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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 9, 2004 as doi:10.1096/fj.04-1714fje. |
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* Cardiovascular Research Center, Massachusetts General Hospital, Boston, Massachusetts, USA;
Massachusetts Institute of Technology, Department of Mechanical Engineering and Center for Cancer Research, Cambridge, Massachusetts, USA; and
Harvard University Bauer Center for Genomics Research, Boston, Massachusetts, USA
1 Correspondence: Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA 02114, USA. E-mail: rhajjar{at}partners.org
SPECIFIC AIMS
Heart failure represents the end stage of familial or acquired cardiac diseases where compensatory mechanisms are no longer able to restore cardiac function and can further accelerate the disease process. A wide array of genes has been shown to be altered in that process and a number of them have been explored in human and animal models of heart failure. Analysis of transcriptional reprogramming in heart failure enables us to explore the events that trigger the cascade of changes that progress to heart failure as well as the compensatory mechanisms that take place to preserve cardiac function.
Beyond the many structural changes, failing hearts show a permanent decline in contractile function. At a cellular level, the functional defect is detected as a reduction in the amplitude of cell shortening, velocities of shortening and relaxation, as well as abnormal intracellular Ca2+ homeostasis. At the molecular level, despite differences in the triggering event, a defect in protein level and function of SERCA2a is a common finding. We have shown that restoration of the defective protein levels of SERCA2a, which regulates the cyclic compartmentalization of Ca2+ upon contraction and relaxation, leads to an improvement in the contractile function in vitro and in vivo. The restoration of Ca2+ homeostasis leading to improved myocardial function is accompanied by improved survival, normalization of metabolism, and triggered intracellular pathways.
Those positive effects may correlate with transcriptional reprogramming underlying the compensatory changes produced by the triggered or primary genetic alteration. We have explored the pattern of gene expression changes that occur in heart failure after restoration of the intracellular Ca2+ homeostasis and contractile function using in vivo adenoviral gene transfer of SERCA2a. Gene transfer is likely to induce a further perturbation not accounted for by the genetic changes linked to overexpression of the targeted gene. Such a "viral" effect was explored by looking at gene expression profiles after adenovirus (Ad.) gene transfer of a reporter gene.
Our aims were to perform transcript profiling in failing rat hearts compared with normal hearts; identify genes up-regulated in heart failure that are then returned to baseline by gene transfer of SERCA2a; identify genes down-regulated in heart failure that are then returned to baseline by gene transfer of SERCA2a, and evaluate the potential confounding effects of viral particles and reporter genes, an important consideration in investigations based on gene transfer using viral vectors
PRINCIPAL FINDINGS
1. 3172 genes were modified by viral infection and eliminated from the analysis
The remaining 1300 genes were unchanged by overexpressing the reporter gene. Of the 1300 genes that remained after eliminating virus effects, 251 transcripts were found differentially expressed by at least 1.2-fold in failing vs. nonfailing rats. Functional analysis of the group of genes showed a pattern similar to that already reported for failing human hearts. The number of genes down-regulated in failure was 124; those up-regulated were 127.
2. Twenty-four genes were found to be down-regulated in failure and restored to nonfailing levels with SERCA2a overexpression. Conversely, 27 genes were up-regulated in failure and restored down to nonfailing levels with SERCA2a overexpression.
3. Among the genes down-regulated upon failure and normalized by SERCA2a, an important structural gene was found: tensin (1.5-fold)
4. Among the genes up-regulated then corrected back to normal is notably tau protein kinase 1 (GSK3ß), which was up-regulated by 1.3-fold upon cardiac failure
GSK3ß is known to protect from cardiac hypertrophy and from apoptosis in the heart.
5. To validate the microarray data, we used quantitative real-time PCR (QRT-PCR)
S18 rRNA was quantitated using QRT-PCR in the presence (positive control) and absence (negative control) of reverse transcriptase to demonstrate the absence of cDNA contamination in all samples and to account for slight differences in RNA concentration in the different samples. Postamplification dissociation curves demonstrated the presence of a single amplification product in the absence of DNA contamination for each set of primers.
CONCLUSIONS AND SIGNIFICANCE
As a consequence of myocardial dysfunction, the expression of several genes within the heart is altered. Genes are under- or overexpressed as a result of the injury leading to adaptive changes aimed to counteract the effect of the insult.
Among the failing down-regulated genes rescued by SERCA2a was the small peptide tensin. This protein localizes to regions of the plasma membrane where the cell attaches to the extracellular matrix. It cross-links actin filaments and contains a Src homology 2 domain often found in molecules involved in signal transduction. Tensin is a substrate of calpain II and part of the integrin complex consisting of structural and signaling proteins serving as a physical link between the extracellular matrix and cytoskeleton, as well as signal transduction. Normalization of tensin gene expression by SERCA2a gene transfer in the failing heart may represent an interesting mechanism for cardioprotection from apoptosis and cell survival signaling pathways in heart failure. Akt was found to be decreased in failing hearts and rescued by overexpression of SERCA2a. Akt is a powerful survival signal in many systems and is activated by several cardioprotective ligand receptor systems including insulin-, IGF-1-, and gp130-signaling pathways. Its rescue probably plays an important role in the survival benefit observed with SERCA2a overexpression.
The increase of the number of genes in heart failure can be either compensatory or causative for the final phenotype of failure. From this analysis it is not possible to distinguish which genes are indeed compensatory vs. causative. Up-regulated genes can have proliferative functions especially in the setting of our model of hypertrophy in transition to heart failure. GSK-3ß in the heart is involved as a target molecule and effector in some pathways in hypertrophy and heart failure and mediates a variety of cell functions. After overexpression of SERCA2a, the normalization of gene expression for GSK3ß may suggest a modulatory response of GSK3ß upon restoration of cardiac function and after normalization of Ca2+ overload pathways in heart failure. A Ca2+-mediated or direct interaction between SERCA2a and the PI3K/GSK3ß pathways has been recently found. In addition, Akt inhibits GSK3ß and, parallel to restoration of GSK 3 from an elevated level, was restored from a low level by overexpression of SERCA2a.
In addition to the known genes, 
30% of transcripts are unknown and appear to participate in cell homoeostasis and normalization of gene expression after the reconstitution of normal levels of SERCA2a in failing heart tissue. These findings may lead to discovery of novel genes of importance for the pathogenesis of heart failure and dissection of the distinct pathways.
We explored how the reconstitution of SERCA2a protein determines modification of gene expression of functionally related proteins. The use of oligonucleotide arrays can help dissect the network of genes related to SERCA2a regulation and function as well as the response to its genetic reconstitution.
Clustering analysis provides a way to group functionally related genes and their change upon the process of cardiac failure and remodeling. Restoring SERCA2a gene and protein level showed a tendency toward reconstituting the pattern of genes involved in cell cycle, proliferation, death, and metabolism.
Despite technological limitations, gene array will find a role in enhancing knowledge of the perturbations occurring upon development of diseases and help clarify the effects of therapeutic interventions. New genes have been identified that may prove important in understanding the mechanisms of heart failure and show potential as therapeutic targets.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1714fje; doi: 10.1096/fj.04-1714fje
This article has been cited by other articles:
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  H. Rupp, T. P. Rupp, and B. Maisch Fatty acid oxidation inhibition with PPAR{alpha} activation (FOXIB/PPAR{alpha}) for normalizing gene expression in heart failure? Cardiovasc Res, June 1, 2005; 66(3): 423 - 426. [Full Text] [PDF]
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