Differential regional gene expression from cardiac dyssynchrony induced by chronic right ventricular free wall pacing in the

mouse
Kenneth C. Bilchick, Sudip K. Saha, Ed Mikolajczyk, Leslie Cope, Will J. Ferguson, Wayne Yu, Steven Girouard and David A. Kass
Physiol. Genomics 26:109-115, 2006. First published 2 May 2006; doi:10.1152/physiolgenomics.00281.2005 You might find this additional info useful... This article cites 27 articles, 17 of which can be accessed free at: http://physiolgenomics.physiology.org/content/26/2/109.full.html#ref-list-1 This article has been cited by 3 other HighWire hosted articles Ion Channel Subunit Expression Changes in Cardiac Purkinje Fibers : A Potential Role in Conduction Abnormalities Associated With Congestive Heart Failure Ange Maguy, Sabrina Le Bouter, Philippe Comtois, Denis Chartier, Louis Villeneuve, Reza Wakili, Kunihiro Nishida and Stanley Nattel Circulation Research, May 8, 2009; 104 (9): 1113-1122. [Abstract] [Full Text] [PDF] Update on the pathophysiological basics of cardiac resynchronization therapy Angelo Auricchio and Frits W. Prinzen Europace, July , 2008; 10 (7): 797-800. [Abstract] [Full Text] [PDF] Update on the pathophysiological basics of cardiac resynchronization therapy Angelo Auricchio and Frits W. Prinzen Europace, May 14, 2008; . [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://physiolgenomics.physiology.org/content/26/2/109.full.html Additional material and information about Physiological Genomics can be found at: http://www.the-aps.org/publications/pg

Downloaded from physiolgenomics.physiology.org on September 20, 2011

This infomation is current as of September 20, 2011.

Physiological Genomics publishes results of a wide variety of studies from human and from informative model systems with techniques linking genes and pathways to physiology, from prokaryotes to eukaryotes. It is published quarterly in January, April, July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2006 by the American Physiological Society. ISSN: 1094-8341, ESSN: 1531-2267. Visit our website at http://www.the-aps.org/.

Physiol Genomics 26: 109 115, 2006. First published May 2, 2006; doi:10.1152/physiolgenomics.00281.2005.

Differential regional gene expression from cardiac dyssynchrony induced by chronic right ventricular free wall pacing in the mouse
Kenneth C. Bilchick,1 Sudip K. Saha,1 Ed Mikolajczyk,3 Leslie Cope,2 Will J. Ferguson,4 Wayne Yu,2 Steven Girouard,3 and David A. Kass1
Division of Cardiology, Department of Medicine, School of Medicine, and 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland; 3Guidant Corporation, St. Paul, Minnesota; and 4Agilent Technologies, Columbia, Maryland
Submitted 12 November 2005; accepted in nal form 19 April 2006
1

Bilchick, Kenneth C., Sudip K. Saha, Ed Mikolajczyk, Leslie Cope, Will J. Ferguson, Wayne Yu, Steven Girouard, and David A. Kass. Differential regional gene expression from cardiac dyssynchrony induced by chronic right ventricular free wall pacing in the mouse. Physiol Genomics 26: 109 115, 2006. First published May 2, 2006; doi:10.1152/physiolgenomics.00281.2005.Routine clinical right ventricular pacing generates left ventricular dyssynchrony manifested by early septal shortening followed by late lateral contraction, which, in turn, reciprocally stretches the septum. Dyssynchrony is disadvantageous to cardiac mechanoenergetics and worsens clinical prognosis, yet little is known about its molecular consequences. Here, we report the inuence of cardiac dyssynchrony on regional cardiac gene expression in mice. Mice were implanted with a custom-designed miniature cardiac pacemaker and subjected to 1-wk overdrive right ventricular free wall pacing (720 beats/min, baseline heart rate 520 620 beats/min) to generate dyssynchrony (pacemaker: 3-V lithium battery, rate programmable, 1.5 g, bipolar lead). Electrical capture was conrmed by pulsed-wave Doppler and dyssynchrony by echocardiography. Gene expression from the left ventricular septal and lateral wall myocardium was assessed by microarray (dual-dye method, Agilent) using oligonucleotide probes and dye swap. Identical analysis was applied to four synchronously contracting controls. Of the 22,000 genes surveyed, only 18 genes displayed signicant (P 0.01) differential expression between septal/lateral walls 1.5 times that in synchronous controls. Gene changes were conrmed by quantitative PCR with excellent correlations. Most of the genes (n 16) showed greater septal expression. Of particular interest were seven genes coding proteins involved with stretch responses, matrix remodeling, stem cell differentiation to myocyte lineage, and Purkinje ber differentiation. One week of iatrogenic cardiac dyssynchrony triggered regional differential expression in relatively few select genes. Such analysis using a murine implantable pacemaker should facilitate molecular studies of cardiac dyssynchrony and help elucidate novel mechanisms by which stress/stretch stimuli due to dyssynchrony impact the normal and failing heart. microarray; heart; activation timing
RECENT STUDIES

