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Gene expression

Post-transcriptional regulation

Translation

Post-translational modifications J

Fig. 5.1 Genome profile as an "end-point" or a "starting point". In the "end-point" approach, the genomic profile is the result of the transmission of extracellular stimuli to the nucleus. In the "starting point" approach, the gene leads to the production of proteins, which needs to be further studied by proteo-mics.

as an integrator of incoming information. Considering that a cell is viable as long as it has a functional nucleus, gene expression profiling is a way to evaluate the nuclear activity of a specific cell type in a specific milieu. We know that, in turn, the increased expression of a gene does not automatically result in the increased expression of the corresponding protein, and that the expression of a specific protein can be regulated by non-transcriptional mechanisms. We also know that a transcript is not necessarily targeted to the translational machinery, as shown recently with the concept of gene silencing by RNA interference [28, 29].

The "end-poinf strategy is a way to better understand the mechanisms of cardiac adaptation. For example, we often refer to the concept of "stress response" to characterize the response of the heart to conditions as diverse as transient ischemia, aortic banding or catecholamines infusion. Although these conditions can all induce the increased expression of "stress" gene markers (such as the atrial natriuretic factor or proto-oncogenes), each of them can activate the expression of specific subsets of genes, such as specific heat-shock proteins, anti-apoptotic proteins, metabolic enzymes, activators of protein translation or others. Therefore, the response of the myocardium to "stress" depends on the context and the stimulus.

Strategies and Limitations of Genome Profiling

Several parameters must be taken into consideration to perform functional genomics and gene profiling. These include, but are not restricted to, a biological problem, a good model and a reliable technique.

The Biological Problem

The biological problem, or the question asked, is usually obscured by the fact that functional genomics is perceived as a "fishing expedition". Although it is true that genomics studies are not always hypothesis-driven, very expensive and time-consuming experiments can rapidly turn into deep frustration if they are conducted in the absence of an underlying biological question. Depending on the question or the hypothesis, two broad approaches can be used, the horizontal approach and the vertical approach (Fig. 5.2). In the horizontal approach, the tools of functional genomics are used to determine how a change in the function of the organ results in a change of gene expression. This approach is horizontal, because it will analyze a large number of genes at one level of investigation (usually the mRNA expression) and lead to the determination of a genomic profile. In the vertical approach, the question asked to functional genomics is what is the function of a specific gene (Fig. 5.2). In that approach, the investigator concentrates on one gene and conducts his experiments by the combination of various techniques

Functional Genomics

How does function affect gene expression?

What is the functior of unknown genes?

Horizontal approach

Vertical approach

Gene profile Cloning

Transgene Interactions Localization Function

Activity Regulation

Fig. 5.2 The horizontal and vertical approaches of functional genomics. The horizontal approach shows how gene expression is affected by function. The vertical approach shows the function of a specific gene product.

(mRNA expression, protein-protein interaction, cellular localization, transfection, genetically-modified animals...). This last approach is used for the characterization of unknown and novel genes, which still represent a substantial part of our genome [30, 31]. Both approaches are complementary. As we will see below, the subtractive hybridization methodology is used to investigate a large array of genes in a specific condition (horizontal approach), but also brings some information about unknown genes, which in turn determines the vertical approach. Whereas the horizontal approach leads to the discovery of new concepts, the vertical approach leads to the discovery of new functions. Combining both approaches provides a link between description and mechanism.

