Regional protein expression is polarized in hearts with dyssynchronous failure due to left ventricular pacing. Specifically, the late-activated lateral left ventricular endocardium shows increased expression of phosphorylated ERK MAP kinase and reduced expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and connexin43 proteins. In contrast, hearts with equivalent systolic dysfunction but preserved synchronization due to right atrial pacing do not exhibit regional differences in protein expression. This suggests mechanical dyssynchrony generates molecular heterogeneity that may worsen chamber function and arrhythmia risk.

1. Deborah DiSilvestre, Elliot R. McVeigh, Gordon F. Tomaselli and David A. Kass
David D. Spragg, Christophe Leclercq, Morteza Loghmani, Owen P. Faris, Richard S. Tunin,
Regional Alterations in Protein Expression in the Dyssynchronous Failing Heart
Print ISSN: 0009-7322. Online ISSN: 1524-4539
Copyright © 2003 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation
doi: 10.1161/01.CIR.0000088782.99568.CA
2003;108:929-932; originally published online August 18, 2003;Circulation.
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2. Regional Alterations in Protein Expression in the
Dyssynchronous Failing Heart
David D. Spragg, MD*; Christophe Leclercq, MD*; Morteza Loghmani, BS; Owen P. Faris, BS;
Richard S. Tunin, MS; Deborah DiSilvestre, MS; Elliot R. McVeigh, PhD;
Gordon F. Tomaselli, MD; David A. Kass, MD
Background—Left ventricular (LV) mechanical dyssynchrony induces regional heterogeneity of mechanical load and is an
independent predictor of mortality and sudden death in heart failure (HF) patients. We tested whether dyssynchrony also
induces localized disparities in the expression of proteins involved with mechanical stress, function, and arrhythmia
susceptibility.
Methods and Results—Eleven dogs underwent tachycardia-induced HF pacing, either from the right atrium or high right
ventricular free wall. Whereas global LV dysfunction was similar between groups, LV contractile coordination assessed
by tagged MRI was markedly dyssynchronous with right ventricular pacing but synchronous with right atrial pacing. In
dyssynchronous failing hearts, the lateral LV endocardium displayed a 2-fold increase in phosphorylated erk
mitogen-activated protein kinase expression (with no change in phospho-p38 or phospho-jnk), a 30% decline in
sarcoplasmic reticulum Ca2ϩ
-ATPase, an 80% reduction in phospholamban, and a 60% reduction in the gap junction
protein connexin43, relative to neighboring myocardial segments. In contrast, hearts from both right atrial–paced HF
dogs and an additional 4 noninstrumented control animals showed minimal regional variability in protein expression.
Conclusions—LV dyssynchrony in failing hearts generates myocardial protein dysregulation concentrated in the
late-activated, high-stress lateral endocardium. Such molecular polarization within the LV creates transmural and
transchamber expression gradients of calcium handling and gap junction proteins that may worsen chamber function and
arrhythmia susceptibility. (Circulation. 2003;108:929-932.)
Key Words: heart failure Ⅲ pacing Ⅲ molecular biology Ⅲ sarcoplasmic reticulum
The pathophysiology of heart failure (HF) involves abnor-
malities of stress response kinase signaling, neurohu-
moral stimulation, excitation-contraction coupling, gap junc-
tion and ion channel function, and chamber remodeling.1,2 In
addition, recent studies have shown that left ventricular (LV)
mechanical coordination is frequently disturbed in HF, par-
ticularly in the setting of interventricular conduction delay.
HF patients with interventricular conduction delay and con-
comitant ventricular dyssynchrony have mortality and sudden
cardiac death rates above those predicted from the apparent
severity of systolic dysfunction, HF etiology, functional class,
or concurrent medical therapy.3,4
Acute mechanical discoordination induced by single-site right
ventricular (RV) or LV pacing depresses systolic function,
worsens myocardial efficiency, and leads to marked increases in
wall stress heterogeneity.5,6 Stress is highest in late-activated
myocardial regions because of both exaggerated stretch in early
systole (secondary to septal contraction) and late systolic con-
traction against increased afterload. Chronic discoordination
leads to chamber remodeling of both early- and late-activated
segments.7 However, its effect on myocardial protein expression
remains unknown. In the present study, we tested the hypothesis
that stress polarization due to dyssynchrony induces regional
expression disparities of key proteins involved with stress
response, muscle mechanics, and electrophysiology. Using HF
models with or without superimposed LV discoordination, we
studied tissue levels of the mitogen-activated protein kinases,
sarcoplasmic reticulum Ca2ϩ
-ATPase (SERCA2a) and phospho-
lamban (PLB), and the gap junction protein connexin43 (Cx43).
