JTCS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Stephen Westaby
Rana Sayeed
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bovill, E.
Right arrow Articles by Shaw, T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Bovill, E.
Right arrow Articles by Shaw, T.
Related Collections
Right arrow Congestive Heart Failure
Right arrow Valve disease

J Thorac Cardiovasc Surg 2008;136:709-716
© 2008 The American Association for Thoracic Surgery

Evolving Technology

Induction by left ventricular overload and left ventricular failure of the human Jumonji gene (JARID2) encoding a protein that regulates transcription and reexpression of a protective fetal program

Esta Bovill, MBBSa,*, Stephen Westaby, MS PhDb, Shiney Reji, PhDa, Rana Sayeed, MDb, Alastair Crisp, MAc, Tony Shaw, PhDa

a Department of Medicine, University College London, London, UK
b John Radcliffe Hospital, Oxford, UK
c Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, UK

Received for publication August 19, 2007; revisions received January 23, 2008; accepted for publication February 15, 2008.

* Address for reprints: Esta Bovill, MBBS, c/o Oxford Heart Centre, John Radcliffe Hospital, Headley Way, Headington, Oxford OX3 9DU, UK. (Email: ebovill{at}doctors.net.uk).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Objective: We identified changes in Jumonji (JARID2) expression in failing human hearts and determined its effects on expressions of atrial natriuretic factor (ANF), myosin light chain 2a (MLC2A), and {alpha} myosin heavy chain (MHCA), genes associated with both human heart failure and the fetal gene program.

Methods: Left ventricular outflow tract cardiac biopsy samples were taken from 31 patients with aortic valvular stenosis. Hearts were grouped according to left ventricular size and function: nonfailing hearts (undilated with good function) and failing hearts (dilated with poor function). Protein levels were determined by Western blotting, and messenger RNA transcript levels by ratiometric reverse transcriptase–polymerase chain reaction. Luciferase assays in HL-2 cells were used to assess effects of Jarid2 on Anf, Mlc2a, and Mhca transcriptions. Chromatin immunoprecipitation was used to detect interaction of JARID2 with specific target-gene promoters.

Results: JARID2 and MHCA expressions were reduced in failing hearts, whereas MLC2A and ANF were increased. In HL-2 cell culture, Jarid2 suppressed Anf and Mlc2a but enhanced Mhca. Jarid2 expression was reduced by cyclic mechanical stress, with concomitant increased Anf and Mlc2a and decreased Mhca expressions, reproducing the expression profile found in decompensated human pressure overload.

Conclusion: Jumonji expression is reduced by mechanical stress in human heart failure from aortic stenosis. JARID2 regulates ANF, MLC2A, and MHCA transcription and contributes to reexpression of the fetal gene program in decompensated aortic stenosis. JARID2 appears important in transcriptional regulation of fetal genes and may emerge as a diagnostic marker for left ventricular decompensation in aortic stenosis.

Abbreviations and Acronyms {alpha}-MHC = {alpha} myosin heavy chain; ANF = atrial natriuretic factor; ANF = human gene for atrial natriuretic factor; Anf = mouse gene for atrial natriuretic factor; β-MHC = β myosin heavy chain; bp = base pairs; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; GFP = green fluorescent protein; JARID2 = human Jumonji gene; Jarid2 = mouse Jumonji gene; kb = kilobase; LV = left ventricle; LVEF = left ventricular ejection fraction; MHCA = human gene for {alpha} myosin heavy chain; Mhca = mouse gene for {alpha} myosin heavy chain; MLC-2 = myosin light chain 2; MLC2A = human gene for myosin light chain 2a; Mlc2a = mouse gene for myosin light chain 2a; mRNA = messenger RNA; PCR = polymerase chain reaction; RT-PCR = reverse transcriptase–polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
The initial response of the heart to hemodynamic overload is cardiac hypertrophy. When overload continues, however, the adaptive hypertrophic changes become deleterious, with progression to decompensated heart failure.1Go Heart failure is accompanied by structural and physiologic changes that include myocyte hypertrophy, interstitial fibrosis, and altered contractile function.2Go The phenotypic changes are mediated by the altered expression of particular classes of genes. These include genes encoding contractile proteins and proteins involved in calcium regulation and excitation–contraction coupling, together with reexpression of the fetal gene program.3Go This involves the ventricular reexpression of fetal and neonatal isoforms of contractile and other proteins, such as atrial natriuretic factor (ANF). These findings have been reported both in heart failure models3,4Go and in patients with heart failure.5-7Go. Within the human myocyte contractile apparatus, {alpha} myosin heavy chain ({alpha}-MHC) is the predominant myosin heavy chain isoform expressed in human ventricular myocardium, but messenger RNA (mRNA) expression of the gene for {alpha}-MHC, MHCA, is greatly reduced in failing hearts.8,9Go. There are also changes in expressions of genes for myosin light chain 2 (MLC-2) in both human heart failure and animal models of the disease.10Go Although several transcriptional regulators of ANF, the gene for MLC-2a (MLC2A), and MHCA expressions have been identified in the hypertrophied and failing heart (eg, GATA4, MEF2, Nkx-2.5), the precise molecular pathways responding to hemodynamic overload and to mechanical stress remain to be defined.11,12Go

