elevated plasma marinobufagenin, an endogenous cardiotonic...

Elevated Plasma Marinobufagenin, An Endogenous Cardiotonic Steroid, Is

Associated with Right Ventricular Dysfunction and Nitrative Stress in Heart Failure

Kennedy et al: Marinobufagenin in Heart Failure

David J. Kennedy, PhD1,3; Kevin Shrestha, MD1; Brendan Sheehey, BS1; Xinmin S. Li, PhD1;

Anuradha Guggilam, DVM, PhD1; Yuping Wu, PhD1; Michael Finucan, BS1; Alaa Gabi, MD1;

Charles M. Medert, BS1; Kristen Westfall, MBA1; Allen Borowski, RDCS1;

Olga Fedorova, PhD5; Alexei Y. Bagrov, MD, PhD5; W.H. Wilson Tang, MD1, 2, 4

1Department of Cellular and Molecular Medicine, 2Center for Cardiovascular Diagnostics and

Prevention, Lerner Research Institute 3Department of Nephrology and Hypertension, Glickman

Urological and Kidney Institute 4Department of Cardiovascular Medicine, Heart and Vascular

Institute, Cleveland Clinic, Cleveland, OH; 5Laboratory of Cardiovascular Science, National

Institute on Aging, National Institutes of Health, Baltimore, MD

Correspondence to W.H. Wilson Tang, MD 9500 Euclid Ave, Desk J3-4 Cleveland, OH 44195 Phone: 216.444.2121 Fax: 216.445.6165 E-mail: [email protected] DOI: 10.1161/CIRCHEARTFAILURE.114.001976 Journal Subject Codes: [110] Congestive heart failure, [115] Remodeling, [33] Other diagnostic testing, [100] Health policy and outcome research, [130] Animal models of human disease, [91] Oxidant stress

Prevention, Lerner Research Institute 3Department of Nephrology and Hyperteennnsioioion,n,n, GGGlililickckckmamaman

Urollogogogicicicalalal aaandnn KKKidii ney Institute 4Department oooff f CaCC rdiovascular Mediiicicicinenn , Heart and Vascula

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Abstract

Background—Plasma levels of cardiotonic steroids (CTS) are elevated in volume-expanded

states such as chronic kidney disease, but the role of these natriuretic hormones in subjects with

heart failure (HF) is unclear. We sought to determine the prognostic role of the CTS

marinobufagenin (MBG) in HF, particularly in relation to long-term outcomes.

Methods and Results—We first measured plasma MBG levels and performed comprehensive

clinical, laboratory, and echocardiographic assessment in 245 HF patients. All-cause mortality,

cardiac transplantation, and HF hospitalization were tracked for 5 years. In our study cohort,

median [interquartile range] MBG was 583 [383-812] pM. Higher MBG was associated with

higher myeloperoxidase (MPO, r=0.42, p<0.0001), BNP (r=0.25, p=0.001), and asymmetric

dimethylarginine (ADMA, r=0.32, p<0.001). Elevated levels of MBG were associated with

measures of worse right ventricular function (RV s’: r= -0.39, p<0.0001) and predicted increased

risk of adverse clinical outcomes (MBG 574 pM: HR 1.58 [1.10-2.31], p=0.014) even after

adjustment for age, gender, diabetes mellitus, and ischemic etiology. In mice, a left anterior

descending coronary artery ligation model of heart failure lead to increases in MBG, while

infusion of MBG into mice for 4 weeks lead to significant increases in MPO, ADMA, and

cardiac fibrosis.

Conclusions—In the setting of heart failure, elevated plasma levels of MBG are associated with

right ventricular dysfunction and predict worse long-term clinical outcomes in multivariable

models adjusting for established clinical and biochemical risk factors. Infusion of MBG appears

to directly contribute to increased nitrative stress and cardiac fibrosis.

Key Words: heart failure, cardiotonic steroids, outcome, nitrative stress, cardiac fibrosis

higher myeloperoxidase (MPO, r=0.42, p<0.0001), BNP (r=0.25, p=0.001), andndnd aaasysysymmmmmmetric

dimethhylyly arargigig nin neee (ADMA, r=0.32, p<0.001). Elevevated levels of MBGBGBG were associated with

mmmeaaasures of wowoworsrsrsee e riighghght t t veveventnn riiicucuculalalarrr fufuuncncnctit ononon (((RRRV s’: r== --0.0.0.393939, p<p<p<0...000000010101))) ana d prprpredededicicicteted d d ininincrcrcreaeaeaseses dRR

iiisksksk of adverse cllliniccalal outtcococomemm s (M(M(MBGBGBG 575774 pMppM: HRHRHR 111.5558 [[1.10-2.3111], p=0== .011414))) eeeveeen aaafttte

adjustment for age, gender, diabbbetetetesee mellitutuus, aaandnn iiiscscscheh mic etiology. In mice, a left anterio

dedescscenendidingngg ccororononarary y y ararteteryryy lligiggatatioion n momodedel l ofof hheaeartrt ffaiailulurere lleaead d toto iincncrereasaseses iin n MBMBG,G,, wwhihilele



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Cardiotonic steroids (CTS) are a class of endogenous volume-sensitive hormones that bind to the

Na/K-ATPase, and include cardenolides (such as digoxin and ouabain) as well as aglycone

bufadienolides (such as telocinobufagin and marinobufagenin [MBG])1, 2. Increased circulating

levels of CTS have been proposed as a compensatory mechanism for natriuresis and vascular

tone in volume-expanded conditions including salt-sensitive hypertension, chronic kidney

disease, and preeclampsia3-5. In these settings, CTS contribute to enhanced natriuresis by

inducing endocytosis of proximal tubule cell Na/K-ATPase. This serves to remove the Na/K-

ATPase from the basolateral membrane and thus reduces the transport of sodium from the

tubular lumen to the blood compartment, thereby increasing sodium excretion6, 7. However, CTS

may exert “off-target” signal transduction effects beyond their direct effects on the sodium

pump2, 8. Hence, chronic stimulation of Na/K-ATPase signaling by CTS has important

implications for not only for the natriuretic response to increased salt load9 but also has been

implicated in pathological adaptation to volume expansion including hypertension, hypertrophy,

and fibrosis10, 11. Accumulation of MBG and other CTS has been documented in a variety of

cardiovascular disease states beyond conditions marked by plasma volume expansion and fluid

retention4, 12-15. Furthermore, myocardial hypertrophy and growth effects of CTS have been

proposed in the setting of hypertension and renal dysfunction16, and supported by both

experimental and clinical data demonstrating the association between an endogenous “digoxin-

like substance" and the development and severity of heart failure (HF)17, 18. Clinical and

experimental evidence from our group and others has also demonstrated the pro-oxidant and pro-

fibrotic effects of these steroid hormones in both cardiac and renal tissue19-23. As the relationship

among MBG and myocardial structure and performance in the contemporary HF population has

not been examined, we hypothesized that an increase in circulating MBG levels may track with

may exert “off-target” signal transduction effects beyond their direct effectsss ononon tthehehe sssododo iui m

pumpp2, 8.. HeHencce,e,e chronic stimulation of Na/KK-ATPase signaling bybyby CTS has importan

mmmplplplications fororor nnnott ooonlnln y y y fofof r thththe e e nanan trtrtriuiuiurerr tititic c c rerr sponoonse tttoo o ininincrcc eaaasssed d d sasasaltltlt llloao dd9 bbbututut aaalssooo hahahasss bebebeene

mmmplpllicated in papaathththologggicccal aadddapapaptationonon ttto voluuummme eeexppannnsisisioon innncluuudiiing hyyyppepertrttennnsion,,, hhyhyppperrrtrophphphy

and fibrosis10, 11. Accumulationnn ooof f f MBG ananand ototothehh r r r CTCTC S has been documented in a variety od

cacardrdioiovavascsculularar ddisiseaeasese sstatatetes s bebeyoyoy ndnd cconondidititionons s mamarkrkeded bby y y plplp asasmama vvololumume e exexpapap nsnsioion n anand d flfluiuidd



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disease severity and provide important prognostic information in the setting of HF. Further, as

MBG in increased in volume overloaded states and associated with diastolic dysfunction in

animal models, we hypothesized that circulating levels would be increased in HF patients with

preserved ejection fraction and associate with echocardiographic indices of diastolic dysfunction.