The Model

A good model of investigation is equally important. Most of the experiments of functional genomics are performed on samples from in vivo studies. This adds to the complexity of the experiments because, in addition to the potential problem of reproducibility and sample-to-sample variation, we must also consider the relevance of the model used and the possibility of inter-species differences. For obvious epidemiological reasons, most of us are interested in determining how the human heart develops and ages, and how it can be injured and repaired. We therefore need to develop and use the animal models which are relevant to clinical problems in the human heart. So far, these questions are mainly addressed in rodent models, which points out to the issue of species differences. A classical example of genomic differences between species is the "fetal gene program". The pioneering experiments of gene expression in the heart pinpointed that a rodent heart submitted to increased workload switches the expression of contractile proteins from an "adult" isoform back to an isoform preferentially expressed in the fetal heart. For instance, the cardiac-specific isoform of alpha-actin is partially replaced by the "fetal" skeletal isoform, whereas the alpha-isoform of myosin heavy chain is partially replaced by the beta-isoform [32-34]. This protein switching results from a switch in the expression of the corresponding genes. It has been shown since that this concept can be extended to other categories of genes (such as genes involved in the regulation of calcium handling and metabolism) [11, 12, 35, 36], and is also reproduced when the workload is decreased rather than increased [13]. However, such "fetal gene program" is hardly reproduced in larger mammals [37]. For instance, the predominant isoform of myosin heavy chain in the adult is already the beta-isoform [38] and other mechanisms of adaptation of contractile performance, such as the phosphorylation or dephosphorylation of myosin light chains [39, 40], is more developed in large mammals. Other adaptive mechanisms are activated in larger mammals that do not necessarily exist in rodents. An obvious reason for the discrepancy between species is that a mouse heart beating at 500 beats per minute has different regulatory mechanisms than a human heart beating at 75 beats per minute, although the overall goal of cardiac function remains the same. Heart rate, cardiac chamber geometry, loading condi tions, mechanisms of calcium handling, coronary anatomy and collaterals are just a few examples of biological and anatomical parameters responsible for species differences. It is therefore crucial to determine the relevance of the model for the problem investigated. Rodents, especially mice, are particularly attractive as "test tubes" to study the effects of one specific gene, either overexpressed or knocked-out, on global gene expression. As shown below, larger mammals can be useful for more complex models of ischemic heart disease or heart failure.

The Technological Approach

One of the first techniques used for gene profiling was the differential display [41-43]. Although elegant and simple, this technique is usually extremely time-consuming because it addresses one gene at a time. Currently, the technology of microarrays is extremely popular. One chip contains most of the expressed transcripts from one species. The sample preparation is relatively easy, provided that some RNA of good quality is extracted from the tissue or the cells. The procedure can be started with as low as 5 ^g total RNA. Processing the chips is a quick and automated process. The downside is that the signal to noise ratio can be weak in some hybridizations, which necessitates to repeat the experiments or to increase the number of samples in the series. Each chip represents a compromise between sensitivity and specificity. The choice between cDNA and oligonucleotide chips is an illustration of this compromise. Also, the analysis of the data is not necessarily easy, as thousands of genes have to be analyzed and clustered [44]. It results that the statistical tests needed to determine significant differences are far more complex than a simple Students t test [45]. Biological variations from sample to sample must be taken into account and several recent studies stressed the importance of repeating the experiments in a representative series of samples [46, 47]. Even more importantly, the investigator must often rely on the use of commercial chips, which limits the possibility to control their quality or content. Cross-contaminations between different clones can not be excluded, and the chips are sometimes printed erroneously [48]. Therefore, even if the technique is powerful and attractive, its use requires a validation of the results by alternative molecular techniques. In addition, these chips are so far available for a restricted number of species only. For these reasons, the subtractive hybridization can be an attractive alternative in specific cases.

Analyzing Gene Expression by Subtractive Hybridization

The subtractive suppression hybridization represents a large-scale, unbiased method for detecting transcriptionally and post-transcriptionally regulated genes, both known and unknown, independently of the prevalence of these transcripts [49]. A fruitful utilization of this technique requires some RNA of excellent quality, a se quencing facility and alternative methods of validation. It can be coupled to the printing and hybridization of microarrays [50].

Methodology

The procedure is summarized in Fig. 5.3. The subtraction is performed between two biological samples from which total RNA and, subsequently, poly-A messenger RNA are extracted. The subtraction is followed by the subcloning of the different subtracted cDNAs. After purification, the clones can be used for sequencing or microarray printing. Querying the sequences in different databases identifies the subtracted genes. The microarrays offer a semi-quantitation of the variation in gene expression between the samples. The results need to be validated by alternative methods, such as Northern blot, quantitative PCR and in-situ hybridization.