We reveal marked dyssynchrony-dependent transmural and tran-
schamber protein expression gradients, suggesting a novel mecha-
nism for the enhanced morbidity seen in discoordinate LV failure.
Methods
Model Generation
Adult mongrel dogs (Bruce Rotz Kennels, Shippensburg, Pa) were
paced (210 to 250 bpm) from the right atrium (RA; nϭ5) or RV free
Received January 27, 2003; de novo received May 27, 2003; revision received July 14, 2003; accepted July 18, 2003.
From the Division of Cardiology, Department of Medicine (D.S., C.L., M.L., R.T., D.D., G.T., D.A.K.), Johns Hopkins University, Baltimore, Md;
and the National Heart, Lung and Blood Institute/Laboratory of Cardiac Energetics (O.F., E.M.), National Institutes of Health, Bethesda, Md.
Dr Kass serves as a consultant to Guidant, Inc, which provided funding for this study.
*These authors contributed equally to the work.
Cine images are available in the online-only Data Supplement at http://www.circulationaha.org.
Correspondence to David A. Kass, Halsted 500, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287. E-mail dkass@jhmi.edu
© 2003 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org DOI: 10.1161/01.CIR.0000088782.99568.CA
929 by guest on February 25, 2013http://circ.ahajournals.org/Downloaded from

3. wall (nϭ6), as described previously,8 or were left uninstrumented
(nϭ4). At terminal study, dogs were anesthetized (10 to 15 mg/kg
thiopental, 1% to 2% isoflurane); LV pressures measured by micro-
manometer (Millar); and the hearts retroperfused with cold cardio-
plegia, excised, dissected into endocardial and mid/epicardial seg-
ments from the interventricular septum and LV lateral wall, and
frozen in liquid nitrogen. All procedures followed USDA guidelines
and the protocol was approved by the Animal Care and Use
Committee of the Johns Hopkins Medical Institutions.
Assessment of Mechanical Dyssynchrony
A subgroup of 4 dogs was studied by magnetic resonance (MR)
imaging to document the effect of activation site on LV coordination.
MR 3D tagged images were obtained (GE Signa 1.5T) during either
RA or RV free-wall stimulation, and mechanical dyssynchrony
indexed by a circumferential uniformity ratio estimate (CURE), as
described previously.9 Perfectly synchronous or dyssynchronous
contraction patterns yield CURE indices of 1 or 0, respectively.
Western Blot Analysis
Frozen myocardium was homogenized in lysis buffer (Cell Signaling
Technology), and equivalent samples (confirmed by coprobing for
glyceraldhyde-3-phosphate dehydrogenase or calsequestrin) were
loaded for gel electrophoresis. After transfer to nitrocellulose,
membranes were blocked and probed overnight at 4°C with primary
antibodies for p-p38, p-erk, p-jnk (Cell Signaling Technology;
1:1000 dilution), PLB, SERCA2a (Affinity BioReagents; 1:5000
dilution), or Cx43 (Chemicon Intl; 1:1000 dilution). Membranes
were incubated with HRP-conjugated secondary antibodies (Cell
Signaling Technology and Upstate Biotechnology; 1:10 000 dilu-
tions) for 1 hour at room temperature. Protein levels were detected
by chemiluminescence and autoradiography, quantified using NIH
ImageJ software, and blot intensity normalized such that lateral
endocardial signal equaled 100 arbitrary units.
Statistical Analysis
Results are expressed as meanϮSEM. Statistical significance was
assessed by ANOVA with post hoc Tukey tests for multiple
comparisons.
