Getting to the heart of the matter: Focus on "Microarray analysis of global changes in gene expression during cardiac myocyte differentiation"

Abeel A. Mangi1, Susan B. Glueck2 and Richard E. Pratt1

1 Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115
2 Deputy Editor, Physiological Genomics

CULTURED CARDIAC MYOCYTES provide a model system for studies of myocyte biology, in particular, studies on hypertrophy and apoptosis. Both neonatal and adult cardiac myocytes have been employed successfully for these studies; however, both cultures present certain experimental difficulties. For example, neonatal cells can be isolated in large numbers and can be cultured for several days or weeks. However, these cells lack many characteristics of adult cardiac myocytes. Moreover, these cells are notoriously difficult to transfect by chemical means and can only be transduced with adenovirus or adenovirus-associated virus. Adult cardiac myocytes have also been employed, but these suffer from many of the same problems seen with neonatal cells. The isolation of adult myocardial cells is difficult, and the yields are low. In addition, the cells can only be cultured for a few days, necessitating continual re-isolation.

Importantly, neither cell culture is suitable for studies of early cardiac differentiation, since both cultures are highly and terminally differentiated. For studies of myocyte differentiation, embryonic stem cells, the totipotent cell lines derived from blastocysts, have been employed. Studies have shown that embryonic stem cells in culture can differentiate into cells of multiple lineages (2). During spontaneous differentiation of embryonic stem cells into embryoid bodies, regions exhibiting synchronous contractions are observed, and these regions can be isolated or dissected and grown in culture. Studies demonstrate that these cells express several cardiac-specific markers such as {alpha}- and ß-myosin, tropomyosin, myosin light chain (MLC), connexin-43, atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) (1, 3, 12, 15, 17). These cells are multinucleated and have been shown to possess appropriate action potentials and calcium response (9, 11, 20). Most importantly, cell cycle withdrawal and terminal differentiation in embryonic stem-cell-derived cardiac myocytes follows a pattern similar to that of cardiac development in vivo (7). This differentiation into cells of cardiac myocyte lineage has been demonstrated with both murine and human embryonic stem cells (18).

Thus, embryonic stem cells appear to be a suitable source of cells for basic biological studies. However, the differentiation of cultured embryonic stem cells into cardiac myocyte lineage cells is not specific but yields cells of multiple lineages (2). Dissection can enrich the population, but this is tedious and still yields mixed cultures. This has been addressed by Klug et al. (8) who demonstrated that embryonic stem cells could be easily transfected with a selectable marker (aminoglycoside phosphotransferase) under the control of the cardiac myosin heavy chain (MHC) promoter. Selection with the aminoglycoside antibiotic, G418, yielded a pure population (>99%) of highly differentiated cardiac myocytes. These cells expressed multiple cardiac markers (MHC, titin, actinin, desmin, ANP, dystrophin, utrophin, MLC-2) and exhibited spontaneous and rhythmic contractions. These characteristics have been reported to be stable for up to a year in culture. Moreover, ultrastructural analysis by transmission electron microscopy demonstrated that these cells exhibited normal adult sarcomeric structure, intercalated discs with desmosomes, fascia adherens, and gap junctions (8, 19). Thus, these selected cells appear to possess all the molecular and ultrastructural characteristics of cardiac myocytes.

Although the ability to select differentiated cardiac myocytes from a culture of embryonic stem cells has certain advantages, the cultures can only be used to study differentiated cells and not the differentiation process itself. During the important early periods of differentiation, the cells destined to become myocytes constitute only a small fraction of the total cells; moreover, the phenotype of the contaminating cells changes constantly due to the selection process. Thus, use of these cultures in studies of differentiation is severely limited.

To be useful in studies of differentiation, the ideal cell line would be one in which the differentiation process is nearly quantitative. The cell line P19, which was cultured from a mouse embryonal carcinoma, is capable of differentiating into a number of different cell types, including cardiac, skeletal, and neuronal, depending upon the treatment. For example, when treated with 1% dimethyl sulfoxide (DMSO), these cells differentiate into cardiac myocytes, albeit with low efficiency (10). However, one line has been subcloned from these cells and shown to differentiate into cardiac myocytes with efficiency greater than 90% (4). These cells, termed P19CL6, express markers of cardiac myocytes such as {alpha}- and ß-cardiac myosin, desmin, and sarcomeric MHC, but not MyoD or myogenin, which are markers of skeletal muscle (4). In addition, these cells exhibit spontaneous contractions that are regulated by muscarinic acetylcholine receptor stimulation but not by nicotinic receptor stimulation, a characteristic of cardiac, but not skeletal, muscle myocytes (4). Komuro and colleagues (5, 6, 13, 14) have used these cells to examine the role of Smads, Tbx-5, and Nkx2.5 in the differentiation process and have discovered a novel protein that they termed Midori (6) which appears necessary for differentiation. Midori was discovered using differential display, demonstrating the usefulness of genomic technologies in the study of myocyte differentiation.

