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J Thorac Cardiovasc Surg 2006;132:1262-1271
© 2006 The American Association for Thoracic Surgery

Surgery for Congenital Heart Disease

Myocardial transcription factors are modulated during pathologic cardiac hypertrophy in vivo

^a University of California, San Francisco, Department of Surgery, San Francisco, Calif
^b University of California, San Francisco, Department of Pediatrics, San Francisco, Calif.

Read at the Eighty-sixth Annual Meeting of The American Association for Thoracic Surgery, Philadelphia, Pa, April 29-May 3, 2006.

Received for publication April 27, 2006; revisions received June 30, 2006; accepted for publication August 10, 2006.

^_* Address for reprints: Anthony Azakie, MD, 513 Parnassus Ave, Room S-549, Box 0117, University of California San Francisco, San Francisco, CA 94143. (Email: azakiet{at}surgery.ucsf.edu).

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion In Vivo Model of... Appendix E1: Chromatin... References

 
OBJECTIVES: In the current study we describe and characterize a novel ovine model of biventricular hypertrophy and heart failure and evaluate the role of selected cardiac transcription factors in the regulation of cardiac gene expression during pathologic hypertrophy in vivo. The cardiac troponin T promoter is used as a model gene.

METHODS AND RESULTS: Transient transfections of ovine cardiomyocytes in culture show that Sp1, transcriptional enhancer factor-1, and myocyte enhancer factor-2 activate cardiac troponin T promoter constructs. Cotransfection of Sp3 inhibits cardiac troponin T promoter activity and represses Sp1-mediated activation of the cardiac troponin T promoter. By chromatin immunoprecipitation, transcriptional enhancer factor-1, myocyte enhancer factor-2, NKX2.5, GATA-4, and Sp factors bind the cardiac troponin T promoter in vivo. To assess the role of cardiac transcription during pathologic hypertrophy, in vivo, we created surgical aorta-pulmonary shunts in utero in fetal lambs. Two weeks after spontaneous delivery, shunted lambs showed failure to thrive, significant biventricular hypertrophy, and heart failure. Shunted hearts had significant increases in myosin and cardiac troponin T protein expression. There was a shift in expression to the high-molecular-weight fetal isoforms. Transcriptional enhancer factor-1, myocyte enhancer factor-2, GATA-4, NKX2.5, and Sp1 transcription factor levels were increased in all heart chambers of shunted animals. Sp3 expression was decreased in shunted ventricles. Immunoprecipitated Sp3 was associated with significant increases in histone acetyl transferase activity and decreases in histone-deacetylase activity.

CONCLUSION: The shunted neonatal lamb is a valid, novel model of pathologic biventricular hypertrophy. During pathologic hypertrophy myocardial transactivators are upregulated while repressors are downregulated.

	Introduction

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Cardiac failure and dysfunction can result from numerous clinicopathologic conditions including hypertension, ischemic heart disease, valvular heart disease, cardiomyopathy, and congenital heart disease. The need for effective broad-based therapy for patients with heart failure far outweighs and has outpaced current therapeutic strategies. Further understanding of the complex cellular mechanisms that lead to heart failure may aid in the development of novel and effective therapies.

Various pathologic signals, stressors, or biomechanical forces elicit different intracellular signaling pathways that ultimately stimulate fetal cardiac gene expression (in children and adults) and pathologic hypertrophy, an early milestone in heart failure.^1,2 Although numerous biomechanical forces and pathologic conditions can result in myocardial dysfunction, it is hypothesized that there is a final common pathway in which transcriptional alterations in sarcomeric gene expression, common to many forms of pathologic hypertrophy (concentric, eccentric), result in contractile dysfunction of the heart. Thus, contractile dysfunction of the heart can be viewed as a transcriptional disorder.^3,4

Transcription factors are nuclear proteins that bind promoter regulatory elements to activate or repress gene expression. The combinatorial interactions between transcription factors and promoter elements that are required for the regulation of cardiac gene expression during normal cardiac development probably operate during pathologic cardiac remodeling and hypertrophy. NKX2.5, myocyte enhancer factor 2 (MEF2), HAND, and GATA transcription factors have been implicated in transcriptional control, pattern formation, and segmental control of cardiac development.^5-8 Using the cardiac troponin T (cTnT) promoter as a model gene, we have shown that transcriptional enhancer factor-1 (TEF-1), MEF2, and Sp family of transcription factors also contribute to the developmental regulation of cardiac gene expression in avian embryonic cardiomyocytes.^9,10 These myocardial transcription factors, which are important for normal growth, development, and maintenance of the cardiac phenotype, may also be implicated during induction of fetal genes and the upregulation of constitutive genes that is seen with pathophysiologic hypertrophy.^2,11