Herein, we examined the relationship among circulating levels of MBG with echocardiographic

parameters, as well as long-term adverse clinical outcomes in patients with HF. We also used an

animal infusion model to demonstrate the contribution of MBG to the clinical phenotype

observed in our human study.

Methods

Study Design and Population. We prospectively enrolled 245 HF patients ( 18 years) seen at the

Cleveland Clinic with a clinical diagnosis of HF and New York Heart Association (NYHA)

functional class I-IV symptoms, who were free of significant renal, hepatic, and valvular

diseases. Study participants were excluded if they experienced any of the following: (1) major

cardiovascular event (myocardial infarction, unstable angina, stroke, transient ischemic attack,

pulmonary embolism) within the preceding 30 days; (2) significant lung diseases, including

chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary arterial hypertension, and

asthma; or (3) major surgery or use of inotropic agents within the past month. We prospectively

followed a composite endpoint including adverse clinical events (all-cause mortality, cardiac

transplantation and HF hospitalization) and all-cause mortality through a process confirmed by

chart review and the Social Security Death Index. Thirteen sex-matched apparently healthy

volunteer participants without a history of heart failure served as non-heart failure controls.

They were prospectively recruited outside of any healthcare institution setting, and did not report

Methods

Study y DeDesis gngng andndd PPopulation. We prospectivelyy eenrnrolled 245 HF patientntntss ( 18 years) seen at the

CCClevvveland ClClClinnnicicic wititthhh a a a clclclini icicicalalal dddiaiaiagngngnosoo isisis ooof f HFFF anddd NNNewewew YoYoYorkrkk HHHeaeaeartrtr AAssssssococociaiaiatitit ononn (((NYNYNYHAHAH

fufuunccctit onal claass III-IVV sssymppptooomsm , whww ooo werrre freeee offf sssiigignnniffficaanntnt renaaal, heheheppapatic, aaandndnd vvvalvvvullla

diseases. Study participants wereree eeexcxx luded ififif thhheyeyey expxpxperee ienced any of the following: (1) majo

cacardrdioiovavascsculularar eeveventnt (((mymyyococarardidialal ininfafarcrctitionon,,, ununststabablele aangngginina,a,, sstrtrookeke, , , trtranansisienent t isischchememicic aattttacackk



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any active medical conditions at the time of blood draw. The study protocol was approved by the

Cleveland Clinic Institutional Review Board and written informed consent was obtained from

each of the study participants prior to their participation in the study.

Biochemical Assays. Blood samples were collected in ethylenediaminetetraacetic acid (EDTA)-

plasma vacutainers at the time of clinical and echocardiographic and hemodynamic evaluation,

were immediately aliquotted and stored at 80°C until analysis. Plasma samples were extracted

for MBG measurements using C18 SepPak cartridges (Waters Inc., Cambridge, MA) and MBG

levels were measured using a competitive fluoroimmunoassay [dissociation enhanced

fluoroimmunoassay (DELFIA)] as previously described22, 24. The MBG DELFIA uses a murine

monoclonal antibody (anti-MBG 4G4) and employs competition between immobilized antigen

(MBG-glycoside-thyroglobulin) and MBG, other cross-reactants, or endogenous cardiotonic

steroids within the sample for a limited number of binding sites on the 4G4 anti-MBG

monoclonal antibodies. Secondary (goat anti-mouse) europium labeled antibody was obtained

from Perkin-Elmer (Waltham, MA). Data on cross reactivity of the MBG antibody have been

reported previously3, 23. For analysis of MBG tissue levels, adrenal glands were homogenized

(TissueLyserII, Quiagen, 5 mm stainless steel beads) and homogenate was extracted with 10 fold

excess of methyl tert-butyl ether before drying under nitrogen gas and resuspension in assay

buffer (50 mM Tris-hydrochloride, 154 mM sodium chloride, 7.7 mM sodium azide, pH 7.8).

Plasma myeloperoxidase (MPO) levels were determined by an enzyme-linked immunosorbent

assay (CardioMPO II test, Cleveland Heart Labs, Cleveland OH). Arginine metabolomic profiles

were quantified by liquid chromatography-electrospray ionization tandem mass spectrometry

(LC-ESI-MS/MS) as previously described25, 26. All other assays including BNP, basic metabolic

fluoroimmunoassay (DELFIA)] as previously described22, 24. The MBG DELFIAIAIA uuuseseess s aa a mumum rine

monoclclononala aantibibbodo y (anti-MBG 4G4) and empplooysyy competition betweweweene immobilized antigen

MMMBBBG-glycoosssidedede-t-t-thyyyrororoglglglobobobuluu ininn) ) ) ananand d MBMM G,G,G, ootheeer crosososs-s-s-rerereacaa taaannnts,s,s, oorrr eeendn ogogogenenenououous ss cacacardrdrdioioiotototoninn crrr

ttteree ooioids within thhhe sssammmpleee ffforo a llimmmiiited nnnumbebber ofofof biinindddinggg sites ooon n thhhe 4GGG44 4 aaanttti-MMMBBBG

monoclonal antibodies. Secondaaryryry ((gogg at antntti-ii mmmououousee))) eueue ropipp um labeled antibody was obtained

frfromom PPererkikin-n ElElmemer r (W(W( alalththamam, , , MAMA).).) DDatata a onon ccrorossss rreaeactctivivitity y y ofof tthehe MMBGBG aantntibibodody y y hahaveve bbeeeenn



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profile and lipid profile were measured on the Architect platform (Abbott Laboratories, Abbott

Park IL). The 4-variable Modification of Diet in Renal Disease (MDRD) equation was used to

calculate estimated glomerular filtration rate (eGFR)27.

Transthoracic Echocardiography. Comprehensive 2-dimensional echocardiography was

performed in standard parasternal and apical views on all participants by an American Society of

Echocardiography registered research sonographer using a Vivid 7 echocardiography machines

(GE Healthcare, Waukesha WI) equipped with a phased-array transducer and following the

American Society of Echocardiography recommendations28, 29. Further detail on

echocardiographic analysis is described in the Supplemental Material.

Animal study. In order to assess MBG levels in a mouse model of post-myocardial infarction

heart failure, left anterior descending (LAD) artery ligation was performed in C57BL6J mice and

plasma MBG was assayed after 4 weeks. Briefly, mice were intubated and ventilated with 60%

oxygen at 120 breaths per minute with an inspiratory pressure of 16 to 18 cm H2O using a rodent

ventilator (Harvard Apparatus). After sternotomy was performed, the left atrium was retracted

for visualization of the proximal LAD using a surgical microscope (Leica M500) and the LAD

was ligated with 10-0 prolene suture. Blanching and dysfunction of the anterior wall verified

LAD ligation. To directly test for a potential contribution of MBG to promotion of cardiac

dysfunction and nitrative stress, osmotic minipumps (Alzet® model 1004) were placed

intraperitoneally in order to deliver MBG (0.1 ug/g/day) or vehicle to mice for 4 weeks similar to

what we have reported in the rat22. Quantitative real-time PCR was used to measure gene

expression with 18S rRNA used as the internal control (TaqMan®, Life Technologies). These

echocardiographic analysis is described in the Supplemental Material.

