Matrix Signaling Subsequent to a Myocardial Infarctionbasictranslational.onlinejacc.org/content/btr/2/5/529...PRECLINICAL RESEARCH Matrix Signaling Subsequent to a Myocardial Infarction

J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 2 , N O . 5 , 2 0 1 7

ª 2 0 1 7 P U B L I S H E D B Y E L S E V I E R O N B E H A L F O F AM E R I C A N C O L L E G E O F

C A R D I O L O G Y F O U N D A T I O N . T H I S I S A N O P E N A C C E S S A R T I C L E U N D E R

T H E C C B Y - N C - N D L I C E N S E ( h t t p : / / c r e a t i v e c o mm o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / ) .

I S S N 2 4 5 2 - 3 0 2 X

h t t p : / / d x . d o i . o r g / 1 0 . 1 0 1 6 / j . j a c b t s . 2 0 1 7 . 0 4 . 0 0 4

PRECLINICAL RESEARCH

Matrix Signaling Subsequent toa Myocardial InfarctionA Proteomic Profile of Tissue Factor Microparticles

Derrick Akpalu,a Gale Newman, PHD,a Mark Brice, PHD,a Mike Powell, PHD,a Rajesh Singh, PHD,a

Alexander Quarshie, MD,b Elizabeth Ofili, MD,b James Fonger, MD,c Nic Chronos, MD,d David Feldman, MD, PHDe,f

VISUAL ABSTRACT

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Akpalu, D. et al. J Am Coll Cardiol Basic Trans Science. 2017;2(5):529–42.

rom the aDepartment of Microbiology, Biochemistry and Immunology, Morehouse School of M

esearch Center, Morehouse School of Medicine, Atlanta, Georgia; cCardioScout LLC, Atlanta,

ake Oconee, Eatonton, Georgia; eDivision of Cardiology University of Cincinnati Medical

epartment of Cardiology, Inselspital Hospital, University of Bern, Switzerland. This work w

onal Institutes of Health (NIH) and National Institute on Minority Health and Health

RO1HL84498-5, NIH/NIMHD 2S21MD000101NIMHD 8U54MD007588-04; the Minority Bio

esearch Initiative for Scientific Advancement program 5R25GM058268; the Research

G12MD007602; and NIH/NHLBI contract grant number R21HL092358. All authors have repor

levant to the contents of this paper to disclose.

HIGHLIGHTS

� The occurrence of an MI activates

production of TFMPs.

� We induced an MI in Yucatan miniswine

and collected plasma samples over a

6-month period post-MI.

� Experimental groups consisted of

infarcted but untreated animals and

infarcted animals treated with CRT plus

b-blocker.

� Using proteomic profiling, we confirm the

heterogeneity of TFMP protein content

with respect to physiological status of the

host temporally.

� Spatially, the contents of the TFMPs

provided information about multiple

entities supplemental to what we

obtained from assessing a set of

8 currently used cardiac biomarkers.

� The results from this study support

recommending TFMP protein content

profiling be used prospectively as a viable

investigative methodology for chronic

ischemic cardiomyopathy to help improve

our understanding of b-adrenergic

receptor signaling after an MI.

edicine, Atlanta, Georgia; bClinical

Georgia; dCardiology Care Clinic of

Center, Cincinnati, Ohio; and the

as supported by the following Na-

Disparities (NIMHD) grants: NIH

medical Research Support of the

Centers in Minority Institutions

ted that they have no relationships

http://crossmark.crossref.org/dialog/?doi=10.1016/j.jacbts.2017.04.004&domain=pdf

http://creativecommons.org/licenses/by-nc-nd/4.0/

http://dx.doi.org/10.1016/j.jacbts.2017.04.004

ABBR EV I A T I ON S

AND ACRONYMS

ADRB1 = b1-adrenergic

receptor

ADRB2 = b2-adrenergic

receptor

AR = adrenergic receptor

ARRB1 = b1-arrestin

BB = b-blocker

cAMP = cyclic adenosine

monophosphate

CRT = cardiac

resynchronization therapy

EDV = end-diastolic volume

EF = ejection fraction

ELISA = enzyme-linked

immunosorbent assay

ESV = end-systolic volume

FACS = fluorescence-activated

cell sorting

GRK = G-protein receptor

kinase

HSP = heat shock protein

HUVEC = human umbilical vein

endothelial cell

LVAd MV = left ventricular

area around the mitral valve at

diastole

LVAs MV = left ventricular

area around the mitral valve at

systole

LVAd PM = left ventricular

area around the papillary

muscle at diastole

LVAs PM = left ventricular area

around the papillary muscle at

systole

MI = myocardial infarction

MP = microparticle

PCR = polymerase chain

reaction

TF = tissue factor

TFMP = tissue factor–bearing

microparticle

TnT = troponin T

All authors

institutions

visit the JA

Manuscript

Akpalu et al. J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 2 , N O . 5 , 2 0 1 7

Matrix Signaling After Myocardial Infarction O C T O B E R 2 0 1 7 : 5 2 9 – 4 2

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SUMMARY

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This study investigated the release and proteomic profile of tissue factor microparticles (TFMPs) prospectively

(up to 6 months) following a myocardial infarction (MI) in a chronic porcine model to establish their utility in

tracking cellular level activities that predict physiologic outcomes. Our animal groups (n ¼ 6 to 8 each)

consisted of control, noninfarcted (negative control); infarcted only (positive control); and infarcted animals

treated with cardiac resynchronization therapy (CRT) and a b-blocker (BB) (metoprolol succinate). The authors

found different protein profiles in TFMPs between the control, infarcted only group, and the CRT þ BB treated

group with predictive impact on the outward phenotype of pathological remodeling after an MI within and

between groups. This novel approach of monitoring cellular level activities by profiling the content of

TFMPs has the potential of addressing a shortfall of the current crop of cardiac biomarkers, which is the inability

to capture composite molecular changes associated with chronic maladaptive signaling in a spatial and temporal

manner. (J Am Coll Cardiol Basic Trans Science 2017;2:529–42) © 2017 Published by Elsevier on behalf of

American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

A dvances in diagnosis and manage-ment of myocardial infarction (MI)have accounted for a decrease in

acute mortality fromMI (1,2). Much, however,remains to be understood about thecellular and molecular mechanisms of MIlongitudinally beyond the initial few daysand weeks.