Recent interest in "transcriptional disorders of the heart" has also implicated cardiac histone acetylation in the development of pathologic cardiac hypertrophy and heart failure. Histone acetyl transferases (HATs) are believed to acetylate histone proteins, relax chromatin, and expose "prohypertrophic" genes for activation by cardiogenic transcription factors.^12,13 Histone deacetylases (HDAC classes I and II) have also been implicated in regulation of myocardial gene expression and cardiac hypertrophy.^4,12,14 Class I HDACs may act as prohypertrophic agonists.^15-17 Thus, isoform-specific inhibitors of class I HDACs and/or cardiac-specific HATs may have an important and specific inhibitory effect on the development of pathologic cardiac hypertrophy, contractile dysfunction, and heart failure.^15-19

Recently, Kee and colleagues¹⁵ have shown that inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding. Similarly, Kong and colleagues²⁰ reported that suppression of class I and II HDACs blunts pressure-overload cardiac hypertrophy. Suppression of ventricular growth was well tolerated in terms of both clinical outcome and cardiac performance measures. Interstitial fibrosis was diminished in hearts treated with HDAC inhibitors, and collagen synthesis in isolated cardiac fibroblasts was suppressed. There was no evidence of cell death or apoptosis. Systolic function in the setting of blunted hypertrophic growth was preserved, as shown by echocardiography and hemodynamic data. The hypertrophy-associated switch of adult and fetal sarcomeric isoforms was attenuated, which likely contributed to the observed preservation of systolic function in HDAC inhibitor-treated hearts. Both these studies suggest that HDAC inhibition is a viable therapeutic strategy that holds promise in the treatment of pressure load-induced heart disease. The effects of HDAC inhibition on volume-loaded heart dysfunction are not known.

The aims of the current study are (1) to biochemically and morphologically characterize a clinically relevant model of pathologic hypertrophy and congestive heart failure and (2) to determine the changes in expression levels and posttranslational modifications of selected cardiac transcription factors. In utero placement of aorta–pulmonary arterial shunts has been reported as a model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs²¹ and is used in this study as a model of pathologic cardiac hypertrophy. Surgically created aorta-pulmonary shunts in fetal lambs produced left ventricular volume overload and right ventricular pressure overload, leading to altered contractile protein gene expression, biventricular hypertrophy, and congestive heart failure. In the shunted lamb model, pathologic cardiac remodeling and deleterious cardiac gene expression occurred in association with changes in the levels and modification states of cardiac-specific transcription factors (GATA-4, DTEF-1, Sp1, Sp3, and NKX2.5). Furthermore, we show that, functionally, TEF-1, MEF2, and Sp1 factors, which bind the cTnT promoter within the context of chromatin, transactivate the cTnT promoter in ovine cardiomyocytes, implicating them as important regulators of the pathologic hypertrophic process.

	Materials and Methods

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Reagents
Media, serum, enzymes, reagents, and materials for tissue culture were purchased from the Cell Culture facility at the University of California, San Francisco. Oligonucleotides were purchased from Operon Technologies Inc (Alameda, Calif) or Invitrogen (Carlsbad, Calif).

Plasmids and Constructs: Tissue Culture, Cell Transfections, and Reporter Gene Assays
The promoter/reporter constructs used in this study consist of 268 nucleotides of the chicken cTnT promoter upstream of the transcription initiation site (Gallus domesticus cardiac isoform of troponin T [TNT] gene, exon 1, and promoter sequence. ACCESSION# M5790). Luciferase reporter constructs were created and subcloned into the plasmid pGL2 (Promega, Madison Wis), as previously described.⁹