AAnAnimmmal studyyy. InInIn ordrdrdererer tttooo asa sesesessssss MMMBGBGBG levevevelelels innn aa mmououousesese mmmodddelel ooof ff popopoststs -mmyoyoyocacacardrdrdiaialll inininfafafarcrcrctititiono

hehehearrrt tt failure,, leffttt aaanterrriooor deesssceene dingngng (LALALAD)) aarrteryyy lligagaatititioon wwwas ppeperrformmmeddd ininn CCC57BLBLBL66J6J mmmice annnd

plasma MBG was assayed after 444 wwweeks. Brrrieiei flflly,y,y, micicice e e were intubated and ventilated with 60%

oxoxygygygenen aat t 121200 brbreaeaththss pepep r r miminunutete wwitith h anan iinnspsppiriratatorory y y prprp esessusurere ofof 116 6 toto 1188 cmcm HH222O O ususining g g a a rorodedenn



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studies were approved by the Cleveland Clinic Institutional Animal Care and Use Committee

and the procedures followed were in accordance with institutional guidelines.

Quantitative Histologic Techniques. Mason’s trichome and picosirius red staining was

performed on deparafinized 5 μm serial heart sections. The sections were mounted under a Leica

DM 2500 microscope and digitized with a QImaging MicroPublisher 5.0 RTV camera. Further

detail on quantitative morphometric analysis is described in the Supplemental Material.

Statistical Analysis. Normally distributed continuous variables were summarized as mean ±

standard deviation if normally distributed, or median (interquartile range [IQR]) if non-normally

distributed. If more than two groups were compared, one-way analysis of variance (ANOVA)

was performed prior to comparison of individual groups with the unpaired Student’s t-test with

Bonferroni’s correction for multiple comparisons. If only two groups of normal data were

compared, the Student’s t-test was used without correction. P values for non-parametric

comparisons or those based on small sample size were performed using the Mann-Whitney test.

Associations between changes in MBG and clinical, biochemical, and echocardiographic

measures were performed using the Spearman's rank correlation method. Clinical risk and time

to clinical adverse events associated with increased MBG levels were assessed by Cox

proportional hazard analysis and the units for hazard ratios represent the dichotomized values of

MBG 574 pM and <574 pM. The optimal sensitivity and specificity cutoff of MBG ( 574 pM)

levels in our cohort was determined using receiver operator characteristic (ROC) curve analyses

in the context of the time to event and the Hazard Ratios reported represent the risk associated

with MBG levels 574 pM. This value was chosen as the optimal ROC cut-point that

tandard deviation if normally distributed, or median (interquartile range [IQR]))) if f f nooon-n-n-nononormrmrmally

distribubuteted.d IIf f momomorer than two groups were compparared, one-way analyssisisis of variance (ANOVA

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maximized sensitivity plus specificity in ROC curve analysis of the nominal logistic regression

fit between MBG modeled as a continuous variable and adverse clinical outcomes modeled as a

nominal dichotomous variable. Survival curves (all-cause mortality, cardiac transplantation or

HF hospitalization) were generated from Kaplan-Meier survival analysis. Continuous net

reclassification improvement (NRI) was used as test of association and integrated discrimination

improvement (IDI) was to measure improvement in model performance. The p values compare

models with and without MBG. The model was adjusted for traditional risk factors, including

age, sex, diabetes mellitus and ischemic etiology. Statistical analysis was performed using

GraphPad Prism®, JMP 10.0 (SAS Institute, Cary, NC), and R 3.1.2 (Vienna, Austria). P values

<0.05 were considered statistically significant.

Results

Subject Characteristics. Baseline characteristics of our study cohort are presented in the Table.

Median MBG level was 583 [IQR 383-812] pM in patients with HF compared to 241 [IQR 212,

281] pM in non-HF controls (n=13, mean age 43 12 years, 64% male, body mass index 27 3,

15% African American). Patients with HF have higher MBG levels when compared to that of

non-HF controls, regardless of whether they have reduced or preserved LVEF (Figure 1).

Furthermore, higher MBG was associated with higher indices of inflammation/oxidative stress

(MPO: r=0.42, p<0.0001), myocardial stress (BNP: r=0.25, p=0.001), and nitrative stress

[asymmetric dimethylarginine (ADMA): r=0.32, p<0.001; symmetric dimethylarginine (SDMA):

r=0.34, p<0.001; and mono methyl arginine (MMA): r=0.40, p<0.0001]. In our study cohort,

there was no significant association between MBG and cystatin C (p=0.485) and estimated GFR

(p=0.345).

<0.05 were considered statistically significant.

ReRR suultss

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Median MBG level was 583 [IQR R R 38383 3-812]] pppM M M ininin ppatatatieieients with HF compared to 241 [IQR 212

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MBG Levels and Myocardial Structure and Performance. Supplemental Table 1 presents the

relationships between MBG levels and echocardiographic parameters of cardiac structure and

performance. In univariate analysis, higher MBG was associated with indices of LV diastolic

function (Mitral deceleration time: r= -0.24, p=0.007) and RV diastolic function (Tricuspid E/e’:

r=0.22, p=0.027; Tricuspid deceleration time: r= -0.38, p=0.002), as well as larger RV size (RV

end-diastolic area: r=0.21, p=0.023). Elevated levels of MBG were associated with measures of

worse RV systolic function (RV s’: r= -0.39, p<0.0001), but not left-sided systolic function

(Supplemental Table 1).

MBG Levels and Prognosis. In our study cohort, 118 patients experienced an adverse event of

death, cardiac transplantation, or HF hospitalization over the 5 year follow-up. When divided as

dichotomous variable according to optimal cut-point (574 pM), elevated MBG was a predictor of

increased risk of 5-year adverse outcomes, with higher MBG predicting increased risk of adverse

clinical events (Hazard ratio 1.58 [95% confidence interval 1.10-2.31], p=0.014, Figure 2A). The

predictive value of MBG remained statistically significant after adjustment for age, gender,

diabetes mellitus, and ischemic etiology, but not eGFR (Supplemental Table 2). Moreover, the

addition of MBG to traditional risk factors such as age, gender, ischemic etiology, and diabetes

resulted in an 33.6% event-specific net reclassification (95% confidence interval 9.3%-57.8%,

p=0.007) and a 3% integrated discrimination improvement (Supplemental Table 3). When

defined by quartiles, this trend was confirmed using Cochran-Amitage test for the trend over

quartiles (p=0.0297), although the trend was not monotonic (Supplemental Table 4). MBG did

not predict events when modeled as a continuous variable (Hazard ratio 1.14 [0.95 – 1.37],

MBG LeLevevels anddd PPrognosis. In our study cohort,,, 1118 patients experiennncec d an adverse event o

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p=0.15, per 1 standard deviation increment 0.62 with MBG modeled as a natural log transformed

continuous variable).

We next selected a subgroup of the HF patients that had serial blood draws available at the time

of their initial presentation in the hospital and 48-72 hours after admission (n=115 and included

35 events) and measured MBG. Rising MGB levels over the course of admission predicted

increased risk of adverse outcomes [% MBG modeled as a continuous variable per standard

deviation increments where 1 standard deviation = 0.91%, Hazard ratio 1.30 (95% confidence

interval 1.04-1.56), p=0.025, per 1 standard deviation increment, Figure 2B].

