Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 32
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Overexpression of HO-1 reduced infarct size [28], whereas infarct size was increased in aHO-1 null hearts [29]. In addition, HO-1 null hearts did not exhibit acute preconditioning, suggesting a role for HO-1 in preconditioning. Also, hearts from micenull for glutathione peroxidase-1 (GSHPx-1) had increased infarcts and poorer recovery of function [30].Overexpression of the anti-apoptotic bcl-2 has also been shown to be cardioprotective [31, 32]. Hearts null for leukocyte-type 12-lipoxygenase did not exhibit preconditioning [32], and hearts from mice null for the prostaglandin-I(2) receptorhad an increase in infarct size [33].
Hearts from mice null for tumor necrosis factor alpha (TNF-a) showed a decrease in infarct size [34], whereas hearts frommice lacking both TNF-a receptor 1 and 2 had an increase in infarct size [35].Animal and human studies have suggested that females show decreased susceptibility to myocardial disease [36–39]. Estrogen and other female hormones lead toaltered expression of numerous genes. Data in the literature reported that femaleshave increased levels of nuclear phospho-Akt which could be important in cardioprotection [40].
Estrogen was also reported to activate phosphatidylinositol-3-kinase (PI3-kinase) [41], and increase expression of eNOS [41], which could have arole in cardioprotection. Because of the large number of estrogen regulated genes,there are many potential candidates for cardioprotective genes in females.1011026 DNA Microarray Gene Profiling6.2.2Potential Mechanisms of CardioprotectionMany of the genes implicated in cardioprotection are transcription factors which,by inducing other genes, could alter expression of many proteins and thereby leadto cardioprotection.
Another major group of genes involved in cardioprotectionare typically classified as stress (or heat shock) proteins. These include HSPs,GSHPx-1, SODs and HO. The antioxidant gene products could enhance protection by a reduction in reactive oxygen species, although it is likely that their protective effect is more complex than this. HSPs have been traditionally thought tobe involved in regulating protein folding and degradation, although recent studiessuggest that they are also involved in nuclear hormone regulation, apoptosis andother functions that could be important in protection [42]. There is also altered expression of several genes involved in growth factor signaling.
Growth factors caninduce cardioprotection by upregulating survival pathways such as PI3-K/Akt andNF-jB. Growth factors can also activate PKC, which has been shown to be cardioprotective. The precise mechanisms by which activation of these genes leads tocardioprotection are still to be elucidated.6.3Is There a Common Set of Cardioprotective Genes?As discussed above, multiple signals can induce cardioprotection. Preconditioningcan be initiated by multiple, diverse signals.
The data so far suggest that there isremarkable similarity in the signaling pathways that lead to cardioprotection by verydiverse initiators. For example, intermittent ischemia, activators of PKC and adenosine agonists all seem to activate similar signaling pathways involving PKC and aKATP channel.
However it is unknown whether there are subtle differences in activation of cardioprotective genes with different initiators. In addition, it is not clearwhether estrogen-mediated protection and preconditioning activate similar cardioprotective genes. Among the important questions that can be addressed by highthrough-put methods such as microarrays are whether there is a common set of cardioprotective genes or whether there are redundant cardioprotective genes. Althoughit is tempting to assume that there is a common set of cardioprotective genes thismay not be the case.
A recent study by Dorn and coworkers [43] used microarrayto examine whether there were common gene changes in hypertrophy that developed in different transgenic models of hypertrophy (cardiac expression of calsequestrin, calcineurin, Gaq, and PKC-e activating peptide). Atrial natriuretic peptide wasthe only common gene changes across the different models of hypertrophy.A related question is whether activation or inactivation of a single gene is cardioprotective? Studies with transgenic mice suggest that alterations in a single genecan lead to cardioprotection, but it is unclear whether these single gene changesresult in additional secondary changes in gene expression, particularly in responseto a stress such as pressure overload or ischemia.6.5 Approaches to Identify Genes Involved in Cardioprotection6.4Effects of Long-term Activation and Dosage of Cardioprotective GenesOne obvious goal of identification of cardioprotective genes is so that strategiescan de devised to activate these genes to induce cardioprotection.
However therecould be different effects with short versus long-term activation of these genes.For example, we have shown that phosphorylation and inhibition of glycogensynthase kinase-3b (GSK-3b) is cardioprotective [44]. However, studies in neonatalmyocytes suggest that phosphorylation and inactivation of GSK-3b can initiate hypertrophy [45, 46].
Also, activation of the PI3K/Akt pathway is suggested to be important in cardioprotection in females [39, 40]; yet long term activation of PI3K isreported to lead to hypertrophy. These data suggest that sustained activation hasdifferent effects than acute activation.Another issue to consider is the level of gene activation or inactivation.
It hasbeen shown that low levels of cardiac specific overexpression of PKC-e are cardioprotective, whereas high levels of PKC-e overexpression lead to hypertrophy [12].Similarly, low level expression of adenosine A3 receptor is cardioprotective,whereas high levels result in a dilated cardiomyopathy [19]. Thus, it is importantto consider the level of gene expression when assessing models of cardioprotection.6.5Approaches to Identify Genes Involved in CardioprotectionThe quest for genes involved in cardioprotection has many facets. From the preceding discussion it is clear that we need to know whether there are commongenes or gene patterns that lead to cardioprotection, or whether different modelsof cardioprotection are mediated by completely different genes or sets of genes.
Itis well recognized in transgenic animal studies that alteration of a single gene canhave very different phenotypes depending on the genetic background [47]. Upregulation of a gene can have different consequences depending on what othergenes are expressed. The ability to examine a large number of genes, to discernsuch differences, is a strength of the microarray technique. Also, there are datasuggesting that altered gene expression can have different effects depending onthe level of overexpression or duration of expression. Given these issues, howdoes one identify genes involved in cardioprotection? One obvious approach is tostudy multiple models of cardioprotection and determine if there are commongenes or gene patterns.
In section 1, we discussed different models of cardioprotection. We will briefly consider how one might use these models in microarraystudies to identify cardioprotective genes.1031046 DNA Microarray Gene Profiling6.5.1PreconditioningPreconditioning is one of the better studied models of cardioprotection. As discussed, preconditioning with brief intermittent period of ischemia and reperfusion has been shown to reduce injury during a subsequent sustained period ofischemia occurring 24 to 72 hours later.
This delayed preconditioning requiresnew gene transcription. One strategy would be to use gene array technology to examine changes in gene expression induced by preconditioning. However, ischemia and reperfusion are likely to lead to many changes in gene expression, andonly a small subset of these changes are likely to be causally important in cardioprotection. Perhaps a more direct strategy would be to treat animals with a cardioprotective agent and examine changes in gene expression. Perfused heart modelsare only viable for a few hours, therefore to study changes in gene expression thatoccur hours after administration of an agonist would require an intact animalmodel.
Although a number of drugs and agonists can be given to a perfusedheart to mimic acute preconditioning, many of these agents are not well-toleratedby the intact animal, and thus it is difficult to examine the change in gene expression occurring over a 24 hour period. Alternatively, one could add agents thatmimic preconditioning to a perfused heart or myocytes and examine acutechanges in gene expression. A recent study used cDNA microarray profiling toidentify changes in gene expression in neonatal rat cardiomyocytes following overnight incubation with IGF-1 [48].
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