Results
Mechanical Dysfunction and Dyssynchrony: RV
Versus RA Pacing Models
RA and RV pacing models generated similar degrees of
global LV dysfunction, as reflected by depression of dP/dtmax
(normal values in anesthetized dog are Ϸ2000 mm Hg/s) and
elevation of LV end-diastolic pressure (Table). However, the
two models generated markedly different degrees of LV
mechanical coordination. Whereas rapid RA pacing pre-
served nearly uniform contraction at all LV regional seg-
ments, high RV free wall pacing induced substantial LV
dyssynchrony, with early interventricular septal contraction
and coincident lateral LV stretch followed by delayed lateral
LV contraction and septal stretch (CURE index; Table). Cine
images based on tagged MRI circumferential strain analysis
depicting this dyssynchrony can be viewed in the online-only
Data Supplement at http://www.circulationaha.org.
Dyssynchronous HF and Regional
Protein Expression
The Figure displays representative Western blots and sum-
mary data for regional protein expression of activated
MAPKs, SERCA2a, PLB, and Cx43 in failing hearts with
preserved (RA pacing) or disturbed (RV pacing) LV coordi-
nation. With RV pacing, p-erk increased nearly 100% in the
posterolateral LV endocardium relative to the other segments
assessed (PϽ0.009). In contrast, p-erk levels did not vary
significantly by region in RA pacing HF models. p-jnk and
p-p38 levels did not vary regionally in either RV- or RA-
paced models. Total levels for all three MAP kinase proteins
were also not significantly different among the four regions
(data not shown).
The late-activated LV endocardium also demonstrated
significant reductions in SERCA2a and PLB protein expres-
sion in dyssynchronous HF. SERCA2a declined Ϸ30% rela-
tive to the other regions (PϽ0.001), and PLB fell even more
(Ϸ80%; PϽ0.001). In contrast, RA pacing did not induce
regional disparities in the expression of either protein. Fi-
nally, the same posterolateral endocardial region displayed
marked downregulation of Cx43 expression (Ϸ60% relative
to other segments; PϽ0.001) in RV-paced dogs. Once again,
this localized abnormality was not observed in HF with
preserved LV coordination, as shown by the RA-paced data.
In noninstrumented control hearts, MAPK and Cx43 levels
did not vary significantly by region. However, both
SERCA2a and PLB levels were moderately and globally
reduced in LV endocardial versus epicardial tissue (by 20%
and 30%, respectively; Pϭ0.03), consistent with prior data.12
This transmural gradient was significantly lower than that
observed in the lateral endocardium of dyssynchronous HF
hearts.
Discussion
The present study provides novel evidence that mechanical
dyssynchrony spatially polarizes ventricular protein expres-
sion, particularly within the late-activated lateral wall, gen-
erating transmural and transventricular gradients in p-erk,
SERCA2a, PLB, and Cx43 levels. Similar expression gradi-
ents were not seen in HF with equivalent global dysfunction
but preserved LV coordination, indicating the importance of
dyssynchrony to this process. We propose that such region-
ally specific molecular polarization may contribute to hetero-
geneous electromechanics and underlie enhanced arrhythmia
susceptibility in HF patients with LV dyssynchrony.
Mechanical load heterogeneity in the setting of LV disco-
ordination was examined by Prinzen et al5 using MR tagged
imaging and stress modeling. The authors predicted that
late-activated ventricular segments were subjected to greatest
stress because of locally enhanced preload (secondary to
early systolic stretch) and afterload (due to late systolic
contraction against high LV cavity pressures), and correlated
LV Dysfunction and Coordination Parameters in RA and RV
Pacing–Induced HF
RV-Paced Dogs
(Dyssynchronous)
(nϭ6)
RA-Paced Dogs
(Synchronous)
(nϭ5) P
dP/dtmax, mm Hg/s 1295Ϯ250 1141Ϯ183 NS
LV end-diastolic pressure,
mm Hg
26Ϯ6 32Ϯ5 NS
CURE index
(dyssynchrony),
dimensionless
0.52Ϯ0.14 0.90Ϯ0.04 Ͻ0.05
930 Circulation August 26, 2003
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4. increased wall stress to increased regional blood flow, nutri-
ent consumption, and hypertrophy.5,6 The endocardium is
particularly subject to stress redistribution because of myo-
cardial fiber orientation, direct cavity pressure load, and a
greater compromise in blood flow.10 Biomechanical stress on
the posterolateral endocardium in dyssynchronous HF is
uniquely elevated, therefore, and may explain the distribution
of molecular abnormalities in our study.