MBG Promotes Cardiac Fibrosis and Nitrative Stress in Animal Models. In order to assess

MBG levels in a mouse model of post-myocardial infarction heart failure, left anterior

descending artery ligation (LADx) was performed in C57BL6J mice. After 4 weeks of LADx we

observed a significant decrease in left ventricular ejection fraction and increases left ventricular

size (Figure 3A and B). We also observed changes in left ventricular homogenate of molecular

markers of cardiac calcium handling [sarcoplasmic reticulum calcium ATPase 2a (SERCA2a)

and sodium calcium exchanger (NCX-1)] and cardiac hypertrophy [beta myosin heavy chain

( MHC) and atrial natriuretic peptide (ANP)] consistent with a heart failure phenotype in this

model (Figure 3C-F). Further, we noted increases in MBG levels in the post-MI heart failure

mice (Figure 3G), with MBG levels observed within the range of values detected among HF

subjects studied (50th percentile 585 pM). We also performed the LAD ligation procedure on a

separate group of mice (n=5) and, after 1 week, isolated and extracted the adrenal glands and

MBG PrPromo ototes CCCardiac Fibrosis and Nitrative SSStrt ess in NN Animal Mododdele s. In order to asses

MBMBMBGGG levelsss iiinnn a a momomousususeee momomodededelll ofofof pppososst-t-t-mymm occcaardialalal iiinfnfnfarctctctiiion n n heheeararart t faaailililururure,e,e, lefefefttt anananteteterirrio

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observed a significant decrease ininn llleeeft ventricicicullararar ejejejectctctioioi n fraction and increases left ventricula

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performed MBG measurement. Here we found that adrenal MBG levels were elevated vs control

mice, but this did not reach statistical significance (Mann-Whitney p value = 0.056, Figure 3H).

To directly test for a potential contribution of MBG to promotion of cardiac dysfunction and

nitrative stress, mice were infused intraperitoneally with MBG (0.1 ug/g/day) which results in

comparable levels to the post-MI heart failure mice (Figure 4A). MBG infusion did not result in

decreased ejection fraction, but was accompanied by a significant increase in left ventricular size

(Figure 3A and B). Importantly, mice infused with MBG demonstrated corresponding increases

in MPO (Figure 4B) as well as increases in several methylated arginine markers of nitrative

stress including ADMA, SDMA, and MMA (Figure 4 C-E). Furthermore, while MBG infused

mice experienced significant left ventricular cardiac fibrosis vs vehicle treated controls, right

ventricular cardiac fibrosis appeared to be increased vs vehicle treated controls but this did not

reach statistical significance (Mann-Whitney p value = 0.219, Figure 5).

Discussion

While elevated CTS have been associated with cardiac hypertrophy and dysfunction in subjects

with hypertension30, 31, hypertrophic cardiomyopathy32, decompensated HF33, 34, and

cardiomyopathy in chronic kidney disease15, 23, this is the first study to our knowledge that

examines the relationship between plasma MBG and cardiac parameters as well as adverse

clinical events in a broad and contemporary cohort of patients with HF. The relationships

between elevated MBG and indices of inflammation/oxidative stress, myocardial stress, and

nitrative stress and the predominantly diastolic dysfunction is consistent with the physiologic

effects of MBG on the myocardium in volume-expanded states. These findings support future

tress including ADMA, SDMA, and MMA (Figure 4 C-E). Furthermore, whiiilele MMMBGBGBG iiinfnfnfused

mice eexpxppere ieencn ededed significant left ventricular carddiai c fibrosis vs vehiclclclee treated controls, righ

veveventnttricular cacaardrddiaiaiac cc fifiibrbrbrosososisisis appppppeaeaearrrededed tttooo bebebe iiincncncreaseed vvsss vevevehihihicle e e trtreaeaeatetedd d cococ ntrororolslsls bbbututu tthihihisss didididdd nonnt

eeeacacch hh statistical sssigggniffiiicaaancee (MMMann-W-WWhhhitttneyyy ppp vallluee === 000 22.219,,, Figugugurre 5)..

DiDiscscusussisionon



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investigations on the potential role of modulation of MBG levels or activity as a novel targeted

therapy in the population of HF patients burdened with significant cardiovascular disease and

death.

Contribution of CTS in Heart Failure. While previous human studies have demonstrated

the detection of elevated CTS in the setting of acute myocardial infarction13, 35, 36 and HF37, the

novel findings in the current study was the relationship between elevated MBG and worsened

right ventricular function (a condition often associated with venous congestion) as assessed by

standard echocardiographic indices as well as adverse clinical outcomes and nitrative stress.

Further, the data from the subgroup of patients who had serial MBG measurements suggests that

MBG levels are dynamic and supports the view that rising levels of MGB during admission

predict worse long-term clinical outcomes.

The relationship between cardiac structure, hemodynamics and CTS has also been

observed in several other cohort studies. Endogenous plasma ouabain levels are elevated in

patients with severely impaired left ventricular function (EF< 30%)34 and demonstrated

significant positive correlation with hemodynamics such as blood pressure as well as cardiac

indices such as left ventricular mass index, left ventricular end diastolic volume, as well as

eccentric remodeling in hypertensive patients30, 31. Both circulating and myocardial tissue CTS

immunoreactivity was positively correlated with left ventricular end-diastolic pressures and

inversely correlated with cardiac index in patients with hypertrophic cardiomyopathy32. The

shift from compensated left ventricular hypertrophy to CHF is not only marked by a three-fold

increase in endogenous plasma ouabain levels in decompensated CHF, but also an increase in the

sensitivity of cardiac Na/K-ATPase to ouabain33, 34. Levels of circulating endogenous ouabain

also predicted HF progression in idiopathic dilated cardiomyopathy patients38 and left ventricular

MBG levels are dynamic and supports the view that rising levels of MGB dududuririringngng aaadmdmdmisisissisis ono

predicicicttt wowoworsrsrseee loongngng-term clinical outcomes.m

The relalalatioononshshshippp bbbeetetweweweenenn cccarrrdididiac sstrrructuuurre, hhhemememodododynnamamamiicics ananand d CTCTCTSSS hahahas s aaalsososo bbbeeeeen

obbbseseservrvrved in n n sess vvverrral ooothhher cococohohohortrt ssstuttudddieees. EnEnEndogggennooususus ppplalalasssma oououababaiiin leeevevevelsll arerere ellevvvateddd in

patients with severely impairedd d leleleftftft vvvenenentrtrtricici ulululararar fffunununctctctioioion n n (E(( F< 30%)34 and demonstrated

iiigngnifififiicicananttt popo isisitititiveve ccororrerellalatititionon wititithhh hhehemomoddydynana imimicscs susu hchch aass blblblooooddd prpresessusurere aass wewellllll aass caca drdrdiiaiacc



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hypertrophy in the setting of end-stage renal disease39. Increased plasma MBG levels parallel the

progression of HF37 and are associated with a “uremic cardiomyopathy” in chronic kidney

disease15, 22, 23. Experimentally, Dahl salt-sensitive rats fed a high-salt diet demonstrated

compensated left ventricular hypertrophy progressing to dilated cardiomyopathy in parallel with

increasing plasma MBG level as well as increased expression and sensitivity of the Na/K-

ATPase -1 to MBG33. Interestingly, in our study, MBG was not significantly associated with

cystatin C or estimated GFR, suggesting factors beyond renal insufficiency that influence MBG

production. As we have previously demonstrated that MBG is synthetized by adrenocortical cells

and that Angiotensin II can be its secretagogue 40, 41 we isolated and extracted the adrenal glands

and performed MBG measurement in a group of mice one week after LADx and noted it was

significantly elevated. This supports the adrenal tissue as a significant contributor to the pool of

circulating MBG in a post-MI heart failure model. Taken together with our findings, these

observations suggest that the elevated CTS levels which accompany edematous states like HF

may promote downstream adverse cardiovascular consequences.