Progressive chronic heart failure and thereduction in cardiac output after an MI causethe activation of neurohormonal responsesand perturbation in long-term adrenergicsignaling, which leads to changes in thesympathetic nervous system (3). b-adrenergicreceptor (AR) activation, in addition toincreasing acute cardiac performance, initi-ates multiple signaling cascades simulta-neously through G-protein receptor kinases(GRKs) and b-arrestin–mediated pathways.With an adaptive upregulation of GRK2, thereis a concordant increase in heart failurephenotype, in part mediated by the depletionof b-AR–mediated inotropic reserve (4–6).Additionally, chronic activation of the sym-pathetic nervous system leads to pathologicalremodeling, necrosis, and apoptosis (7). Two

important aspects in the treatment of chronic heartfailure with pathological remodeling include the use ofb-blockers (BBs) and cardiac resynchronization ther-apy (CRT) (4–6). Together these interventions improve

test they are in compliance with human studies committe

d Food and Drug Administration guidelines, including patien

: Basic to Translational Science author instructions page.

ceived November 17, 2016; revised manuscript received Febru

symptoms and enhance left ventricular function whileslowing down the progression of maladaptive remod-eling and improving morbidity and mortality inappropriately selected patients (4–6).

Previous investigations revealed an elevationof microparticle (MP) levels in patients with cardio-vascular diseases, specifically those with acutecoronary syndromes (8–11). MPs are small vesiclesreleased from the plasma membrane of cells such asplatelets, leukocytes, erythrocytes, endothelial cells,and muscle cells; they contain cell surface proteinsalong with cytoplasmic components of their cells oforigin (8,9,12–15). MPs produced as a result of humanatherosclerotic plaque formation possess tissue factor(TF) activity along with an outer membrane composedof phosphatidylserine for prohemostatic activity(8,9,11,12,16,17). In patients with various forms of car-diovascular disease, circulating MPs cause endothelialcell dysfunction (9,11,12,16,18–20) and act as a keydriver of atherosclerosis (9,12,13,15,21). In addition toensuring hemostasis, TF plays a cell signaling role bypromoting pleiotropic inflammatory responses. Hith-erto, the reports of the elevation of tissue factor (TF)microparticles (MPs) in patients have been of quanti-tative observations, with no studies describing theprotein content of TFMPs over the long term. This studyinvestigated the release and proteomic profile of TFMPsprospectively after an MI in a chronic porcine model.

The majority of the current models of post-MIsignaling are in smaller animal models with limited

es and animal welfare regulations of the authors’

t consent where appropriate. For more information,

ary 27, 2017, accepted April 4, 2017.

http://creativecommons.org/licenses/by-nc-nd/4.0/

http://www.basictranslational.onlinejacc.org/content/instructions-authors

FIGURE 1 Study Schedule and Sample Collection Plan

CRT ¼ cardiac resynchronization therapy.

J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . 2 , N O . 5 , 2 0 1 7 Akpalu et al.O C T O B E R 2 0 1 7 : 5 2 9 – 4 2 Matrix Signaling After Myocardial Infarction

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duration. For a variety of reasons, investigatorstypically follow signaling for a few hours to severalweeks. Because humans usually do not developpathological remodeling or heart failure in days orweeks, the purpose of this study was to identifyadditional mechanisms involved in progressive heartfailure over time. An approach that enables theobservation of both spatial (membrane to the nu-cleus) and temporal signaling in a large animal modelwould enhance our understanding of the multifac-eted post-ischemic processes. Investigators haveestablished that signaling pathways seldom functionin isolation but are part of a matrix and involve theactivation of multiple pathways at different time in-tervals (22). Hence, we propose that evaluation ofdiscrete signaling pathways for a moment in timemight not adequately capture the complex signalingobserved in humans.

METHODS

ANIMAL MODEL OF MI. All animal studies wereapproved by the St. Joseph’s Translational ResearchInstitute and Morehouse School of Medicine Institu-tional Animal Care and Use Committee and complywith The Guide for the Care and Use of LaboratoryAnimals (8th edition, 2011). The animals were housedindividually and had access to food and water adlibitum to exceed all animal standards. Eight-month-old female Yucatan miniswine were infarcted by useof a collagen plug procedure (23). Two and a halfmonths after the infarct, animals were placed in 1 of 2experimental groups. Pigs in the first group (n ¼ 8)had a collagen plug–induced MI and survived butreceived no treatment, whereas pigs in the secondgroup (n ¼ 6) were MI survivors treated with CRT plusa BB (metoprolol). The third group of female controlpigs (n ¼ 8) had no infarction or treatments. Plasma

was collected at 2.5, 3.5, 4.5, and 6 months afterinfarction (Figure 1). All infarcted animals were killedat 6 months.

MI PROCEDURE. All animals were intubated, and theear or external jugular veinwas cannulated tomaintainadequate anesthesia throughout the procedure. Theanimals (weighing 45 to 55 kg each) were sedated withketamine combined with xylazine and maintainedon isoflurane (0% to 5%). Buprenorphine or morphinewas given perioperatively. Arterial pressure wasmonitored by accessing a femoral artery with anappropriately sized vascular introducer sheath.Heparin (150 U/kg) was administered, and 10 to 30 minlater, activated clotting time was checked to confirm itwas >300 s. An appropriately sized guiding catheterwas introduced and advanced to the ostium of the leftmain coronary artery, and contrast angiography wasperformed. An infusion catheter was advancedthrough the guiding catheter into the midportion ofthe left anterior descending coronary artery. At thediscretion of the operator, z2 ml of collagen wasinjected in 0.2-ml increments to occlude the majorityof the left anterior descending coronary artery.Additional buprenorphine was given for immediatepost-operative analgesia 1 to 2 times per day for 1 to5 days after infarction.

