electrophysiologic remodeling of the left ventricle in ... · sulaiman surie, md,‡ antoni c. g...

Journal of the American College of Cardiology Vol. 59, No. 24, 2012© 2012 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00

Electrophysiologic Remodeling of the Left Ventriclein Pressure Overload-Induced Right Ventricular FailureMaxim Hardziyenka, MD, PHD,*† Maria E. Campian, MD,* Arie O. Verkerk, PHD,*Sulaiman Surie, MD,‡ Antoni C. G. van Ginneken, PHD,* Sara Hakim, BSC,§André C. Linnenbank, PHD,* H.A.C.M. Rianne de Bruin-Bon, BSC,� Leander Beekman, BSC,*Mart N. van der Plas, MSC,‡ Carol A. Remme, MD, PHD,* Toon A. B. van Veen, PHD,§Paul Bresser, MD, PHD,*‡ Jacques M. T. de Bakker, PHD,*†§ Hanno L. Tan, MD, PHD*�

Amsterdam and Utrecht, the Netherlands

Objectives The purpose of this study was to analyze the electrophysiologic remodeling of the atrophic left ventricle (LV) inright ventricular (RV) failure (RVF) after RV pressure overload.

Background The LV in pressure-induced RVF develops dysfunction, reduction in mass, and altered gene expression, due toatrophic remodeling. LV atrophy is associated with electrophysiologic remodeling.

Methods We conducted epicardial mapping in Langendorff-perfused hearts, patch-clamp studies, gene expression studies,and protein level studies of the LV in rats with pressure-induced RVF (monocrotaline [MCT] injection, n � 25;controls with saline injection, n � 18). We also performed epicardial mapping of the LV in patients with RVF af-ter chronic thromboembolic pulmonary hypertension (CTEPH) (RVF, n � 10; no RVF, n � 16).

Results The LV of rats with MCT-induced RVF exhibited electrophysiologic remodeling: longer action potentials (APs) at90% repolarization and effective refractory periods (ERPs) (60 � 1 ms vs. 44 � 1 ms; p � 0.001), and slowerlongitudinal conduction velocity (62 � 2 cm/s vs. 70 � 1 cm/s; p � 0.003). AP/ERP prolongation agreed withreduced Kcnip2 expression, which encodes the repolarizing potassium channel subunit KChIP2 (0.07 � 0.01 vs.0.11 � 0.02; p � 0.05). Conduction slowing was not explained by impaired impulse formation, as AP maximumupstroke velocity, whole-cell sodium current magnitude/properties, and mRNA levels of Scn5a were unaltered. In-stead, impulse transmission in RVF was hampered by reduction in cell length (111.6 � 0.7 �m vs. 122.0 � 0.4 �m;p � 0.02) and width (21.9 � 0.2 �m vs. 25.3 � 0.3 �m; p � 0.002), and impaired cell-to-cell impulse transmission(24% reduction in Connexin-43 levels). The LV of patients with CTEPH with RVF also exhibited ERP prolongation(306 � 8 ms vs. 268 � 5 ms; p � 0.001) and conduction slowing (53 � 3 cm/s vs. 64 � 3 cm/s; p � 0.005).

Conclusions Pressure-induced RVF is associated with electrophysiologic remodeling of the atrophic LV. (J Am Coll Cardiol2012;59:2193–202) © 2012 by the American College of Cardiology Foundation

Published by Elsevier Inc. doi:10.1016/j.jacc.2012.01.063

Right ventricular (RV) failure (RVF) secondary to chronicpressure overload is an important determinant of survival inpatients with chronic thromboembolic pulmonary hyperten-sion (CTEPH) and other forms of pulmonary arterialhypertension (PAH) (1,2). These patients develop not only

From the *Heart Failure Research Center, Academic Medical Center, University ofAmsterdam, Amsterdam, the Netherlands; †Interuniversity Cardiology Institute ofthe Netherlands, Utrecht, the Netherlands; ‡Department of Pulmonology, AcademicMedical Center, University of Amsterdam, Amsterdam, the Netherlands; §DivisionHeart and Lungs, Department of Medical Physiology, University Medical CenterUtrecht, Utrecht, the Netherlands; and the �Department of Cardiology, AcademicMedical Center, University of Amsterdam, Amsterdam, the Netherlands. Dr. Tanwas supported by the Royal Netherlands Academy of Arts and Sciences (KNAW) andthe Netherlands Organization for Scientific Research (NWO, grant ZonMW Vici918.86.616). All other authors have reported that they have no relationships relevantto the contents of this paper to disclose.

