Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 29
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So far, most studies have been performed in models of myocardial infarction and remodeling [50, 56, 57]. In these studies, the induction of irreversibleischemia mainly affects the expression of genes involved in the synthesis of cyto-5.4 Subtractive Hybridization of Myocardial Ischemiaskeletal proteins and of the extracellular matrix. These studies will lead to a betterunderstanding of the impact of drug therapy (such as angiotensin-converting enzyme inhibitors) on cardiac remodeling [57].
However, it will be important in thefuture to explore the adaptation to reversible ischemia in these models, to betterelucidate the potential protective mechanisms that the heart can develop beforeischemia becomes irreversible. Such work will be equally important to developnew therapeutic strategies.5.4Subtractive Hybridization of Myocardial Ischemia5.4.1Myocardial StunningMyocardial stunning refers to the myocardial dysfunction that follows an acuteepisode of ischemia [58, 59].
A preserved ultrastructure of the myocardium andthe absence of cell loss characterize this form of non-lethal, fully reversible ischemia [60]. These characteristics are in striking contrast to other models of ischemia-reperfusion, in which both necrosis and apoptosis are observed [61, 62]. Thesyndrome of stunning is highly prevalent in different etiologies of clinical ischemic coronary artery disease, including stable or unstable angina pectoris, myocardial infarction, chronic multivessel disease, and post-surgical dysfunction [63, 64].Due to the major prevalence of ischemic heart disease, stunning is of paramountclinical importance because it corresponds to a condition in which myocardial viability is maintained.Although it was previously thought that the ventricular dysfunction induced byischemia could not be brought back to a normal contractile state, it is now clearlydemonstrated that the viability of dysfunctional myocardium is preserved [65].
Unraveling the molecular mechanisms of cardioprotection in stunned myocardiumcan open new avenues to salvage dysfunctional cardiac tissue and prevent cardiaccell loss. Especially, a better understanding of the mechanisms by which the molecular and cellular adaptations maintain cell survival should open new therapeutic opportunities. These mechanisms remain largely unknown in large mammalian models. Yet, the models of ischemic heart disease in large mammals, especially the swine, are most clinically relevant. This is due to the fact that the swineheart and the human heart share the same geometry, heart rate, coronary anatomy and absence of collaterals.5.4.2Genomic Profile of Myocardial StunningThe absence of irreversible cellular damage in stunned myocardium may correspondeither to an increased resistance of the heart to ischemia, or to the absence of stimulitriggering the pathways of cell death. The latter possibility is of low probability, be-91925 Genomics by Subtractive Hybridizationcause even mild episodes of ischemia/reperfusion can activate different intracellularstress pathways leading to cell death [66–69].
The subtractive hybridization represents a good technique to investigate which cardioprotective mechanisms are activated in the ischemic heart. One hypothesis to be tested by gene profiling is whethermyocardial stunning triggers the coordinated expression of different sets of genesacting to protect the myocardium against irreversible damage.These experiments were performed in a swine model of regional low-flow ischemia, in which the blood flow through the left anterior descending coronary arteryis decreased by about 50% during 90 minutes [70]. This ischemic episode is followedby full reperfusion. Despite the normalization of blood flow after reperfusion, thecontractile function remains depressed, which reflects myocardial stunning.
A fullfunctional recovery is typically observed after 48–72 h, and pathological examinationof this myocardium does not show any necrosis or apoptosis. We used this model ofreversible ischemia to investigate whether the protection of the myocardium againstirreversible damage correlates with a specific gene profile [52]. A subtractive hybridization was therefore performed between the post-ischemic, stunned myocardiumand the remote, normal myocardium. Because this is a model of regional ischemia,stunned and normal areas can be compared within the same hearts. In these specificexperiments, the contamination of mitochondrial DNA after two rounds of hybridization was about 35%. After database query, 60% of the nuclear-encoded sequencescorresponded to known gene products.
The remaining 40% could be divided equallybetween known sequences of unknown function and sequences with no match. Interestingly, we found that more than 30% of the genes that were upregulated instunned myocardium are involved in different mechanisms of cell survival, including: resistance to apoptosis, cytoprotection (”stress response”) and cell growth. Manyof them had been implied previously in the survival of other cell types but had notbeen described before in the heart. In particular, we found an induction in stunnedmyocardium of several genes not expressed in the normal myocardium and whichparticipate to the development and growth of different forms of tumors. This illustrates the power of the subtractive hybridization to pull out “unexpected” genes andprofiles. An example of the validation of these results is shown on Fig.