CRT AND BB THERAPY. Biventricular pacing deviceswere implanted as described previously (24). Briefly,pigs that had an MI were treated with CRT and BBtherapy (metoprolol succinate, Novartis, Basel,Switzerland) titrated to 2 mg/kg over several weeks,from 2.5 months post-infarction until termination at6 months. This dosing was intended to emulate thedosing used in MERIT-HF (Metoprolol CR/XL Ran-domized Intervention Trial in Congestive Heart Fail-ure) (25). To achieve a biventricular capture rate>95%, pacing was obtained by simultaneous left



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lateral wall (via coronary sinus) and right ventricularseptal wall pacing. Placement was ensured by fluo-roscopy at the time of placement, and fluoroscopywas used at post-operative 3 days and at 1 month toensure capture.

BLOOD SAMPLE COLLECTION, STORAGE, AND

NECROPSY. Blood samples (7 to 8 ml) were collectedinto ethylenediaminetetraacetic acid tubes (BDVacutainer Systems, Franklin Lakes, New Jersey).The plasma samples were centrifuged at 2,000 � g (20min) to remove any residual platelets, cell debris, andprecipitates. Platelet-poor plasma was stored frozenat �80�C in 1-ml aliquots until one-time use at anal-ysis. Plasma samples were subjected to no furtherfreeze-thaw cycles.

QUANTITATION ASSAYS (HIGH-SENSITIVITY

TROPONIN T, b1-AR, b2-AR, b-ARRESTIN-1, cAMP,

EPINEPHRINE, NOREPINEPHRINE, AND GRK2). Tomeasure b1-AR (ADRB1), b2-AR (ADRB2), b1-arrestin(ARRB1), and cyclic adenosine monophosphate(cAMP) levels, porcine hearts were harvested whenthe animals were killed 6 months after the infarction.Harvested cardiac tissue was rinsed with phosphate-buffered saline and quickly frozen in liquid nitro-gen. Frozen tissue was then placed in RNAlater RNAStabilization Reagent (Qiagen, Valencia, California)and stored at �80�C. Frozen ventricle samples werehomogenized in protein lysate buffer before beingcentrifuged for 20 min at 2,000 rpm. The supernatantwas assayed for ARRB1, ADRB1, ADRB2, and cAMPcontent by enzyme-linked immunosorbent assay(ELISA) (MyBioSource Inc., San Diego, California) inaccordance with the manufacturer’s protocol. Real-time polymerase chain reaction (PCR) was used tomeasure levels of GRK2 (Qiagen OneStep real-timePCR and HotStarTaq Plus Master Mix kit). Harvestedcardiac tissue samples were analyzed for fibrosis in-tensity using a commercially available trichromestain (Masson) kit following the assay proceduredetailed by Sigma-Aldrich (St. Louis, Missouri).

Plasma samples were collected and used to deter-mine the levels of troponin T (TnT) by ELISA (LifeSciences Advanced Technologies, Inc., St. Petersburg,Florida) following the manufacturer’s protocol. Con-centrations of epinephrine and norepinephrine weredetermined with plasma samples by ELISA at all 4time points post-infarction. (MyBioSource Inc.).

MP ISOLATION AND LABELING. Platelet-poor plasmawas centrifuged at 20,000 � g (30 min) to pellet theMPs. The MP pellet was washed by resuspension inHank’s balanced salt solution (Sigma-Aldrich). Thewashed pellet was incubated in 200 ml of bindingbuffer with saturating concentrations (20 ml) of

fluorescein isothiocyanate–conjugated annexin Vantibody (Clontech, Mountain View, California) andphycoerythrin-conjugated TF antibody (Abcam, Cam-bridge, Massachusetts) for 1 h on ice in the dark. Afterlabeling, the MPs were washed by resuspension withHank’s balanced salt solution to remove excess un-bound antibody by pelleting at 20,000 � g for 30 minand resuspending in 500 ml of Hank’s solution beforefluorescence-activated cell sorting (FACS) analysis.

FLOW CYTOMETRY SORTING OF MPs. Analysis ofTFMPs was performed with 2-color flow cytometry ona BD FACS Aria II equipped with the FACS DIVAsoftware, version 6.1.2 (BD Biosciences, San Jose,California). The MPs were analyzed with logarithmicmeasurements for forward scatter and side scatter.TFMPs were sorted based on double positivity for TFantigen and annexin V, which identifies TFMPs. Thesorted TFMPs were centrifuged at 100,000 � g torecover all MPs in the sample for proteomic analysis.

MASS SPECTROMETRY ANALYSIS. The samples wereacetone precipitated, rehydrated in trypsin digestbuffer (50 mmol/l ammonium bicarbonate), reducedin dithiothreitol 10 mmol/l at 56�C for 30 min, andalkylated with iodoacetic acid (15 mmol/l) for 30 minat room temperature in the dark. Samples weretrypsin digested 20 (ng/ml) for 4 h at 37�C, and beforeanalysis, they were acidified by formic acid to a con-centration of 0.1%. Spectra were collected withXcalibur 2.2 software (Thermo Fisher Scientific,Waltham, Massachusetts) using a threshold of 200counts/hits. Spectra were then searched with Prote-ome Discoverer 3.0 software (Thermo Fisher Scienti-fic). Each sample produced 1 or 2 significant hits asdetermined by false discovery rates of 1.0% and 5.0%using a corresponding reverse database.

Porcine peptide sequences detected were exportedfrom ProteoIQ software version 2.3.08 (NuSep, Inc.,Bogart, Georgia) into protein BLAST, Blastp (NationalCenter for Biotechnology Information) and werechecked against the human protein database todetermine porcine/human protein homologs. Onlyhuman proteins with >85% homology were used forthe analysis. This conversion was necessary becausePathway Studio (Elsevier, Atlanta, Georgia) only usesproteins from humans, mice, and rats in its analysis.The resultant homologous human protein Entrez IDnumbers were uploaded into Pathway Studio 10 forpathway analysis.

PATHWAY AND FUNCTIONAL ENRICHMENT ANALYSIS.