but were largely asymptomatic led to a greater incidence of worsening or new onset heart failure (26). Clinical studies employing biventricular stimulation (cardiac resynchronization therapy) to treat dyssynchrony have revealed signicant benets in both morbidity and mortality (3, 4). Although the mechanical aspects of left ventricular dysfunction due to dyssynchrony have been well studied, the impact of dyssynchrony on molecular signaling remains ill dened. Studies have reported regional changes in calcium handling and stress kinase proteins as well as connexin 43, but these data reect a small targeted sampling of potential changes that might occur (20). There have been no broad-based analyses of gene expression alterations from dyssynchrony. This has largely stemmed from the lack of a suitable animal model and, for clinical studies, the lack of appropriate regional tissue and difculties in interpreting changes without adequate controls. Besides the human, the mammalian species most often utilized for genetic array analysis is the mouse. However, cardiac dyssynchrony in mice has not been previously studied, largely due to technical difculties. To this end, we developed a custom miniature implantable mouse pacemaker capable of stimulating the murine heart at rates varying from 400 to 1,200 beats/min. In this study, we employed right ventricular free wall stimulation to generate cardiac dyssynchrony and then examined its effects on regional gene expression using differential microarray analysis.
METHODS

Downloaded from physiolgenomics.physiology.org on September 20, 2011

have shown that dyssynchronous contraction of the left ventricle is a prominent and independent risk factor for worsened cardiovascular morbidity and mortality in patients with dilated heart failure (2). This pathophysiology occurs both from native conduction delay such as a left bundle branch block and articial right ventricular pacing. Indeed, right ventricular pacing in patients who had reduced systolic function

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org). Address for reprint requests and other correspondence: D. A. Kass, Dept. of Medicine, Johns Hopkins Univ. School of Medicine, Ross Bldg. 835, 720 Rutland Ave., Baltimore, MD 21205 (e-mail: dkass@jhmi.edu).

Miniature implantable mouse pacemaker. Figure 1A shows the newly developed mouse pacemaker. The body of the pacemaker is 1.2 cm in diameter, weighs 1.5 g, and includes a 3-V lithium battery. The pacemaker is soldered to a 5-cm unipolar lead with a 1.75-turn solid platinum helix tip for tissue contact. A bipolar conguration is used to minimize thoracic muscle stimulation, with a 6-cm segment of 32gauge insulated copper magnet wire soldered to the body and then wrapped around the main lead to terminate close to the distal helix. The pacemaker is painted with insulating material so that current ows between the helix and parallel wire rather than back to the pacemaker body via conductive tissues. To preserve battery life further, a resistor is connected in series with the helical lead. The pacemaker has four rate settings: 450, 550, 720, and 1,050 beats/min, with the rate adjusted by touching a magnet to the pacemaker body. Before implantation, the rate is set to 720 beats/min, and rate and output voltage are conrmed by oscilloscope. Implant procedure. The protocol was approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Wild-type mice (C57Bl/6, 12 wk) were anesthetized with etomidate (10 mg/kg), intubated, and mechanically ventilated (6.7 l/g at 120 breaths/min). Anesthesia was maintained with inhaled isourane (2%) while the electrocardiographic monitoring was performed. A ventral
109

1094-8341/06 $8.00 Copyright 2006 the American Physiological Society

110

REGIONAL GENE EXPRESSION IN MURINE CARDIAC DYSSYNCHRONY

Fig. 1. A: bipolar implantable mouse pacemaker compared with the size of a 19-gauge needle; B: mouse with pacemaker implanted in the abdomen (arrow).

Downloaded from physiolgenomics.physiology.org on September 20, 2011

midline incision exposed the chest wall and abdomen, and abdominal subcutaneous tissue was dissected to create a pocket for the pacemaker. The right ventricular free wall was exposed by right anterolateral thoracotomy. After pericardial dissection, the pacemaker lead was advanced through the thoracotomy, placed perpendicular to the surface of the right ventricle, and xed to the heart tissue by 134 revolutions of the distal screw lead. Capture was conrmed by electrocardiogram, and hemodynamic assessment (Fig. 2, A and B) was used to conrm stable mechanical function (Doppler aortic ow) at the 720 beats/min stimulation rate. The thoracotomy was closed with 6-0 Proline, and the site of lead entrance into the chest was sealed with glue to preserve an appropriate lead angle and position. The thorax was then evacuated by needle suction. After reconrmation of capture by Doppler ow at varying pacing rates, the device was set to 720 beats/min. The pacemaker can was placed subcutaneously in the abdomen (Fig. 1B), and the skin closed with 5-0 silk. The animal was then allowed to recover. Successful implantation and capture for a period of at least 1 wk were achieved in 60% of mice. Postoperative evaluation and terminal study. Gene expression analysis was performed after 1 wk of pacing, a time when sustained capture was assured and documented by electrocardiographic and hemodynamic monitoring. At this time, animals (n 4) were anesthetized and intubated, pacemaker capture was reconrmed, and the heart was rapidly removed and washed in cold saline. The left ventricle was isolated and divided into septal and lateral wall halves