The human Jumonji gene (JARID2) encodes a 1234–amino acid protein homologous to human retinoblastoma-binding protein 2.13Go JARID2 contains nuclear localization signals and an AT-rich interaction domain for DNA binding similar to other transcription factors, such as dead ringer (Dmel\retn) in Drosophila and SWI1 in yeast,14,15Go but it does not have a well-defined consensus binding sequence.16,17Go JARID2 has an exclusively nuclear distribution and has recently been identified as a transcriptional regulator of ANF,16Go {alpha}-MHC,18Go and cyclin D1.19-21Go

The mouse Jumonji gene (Jarid2) has been identified as an important factor for normal cardiac development in mice22,23Go; Jarid2 is expressed in cardiac and vascular tissues in adult mice, especially in the interventricular septum, ventricular free wall, and outflow tract. In Jarid2 –/– mice, malformations are found in the interventricular septum and outflow tract (ventricular septal defect and double-outlet right ventricle), and the expression patterns of several cardiac-specific genes are disrupted.22,23Go Ventricular ANF expression normally declines rapidly just before birth, whereas atrial expression is maintained,24Go but ANF expression remains high in the Jarid2 –/– murine ventricle.22Go Similarly, ventricular MLC2A expression falls during normal cardiac chamber development25Go but persists in Jarid2 –/– hearts. There is failure of the normal switching of β myosin heavy chain (β-MHC) to {alpha}-MHC in the murine ventricle near birth, with higher ventricular β-MHC levels and reduced MHCA expression in Jarid2 –/– mice.22Go

In view of these findings, we considered that JARID2 expression might be altered in patients with valvular heart disease and that JARID2 might contribute to the regulation of ANF, MLC2A, and MHCA. Patients with aortic stenosis manifest a wide spectrum of left ventricular (LV) function, from normal to severely impaired. In this study, our objectives were to elucidate the molecular response of human LV myocardium to increased afterload in aortic stenosis and to explore the diagnostic potential of these changes to influence the timing of cardiac surgery before hypertrophy devolves into dilatation. We elected to investigate several intuitively logical genes that are associated both with an increase in afterload in experimental animals and with altered cell-cycle influence (eg, ventricular septal defects) in gene-knockout mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Procurement of Human Myocardial Tissue
Under a protocol approved by the Oxford Radcliffe Research Ethics Committee and after informed consent, human myocardial biopsy samples were obtained from the LV outflow tract of selected patients undergoing isolated aortic valve replacement for aortic stenosis (ie, with pressure-overload cardiac hypertrophy). The specimens were obtained from the endocardium of the subvalvular region, away from the conduction tissue and in an area accessible by transcatheter endocardial biopsy. Patients with associated coronary artery disease were excluded. All patients underwent preoperative transthoracic echocardiography to determine LV ejection fraction (LVEF) and LV dimensions.26Go Study patients were selected to fall clearly into one of two groups on the basis of the echocardiographic assessment of LV function. The failing heart group (n = 15) had reduced LVEF and increased LV dimensions; the nonfailing group (n = 16) had preserved LVEF and normal LV dimensions (Go Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1 Preoperative echocardiographic assessment and New York Heart Association functional class of patients
 
Western Blotting for JARID2 and ANF
Myocardial biopsy samples were frozen and ground under liquid nitrogen and stored at –80°C. Protein was extracted into a urea buffer (9-mol/L urea, 2% sodium dodecylsulfate, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 0.5% Triton X-100 [The Dow Chemical Company, Midland, Mich], 20–mmol/L dithiothreitol, and 50-mmol/L tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4), and the concentration of each sample was determined by Bradford assay (Sigma–Aldrich Co, St Louis, Mo). Human JARID2 gene product in myocardial protein isolates was detected by Western blotting. Sodium dodecylsulfate–polyacrylamide gels were loaded with 100-µg aliquots of protein for electrophoresis and blotting. Blots were probed with a rabbit polyclonal primary antibody raised against a synthetic peptide of human JARID2 (accession NP004964; Eurogentec Ltd, Hythe, UK) that detects a predicted 145-kDa band for JARID2, a mouse anti–human ANF antibody (Abcam plc, Cambridge, UK) was used to detect ANF, and gel loading was confirmed by reprobing blots with a β-actin antibody (Abcam). Blots were probed with a horseradish peroxidase–labeled secondary antibody (Dako A/S, Glostrup, Denmark) and analyzed by chemiluminescence (Amersham Biosciences Europe GmbH, Freiburg, Germany) on a densitometer (Bio-Rad Laboratories Inc, Hercules, Calif) to allow calculation of the relative intensity of each band with proprietary software.