1. Experimental samples. Experiments are usually started from frozen tissue. A comparison can be made between an experimental heart and a sham, or, for larger species, between two areas of the same hearts. The last choice allows a paired comparison of the data. About 200-400 mg of tissue is usually enough to collect the amount of RNA needed for the experiment. Compared to other tissues, the mRNA content per gram of cardiac tissue is relatively low. It is recom-

Fig. 5.3 Overview of the different steps of the subtractive hybridization. See text for details.

Total RNA extraction 1

Poly-A RNA extraction 1

Double-stranded DNA synthesis 1

Subtractive hybridization 1

Subcloning

Validation Sequencing Microarrays

Tissue sample

mended to pool several samples from each group, to ensure that the subtraction is well representative of the condition tested.

2. RNA isolation. This step is probably the most important of the whole procedure, as all the information gathered from the subtraction tightly depends on the quality of the starting material, which is the poly-A RNA. Total RNA extraction is usually performed by the phenol/chloroform extraction [51] and does not require further purification. RNAse-free conditions are mandatory to preserve the structure and the full length of the RNA as well as possible. Purification of the poly-A mRNA requires more attention. Different techniques are available, some insisting more on the exclusion of the contaminating ribosomal RNA, some insisting more on the recovery of rare transcripts. The latter choice is probably the best in this case, to ensure that the library to be subtracted contains as many different transcripts as possible. A contamination by ribosomal RNA will usually not jeopardize the experiment. The quality of the RNA must be verified by the 260/280 absorbance ratio, by Northern blot or by automated methods.

3. Subtractive hybridization. The subtraction between two libraries begins by the reverse transcription of the poly-A mRNA into double-stranded DNA (Fig. 5.4). The reverse transcription is usually performed with an oligo-dT primer, rather than random hexamers, to increase the length of the products. Because of this priming method, the need of good-quality poly-A RNA is self-explanatory. The DNA from both samples is subsequently digested by a 4 base-cutter restriction

Fig. 5.4 Principle of the subtractive hybridization and amplification of the subtracted products. See text for details.

enzyme (Rsa I, typically). One of the two libraries is then ligated to specific adapters, before the two preparations are hybridized together. Two successive rounds of hybridization are usually performed. The duration of the hybridization is a crucial aspect of the experiment for the following reason. The hybridization between two complementary strands of DNA is proportional to the concentration (Co) of the strands and the time (t) of hybridization. The product of these two factors (Cot) is specific for each sample of DNA sample. Because of the Cot principle, the more abundant transcripts will hybridize faster. As the subtraction does not affect the concentration (Co) of the cDNAs, adjusting the time of hybridization (t) is a mechanism of normalization, to ensure that all the transcripts (rare or abundant) will hybridize relatively equally. Because of this normalization, the chance to find a transcript in the subtracted library after the experiment is relatively independent of its original abundance. The DNA fragments that do not hybridize correspond to those which are expressed differently in the two preparations (Fig. 5.4). These gene products are further amplified by PCR using a single primer pair that recognizes the adapters, and subcloned in a vector system. The plasmid can be used for sequencing, microarrays, Northern blot or Dot blot (Fig. 5.4). The adapters are ligated to one cDNA library only. For instance, if the adapters are ligated on the cDNA library from an isch-emic heart (labeled "tissue of interest' in Fig. 5.4) and if this library is subsequently subtracted from a library from normal myocardium, the experiment will show the genes that are overexpressed in ischemic myocardium. Reciprocally, the adapters can be ligated to the library from normal heart, to show the genes that are downregulated in the ischemic territory. A complete hybridization therefore includes a "forward" and a "reverse" library to show both the up-regulation and downregulation of genes in one condition compared to another.