Pathway Studio version 10 (PS10) with Disease Fx andChem Fx cartridges was used to analyze the proteo-mic data between the different treatment groups andan analysis of cell processes, disease processes, and


533

functional classes determined for the identified pro-teins. Functional enrichment analysis was conductedwith FunRich (26) version 2.1.2 (27).

STATISTICAL ANALYSIS. SigmaPlot version 12 wasused for the statistical analysis of MP counts, ELISA,and PCR data. Where nonparametric methods ofanalysis were applied, data are summarized usingmedian (1st quartile, 3rd quartile); otherwise data areexpressed as mean � SD. Comparisons were madeusing nonparametric tests, Kruskal-Wallis 1-wayanalysis of variance and Mann-Whitney rank sumtest, as appropriate. Dunn’s method was applied tothe probability values whenever multiple compari-sons arose. To compare TFMP count changes acrosstime within treatments groups, the Wilcoxonmatched-pairs signed ranks method was applied. The1-way repeated-measures analysis of variancemethod was applied to the TnT data with multiplepairwise comparison involving the Holm-Sidakmethod applied. All tests were 2-sided, withp < 0.05 considered significant.

RESULTS

HEMODYNAMIC, CARDIAC DIMENSION, AND

FIBROSIS INTENSITY OBSERVATIONS. Assessmentsof end-diastolic volume (EDV), end-systolic volume(ESV), and ejection fraction (EF) were made at base-line, 1 week post-infarction, and at study termination.Overall, after the MI, there were increases in EDV andESV with decreasing EF in both infarcted groups ofanimals. There were no statistically significant dif-ferences between groups at baseline or at 1 week post-infarction. Baseline EDVs for the CRTþBB–treatedanimals and for infarcted, untreated animals aver-aged 49.25 � 20.47 ml and 53.10 � 15.67 ml, respec-tively (p ¼ 0.722). Median ESV values for the treatedversus untreated animals were 17.86 ml (15.35 ml,21.223 ml) and 20.47 ml (12.49 ml, 25.43 ml), respec-tively (p ¼ 0.931). Baseline EF for CRTþBB–treatedversus infarcted, untreated animals averaged 59.78 �5.32% and 61.85 � 2.52%, respectively (p ¼ 0.448).

Post-infarction EDV values for CRTþBB–treatedversus infarcted, untreated animals averaged73.61 � 14.60 ml and 85.86 � 16.67 ml, respectively(p ¼ 0.205). At the same time point, ESV values forCRTþBB–treated versus infarcted, untreated animalsaveraged 50.67 � 11.96 ml versus 56.93 � 9.88 ml,respectively (p ¼ 0.375). The assessed EF for theCRTþBB–treated and infarcted, untreated groupsafter infarction averaged 27.22 � 5.18% and 24.15 �1.44%, respectively (p ¼ 0.238).

At termination of the study, there was a statisti-cally significant difference (p ¼ 0.046) in mean EDV

values between the 2 infarcted groups (CRTþBB 112 �35.35 ml vs. MI only 151.03 � 13.14 ml). At the sametime point, there was a noticeable trend of increasingESV in both groups; however, the increase in ESVvalues was comparatively more pronounced in theuntreated group than in the CRTþBB–treated animals.ESV values for the CRTþBB versus MI-only animals atthis time point were 70.78 � 28.03 ml versus 104.63 �18.95 ml (p ¼ 0.069). At study termination, EF valuesfor the CRTþBB–treated animals were relatively bet-ter than for the infarcted, untreated animals, with thedifference approaching statistical significance (p ¼0.0527) 3.5 months after treatment was initiated inthe treatment group. The values were 37.905 �6.036% versus 30.865 � 5.874% for the CRTþBB andMI-only groups, respectively.

Cardiac dimension measurements were conductedwith echocardiograms at baseline, 1 week post-infarction, and after necropsy. Predictably, after theinfarction, there was a noticeable increase in cardiacdimensions for all animals, as observed in left ven-tricular area around the mitral valve at diastole (LVAdMV) and systole (LVAs MV). Additionally, cardiacenlargement was observed in the area around thepapillary muscle at diastole (LVAd PM) and systole(LVAs PM). In all groups, there was an increase in thesecardiac measurements between the 1-week post-infarction assessment time point and study termina-tion. Comparison of the cardiac dimensions betweenthe 2 infarcted groups at study termination showed nostatistical differences with respect to LVAd MV, LVAsMV, and LVAs PM; however, there was a statisticallysignificant difference (p ¼ 0.009) in LVAd PM valuesbetween the CRTþBB–treated versus MI-only groups.

Extent of fibrosis in the harvested infarcted heartswas also determined on a scale of 1 to 4 (0 ¼ normal,1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, and 4 ¼ severe),normalized to samples from normal, uninfarctedporcine hearts. Seven cardiac regions were examined:right ventricle lateral wall; septum; border; infarct;normal; left atrium; and right atrium. Briefly, the re-sults revealed grade 3 to 4 fibrosis in the septum,border, and infarct regions of the hearts of bothinfarcted animal groups. In the right lateral wall, leftatrium, and right atrial regions of the harvested car-diac tissues, normal to mild fibrosis was observed.TFMP COUNTS. At 2.5 months post-infarction, therewas no significant difference in TFMP counts betweenthe experimental groups (Figure 2). The averageTFMP counts per 10,000 events at 2.5 months were1,806 � 423 for the control group, 1,281 � 909 for theinfarcted, untreated pigs, and 1,233 � 752 for theCRTþBB–treated group. Over the remaining timepoints, the counts of the infarcted, untreated animals

FIGURE 2 Flow Cytometric Enumeration of TFMP Counts per 10,000 Events

Tissue factor microparticles (TFMPs) increased in the infarcted animals over time, but

there were no statistically significant differences in the numbers of TFMPs.

BB ¼ b-blocker; CRT ¼ cardiac resynchronization therapy; MI ¼ myocardial infarction;

MP ¼ microparticles; TF ¼ tissue factor.



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continued to increase to 3,156 � 1,360, whereas thecounts of the treated animals tracked between 1,233 �752 and 2,173 � 1,405. Statistically, there were nosignificant differences in the numbers of MPs withinor between the groups at any time points.