Ratiometric Reverse Transcriptase–Polymerase Chain Reaction for MHCA and MLC2A mRNA Expressions
Total RNA was extracted from myocardial biopsy samples with the QIAGEN RNeasy extraction protocol (QIAGEN Ltd, Crawley, United Kingdom), and 1 µg total RNA was reverse transcribed according to the manufacturer reverse transcriptase–polymerase chain reaction (RT–PCR) protocol with oligodeoxythymidine primers (Promega Corporation, Madison, Wis); PCR primer sequences and conditions are shown in Go Table 2. A fragment of 272 base pairs (bp) at the 3' end of human MLC2A (accession M94547) and a 296-bp fragment from the 3' end of human MHCA (accession D00943) were amplified, and the identities of the products were confirmed by sequencing. Amplified sequences were separated by gel electrophoresis and analyzed on a GeneGnome imaging system (Syngene; Synoptics Ltd, Cambridge, UK) to measure band intensity against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression within each sample.27Go


View this table:
[in this window]
[in a new window]

  		Table 2 Primer sequences and polymerase chain reaction conditions
 
Promoter–Luciferase Constructs and Luciferase Assays
The 2-kilobase (kb) MLC2A promoter was produced by PCR amplification of a described promoter region28Go from genomic DNA of HEK293 cells and insertion of the amplified sequence into the BamHI and EcoRI sites of pGL3 Basic Vector (Promega). The 5.6-kb MHCA promoter was provided by Dr J. Gulick and Prof J. Robbins (The Cleveland Clinic, Cleveland, Ohio),29Go and the 3.7-kb ANF promoter–luciferase construct was supplied by Prof M. Nemer (Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada).11Go Luciferase assays were performed by cotransfecting equimolar amounts of each construct and 40 pg of pRL-CMV Vector (Promega_ into HL-2 cells with the Effectene reagent (QIAGEN); vector-only control experiments were performed concurrently. Cell lysates were assayed for Photinus and Renilla luciferase activities after 48 hours with the Promega dual-luciferase assay system; Photinus luciferase activity was normalized against Renilla activity to control for variable transfection efficiencies.

Modified Chromatin Immunoprecipitation of jmj–Promoter Complexes
HL-2 cells were cotransfected with equimolar amounts of promoter–luciferase construct and jmj–green fluorescent protein (GFP)22Go or GFP vector alone, and a modified chromatin immunoprecipitation assay was performed as previously described30Go after 48 hours. For each precipitation reaction, a third of the lysate was used as a positive control, and total DNA was ethanol precipitated. Another third of the lysate was diluted 10-fold, and protein–DNA complexes were precipitated by an anti-GFP antibody (Upstate Biotechnology, Inc, Lake Placid, NY) and protein A–agarose. The remaining third of the lysate was processed in parallel with an irrelevant (anti-Myc) antibody. PCR amplification of the luciferase gene was performed on the washed immunoprecipitated complexes.30Go

Jarid2 Expression in Stressed HL-2 Cells
Murine HL-2 cells, derived from HL-1 cells, were kindly donated by Dr William Claycomb (Louisiana State University Medical Center, New Orleans, La) and grown in Claycomb media (JRH Biosciences, Lenexa, Kan) as previously described.31Go Cells were subcultured into gelatin- and fibronectin-coated FlexerCell plates (Flexcell International Corporation, Hillsborough, NC) for 24 hours, and then confluent layers of cells were subjected to 7.5% stretch at 60 Hz for up to 24 hours on a FlexerCell-4000 tension system (Flexcell). Experiments were performed in triplicate on three separate occasions. Cells were harvested by trypsinization and centrifugation, then washed in ice-cold phosphate-buffered saline solution before total RNA extraction with the QIAGEN RNeasy kit or total protein extraction into Radio Immuno Precipitation Assay (RIPA) buffer. RT–PCR was performed on 1-µg aliquots of total RNA with the primers and conditions shown in Table 2 to amplify 3'-terminal fragments of murine Jarid2 (458 bp), Mlc2a (342 bp), and Mhca (361 bp). Amplicon identity was confirmed by sequencing. GAPDH RT-PCR was performed on the same samples, and relative target mRNA expressions were calculated as before.

