiols [61,73] that is likely to be mostly

related to the oxidation of Cys34 in HSA. In addition, the increase in plasma carbonyls was

documented without identifying the proteins involved [75].

Even if direct evidence of albumin oxidation is obtained, due to its exchange with the

interstitial space it would be difficult to understand whether the observed changes are

generated by cardiac cells (and/or other tissues) or occur directly within the blood. This is

a relevant issue when effects of antioxidant or protective interventions are considered. In

fact, the important goal is to reduce oxidative stress in the injured organ. A decrease in the

levels of oxidation biomarkers in plasma is important only if it reflects reliably the reduction

of oxidative stress in the failing heart. In addition, components present in only plasma (i.e.,

not released from failing cardiomyocytes or diseased vessels) should be characterized to

understand whether and how much plasma oxidation follows oxidative stress generated

within failing hearts.

In this respect, also experimental studies fell short of relating oxidative events in plasma

with oxidation of intracellular components that are relevant for ensuing contractile

dysfunction. Our findings described below are aimed at filling these blanks. After

identifying novel targets of oxidative stress in plasma, we related their changes with both

the occurrence of oxidation in myofibrillar proteins and contractile dysfunction in a model of

heart failure, such as rapid atrial pacing in rabbit, devoid of ischemia.

Identification of novel proteins as biomarkers of heart failure independent of

coronary heart disease

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Evidence of oxidative stress in pacing-induced HF model

The aim of our study was to find new specific biomarkers for HF, easily monitored in a

clinical setting. HF was induced in a rabbit model by rapid left ventricular (LV) pacing for 3

weeks. A total of 8 sham-operated rabbits and 7 HF rabbits were enrolled in this study.

Heart morphology and function are significantly modified after 3 weeks of LV pacing. HF

was evident from clinical signs, such as a strong decrease of LV fractional shortening (FS)

and a significant increase of hypertrophy and apoptosis. In an additional group, rabbit

received vitamins C and E for 3 weeks without (Sham-Vit, n=3) or with LV pacing (HF-Vit,

n=6). Administration of antioxidant vitamins during the 3 weeks of pacing significantly

decreased LVFS and reduced cardiomyocyte cross-sectional area and the number of

TUNEL-cardiomyocyte.

In our previous studies we developed quantitative assessment of oxidation of myofibrillar

proteins, in particular actin and tropomyosin, to detect the progression of HF in several

animal models as well as in human HF [55,58,79]. In this rabbit model the degree of

oxidative modifications of myofibrillar proteins is assessed using the oxyblot technique, a

method to measure protein carbonylation. Figure 1 shows that actin carbonylation is

significantly increased in hearts subjected to pacing induced HF compared to sham

operated rabbits, confirming our previous finding in other cardiac disease models. The

degree of actin carbonylation, measured by densitometric analysis, is approximately 50%

higher in HF compared to sham-operated rabbits (p=0.0043) (Fig.1 panel B). This result

confirms both the occurence of HF and the presence of oxidative stress in the myocardium

of this rabbit model.

Two novel markers of oxidative stress in plasma

To assess whether plasma proteins could be subjected to carbonylation, plasma samples

were collected from HF and sham-operated rabbits. To detect protein carbonylation the

plasma proteins were subjected to DNP-derivatization and then analyzed by immunoblot

probed with anti-DNP antibody. The representative 2D gel probed with anti-DNP antibody

in Figure 2 (panel A) shows a strong increase in plasma protein carbonylation in HF

compared to sham-operated rabbits, especially at the level of two proteins with a

molecular weight around 80 kDa. The identity of two major carbonylated spots was

confirmed using proteomic analysis as histidine-rich glycoprotein (HRG) and

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serotransferrin (TRF), the upper and the lower spot respectively in the figure 2, panel A. In

order to quantify the amount of HRG and TRF carbonylation in plasma HF we used

western blot analysis: after incubation with anti-DNP antibody the membranes are stained

with anti-HRG and anti-TRF antibodies (Fig. 2 panel B). Densitometric analysis illustrated

in Figure 2 (panel C) shows that the amount of carbonylation in both the plasma proteins

was significantly increased in HF rabbits (a raise of 2.9 fold and 1.5 fold in HF vs sham-

operated group, HRG and TRF respectively, p<0.05). Administration of antioxidant

vitamins C and E throughout the three weeks of LV pacing completely and significantly

abolished the increase in plasma HRG and TRF carbonylation (Figure 2, panel C).