4. Sequencing and database query. Although the hybridization in itself can be achieved in less than a week, the identification of the subcloned genes is more labor-intensive. Nowadays, the sequencing work is greatly facilitated by the use of automated sequencers using a 96-well or 384-well format. Some investigators prefer to verify first the quality of the library by microarrays. The sequencing also determines the quality of the library by the extent of the different gene products found, and by the redundancy of the same products. Finding new products by sequencing a library follows an exponential curve. The first batches of sequences bring many different gene products, then further sequencing shows the same products coming back again and again. It is reasonable to interrupt the sequencing when the last batch of clones does not bring more than 10% of new information (for instance, if less than 10 new gene products are found in a 96-well sequencing plate). Also, if a specific gene is highly regulated (i.e., if the concentration of the transcript between the two conditions tested shows more than a 10-fold difference), this specific product has a high probability to be sequenced more often.

5. Microarrays. Microarrays represent a reliable technique to verify at a glance which clones from the library are truly regulated. It consists in spotting all the clones isolated on nylon membranes or glass chips and to hybridize them with labeled RNA from the different experimental groups tested. The labeling can be radioactive (using 33P isotope instead of 32P for a better resolution) or nonradioactive (using fluorescent dyes or chemiluminescence). The isotopic method is limited in its sensitivity. This technique therefore offers some information about the magnitude of changes in expression for each gene product. These chips can also be hybridized with labeled fragments of the mitochondrial genome. By this approach, it is possible to define which clones correspond to mitochondrial DNA, instead of nuclear-encoded genes. This strategy may greatly reduce the number of clones to sequence. 6. Validation. Whatever the results of the hybridization and the identity of the isolated clones, a method of validation is always required. It is necessary to rely on methods that truly quantify the changes in expression of one specific gene of interest. One of the most popular methods is the Northern blot, because the probe is already available in the form of the clone sequenced from the library. The average size of the cDNA fragments obtained during the subtraction (0.6 to 1.2 Kb) corresponds to the recommended size of a usual probe for Northern blot. In addition, the tissue localization of the transcript can be validated further by in-situ hybridization. Again, the subcloned DNA fragment can be used as a probe, although some investigators prefer to use oligo probes. Finally, the quantitative PCR (using specific fluorogenic probes or as a screening technique with the SybrGreen) will be the preferred choice when analyzing a large number of samples, when making a time-course, or when the RNA supply is limited.

Advantages and Disadvantages

The main advantage of the subtractive hybridization consists in its unbiased nature. Potentially, all the genes regulated by a specific condition can be found. This requires a good preparation of RNA and an extensive work of subcloning and sequencing. Ischemia/reperfusion, cardiac hypertrophy and heart failure, in which changes in both workload and ventricular function are found, can affect dramatically the expression of genes in the heart and can trigger the expression of genes that are not normally expressed in myocardium. The subtractive hybridization is therefore an excellent tool for discovery. It can show "unexpected" gene profiles, or lead to the discovery and characterization of novel genes. The technique is therefore excellent for both the horizontal and vertical approaches described above. It starts by a genomic profiling and then, depending on the identity of the clones, can lead to an in-depth analysis of specific products. Another advantage is that the experiment does not depend on the species studied. The different genome projects have substantially increased the number of sequences available in public databases, so that querying sequences from swine, dog or even woodchuck tissues is not a problem in practice.

The inconvenience of the subtractive hybridization is that, due to its random nature, interesting clones can be "missed". The technique is not as comprehensive as a DNA chip (although all the clones collected from a subtraction can be spotted on a heart-specific, custom-made chip). However, the chance to find a specific gene is proportional to the magnitude of its upregulation in one group compared to the other. Another inconvenience is the requirement of an access to a dedicated sequencing facility. Finally, especially in the heart, it is impossible to avoid the "contamination" of the library by non-nuclear genes from the mitochondrial DNA. Due to the large amount of mitochondria in the cardiomyocyte, such contamination can represent 20-50% of the overall library [50, 52]. This contamination can be limited by performing additional rounds of hybridization, at the expense of loosing rarely expressed nuclear transcripts. The mitochondrial genome includes the genes coding for the subunits 1 to 6 of NADH dehydrogenase, the subunits 1 to 3 of cytochrome oxidase, the ribosomal RNAs 12S and 16S, the cytochrome b and the subunits 6 and 8 of ATP synthase. A last inconvenience is that the subtraction requires to start with 2-3 ^g of poly-RNA, which may represent about 50 ^g of total RNA in the heart. There are some experimental conditions, especially the genomic profiles during development, in which such amount can hardly be obtained.