that were further trimmed to provide septal and lateral free wall samples. The tissue was rapidly frozen in liquid nitrogen and stored for subsequent analysis. In addition to tissue from dyssynchronous mice, myocardium from normal controls (synchronous contraction, n 4) was also obtained using the same procedure. Physiological studies. Chronic left ventricular dyssynchrony was documented by two-dimensional guided M-mode echocardiography (Sequoia C256, Siemens, 15-MHz linear array transducer) in several animals. In addition, the impact of right ventricular pacing-induced dyssynchrony on left ventricular chamber function was assessed by in vivo pressure-volume analysis (7) in 10 additional mice. For this assessment, anesthetized animals underwent a limited thoracotomy, after which a 1.4-Fr pressure-volume catheter A (SPR-719, Millar) was inserted into the heart via an apical stab and advanced to the aortic root. The catheter was interfaced with a custom-designed stimulator/analyzer, and pressure-volume data were recorded at 2 kHz for analysis. RNA isolation and microarray design. Total RNA was extracted with TRIzol reagent from left ventricular segments, and septal and lateral isolates from the four dyssynchronous mice were kept separate. RNA samples from the septal and lateral left ventricular walls of four synchronous control mice were pooled into a control septal and lateral sample. Functional genomic analysis was conducted on these 10 RNA samples (8 from dyssynchronous mice and 2 from control mice) with uorophore reversal, such that each sample was assayed on two different microarrays. Corresponding septal and lateral samples from

Fig. 2. Pulsed-wave Doppler aortic ow signal conrms the mechanical capture in conjunction with an electrocardiogram (ECG) showing right ventricular (RV) pacing.

Physiol Genomics VOL

26

www.physiolgenomics.org

REGIONAL GENE EXPRESSION IN MURINE CARDIAC DYSSYNCHRONY

111

the same left ventricle were paired on the same microarray to achieve a direct comparison of the relative gene expression in different regions of the same heart. Agilent Mouse Oligo microarray slides (G4121A, Agilent Technologies; Palo Alto, CA) containing 20,000 probes were used. The 60-mer oligonucleotide probes were oriented in the sense (5 3 ) direction and spotted using Agilents SurePrint fabrication technology, which utilizes an industrial-scale inkjet printer that synthesizes oligonucleotide probes in situ on glass wafers, which are then scribed onto bar-coded 1 3-in. glass slides. A complete probe list (G4121A) may be found online at http://www.chem.agilent.com/cag/bsp/ oligoGL/011978_D_GeneList_20050310.html. Microarray analysis. Gene expression data were obtained using Agilent G2567AA Feature Extraction software, using defaults for all parameters except ratio terms, which were changed according to the Agilent protocol to t the direct labeling procedure. Files and images, including error values and P values, were exported from the Agilent Feature Extraction software and loaded into Rosetta Resolver (version 3.2, build 3.2.2.0.33, Rosetta Biosoftware; Kirkland, WA). Four arrays for each sample pair, including uorophore reversals, were combined into ratio experiments in Rosetta Resolver (21). Intensity plots were produced for each ratio experiment, and genes specically output by this software were considered signature genes if P 0.01. P values reecting both microarray slide and biological variation were calculated using the Rosetta Resolver error model (21). An alternate analysis was performed as follows. Agilent analysis was replaced with standard cDNA methods for within microarray normalization and background correction, and minor intermicroarray normalization was performed. Fluorophore swap pairs were averaged, and each gene was tested for differential expression using the significance analysis of microarrays (SAM) method (25). PCR conrmation. cDNA was synthesized from paced and pooled control RNA samples using TaqMan Reverse Transcription Reagents (Applied Biosystems). Real-time quantitative PCR (qPCR) was performed using TaqMan Universal PCR MasterMix and an ABI Prism 7700 Sequence Detector (Applied Biosystems) to conrm the results of the microarray. The following primer-probe mixes were used: dickkopf homolog 3 (dkk-3), matrilin-1 (COMP), myosin binding protein H (MyBP H), myosin regulatory light chain A, osteopontin, latent transforming growth factor- binding protein (LTBP)-2, and myosin binding protein C (MyBP C). The comparative cycle threshold method ( Ct) was used to calculate septal cDNA expression relative to lateral cDNA expression, which was normalized using GAPDH

cDNA expression for each sample. Paced and pooled control septalto-lateral fold changes were then compared. Sham pacemaker study. To rule out effects of the surgical pacemaker implantation itself on regional gene expression, sham pacemakers were constructed that were identical to the functional pacemakers except that the battery was insulated from the attached pacemaker lead. These devices were then implanted in four additional mice (C57/Bl6, 12 wk) using the same surgical techniques as in the active-pacing animals. One week after the surgery, hearts were harvested in an identical manner, and qPCR was performed with the same seven probes listed above with a GAPDH probe for normalization. Regional expression for each gene was contrasted to mice with functional pacemakers and the pooled control sample.
RESULTS