Hearts were harvested from neonatal lambs and cardiomyocyte cultures established by standard techniques.¹⁰ The differentiation state of cultured cardiomyocytes was determined by light microscopy, presence of beating cardiomyocytes, immunocytochemistry for MF20, and Western blotting for myosins, cTnT, GATA-4, MEF2, and NKX2.5. Plasmid constructs were transfected according to the manufacturer’s protocol (Effectene Transfection kit; Qiagen, Valencia, Calif) with minor modifications.⁹ Reporter gene activity was determined with the Dual Luciferase reporter assay (DLR-Promega, Madison, Wis). Firefly light intensity was read on a Luminometer TD-20/20, DLR Ready (Turner Designs Instrument, Sunnyvale, Calif). Cotransfection with 20 ng phRL-TK reporter was used to standardize for transfection variability. Cotransfection efficiency of Sp, MEF2, and DTEF-1 expression constructs was verified with pSV-ß-galactosidase (Promega, Madison Wis) and/or pAAV-GFP staining. Reporter gene activity was expressed as mean ± standard deviation. Statistical comparisons were performed by paired t tests.

Nuclear Extracts
Cardiac muscle was harvested from 2-week-old lambs (control and shunted) at 4°C. Nuclear extracts were prepared by standard methods (Active Motif, Inc, Carlsbad, Calif).

Western Blot Analysis and Densitometry
Total protein concentration was quantitated with the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, Calif). An equal amount of protein was loaded in each lane for Western blots: sodium dodecylsulfate–polyacrylamide gel electrophoresis was used to separate nuclear proteins (10 µg) on a 10% gel and followed by transfer to a polyvinylidene difluoride membrane (Amersham Biosciences, GE Healthcare Bio-Sciences Corp, Piscataway, NJ). Membranes were then blocked and incubated with antibody as previously described.⁹ Reactive bands were visualized with the SuperSignal West Femto Maximum Sensitivity Substrate Kit (Pierce, Rockford, Ill) and Kodak 440CF image station (Kodak, New Haven, Conn). The image was selected and analyzed with the public domain program NIH Image, and band intensity was quantified. Parallel sodium dodecylsulfate gels and immunoblots of ß-actin and glyceraldehyde-3-phosphate dehydrogenase controls were performed to verify sample integrity and loaded quantity.

Surgical Preparations and Care: Ewes
Pregnant, mixed-breed Western ewes (135-140 days’ gestation; term = 145 days) were operated on under sterile conditions. Through a left lateral fetal thoracotomy, an 8.0-mm polytetrafluoroethylene vascular graft (2-mm length; W. L. Gore & Associates, Inc, Flagstaff, Ariz) was sewn between the ascending aorta and main pulmonary artery. All lambs were spontaneously delivered and were instrumented as previously described.²¹

Heart Tissue Preparation
Whole heart and chamber-specific weights were measured in all shunted and control specimens. Samples of right atrium, right ventricle, left atrium, left ventricle, and ventricular septum were excised, weighed, and used to make fresh nuclear preparations. Whole tissue and excess nuclear preparations were snap-frozen in liquid nitrogen. Samples were stored at –70°C until used for analysis.

Determination of DNA Content
DNA content was determined by standard methods (Genomic DNA mini-prep; Bay Gene Inc, San Francisco, Calif). Values were normalized to gram of cardiac muscle.

Statistical Analysis
Comparisons between shunt and age-matched controls were made by an unpaired t test.

All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco. All animals were put to death by appropriate methods as described in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

	Results

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In Utero Placement of Aorta–Pulmonary Shunts Results in Growth Failure, Four-chamber Cardiomegaly, and Biventricular Hypertrophy
Shunted lambs showed clinical evidence of failure to thrive. Compared with control animals, shunted lambs were tachypneic and demonstrated less activity and lower tolerance to stress after 1 week of age. By 2 weeks of age, shunted lambs had significantly slower weight gain than controls. The hearts of shunted lambs were significantly larger than those of controls (Figure 1, Table 1, and Figure E1). All four chambers showed substantial increases in mass without significant increases in DNA content, suggesting that hypertrophy and not hyperplasia was the predominant stress response. The left ventricle was dilated and showed evidence of eccentric hypertrophy, while the right ventricle was predominantly thick-walled with evidence of concentric hypertrophy.

View larger version (87K): [in this window] [in a new window]   Figure 1. Gross pathology of shunted hearts at 2 weeks of age. Left panel, Photograph of shunted heart. Right panel, Photograph of control heart.

View larger version (12K): [in this window] [in a new window]   Figure E1. Shunted hearts showed 4-chamber cardiomegaly.