Statistical Analysis
Data are expressed as a mean ± SD. Groups were compared with Student t test or analysis of variance as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Jumonji Expression Reduction in Human Heart Failure
JARID2 protein was reduced by 60% (P < .005) in failing hearts relative to nonfailing hearts (Go Figure 1, A and B). Ventricular ANF protein was expressed only in failing hearts (Figure 1, A). MLC2A mRNA was not detectable by RT–PCR in nonfailing hearts but was present in failing hearts (Figure 1, A). There was a 42% decrease (P < .005) in MHCA mRNA expression in failing hearts relative to nonfailing hearts (Figure 1, A and C).


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Figure 1. Jumonji (JARID2) levels are decreased in failing human ventricular biopsy samples. A, Representative Western blots (top panel) and reverse transcriptase–polymerase chain reaction (RT-PCR) gels (lower panel) from nonfailing and failing hearts. Jumonji protein expression is reduced in ventricular biopsy samples from failing hearts relative to nonfailing hearts; conversely, atrial natriuretic factor (ANF) is not detectable in nonfailing ventricles but is present in failing hearts. Reverse transcriptase–polymerase chain reaction for {alpha} myosin heavy chain ({alpha}MHC) messenger RNA shows decreased expression in failing hearts, whereas myosin light chain 2a (MLC2a) mesenger RNA is reexpressed in ventricular tissue from failing hearts. B, Western blot densitometry shows 60% decrease in Jumonji protein levels in failing relative to nonfailing hearts. Asterisk indicates P < .005. C, Ratiometric reverse transcriptase–polymerase chain reaction shows 42% decrease in {alpha} myosin heavy chain messenger RNA expression in failing hearts. Asterisk indicates P < .005. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

 
Jumonji Regulation of Anf, Mlc2a, and Mhca Expressions in a Dose-Dependent Manner
In murine HL-2 cell cotransfection experiments, Jumonji induced dose-dependent suppression of Anf (Go Figure 2, A, left columns) and Mlc2a (Figure 2, B) expressions. In contrast, Jumonji enhanced Mhca expression in a dose-dependent manner (Figure 2, C).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Figure 2. Murine gene Jarid2 mediates suppression of atrial natriuretic peptide (ANF) and myosin light chain 2a (MLC2a) gene transcription but promotion of {alpha} myosin heavy chain (aMHC) gene transcription. Control level of transcription of each promoter–luciferase construct in presence of 0.25 pmol empty vector is set at 100, and other values are expressed relative to control. Increasing amounts of transfected plasmid from 0.1 to 0.25 pmol are denoted by black wedges. Jarid2 induces dose-dependent supression of ANF (left column) and MLC2a (central column) transcription but dose-dependent activation of aMHC transcription (right column). Data shown represent at least three independent experiments, each performed in triplicate. Asterisk indicates P < .005 versus control. pEGFP, Plasmid enhanced green fluorescent protein.

 
Jumonji interaction With Anf, Mlc2a, and Mhca Promoters in HL-2 cells
With a modified chromatin immunoprecipitation assay, Jarid2–GFP was immunoprecipitated with target promoter–luciferase constructs. PCR for luciferase in precipitated protein–DNA complexes confirmed that Jarid2–GFP binds to Anf, Mlc2a, and Mhca promoters (Go Figure 3).


Figure 3
View larger version (37K):
[in this window]
[in a new window]

  		Figure 3. Coimmunoprecipitation of jmj–green fluorescent protein (GFP) complex with atrial natriuretic factor (ANF), myosin light chain 2a (MLC-2a), and {alpha} myosin heavy chain ({alpha}MHC) promoters. An anti–green fluorescent protein antibody was used to precipitate jmj–promoter complexes formed by cotransfection of Jarid2–GFP, and target promoter–luciferase construct into HL-2 cells and polymerase chain reaction for luciferase (Luc) confirmed Jarid2–GFP binding to target promoter; target promoters are for atrial natriuretic factor, myosin light chain 2a, and {alpha} myosin heavy chain. Negative controls (neg. cont.) are GFP plasmid vector only and irrelevant antibody (anti-Myc); cell lysates (Input) are used as positive controls.