Dyssynchrony in the intact mouse. Figure 3 shows left ventricular dyssynchrony induced by right ventricular free wall pacing and assessed by M-mode echocardiography. Also shown for comparison is the M-mode echocardiogram in a mouse with normal contraction, showing the septal and lateral left ventricular walls contracting simultaneously. This would be expected with normal sinus rhythm or atrial pacing because the pacing impulse from the atrium is conducted through the atrioventricular node and the ventricles are activated simultaneously. In contrast, septal contraction was earlier in the left ventricles with right ventricular free wall pacing. The corresponding electrocardiograms from mice with atrial pacing and right ventricular pacing are also reproduced in Fig. 3. Figure 4 shows an example of pressure-volume loops for normal sinus rhythm and right ventricular free wall pacing in the mouse, revealing a reduction in overall left ventricular function due to dyssynchrony. Compared with atrial pacing, right ventricular free wall stimulation lowered the mean peak rate of pressure rise (dP/dtmax) by 14 8% (P 0.001), stroke volume by 19 13% (P 0.01), and stroke work by 22 11% (P 0.005). The heart weights of mice after 2 wk of right ventricular free wall pacing were similar to those of controls. Microarray analysis. The full dataset is MIAME compliant and may be accessed at the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.

Downloaded from physiolgenomics.physiology.org on September 20, 2011

Fig. 3. ECG and M-mode echocardiogram (Echo) in the mouse with atrial pacing versus RV free wall pacing. Dyssynchrony is demonstrated in the mouse with RV free wall pacing with early septal shortening and late lateral shortening (arrows).

Physiol Genomics VOL

26

www.physiolgenomics.org

112

REGIONAL GENE EXPRESSION IN MURINE CARDIAC DYSSYNCHRONY

Fig. 4. Pressure-volume loops in the mouse with a pacemaker implanted in the RV (RVP) compared with those in a mouse in normal sinus rhythm (NSR).

nlm.nih.gov/geo/submission/geosubmi.cgi). RNA isolated from all hearts was analyzed by spectrophotometry and found to be of high quality. Of the 20,000 genes studied, the Agilent analysis revealed only 18 genes with signicant (P 0.01) differential expression between the left ventricular septal and lateral walls of a magnitude of 1.5-fold or greater, each being normalized for corresponding regional disparities (if any) in control synchronous hearts (Table 1). By the SAM method with a false discovery rate of 21% and a 1.5-fold threshold, 10 signicant genes were identied, most of them among the original 18 already identied. The vast majority (16/18) of the genes identied by the Agilent method had septal expression increased over lateral expression. Relative changes in gene expression for the seven genes with prominent disparities identied by the Agilent method were conrmed by qPCR, and excellent correlations were obtained between qPCR and Agilent analysis for all genes tested (Table 2). Regional expression changes for dkk-3, myosin regulatory light chain A, matrilin-1, and MyBP H were greater than 5-fold by qPCR, whereas changes of 3.7-, 2.4-, and 1.6-fold were observed for osteopontin, LTBP-2, and MyBP C, respectively.

Downloaded from physiolgenomics.physiology.org on September 20, 2011

Table 1. Signicant genes in dyssynchronous hearts compared with synchronous control hearts
Lateral-to-Septal Fold Change Accession No. D hearts C hearts D Fold Change Compared with C Fold Change

Myosin regulatory light chain A Dkk-3 RIKEN (1500015O10Rik) Matrilin-1 (COMP) MyBPH (similar) Angiopoietin-like factor Osteopontin Myosin essential light chain A Proteoglycan 3 RIKEN (1110018M03Rik) Irx6 Myosin heavy chain 7 Protein C17 FAST MyBP C Wnt inhibitor factor 1 LTBP-2 RIKEN (1200004E24) RIKEN A330041G23 Matrix carboxyglutamate protein Ankyrin-like repeat protein Thrombospondin 4 Deiodinase type II Myotilin Clone IMAGE:5355604 Serpinf1 -2-HS-glycoprotein Methionine synthase act protein Platelet-derived growth factor receptor Scleraxis Neuritin Ventricular alkali myosin light chain Kcna1 Leucine-rich repeat protein RIKEN (3110023K12Rik) NHE2 Calponin 1 Protein tyrosine kinase 9

NM_022879 AK004853 NM_024283 NM_016685 AK004148 BC023373 NM_009263 NM_010858 NM_021400 NM_026271 AF165986 NM_080728 AK076770 AK029282 NM_011915 NM_013589 BC025879 BC019641 BC009120 AK009959 NM_011582 NM_010050 NM_021484 BC023221 NM_011340 NM_013465 AK085987 AK004179 S78079 AK003046 X67685 NM_010595 AK044492 NM_026523 AK077026 NM_009922 NM_008971

1/17.3 1/9.5 1/7.5 1/6.2 1/5.1 1/4.2 1/3.1 1/2.7 1/2.6 1/2.5 1/2.3 1/2.0 1/2.0 1/1.9 1/1.9 1/1.8 1/1.8 1/1.8 1/1.8 1/1.7 1/1.7 1/1.7 1/1.6 1/1.6 1/1.6 1/1.5 1/1.5 1/1.5 1/1.5 1/0.7 1/0.6 1/0.6 1/0.6 1/0.6 1/0.6 1/0.5 1/0.4

1/1.2 1/1.5 1/1.2 1/1.4 1/0.9 1/1.2 1/0.9 1/1.4 1/1.2 1/1.1 1/2.9 1/1.8 1/1.0 1/1.2 1/2.0 1/1.1 1/1.4 1/1.0 1/1.0 1/1.4 1/0.9 1/1.2 1/1.2 1/1.2 1/1.1 1/1.5 1/1.4 1/1.0 1/1.5 1/0.5 1/1.4 1/0.6 1/0.8 1/0.6 1/0.7 1/0.6 1/0.9