Sarcomeric Protein and Myocardial Transcription Factor Levels Are Altered During Pathophysiologic Shunting in the Neonatal Ovine Heart
Western blots of shunted (n = 5) and control (n = 5) hearts were analyzed by band densitometry. The hearts of 2-week-old shunted lambs showed significant increases in myosin and cTnT expression (Figure 2 and Figure 3, A and B). Fetal, high-molecular-weight isoforms of cTnT were upregulated, consistent with the activation of a pathologic hypertrophic program (Figure 2). The increase in sarcomeric protein expression and associated fetal isoforms was seen in all four chambers. Associated with the increase in sarcomeric proteins, a variety of cardiac transcription factors known to govern cardiac gene expression during embryogenesis, growth, and development were upregulated (Figures 2 and 3). TEF-1 factors, which regulate muscle gene promoters (eg, cTnT, - and ß-myosin heavy chains, tropomyosins, ß-acetylcholine receptor, and -actin genes) through muscle CAT sites, were significantly upregulated in the right side of the heart (Figure 3, C). There was a trend to increased expression in the left atrium and left ventricle. Both GATA-4 and NKX2.5 levels were significantly increased in all four chambers (Figure 3, D and E). The magnitude of the increase in NKX2.5 levels, although statistically significant, was small (Figure 3, E). Densitometric analysis of Sp factor expression showed that Sp1, a transcriptional activator of cardiac promoters, was increased in all four chambers; Sp3, a transcriptional repressor of cTnT promoter constructs, was significantly downregulated during pathologic hypertrophy.

View larger version (16K): [in this window] [in a new window]   Figure 2. Characteristic Western blot of sarcomeric proteins and selected transcription factors in 2-week-old control and shunted lambs. Antibody used for Western blot analysis is listed on left. MF20 (antimyosin) and cTnT levels are increased in shunted animals, consistent with the hypertrophy and increased mass seen on gross pathologic examination. Fetal/embryonic isoforms of cTnT are expressed in the hypertrophied, shunted hearts. There are associated increases in DTEF-1, MEF2 (not shown), GATA4, NKX2.5, and Sp1 levels and concomitant decreases in Sp3 levels within the cardiac chambers (see Figure 5). ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control proteins are comparable in shunted and control specimens. RA, Right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; Sept, interventricular septal muscle; MW (kDa), molecular weight in kilo-Daltons.

View larger version (30K): [in this window] [in a new window]   Figure 3. Bar graphs of densitometric analysis of selected myocardial transcription factors during pathologic biventricular hypertrophy in neonatal lambs. Bands seen on Western blots of myocardial specimens from hearts of control (n = 5) and shunted (n = 5) neonatal lamb were analyzed by quantitative densitometry. Myosins and cardiac troponin protein expression are increased in all four chambers. TEF-1 proteins are primarily increased in the right side of the heart whereas NKX 2.5, GATA-4, and Sp1 are upregulated and Sp3 is downregulated in all four cardiac chambers. A, Myosin expression detected with MF20 antibody. B, Cardiac troponin T (cTnT). C. TEF-1 proteins. D, GATA-4. E, NKX2.5. F, Sp1. G, Sp3. ß-actin (H) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (I) were used as controls. RA, Right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; Septum, interventricular septal muscle.

View larger version (134K): [in this window] [in a new window]   Figure E2. Immunohistochemistry of sheep myocardium double-stained with MF20 (red) antibody and Sp1 antibody (green immunofluorescence).

View larger version (117K): [in this window] [in a new window]   Figure E3. Immunohistochemistry of sheep myocardium double-stained with MF20 (red) antibody and Sp3 antibody (green immunofluorescence).

View larger version (141K): [in this window] [in a new window]   Figure E4. Immunohistochemistry of sheep myocardium double-stained with MF20 (red) antibody and DTEF-1 antibody (green immunofluorescence).