In this online release of Physiological Genomics, Peng et al. (Ref. 16; see page 145 in this release) have furthered these studies by the use of microarray analysis, using P19CL6 cells induced to differentiate with 1% DMSO. To confirm the successful differentiation of these cells into cardiac myocytes, the authors demonstrated the expression of the myocyte-specific markers MF20, MLC2v, GATA-4, and MEF2C. Moreover, electrophysiological studies with patch clamping revealed action potentials characteristic of cardiac myocytes. For expression array analysis, they employed spotted cDNA arrays consisting of 8,956 mouse cDNAs of known sequences; the probes for hybridization were reverse-transcribed using RNA from DMSO-treated cells, or from undifferentiated cells as a reference.

For the analysis, cells were harvested daily for 10 days following addition of DMSO. Peng et al. employed stringent criteria for calling genes as differentially expressed, requiring greater than twofold differences in expression over three consecutive time periods. Using these criteria, 541 genes were upregulated [224 known and 317 unknown expressed sequence tags (ESTs)], and 469 were downregulated (234 known and 235 unknown ESTs). Having demonstrated that treatment with DMSO for a minimum of 4 days was required for differentiation and initiation of contractions, the authors were particularly interested in genes whose expression was altered during this time period. Analysis revealed that a surprisingly low number of genes were upregulated during this period, with 29 transcripts present at >2-fold above control. Of these, 16 were unknown ESTs.

By nature, microarray analysis leads to the generation, rather than the testing, of hypotheses. Several interesting stories come from these present studies and will form the basis for further studies. For example, P19CL6 cells, like the P19 cells from which they are derived, are pluripotent and can differentiate into several different lineages. One might speculate that commitment to one lineage would be accompanied by the parallel inhibition of the other lineage pathways. Indeed, Tbx-6, a transcription factor that represses neural cell differentiation, was induced during the DMSO-dependent cardiac myocyte differentiation. Similarly, Twist, a direct activator of MEF2 and an inhibitor of MyoD transactivation, was also induced. Such hypotheses are intriguing and easily tested. However, the role of differentially expressed gene products in such studies is not always as easy to interpret, and microarray analysis very often produces seemingly counterintuitive results. For example, the Wnt-Frizzled signaling pathways are thought to suppress or inhibit cardiac differentiation, yet in this study, several components of this pathway are upregulated.

Examples such as this demonstrate the difficulties and challenges faced by those studying physiological genomics. In the past, the paradigm was to identify one candidate gene and to explore the consequences of the differential expression of this gene on the phenotype of interest. This approach has contributed greatly to our understanding of cardiac cell biology, but, as this and other studies demonstrate, that understanding is incomplete. Now we are faced with the daunting challenge of demonstrating the contribution of many gene products toward a phenotype. As an example of this challenge, this current study yielded conservatively ~1,000 genes (541 upregulated and 469 downregulated) whose products exhibited differential abundances at some point during the course of the study. The challenge is even more difficult when one considers that less than one-third of the genome was analyzed in this study and that for a true understanding of the biology of cardiac myocyte differentiation, these genes should be tested not only singly but also in combination.

How can these challenges be met? As pointed out by Peng et al., the key phase in this particular model may occur within the first few days, a time during which continued DMSO treatment is required for differentiation. After this phase, DMSO can be withdrawn and the cells will still progress toward differentiation, suggesting that alterations in gene expression that occur in a DMSO-dependent manner early are necessary and sufficient to drive differentiation in a DMSO-independent manner in the later phases. Thus, focusing on the genes that exhibit differential patterns of expression during these first few days may be the key to finding the early determinants of myocyte differentiation. Peng et al. found that surprisingly few genes were differentially expressed during this early phase. They suggest that this number is quite manageable, and indeed, with an appropriately designed screening and verification strategy, the role of these genes in the differentiation of P19CL6 cells can be quickly defined.

Based on this study as well as previous reports, it appears that the P19CL6 cell line may prove to be a useful tool in the identification and analysis of the determinants of myocyte differentiation. Moreover, we anticipate that these cells may also find utility in testing candidate gene products for involvement in cardiac diseases such as hypertrophy and failure.

FOOTNOTES

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. A. Mangi, Dept. of Medicine, Thorn 1309, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115 (E-mail: amangi{at}partners.org).

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