1/14.8* 1/6.3* 1/6.1* 1/4.3* 1/5.7* 1/3.5* 1/3.6* 1/2.0* 1/2.3* 1/2.3* 1/0.8 1/1.1 1/2.0* 1/1.6* 1/0.9 1/1.7* 1/1.3 1/1.7* 1/1.8* 1/1.2 1/2.0* 1/1.5 1/1.3 1/1.3 1/1.4 1/1.1 1/1.1 1/1.5 1/1.0 1/1.3 1/0.5* 1/1.0 1/0.7 1/1.0 1/0.8 1/0.8 1/0.5*

*The 18 genes with a 1.5-fold increase or decrease in lateral gene expression compared with septal gene expression in dyssynchronous (D) hearts after normalization for the corresponding fold change in synchronous control mice (C). Dkk-3, Dickkopf homolog 3; MyBP, myosin binding protein; Irx6, iroquosis homobox protein 6; LTBP, latent transforming growth factor- binding protein; Kcna1, K voltage-gated channel, member 1; NHE2, Na /H exchanger 2. Physiol Genomics VOL
26

www.physiolgenomics.org

REGIONAL GENE EXPRESSION IN MURINE CARDIAC DYSSYNCHRONY

113

Table 2. Subset of signicant PCR-conrmed genes in dyssynchronous hearts after normalization to synchronous controls
Lateral-to-Septal Fold Change in D (Normalized to C) Gene Function Microarray qPCR Expression in D Versus C Septal Lateral

Myosin regulatory light chain A Dkk-3 Matrilin-1 (COMP) MyBP H (similar) Osteopontin LTBP-2 MyBP C (skeletal, fast)

Normally suppressed, inducible by MEF2A, GATA4, and SRF Proliferation/differentiation of cardiac progenitor cells Matrix remodeling-stress responsive Expressed in Purkinje ber myocytes Matrix remodeling-stretch/stress responsive Growth and matrix remodeling Growth and hypertrophy

1/14.8 1/6.3 1/4.3 1/5.7 1/3.6 1/1.7 1/1.6

1/ 15 1/5.6 1/6 1/5.6 1/3.7 1/2.4 1/1.6

11 11 1 11 11 1

222 22 22 22 2 2

The lateral-to-septal fold changes represent the ratio of two ratios: the ratio of lateral-to-septal gene expression in dyssynchronous hearts divided by the ratio of lateral-to-septal gene expression in synchronous control hearts. qPCR, quantitative PCR; MEF2A, myocyte enhancer factor 2A; GATA4, GATA-binding protein 4; SRF, serum response factor.

Downloaded from physiolgenomics.physiology.org on September 20, 2011

These seven genes were also of particular interest due to their potential impact on cardiac structure and function. They included genes coding proteins involved with myocardial stretch response (matrilin-1 and osteopontin), matrix remodeling (matrilin-1, osteopontin, and LTBP-2), stem cell differentiation to myocyte lineage (dkk-3), and Purkinje ber differentiation (MyBP H). As shown in Table 2, differential expression for most genes was the result of both an increase in septal expression and a decrease in lateral wall expression (compared with the synchronous pooled septal and lateral left ventricular samples). Such analysis was possible because gene expression by qPCR was measured relative to the housekeeping gene GAPDH. As shown in Table 3, regional gene expression ratios as measured by qPCR in mice with sham pacemakers implanted in the right ventricle (n 4) were close to unity and thus similar to that obtained in the pooled control sample. Regional expression ratios in dyssynchronous right ventricular free wallpaced mice (shown here without normalization to the pooled control) were statistically different from the sham results.
DISCUSSION

We developed a new experimental system for the study of spatial and temporal gene expression changes in the presence of mechanical dyssynchrony. By combining a novel miniature Table 3. Direct comparison of signicant genes in mice with functional and nonfunctional pacemakers implanted in the RV
RVP PCR (n 4) Sham PCR (n 4) Pooled Control PCR

Myosin regulatory light chain A Dkk-3 Matrilin-1 MyBP H Osteopontin LTBP-2 MyBP C

1/ 15 1/8.6* 1/7.2 1/7.6 1/3.1* 1/2.5 1/2.4

1/1.1 1/0.9 1/1.0 1/1.1 1/0.9 1/0.9 1/1.3

1/1.4 1/1.5 1/1.2 1/1.4 1/0.8 1/1.0 1/1.5

Data from mice implanted with functional right ventricular (RV) pacemakers (RVP) are provided without normalization to the pooled control to facilitate the three-way comparison. Note that the fold changes expressed in Table 2 (qPCR results) are equivalent to the ratios of the fold changes for the RVP and pooled control PCR data in this table. Sham animals were implanted with nonfunctional pacemakers. *P 0.001 vs. Sham; P 0.005 vs. sham; P 0.01 vs. sham; P 0.02 vs. sham. Physiol Genomics VOL
26