View larger version (20K): [in this window] [in a new window]   Figure E5. Transcriptional complexes bind the cTnT promoter in vivo. Polymerase chain reaction using primers specific for cTnT promoter on DNA purified after chromatin immunoprecipitation. After cross-linking and sonication, chromatin complexes from neonatal sheep heart nuclear preparations were purified. Chromatin was incubated with antibodies to DTEF-1, MEF2, GATA-4, Nkx2.5, Sp1, Sp3, poly-ADP-ribose polymerase (PARP), poly-ADP-ribose (PAR), ß-actin, cTnT, and preimmune serum. Separation of immune complexes by elution from protein A/G/agarose beads was followed by reversal of protein-DNA crosslinks and DNA purification. Lane A depicts chromatin input DNA plus antibody. Lanes A-E include the ChIP "output" DNA for polymerase chain reactions. Immunoprecipitation of chromatin preparations was performed with antibodies listed on the left-sided column. Lane F contains ChIP buffer as the "DNA source." Lane G contains the control, "input DNA" to verify the quality of nuclear preparations. a, Output DNA; b, input DNA; c, polymerase chain reaction buffer and primers.

View larger version (15K): [in this window] [in a new window]   Figure 4. Luciferase reporter gene activity driven by –268cTnT promoter constructs that are transiently transfected in primary cultures of neonatal ovine cardiomyocytes. Baseline –268cTnT relative activity is increased when Sp1, DTEF-1A or B, and MEF2C are cotransfected. Cotransfection of Sp3 results in repression of baseline cTnT promoter activity. Sp3 inhibits Sp1-mediated gene activation. MEF2 and DTEF-1 have a synergistic effect on reporter gene activity. Horizontal axis, Cotransfected expression constructs. Vertical axis, Relative reporter gene activity indexed to TK-driven reporter to control for transfection variability. Cells alone, pGL2 basic vector, and pcDNA4HisMax expression constructs have undetectable activity (not shown). Anti-luciferase staining of cardiomyocyte cultures shows reporter gene expression restricted to myocyte population.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion In Vivo Model of... Appendix E1: Chromatin... References

	In Vivo Model of Pathologic Cardiac Hypertrophy

Top Abstract Introduction Materials and Methods Results Discussion In Vivo Model of... Appendix E1: Chromatin... References

 
Murine models of cardiac hypertrophy are favorable because the mouse is amenable to transgenic manipulation, allowing for the identification of molecular mechanisms of pathophysiologic processes. Aortic banding or constriction in the mouse, which has a heart rate of 500 beats/min and chamber dimensions in the millimeter range, is a commonly used animal model for the study of pathologic hypertrophy. Echocardiographic or hemodynamic analysis of murine models, although well established, requires special equipment and expertise. Compared with mouse models of pathologic hypertrophy, the ovine model has some advantages. It is amenable to standard echocardiographic and catheterization techniques and is not limited to the study of only pressure loading of the left side of the heart, but can be used to study other biomechanical stressors including volume loading of the heart. Furthermore, pathologic hypertrophy can be generated in either the left or right ventricle, or both, thus allowing for the determination of common or asymmetric transcriptional responses of either side of the heart.

The ovine model used in this study, that is, in utero placement of a large aorta-pulmonary shunt in fetal lambs, produces significant left-to-right shunting and increases in pulmonary blood flow and pulmonary hypertension. The left ventricular myocardium is stressed by persistent and significant volume loading, while the right ventricle eventually experiences pressure loading. This model is clinically relevant and mimics some congenital heart defects, such as patent ductus arteriosus, large ventricular septal defects, and aorta-pulmonary windows. Furthermore, volume loading of the left ventricle is also clinically seen with valvular heart disease such as aortic and mitral insufficiency. Right ventricular pressure overload is also commonly seen clinically, as in pulmonary hypertension, tetralogy of Fallot, and pulmonary stenosis. Last, the model mimics the surgically created aorta–pulmonary shunts used for the management of single ventricle anomalies with inadequate pulmonary blood flow.

The central features of the cardiomyocyte hypertrophic response include increases in contractile protein content, induction of contractile protein isoforms, and expression of embryonic markers.¹¹ The observed protein biochemical changes in this model are consistent with expected changes in sarcomeric protein expression. There is an upregulation of constitutively expressed genes (myosins), as well as induction of fetal contractile and noncontractile cardiac genes (high-molecular-weight troponin isoforms and atrial natriuretic factor/brain natriuretic peptide [data not shown]). Proto-oncogenes and other members of the immediate-early gene programs, also known to be activated during hypertrophy, were not evaluated in this study, nor were regulators of cell survival and apoptosis.¹¹

