Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 20
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The genes were sorted according to their functional groups and the portrait thus produced showed generegulatory processes during viremic, inflammatory, and healing phases of themyocarditic process. The same group also utilized differential mRNA displaymethod to assess gene expression at the transcription level in a mouse enteroviralmodel, and found 2 up-regulated (Mus musculus inducible GTPase, mouse mitochondrial hydrophobic peptide) and 3 down-regulated (mouse b-globin, Homo sapiens cAMP-regulated response element binding protein binding protein, Mus53543 Heart Failure: A Genomics Approachmusculus Nip21 mRNA) candidate genes.
Microarray studies in artery and veinhave also yielded differential gene expression profiles. In one study using cDNAarray analysis to explore the genes in the vasculature system, Adams et al. [32] described a set of 68 genes that were consistently differentially expressed in aorticmedia as compared with vena cava media.In the first reported human microarray study in end stage heart failure, Yang et al.[33] examined gene expression in 2 failing human hearts using oligo-based arrays.The investigators used high density oligonucleotide arrays to investigate failingand nonfailing human hearts (end stage ischemic and dilated cardiomyopathy). Similar changes were identified in twelve genes in both types of heart failure, which theauthors maintain, indicate that these changes may be intrinsic to heart failure.
Theyfound altered expression in cytoskeletal and myofibrillar genes, in genes involved indegradation and disassembly of myocardial proteins, in metabolism, in protein synthesis and genes encoding stress proteins. While the “Affychip” in this study offers acarefully-controlled systematic method of analysis, its current lack of user flexibilityin its design hinders novel gene discovery currently available in tissue-specific arrays.Our laboratory has taken a different approach to microarray technology. Takingadvantage of our vast previously-acquired resources, we have constructed a firstgeneration custom-made cardiovascular-based cDNA microarray, which we termthe “CardioChip” [34].
Its practicality and flexibility has allowed us to conceptualize the molecular events surrounding end-stage heart failure. The current Cardiochip contains 10,368 redundant and randomly sequenced expressed sequencetags, derived from several human heart and artery cDNA libraries.
The Cardiochiphas been used to develop a profile of previously-suspected candidates involved inmolecular events surrounding the pathology of heart failure; more importantly, thismethod identifies novel candidates that may, with further verification at the functional level, be responsible for contributing to the demise of myocardial function.In our recent study of dilated cardiomyopathy (DCM), the Cardiochip verifiedseveral expected candidate genes and identified some novel candidates [35]. Atrialnaturietic peptide showed an intense level of up-regulation across the DCM patient samples, confirming at the microarray level its pivotal role as a circulatingmarker of cardiac muscle stress [36]. Indeed, its presence in our analysis lent a degree of credence to the validity of our study.
Despite its significant up-regulationversus non-failing samples, the level of atrial naturietic peptide was highly variable among the patients.In addition, we observed a consistent up-regulation of selected sarcomeric andextracellular matrix proteins (i.e., b-myosin heavy chain, a-actinin, a-cardiac actin,troponin I, tropomyosin, collagen, etc.). Evidence in knockout mice and humanstudies has offered insight into the putative role of these proteins in maintainingsarcomeric integrity [34–43]. Mutations of proteins associated with a-actinin,namely MLP, cardiac a-actin, desmin and titin, have been shown to be present incertain forms of human DCM [44–48].
Ambiguities exist in the literature regarding the expression of collagen and other members of the extracellular matrix;nonetheless, regulation of the extracellular matrix is important in the formationof fibrosis and impaired contractile function [49, 50].3.3 Genomic Approach to Heart FailureCalcium signaling has recently become an important area of interest in the investigation of heart failure [51]. A decrease in calcium cycling genes has beenshown to result in reduced contractility in mice whose b-adrenergic stimulation isblunted leading to decreased phospholamban phosphorylation [33].
Ca2+ATPase iskey in regulating contractility, and its ~2-fold average down-regulation in ourDCM samples lends credence to its involvement. This is supported by a recentstudy in which the transfer of the Ca2+ATPase gene into the rat myocardium prevents certain features of heart failure [52]. The presence of Ca2+/calmodulin-dependent kinase in our analysis, despite showing only about 1.1-fold down-regulation, is particularly intriguing, as it is known to phosphorylate phospholamban[53]. In addition, inositol 1,4,5-triphosphate receptor (a member of the calciumchannel family) which may be responsible for calcium release from intracellularstores, [54] was also significantly down-regulated (1.86-fold).
Inositol 1,4,5-trisphosphate 3-kinase was recently cloned [55] and may be another key component inthis regulation (1.86-fold). Our findings suggest that the role of Ca2+ signalingdown-regulation may be of crucial significance in the evolution of heart failureand would warrant further investigation.A number of novel ESTs were also identified from our study to be differentiallyregulated.