murine implantable pacemaker to create programmable dyssynchrony and microarray/PCR methods for quantifying regional gene expression, we demonstrated prominent regional changes in gene expression between the lateral and septal left ventricular walls. The genes that showed signicant regional expression heterogeneity (a relatively small number compared with the total number of genes tested) are involved in important processes such as matrix remodeling, stem cell differentiation, myocardial stress responses, and hypertrophy. Cardiac dyssynchrony, whether due to a left bundle branch block or iatrogenically induced by right ventricular free wall pacing, induces regional changes in mechanical function, loading, and metabolic requirements for the myocardium. Early shortening occurs in the septum, leading to greater prestretch (preload) in the lateral wall. Late shortening of the lateral wall occurs at somewhat higher stress but also leads to systolic rightward stretch of the septum. Studies have revealed corresponding acute increases in lateral versus septal perfusion and glucose metabolism. In a canine model of heart failure with dyssynchrony, we found regional protein expression of calciumhandling proteins [sarco(endo)plasmic reticular Ca2 -ATPase and phospholamban] and gap junction protein connexin 43 to be lower in the lateral wall than the septal wall, whereas the stress response MAPK (ERK1/2) was enhanced (20). However, this was not observed in dyssynchronous nonfailing hearts (19). The present model is closer to the latter condition, and our results point to different signaling such that the septal stretch may be more signicant to localizing gene expression than lateral loading in this setting. Importantly, there have been no prior systematic broad surveys of the molecular changes associated with dyssynchrony. A remarkable aspect of our ndings is that only 0.1% of the 22,000 genes assayed (representing nearly all the presently characterized and named genes in the mouse genome) revealed signicant regional changes relative to synchronous controls. Among these, several had changes of greater than vefold by qPCR. We were able to rule out that these regional changes were due to intrinsic differences between septum and lateral walls by employing pooled synchronous controls in the analysis. The expression ratio therefore represents the ratio of expression in lateral versus septal walls for dyssynchronous hearts normalized to the same ratio in synchronous hearts. For most of the signicant genes, the synchronous heart regional ratios were very close to unity. The lack of signicant changes
www.physiolgenomics.org

114

REGIONAL GENE EXPRESSION IN MURINE CARDIAC DYSSYNCHRONY

in many genes was not due to marked scatter among individual samples but rather to consistent minimal differences in expression. This nding indicates that there was a high level of signal relative to noise and that targeted signaling is involved. The broad functions of differentially expressed genes included stretch responses, matrix remodeling, stem cell differentiation to myocyte lineage, and Purkinje ber differentiation. Among them was osteopontin, which belongs to a family of matricellular proteins that are important for helping the heart respond to hemodynamic stress by modulating cell function and cell-matrix interactions (16). Osteopontin is regulated by Akt kinase (28) and is primarily expressed by cardiomyocytes in the heart (8). It modulates myocardial hypertrophy in response to chronic pressure overload in mice (27), promoting brosis by protecting cardiac broblasts from cell death (29). It is also involved in vascular remodeling, inammation, and lipid metabolism (22). Osteopontin-decient mice have exaggerated left ventricular dilation and reduced collagen deposition after a myocardial infarction, further suggesting that osteopontin has a benecial effect on left ventricular remodeling in animals coping with stress (24). Another differentially expressed gene was matrilin-1. Matrilins are involved in matrix remodeling and response to stress. Matrilin-1, formerly known as cartilage oligomeric matrix protein (13), contains von Willebrand factor A and EGF domains, can form lamentous networks with or without collagen and is rst expressed in the mouse ventricular myocardium and endocardium during the second week postconception (17). Heterogeneous expression of LTBP-2 was also observed. LTBPs belong to the brillin-LTBP family of extracellular matrix proteins and are important for the appropriate secretion and folding of tissue growth factor- proteins. They are potent regulators of extracellular matrix formation with important immunomodulatory and regulatory roles for cell growth (15). Dkk-3 was differentially expressed with upregulation in the septum and downregulation in the lateral free wall of dyssynchronous left ventricles. The dkk family of genes, originally studied in Xenopus embryogenesis, inhibit Wnt signaling (12). Dkk-3 is expressed in many adult tissues, including the ventricular myocardium. The related protein dkk-1 plays an important role in adult stem cell proliferation through Wnt-5a antagonism (9, 10), suggesting another possible inuence of this family of genes on cardiac growth and differentiation. Two MyBPs, MyBP H and MyBP C, also had signicant differences in regional gene expression. Interestingly, recent experiments in chicken hearts have shown that skeletal musclespecic MyBP H is present exclusively in myobril bands within Purkinje bers (1), and its upregulation may be in response to the altered stimulation timing. MyBP C, a thick lament-associated protein localizing to the cross-bridge-containing C zones of striated muscle sarcomeres, is a substrate of cAMP kinase and plays an important role in the regulation of cross-bridge cycling and contraction (6). Clinical interest in C protein is due, in part, to mutations in this protein associated with hypertrophic cardiomyopathy. Finally, signicant regional differences in the expression of regulatory myosin light chain A were observed. Both regulatory and essential myosin light chain A are expressed in the left ventricle during development but are only expressed in adult left ventricles in the presence of heart disease. For example,
Physiol Genomics VOL
26

