Analysis of gene expression in cardiac development and disease

grantor: University of TorontoLarge-scale partial sequencing of randomly selected cDNA clones to generate expressed sequence tags (ESTs) is an efficient means of discovering novel genes and characterizing transcription in different tissues. To characterize gene expression and its changes in cardiac development and disease, an EST project was initiated using adult and fetal human heart cDNA libraries. Generation of 3,874 ESTs from an adult heart library, representing the first catalogue of genes expressed in the cardiovascular system, revealed that approximately half of all transcripts represented genes of unknown function. Analysis of the remaining half of transcripts representing known genes demonstrated expression patterns consistent with physiologic function of the heart. Comparison of these patterns with those derived from 2,244 ESTs generated from fetal heart, and with patterns obtained from other tissues, found several differences in gene expression between fetal and adult heart suggestive of a rapidly growing, less differentiated phenotype in the fetal heart relative to the adult heart. Further acquisition of larger numbers of ESTs from nine cardiac cDNA libraries, coupled with development of new strategies for data analysis, allowed for detailed prediction of individual genes exhibiting differential expression in cardiac development and disease. These strategies were applied to analyze gene expression in hypertrophic failing heart, and to study the expression of cell cycle regulators in cardiac development and hypertrophy. In the former analysis, a total of 64 genes was predicted to be differentially expressed in cardiac hypertrophy. Supporting the validity of these predictions, RT-PCR analysis of 12 of these genes confirmed 11 to be differentially expressed. In the latter analysis, transcripts of cell cycle regulators were suggested to be differentially expressed between fetal and adult hearts. Subsequent ' in vitro' analyses confirmed down-regulation of S- and G2/M-phase regulators in adult heart relative to fetal heart, and also found re-induction of several S-phase (e.g., cyclin A, PCNA), but not G2/M-phase (e.g., cyclin B, cdc2), regulators in hypertrophy. These results extend, at a molecular level, current understanding of cardiovascular function, while suggesting several new avenues of investigation. Further, they demonstrate the power of EST-based expression analyses for exploring questions of cardiovascular biology and medicine.Ph.D