Take Heart in the Age of “Omics”

• transcriptome
• heart development
• heart progenitors and stem cells

The advent of access to comprehensive sets of information (ie, genome, transcriptome, proteome, interactome, phenome, and localizome) has brought a new way of global thinking to biological questions, and analysis using these sets is increasingly the choice for many investigations. Although these approaches may appear as a substitute for the more traditional “reductionist” approach that tackles 1 or few genes or gene products at 1 time, they are, in reality, complementary and have the potential to greatly enhance the traditional approach. The “omic,” or comprehensive, approach can yield mountains of new information in a relatively short time. Progress in applying the “omic” approach is still in its early exponential phase and does not look like it will plateau any time soon.

An “omic” approach has now been used in the study of heart development. The results provide additional insights into the complexity and number of molecules that orchestrate the development and maintenance of multiple cell types and intricate physiology and pathology of the heart. Transcriptome, or expression profiling of certain cells, groups of cells, or tissues at defined stages and conditions, has been one of the earliest strategies applied to study of the heart. However, using the transcriptome as a tool has several pitfalls that must be acknowledged, including false-negatives and false-positives, frequently ascribed to problems with technical reproducibility. Transcriptome analysis also carries conceptual weaknesses, in that altered expression is not always causally related to a phenotype or process because a change in transcription may not necessarily register as a change in protein synthesis. Furthermore, gene regulation is not achieved only at the transcriptional level. Several other strategies such as posttranscriptional, translational, or posttranslational modifications may also play significant roles. Thus, several approaches are needed for more accurate conclusions.

Microarrays, or microchips, were first used to analyze the transcriptional profile of adult heart in normal and pathological conditions (reviewed by Cook and Rosenzweig)¹ such as hypertrophy,^2,3 cardiac infarction,⁴ and heart failure.^5,6 Cell lines that can be induced to differentiate into cardiomyocytes have also provided valuable information.⁷ More recently, microarray analysis has also been extended to the study of heart development.⁸

The article by Masino et al, presented in this issue of Circulation Research,⁹ has taken advantage of transgenic technology to specifically mark and then isolate presumptive myocardial cells for transcriptome analysis at different stages of induction and differentiation. To identify myocardial cells specifically, the authors made a mouse expressing enhanced yellow fluorescent protein under control of a cardiac-specific enhancer constructed from the Nkx2.5 promoter region. This enhancer of Nkx2.5 is expressed in myocardial progenitor cells starting from the crescent stage (E7.75) and remains active in both the heart tube (E8.5) and looped heart tube (E9.5) stages. Although the Nkx2.5 enhancer does not necessarily identify all of the myocardial progenitors, it certainly provides access to a reasonably representative group.

Masino et al isolated enhanced yellow fluorescent protein-positive myocardial cells at the 3 stages mentioned using fluorescence-activated cell sorting with >95% purity. This approach has the advantage of restricting transcriptome analysis to a relatively pure cell type without mixing cells that usually contaminate myocardial preparations (ie, blood and endothelial cells).

The RNA isolated from selected cells was amplified using a T7-based system and hybridized to Affymetrics GeneChips. Analyses showed several hundred genes were enriched in the myocardial cells (≥2-fold) compared with age-matched, non-cardiac embryonic cells. The expression profile included many genes known to be important in myocardial development, confirming the validity of this approach.

Comparison of the enriched genes at the 3 stages analyzed showed strong overlap (>40%) between heart tube and looped heart stages, but <5% genes were enriched with the crescent stage. These results indicate that there are many specific genes that are involved in early heart development that await more detailed molecular and functional analysis.

An unexpected finding showed a set of genes known to be important in the hematopoietic/vascular program were strongly enriched at the heart crescent stage. These results support the idea of a common history of the two lineages and explain their ability to switch between these 2 cell fates, seen both in cell lines¹⁰ and in vivo (reviewed by Olson).¹¹ The authors further compared the expression profile of the early developing myocardium with 2 different cell types: embryonic stem cells and adult mouse cardiomyocytes. Cardiac regulators and structural genes were enriched when compared with embryonic stem cells, as expected, and unchanged, or even reduced, when compared with adult mouse cardiomyocytes. Such comparisons facilitate understanding the behavior of potential myocardial stem cell populations. A detailed knowledge of the expression profile of heart precursors can establish criteria for the selection of stem cells for transplantation and reduce the possibility of unwanted fates that a stem cell population may undertake.

Masino et al substantiated the microarray data using RT-PCR and in situ hybridization. Several of the candidate genes were confirmed to be expressed at the right stages and in the right cells.

The task now is to best apply these new comprehensive approaches to understand the heart. Additional “transcriptomic” and other “omic” analyses will further advance heart research. For example, different early heart markers can be helpful to verify the available data, add other perhaps non-Nkx2.5 expressing myocardial populations, and ultimately complete the picture of early myocardial gene expression. Protein profiling (proteome) of the heart, more comprehensive analysis of gene function by knocking down or out of candidate genes (phenome), protein-protein interaction (interactome), and protein cellular and subcellular localization (localizome) are all needed for a deeper understanding of the development, physiology, and pathology of the heart. Each technique will collectively contribute to the diagnosis, prevention, and therapy of the great number of heart diseases.