left ventricular expression of essential myosin light chain A has been shown to increase in hearts with left ventricular hypertrophy (14) and decrease after surgical reduction of aortic stenosis in pressure-loaded hearts (23). Reconstitution of the protein with ventricular myosin increases actin-activated myosin ATPase activity, suggesting that contractility may be improved in vivo (11). Persistent left ventricular expression of regulatory myosin light chain A has been described in mice decient in the retinoid X receptor- (5). Decreased ventricular expression of regulatory myosin light chain A is thought to be due to repressed transcription, although the exact mechanism is unclear. The regulatory myosin light chain A promoter has binding sites for myocyte enhancer factor 2A, GATA-binding protein 4, serum response factor, and retinoic acid, so there are many possible candidates for reactivation (18). The septal/ lateral wall disparities consistently observed in dyssynchronous mice but not in synchronous mice make atrial contamination unlikely. Several limitations of this study should be noted. The present pacemakers are not constant-current devices. As a result, any short circuiting that might form due to brosis or uid can drain the battery. We initially tried longer pacing periods but found that pacing capture was less predictable after 10 14 days. Thus our model is one of the early changes occurring in cardiac dyssynchrony without overt heart failure. Longer-term dyssynchrony and dyssynchrony superimposed on a failing heart could well involve more and different genes. Moreover, although a mouse model of dyssynchrony has advantages, including tight control over genetic background, improved signal-to-noise ratios in studying expression responses, and a potential to use transgenic and knockout models, it does not necessarily reect dyssynchrony responses for a human heart. In conclusion, cardiac dyssynchrony, itself, induces regional changes in gene expression that are consistent with early stages of cardiac remodeling. The present study serves to establish the feasibility of such studies, highlights novel genes involved in regional changes in activation timing, and indicates the apparent role of late septal systolic stretch in this behavior. Whether the spatial patterns of gene expression are sustained over longer periods of dyssynchrony, are accompanied by additional changes, and are altered by states of cardiac failure states remains the focus of ongoing investigations.
ACKNOWLEDGMENTS The authors thank Drs. Eiki Takimoto and Hunter Champion for the technical assistance in this project. GRANTS This study was supported by National Heart, Lung, and Blood Institute Grants T32-HL-07227, PO1-HL-077180, and PO1-HL-59408 (to D. A. Kass), a grant from the Guidant Corporation, and the Abraham and Virginia Weiss Professorship (to D. A. Kass). DISCLOSURES D. A. Kass is a consultant and scientic advisory board member for the Guidant Corporation. S. Girouard and E. Mikolajczyk are employees of Guidant Corporation. REFERENCES 1. Alyonycheva T, Cohen-Gould L, Siewert C, Fischman DA, and Mikawa T. Skeletal muscle-specic myosin binding protein-H is expressed www.physiolgenomics.org

Downloaded from physiolgenomics.physiology.org on September 20, 2011

REGIONAL GENE EXPRESSION IN MURINE CARDIAC DYSSYNCHRONY in Purkinje bers of the cardiac conduction system. Circ Res 80: 665 672, 1997. Baldasseroni S, De Biase L, Fresco C, Marchionni N, Marini M, Masotti G, Orsini G, Porcu M, Pozzar F, Scherillo M, and Maggioni AP. Cumulative effect of complete left bundle-branch block and chronic atrial brillation on 1-year mortality and hospitalization in patients with congestive heart failure. A report from the Italian network on congestive heart failure (in-CHF database). Eur Heart J 23: 16921698, 2002. Bristow MR, Saxon LA, Boehmer J, Krueger S, Kass DA, De Marco T, Carson P, DiCarlo L, DeMets D, White BG, DeVries DW, and Feldman AM. Cardiac-resynchronization therapy with or without an implantable debrillator in advanced chronic heart failure. N Engl J Med 350: 2140 2150, 2004. Cleland JG, Daubert JC, Erdmann E, Freemantle N, Gras D, Kappenberger L, and Tavazzi L. The effect of cardiac resynchronization on morbidity and mortality in heart failure. N Engl J Med 352: 1539 1549, 2005. Dyson E, Sucov HM, Kubalak SW, Schmid-Schonbein GW, DeLano FA, Evans RM, Ross J Jr, and Chien KR. Atrial-like phenotype is associated with embryonic ventricular failure in retinoid X receptor alpha / mice. Proc Natl Acad Sci USA 92: 7386 7390, 1995. Flashman E, Redwood C, Moolman-Smook J, and Watkins H. Cardiac myosin binding protein C: its role in physiology and disease. Circ Res 94: 1279 1289, 2004. Georgakopoulos D and Kass D. Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart. J Physiol 534: 535545, 2001. Graf K, Do YS, Ashizawa N, Meehan WP, Giachelli CM, Marboe CC, Fleck E, and Hsueh WA. Myocardial osteopontin expression is associated with left ventricular hypertrophy. Circulation 96: 30633071, 1997. Gregory CA, Singh H, Perry AS, and Prockop DJ. The Wnt signaling inhibitor dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 278: 2806728078, 2003. Horwitz EM. Dkk-1-mediated expansion of adult stem cells. Trends Biotechnol 22: 386 388, 2004. Khalina YN, Bartsch H, Petzhold D, Haase H, Podlubnaya ZA, Shpagina MD, and Morano I. Reconstitution of ventricular myosin with atrial light chains-1 improves its functional properties. Acta Biochim Pol 2005. Monaghan AP, Kioschis P, Wu W, Zuniga A, Bock D, Poustka A, Delius H, and Niehrs C. Dickkopf genes are co-ordinately expressed in mesodermal lineages. Mech Dev 87: 4556, 1999. Paulsson M and Heinegard D. Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage. Biochem J 183: 539 545, 1979. Ritter O, Luther HP, Haase H, Baltas LG, Baumann G, Schulte HD, and Morano I. Expression of atrial myosin light chains but not alphamyosin heavy chains is correlated in vivo with increased ventricular function in patients with hypertrophic obstructive cardiomyopathy. J Mol Med 77: 677 685, 1999. Saharinen J, Hyytiainen M, Taipale J, and Keski-Oja J. Latent transforming growth factor-beta binding proteins (LTBPs)structural extracellular matrix proteins for targeting TGF-beta action. Cytokine Growth Factor Rev 10: 99 117, 1999.