Manuscript received October 2, 2011; revised manuscript received January 4, 2012,accepted January 10, 2012.

RV dysfunction, but also left ventricular (LV) dysfunction(3,4). LV diastolic filling is diminished, because diastolicLV peak filling rate is related to RV ejection fraction (5).

See page 2203

We recently reported in CTEPH patients with RVF and inan experimental model of RVF (monocrotaline [MCT]-induced PAH in rats) that this is associated with a reductionin LV mass and an altered gene expression profile of the LV,which is most likely due to atrophic remodeling of the LV(6). Because unloading and/or atrophy of the LV is associ-ated with electrophysiologic remodeling (7), we hypothe-sized that electrophysiologic remodeling of the LV alsooccurs in PAH-induced RVF. We therefore studied electro-physiologic properties and gene expression profiles of the LV
in rats with MCT-induced RVF. Moreover, we investigated

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2194 Hardziyenka et al. JACC Vol. 59, No. 24, 2012LV Electrophysiology in RV Failure June 12, 2012:2193–202

whether CTEPH patients withRVF exhibited similar electro-physiologic changes in the LV.

Methods

Animal Studies

Investigations conformed withthe Guide for the Care and Use ofLaboratory Animals published bythe U.S. National Institutes ofHealth (NIH Publication No.85-23, revised 1996). Animalswere cared for in accordance withinstitutional guidelines for thecare and use of laboratory ani-mals. Twenty-five 7-week-oldmale Wistar rats received an in-traperitoneal injection of MCT(60 mg/kg) to induce PAH andRVF. Eighteen control animalswere injected intraperitoneallywith the same volume of saline (3ml/kg). As reported previously(8), MCT injection leads to sub-sequent disease stages, culminat-ing in RVF. To recognize when arat reached the RVF stage, weused clinical criteria: body weight

loss and �1 of the following: dyspnea, hypothermia, cya-nosis, and lethargy. Rats were sacrificed when they devel-oped RVF (RVF rats) or at the same time after salineinjection (control rats).Echocardiographic and electrocardiographic analysis.Echocardiographic and electrocardiographic (ECG) (limb leads)recordings were made just before sacrifice. Echocardiographicanalysis of the RV and LV was conducted as reported previously(8,9). LV volumes were estimated on the basis of the short-axisarea–length method (10). Cardiac output was calculated as LVstroke volume multiplied by heart rate. RR interval and QRS/QTduration were analyzed in ECG lead II. Because the diagnosis ofRVF was determined on the basis of clinical, rather than echo-cardiographic criteria, it was not necessary to perform echocardi-ography in all animals. Accordingly, we conducted echocardiog-raphy in 20 rats.Epicardial mapping. Hearts were harvested immediatelyfter sacrifice, and perfused according to the Langendorffechnique at constant pressure (70 cm water) with Tyrode’solution (37°C) containing: sodium chloride 130 mM, potas-ium chloride 5.6 mM, calcium chloride 2.2 mM, magnesiumhloride 0.55 mM, sodium bicarbonate 24.2 mM, and glucose1.1 mM (pH 7.4 with 95% oxygen and 5% carbon dioxide)11). Extracellular electrograms of the LV and RV wereecorded with a 208-point multielectrode array, mounted onmicromanipulator. Electrodes were silver wires of 0.1 mm

Abbreviationsand Acronyms

AP � action potential

BNP � brain-typenatriuretic peptide

CTEPH � chronicthromboembolic pulmonaryhypertension

Cx-43 � Connexin-43

ECG � electrocardiographic

ERP � effective refractoryperiod

INa � whole-cell sodiumcurrent

Ito � transient outwardpotassium current

LV � left ventricle

MCT � monocrotaline

PAH � pulmonary arterialhypertension

RT-PCR � real-timepolymerase chain reaction

RV � right ventricle

RVF � right ventricularfailure

TAPSE � tricuspid annulusplane systolic excursion

iameter and arranged in a 16 � 13 grid at 0.5-mm interelec-

rode distances. Recordings were made during stimulationrectangular pulses of 2-ms duration and twice diastolic thresh-ld) from the center of the grid at a basic cycle length of 200s. The effective refractory period (ERP) (the coupling inter-

al of the shortest premature stimulus that failed to achieveocal capture) was determined for each ventricle separately asollows. Every eighth stimulus was followed by 1 prematuretimulus. Starting at 180 ms, the coupling interval of theremature stimulus was reduced in steps of 10 ms untilonduction block. Then, starting from the coupling interval ofhe last conducted premature stimulus, the coupling interval ofhe premature stimulus was reduced in steps of 1 ms untilRP. We constructed activation maps from activation times

12), and calculated longitudinal and transversal conductionelocities (11) (Fig. 1). The susceptibility to tachyarrhythmiasas tested as reported previously (13).yocyte isolation and measurement of cell dimensions.