Verification with quantitative real-time RT-PCR confirmed this expression. It is an intriguing prospect that these among other transcripts, after fulllength sequencing, represent novel cardiac-specific genes encoding proteins thatare potentially key to solving the puzzle of the molecular pathophysiology of heartfailure.
Indeed, our microarray analysis not only serves as a genomic model for amore complete understanding of DCM, but also as a focused target for possibletherapeutic interventions specific to the cardiac tissue. Investigations are currentlyunderway to elucidate the function of these candidates.In a similar study [56] our laboratory developed comparative microarray portraits of DCM and hypertrophic cardiomyopathy (HCM). Overall, our resultsshowed that 192 genes were highly expressed in both DCM and HCM (atrial natriuretic peptide, CD59, decorin, elongation factor 2 and heat shock protein 90)and that 51 genes were downregulated in both conditions (elastin, sarcomeric/reticulum Ca2+ -ATPase).
Differentially expressed genes as determined quantitativelyby RT-PCR (Fig. 3.3) included a B-crystallin, antagonizer of myc transcriptional activity, beta dystrobrevin, calsequestrin, lipocortin and lumican). What this studyshows is that although having similar clinical features, the gene defects leading toDCM and HCM differ.
DCM, a cytoskeletalopathy, and HCM, a sarcomyopathy,are common forms of cardiomyopathy that result in end stage heart failurethrough different remodelling and molecular pathways. Our microarray portrait ofDCM demonstrated that more genes involved in cell and organism defence wereupregulated, especially immune system response genes. By contrast, protein andcell expression genes were downregulated. Hypertrophic processes are evident inthe increase in ribosomal genes upregulated in HCM, whereas cell signalling andcell structure genes were downregulated.These reports describe the most informative cDNA microarray-based analysis ofend-stage heart failure derived from DCM and HCM currently available.
These in-55563 Heart Failure: A Genomics ApproachReal-time RT-PCR confirmed commonly up-regulated or down-regulated (A)and differentially expressed (B) genes in DCMand HCM. The fold change is displayed asrelative to normalized normal adult heartsamples. * denotes p < 0.05, #: p < 0.01. Theatrial natriuretic peptide was increased morethan 20-fold in both DCM and HCM, notshown in the bar graph.
Calpain: calcium actiFig. 3.3vated neutral protease, EF2: elongation factor2, HSP 90: heat shock protein 90, SOD: copper/zinc superoxide dismutase, SERCA: sarcoplasmic/reticulum calcium-ATPase, B-cryst: aB-crystallin, CASQ: calsequestrin, MALC: atrialmyosin alkali light chain, BDTN: b-dystrobrevin, Mad: antagonizer of myc transcriptionalactivity, and TRR: thioredoxin reductase.3.4 Conclusionvestigations are not exhaustive in that they do not attempt to fully characterize themolecular basis of heart failure. Their intention is to provide a preliminary portrait of global gene expression in complex cardiovascular disease using cDNA microarray and QRT-PCR technology, and to highlight the effectiveness of our everevolving platform for gene discovery.As these intriguing findings show, microarray data offer a holistic view of theinterrelated gene network during disease processes.
Genes which are either overexpressed or under-expressed in a diseased tissue or organ present prima facie evidence that they are involved in disease pathogenesis. Using the genomicapproach, previously unrecognized alterations in the expression of specific genescan be identified and novel genes can also be discovered, leading to a clearer understanding of the gene pathways. However, nature functions by integration, andproteins do not work in isolation but are instead involved in interrelated networks. The challenge of genomics lies not only in identifying genes, but also inunderstanding their function, the latter also referred to as “functional genomics”.In the short term, the goal is to assign putative function to each of the genesusing systematic, high-throughput approaches to the database.
As functional information accumulates, the knowledge gap will be filled out in the form of expression profile studies, protein microcharacterization and their post-translationalmodifications, protein-protein interactions, computational approaches, and the response to loss of function by mutation [57]. Unraveling these networks and theirinteractions will be vital to an integrated mapping between genotype and phenotype. Gene chip technology may eventually be used to diagnose, stage or classifyclinical conditions by detecting genetic markers associated with disease states inbiopsy or blood samples.
Indeed, gene expression microarray technology is apowerful tool with enormous potential in the years to come.3.4ConclusionHeart failure is a complex syndrome. It may involve either the right or left ventricle, progress from compensated to decompensated stages, result from ischemic ornonischemic etiology, affect mainly systolic or diastolic function, and pertain tohigh cardiac output or low cardiac output status. The mechanism by which its genetic machinery controls these responses remains to be fully elucidated.
The complexity of this disease raises numerous questions. Do all kinds of heart failureshare a common final pathway? The transition from compensated cardiac hypertrophy to decompensated heart failure is accompanied by marked changes in theexpression of an array of genes in the heart. What is the marker for early myocardial decompensation and could we intervene in its progression? The genome-wideapproach will help us to integrate our current understanding of the pathophysiological pathways associated with heart failure.
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