Mechanisms Linking CTS to Cardiovascular Pathology in Heart Failure. In addition to

their well known effects on the ion transporting functions of the Na/K-ATPase, CTS also bind to

and initiated signaling through a non-pumping pool of the Na/K-ATPase which reside in

caveolae6. CTS confer a conformational change to the Na/K-ATPase that releases the Src kinase

domain, thus activating Src kinase and multiple downstream targets2, 8, 11. This novel Na/K-

ATPase-mediated signaling is responsible for a variety of key cellular roles involving cell

growth/hypertrophy, reactive oxygen species production, and collagen synthesis6.

In the present study we demonstrate for the first time that increased circulating levels of

MBG in human heart failure associate with elevations in markers of inflammation and nitrative

and performed MBG measurement in a group of mice one week after LADx aaanddd nnnotototededed iiittt wa

ignifificicicananantltlt y yy elee evevvataa ed. This supports the adrenal ttisisissuerr as a significant ccconoo tributor to the pool o

cicicircccuulating MBMBMBGG G innn aaa pppososost-t MIMIMI hhheeeararart tt fafafaillururure mooodeel. TTTakakakenenen togogogetetethhhererr wwwiti h ououourrr fififindndn ininingsgsgs, , , thththesee e

obobbseeervr ations sssuggggggest ttthhaat thhhe e elee evatatatededd CCCTSS leeevellls whwhhicicichh aacaccompmpmpany eededememmatttouo s ssstaatatess llikee HHHF

may promote downstream adverssee cccarara did ovovasascucuculaaar r r cococ nsnsnseqeqequeuencceses. .

MeMeMechchchanananisisismsmsms LLLinininkikikingngng CCCTSTSTS tttooo CaCaCardrdrdioioiovavavascscsculululararar PPPatatathohohololologygygy iiinnn HeHeHearararttt FaFaFailililururureee. IIInnn adadaddididitititiononon tttooo



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stress including MPO and the methylated arginine metabolites ADMA, SDMA, and MMA.

Using an animal model of post-MI heart failure, we also demonstrate a significant increase in

plasma MBG 4 weeks after ligation of the left anterior descending artery in mice. Further,

infusion of mice with MBG, which results in similar circulating levels as those seen in both

human and experimental heart failure, recapitulated the increases in MPO as well as ADMA,

SDMA, and MMA seen in our human heart failure study.

The mechanism whereby MBG may increase methylated arginine metabolites is unclear.

We and others have shown that cardiotonic steroids such as MBG and ouabain increase ROS and

inflammatory cytokines in cardiac42 and renal43 cell types, and also decrease NO bioavailability

without changes in eNOS expression44. Inflammation and increased oxidant stress can

significantly impact methyltransferases necessary for arginine methylation, the proteases

involved in release of free methylargnine metabolites, and the catabolic dimethylarginine

dimethylaminohydrolases responsible for metabolism of ADMA45. Thus, it is possible that some

of the observed associations between methylated arginine metabolites and MBG may occur in

part via ROS mediated perturbations in these enzymatic pathways.

The association of CTS with markers of inflammation and nitrative stress is not however

without precedent. We have previously reported in the rat that treatment of both cardiac

myocytes and isolated perfused hearts with the CTS ouabain yielded increased nitrative

modification and decreased activity of cardiac calcium handling proteins as well as diastolic

dysfunction46. We have also shown that ouabain induces increases in inflammatory cytokine

expression from both macrophage and renal proximal tubular cell types43. Our findings are also

in parallel with animal models such as partial (5/6th) nephrectomy showing elevations in

circulating MBG levels that stimulate systemic oxidant stress, oxidative modification and

without changes in eNOS expression44. Inflammation and increased oxiidadadantnn ssstrtrtresesesss s cacan

ignififficicicananantltltly y y immmpapapact methyltransferases necessasasaryryry for arginine metetethyhh lation, the protease

nnnvooolved in reeeleleleaaase e ofofof fffreeee mememethththylylylarara gggnininne ee metttabbboliliitetetesss, aaanddd thehehe cccatatatababa olicicic dddimimimetete hyhyhylalalargrgrgininininini ea

didiimememethylaminnnohohhydydydrolaaaseees reeespopoponsibibblel fooor mmmetttabolissm m m ofofof ADMDMD AAA4555. Thuuus,, it iiis popp ssibibibllle ttthhhat sooommme

of the observed associations betweweweenenen mememethththylylylatata ededed aaargrgrginininininineee mememetatat bolites and MBG may occur in

papapartrtrt vvviaiaia RRROSOSOS mmmededediaiaiateteteddd pepepertrtrturururbababatititionononsss ininin ttthehehesesese eeenznznzymymymatataticicic pppatatathwhwhwayayaysss.



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fibrosis of cardiac tissue and cardiac dysfunction in the rat19, 22, 47. This cardiac phenotype can

also be recapitulated by infusion of MBG. In contrast, both active and passive immunization

against MBG as well as lowering circulating MBG levels via adrenalectomy significantly reduce

the oxidant stress and cardiac dysfunction independent of changes in blood pressure.

In our study, mice infused with MBG also demonstrated significant increases in cardiac

fibrosis. MBG and ouabain have both been shown to increase [3H]proline incorporation in

addition to collagen expression (both mRNA and protein) in cardiac and renal fibroblast cell

types20 and these effects were blocked by pharmacological antagonism of the TGF beta

pathway19. We have also noted that decreases in Fli-1 (a negative regulator of collagen synthesis)

expression appear to be necessary for MBG to induce increases in collagen in several types of

fibroblasts (cardiac, renal, and dermal). Additionally, MBG induces translocation of PKCdelta

from the cytosol to the nucleus in a PLC dependent manner, and this translocation of PKCdelta

causes the phosphorylation and subsequent degradation of Fli-120. In several fibroblast cell types,

CTS also stimulate Na/K-ATPase and oxidant signaling which induce collagen production19, 22,

43,. These signaling pathways are not only significantly attenuated by oxidant scavenging and

inhibition of Src kinase, but also through competitive inhibitory mechanisms induced by

spironolactone and canrenone binding to the Na/K-ATPase19, 22, 48, 49. Thus, the pro-inflammatory

and pro-fibrotic CTS-Na/K-ATPase signaling axis may provide a novel therapeutic target in

settings such as HF where elevated CTS induce inflammation and cardiac fibrosis.

Study limitations. Despite being the largest study to our knowledge of reporting the

relationship between clinical outcomes and MBG in HF, the current study is still limited in the

relatively small number of patients as well as the selection bias that potentially confounds

interpretation of such cohort studies. We did not have sufficient data to analyze central venous

expression appear to be necessary for MBG to induce increases in collagen in sseveve eeeralalal tttypypypesese o

fibrobbblalalastststss s (c(c(cardididiacaa , renal, and dermal). Additionananallll y, MBG induces trararannnslocation of PKCdelta

frfrfrommm the cytosololol ttto thththee e nununuclcc euuus s s ininin aaa PPPLCLCLC dddepepe eeendeeennnt mmmananannenener,r,r, andndnd ttthihihis trtrtranannslocococatatatioioion n n offf PPPKCKCKCdededeltl a

caaausususesee the phohohosppphhooryllaattion aaandndnd subssseqeqe ueueuent dddegeggraddadatttionnn ooof FFFliii-12000. IIIn sevvverralll fibibibrobbllalasstst cccellll tyyypeees

CTS also stimulate Na/K-ATPasesee aaandndnd oooxixixidadadantnn sssigigignanan lililingngng wwwhihihichchch induce collagen production19, 22

3,. ThThTheseseseee sisisigngngnalalalinininggg papapathththwawawaysysys aaarerere nnnototot ooonlnlnlyyy sisisigngngnififificicicananantltltlyyy atatattetetenununuatatatededed bbbyyy oxoxoxiiiffffff dadadantntnt ssscacacavevevengngnginininggg anananddd



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pressure or other indices of right ventricular function, thus the measurements indicating

worsened RV function are based solely on echocardiographic indices. Our Cox proportional

hazard analyses with multiple covariables was limited by lack of power due to missing data.