V and RV myocytes of 7 RVF rats and 4 control rats weresolated enzymatically (14), and the length and width of 50

Figure 1Method to Determine EffectiveRefractory Period and ConductionVelocity in Rats (Epicardial Mapping: Methods)

(A) Examples of determination of effective refractory period during programmedstimulation. (B) Examples of subepicardial activation maps during constant pacingfrom the center of the multielectrode array at basic cycle length of 200 ms; arrowsindicate direction of longitudinal conduction wavefront (L) and transversal conduc-tion wavefront (T). CTRL � control; LV � left ventricle; RV � right ventricle; RVF �

right ventricular failure; S1 � stimulus applied at basic cycle length of 200 ms;S � premature stimulus with a coupling interval at the refractory period.
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2195JACC Vol. 59, No. 24, 2012 Hardziyenka et al.June 12, 2012:2193–202 LV Electrophysiology in RV Failure

randomly selected viable rod-shaped myocytes were mea-sured (3-�m resolution).Cellular electrophysiology. Action potentials (APs) andwhole-cell sodium current (INa) were recorded using stan-

ard methods. Voltage control, data acquisition, and anal-sis were accomplished using custom-made software. INa

signals were low-pass filtered (cutoff 5 kHz) and digitized at20 kHz. APs were filtered and digitized at 10 and 40 kHz,respectively. Cell membrane capacitance (Cm) was estimateds described previously (15). Series resistance was compensatedo reduce resistance voltage errors and increase the temporalesolution by 80% in a whole-cell voltage clamp. APs wereecorded at 36°C � 0.2°C with the perforated patch-clamp

technique. INa was recorded at 20°C with the whole-cellruptured patch-clamp technique.Histology. LV and RV tissues were excised from thepositions where the multielectrode recordings were made.These tissues were obtained from the same positions in allanimals (i.e., the LV and RV free walls). These tissues werefixed in formalin (RVF rats, n � 4 and control rats, n � 4).Seven-micrometer-thick sections were cut parallel to theepicardium, stained with Picro-sirius Red, and examined bylight microscopy (10� magnification) for collagen. Theamount of collagen in the recording area was determinedusing the Image-Pro Plus software package (version 5.02,Media Cybernetics, Bethesda, Maryland) after digitizing 6randomly selected fields per section with a slide scanner.Western blots and immunohistochemistry. Total cellularprotein was isolated from the LV and RV free walls fromthe positions where the multielectrode recordings weremade (RVF rats, n � 5 and control rats, n � 5). StandardSDS-PAGE and Western blot analyses were conductedusing antibodies directed against the Connexin-43 protein(Cx-43, Transduction). To compare the expression levelsof Cx-43 between RVF and control hearts, the pooledsamples were separated on gel (25 �g total cellularprotein per sample) and blotted onto nitrocellulose. Theoptical densities of the obtained signals were quantified(ImageQuant software, Molecular Dynamics, Sunnyvale,California) and corrected for possible deviations in theamount of transferred total protein. For this, reversiblePonceau-S staining of the blots was performed, afterwhich signals were digitized by scanning at 400 dpi.Cryosections (10-�m thickness) were sliced perpendicu-lar to the epicardial surface from biopsies rapidly frozenin liquid nitrogen. Thus, each section encompassed theentire ventricular wall. Immunohistochemistry was per-formed as described previously (13).Quantitative real-time polymerase chain reaction. RNAwas isolated from LV samples of 4 RVF and 5 control ratsfrom the positions where the multielectrode recordings weremade. mRNA expression levels of the main subunits of thecardiac sodium channel (Scn5a), the L-type calcium channel(Cacna1c), and the various potassium channels (Kcnip2,Kcnd2, Kcnd3, Kcna4, Kcnq1, Kcne1) were quantified using
the LightCycler system for real-time polymerase chain
reaction (RT-PCR) (Roche Applied Science, Basel, Swit-zerland). Quantitative RT-PCR data were analyzed withthe LinRegPCR program (16). All samples were processedin triplicate and expression levels were normalized to glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH).