These results show, for the first time, the occurrence of plasma protein carbonylation in a

HF model, identifying two new major targets of oxidative stress in the plasma. Our finding

demonstrates that antioxidant treatment completely rescues protein plasma carbonylation,

keeping the degree of HRG and TRF carbonylation to physiological values. Moreover, this

evidence is entirely novel in the case of HRG carbonylation, demonstrating a new post-

modification at the level of this protein. Unlike HRG, TRF has been demonstrated to be

carbonylated in several stress-related diseases, including cancer, abdominal aortic

aneurysm, and Alzheimer disease and in aging mice [80-82]. Moreover, Guidi et al.

demonstrated a decrease in TRF carbonylation after regular physical exercise, a condition

that leads to cardiovascular protection reducing risk of obesity and of diabetes [83]. TRF is

an iron (Fe3+)-binding and -transporting protein. Thanan et al. suggested that the

accumulation of iron into a carcinoma tissue model is a result of the accumulation and

dysfunction of the iron transporter TRF due to carbonylation [84].

HRG and TRF carbonylation correlated with actin carbonylation and contractile

dysfunction in rabbit HF

Next, we aimed to demonstrate that the increased oxidative stress inside the heart is significantly

correlated with the occurrence of oxidative stress in plasma proteins. In our previous work we

correlated myofibrillar protein carbonylation with contractile dysfunction in human HF. However,

the process occurring within cardiomyocytes has not been related to oxidative stress in plasma

that can be easily monitored in both experimental and clinical settings. Here, we correlated HRG

and TRF carbonylation levels with actin carbonylation that, as we previously demonstrated, plays

a relevant role in contractile failure. Our findings demonstrated a positive linear correlation

between HRG and TRF to actin carbonylation (R2=0.7188 and R2= 0.4539, respectively) (Fig. 3).

14

HF is well established in causing contractile impairment, as shown by a strong reduction of FS

after three weeks of LV-pacing (table 1). In this work, we provided evidence of a correlation

between HRG and TRF plasma carbonylation and contractile dysfunction. Figure 4 (panel A)

shows a negative correlation between both plasma protein carbonylation and the decrease in FS

that occurs in hearts subjected to pacing induced HF model (R2=0.518 and R2= 0.517, for HRG

and TRF respectively). Of interest, figure 4 (panel B) shows that the loss in HRG and TRF

carbonylation after antioxidant treatment correlates with the higher values of LVFS that we

detected in HF-vitamin treated rabbits.

Taken together, these findings propose a strong rationale to exploiting protein plasma oxidation

as a marker to evaluate both HF diagnosis and, more importantly, to improve the efficacy of the

therapeutic interventions. We found two possible biomarkers associated with rabbit HF model,

focusing the attention on plasma-associated markers rather than on tissue-associated markers

because the application of circulating biomarkers in diagnostic laboratories would be relatively

simple. Further study is required to confirm the potential and the clinical role of HRG and TRF

carbonylation in plasma HF and to further investigate whether and how these proteins participate

in this disease in other HF animal models as well as in human HF.

Methods

The present study was approved by the bioethical committee of the district of Düsseldorf,

Germany, and all animals were treated and cared for in accordance with the Guide for the

Care and Use of Laboratory Animals published by the US National Institutes of Health

(NIH publication No. 85-23, revised 1996).

Experimental model

Male Chinchilla bastard rabbits (Charles River, Kisslegg, Germany) weighing 3-4 kg were

anesthetized, and a pacing lead was sutured onto the apical region of the left ventricle.

The pacing lead was connected to a pacemaker (Medtronic, Düsseldorf, Germany), which

was implanted subcutaneously as previously described [79]. After euthanasia of the

rabbits the hearts were quickly removed and stored in liquid nitrogen until use. Plasma

samples were collected by puncture of the LV at the end of the study and stored at -80oC.

