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European Journal of Heart Failure 2008 10(12):1177-1180; doi:10.1016/j.ejheart.2008.08.008
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© 2008 European Society of Cardiology

Gene expression profiling of human hibernating myocardium: Increased expression of B-type natriuretic peptide and proenkephalin in hypocontractile vs normally-contracting regions of the heart

Sanjay K. Prasada,b, Angela Clerka, Timothy E. Cullingforda, Alexander W.Y. Chena, Timothy J. Kempa, Timothy M. Cannellb, Martin R. Cowiea,* and Mario Petrouc

a NHLI Division, Faculty of Medicine, Imperial College London London, UK
b Cardiovascular Magnetic Resonance Unit, Royal Brompton and Harefield NHS Trust London, UK
c Department of Cardiac Surgery, Royal Brompton and Harefield NHS Trust London, UK

* Corresponding author. NHLI Division, Faculty of Medicine, Imperial College London, Dovehouse Street, London SW3 6LY, UK. Tel.: +44 20 7351 8856/8164; fax: +44 20 7351 8148. E-mail address: m.cowie{at}imperial.ac.uk (M.R. Cowie).


   Abstract
Top
 Abstract
1. Background and aim
 2. Methods
 3. Results
 4. Conclusion
 Appendix A. Supplementary data
 References
 
A greater understanding of the molecular basis of hibernating myocardium may assist in identifying those patients who would most benefit from revascularization. Paired heart biopsies were taken from hypocontractile and normally-contracting myocardium (identified by cardiovascular magnetic resonance) from 6 patients with chronic stable angina scheduled for bypass grafting. Gene expression profiles of hypocontractile and normally-contracting samples were compared using Affymetrix microarrays. The data for patients with confirmed hibernating myocardium were analysed separately and a different, though overlapping, set (up to 380) of genes was identified which may constitute a molecular fingerprint for hibernating myocardium. The expression of B-type natriuretic peptide (BNP) was increased in hypocontractile relative to normally-contracting myocardium. The expression of BNP correlated most closely with the expression of proenkephalin and follistatin 3, which may constitute additional heart failure markers. Our data illustrate differential gene expression in hypocontractile and/hibernating myocardium relative to normally-contracting myocardium within individual human hearts. Changes in expression of these genes, including increased relative expression of natriuretic and other factors, may constitute a molecular signature for hypocontractile and/or hibernating myocardium.

Key Words: Genes • Hibernation • Myocardium • Natriuretic peptides

Received December 10, 2007; Revised May 1, 2008; Accepted August 14, 2008


   1. Background and aim
Top
 Abstract
 1. Background and aim
2. Methods
 3. Results
 4. Conclusion
 Appendix A. Supplementary data
 References
 
Myocardial revascularization is not always successful in restoring ventricular function. The global changes in gene and protein expression which occur in hibernating myocardium compared with normally-contracting myocardium remain to be established. A greater understanding of the molecular basis of hibernating myocardium may assist in identifying those patients who would most benefit from revascularization [1,2]. We therefore compared the gene expression profiles in heart biopsies from hypocontractile and normally-contracting myocardium (identified by CMR) using Affymetrix Human Genome microarrays (>47,000 transcripts). The focus was on patients with chronic stable angina and only mild impairment of left ventricular function, to reduce the influence of adaptive changes in the remote, normally-contracting region.


   2. Methods
Top
 Abstract
 1. Background and aim
 2. Methods
3. Results
 4. Conclusion
 Appendix A. Supplementary data
 References
 
Patients with chronic stable angina scheduled for CABG were selected. Hibernating myocardium was identified using late-enhancement CMR [3,4]. Heart biopsies were collected for each patient (n=11) at the time of CABG from a previously determined segment with evidence of hibernation and a paired sample from a segment with normal viability and wall motion (Online supplement, Table 1). Total RNA was extracted (Online supplement, Table 2). Fragmentation of antisense cRNA and hybridisation to Affymetrix Human Genome U133 plus 2.0 arrays were performed.

Log10 values were used for the analysis of the hybridisation data with minimum values set at 0.01. The data were normalised per array (to the 50th percentile) and the samples were paired (the value for each gene in each hypocontractile sample was normalised to that of the normally-contracting sample from the same patient). Genes were selected for further analysis if present in at least 50% of normal samples or 50% of hibernating samples. The expression of BNP, ANP and proenkephalin mRNAs was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) for the paired samples.

Gene and condition clustering, and Standard correlations (Pearson correlation about zero) were performed with GeneSpring 7.2. For selected transcripts, the identities of genes corresponding to the sequences for individual probe sets were confirmed using BLAST (basic local alignment search tool). RNA was isolated from cardiac myocytes and cDNA prepared as previously described [8]. RT-PCR was performed using specific primers for GAPDH, ANP, BNP and proenkephalin.