Genomics of Myocardial Ischemia

The discovery that gene expression can be affected by changes in workload [21, 32] rapidly led to the investigation of the adaptation of myocardial gene expression in the ischemic heart. Schapef s laboratory provided pioneering studies on alterations in gene expression in large mammalian models of ischemia/reperfusion. These studies were mainly designed to follow the time-course of changes in genes coding for proto-oncogenes and calcium-handling proteins [53-55]. The proto-on-cogenes were chosen because they represent a rapid mechanism of transcriptional adaptation to stress, whereas calcium-handling genes were studied to correlate their expression to the prolonged dysfunction that follows ischemia. Another goal was to investigate whether repetitive episodes of ischemia/reperfusion inducing preconditioning are accompanied by a change in gene expression. Several proto-oncogenes (such as c-fos or junB) showed an increase either during ischemia or reperfusion. Similarly, the Ca2+-ATPase and calsequestrin were found to increase during post-ischemic dysfunction. These studies were the first to show that even short episodes of ischemia-reperfusion could affect myocardial gene expression. Also, they showed the possibility to investigate myocardial gene expression in large mammalian models of ischemic heart disease. However, the experimental design of this work adopted a "vertical approach", which precluded the genomic profiling of these hearts. Because only limited subsets of genes were investigated, the global impact of these changes could hardly be determined.

More recently, the technology of microarrays has been used in several rodent models to offer a more comprehensive view of the genomic adaptation to ischemia. So far, most studies have been performed in models of myocardial infarction and remodeling [50, 56, 57]. In these studies, the induction of irreversible ischemia mainly affects the expression of genes involved in the synthesis of cyto-

skeletal proteins and of the extracellular matrix. These studies will lead to a better understanding of the impact of drug therapy (such as angiotensin-converting enzyme inhibitors) on cardiac remodeling [57]. However, it will be important in the future to explore the adaptation to reversible ischemia in these models, to better elucidate the potential protective mechanisms that the heart can develop before ischemia becomes irreversible. Such work will be equally important to develop new therapeutic strategies.

Subtractive Hybridization of Myocardial Ischemia

Myocardial Stunning

Myocardial stunning refers to the myocardial dysfunction that follows an acute episode of ischemia [58, 59]. A preserved ultrastructure of the myocardium and the absence of cell loss characterize this form of non-lethal, fully reversible ischemia [60]. These characteristics are in striking contrast to other models of isch-emia-reperfusion, in which both necrosis and apoptosis are observed [61, 62]. The syndrome of stunning is highly prevalent in different etiologies of clinical isch-emic 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 paramount clinical importance because it corresponds to a condition in which myocardial viability is maintained.

Although it was previously thought that the ventricular dysfunction induced by ischemia could not be brought back to a normal contractile state, it is now clearly demonstrated that the viability of dysfunctional myocardium is preserved [65]. Unraveling the molecular mechanisms of cardioprotection in stunned myocardium can open new avenues to salvage dysfunctional cardiac tissue and prevent cardiac cell 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 swine heart and the human heart share the same geometry, heart rate, coronary anatomy and absence of collaterals.