Similarly, when defined by quartiles, while the trend between MBG and worse clinical outcomes

was similar to that obtained by the optimal cutpoint analysis, it was not statistically significant.

Nevertheless, our study examines for the first time the contribution of MBG to incident

cardiovascular outcomes in a cohort of patients with HF and demonstrates a novel association

between CTS and markers of inflammation and nitrative stress. We would emphasize that the

purpose of this study was to demonstrate underlying physiology rather than to propose MBG as a

biomarker as it tracks with worsening renal function. The increased inflammation, nitrative stress

and cardiac fibrosis seen with elevations in MBG may be an important mechanism of

cardiovascular dysfunction in patients with heart failure and requires further investigation.

Conclusion

Elevated levels of MBG are associated with indices of worse cardiac dysfunction as well as

increased risk for development of adverse clinical outcomes in patients with HF even after

multivariable models adjustment for established clinical risk factors. Similarly, changes in MBG

levels over time appear to be of prognostic benefit in HF patients, and infusion of MBG in an

animal model appears to directly contribute to increased nitrative stress, cardiac fibrosis and

dysfunction. Thus, MBG may serve as an important therapeutic target in patients with heart

failure.

biomarker as it tracks with worsening renal function. The increased inflammationnn, nninitrtrtratatativiviveee stss res

and cardiac fibrbrrosis seen with elevations in MMBG may be an impmm ortant mechanism o

caaardddiovascularaa dddysysysfuuncncnctitit ononon in papapatititienenentsss wwwitii h hh hhheaeaart fffaiilureee aaandndnd rrreqqquiuiuireeesss fuuurtrtrthehh r innnvevevestststigigigattioioion.nn

Conclusion

ElElevevatateded llevevelels s ofof MMBGBG aarere aassssocociaiateted d wiwithth inindidiceces s ofof wwororsese ccarardidiacac ddysysy fufuncnctitionon aas s wewellll aass



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Sources of Funding

This research was supported by grants from the National Institutes of Health (NIH,

R01HL103931) and the NIH/Office of Dietary Supplements (P20HL113452), and the Cleveland

Clinic Clinical Research Unit of the Case Western Reserve University CTSA (UL1TR 000439).

Dr. Kennedy was supported by an American Heart Association Scientist Development Grant

14SDG18650010. Drs. Fedorova and Bagrov are supported by Intramural Research Program,

National Institute on Aging, National Institutes of Health. Mass spectrometry studies were

performed on instruments housed in a facility supported in part by a Center of Innovations

Award by AB SCIEX.

Disclosures

None.

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37. Fridman AI, Matveev SA, Agalakova NI, Fedorova OV, Lakatta EG and Bagrov AY. Marinobufagenin, an endogenous ligand of alpha-1 sodium pump, is a marker of congestive heart failure severity. J Hypertens. 2002;20:1189-94.

38. Pitzalis MV, Hamlyn JM, Messaggio E, Iacoviello M, Forleo C, Romito R, de Tommasi E, Rizzon P, Bianchi G and Manunta P. Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy. Eur J Heart Fail. 2006;8:179-86.

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40. Fedorova OV, Agalakova NI, Talan MI, Lakatta EG and Bagrov AY. Brain ouabain stimulates peripheral marinobufagenin via angiotensin II signalling in NaCl-loaded Dahl-S rats. Journal of Hypertension. 2005;23:1515-23.

41. Dmitrieva RI, Bagrov AY, Lalli E, Sassone-Corsi P, Stocco DM and Doris PA. Mammalian bufadienolide is synthesized from cholesterol in the adrenal cortex by a pathway that Is independent of cholesterol side-chain cleavage. Hypertension. 2000;36:442-8.

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43. Kennedy DJ, Chen Y, Huang W, Viterna J, Liu J, Westfall K, Tian J, Bartlett DJ, Tang WH, Xie Z, Shapiro JI and Silverstein RL. CD36 and Na/K-ATPase-alpha1 form a proinflammatory signaling loop in kidney. Hypertension. 2013;61:216-24.

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48. El-Okdi N, Smaili S, Raju V, Shidyak A, Gupta S, Fedorova L, Elkareh J, Periyasamy S, Shapiro AP, Kahaleh MB, Malhotra D, Xie Z, Chin KV and Shapiro JI. Effects of cardiotonic steroids on dermal collagen synthesis and wound healing. J Appl Physiol. 2008;105:30-6.

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41. Dmitrieva RI, Bagrov AY, Lalli E, Sassone-Corsi P, Stocco DM and Doris PA... MaMaMammmmmmalaaliaianbufadienolide is synthesized from cholesterol in the adrenal cortex by a pathwwwayayy tthahahat t t IsIsIs independent of cholesterol side-chain cleavage. Hypertension. 2000;36:442-8.

42. Priyiyyadadarshshi S,S,S, Valentine B, Han C, Fedorova OVOVV, Bagrov AY, Liu JJ, , , PeP riyasamy SM, KeKeKennn edededy yy D,D, MMMalhotra D, Xie Z and Shapiro JIJIJI. EEffect of green n n tet a exexxtrtrtract on cardiachhhypertrophphphy y y fofofollowowowinining g g 5/55 6 6 nenenephphphrerer ctctctomomomy y y ininin the raat. KiKiKidndndneyeyey Intntnt. 202020030303;6;6;63:33 177858585-9-9-90.0.0.

434 . Kennedy DJJ,,, CCChennn YYY, HuHuuaanang W,WW VVViteeernaaa JJJ, Liuuu JJJ, WWWeesestftftfallll K,K,, TTTian JJJ, BBBararrtlllett DJDJDJ, TaTaanggg WWWHH,H XXiX e Z, Shaapiiirooo JI aanandd Sillvlveererstein n n RRLL... CDDD36 annnd Na/a/a/K-K-K ATATATPassse---alphaaa1 fooormmm a prprproioioinfnfnflalalammmmmmaatatororory y y sisisigngngnalaa inng g g lololoopopop in kikikidndndneyeyey... HyHyHypepepertrtrtenenensisisiononon. 20202013133;6;6;61:1:1:2121216-6-6-242424.

44. Wenceslau CF, Davel AP, Xaaavivivieree FE and d d RoRoossssssono i i i LVLVL . Longgg-term ouabain treatment impairsvascular function in resistanceee aaartrtrterererieieiess.s. JJ J VVVascscsc RRReseses.. 202020111111;4;4;48:8:8:316-26.

4545. . PoPopepep AAJ,J,, DDruruhahan n L,L,, GGuzuzmaman n JEJE,,, FoForbrbeses SSP,P,, MuMururugegeg sasan n V,V,, LLu u D,D,, XXiaia YY, , , ChChicicoioinene LLG,G,, Parinandi NL and Cardounel AJ Role of DDAH-1 in lipid peroxidation product-mediated



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Table. Baseline Characteristics (n=245).