Development of heart failure

15

HF was induced by rapid LV pacing (400 bpm) for 3 weeks (HF, n=7). Eight sham-

operated rabbits served as controls (sham). In two additional groups rabbits received

either placebo or vitamins C (100mg/kg/day per os) and E (200mg/day per os) without

(sham-vit, n=3) or with rapid LV pacing (HF-vit, n=6).

Clinical parameters

Heart rate and LV function were measured as previously described [85]. LV fractional

shortening (LVFS) was calculated using this equation: ((LV end-diastolic diameter –end-

systolic diameter)/ LV end-diastolic diameter X 100).

Apoptosis was assessed by means the TdT-mediated dUTP nick end labeling (TUNEL)

technique (In Situ Death Detection Kit, La Roche Diagnostic, Mannheim, Germany) and

the TUNEL-positive nuclei were counted using fluorescence microscopy (Leica DMLB,

Bensheim, Germany). Cardiomyocyte cross-sectional area was measured staining the

tissue sections with haematoxylin and eosin. Fibrosis was measured in random fields by

planimetry and the degree of tissue damage was expressed as percentage of the entire

field of view.

Protein extraction

Heart protein extraction was performed as previously reported [55]. Briefly, heart samples

were homogenized in ice-cold PBS, pH 7.2 containing 5 mM EDTA and then were

centrifuged at 12 000 x g for 10 min at 4°C. The resulting pellet was resuspended in

sample buffer (2% SDS, 5% glycerol, 125 mM Tris–HCl, and 10% -mercaptoethanol pH

6.8). Plasma samples were stored in liquid nitrogen and then were diluted (1:10) in ice-

cold PBS, pH 7.2. Protein concentration was determined by Bradford assay (Bio-Rad) both

for the heart and the plasma samples.

Oxyblot technique (protein carbonylation)

Total myocardial protein and plasma proteins carbonylation was measured using the

Oxyblot Protein Oxidation Detection Kit (Chemicon, USA) according to the manufacturer’s

protocol. Briefly, after protein quantification, carbonyl groups were derivatized by reaction

with 2,4-dinitrophenylhydrazine for 15 minutes at room temperature. Dinitrophenyl-(DNP-)

derivatized proteins were then separated using SDS-PAGE and transferred to a

nitrocellulose membrane. Membranes were incubated overnight at 4°C with anti-DNP

antibody (1:150 dilution), washed three times with TBS-Tween and incubated with

16

secondary goat anti-rabbit/horseradish peroxidase antibody (1:300) for 1 hour at room

temperature. Membranes were incubated with ECL reagent. Bands were visualized using

a Kodak X-Omat film processor. Successively, the membranes were stripped and probed

with anti--sarcomeric actin 5C5 clone (Sigma, USA), anti-HRG (Abcam) and anti- TRF

(GenWay Biotech, Inc.) antibodies. Quantification of actin, TRF and HRG carbonyl

modification was made by the ratio between the densitometric values of the corresponding

band in the oxyblot or of the bands in the Red Ponceau and those of the bands stained

with the corresponding antibody, analyzed using the ImageJ software (NIH, USA).

2 Dimension Electrophoresis

Plasma proteins (150 μg) were solubilized in a lysis buffer containing 8 M urea, 4%3-[(3-

Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM dithiothreitol

(DTT) and 2.0% 4–7 nonlinear immobilized pH gradient (IPG) buffer.

The proteins were resolved by isoelectric focusing on 11-cm immobilized pH gradient

strips (pH 3-10). The IPG-strips were rehydrated at 30v for 10 hr and were focused

according to the following conditions:

Step 1:250V for 250vhr

Step 2: 500V for 500vhr

Step 3: 1000V for 1000vhr

Step 4: 8000V “Gradient” for 1hr

Step 5: 8000V for 66,667vhr

Step 6: 8000V for 1hr

After focusing, the IPG-stips were equilibrated for 10 min in 6 M urea, 30% glycerol, 2%

Sodium Dodecyl Sulfate (SDS), 0.05 M Tris–HCl, pH 6.8, 2% DTT, and subsequently for

10 min in the same urea/SDS/Tris buffer solution but substituting the 2% DTT with 2.5%

iodoacetamide. After IEF, IPG-strips were subjected to in-strip DNP derivatization

(derivatization of protein carbonyls) for 10 min.

