Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in developed countries. Underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown but are expected to result from causal alterations in gene and protein expression. Proteomic technology now allows us to examine global alterations in protein expression in the diseased heart and can provide new insights into cellular mechanisms involved in cardiac dysfunction. The majority of proteomic investigations still use 2D gel electrophoresis (2-DE) with immobilized pH gradients to separate the proteins in a sample and combine this with mass spectrometry (MS) technologies to identify proteins. In spite of the development of novel gel-free technologies, 2-DE remains the only technique that can be routinely applied to parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. It can resolve >5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect <1 ng of protein per spot. Furthermore, 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. The use of proteomics to investigate heart disease should result in the generation of new diagnostic and therapeutic markers. In this article, we review the current status of proteomic technologies, describing the 2-DE proteomics workflow, with an overview of protein identification by MS and how these technologies are being applied to studies of human heart disease.

This chapter contains sections titled: Heart Proteomics Heart 2-D Protein Databases Dilated Cardiomyopathy Animal Models of Heart Disease Subproteomics of the Heart Mitochondria PKC Signal Transduction Pathways Proteomics of Cultured Cardiac Myocytes Proteomic Characterization of Cardiac Antigens in Heart Disease and Transplantation Markers of Acute Allograft Rejection Vessel Proteomics Proteomics of Intact Vessels Proteomics of Isolated Vessel Cells Laser Capture Microdissection Concluding Remarks

Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in developed countries. Underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown but are expected to result from causal alterations in gene and protein expression. Proteomic technology now allows us to examine global alterations in protein expression in the diseased heart and can provide new insights into cellular mechanisms involved in cardiac dysfunction. The majority of proteomic investigations still use 2D gel electrophoresis (2-DE) with immobilized pH gradients to separate the proteins in a sample and combine this with mass spectrometry (MS) technologies to identify proteins. In spite of the development of novel gel-free technologies, 2-DE remains the only technique that can be routinely applied to parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. It can resolve 5000 proteins simultaneously ( 2000 proteins routinely) and can detect 1 ng of protein per spot. Furthermore, 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. The use of proteomics to investigate heart disease should result in the generation of new diagnostic and therapeutic markers. In this article, we review the current status of proteomic technologies, describing the 2-DE proteomics workflow, with an overview of protein identification by MS and how these technologies are being applied to studies of human heart disease. (Circ Res. 2006;98:309-321.)

Two-dimensional gel electrophoresis (2-DE) combined with protein identification by mass spectrometry (MS) is currently the method of choice in the majority of proteomic projects. Novel gel-free technologies have been developed but 2-DE remains the technique of choice for quantitative expression profiling of large sets of complex protein mixtures such as whole cell/tissue lysates. Solubilized proteins are separated in the first dimension according to their charge properties (isoelectric point, pI) by isoelectric focusing (IEF) under denaturing conditions, followed by their separation in the second dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to their relative molecular mass (Mr). 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect less than 1 ng of protein per spot. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or posttranslational modifications. In this chapter we describe the various steps in the 2-DE proteomics workflow, namely sample preparation, solubilization, 2-D gel electrophoresis, protein detection and visualization, and protein identification by mass spectrometry. The use of 2-DE in conjunction with laser microdissection microscopy is presented and discussed.

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.

Proteomics generates information on expressed proteins, and laser microdissection (LMD) is a method that allows enrichment of specific cell types from complex heterogeneous tissue. Together they provide a powerful tool for functional genomic research. Here, we have investigated (i) the effects of fixation and staining on cardiac proteins separated by two‐dimensional gel electrophoresis (2‐DE) and (ii) feasibility of using LMD to separately prepare myocytes and blood vessels for 2‐DE gel analysis. This is the first such study of human heart. The effect of fixation (ethanol or acetone), staining with haematoxylin and eosin in the presence and absence of xylene, and antibody staining was investigated. Proteins were separated by 2‐DE and spots detected by silver staining. Quantitative spot analysis showed that contractile proteins were preserved under all conditions, and no significant differences were found when the groups studied were compared with the control group. However, there were differences in the visual quality of the gel patterns. LMD provided enough protein from blood vessels and myocytes to run one large‐format (18×24 cm) 2‐D gel for each subset of cells. Collection of this material took 70 h (≈︁ 2800 blood vessels and 17 000 myocytes) and resulted in tissue‐specific gel patterns for these two structures. In conclusion, the use of haematoxylin and eosin staining without xylene provided the best morphology and did not significantly affect protein spot number.

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.