TFMP PROTEOMIC PROFILE. Mass spectrometry re-sults showed that the proteomic content of TFMPschanged over time within and between groups. Forthe CRTþBB–treated animals, there were 177, 125, and137 proteins detected in the TFMPs collected at the3.5-, 4.5-, and 6-month time points, respectively. Inthe infarcted, untreated group, we observed 196, 207,and 65 proteins at the 3.5-, 4.5-, and 6-month timepoints, respectively. The control group animals had202 different proteins detected at the single timepoint assessed. The differences and similarities of theproteomic profiles were evaluated for functionalenrichment using the FunRich tool and the PathwayStudio software as further described in “MolecularFunction Analyses of TFMP proteins.”

On the basis of the sites of expression of theidentified proteins, TFMPs were determined to befrom heart muscle, plasma, blood vessels, and humanumbilical vein endothelial cells (HUVECs), as shownin Table 1. The observed high number of proteinsmapped to HUVECs is a consequence of the abun-dance of model systems involving HUVECs for theidentification of endothelial cell function in pub-lished literature (28–30). FunRich and other molecu-lar analysis tools rely on published literature for thegeneration of their output. This observation, howev-er, points to the relevance of these molecular entitiesin the post-MI state.

MOLECULAR FUNCTION ANALYSES OF TFMP

PROTEINS. To further characterize the identifiedTFMP proteins and to explore for temporal differ-ences, the standalone open-access tool FunRich andPathway Studios version 10 software were jointly usedto determine their molecular functions. Comparedwith the other 2 groups, the CRTþBB–treated group atthe 3.5-month time point had a 6-fold increase in thenumber of proteins associated with ATPase activityand a lower percentage of proteins functioning asextracellular matrix structural constituent (Figure 3).The control group had 2.5 times and 4 times as manyproteins related to ubiquitin-specific protease activityas did the infarcted, untreated animals and theCRTþBB–treated animals, respectively. With regardto G-protein–coupled receptor activity at the 3.5-month time point, the infarcted, untreated grouphad the most activity, with nearly a 10- and 5-foldincrease in the number of proteins compared withthe control and CRTþBB–treated animals,respectively.

At the 4.5-month time point, the TFMP proteinsdiffered in 3 main functional classes between theexperimental groups: structural constituents of thecytoskeleton, ATPase activity, and intracellularligand-gated ion channel activity (Figure 4). Therewas a considerable difference in the structural con-stituents of the cytoskeleton between the CRTþBB–treated animals and the control animals, in the ratioof z1:35 (Figure 4). The infarcted, untreated animalshad no detectible proteins functioning as structuralconstituents of the cytoskeleton. With regard toATPase activity, the CRTþBB group recorded thehighest percentage, with nearly a 2-fold increasecompared to the control group and z1.5 times thenumber observed in the infarcted, untreated group.The CRTþBB group had no detectable proteins func-tioning in intracellular ligand-gated ion channel ac-tivity, but there was a 4-fold increase in the infarcted,untreated group compared with the control group.

By the 6-month time point, CRTþBB treatment wasassociated with anz4-fold increase in ATPase proteincontent compared with controls, whereas theinfarcted, untreated animals had 3 times as manyATPase proteins as the control animals. There were noproteins associatedwith extracellular ligand-gated ionchannel activity detected in the CRTþBB group,although in the infarcted, untreated animals, therewas a 3-fold increase compared with the control group.The infarcted animals had higher levels of extracel-lular matrix structural constituents than the controlgroup animals (Figure 5). Additionally, the infarcted,untreated animals had a higher percentage of proteinsassociated with ubiquitin protease activity than the

TABLE 1 FunRich Analysis of Site of Expression of TFMP Protein

Treatment andSite of Expression

No. of Genesin Dataset

Uncorrectedp Value

(Hypergeometric Test)

Storey andTibshirani Method

q Value

Control

Heart muscle 72 0.15 0.07

Plasma 156 0.00 0.00

Blood vessels 8 0.00 0.00

HUVECs 163 0.00 0.00

Untreated 3.5 months


Plasma 138 0.00 0.00


HUVECs 143 0.01 0.00

Untreated 4.5 months


Plasma 158 0.00 0.00


HUVECs 172 0.00 0.00

Untreated 6 months


Plasma 52 0.00 0.02


HUVECs 52 0.03 0.09

CRTþBB 3.5 months


Plasma 142 0.00 0.00


HUVECs 141 0.00 0.00

CRTþBB 4.5 months


Plasma 106 0.00 0.00


HUVECs 98 0.00 0.00

CRTþBB 6 months


Plasma 112 0.00 0.00


HUVECs 109 0.00 0.00

BB ¼ b-blocker; CRT ¼ cardiac resynchronization therapy; HUVECs ¼ human umbilical veinendothelial cells; TFMP ¼ tissue factor–bearing microparticles.


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CRTþBB–treated group (1:8 ratio) and an z50% in-crease compared with the control group animals.TFMP PROTEIN CONTENT AND POST-MI DISEASE STATUS.

To further understand the association between diseasestatus and TFMP protein content, Pathway Studioanalysis was conducted. Evaluations were based onfunctional associations with known active cell pro-cesses post-MI, such as apoptosis, oxidative stress,and inflammation, with adjustments made for theadministration of the BBmetoprolol where applicable.

At the 3.5-month time point (SupplementalFigure 1), TFMP proteins in the treated group wereassociated with cardiac cell antiapoptosis and facili-tation of cardiomyocyte function. These TFMP pro-teins included sarcoplasmic/endoplasmic reticulumCa2þ-ATPase, SERCA proteins (ATP2A2), ephrin, andhexokinase 1 and 2 (HK I, HK II). Another cardiac cell–enhancing factor detected in the treated group butnot the infarcted, untreated group or control groupwas guanylate cyclase A. Conversely, at the 3.5-month time point, hypoxia-driven factors such ashypoxia-inducible factor-1a, hypoxia-induciblefactor-2a, and FLT-1 (Fms-related tyrosine kinase-1)were detected in the infarcted, untreated animals.

