Large-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.

This book presents both cutting-edge and established methods for studying cardiac gene expression. The protocols provide a template for solid research, and cover the process through screening, analysis, characterization, and functional confirmation of novel genes or known genes with a new function. The concluding section of the book highlights methods that facilitate overexpression or cardiac-specific targeted gene deletion.

The heart is the first organ to function in development and continues to beat for seventy or more years in an adult's life. Cardiogenesis therefore is no simple task; genes have to be precisely regulated to meet the needs of a developing heart. ATP-dependent chromatin remodeling provides an important mechanism to regulate gene expression. Specifically, Brg1-associated factor, or the BAF, complexes, are crucial in heart development. Endocardial Brg1 represses the expression of a metalloproteinase, ADAMTS1, in order to allow sufficient cardiac jelly expansion for trabecular development. In addition, Brg1 functions in the myocardium to repress VEGFA to prevent the ectopic formation of coronary vasculature from the epicardium in a non-cell autonomous manner. And lastly, Brg1 serves as a bridge linking embryonic development and adult cardiomyopathies. Brg1 functions in the myocardium to keep the cardiomyocytes in a proliferating state through promoting BMP10 and repressing a cyclin-dependent kinase inhibitor p57kip2. Without Brg1, cardiomyocytes cease cell division, mature, and express adult form of myosin heavy (MHC) chain gene. Brg1 is normally turned off in adult life; however, following cardiac stress it is reactivated and turns on embryonic fetal program characterized by re-induction of embryonic MHC expression. Preventing Brg1 re-expression can repress cardiac hypertrophy and restore adult MHC expression. Furthermore, Brg1physically interacts with other chromatin remodeling enzymes such as histone deacetylases and poly-ADP ribose polymerases to control expression of MHC genes and regulate cardiomyocyte differentiation. In all, ATP-dependent chromatin remodeling plays important roles in heart development and disease and may provide a suitable therapeutic target for human cardiomyopathies in the future.

Jarid2 (Jumonji A/T-rich interaction domain 2) is an essential factor for normal heart development. Deletion of Jarid2 in mice results in cardiac malformations recapitulating human congenital cardiac disease, and dysregulation of gene expression during development. However, the cardiac-specific developmental function and the precise epigenetic regulation of gene expression by Jarid2 remain to be elucidated. Here I demonstrate that cardiac-specific deletion of Jarid2 in the developmental or postnatal myocardium causes cardiac malformations and functional defects. I employed three different cardiac-expressing Cre transgenic mice. Deletion of Jarid2 by Nkx2.5-Cre (Jarid2Nkx) caused cardiac malformations including ventricular septal defects, thin myocardium, hypertrabeculation, increased cardiac jelly and neonatal lethality. In contrast, later deletion of Jarid2 in mice with cTnt-Cre or [alpha]MHC-Cre transgenic lines did not cause gross abnormalities in development. By employing combinatorial genome-wide approaches and molecular analyses, I show that Jarid2 is required for PRC2 occupancy and H3K27me3 on the Isl1 locus, leading to proper repression of the target gene expression during cardiac development. Jarid2 represses neural gene expression, cardiac jelly, and several important factors such as Isl1 and Bmp10, all of which are crucial for normal ventricular development. Thus, early deletion of Jarid2 in the myocardium results in dysregulation of gene expression and developmental defects later in development. Interestingly, deletion of Jarid2 by [alpha]MHC-Cre (Jarid2[alpha]MHC) resulted in complete lethality by 9 months of age with dilated cardiomyopathy. Jarid2[alpha]MHC mice showed an increase in fetal gene expression, such as Tnni1 and Acta2 at neonatal stages. By performing RNA-seq and pathway analyses on Jarid2[alpha]MHC postnatal hearts, we discovered that Jarid2[alpha]MHC hearts showed marked changes in heart failure associated with genes and metabolism. Further, a set of genes such as heart failure related genes were already dysregulated in neonatal Jarid2[alpha]MHC hearts, which may be causal for heart failure later. Therefore, Jarid2 is also required for myocardial maturation and maintaining cardiac function in adult stages. These studies reveal the critical roles of Jarid2 in the myocardial development. Jarid2 is necessary to establish correct epigenetics on the target genomic loci during a narrow developmental window, which is prior to differentiation of cardiac progenitors into cardiomyocytes and maturation of cardiomyocytes.