PKC-delta is believed to play an essential role in cardiomyocyte growth. In the present study, we investigated the effect of PKC-delta on cardiac metabolism using PKC-delta knockout mice generated in our laboratories. Proteomic analysis of heart protein extracts revealed profound changes in enzymes related to energy metabolism: certain isoforms of glycolytic enzymes, e.g., lactate dehydrogenase and pyruvate kinase, were absent or decreased, whereas several enzymes involved in lipid metabolism, e.g., phosphorylated isoforms of acyl-CoA dehydrogenases, showed a marked increase in PKC-delta(-/-) hearts. Moreover, PKC-delta deficiency was associated with changes in antioxidants, namely, 1-Cys peroxiredoxin and selenium-binding protein 1, and posttranslational modifications of chaperones involved in cytoskeleton regulation, such as heat shock protein (HSP)20, HSP27, and the zeta-subunit of the cytosolic chaperone containing the T-complex polypeptide 1. High-resolution NMR analysis of cardiac metabolites confirmed a significant decrease in the ratio of glycolytic end products (alanine + lactate) to end products of lipid metabolism (acetate) in PKC-delta(-/-) hearts. Taken together, our data demonstrate that loss of PKC-delta causes a shift from glucose to lipid metabolism in murine hearts, and we provide a detailed description of the enzymatic changes on a proteomic level. The consequences of these metabolic alterations on sensitivity to myocardial ischemia are further explored in the accompanyingpaper (20).

PROTEOMICSVolume 4, Issue 5 p. 1519-1519 ErratumFree Access Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue (Proteomics 2004, vol. 4, issue 1, pp. 27–30) Kyla Pennington, Corresponding Author Kyla Pennington k.pennington@iop.kcl.ac.uk Department of Psychological Medicine,PO Box 052, Section of Neuropathology and Psychiatry, Department of Psychological Medicine, Institute of Psychiatry, 1 Windsor Walk, Denmark Hill, SE5, UK Fax: +44-207-848-5109===Search for more papers by this authorEmma McGregor, Emma McGregor Proteome Sciences,Search for more papers by this authorClare L. Beasley, Clare L. Beasley Department of Psychological Medicine,Search for more papers by this authorIan Everall, Ian Everall Department of Psychological Medicine,Search for more papers by this authorDavid Cotter, David Cotter Department of Psychological Medicine,Search for more papers by this authorMichael J. Dunn, Michael J. Dunn Department Neuroscience, Institute of Psychiatry, King's College London, UKSearch for more papers by this author Kyla Pennington, Corresponding Author Kyla Pennington k.pennington@iop.kcl.ac.uk Department of Psychological Medicine,PO Box 052, Section of Neuropathology and Psychiatry, Department of Psychological Medicine, Institute of Psychiatry, 1 Windsor Walk, Denmark Hill, SE5, UK Fax: +44-207-848-5109===Search for more papers by this authorEmma McGregor, Emma McGregor Proteome Sciences,Search for more papers by this authorClare L. Beasley, Clare L. Beasley Department of Psychological Medicine,Search for more papers by this authorIan Everall, Ian Everall Department of Psychological Medicine,Search for more papers by this authorDavid Cotter, David Cotter Department of Psychological Medicine,Search for more papers by this authorMichael J. Dunn, Michael J. Dunn Department Neuroscience, Institute of Psychiatry, King's College London, UKSearch for more papers by this author First published: 21 April 2004 https://doi.org/10.1002/pmic.200490026AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume4, Issue5Special Issue: PROTEOMIC FORUM 2003 Proceedings of the International Meeting on Proteome Analysis, Munich, Germany 14-17 September 2003May 2004Pages 1519-1519 RelatedInformation

Chapter 11 Proteomics, A Step beyond Genomics: Applications to Cardiovascular Disease Emma McGregor, Emma McGregor Proteome Sciences plc, Institute of Psychiatry, Kings College, De Crespigny Park, London, SE5 8AF, UKSearch for more papers by this authorMichael J. Dunn, Michael J. Dunn [email protected] Department of Neurosciences, Institute of Psychiatry, Kings College, De Crespigny Park, London, SE5 8AF, UKSearch for more papers by this author Emma McGregor, Emma McGregor Proteome Sciences plc, Institute of Psychiatry, Kings College, De Crespigny Park, London, SE5 8AF, UKSearch for more papers by this authorMichael J. Dunn, Michael J. Dunn [email protected] Department of Neurosciences, Institute of Psychiatry, Kings College, De Crespigny Park, London, SE5 8AF, UKSearch for more papers by this author Book Editor(s):Jennifer E. van Eyk, Jennifer E. van Eyk Department of Physiology, Queen's University, 429 Botterell Hall, Kingston, Ontario K7L 2N6, Canada. http://meds.queensu.ca/medicine/physiol/about.htmlSearch for more papers by this authorMichael J. Dunn, Michael J. Dunn Department of Neurosciences, Institute of Psychiatry, Kings College, De Crespigny Park, London, SE5 8AF, UKSearch for more papers by this author First published: 21 February 2003 https://doi.org/10.1002/352760152X.ch11 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary Introduction Protein Solubilization Protein Separation Protein Detection/Visualisation Protein Identification Bioinformatics Cardiovascular Proteomics Heart 2DE Protein Databases Dilated Cardiomyopathy Animal Models of Heart Disease Proteomic Characterization of Cardiac Antigens in Heart Disease and Transplantation Summary References Proteomic and Genomic Analysis of Cardiovascular Disease RelatedInformation