At the 4.5-month time point, repair and structuraltissue formation proteins were found in the CRTþBB–treated group (Supplemental Figure 2). These proteinswere keratin, desmin, actin, vimentin, myosin, and b-catenin. Compared with the treated pigs, there werefewer myosin subtypes observed in the infarcted, un-treated group. Additionally, proteins that enhancecardiac cell performance, such as sodium/potassium-transporting ATPase subunit alpha-1 and -2 (ATP1A1,ATP1A2), and proteins of ryanodine receptors 2 and 3(RYR2, RYR3) were observed in the untreated group.Furthermore, in both infarcted groups, factors that canpromote inflammation were detected; however, inaddition to thrombin (F2), TF, and putative serineproteases (PRSS), which were detected in the treatedgroup, nuclear factor-kappa-B1 (NF-KB1) and ADAMmetallopeptidase domain 17 (ADAM17) were detectedin the infarcted, untreated group.

By the 6-month time point, TFMPs in the treatedgroup contained cytoprotective heat shock proteins(HSPs) such as HSPA1A, HSPA1B, and HSPA5 (Figure 6).HSPwas not detected in any other group. In addition tothe cytoprotective proteins, pigs in the treated groupcontained proteins required for the maintenance ofcardiac function (ATP1A1 and ATP1A2). There were nounique proteins or functional patterns observed in theinfarcted, untreated animals at this time point.HIGH-SENSITIVITY CARDIAC TnT ASSESSMENT. Serumlevels of TnT were assessed between groups (Figure 7).Area under the curve analyses for the period from

2.5 to 6 months post-MI were conducted. ANOVA washighly statistically significant across all comparisons:MI versus control, p < 0.001; MI versus CRTþBB,p <0.002; and CRTþBB versus control, p < 0.002.

ADRB2, cAMP, AND GRK2 CONCENTRATIONS. Themean concentration of ADRB2 in myocardial tissue inthe control group animals was 0.681 � 0.056 ng/mlcompared to 0.205 � 0.086 ng/ml in the infarcted,untreated group and 0.22 � 0.223 ng/ml in theCRTþBB–treated group (Supplemental Figure 3).Statistically, there were differences in the averageconcentration between the infarcted but untreatedgroup and the control group (p ¼ 0.003) and betweenthe CRTþBB–treated group and the control group





FIGURE 3 Molecular Function Analysis of TFMP Protein Content at 3.5 Months Post-MI

25.0%

20.0%

15.0%

10.0%

5.0%

0.0%

22.0%21.0%

15.6%

1.1%

6.0%

1.1%

4.3%

1.8% 1.8%

10.5% Control

CRT+BB

MI Only

1.1% 1.1%

Gen

es (%

)

Molecular Function

Extracellular MatrixStructural Constituent

ATPase Activity Ubiquitin-specific ProteaseActivity

G-protein Coupled ReceptorActivity

Functional differences between groups at the 3.5-month time point using FunRich software are shown. Abbreviations as in Figure 2.



536

(p ¼ 0.003). There were no statistically significant dif-ferences between the infarcted, untreated group andthe treated group. Median cAMP tissue concentrationfor the treated group was 29.778 mg/ml compared with17.174 mg/ml in the infarcted group (SupplementalFigure 4), which was statistically significantlydifferent from the control group (CRTþBB vs. control:p ¼ 0.05; MI vs. control: p ¼ 0.05). GRK2 concentra-tions using quantitative PCR analysis (SupplementalFigure 5) from the myocardial tissue at study

FIGURE 4 Molecular Function Analysis of TFMP Protein Content at

20.0%

18.0%

16.0%

14.0%

12.0%

10.0%

8.0%

6.0%

4.0%

2.0%

0.0%

Gen

es (%

)

Molecular Fu

0.5%0.0%

1.1%

2.5%

17.8%

ATPase AcStructural Constituent ofCytoskeleton

Functional differences between groups at the 4.5-month time point usin

termination showed a statistically significant differ-ence (p ¼ 0.013) between the treated and untreatedanimals (0.471 � 0.349 RFU/18S [relative fluorescenceunits/18S ribosomal RNA] and 1.321 � 0.455 RFU/18S,respectively).ADRB1, ARRB1, EPINEPHRINE, AND NOREPINEPHRINE

CONCENTRATIONS POST-INFARCTION. ADRB1 concen-trations in cardiac tissue at 6 months after infarctionshowed levels as follows: treated group > controlgroup > infarcted, untreated group, with mean

4.5 Months Post-MI

nction

1.5%0.5%

0.0%

2.0%

tivity Intracellular Ligand-gated lon ChannelActivity

Control

CRT+BB

MI Only

g FunRich software are shown. Abbreviations as in Figure 2.





FIGURE 6 Pathway Studio Analysis of Proteomic Content of TFMPs at 6 Months in the Infarcted Groups Compared With

Control Group Profile

(A) Heat shock proteins (HSPs) associated with post-ischemic conditioning detected in CRTþBB–treated animals only. Metoprolol administration enhanced levels of

HSPA1A. (B) Factors related to cardiac function improvement were detected in treated animals 6 months after infarction, but no such observation made in the

infarcted, untreated animals.

FIGURE 5 Molecular Function Analysis of TFMP Protein Content at 6 Months Post-MI

30.0%

25.0%

20.0%

15.0%

10.0%

5.0%

0.0%

Gen

es (%

)

ATPase ActivityExtracellular MatrixStructural Constituent

Extracellular Ligand-GatedIon Channel Activity

Ubiquitin-Specific ProteaseActivity

Molecular Function

22.0%

27.6%25.8%

1.1%

4.7%3.2%

2.2%

0.0%

6.5%

4.3%

6.5%

0.8%

Control

CRT+BB

MI Only

Functional differences between groups at the 6-month time point using FunRich software are shown. Abbreviations as in Figure 2.


537

FIGURE 7 Serum Troponin T Levels Over the 6-Month Period Post-Infarction

Showing Chronic Myocardial Cell Death in Infarcted Animals Over the

6-Month Study Duration

2.01.81.61.41.21.00.80.60.40.20.0

CRT+ BB

2.5 mth

Con

cent

ratio

n of

Car

diac

Tro

poni

n T

(ng/

mL)

Avg 3.5mth Avg 4.5mth Avg 6 mth Avg

Untreated

Control

The differences in mean values between the infarcted groups became statistically

significant at the 6-month time point (p ¼ 0.008). Avg ¼ average; mth ¼ month; other

abbreviations as in Figure 2.