Variable Value

Demographics:

Age (years) 58 ± 13

Male gender, n (%) 164 (67%)

Body mass index (kg/m2) 30 ± 6

African American, n (%) 45 (18%)

Heart failure history:

Ischemic etiology, n (%) 92 (39%)

NYHA class III or IV, n (%) 95 (66%)

Co-morbidities:

Hypertension, n (%) 130 (54%)

Diabetes mellitus, n (%) 92 (38%)

Echocardiographic indices:

LV mass index (g/m2) 159 ± 57

LV end-diastolic volume index (mL/m2) 97 ± 42

LV ejection fraction (%) 31 ± 13

Mitral E/e’ ratio 22 ± 12

Mitral E/A ratio 1.9 ± 1.0

Mitral DT (ms) 160 ± 52

Tricuspid E/e’ ratio 7.1 ± 3.8

LA volume index (mL/m2) 41 ± 17

RV end-diastolic area (cm2) 26 ± 9

NYHA class III or IV, n (%) 95 (((666666%)%)%)

CoCoCo-m-m-morororbibibididitititiesee :

Hypertenenensssioonon, n nn (%(%(%))) 131313000 (5(5(54%4%4%)))

Diabbetetetesee mmmellitttusss, n (%)%)%) 929292 (3888%%%)

Echocardiographic indices:

LVLVLV mass iiindddex (((g///m222))) 151515999 ±±± 575757



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RV s’ (cm/s) 9 ± 3

RV Fractional area change 34 ± 12

Tricuspid DT (ms) 189 ± 61

RA volume index (mL/m2) 34 ± 18

Medications:

Angiotensin converting enzyme inhibitors or angiotensin

receptor blockers, n (%)

143 (61%)

Beta-blockers, n (%) 171 (73%)

Spironolactone, n (%) 87 (37%)

Loop diuretics, n (%) 151 (64%)

Digoxin, n (%) 70 (30%)

Laboratory data:

MBG (pM) 583 [383, 812]

Myeloperoxidase (pM) 124 [77- 253]

BNP (pg/mL) 431 [97, 1417]

eGFR (mL/min/1.73m2) 71 ± 32

Cystatin C (mg/L) 1.85 [1.31 – 2.61]

ADMA ( M) 1.05 [0.82 – 1.33]

SDMA ( M) 1.70 [1.05 – 2.96]

MMA (nM) 50 [34 – 92]

Abbreviation: NYHA, New York Heart Association; LV, left ventricular; RV, right ventricular; MBG, marinobufagenin (MBG); eGFR, estimated glomerular filtration rate; BNP, B-type natriuretic peptide, ADMA, Asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; MMA, mono methyl arginine.

Loop diuretics, n (%) 151 (6(6(64%4%4%)))

DiDiDigogogoxixixin, nnn (%) 70 (30%)

LLLaboratoryyy dddaaata:a:a:

MBG G G (p(( M)M)M) 58883 [3[ 8333, 8881222]

Myeloperoxidase (pM) 124 [77- 253]

BNBNBNPPP (((p //g/ LLmL))) 434343111 [9[9[9777, 1114141417]7]7]



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Figure Legends

Figure 1. Marinobufagenin levels in heart failure. Comparison of plasma marinobufagenin

levels between non-heart failure (non-HF) participants and HF participants with reduced ( 40)

or preserved (>40) left ventricular ejection fraction (EF). ** p <0.001 vs control, by Student’s

t-test with Bonferroni’s correction.

Figure 2. Kaplan-Meier analysis of major adverse cardiac events (MACE) in participants

with heart failure. (A) Heart failure patients (n=245) stratified according to optimal cutoff for

plasma marinobufagenin (MBG) as follows: “Low” MBG (<574 pM) or “High” MBG ( 574

pM). (B) Subgroup analysis of heart failure patients (n=115) with serial blood draws from

admission to pre-discharge at 48-72 hours stratified according to optimal cutoff for change in

plasma MBG as follows: “decreasing” MBG (<5%) or “increasing” MBG ( 5%).

Figure 3. Elevated MBG levels contribute to nitrative stress. Echocardiographic measures of

cardiac ejection fraction (A) and Diastolic Left Ventricular Internal Dimension (LVIDd) (B)

after 4 weeks of either LAD ligation (LADx) or MBG infusion. Gene expression of calcium

handling proteins sarcoplasmic reticulum calcium ATPase (SERCA2a) (C) and sodium calcium

exchanger (NCX-1) (D), and hypertrophic markers beta myosin heavy chain ( MHC) (E) and

atrial natriuretic peptide (ANP) (F) after 4 weeks of LADx. Plasma MBG (G) levels are

increased 4 weeks after LAD ligation in a post myocardial infarction heart failure model. (H)

Adrenal tissue MBG levels 1 week after LAD ligation. P values were calculated using the Mann-

Whitney U test.

pM). (B) Subgroup analysis of heart failure patients (n=115) with serial bllooooooddd drdrdrawawaws s s frf om

admisssioion n too preee-ddischarge at 48-72 hours stratififiede according to optiimamm l cutoff for change in

plplplasssma MBGGG aasss fofofollowowows:s:s: ““dedd crcrreaeaeasisisingngng”” MMMBGBGG (((<5< %%%) or “i“iincncncrerereasininin ”g”g” MMMBGBGBG ( 555%)%)%)...

Figure 3. Elevated MBG levels s s cococontn ributeee to o nininitrt atatativivive stress. Echocardiographic measures o

cacardrdiaiac c ejejjecectitionon ffraractctioion n (A(A( ))) anand d DiDiasastotolilic c LeLeftft VeVentntriricuculalar r InInteternrnalal DDimimenensisionon (((LVLVIDIDd)d)) (((BB



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Figure 4. Elevated MBG levels contribute to nitrative stress. After 4 week infusion of MBG,

plasma levels of MBG (A) and MPO (B), as well as methylated arginine metabolites asymmetric

dimethylarginine (ADMA, C), symmetric dimethylarginine (SDMA, D), and mono methyl

arginine (MMA, E) are increased vs vehicle treated mice. P values were calculated using the

Mann-Whitney U test.

Figure 5. Elevated MBG levels contribute to fibrosis. Representative picosirius red histology

(A) and quantitative morphometry (B) from mouse hearts after 4 week MBG infusion. Scale bar

represents 100 um. P values were calculated using the Mann-Whitney U test.



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Alexei Y. Bagrov and W.H. Wilson TangMichael Finucan, Alaa Gabi, Charles M. Medert, Kristen Westfall, Allen Borowski, Olga Fedorova,

David J. Kennedy, Kevin Shrestha, Brendan Sheehy, Xinmin S. Li, Anuradha Guggilam, Yuping Wu,Right Ventricular Dysfunction and Nitrative Stress in Heart Failure

Elevated Plasma Marinobufagenin, An Endogenous Cardiotonic Steroid, Is Associated with

Print ISSN: 1941-3289. Online ISSN: 1941-3297 Copyright © 2015 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation: Heart Failure published online August 14, 2015;Circ Heart Fail.

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1

SUPPLEMENTAL MATERIAL

Quantitative Histologic Techniques. Mason’s trichome and picosirius red staining was

performed on deparafinized 5 µm serial heart sections. The sections were mounted

under a Leica DM 2500 microscope and digitized with a QImaging MicroPublisher 5.0

RTV camera. Quantitative morphometric analysis was performed on fields (at least 8

from each animal) and the collagen volume was determined using automated and

customized algorithms/scripts for batch analysis (ImageIQ Inc., Cleveland, OH) written

for Image Pro Plus 7.0. Briefly, a set of representative images were chosen that

demonstrated a wide range of staining intensities and prevalence. In an automated

script, these "training" images were loaded one after another prompting the user to

delineate pixels representing positive collagen staining using an interactive color picking

tool. An iterative color profile or classifier was generated and subsequently applied to all

images in a given directory using a fully automated algorithm. Positive pixels, as defined

by the color profile, were segmented and summed to provide positive staining area.