   3. Results
Top
 Abstract
 1. Background and aim
 2. Methods
 3. Results
4. Conclusion
 Appendix A. Supplementary data
 References
 
Of the probe sets analysed, the expression of 354 was significantly different (FDR<0.05; >1.5-fold) in hypocontractile vs normally-contracting myocardium (Group A transcripts). Using these transcripts, the samples clustered largely according to myocardial contractility (Online supplement, Fig. 1A and B). The greatest increase in expression in hypocontractile vs normally-contracting myocardium was for BNP mRNA (7.61±5.23 fold increase) which was elevated in hypocontractile myocardium (Fig. 2B). Of 20 genes with standard correlation of >0.85, the expression of mRNAs for proenkephalin (PENK) and follistatin-like 3 (FSTL3) (both of which encode secreted, soluble peptides) correlated most closely with BNP expression (Online supplement, Fig. 2D). The correlation for proenkephalin was greater than that of ANP (Online supplement, Fig. 2C and E), suggesting that, like BNP, proenkephalin and follistatin-like 3 may serve as markers of ischaemic heart disease and contractile dysfunction. Consistent with the microarray data, the expression of BNP, ANP and proenkephalin on w RT-PCR was increased in hypocontractile vs normally-contracting samples from patients 2, 5 and 7 (Online supplement, Fig. 2F).


Figure 01
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  		Fig. 2 A, Classification of genes for proteins of known/probable function identified with significantly different (FDR<0.05; >1.5-fold change) mRNA expression in hypocontractile vs normally-contracting myocardium in all patients (Group A transcripts; for identities, see Online supplement, Table 3). B and C (online), Expression levels for BNP (panel B) and ANP (panel C online) in normally-contracting and hypocontractile myocardium (samples from individual patients are linked). D (online), Correlation of expression of Group A transcripts (FDR<0.01; >2-fold difference in expression in hypocontractile vs normally-contracting myocardium; Standard correlation >0.85 compared with BNP) with BNP. The heatmap shows expression levels of unpaired samples ranging from zero (cyan) through 1 (black) to 30 (red) normalised Log10 fluorescence units. The probe set identity is shown on the left, with the gene symbol and standard correlation with BNP expression on the right. E (online), Linear regression analysis of the expression of proenkaphalin (upper panel) or ANP (lower panel) compared with BNP. F (online), Expression of BNP, ANP, proenkephalin or GAPDH mRNAs in hypocontractile (H) or normally-contracting (N) myocardium from patients 1, 2, 5, 6 and 7 assessed by RT-PCR. No RT = no reverse transcriptase control. The position of the 600 bp marker is shown on the left.

 
In order to assess the changes in gene expression associated with hibernating myocardium (i.e. that with reversible dysfunction), the data for the five patients known to have improved ventricular function following CABG were analysed separately. Interestingly, a greater number of significant (FDR<0.05; >1.5-fold difference between hibernating and normally-contracting myocardium) changes was identified than with the full patient cohort, including 227 mRNAs (206 different genes) for proteins of known or probable function (Fig. 3A; Online supplement, Table 6). The greatest difference between Group A transcripts (i.e. the full patient cohort) and Group IF transcripts (i.e. patients with improved LV function) was an increase in the number of transcriptional regulators with decreased expression in hibernating vs normally-contracting myocardium (Figs. 2A and 3A).


Figure 02
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  		Fig. 3 A, Classification of genes for proteins of known/probable function identified with significantly different mRNA expression (FDR<0.05; >1.5-fold change) in hibernating vs normally-contracting myocardium in patients with improved left ventricular function following CABG (Group IF transcripts; for identities, see Online supplement, Table 6). B, (online) Venn diagram comparing Group A and Group IF transcripts (Online supplement, Tables 3-5 and 6-8, respectively). C (online), Condition clustering of patients according to Group IF transcripts. The heatmap shows the expression levels of transcripts in hibernating myocardium relative to normally-contracting myocardium (i.e. paired samples) ranging from zero (cyan) through 1 (black) to 6 (red) normalised Log10 fluorescence units. Patient numbers (1-11) are shown.

 
Surprisingly (given that the n value is smaller), ~40% of the probe sets for Group IF transcripts were not represented in Group A transcripts (Online supplement, Fig. 3B). Furthermore, whereas 112 (~32%) of the probe sets of Group A genes were significant at FDR<0.01 (Online supplement, Tables 6-8), for Group IF genes, 342 (~85%) probe sets were significant at FDR<0.01 (Online supplement, Tables 3-5). These data suggest that the subset of patients with improvement in left ventricular function represented a more homogeneous group with respect to gene expression. This was highlighted by condition clustering of the patients on the basis of the relative expression of Group IF transcripts in hypocontractile vs normally-contracting myocardium: (Online supplement, Fig. 3C).


