MOTIVATION: Several statistical methods that combine analysis of differential gene expression with biological knowledge databases have been proposed for a more rapid interpretation of expression data. However, most such methods are based on a series of univariate statistical tests and do not properly account for the complex structure of gene interactions. RESULTS: We present a simple yet effective multivariate statistical procedure for assessing the correlation between a subspace defined by a group of genes and a binary phenotype. A subspace is deemed significant if the samples corresponding to different phenotypes are well separated in that subspace. The separation is measured using Hotelling's T(2) statistic, which captures the covariance structure of the subspace. When the dimension of the subspace is larger than that of the sample space, we project the original data to a smaller orthonormal subspace. We use this method to search through functional pathway subspaces defined by Reactome, KEGG, BioCarta and Gene Ontology. To demonstrate its performance, we apply this method to the data from two published studies, and visualize the results in the principal component space.

An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiomyocytes, the transcription factor Gata4 is required for agonist-induced hypertrophy. We hypothesized that, in the intact organism, Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed up-regulation of genes associated with apoptosis and fibrosis, including members of the TGF-beta pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure.

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart disease. At localized swellings of extracellular matrix known as the endocardial cushions, the endothelial lining of the heart undergoes an epithelial to mesenchymal transition (EMT) to form the mesenchymal progenitors of the AV valves. Further growth and differentiation of these mesenchymal precursors results in the formation of portions of the atrial and ventricular septae, and the generation of thin, pliable valves. Gata4, which encodes a zinc finger transcription factor, is expressed in the endothelium and mesenchyme of the AV valves. Using a Tie2-Cre transgene, we selectively inactivated Gata4 within endothelial-derived cells. Mutant endothelium failed to undergo EMT, resulting in hypocellular cushions. Mutant cushions had decreased levels of Erbb3, an EGF-family receptor essential for EMT in the atrioventricular cushions. In Gata4 mutant embryos, Erbb3 downregulation was associated with impaired activation of Erk, which is also required for EMT. Expression of a Gata4 mutant protein defective in interaction with Friend of Gata (FOG) cofactors rescued the EMT defect, but resulted in a decreased proliferation of mesenchyme and hypoplastic cushions that failed to septate the ventricular inlet. We demonstrate two novel functions of Gata4 in development of the AV valves. First, Gata4 functions as an upstream regulator of an Erbb3-Erk pathway necessary for EMT, and second, Gata4 acts to promote cushion mesenchyme growth and remodeling.

Cardiac hypertrophy is a complex and nonhomogenous response to various stimuli. In this study, we used high-density oligonucleotide microarray to examine gene expression profiles during physiological hypertrophy, pathological hypertrophy, and heart failure in Dahl salt-sensitive rats. There were changes in 404/3,160 and 874/3,160 genes between physiological and pathological hypertrophy and the transition from hypertrophy to heart failure, respectively. There were increases in stress response genes (e.g., heat shock proteins) and inflammation-related genes (e.g., pancreatitis-associated protein and arachidonate 12-lipoxygenase) in pathological processes but not in physiological hypertrophy. Furthermore, atrial natriuretic factor and brain natriuretic protein showed distinctive changes that are very specific to different conditions. In addition, we used a resampling-based gene score-calculating method to define significantly altered gene clusters, based on Gene Ontology classification. It revealed significant alterations in genes involved in the apoptosis pathway during pathological hypertrophy, suggesting that the apoptosis pathway may play a role during the transition to heart failure. In addition, there were significant changes in glucose/insulin signaling, protein biosynthesis, and epidermal growth factor signaling during physiological hypertrophy but not during pathological hypertrophy.

SUMMARY: To increase compatibility between different generations of Affymetrix GeneChip arrays, we propose a method of filtering probes based on their sequences. Our method is implemented as a web-based service for downloading necessary materials for converting the raw data files (*.CEL) for comparative analysis. The user can specify the appropriate level of filtering by setting the criteria for the minimum overlap length between probe sequences and the minimum number of usable probe pairs per probe set. Our website supports a within-species comparison for human and mouse GeneChip arrays. AVAILABILITY: http://www.crosschip.org

Accurate and rapid identification of perturbed pathways through the analysis of genome-wide expression profiles facilitates the generation of biological hypotheses. We propose a statistical framework for determining whether a specified group of genes for a pathway has a coordinated association with a phenotype of interest. Several issues on proper hypothesis-testing procedures are clarified. In particular, it is shown that the differences in the correlation structure of each set of genes can lead to a biased comparison among gene sets unless a normalization procedure is applied. We propose statistical tests for two important but different aspects of association for each group of genes. This approach has more statistical power than currently available methods and can result in the discovery of statistically significant pathways that are not detected by other methods. This method is applied to data sets involving diabetes, inflammatory myopathies, and Alzheimer's disease, using gene sets we compiled from various public databases. In the case of inflammatory myopathies, we have correctly identified the known cytotoxic T lymphocyte-mediated autoimmunity in inclusion body myositis. Furthermore, we predicted the presence of dendritic cells in inclusion body myositis and of an IFN-alpha/beta response in dermatomyositis, neither of which was previously described. These predictions have been subsequently corroborated by immunohistochemistry.