The strips were loaded onto a gel and the proteins separated by electrophoresis on SDS-

PAGE. The membranes transferred to a nitrocellulose membrane and were incubated with

anti-DNP antibody as described in the paragraph above.

Mass spectrometry analysis

Plasma proteins were subjected to 2-dimensional electrophoresis followed by DNP

derivatization and a separation by SDS-PAGE. The gels were stained with colloidal

17

Coomassie Blue G-250 (Sigma) and the two interested bands were cut and sent to the

proteomic core facilities VIMM, University of Padua (Italy) for protein identification.

Statistics

Data are expressed as mean values±SEM. Cardiomyocyte cross-sectional area, TUNEL-

positive cardiomyocite and plasma protein carbonylaiton were compared between

untreated and treated sham and HF rabbits using one-way ANOVA. Actin carbonylation

between sham and HF rabbits was compared by two-tailed unpaired Student’s t-test.

Linear regression was performed to determine correlation between plasma protein

carbonylation and LVFS parameter. All statistical analysis was performed using Prism

program and values of P < 0.05 were considered significant.

18

Acknowledgements

This work was supported by the COST Action "EU-ROS" BM1203 (to R.S and F.D.L.) and

grants from the University of Padova, Fondazione Cariparo, and CNR (to F.D.L.).

19

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Figure legends

Fig.1. Actin carbonylation is increased in HF.

Septum proteins extract from biopsies of HF and Sham operated rabbits were subjected to

DNP-derivatization to detect protein carbonylation and then analyzed by immunoblot probed

with anti-DNP antibodies. (A) Red Poceau staining was performed for protein loading. Actin was

identified by immunoblot. (B) The Oxidation Index is given by the ratio between the

densitometric values of the actin band in the oxyblot and those of the corresponding bands

stained with Red Ponceau. * p<0.05 vs Sham. Sham: sham-operated rabbits (n=8); HF: heart

failure rabbits 3 weeks (n=7); Mean ± SEM.

Fig.2. Plasma protein carbonylation is increased in HF rabbits.

(A) Plasma proteins were extracted and subjected to 2D gel electrophoresis followed by oxyblot

analyses. (B) Plasma proteins from HF, Sham-operated, and HF and Sham-operated pretreated

with vitamins C and E rabbits were subjected to DNP-derivatization to detect protein

carbonylation and then analyzed by immunoblot probed with anti-DNP antibodies. Red Poceau

staining was performed for protein loading. (C) The densitometric analysis is given by the ratio

between the densitometric values of the STF or HRG bands in the oxyblot and the albumin band

stained with Red Ponceau. * p<0.05 vs. Sham, # p<0.05 vs HF. Sham: sham-operated rabbits

(n=8); Sham-Vit: sham-operated rabbits receiving vitamins C and E (n=3); HF: heart failure

rabbits (n=7); HF: heart failure rabbits receiving vitamins C and E (n=6); Mean ± SEM.

Fig.3. Relationship between HRG and STF carbonylation with Actin carbonylation in

plasma HF samples.

Sham: sham-operated rabbits (n=6); HF: heart failure rabbits, 3 weeks (n=7);

Fig.4. Carbonylation of HRG and STF correlates with contractile dysfunction.

(A) The degree of HRG and STF carbonylation correlates with the decrease in fractional

shortening during HF. (B) The degree of HRG and STF carbonylation in HF rabbits receiving

vitamins C and E treatment shows a linear trend line with the improvement in fractional

shortening. Sham: sham-operated rabbits (n=8); HF: heart failure rabbits (n=7); HF: heart failure

rabbits receiving vitamins C and E (n=6)

Figure legends

Figure 1Click here to download high resolution image

Figure 2Click here to download high resolution image

Figure 3Click here to download high resolution image

Figure 4Click here to download high resolution image