 
Reductions of Jarid2 and Mhca Expressions but Increases of Anf and Mlc2a Expressions With Mechanical Stress
Expression of mRNA in stressed HL-2 cell layers was assessed by ratiometric RT–PCR. Mechanical stress induced a time-dependent reduction in Jarid2 expression but had no effect on GAPDH (Go Figure 4, A): Jarid2 expression was decreased by 85% after 24 hours with mechanical stress relative to baseline (P < .01, Figure 4, B). Changes in Jarid2 expression were accompanied by increased ANF protein and MLC2A mRNA levels and decreased MHCA expression (Figure 4, A).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		Figure 4. Gene Jarid2 is supressed by mechanical stress. A, reverse transcriptase–polymerase chain reactions for Jarid2, myosin light chain 2a (MLC2a), {alpha} myosin heavy chain ({alpha}MHC), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with Western blotting for atrial natriuretic protein (ANF), were performed on extracts of HL-2 cells subjected to mechanical stress up to 24 hours. These gels show time-dependent reductions in Jarid2 and {alpha} myosin heavy chain messenger RNA expressions and time-dependent increases in myosin light chain 2a messenger RNA and atrial natriuretic factor protein expressions. B, Ratiometric reverse transciptase–polymerase chain reaction shows that Jarid2 expression decreases with longer duration of mechanical stress. Asterisk indicates P < .005 relative to baseline (repeated measures analysis of variance).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
We studied JARID2 expression in the failing human heart, in which altered ventricular ANF, MLC2A, and MHCA expressions are well recognized. We also examined the interactions of Jarid2 with specific target promoters and other transcription factors in murine cell culture. In patients with aortic stenosis, we have shown the reexpression of a fetal gene program in cardiac myocytes subjected to increased afterload.

Limitations of the Study
All patients with aortic stenosis have LV hypertrophy develop, so their myocardium cannot be considered normal even when ventricular function is preserved. As such, it may not be possible to extrapolate our findings to all cases of heart failure. For this clinical study, we selected patients in whom well-preserved LV function could clearly be differentiated from those with LV dilatation and heart failure in response to afterload.

Regulation of MHCA Transcription by JARID2
The protein {alpha}-MHC has higher adenosine triphosphatase activity than does β-MHC and produces faster shortening velocity of cardiac myofibers. Lower ventricular systolic resistance and greater contractile force are observed in hearts with {alpha}-MHC as the predominant myosin heavy chain isoform.32,33Go. The {alpha}-MHC levels are greatly reduced in failing hearts, regardless of etiology, and this has been proposed as an important factor for reduced myocardial contractility in heart failure.8,34Go Several regulatory elements and their binding factors, including GATA4 and MEF2A, that promote cardiac expression of MHCA have been identified,35Go but molecular mechanisms underlying the suppression of MHCA expression in heart failure remain unknown. We have shown that JARID2 interacts with the MHCA promoter to activate MHCA transcription in a dose-dependent manner. Our results differ from those of Kim and associates,18Go who used a small promoter fragment (368 bp) to show transcriptional suppression by JARID2, whereas we used a much larger fragment (>5 kb). The suppression apparent on the minimal promoter may not be significant when a larger regulatory region is used. We also have shown that JARID2 expression is decreased in human heart failure, and this may contribute to the reduction in {alpha}-MHC levels seen in failing hearts. This finding is compatibile with the reduced ventricular Mhca expression found in Jarid2 –/– mice.22Go Thus JARID2 has a role in the regulation of MHCA gene expression.

Regulation of MLC2A Transcription by JARID2
The protein MLC-2a is seen throughout the fetal heart but is restricted to the atria in later development.36Go MLC2A expression in the ventricular myocardium instead of MLC2V produces a dilated cardiomyopathy characterized by impaired contractility and reduced ventricular ejection fraction.37Go It appears that the restriction of MLC2A expression to the adult atrium is achieved through suppression of ventricular MLC2A transcription.38Go We have shown that JARID2 suppresses MLC2A transcription, suggesting that JARID2 contributes to the normal mechanism for suppression of ventricular MLC2A expression. We have also shown low-level MLC2A expression in the ventricular myocardium of the failing hearts; this may be related to reduced jmj levels.