BACKGROUND: One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform. RESULTS: We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis. CONCLUSIONS: Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different generations of microarrays can be combined more effectively.

Insulin-like growth factor 1 (IGF1) was considered a potential candidate for the treatment of heart failure. However, some animal studies and clinical trials have questioned whether elevating IGF1 chronically is beneficial. Secondary effects of increased serum IGF1 levels on other tissues may explain these unfavorable results. The aim of the current study was to examine the role of IGF1 in cardiac myocytes in the absence of secondary effects, and to elucidate downstream signaling pathways and transcriptional regulatory effects of the IGF1 receptor (IGF1R). Transgenic mice overexpressing IGF1R in the heart displayed cardiac hypertrophy, which was the result of an increase in myocyte size, and there was no evidence of histopathology. IGF1R transgenics also displayed enhanced systolic function at 3 months of age, and this was maintained at 12-16 months of age. The phosphoinositide 3-kinase (PI3K)-Akt-p70S6K1 pathway was significantly activated in hearts from IGF1R transgenics. Cardiac hypertrophy induced by overexpression of IGF1R was completely blocked by a dominant negative PI3K(p110alpha) mutant, suggesting IGF1R promotes compensated cardiac hypertrophy in a PI3K(p110alpha)-dependent manner. This study suggests that targeting the cardiac IGF1R-PI3K(p110alpha) pathway could be a potential therapeutic strategy for the treatment of heart failure.

Mouse models mimicking human diseases are important tools in trying to understand the underlying mechanisms of many disease states. Several surgical models have been described that mimic human myocardial infarction (MI) and pressure-overload-induced cardiac hypertrophy. However, there are very few detailed descriptions for performing these surgical techniques in mice. Consequently, the number of laboratories that are proficient in performing cardiac surgical procedures in mice has been limited. Microarray technologies measure the expression of thousands of genes simultaneously, allowing for the identification of genes and pathways that may potentially be involved in the disease process. The statistical analysis of microarray experiments is highly influenced by the amount of variability in the experiment. To keep the number of required independent biological replicates and the associated costs of the study to a minimum, it is critical to minimize experimental variability by optimizing the surgical procedures. The aim of this publication was to provide a detailed description of techniques required to perform mouse cardiac surgery, such that these models can be utilized for genomic studies. A description of three major surgical procedures has been provided: 1) aortic constriction, 2) pulmonary artery banding, 3) MI (including ischemia-reperfusion). Emphasis has been placed on technical procedures with the inclusion of thorough descriptions of all equipment and devices employed in surgery, as well as the application of such techniques for expression profiling studies. The cardiac surgical techniques described have been, and will continue to be, important for elucidating the molecular mechanisms of cardiac hypertrophy and failure with high-throughput technology.

Alterations in brain high-energy phosphate metabolism, determined by in vivo magnetic resonance spectroscopy (MRS), have been reported in subjects with a number of brain disorders including major depression, schizophrenia, and substance abuse. It is not clear to what extent these changes can be modified by pharmacological or nutritional means. To address this possibility, we evaluated changes in brain chemistry that were associated with oral creatine (Cr) administration. We hypothesized that oral Cr supplementation, by increasing brain creatine and high-energy phosphate stored in phosphocreatine, would result in an increase in the creatine resonance, as measured using proton 1H-MRS, and a decrease in the beta-nucleoside triphosphate (NTP) peak and an increase in the phosphocreatine (PCr) peak, as measured by phosphorus 31P-MRS, in brain of healthy human subjects. Fifteen healthy male subjects (age=22.9+/-2.2; body mass index=22.9+/-1.7), who were without any axis I disorders or physical or neurological illness, were recruited. Ten subjects took creatine-monohydrate, 0.3 g/kg/day for the first 7 days and 0.03 g/kg/day for the next 7 days (creatine group). Five comparison subjects took equivalent amounts of sucrose as placebo (placebo group). Both 1H- and 31P-MRS scans were acquired at baseline, as well as at day 7 and day 14 of oral supplementation. 1H-MRS: Water suppressed localized spectra were acquired using a single-voxel (1.5 cm x 2 cm x 2 cm) proton MRS PRESS sequence in the left frontal lobe. 31P-MRS: Phosphorus spectral data were recorded from a 5-cm-thick axial brain slice using a short-TE slice selective spin-echo pulse sequence. The creatine group had significantly increased brain creatine levels (8.1% and 9.3%, in creatine/N-acetyl aspartate and creatine/choline ratios, respectively) compared to the placebo group over the 2-week period. The creatine group had significantly decreased beta-NTP levels (7.8%) and marginally increased PCr (3.4%) over the same period. In addition, the brain inorganic phosphate level increased over the same period in the creatine group (9.8%). The current study is the first multinuclear (1H and 31P) MRS study to evaluate changes in brain high-energy phosphate metabolism following oral creatine supplementation in healthy human subjects. These findings suggest the possibility of using oral creatine supplementation to modify brain high-energy phosphate metabolism in subjects with various brain disorders, including major depression, schizophrenia, cocaine and opiate abuse, where alterations in brain high-energy phosphate metabolism have been reported.