538

concentrations of 0.133 ng/ml, 0.027 ng/ml, andbelow limits of detection, respectively. There were nosignificant differences in the mean concentrationsof ARRB1, epinephrine, and norepinephrine betweenthe experimental groups in our study; however, therewas the expected elevation in norepinephrine andepinephrine concentrations in the infarcted animalscompared with the control group at all time points.

DISCUSSION

In heart failure patients with severe systolicdysfunction and asynchronous contraction, CRT isan established, effective adjunctive therapy whenpaired with BB. To adequately emulate the humancondition, we used an animal model with decreasedEF and asynchrony. The CRTþBB regimen improvesleft ventricular function and prognosis, whereasmetoprolol reduces infarct size (31,32). What is notfully characterized is the long-term signaling impactof b1-adrenergic blockade on the infarcted heart.

Reports indicate that many of the cellular re-sponses after an MI, such as fibrous tissue deposition,occur within the first few days to weeks of the post-infarction period (33–35). The degree of fibrosis(grade 3 or 4) observed in the septal area, border, andinfarct regions of the hearts of both groups ofinfarcted pigs was in line with the expectation ofstructural changes (fibrous tissue deposition) re-ported previously (36). Additionally, the morpholog-ical changes in cardiac dimensions showed that thisstudy also depicted the expected morphologicalalterations observed in clinical settings (37) and

confirmed that the experimental procedure success-fully induced the MI and mimicked post-MI observa-tions. Furthermore, the hematologic observationsdepicted the functional consequences of infarction,as well as treatment, with respect to time. Collec-tively, the structural, morphological, and functionalterations observed are evidence of pathologicalremodeling in response to infarction. Clinically, theobservation of improved EF and the differences inEDV and ESV between the infarcted animals point to atrend toward better outcomes in our treatment groupanimals than in the infarcted, untreated animals. Thisobservation is in line with general clinical observa-tions of better prognosis with treatment with CRTþBBand also aligns with the observed decline in TnTlevels with treatment in our study. Results from thisstudy therefore confirmed the documented beneficialeffects of b1 blockade in improving outcomes (31,32).More importantly, the proteomic profile of the TFMPsprovided insights into the long-term impact ofmetoprolol usage on active signaling entities of thesurviving cardiomyocytes. First, the inability todifferentiate experimental groups in the long-termpost-MI stage using TFMP numbers confirmed ourview that the more relevant information is the pro-teomic profile of the TFMPs. Second, our findingsdemonstrate unique signatures for each infarcted pigcohort associated with either adaptive or healingchanges, as well as progressive pathological remod-eling or maladaptive signaling leading to worseningheart failure. Additionally, the proteomic profile ofthe TFMPs provided a matrix view of the spatial andtemporal changes and differences in signaling en-tities between the study groups. We infer from ourTFMP proteomic data and Pathway Studio analysesthat the improved prognosis of the CRTþBB–treatedanimals proceeded in a stagewise manner via thefollowing general modalities: 1) cardiomyocyte sur-vival and function enhancement; 2) structural repair;and 3) cytoprotection and post-ischemic conditioning(Figures 8 and 9).

A month after treatment initiation, cardiomyocytesurvival and function enhancing factors were detec-ted as depicted by Supplemental Figure 1A. Collec-tively, the listed proteins confer protective propertiesto the myocardium by promoting chemomechanicaland antiapoptotic properties (38) while attenuatingchronic cardiac remodeling (39–41). The detection ofSERCA proteins in the CRTþBB–treated group isimportant, because metoprolol has been reported torestore cellular levels of SERCA2a (42,43). The detec-tion of cardiomyocyte survival factors perhaps ex-plains the trend toward the decrease in levels of TnTin the treated group compared with the infarcted,


FIGURE 8 Summary Observations Based on Proteomic Profile of TFMPs From CRTþBB–Treated Animals From Treatment

Initiation Until End of Study

Abbreviations as in Figure 2.


539

untreated animals. In the absence of treatment, thedetection of hypoxia-driven factors (SupplementalFigure 1B) predicted worse outcomes for theinfarcted, untreated animals, as confirmed by the re-sults of the currently used biomarkers. At stage2 (2 months after treatment initiation), a wide range ofstructural or repair proteins were present in theTFMPs of the treated group (Supplemental Figure 2).

FIGURE 9 Summary Observations Based on Proteomic Profile of TFM

Abbreviations as in Figure 2.

This was in contrast to the observed narrower array ofstructural proteins detected in the infarcted, un-treated group. Additionally, the differences in thestructural proteins and the late detection of chemo-mechanical proteins could be evidence of delayed andperhaps ineffective cellular signaling in the absenceof treatment. The observed wide range of structuralproteins in the treated animals could be indicative of

Ps From Untreated Animals From 3.5 Months Until End of Study






540

ongoing or incipient repair and healing processestaking effect in the treated group that contributed tothe reported resolution of cardiac cell damage. Sixmonths after infarction, there was an indication ofpost-ischemic conditioning and cytoprotection, asevidenced by the presence of HSP proteins in thetreated group (Figure 6). HSP was not detected in anyother group, and its detection in the treated group is aconsequence of metoprolol administration (44). Thisis relevant because HSP overexpression attenuatesmyocardial apoptosis (45,46), and it confirms theactivation of cytoprotective processes months afterCRTþBB treatment initiation.