Regulation of ANF Transcription by JARID2
During fetal development, ANF is expressed in both atria and ventricles, but it is restricted to the atria near birth.39Go The minimal mammalian Anf promoter sufficient for correct chamber-specific expression in mice40Go contains binding sites for SRF, GATA4, and Nkx-2.5 and was included in the construct used in this study. ANF expression is regulated by Nkx-2.5 and GATA4, and mutation of either binding site results in a failure of ANF transcription.38Go This finding is in agreement with those of previous studies with cultured cardiomyocytes, which showed that mutation of the NKE resulted in upregulation of ANF promoter activity in ventricular cells.12Go Small and Kreig38Go interpreted these results as an indication that the GATA and NKE sites are recognition sequences for an unidentified suppressor of ventricular ANF expression. Transgenic mice that overexpress myocardial Nkx-2.5 show persistent ventricular ANF expression,41Go suggesting that ventricular Nkx-2.5 competes with a putative suppressor factor. Studies of the ANF promoter suggest that, as with MLC2A, atrial-restricted expression of ANF is achieved through inhibition of ventricular expression. Our group and others16Go have independently identified JARID2 as a suppressor of ANF transcription. We have shown that a decrease in JARID2 levels, as found after mechanical stress and in human heart failure, is associated with increased ANF expression. This observation, together with the finding that JARID2 associates with the ANF promoter in vitro, strongly suggests that increased ANF expression in failing hearts is at least partly the result of removal of suppression by declining JARID2.

Regulation of Jarid2 Levels by Mechanical Stress
Heart failure may follow hemodynamic overload. Myocytes are direct sensors of hemodynamic load that may be mimicked in vitro by subjecting cultured cell layers to mechanical stress. Murine HL-2 cells subjected to mechanical stress in vitro showed a striking and significant decrease (85%) in Jarid2 levels. RT–PCR of stressed cell extracts showed increased Mlc2a and decreased Mhca expressions. Western blotting of parallel samples showed increased ANF in stressed cells. This is consistent with our observations that Jarid2 suppresses Mlc2a and ANF expressions while activating Mhca expression, and it is also consistent with the upregulation of ANF expression by mechanical stress.

Decreased Jumonji Protein Expression in Failing Human Hearts
JARID2 is a factor responsive to mechanical stress: this led us to examine JARID2 in pressure-overload induced heart failure. Immunoblot analysis showed decreased JARID2 protein product in LV biopsy samples from failing hearts relative to samples from nonfailing hearts with preserved LV function. These changes in protein level are consistent with those observed for mRNA in stressed cultured cells. We speculate that the stimuli for reduced JARID2 expression are chamber dilatation and increased wall stress, and reduced JARID2 expression in turn leads to misexpressions of ANF, MLC2A and MHCA.


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
This study has demonstrated a previously unrecognized regulatory function of JARID2 with possible roles in regulating ANF, MLC2A, and MHCA genes in heart failure. We have shown that murine Jarid2 expression is downregulated in response to mechanical stress and that this is associated with decreased Mhca expression but increased Mlc2a and Anf expressions. JARID2 is a crucial factor for the regulation of ANF, MLC-2a and {alpha}-MHC proteins. Normal ANF and MLC2A expressions may be controlled by a balance among the actions of JARID2, GATA4, and Nkx-2.5; in the failing human heart, reduced JARID2 expression thus contributes to increased ANF and MLC2A expressions and to decreased MHCA expression, which may ultimately contribute to the contractile insufficiency.

Future Work
It may be that the LV responds to increased afterload by reexpressing the fetal gene program. Our study suggests that this occurs at or after the point at which the LV begins to dilate, and that human JARID2 is an important transcriptional regulator central to the timing of this breaking point. This suggests we might perform JARID2 characterization by endomyocardial biopsy during the assessment of the severity of aortic stenosis (while LV function remains well preserved). When JARID2 or cell cycle or fetal reprogramming genes are expressed, the cardiologist could argue that the time for aortic valve replacement has arrived. A panel of fetal reprogramming genes could be used to pinpoint the breaking point at which LV hypertrophy switches to LV dilatation. Genetic studies could thus be a useful clinical tool when recommending the timing of aortic valve replacement.

We now aim to study a full range of patients with aortic stenosis and regurgitation. We plan to use logistic regression analysis to further define the relationship between JARID2 and LVEF.


   Acknowledgments
 
We thank Prof M. Nemer (Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada), Dr J. Gulick and Prof J. Robbins (The Cleveland Clinic, Cleveland, Ohio), and Prof G. Lyons and Dr Y. Lee (University of Wisconsin School of Medicine and Public Health, Madison, Wis) for their kind gift of plasmids.