The finding that TEF-1, NKX2.5, Sp1, and GATA factors are upregulated during the cardiac hypertrophic response is consistent with their chromatin binding and transactivating properties. Likewise, downregulation of Sp3 during pathologic hypertrophy is consistent with its inhibitory effects on cTnT promoter activity. These data suggest that the increase in sarcomeric proteins and induction of embryonic cardiac genes seen during pathologic hypertrophy are preceded and regulated by increases in myocardial transactivators, decreases in cardiac gene repressors, and induction of posttranslational modifications that modulate transcription factor function. These data do not provide direct evidence that TEF-1, MEF2, NKX2.5, GATA-4, and Sp1 upregulation and Sp3 downregulation, in vivo, are mechanistically responsible, in part, for the changes in sarcomeric protein expression that are seen during pathologic cardiac hypertrophy in the ovine model. Further studies are warranted to investigate these important mechanisms.

The TEF-1 family of transcription factors has been implicated in muscle gene regulation.²⁷ We have previously cloned and characterized DTEF-1, a cardiac-enriched family member that regulates cardiac gene expression and is expressed in the early embryo.^9,10 During volume loading of the left ventricle and pressure loading of the right ventricle, TEF-1 polypeptides are upregulated to a greater degree in the right side of the heart. TEF-1 upregulation during biomechanical stress may therefore be an asymmetric, chamber-specific response or may act as a pressure-responsive rather than volume-responsive transactivator. Poly-ADP-ribose polymerase, a known coactivator of TEF-1,²⁸ has been implicated in myocardial cell hypoxic-ischemic injury and cardiomyocyte survival. In this model, we have also found that DTEF-1 becomes heavily ADP-ribosylated (data not shown), a process amenable to inhibition and potential target for pharmacotherapy of heart failure.²⁹

The Sp family of transcription factors is important in the assembly of the basal transcriptional machinery, potentially acting as a central and widespread regulatory network in pathologic hypertrophy. We have previously shown that Sp1 and Sp3 have counterregulatory functions in avian myocytes.³⁰ Although Sp3 expression is downregulated during pathologic hypertrophy, we have found that Sp3-associated HAT activity is increased and HDAC activity is significantly decreased (Figures E6 and E7). Hence, acetylation of Sp3 may convert it from a repressor to a transcriptional activator during the induction of pathologic cardiac hypertrophy and heart failure.

View larger version (11K): [in this window] [in a new window]   Figure E6. Histone acetyl transferase (HAT) activity associated with immunoprecipitated Sp3 is upregulated during pathologic cardiac hypertrophy.

View larger version (9K): [in this window] [in a new window]   Figure E7. Histone deacetylase activity associated with immunoprecipitated Sp3 is decreased during pathologic hypertrophy.

	Appendix E1: Chromatin Immunoprecipitation Supplement

Top Abstract Introduction Materials and Methods Results Discussion In Vivo Model of... Appendix E1: Chromatin... References

 
Method: Chromatin Immunoprecipitation (ChIP)
All ChIP assays were performed on the basis of the protocols of the chromatin immunoprecipitation assay kit (Upstate, Lake Placid, NY) [Azakie, 2005—AJP]. Five micrograms of primary antibody against DTEF-1, GATA-4, MEF2, NKX2.5, Sp1, Sp3, PARP, or PAR was added to a 500 µL chromatin sample for the immunoprecipitation reaction. ChIP dilution buffer and preimmune rabbit serum were used as controls for nonspecific interactions and DNA contamination. Anti-ß-actin and anti-cTnT antibodies were used as negative controls.

Polymerase Chain Reaction
After DNA purification, samples were subjected to polymerase chain reaction with primers designed for the chick heart cTnT promoter (cTnT-268) as follows: upper primer: 5'GCTGGCTGGCTTGTGTCA-3'; lower primer: 5'-CTTGGGGGGCAGAGGCTTT-3'.

The primers used for polymerase chain reaction were designed by a primer analysis software (Oligo 6.8; Molecular Biology Insights, Inc, Cascade, Colo). The amplified polymerase chain reaction product is 265 bp.

	Acknowledgments

 
We thank Leonard Moon for assistance with the manuscript and figures.

	Footnotes

 
^_* A.A. is supported by a joint program grant from the National Heart, Lung, and Blood Institute (1 K08 HL72774-01) and The Thoracic Surgery Foundation for Research and Education. This work was supported in part by grant NIH-R01- HL61284 (J.F.).

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