Patient Studies

We studied 26 CTEPH patients who were included in aprevious study from our group (17). For the present study, wedivided them into 2 groups: patients with RVF (n � 10) andpatients without RVF (n � 16). RVF was defined as tricuspidannulus plane systolic excursion (TAPSE) �16 mm (18). Allsubjects gave written informed consent. Investigations wereapproved by the local institutional review board.Echocardiography. Echocardiographic data of the wholecohort were presented in a previous study (17). In this study,we compared the echocardiographic data between RVF andnon-RVF patients.Epicardial mapping. All patients underwent in situ epi-cardial mapping of the LV lateral wall during nativecirculation at pulmonary endarterectomy (body core tem-perature 36.0°C to 36.5°C) as described previously (17).Using a multielectrode grid (144 or 126 electrodes, 1 � 1-or 0.4 � 0.4-mm interelectrode distance, respectively), we deter-mined LV ERP by programmed electrical stimulation from thecenter of the grid, and calculated longitudinal and transversalconduction velocities from ellipsoid activation patterns.Statistical analysis. Statistical analysis was performed us-ing SPSS software (version 16.0.0, SPSS, Inc., Chicago,Illinois). All data are presented as mean � SEM. Groups werecompared using unpaired 2-tailed Student t tests, Mann-Whitney U nonparametric test, or chi-square test when appro-priate. A p value �0.05 was considered statistically significant.

Results

Animal Studies

Echocardiographic and autopsy findings. RVF occurredat 26 � 2 (range, 22 to 30) days after MCT injection.Table 1 shows echocardiographic and morphometric vari-ables of control and RVF rats. Compared with control rats,RVF rats had a significantly thicker RV wall, a dilated RVwith reduced contractility (TAPSE), and an abnormalechocardiographic pattern of RV diastolic filling (lowertricuspid E/A ratio). Diastolic filling of the LV was alsoaltered (reduced mitral E/A ratio and LV end-diastolicvolume). This was associated with significantly diminishedLV stroke volume and cardiac output. These echocardio-graphic data were in agreement with autopsy findings;compared with control rats, RVF rats had significantlyreduced body, liver, and LV weights, and increased lung andRV weights. Moreover, RVF rats had pleural effusion,whereas control rats did not.Electrocardiographic and epicardial mapping. RVF rats(n � 6) had significantly longer QRS and QT durations
than control rats (n � 4, 34 � 1 ms vs. 28 � 1 ms; p �

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0.004 and 120 � 10 ms vs. 64 � 4 ms; p � 0.003,espectively). RR interval was not significantly differentetween RVF and control rats (196 � 14 ms vs.154 � 16s; p � 0.1). LV ERP was significantly longer in RVF rats

than in control rats (Fig. 2A), whereas LV longitudinalconduction velocity was significantly reduced (Fig. 2B). Inthe RV, ERP was also longer in RVF rats than in controlrats (Fig. 2A). In contrast to the reduction in longitudinalconduction velocity in the LV of RVF rats, longitudinal andtransversal conduction velocities in the RV were higher inRVF rats than in control rats (Figs. 2B and 2C). Tachyar-rhythmias were noninducible during programmed electricalstimulation, neither in RVF rats, nor in control rats.Cellular electrophysiology. To establish the cellular basisof ERP prolongation and reduction in longitudinal conduc-tion velocity, we conducted electrophysiologic studies inisolated LV and RV myocytes. Figure 3A shows represen-tative APs from a LV and RV myocyte of a control rat anda RVF rat. LV and RV myocytes of RVF rats hadsignificantly longer APs at 90% repolarization at all pacingrates tested (Figs. 3B and 3C). In contrast, no significantdifferences in maximal upstroke velocity were observedbetween RVF and control rats. Similarly, no differences inresting membrane potential or AP amplitude were found(data not shown). In further support of the notion thatconduction slowing was not explained by changes in ioniccurrents, patch-clamp studies of INa revealed that mean INadensities and voltage dependence of INa steady-state activa-tion/inactivation in LV myocytes were not different between

Echocardiographic andMorphometric Variables of Rats at SacrificeTable 1 Echocardiographic andMorphometric Variables of Rats at Sacrifice

Control(n � 18)