5.5 for theplasminogen activator inhibitor-1 (PAI-1), a serpin with anti-apoptotic properties[71]. A Northern blot on ischemic and normal samples from five different heartsshowed a reproducible and robust induction of this gene during ischemia. It couldbe confirmed by in-situ hybridization that this induction occurred in cardiomyocytes. Finally, the measurement of PAI-1 by quantitative PCR shows that the expression of this gene starts to increase during ischemia but peaks during post-ischemicstunning and returns back to normal after 12 hours, when the contractile functionrecovers. The sensitivity of the quantitative PCR also allows to measure separatelythe sub-endocardium from the sub-epicardium, to show that the gene response isof higher amplitude in the sub-endocardium, where the flow reduction is the mostimportant.
Remarkably, this transmural difference was found for all the genes measured [52]. There is therefore a gradient of gene response that matches the gradientof flow reduction, which shows that the nuclear response is not an “all or nothing”phenomenon but is proportional to the intensity of the initial stimulus.5.4 Subtractive Hybridization of Myocardial IschemiaMethods of validation of the subtractive library. This example illustrates the regulation of PAI-1 RNA in stunned myocardium.Panel A shows the reproducibility of the induction of PAI-1 in five different samplesfrom stunned territory. The correspondingcontrol (remote) area in the same hearts doesnot show any signal.
Panel B shows by in-situhybridization that PAI-1 induction occurs incardiomyocytes. Panel C shows by quantitative PCR the time-course of PAI-1 inductionduring stunning, with a maximal increase atone hour reperfusion and a normalization atFig. 5.55.4.3Chasing Novel GenesAs mentioned above, about 20% of the gene products found in the subtractive hybridization of stunned myocardium in the pig did not recall any known sequencein public databases. This last group is particularly interesting, because it containsthe novel genes. However, because the cDNA synthesis starting the subtraction experiments is primed by oligo-dT, an “unknown” sequence can also correspond tothe 3' UTR of a known gene. This can be due to two reasons.
First, many se-93945 Genomics by Subtractive Hybridizationquencing laboratories are interested in cloning only the coding sequence of thegenes (”ORFome”), and the 3'UTR is left undetermined. Second, the 3' UTR isthe region of the transcript that varies the most within and between species (alternative splicings, regulatory elements, duplications and deletions). Therefore,characterizing a “novel” gene remains a challenge. Especially, when many sequences from a subtraction library seem to be “novel”, it can be difficult to choosewhich are those with the highest priority for further investigation.
Different criteria can be applied to answer that question.· Sequence length. It is better to start with a long sequence from the subtractivehybridization. Considering two gene products that do not match any known sequence, a fragment of 1.2 Kb is statistically more likely to be novel than a fragment of 250 base pairs.· Tissue specificity. This is preferable to improve the originality of the work. Thetissue selectivity can be assessed easily with a multi-tissue Northern blot. Agene that is expressed only in the heart is probably more interesting than agene that is expressed ubiquitously.
The Northern blot will also determine thesize of the transcript and thereby will predict the amount of work needed toachieve a full-length cloning.· Regulated gene. It is preferable to concentrate on true positives, i.e., genesshowing a differential expression between the two experimental groups. Oncean interesting target has been identified, the different methodologies of the“vertical approach” (Fig. 5.2) can be applied for a full characterization of thetranscript and the corresponding protein. This approach can be applied not onlyfor the novel genes, but also for known genes that had not been described inthe heart before.5.5SummaryThe regulation of myocardial gene expression is highly sensitive to any extracellular or intracellular stimulus that affects contractile function.
The recent development of powerful technologies allows to study the broad genomic profile of theheart in various experimental conditions. Although mainly investigated in rodents, gene regulation in the heart needs to be elucidated further in large mammalian models that reproduce the different forms of cardiac disease in humans.The biological questions addressed in these models require the use of appropriatetechniques. The use of the subtractive hybridization is particularly attractive because it can be applied to any species. The strength of the subtractive hybridization relies on its unbiased nature and its power to extract even low-abundancetranscripts.
In addition, the subtraction experiments reveal “unexpected” gene profiles and represent a starting point for the characterization of novel genes.5.7 References5.6AcknowledgementsI am particularly grateful to Stephen F. Vatner for his support and for developingthe animal models used in these experiments. I also express my deepest gratitudeto James E. Tomlinson who initiated me to the molecular techniques of subtractive hybridization. I also thank Raymond K.
Kudej, Song-Jung Kim, Dorothy E.Vatner, Vinciane Gaussin, James N. Topper, Junichi Sadoshima and Maha Abdellatif for their collaboration and fruitful discussions, as well as Erika Thompson,Anna Zajac and Li Wang for their expert technical assistance.5.7References12345678Nadal-Ginard, B., Mahdavi, V. Molecular basis of cardiac performance: plasticity of the myocardium generated throughprotein isoform switches. J.
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