The results from the biomarkers used confirm animproved prognosis for the treated animals and sup-port the investigational and interventional opportu-nity offered by TFMP proteomic profiling post-MI.This was in line with a decline in TnT levels in thetreated animals, whereas chronic troponin leakresulting from the death of cardiomyocytes persistedin the infarcted, untreated animals. By having lesscell death characterized as a smaller troponin leak,these data suggest that treatment with CRTþBBgradually attenuated cell death in the treated group.The observed increase in ADRB1 levels in the treatedgroup and its decrease in the infarcted animals trackswith reports of increasing b-AR expression after theadministration of metoprolol (47,48) and providesfurther evidence of the long-term cellular effects ofmetoprolol administration. This observation mightsuggest that chronic b1-blockade is associated withrecirculation of ADRB1 receptors to the car-diomyocyte surface as the levels of ischemic stressorsdecline and the surviving cardiomyocyte recovers,possibly via a negative feedback loop. The lowerlevels of ADRB2 in both infarcted groups are inagreement with published data proposing that mostprotective adrenergic signaling is mediated via b2-receptors and Gi signaling. This explains the observeddecreased b-AR expression along with loweredsensitivity in the aftermath of an MI (49). Further-more, impaired intracellular Ca2þ handling in thefailing heart has been ascribed to either a decreasedexpression of SERCA2a or a shift in the interactionbetween phospholamban and SR Ca2þ-ATPase activity(50,51). Additionally, in the failing heart, there is acorresponding increase in SR Ca2þ ATPase activitywith increasing cAMP concentrations (51). Thisperhaps accounts for the observed cAMP increase inour infarcted animals compared with the control an-imals. Because metoprolol treatment restores cAMP-dependent inotropic effects independent of b-AR(52), this might explain the observed higher levels ofcAMP in the treated animals than in the other groups.

The elevated epinephrine and norepinephrine levelsin our infarcted group animals compared with thecontrol animals are in line with expectations in theacute phase of an MI but highlight the ensuingcellular derangement chronically post-MI. Finally,the observed elevation of GRK2 levels underscoresthe heart’s decreased contractile function by theblunting of procontractile signaling of the b-ARs (53).

The composite of these biomarker evaluations andthe proteomic profiling of the TFMPs showed thattreatment with CRTþBB resulted in improvement inoutcome. Using pathway and functional analyses, theproteomic profile of TFMPs coupled the detection ofdiverse signaling mediators with temporal occurrenceto disease status and allowed the prediction ofoutcome in the post-MI setting. Most importantly, theobserved TFMP proteomic profile provided informa-tion of additive value to what was obtained by anal-ysis of 8 current biomarkers in our chronic ischemiccardiomyopathy model.

This study focused on chronic ischemic cardiomy-opathy in a porcine model of ischemic cardiomyopa-thy and did not examine the utility of the TPMPproteomic profile in the acute phase. Additionally, inour study, MI was induced by use of a collagen plugand therefore might have missed prelude molecularand cellular information that preceded the incidenceof an MI. This, however, does not minimize thedemonstration of the utility of TFMP protein contentas a cellular methodology to be used to further ourunderstanding of signaling after an MI. These resultsmay not be completely generalizable, but the wealthof information obtained from this study suggests thata follow-on translational study examining the utilityof profiling TFMPs in the acute phase is warranted asa next step. Furthermore, given the amount of in-formation obtained from the proteomic profile ofTFMPs, it would be interesting to determine whetherprofiling of the entire MP population would provideadditional information. Another necessary next stepin this line of inquiry must involve quantitativemethods to explore levels of candidate proteins.Furthermore, it would be important to establishrelevant flow cytometry thresholds of TFMP countsthat would yield levels of putative proteins thatwould allow for assessment in clinical settings.

CONCLUSIONS

The composite of the findings of this study is inagreement with previous studies and clinical obser-vations that treatment with CRTþBB after an MIleads to better outcomes. More importantly, ourfindings, while confirming the heterogeneity of TFMP

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: Current models

of post-MI signaling involve small animals with monitoring for a

few hours to days. Usually, only a few proteins are assessed,

which does not adequately depict the pathophysiology in

humans. TFMP proteomic profiling after an MI enables observa-

tion of both spatial and temporal events in multiple signaling

mechanisms in a long-term mini-swine model. Thus, a dynamic

evaluation of active signaling pathways using TFMP proteomic

profiling would be a valuable source of molecular markers to

investigate bAR signaling post-MI.

TRANSLATIONAL OUTLOOK: A chronic animal model,

6 months post-MI, might more closely emulate human disease

and provide greater clinical translation to the human condition of

the long-term consequences of an MI. In addition to spatial

signaling (membrane to the nucleus), this study assessed chronic

changes in signaling longitudinally, rather than at a single time

point, using the proteomic profile of TFMPs. Thus, we demon-

strated that time and intracellular or spatial signaling impact the

observed outward phenotype of pathological remodeling after

an MI within and between groups.


541

protein content, also demonstrate that proteomicprofiling of TFMPs from both the infarcted and non-infarcted pigs captured the diversity of the proteincontents between and within groups temporally.Changes in the identified proteins within andbetween groups corresponded to relevant changes inmolecular function and reflected the physiologicalstatus of the host. Spatially, the contents of theTFMPs displayed a variety of proteins and providedadditional information on multiple entities supple-mental to what we obtained from assessing 8 of thecurrent cardiac biomarkers. Additionally, for bothinfarcted groups, there was a noticeable time differ-ence in the detected proteins that predictablyreflected active, ongoing cellular activities. There-fore, results of this study support recommendingTFMP protein content profiling prospectively as aviable investigative methodology for chronicischemic cardiomyopathy that could help improveour understanding of bAR signaling after an MI. Theability to dynamically capture active signaling path-ways instead of tracking the presence or absence of asingle molecular entity, as typified by the currentgroup of biomarkers, would provide pertinentanswers to questions about bAR signaling pathwayspost-MI and in heart failure.

ACKNOWLEDGMENTS The authors would like toexpress their gratitude to Jane Chu, Mahfuz Khan,Irena Brandt, and Kayla Jackson for their contribu-tions in sample and data generation for this study.

ADDRESS FOR CORRESPONDENCE: Dr. David S.Feldman, Division of Cardiology, University of CincinnatiMedical Center, 234 Goodman Street, Cincinnati,Ohio 45221. E-mail: [email protected].

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KEY WORDS bAR signaling, chronicischemic cardiomyopathy, matrix signaling,myocardial infarction, tissue factor-bearingmicroparticles, Yucatan mini swine

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