RVF(n � 20) p Value

Echocardiographic variables

Right ventricle

Free-wall thickness, mm 0.62 � 0.01 1.11 � 0.03 �0.001

End-diastolic diameter, mm 4.1 � 0.1 6.8 � 0.2 �0.001

Tricuspid E/A ratio 0.7 � 0.1 0.2 � 0.1 �0.001

Tricuspid annulus plane systolicexcursion, mm

3.0 � 0.2 1.6 � 0.1 �0.001

Left ventricle

Mitral E/A ratio 1.22 � 0.0 0.85 � 0.1 0.001

End-diastolic volume, �l 379 � 6 324 � 7 �0.001

End-systolic volume, �l 130 � 4 134 � 4 0.48

Stroke volume, �l 249 � 5 190 � 6 �0.001

Ejection fraction, % 66 � 1 59 � 1 �0.001

Cardiac output, ml/min 93 � 2 54 � 2 �0.001

Morphometric variables

Body weight, g 350 � 16 284 � 12 �0.001

RV weight 0.23 � 0.00 0.41 � 0.01 �0.001

LV weight 1.13 � 0.27 0.95 � 0.29 �0.001

RV weight/LV weight ratio 0.2 � 0.0 0.4 � 0.0 �0.001

Lung weight, g 1.3 � 0.1 2.3 � 0.2 �0.001

Liver weight, g 15 � 1 11 � 1 �0.001

Values are mean � SD.LV � left ventricular; RV � right ventricular; RVF � right ventricular failure.

RVF and control rats (Table 2, Fig. 4). In RV myocytes,

V1/2 of activation was �9 mV more negative in RVF thanin control rats (p � 0.05), whereas voltage-dependentinactivation was not different (Table 2, Fig. 4).Morphometric and tissue properties, protein levels, andmRNA expression patterns. Given that changes in con-duction velocity may occur on the basis of altered celldimensions (reduced conduction velocity at smaller myocytesize [15]) and/or changes in cell-to-cell impulse transmis-sion, we next studied cell dimensions, protein levels ofCx-43, and tissue properties. LV myocytes were shorter and

Figure 2 Refractory Periods and Conduction Velocitiesin Rats (Epicardial Mapping: Results)

(A) Effective refractory period (ERP), (B) longitudinal conduction velocity (CV),and (C) transversal CV in right ventricle (RV) and left ventricle (LV) of right ven-tricular failure (RVF) and control (CTRL) rats.

narrower in RVF rats than in control rats (111.6 � 0.7 �m

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vs. 122.0 � 0.4 �m ; p � 0.02, and 21.9 � 0.2 �m vs. 25.3 � 0.3�m; p � 0.002, respectively) (Fig. 3D), although theirmembrane capacitances were similar (30 myocytes of RVFrats, 144 � 6 pF vs. 35 myocytes of control rats, 143 � 7 pF;p � 0.9). Compared with control rats, RV myocytes of RVFrats were wider (27.7 � 0.5 �m vs. 23.1 � 0.3 �m; p � 0.015)nd had larger membrane capacitance (166 � 9 pF [26yocytes] vs. 132 � 6 pF [46 myocytes], p � 0.002),hereas their lengths were not different (108.4 � 0.9 �m vs.

110.9 � 0.5 �m; p � 0.9) (Fig. 3D). Figure 5A showsouble labeling of Cx-43 and alpha-actinin. Cx-43 expres-

Figure 3 APDs in Rats

(A) Action potential durations (APDs) at 2.5-Hz pacing rate in isolated right ventric(CTRL) rats. (B) AP duration (APD) at 20%, 50%, and 90% repolarization (APD20, AIsolated RV and LV myocytes of RVF and CTRL rats.

Sodium Current PropertiesTable 2 Sodium Current Properties

RV LV

Control(n � 10)

RVF(n � 8)

Control(n � 8)

RVF(n � 8)

Current density,pA/pF

�86 � 11 �66 � 8 �76 � 11 �66 � 10

Inactivation

V1/2, mV �88.1 � 3.2 �95.5 � 2.7 �92.8 � 3.0 �89.6 � 4.0

k, mV �6.7 � 0.5 �6.9 � 0.5 �6.6 � 0.6 �7.0 � 0.4

Activation

V1/2, mV �52.0 � 2.5 �60.8 � 1.8* �58.0 � 3.0 �58.5 � 2.2

k, mV 4.0 � 0.3 4.0 � 0.5 4.6 � 0.5 4.0 � 0.5

Values are mean � SD. *p �0.05 versus control.

ek � slope factor of (in)activation; V1/2 � half-maximal voltage of (in)activation; other

abbreviations as in Table 1.

ion was homogeneous and aligned along the intercalatedisc, and, to a lesser extent, along the lateral myocyteorders. Cx-43 expression in the LV was less dense in RVFats than in control rats, but remained homogeneous. In theV, Cx-43 expression was denser in RVF rats than in control

ats. Quantification of Cx-43 protein expression by Westernlotting indicated a 24% decrease in the LV and a 30% increasen the RV of RVF rats (signals were corrected for differences inhe total amount of transferred protein) (Fig. 5B). The amountf interstitial collagen deposition was increased in the RV, butot the LV, of RVF rats compared with controls (Fig. 5C).Finally, we conducted quantitative PCR analysis in LVyocytes of the major ion channels that constitute the ratV AP. This analysis revealed that mRNA expression levelsf Kcnip2 (which encodes KChIP2, an auxiliary subunit ofhe transient outward potassium current, Ito) were signifi-antly reduced in RVF rats, in accordance with the observedP/ERP prolongation. In contrast, expression of otherotassium, sodium, and calcium channel subunits remainednchanged (Fig. 5D).