   Footnotes
 
Supported by research grants from the British Heart Foundation (E.B., S.W., and T.S.), the Artificial Heart Fund (S.W. and T.S.), and the Oak Foundation (T.S.).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

  1. Katz AM. Heart failure: a hemodynamic disorder complicated by maladaptive proliferative responses. J Cell Mol Med 2003;7:1-10.[Medline]
  2. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev 1999;79:215-262.[Abstract/Free Full Text]
  3. Chien KR, Knowlton KU, Zhu H, Chien S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J 1991;5:3037-3046.[Abstract]
  4. Vesely DL. Atrial natriuretic peptides in pathophysiological diseases. Cardiovasc Res 2001;51:647-658.[Free Full Text]
  5. Drexler H, Hanze J, Finckh M, Lu W, Just H, Lang RE. Atrial natriuretic peptide in a rat model of cardiac failure. Atrial and ventricular mRNA, atrial content, plasma levels, and effect of volume loading. Circulation 1989;79:620-633.[Abstract/Free Full Text]
  6. Sugawara A, Nakao K, Morii N, Yamada T, Itoh H, Shiono S, et al. Synthesis of atrial natriuretic polypeptide in human failing hearts. Evidence for altered processing of atrial natriuretic polypeptide precursor and augmented synthesis of β-human ANP. J Clin Invest 1988;81:1962-1970.[Medline]
  7. Tsuchimochi H, Kurimoto F, Ieki K, Koyama H, Takaku F, Kawana M, et al. Atrial natriuretic peptide distribution in fetal and failed adult human hearts. Circulation 1988;78:920-927.[Abstract/Free Full Text]
  8. Nakao K, Minobe W, Roden R, Bristow MR, Leinwand LA. Myosin heavy chain gene expression in human heart failure. J Clin Invest 1997;100:2362-2370.[Medline]
  9. Lowes BD, Minobe W, Abraham WT, Rizeq MN, Bohlmeyer TJ, Quaife RA, et al. Changes in gene expression in the intact human heart. Downregulation of {alpha}-myosin heavy chain in hypertrophied, failing ventricular myocardium. J Clin Invest 1997;100:2315-2324.[Medline]
  10. Walsh R, Henkel R, Robbins J. Cardiac myosin heavy- and light-chain gene expression in hypertrophy and heart disease. Heart Failure 1991;6:238-243.
  11. Morin S, Charron F, Robitaille L, Nemer M. GATA-dependent recruitment of MEF2 proteins to target promoters. EMBO J 2000;19:2046-2055.[Medline]
  12. Durocher D, Chen CY, Ardati A, Schwartz RJ, Nemer M. The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium. Mol Cell Biol 1996;16:4648-4655.[Abstract/Free Full Text]
  13. Fattaey AR, Helin K, Dembski MS, Dyson N, Harlow E, Vuocolo GA, et al. Characterization of the retinoblastoma binding proteins RBP1 and RBP2. Oncogene 1993;8:3149-3156.[Medline]
  14. Gregory SL, Kortschak RD, Kalionis B, Saint R. Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins. Mol Cell Biol 1996;16:792-799.[Abstract/Free Full Text]
  15. Peterson CL, Herskowitz I. Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global activator of transcription. Cell 1992;68:573-583.[Medline]
  16. Kim TG, Chen J, Sadoshima J, Lee Y. Jumonji represses atrial natriuretic factor gene expression by inhibiting transcriptional activities of cardiac transcription factors. Mol Cell Biol 2004;24:10151-10160.[Abstract/Free Full Text]
  17. Kim TG, Kraus JC, Chen J, Lee Y. JUMONJI, a critical factor for cardiac development, functions as a transcriptional repressor. J Biol Chem 2003;278:42247-42255.[Abstract/Free Full Text]
  18. Kim TG, Jung J, Mysliwiec MR, Kang S, Lee Y. Jumonji represses {alpha}-cardiac myosin heavy chain expression via inhibiting MEF2 activity. Biochem Biophys Res Commun 2005;329:544-553.[Medline]
  19. Toyoda M, Shirato H, Nakajima K, Kojima M, Takahashi M, Kubota M, et al. jumonji downregulates cardiac cell proliferation by repressing cyclin D1 expression. Dev Cell 2003;5:85-97.[Medline]
  20. Ohno T, Nakajima K, Kojima M, Toyoda M, Takeuchi T. Modifiers of the jumonji mutation downregulate cyclin D1 expression and cardiac cell proliferation. Biochem Biophys Res Commun 2004;317:925-929.[Medline]
  21. Takahashi M, Kojima M, Nakajima K, Suzuki-Migishima R, Takeuchi T. Functions of a jumonji-cyclin D1 pathway in the coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain. Dev Biol 2007;303:549-560.[Medline]