Several investigations have examined regional changes in
protein expression in HF. Using a canine model of LV
hypertrophy, Dosch et al11 reported higher atrial natriuretic
peptide levels in LV basal and midwall segments, with peak
expression in basal endocardium. Prestle et al12 described
SERCA2a and PLB transmural expression gradients in failing
human hearts, with Ϸ25% lower levels in the LV free wall
endocardium. The present study supports the latter findings,
but by contrasting models of LV failure differing only in
degree of mechanical LV coordination, firmly identifies local
biomechanical input (rather than systemic hemodynamics or
neurohumoral signaling) as a key regulator of protein expres-
sion. We believe this is the first study providing a possible
molecular mechanism for the enhanced arrhythmia suscepti-
bility seen in dyssynchronous HF.
We did not test the functional impact of the molecular
abnormalities observed, as this will require extensive isolated
myocyte analysis from different layers and regions of the
heart. However, prior data support such changes as poten-
tially important. For example, increased SERCA2a/PLB lev-
els have been associated with improved SERCA2a-mediated
calcium sequestration and enhanced systolic function.13 In
this regard, the increase in the lateral endocardium
SERCA2a/PLB ratio (Ͼ3-fold) may reflect a regionally
targeted adaptive mechanism to counter higher stress. Ele-
ments of the PLB promoter (eg, GATA box) are responsive to
biomechanical stretch and might explain the disparate
changes seen in septal (stretched) versus lateral (higher-
stress) SERCA2a/PLB expression.14,15 To our knowledge,
however, specific regulators of SERCA2a and PLB that
might drive reduced expression in response to mechanical
stress have not been identified. Cx43 downregulation has
been linked with arrhythmia susceptibility in a variety of
models, likely because of unidirectional conduction delay and
Histograms and representative Western
blots detailing regional expression of ac-
tivated MAPKs (A), calcium-handling pro-
teins (B), and Cx43 (C), in RA-paced and
RV-paced models. Expression is pre-
sented in arbitrary units (A.U.).
Septum-RV and Septum-LV indicate the
RV- and LV-facing aspects of the inter-
ventricular septum, respectively. Note
the increased scale used to display p-erk
expression, and that sample blot for RV
p-p38 shows duplicate lanes for each
myocardial segment. p-erk was signifi-
cantly increased in RV-paced lateral
endocardium, relative to other myocar-
dial segments (*PՅ0.05 vs Septum-RV
and LV-Lateral Wall Mid/Epicardium;
Pϭ0.01 vs Septum-LV), whereas
SERCA2a (†PϽ0.02 vs Septum-LV and
Septum-RV; PϽ0.001 vs LV-Lateral Wall
Mid/Epicardium), PLB (‡PϽ0.001 vs
Septum-RV and LV-Lateral Wall Mid/Epi-
cardium; PϽ0.02 vs Septum-LV), and
Cx43 (¶PϽ0.01 vs Septum-LV and
Septum-RV; PϽ0.001 vs LV-Lateral Wall
Mid/Epicardium) were relatively
decreased.
Spragg et al Dyssynchrony and Myocardial Protein Changes 931
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5. facilitated reentry.16,17 Furthermore, MAPK activation and
Cx43 downregulation may be connected, as constitutively
activated p-jnk potently reduces Cx43 levels in mouse myo-
cardium, and p-erk has been implicated in Cx43 downregu-
lation in liver epithelial cells.18,19 Ongoing studies are ad-
dressing the physiological importance of the expression
changes revealed in the present study, as well as expanding
the analysis by means of subproteomic and transcriptome
analysis.
In conclusion, LV mechanical dyssynchrony superimposed
with tachycardia-induced HF induces marked regional het-
erogeneity of protein expression at the site of greatest
hemodynamic load. These observations raise the possibility
that cardiac resynchronization therapy may not only improve
ventricular mechanics, but also modulate regional myocardial
protein expression. Further studies with more chronic HF
models are needed to determine the extent to which cardiac
resynchronization can reverse molecular polarization and
impact net cardiac function and arrhythmia susceptibility.
Acknowledgments
This work was supported by grants from the National Institutes of
Health (P50-HL52307) and from Guidant, Inc.
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