288 Current Genomics, 2010, Vol. 11, No. 4 tal mouse cardiac myocyte culture was performed as previ- ously described [1]. The purity of each preparation was veri- fied by immunostaining with an antibody against sarcomeric myosin (MF-20, Developmental Studies Hybridoma Bank) to be greater than 95%. To induce hypertrophy, neonatal myocytes in serum free medium were exposed to media with 20% fetal bovine serum for 24-48 hours. Hypertrophy was assessed at 48 hours by immunostaining cells with a mono- clonal antibody against sarcomeric myosin (MF-20, Devel- opmental Studies Hybridoma Bank), digital image capture and cell size planimetry as previously described [1]. To iden- tify important transcriptional pathways involved in hypertro- phy, RNA was harvested 24 hours after treatment as de- scribed below. RNA Isolation and Microarray Analysis RNA was isolated from cultured myocytes by extraction with a commercially available kit (Qiagen) according to the manufacturer’s instructions. For microarray hybridization, RNA was processed for hybridization to Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays through a core facil- ity in the Functional Genomics Laboratory at the Center for Ecogenetics and Environmental Health at the University of Washington, as previously described [16] and according to the manufacturer’s instructions. The number of independent cultures that were harvested for RNA and processed for each condition is as follows: WT no serum, 5; WT with serum, 7; TG no serum, 6; TG with serum, 7. Raw microarray data was processed with Affymetrix® Expression Console™ Software using Robust Multichip Analysis (RMA). Normalized data was analyzed first by single gene analysis and then by Gene Set Analysis [17], a modification of Gene Set Enrichment Analysis [18]. Selected genes were validated by qRT-PCR as previously described [19]. A full description of the Gene Set Analysis method is available online at http://www-stat.stanford.edu/~tibs/GSA/. Gene sets for analysis were obtained from the Gene Ontol- ogy Project (www.geneontology.org). We focused specifi- cally on gene sets organized according to Biological Process (BP; Gene Ontology). Gene sets were considered to be sig- nificantly altered when their statistical p values and false discovery rates as determined by the software were <0.001. All microarray data have been deposited in the Gene Expres- sion Omnibus Database under accession number GSE14288. RESULTS Cardiac Myocytes that Overexpress CHF1/Hey2 are Resistant to Serum Induced Hypertrophy In Vitro We previously reported that overexpression of CHF1/Hey2 in myocytes suppresses hypertrophy induced by phenylephrine in vivo and in vitro [1]. To verify that this resistance to hypertrophy is a general phenomenon in re- sponse to diverse stimuli, we induced hypertrophy of cul- tured WT and TG cardiac myocytes with 20% serum, a po- tent inducer of hypertrophy. As shown in Fig. (1), WT myo- cytes developed increased cell size within 48 hours, as ex- pected. TG myocytes, in contrast, did not develop increased cell size. These findings are consistent with our earlier pub- lished results and provide the rationale for the transcriptional profiling studies outlined below. Yu et al. Fig. (1). Overexpression of CHF1/Hey2 in cardiac myocytes sup- presses serum-induced hypertrophy in vitro. Cultured WT and TG myocytes were treated with either vehicle or 20% FCS for 48 hours and then immunostained with MF-20 antibody against sarcomeric myosin to distinguish myocytes from contaminating fibroblasts. Cell area was calculated for 50 cells per experiment for three inde- pendent experiments by digital image capture and planimetry. Sta- tistical comparison of average cell size was assessed by a student’s t-test with statistical significance defined as p < 0.05. Transcriptional Profiling of Wild Type and Transgenic Myocytes Before and After Serum Stimulation Reveals Alterations in Specific Gene Sets that are Both Serum- Independent and Serum-Dependent To identify the potential downstream targets of CHF1/Hey2 that may play a role in the myocyte hypertro- phic response, we analyzed transcriptional profiles from wild type and transgenic myocytes before and after serum stimu- lation. Both single gene analysis (SGA) as well as cate- gory/pathway analysis was performed. SGA assesses statisti- cal significance of each gene on the array separately. This approach identified only one gene (CHF1/Hey2, p value < 0.0001, log2FC = 1.58, ~ 3 fold increased, data not shown) as differentially expressed in unstimulated CHF1/Hey2 trans- genic versus wildtype cells. This finding is consistent with our earlier published results that myocardial overexpression of CHF1/Hey2 does not result in any significant baseline phenotype [1]. Analysis of gene expression profiles in serum stimulated cells revealed several individual genes that show signifi- cantly changed expression, defined as a p value < 0.0001, as shown in Table 1. CHF1/Hey2 is significantly changed, as expected. Other genes include Il1f6, Cecr2, Tmc4, Ndufs5, Ndufv3 and Efcab2. IL1F6 is a proinflammatory cytokine, CECR2 is a transcription factor that interacts with chromatin remodeling proteins, while TMC4 is a transmembrane pro- tein of unknown function. NDUFS5 and NDUFV3 are com- ponents of the mitochondrial respiratory chain NADH com- plex I, while EFCAB2 is a putative calcium binding protein of unknown function. To validate some of these findings, we performed qRT-PCR for CHF1/Hey2, Ndufs5, Nudfv3 and Efcab2. As shown in Fig. (2), the mRNA levels are accu- rately reflected by the microarray data. The roles of these potential target genes in cardiac hypertrophy, to the best of our knowledge, are unknown, however.