atient Studies

atient characteristics and echocardiographic analysis.atient characteristics are summarized in Table 3. All patientssed oral anticoagulants for at least 3 months, 7 used the dual

and left ventricle (LV) myocytes of right ventricular failure (RVF) and controlAPD90) at 2.5 Hz. (C) AP duration at 90% repolarization (APD90) at 1 to 8 Hz. (D)

le (RV)PD50,

ndothelin receptor antagonist bosentan, and none used anti-

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arrhythmic drugs (including calcium channel blockers andbeta-blockers). There were no significant differences in ageand/or gender between patients without RVF (n � 16) and

ith RVF (n � 10) (56 � 4 years vs. 61 � 3 years; p � 0.3;nd 7 women/9 men vs. 6 women/4 men; p � 0.7). No

patient had coronary artery disease at coronary angiography.Patients with RVF had, on average, longer PQ and QTcdurations, higher mean pulmonary arterial pressure, rightatrial pressure, and pulmonary capillary wedge pressure, aswell as larger total pulmonary resistance. In contrast, theyhad smaller RV stroke volume and cardiac output. Echo-cardiographic findings are summarized in Table 4. Patientswith RVF had larger RV end-diastolic diameter with lowerindexes of early diastolic relaxation (E=) and contractility(TAPSE). They also had significantly lower LV end-diastolic diameter and volume, along with lower early LVdiastolic relaxation velocity and LV ejection fraction, andmore prominent leftward shift of the interventricular sep-tum (LV early diastolic eccentricity index).Electrocardiographic analysis and epicardial mapping.Patients with RVF had significantly longer PQ and QTcduration than patients without RVF (Table 3). LV ERP inpatients with RVF was significantly longer than in patientswithout RVF (Figs. 6A and 6B), and longitudinal conduc-tion velocity was significantly reduced (Figs. 6C and 6D).

Discussion

The atrophic LV of rats with MCT-induced RVF exhibitedevidence of electrophysiologic remodeling (i.e., longer AP/ERPand slower conduction velocity). AP/ERP prolongation wasconsistent with reduced expression of Kcnip2. Conduction slowing

as not explained by impaired impulse formation, as AP maxi-um upstroke velocity, INa magnitude/properties, and mRNA

Figure 4 Sodium Channel Activation/InactivationProperties in Rats (Sodium Channel Properties)

Average activation/inactivation curves of sodium current. Abbreviations as in Figure 1.

evels of Scn5a were unaltered. Instead, impulse transmission was A

hampered, both by reduction in cell size and by impaired cell-to-cell impulse transmission (reduced Cx-43 levels). AP/ERP pro-longation and conduction slowing of the LV were also found inCTEPH patients with RVF.LV function in RVF due to chronic RV pressure overload.Clinical (3,5,19,20) and experimental (21) studies demon-strated that diastolic filling of the LV is impaired in chronicPAH. LV filling rate relates to RV ejection fraction (5).Accordingly, we found that LV early diastolic relaxationvelocity and LV end-diastolic volume were reduced inpatients with RVF. Similarly, LV diastolic filling wasimpaired in RVF rats. Aside from abnormal LV diastolicfilling, LV systolic function (LV stroke volume and ejectionfraction) was affected in CTEPH patients with RVF and inRVF rats. These findings agree with previous studies thatshowed diminished LV contractility in patients with chronicRV pressure overload (4,20). Moreover, in a previous study(6), we found that LV mass was reduced in CTEPHpatients with chronic RV failure, but that LV mass normal-ized after pulmonary endarterectomy. This finding suggeststhat reduced LV systolic function in the setting of RVfailure may be due to a secondary adaptation with abnormalLV underfilling and mechanical unloading.Possible mechanisms of LV electrophysiologic remodelingduring RVF. The electrophysiologic changes in the LVduring RVF that we observed both in patients and rats(AP/ERP prolongation and conduction slowing) were sim-ilar to changes occurring in the LV during LV failure(22,23). Moreover, a recent study showed that (sub)epicar-dial LV monophasic APs are prolonged in MCT rats withRV hypertrophy (24). Various neurohumoral and mechan-ical factors may affect LV electrophysiology in RVF afterchronic PAH (3,25–27). In the present study, Kcnip2mRNA expression levels were reduced. This was predictedto result in Ito reduction, consistent with previous reportshat linked hypertrophy and/or failure to reduced KChIP2xpression and Ito density (24,28–30). Although not mea-