   4. Conclusion
Top
 Abstract
 1. Background and aim
 2. Methods
 3. Results
 4. Conclusion
Appendix A. Supplementary data
 References
 
Our data of patients with only mild left ventricular impairment indicate that, even within a single heart, there is generally increased expression of BNP mRNAs in the hypocontractile area relative to normally-contracting myocardium (Fig. 2B). Secondly, in addition to BNP we identified a number of potential markers of hypocontractile myocardium including proenkephalin, the expression of which was increased in hypocontractile myocardium and correlated with BNP (Online supplement, Fig. 2D and E). We suggest that at least a subset of these markers represents a "fingerprint" or molecular signature for hibernation [5-11]. However, since none of the transcripts was consistently expressed in hypocontractile myocardium but absent from normally-contracting myocardium, we cannot propose any individual gene as a marker for hypocontractile/hibernating myocardium [12-16]


   Appendix A. Supplementary data
Top
 Abstract
 1. Background and aim
 2. Methods
 3. Results
 4. Conclusion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejheart.2008.08.008.


   Acknowledgements
 
We thank the microarray centre for conducting the hybridisations. This work was funded by the British Heart Foundation (grant no.PG/03/014/15059), and the Royal Brompton and Harefield NHS Trust Clinical Research Committee (grant no. CRC C/03/014). We also thank CORDA for the support of the CMR facility at the Royal Brompton Hospital.


   References
Top
 Abstract
 1. Background and aim
 2. Methods
 3. Results
 4. Conclusion
 Appendix A. Supplementary data
 References
 

  1. Depre C., Vatner S.F. Mechanisms of cell survival in myocardial hibernation. Trends Cardiovasc Med (2005) 15:101–110.[CrossRef][Web of Science][Medline]
  2. Camici P.G., Rimoldi O.E. The contribution of hibernation to heart failure. Ann Med (2004) 36:440–447.[CrossRef][Web of Science][Medline]
  3. Kim R.J., Wu E., Rafael A., et al. The use of contrast-enhanced magnetic resonance imaging to identify reversible myocardial dysfunction. N Engl J Med (2000) 343:1445–1453.[Abstract/Free Full Text]
  4. Underwood S.R., Bax J.J., vom Dahl J., et al. Imaging techniques for the assessment of myocardial hibernation. Report of a Study group of the European Society of Cardiology. Eur Heart J (2004) 25:815–836.[Abstract/Free Full Text]
  5. Elsasser A., Vogt A.M., Kostin S., et al. Human hibernating myocardium is jeopardized by apoptotic and autophagic cell death. J Am Coll Cardiol (2004) 43:2191–2199.[Abstract/Free Full Text]
  6. Depre C., Kim S.-J., John A.S., et al. Program of cell survival underlying human and experimental hibernating myocardium. Circ Res (2004) 95:433–440.[Abstract/Free Full Text]
  7. Depre C., Tomlinson J.E., Kudeh R.K., et al. Gene program for cardiac cell survival induced by transient ischemia in conscious pigs. Proc Natl Acad Sci U S A (2001) 98:9336–9341.[Abstract/Free Full Text]
  8. Kemp T.J., Causton H.A., Clerk A. Changes in gene expression induced by H2O2 in cardiac myocytes. Biochem Biophys Res Commun (2003) 307:416–421.[CrossRef][Web of Science][Medline]
  9. Margulies K.B., Matiwala S., Cornejo C., Olsen H., Craven W.A., Bednarik D. Mixed messages: transcription patterns in failing and recovering human myocardium. Circ Res (2005) 96:592–599.[Abstract/Free Full Text]
  10. Chen Y., Park S., Li Y., et al. Alterations of gene expression in failing myocardium following left ventricular assist device support. Physiol Genomics (2003) 14:251–260.[Abstract/Free Full Text]
  11. Birks E.J., Hall J.L., Barton P.J.R., et al. Gene profiling changes in cytoskeletal proteins during clinical recovery after left ventricular-assist device support. Circulation (2005) 112:57–64.
  12. Kittleson M.M., Hare J.M. Molecular signature analysis: using the myocardial transcriptome as a biomarker in cardiovascular disease. Trends Cardiovasc Med (2005) 15:130–138.[CrossRef][Web of Science][Medline]
  13. Sharma U.C., Pokharel S., Evelo C.T., Maessen J.G. A systematic review of large scale and heterogeneous gene array data in heart failure. J Mol Cell Cardiol (2005) 38:425–432.[CrossRef][Web of Science][Medline]
  14. McCarthy J.J., Parker A., Salem R., et al. Large scale association analysis for identification of genes underlying premature coronary heart disease: cumulative perspective from analysis of 111 candidate genes. J Med Genet (2004) 41:334–341.[Abstract/Free Full Text]
  15. Kragelund C., Gronning B., Kober L., Hildebrandt P., Steffensen R. N-terminal pro-B-type natriuretic peptide and long-term mortality in stable coronary heart disease. N Engl J Med (2005) 352:666–675.[Abstract/Free Full Text]
  16. Ndrepepa G., Braun S., Niemoller K., et al. Prognostic value of N-terminal pro-brain natriuretic peptide in patients with chronic stable angina. Circulation (2005) 112:2102–2107.[Abstract/Free Full Text]

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