Genomic Profile of Myocardial Stunning

The absence ofirreversible cellular damage in stunned myocardium may correspond either to an increased resistance of the heart to ischemia, or to the absence of stimuli triggering the pathways of cell death. The latter possibility is of low probability, be cause even mild episodes of ischemia/reperfusion can activate different intracellular stress 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 whether myocardial stunning triggers the coordinated expression of different sets of genes acting 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 artery is decreased by about 50% during 90 minutes [70]. This ischemic episode is followed by full reperfusion. Despite the normalization of blood flow after reperfusion, the contractile function remains depressed, which reflects myocardial stunning. A full functional recovery is typically observed after 48-72 h, and pathological examination of this myocardium does not show any necrosis or apoptosis. We used this model of reversible ischemia to investigate whether the protection of the myocardium against irreversible damage correlates with a specific gene profile [52]. A subtractive hybridization was therefore performed between the post-ischemic, stunned myocardium and 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 specific experiments, the contamination of mitochondrial DNA after two rounds of hybridization was about 35%. After database query, 60% of the nuclear-encoded sequences corresponded to known gene products. The remaining 40% could be divided equally between known sequences of unknown function and sequences with no match. Interestingly, we found that more than 30% of the genes that were upregulated in stunned myocardium are involved in different mechanisms of cell survival, including: resistance to apoptosis, cytoprotection ("stress response") and cell growth. Many of them had been implied previously in the survival of other cell types but had not been described before in the heart. In particular, we found an induction in stunned myocardium of several genes not expressed in the normal myocardium and which participate to the development and growth of different forms of tumors. This illustrates the power of the subtractive hybridization to pull out "unexpected" genes and profiles. An example of the validation of these results is shown on Fig. 5.5 for the plasminogen activator inhibitor-1 (PAI-1), a serpin with anti-apoptotic properties [71]. A Northern blot on ischemic and normal samples from five different hearts showed a reproducible and robust induction of this gene during ischemia. It could be confirmed by in-situ hybridization that this induction occurred in cardiomyo-cytes. 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-ischemic stunning and returns back to normal after 12 hours, when the contractile function recovers. The sensitivity of the quantitative PCR also allows to measure separately the sub-endocardium from the sub-epicardium, to show that the gene response is of higher amplitude in the sub-endocardium, where the flow reduction is the most important. Remarkably, this transmural difference was found for all the genes measured [52]. There is therefore a gradient of gene response that matches the gradient of 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.

Fig. 5.5 Methods 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 samples from stunned territory. The corresponding control (remote) area in the same hearts does not show any signal. Panel B shows by in-situ hybridization that PAI-1 induction occurs in cardiomyocytes. Panel C shows by quantitative PCR the time-course of PAI-1 induction during stunning, with a maximal increase at one hour reperfusion and a normalization at

Fig. 5.5 Methods 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 samples from stunned territory. The corresponding control (remote) area in the same hearts does not show any signal. Panel B shows by in-situ hybridization that PAI-1 induction occurs in cardiomyocytes. Panel C shows by quantitative PCR the time-course of PAI-1 induction during stunning, with a maximal increase at one hour reperfusion and a normalization at

Chasing Novel Genes

As mentioned above, about 20% of the gene products found in the subtractive hybridization of stunned myocardium in the pig did not recall any known sequence in public databases. This last group is particularly interesting, because it contains the novel genes. However, because the cDNA synthesis starting the subtraction experiments is primed by oligo-dT, an "unknown" sequence can also correspond to the 3' UTR of a known gene. This can be due to two reasons. First, many se quencing laboratories are interested in cloning only the coding sequence of the genes ("ORFome"), and the 3'UTR is left undetermined. Second, the 3' UTR is the 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 choose which 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 subtractive hybridization. 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. The tissue selectivity can be assessed easily with a multi-tissue Northern blot. A gene that is expressed only in the heart is probably more interesting than a gene that is expressed ubiquitously. The Northern blot will also determine the size of the transcript and thereby will predict the amount of work needed to achieve a full-length cloning.

• Regulated gene. It is preferable to concentrate on true positives, i.e., genes showing a differential expression between the two experimental groups. Once an interesting target has been identified, the different methodologies of the "vertical approach" (Fig. 5.2) can be applied for a full characterization of the transcript and the corresponding protein. This approach can be applied not only for the novel genes, but also for known genes that had not been described in the heart before.

Summary

The 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 the heart 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 appropriate techniques. 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-abundance transcripts. In addition, the subtraction experiments reveal "unexpected" gene profiles and represent a starting point for the characterization of novel genes.

Acknowledgements

I am particularly grateful to Stephen F. Vatner for his support and for developing the animal models used in these experiments. I also express my deepest gratitude to 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 Abdella-tif for their collaboration and fruitful discussions, as well as Erika Thompson, Anna Zajac and Li Wang for their expert technical assistance.

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