Total tissue area was determined by extracting the "saturation" channel, applying a lo-

pass filter, and thresholding the result. Any area within the general tissue boundary that

was empty (i.e. white) was removed by converting the original image to grayscale and

applying a fixed threshold for non background pixels on adequately white-balanced

images. Finally, total tissue area and total stained area were exported to Excel. For

post-processing verification, segmented regions were superimposed onto the original

image (green outlines) for each image analyzed.

2

Transthoracic Echocardiography. Comprehensive 2-dimensional echocardiography was

performed in standard parasternal and apical views on all participants by an American

Society of Echocardiography registered research sonographer using a Vivid 7

echocardiography machines (GE Healthcare, Waukesha WI) equipped with a phased-

array transducer. Standard 2-dimensional and Doppler data, triggered to the QRS

complex, was digitally stored in a cine-loop format. The Simpson biplane method was

used to measure left ventricular ejection fraction (LVEF), left ventricular end-systolic

volume index (LVESVI), and left atrial volume index (LAVI). Ventricular volume and

mass measurements were indexed to body surface area. Right ventricular systolic

pressure (RVSP) was calculated from the Doppler estimated tricuspid valve regurgitant

jet velocity using the Bernoulli equation. Assessment and classification of diastolic

parameters, mitral inflow patterns, and quantification of mitral regurgitation were made

following the American Society of Echocardiography recommendations1, 2. Small animal

echocardiography was performed using a Vivid 7 ultrasound machine (GE Medical)

equipped an il3L linear probe operated at 14 MHz in order to obtain 2-D (parasternal

long- and short-axis views) and M-mode images as we have previously published3, 4. All

echocardiographic measurements were averaged over three cardiac cycles.

3

Supplemental Table 1. Univariate correlations between marinobufagenin (MBG) and clinical and echocardiographic characteristics MBG (pM)

Spearman’s r p-value

Age (years) 0.06 0.392

Body mass index (kg/m2) 0.02 0.768

Echocardiographic Indices

LV Structure

LV mass index (g/m2) 0.15 0.073

LVEDVi (mL/m2) -0.05 0.552

LV Systolic function

LV ejection fraction (%) -0.04 0.585

LV Diastolic function

Mitral E/e’ ratio

Mitral E/A ratio

0.13

0.12

0.136

0.202

Mitral DT (ms) -0.24 0.007

LA volume index (mL/m2) -0.04 0.551

RV Structure

RV end-diastolic area (cm2) 0.21 0.023

RV Systolic function

RV s' (cm/s) -0.39 <0.0001

RV Fractional area change -0.42 <0.0001

RV Diastolic Function

Tricuspid E/e' ratio 0.22 0.027

4

Tricuspid DT (ms) -0.38 0.002

RA volume index (mL/m2) 0.22 0.013

Laboratory Data

Myeloperoxidase (pM) 0.42 <0.0001

BNP (pg/mL) 0.25 0.001

eGFR (mL/min/1.73m2) -0.06 0.345

Cystatin C (mg/L) 0.06 0.485

ADMA (μM) 0.32 <0.001

SDMA (μM) 0.34 <0.001

MMA (μM) 0.40 <0.0001

Abbreviation: ADMA, Asymmetric dimethylarginine; SDMA, symmetric dimethylarginine;

MMA, monomethyl arginine

5

Supplemental Table 2. Cox proportional hazards analyses of adverse long-term

clinical outcomes.

HR (95% CI) p-value

MBG 1.58 (1.10 – 2.31) 0.014

Adjusted for age, male gender 1.55 (1.07 – 2.26) 0.019

Adjusted for age, gender, LVEF 1.51 (1.04 – 2.21) 0.031

Adjusted for age, gender, LVEF, ischemic etiology 1.48 (1.01 – 2.17) 0.042

Adjusted for age, gender, ischemic etiology, diabetes

mellitus

1.50 (1.04 – 2.19) 0.031

Adjusted for age, gender, smoking, diabetes mellitus 1.47 (1.02 – 2.14) 0.040

Adjusted for eGFR 1.43 (0.99 – 2.08) 0.060

Adjusted for ischemic etiology 1.53 (1.06 – 2.24) 0.023

Adjusted for mitral E/e' ratio 1.65 (0.97 – 2.87) 0.066

Adjusted for RV s' 1.36 (0.76 – 2.51) 0.305

Adjusted for tricuspid E/e' 1.36 (0.77 – 2.46) 0.290

Adjusted for BNP 1.40 (0.87 – 2.31) 0.169

*HRs represent the risk associated with MBG levels ≥ 574 pM

6

Supplemental Table 3. Net reclassification analysis 5

Discrimination Analysis Percentage 95% CI P-value

AUC

Model with MBG 60.7% 0.33

Model without MBG 57.8%

IDI 3% <0.001

NRI 33.6% 9.3%-57.8% 0.007

Events correctly reclassified 41.9%

Non-events correctly reclassified -8.3%

Abbreviation: AUC, Area under the curve; IDI, integrated discrimination improvement;

NRI, net reclassification index; CI, confidence interval.

Both models were adjusted for age, gender, ischemic etiology, and diabetes. The risk of

mortality was estimated using the Cox model.

7

Supplemental Table 4. Cox proportional hazards analyses by quartiles for adverse

long-term clinical outcomes.

HR (95% CI) P-value

MBG Quartile 4 vs Quartile 1 1.57 (0.91 – 2.75) 0.102



8

Supplemental References

1. Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, Waggoner AD, Flachskampf FA, Pellikka PA and Evangelista A. Recommendations for the evaluation of left ventricular diastolic function by echocardiography. J Am Soc Echocardiogr. 2009;22:107-33.

2. Zoghbi WA, Enriquez-Sarano M, Foster E, Grayburn PA, Kraft CD, Levine RA, Nihoyannopoulos P, Otto CM, Quinones MA, Rakowski H, Stewart WJ, Waggoner A and Weissman NJ. Recommendations for evaluation of the severity of native valvular regurgitation with two-dimensional and Doppler echocardiography. J Am Soc Echocardiogr. 2003;16:777-802.

3. Kennedy DJ, Vetteth S, Periyasamy SM, Kanj M, Fedorova L, Khouri S, Kahaleh MB, Xie Z, Malhotra D, Kolodkin NI, Lakatta EG, Fedorova OV, Bagrov AY and Shapiro JI. Central role for the cardiotonic steroid marinobufagenin in the pathogenesis of experimental uremic cardiomyopathy. Hypertension. 2006;47:488-95.

4. Kennedy DJ, Elkareh J, Shidyak A, Shapiro AP, Smaili S, Mutgi K, Gupta S, Tian J, Morgan E, Khouri S, Cooper CJ, Periyasamy SM, Xie Z, Malhotra D, Fedorova OV, Bagrov AY and Shapiro JI. Partial nephrectomy as a model for uremic cardiomyopathy in the mouse. Am J Physiol Renal Physiol. 2008;294:F450-4.

5. Pencina MJ, D'Agostino RB, Sr. and Steyerberg EW. Extensions of net reclassification improvement calculations to measure usefulness of new biomarkers. Stat Med. 2011;30:11-21.

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