115

2.

3.

4.

5.

6. 7. 8. 9. 10. 11.

12. 13. 14.

15.

16. Schellings MW, Pinto YM, and Heymans S. Matricellular proteins in the heart: possible role during stress and remodeling. Cardiovasc Res 64: 24 31, 2004. 17. Segat D, Frie C, Nitsche PD, Klatt AR, Piecha D, Korpos E, Deak F, Wagener R, Paulsson M, and Smyth N. Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart. Matrix Biol 19: 649 655, 2000. 18. Small EM and Krieg PA. Molecular regulation of cardiac chamberspecic gene expression. Trends Cardiovasc Med 14: 1318, 2004. 19. Spragg DD, Akar FG, Helm RH, Tunin RS, Tomaselli GF, and Kass DA. Abnormal conduction and repolarization in late-activated myocardium of dyssynchronously contracting hearts. Cardiovasc Res 67: 77 86, 2005. 20. Spragg DD, Leclercq C, Loghmani M, Faris OP, Tunin RS, DiSilvestre D, McVeigh ER, Tomaselli GF, and Kass DA. Regional alterations in protein expression in the dyssynchronous failing heart. Circulation 108: 929 932, 2003. 21. Stoughton R and Dai H. USPTO Patent Full-Text and Image Database. Statistical Combining of Cell Expression Proles. US Patent No. 6351712 (online). http://patft.uspto.gov/netacgi/nph-Parser?Sect1 PTO1&Sect2 HITOFF&d PALL&p 1&u %2Fnetahtml%2FPTO%2Fsrchnum.htm& r 1&f G&l 50&s1 6351712.PN.&OS PN/6351712&RS PN/6351712 [22 May 2006]. 22. Strom A, Franzen A, Wangnerud C, Knutsson AK, Heinegard D, and Hultgardh-Nilsson A. Altered vascular remodeling in osteopontin-decient atherosclerotic mice. J Vasc Res 41: 314 322, 2004. 23. Sutsch G, Brunner UT, von Schulthess C, Hirzel HO, Hess OM, Turina M, Krayenbuehl HP, and Schaub MC. Hemodynamic performance and myosin light chain-1 expression of the hypertrophied left ventricle in aortic valve disease before and after valve replacement. Circ Res 70: 10351043, 1992. 24. Trueblood NA, Xie Z, Communal C, Sam F, Ngoy S, Liaw L, Jenkins AW, Wang J, Sawyer DB, Bing OH, Apstein CS, Colucci WS, and Singh K. Exaggerated left ventricular dilation and reduced collagen deposition after myocardial infarction in mice lacking osteopontin. Circ Res 88: 1080 1087, 2001. 25. Tusher VG, Tibshirani R, and Chu G. Signicance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98: 5116 5121, 2001. 26. Wilkoff BL, Cook JR, Epstein AE, Greene HL, Hallstrom AP, Hsia H, Kutalek SP, and Sharma A. Dual-chamber pacing or ventricular backup pacing in patients with an implantable debrillator: the Dual Chamber and VVI Implantable Debrillator (DAVID) Trial. JAMA 288: 31153123, 2002. 27. Xie Z, Singh M, and Singh K. Osteopontin modulates myocardial hypertrophy in response to chronic pressure overload in mice. Hypertension 44: 826 831, 2004. 28. Zhang G, He B, and Weber GF. Growth factor signaling induces metastasis genes in transformed cells: molecular connection between Akt kinase and osteopontin in breast cancer. Mol Cell Biol 23: 6507 6519, 2003. 29. Zohar R, Zhu B, Liu P, Sodek J, and McCulloch CA. Increased cell death in osteopontin-decient cardiac broblasts occurs by a caspase-3independent pathway. Am J Physiol Heart Circ Physiol 287: H1730 H1739, 2004.

Downloaded from physiolgenomics.physiology.org on September 20, 2011

Physiol Genomics VOL

26

www.physiolgenomics.org