sured in this study, elevated angiotensin II levels may causeboth Ito reduction (26,31) and reduction in Cx-43 expres-ion (32). Similarly, endothelin-1 may play a role, as itaused AP prolongation in an experimental study (33),hereas its plasma levels were augmented in CTEPHatients (25) and MCT-treated rats (34). Alternatively,nderfilling may impair LV diastolic function in patientsith CTEPH (3) and MCT-injected rats with RV hyper-

rophy and failure (21). Mechanical unloading reduces cellimensions (35,36), regardless of enhanced neurohumoraltimulation (35), and results in AP prolongation (7). In ourtudy, length and width of LV myocytes of RVF rats weremaller than those in control rats, and LV weight waseduced, similar to previous studies (27,37,38). Reduced cellimensions may in part explain the reduction in longitudi-al conduction velocity (11,39).ifferences in electrophysiologic remodeling between

he left and right ventricles. We found that changes in
P/ERP during RVF were qualitatively similar in the RV


and LV, whereas changes in conduction velocities weredivergent between the RV and LV. Moreover, AP/ERP

Figure 5 Expression and Distribution of Connexin-43 and MajorSarcolemmal Ion Channels in Rats (Connexin-43 and

Expression and distribution of Connexin-43 (Cx-43) and interstitial collagen depositionfailure (RVF) and control (CTRL) rats. (A) Immunolabeling of Cx-43 (green, upper pane(upper panel) and quantified Cx-43 expression in pooled samples (lower panel). (C) Ptent (lower panel). (D) Quantitative real-time polymerase chain reaction results indicaand potassium channel subunits (Kcnip2, Kcnd2, Kcnd3, Kcna4, Kcnq1, Kcne1). GAP

prolongation was more prominent in the RV than in the

LV. The resulting dispersion in electrophysiologic proper-ties between the RV and LV could have predisposed to

r Ion Channels)

RNA expression levels in left ventricle (LV) and right ventricle (RV) in right ventriculardouble labeling of Cx-43 (green) and �-actinin (red, lower panel). (B) Western blotrius Red staining of collagen (upper panel) and quantified interstitial collagen con-NA expression levels of sodium channel (Scn5a), L-type calcium channel (Cacna1c),

glyceraldehyde-3-phosphate dehydrogenase.

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arrhythmias by generating a substrate for reentry. However,

tion; ot


we failed to induce arrhythmias in RVF rats or control rats.This may be, in part, due to prolongation of the excitationwave (the mathematical product of conduction velocity andERP) (40) found in the present study (data not shown),along with the preservation of the homogeneous distribu-tion of Cx-43.Study limitations. First, LV myocardial biopsies were nottaken from CTEPH patients to assess cell and/or tissuemorphology and study cellular electrophysiology. Instead,in-depth analysis of electrophysiologic properties of the LV

Characteristics of Chronic Thromboembolic PulmTable 3 Characteristics of Chronic Thrombo

N

6-min walking distance, m 4

NYHA functional class, I/II/III/IV

Plasma BNP level, pmol/l

Systolic arterial pressure, mm Hg 1

Diastolic arterial pressure, mm Hg

Mean arterial pressure, mm Hg

Electrocardiography

PQ duration, ms 1

QRS duration, ms

QTc duration, ms 4

Right bundle branch block, n

Catheterization

Mean pulmonary arterial pressure, mm Hg

Total pulmonary resistance, dynes/s/cm�5 5

RV stroke volume, ml

Right atrial pressure, mm Hg 6

Cardiac output, l/min 5

Pulmonary capillary wedge pressure, mm Hg 8

Values are mean � SEM, n, or median (interquartile range).BNP � B-type natriuretic peptide; NYHA � New York Heart Associa

Echocardiographic Measurements in ChronicThromboembolic Pulmonary Hypertension PatienTable 4 Echocardiographic Measurements iThromboembolic Pulmonary Hyperte

Heart rate, beats/min

Right ventricle, diastole

RV early diastolic relaxation velocity (E=), cm/s

RV end-diastolic diameter, mm

Right ventricle, systole

Tricuspid regurgitation grade, 0/I/II/III

Pulmonary artery systolic pressure, mm Hg

Tricuspid annulus plane systolic excursion, mm

Left ventricle, diastole

LV early diastolic eccentricity index

LV early diastolic relaxation velocity (E=), cm/s

LV end-diastolic diameter, mm

LV end-diastolic volume, m

Left ventricle, systole

LV end-systolic volume, ml

Ejection fraction, %

LV stroke volume, ml

Values are mean � SD or n.Abbreviations as in Table 1.