  22. Lee Y, Song AJ, Baker R, Micales B, Conway SJ, Lyons GE. Jumonji, a nuclear protein that is necessary for normal heart development. Circ Res 2000;86:932-938.[Abstract/Free Full Text]
  23. Takeuchi T, Kojima M, Nakajima K, Kondo S. jumonji gene is essential for the neurulation and cardiac development of mouse embryos with a C3H/He background. Mech Dev 1999;86:29-38.[Medline]
  24. Argentin S, Ardati A, Tremblay S, Lihrmann I, Robitaille L, Drouin J, et al. Developmental stage-specific regulation of atrial natriuretic factor gene transcription in cardiac cells. Mol Cell Biol 1994;14:777-790.[Abstract/Free Full Text]
  25. Lyons GE. In situ analysis of the cardiac muscle gene program during embryogenesis. Trends Cardiovasc Med 1994;4:70-77.[Medline]
  26. Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 1978;58:1072-1083.[Abstract/Free Full Text]
  27. Kemp TJ, Sadusky TJ, Simon M, Brown R, Eastwood M, Sassoon DA, et al. Identification of a novel stretch-responsive skeletal muscle gene (Smpx). Genomics 2001;72:260-271.[Medline]
  28. Doevendans PA, Bronsaer R, Lozano PR, Kubalak S, van Bilsen M. The murine atrial myosin light chain-2 gene: a member of an evolutionarily conserved family of contractile proteins. Cytogenet Cell Genet 2000;90:248-252.[Medline]
  29. Gulick J, Hewett TE, Klevitsky R, Buck SH, Moss RL, Robbins J. Transgenic remodeling of the regulatory myosin light chains in the mammalian heart. Circ Res 1997;80:655-664.[Abstract/Free Full Text]
  30. Wu WS, Vallian S, Seto E, Yang WM, Edmondson D, Roth S, et al. The growth suppressor PML represses transcription by functionally and physically interacting with histone deacetylases. Mol Cell Biol 2001;21:2259-2268.[Abstract/Free Full Text]
  31. Claycomb WC, Lanson Jr. NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A 1998;95:2979-2984.[Abstract/Free Full Text]
  32. Shroff SG, Motz W. Left ventricular systolic resistance in rats with hypertension and hypertrophy. Am J Physiol 1989;257(2 Pt 2):H386-H394.[Medline]
  33. Shroff SG, Naegelen D, Clark WA. Relation between left ventricular systolic resistance and contractile rate processes. Am J Physiol 1990;258(2 Pt 2):H381-H394.[Medline]
  34. Miyata S, Minobe W, Bristow MR, Leinwand LA. Myosin heavy chain isoform expression in the failing and nonfailing human heart. Circ Res 2000;86:386-390.[Abstract/Free Full Text]
  35. Molkentin JD, Kalvakolanu DV, Markham BE. Transcription factor GATA-4 regulates cardiac muscle-specific expression of the {alpha}-myosin heavy-chain gene. Mol Cell Biol 1994;14:4947-4957.[Abstract/Free Full Text]
  36. Kubalak SW, Miller-Hance WC, O'Brien TX, Dyson E, Chien KR. Chamber specification of atrial myosin light chain-2 expression precedes septation during murine cardiogenesis. J Biol Chem 1994;269:16961-16970.[Abstract/Free Full Text]
  37. Chen J, Kubalak SW, Minamisawa S, Price RL, Becker KD, Hickey R, et al. Selective requirement of myosin light chain 2v in embryonic heart function. J Biol Chem 1998;273:1252-1256.[Abstract/Free Full Text]
  38. Small EM, Krieg PA. Molecular regulation of cardiac chamber-specific gene expression. Trends Cardiovasc Med 2004;14:13-18.[Medline]
  39. Zeller R, Bloch KD, Williams BS, Arceci RJ, Seidman CE. Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev 1987;1:693-698.[Abstract/Free Full Text]
  40. Knowlton KU, Rockman HA, Itani M, Vovan A, Seidman CE, Chien KR. Divergent pathways mediate the induction of ANF transgenes in neonatal and hypertrophic ventricular myocardium. J Clin Invest 1995;96:1311-1318.[Medline]
  41. Takimoto E, Mizuno T, Terasaki F, Shimoyama M, Honda H, Shiojima I, et al. Up-regulation of natriuretic peptides in the ventricle of Csx/Nkx2-5 transgenic mice. Biochem Biophys Res Commun 2000;270:1074-1079.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Stephen Westaby
Rana Sayeed
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bovill, E.
Right arrow Articles by Shaw, T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Bovill, E.
Right arrow Articles by Shaw, T.
Related Collections
Right arrow Congestive Heart Failure
Right arrow Valve disease

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ANN THORAC SURG ASIAN CARDIOVASC THORAC ANN EUR J CARDIOTHORAC SURG
J THORAC CARDIOVASC SURG ICVTS ALL CTSNet JOURNALS