was conducted in the MCT rat model of RVF. Second, weassessed refractoriness and conduction velocities in animalsduring Langendorff perfusion. Therefore, we cannot excludethat acute changes in LV loading conditions influenced theelectrophysiologic properties of the LV due to mechano-electrical feedback (41). However, we also found ERPprolongation and LV longitudinal conduction slowing inCTEPH patients with RVF whose native circulation wasmaintained during recording. Moreover, ERP prolongationand LV conduction slowing during Langendorff perfusion,

ry Hypertension Patientslic Pulmonary Hypertension Patients

(n � 16) RVF (n � 10) p Value

6 357 � 33 0.003

/8/1 0/1/5/4 0.052

98 � 20 0.001

(20) 114 (122)

116 � 5 0.04

73 � 2 0.26

87 � 3 0.10

184 � 10 0.015

107 � 6 0.14

457 � 9 �0.001

2 2 1.00

53 � 3 �0.001

6 1,272 � 135 �0.001

43 � 5 �0.001

.7 13.6 � 2.0 0.004

.3 3.5 � 0.2 �0.001

.3 (n�9) 13.7 � 2.2 (n�7) 0.042

her abbreviations as in Table 1.

onicn Patients

RVF (n � 16) RVF (n � 10) p Value

78 � 2 84 � 5 0.148

6.8 � 0.6 4.3 � 0.7 0.016

39 � 2 50 � 4 �0.001

1/9/6/0 0/0/3/7 0.001

49 � 5 89 � 6 �0.001

2.8 � 0.6 12.3 � 0.8 �0.001

.29 � 0.05 1.76 � 0.11 �0.001

7.9 � 0.6 5.9 � 0.5 0.03

40 � 2 30 � 2 �0.001

08 � 6 87 � 6 0.021

40 � 3 40 � 5 0.96

63 � 2 55 � 3 0.018

68 � 3 47 � 4 �0.001

onaembo

o RVF

98 � 2

0/7

11 � 4

5

31 � 4

77 � 2

95 � 3

56 � 5

95 � 5

18 � 5

34 � 3

36 � 7

73 � 4

.1 � 0

.7 � 0

.2 � 1

tsn Chrnsio

No

2

1

1


and AP prolongation in isolated myocytes support thenotion that the observed electrophysiologic remodeling ismainly explained by impact of chronic RV pressure over-load. This finding is in agreement with a previous study (41)in a normal canine heart, in which only acute LV volumeloading, but not an acute increase in LV afterload changedAP duration. Similarly, acute LV volume loading resulted inconduction slowing in isolated rabbit hearts (41). Anotherstudy limitation was that epicardial mapping in CTEPHpatients was performed under general anesthesia whenthe pericardium was open. Although a previous studyshowed that pericardium-mediated interventricular inter-action in patients with CTEPH is negligible because ofpericardial adaptation to slowly progressive pulmonaryhypertension (42), intrapericardial pressures may wellhave been increased in the present study, contributing toLV underfilling. If so, opening of the pericardium mighthave led to changes in interventricular interaction thataffected the electrical properties of the LV due to

Figure 6 Refractory Periods and Conduction Velocities in Patie

(A) Examples of determination of effective refractory period (ERP) during programmfailure (RVF) and without RVF. (C) Examples of subepicardial activation maps durin600 ms; arrows indicate direction of longitudinal conduction wavefront (L) and trawithout RVF. S1: stimulus applied at basic cycle length of 600 ms; S2: premature

mechanoelectrical feedback.

Conclusions

Electrophysiologic remodeling occurs in the atrophic LV ofCTEPH patients with RVF and in the MCT rat model ofPAH-induced RVF. This is associated with abnormal LVdiastolic filling.

AcknowledgmentsThe authors gratefully thank Dr. Antonius Baartscheer andCees A. Schumacher for their supervision during the cardiacmyocyte isolation.

Reprint requests and correspondence: Dr. Hanno L. Tan, Depart-ment of Cardiology, Academic Medical Center, Meibergdreef 9,1105 AZ Amsterdam, the Netherlands. E-mail: [email protected].

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Key Words: electrophysiologic remodeling y pulmonary hypertension yight ventricular failure.

APPENDIX

For an expanded Methods section,
please see the online version of this article.

electrophysiologic remodeling of the left ventricle in ... · sulaiman surie, md,‡ antoni c. g...

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