Cardiac Protein Gene Expression

Characterization of cardiac-specific gene expression permits confirmation of Western blotting or immunohistochemical staining evaluation of protein levels, as well as allowing evaluation of cellular differentiation and time-dependent de-differentiation in culture. Specific gene expression can be quantified by Northern blot analysis. As the level of contractile proteins within the cultured cells decreases with time, RT-PCR may be employed in order to permit amplification of increasingly weak signals. Both of these techniques yield an aggregate estimate of gene expression in a population of cells in culture, similar to Western blot analysis of protein levels. Neither technique allows identification of specific cells, or even of the percentage of cells that are expressing a particular gene. In order to do this, in situ hybridization using a radiolabelled probe against a specific contractile protein mRNA must be employed.

3.5 Cellular Ultrastructure

Electron microscopy can identify cardiomyocytes by revealing the intracellular contractile apparatus. The progressive changes in intracellular structure, particularly in myotubules, can be monitored, and these data, combined with techniques evaluating cardiac-specific gene expression, permit characterization of the time course of cellular de-differentiation in vitro. The most significant limitation of this technique is the ability to study only a relatively small number of cells.

3.6 Biochemical Identification

Biochemical markers of myocardial tissue, including creatine kinase mass and activity, CK-MB subunit mass and activity, and myoglobin levels can be used for identification of cultured cells, as well as the evaluation of cell de-differentiation.

4. CULTURE TECHNIQUES 4.1 Cell Purification

A crucial step in the process of culturing cardiomyocytes is cell purification. Cells freshly isolated from myocardium include a mixture of cardiomyocytes, vascular endothelial cells, smooth muscle cells and myocardial fibroblasts. If the cardiomyocytes are not purified, fibroblasts will rapidly overgrow the cultures and become the dominant cell type. Purification is best carried out in the initial stages of cell culture and can be accomplished by pre-plating, dilutional cloning, chemical selection, or a combination of these techniques.

The pre-plating technique is useful for reducing contamination by fibroblasts, taking advantage of the fact that cardiomyocytes require more time to attach to a culture dish than do other cell types. The freshly isolated cells are plated on culture dishes and cultured for 2 hours in a humidified 5% CO2 incubator at 37°C. The supernatant containing the suspended cells is then transferred into another dish for further culturing. Because many of the fibroblasts have attached to the initial plate and are therefore not transferred with the supernatant, this second culture will be relatively depleted in fibroblasts. The continuing cultures are maintained at 37°C in humidified 5% CO2.

Another technique by which cardiomyocytes can be purified is dilutional cloning. With this technique, the isolated cells are seeded at a density of 50100 cells per 100 mm diameter dish and cultured at 37°C in 5% CO2. When the cells are initially seeded at low density, the isolated cells form individual colonies after approximately 1 -2 weeks in culture. At this time, colonies of cardiomyocytes (identified morphologically by light microscopy) can be picked up with a sterile Pasteur pipette and transferred to new culture dishes. If any fibroblasts are adjacent to a cardiomyocyte colony of interest, the fibroblasts are injured with a sterile needle under microscopic guidance and subsequently undergo necrosis. A potential disadvantage of this clonal dilution technique, however, is that it selects the fastest growing cardiomyocytes. If these cells have somehow upregulated their growth kinetics, they may not be representative of the general population of cardiomyocytes.

It is also possible to inhibit the growth of fibroblasts in culture by chemical means. Eppenberger-Eberhardt et al. have reported that the addition of cytosine arabinoside can inhibit fibroblast proliferation in culture [44]. However, because the actions of cytosine arabinoside may not be specific only to fibroblasts, potential effects on other cell types must be considered.

4.2 Cell Culture

In cell culture systems, rod-shaped cardiomyocytes isolated from adult myocardium do not divide, and they undergo extensive morphological changes [24, 33, 34, 45]. During the first 1 to 3 days in culture, 80-90% of the cardiomyocytes become rounded and lose their cylindrical rod-like shape. Disorganization of myofibrils takes place at this stage of culture. These cells demonstrate resorption of Z-lines and rapid disorganization of the contractile apparatus.

In contrast, fetal cardiomyocytes can grow in the early stages of culture [23]. During this process, the cells disassemble and reorganize their myofibrils. Cultures of beating cardiomyocytes can be obtained. With time, these fetal cardiomyocytes undergo differentiation, and after a few generations, lose their ability to reorganize their myofibrils. At this stage, many of these cultured cells will no longer contract spontaneously. However, even the non-contracting cells still contain cardiac-specific contractile proteins and retain many of the characteristics of normal cardiomyocytes in vivo.

Spherical cardiomyocytes isolated from pediatric and adult myocardium are differentiated and can grow in vitro. Although these cells do not demonstrate spontaneous contractile activity in vitro, they possess many of the characteristics of normal cardiomyocytes in vivo. The properties include immunohistochemical staining, Northern blot analysis of cardiomyocyte-specific contractile proteins and biochemical assays of creatine kinase MB fraction activity and mass [21, 32]. These cultured cells do, however, undergo phenotypic modulation compared to their in vivo counterparts. In addition to the morphological changes, which include the formation of pseudopods and development of a stellate appearance, the intracellular contractile apparatus becomes progressively more disorganized and the myofibrils appear less distinct. The intracellular content of contractile proteins also decreases with increasing time in culture. The intensity of the immunofluorescent staining for contractile proteins, such as myosin heavy chain or myosin light chain, in three month old cells is significantly less than that in freshly isolated cells, and even weaker in six month old cells [21].

In any cardiomyocyte culture, the culture medium is essential for maintenance of cell morphology, growth characteristics and phenotypic modulation. For example, the concentration of serum in the culture medium determines the phenotype of the cultured cardiomyocytes. Cardiomyocytes are normally cultured in medium containing 10% FBS [46]. In this medium, fetal cardiomyocytes demonstrate little, if any, proliferation, and left unchecked myocardial fibroblasts may eventually overgrow this culture. Nag and Cheng [33] reported mitosis and cell division in adult rat cardiomyocytes during the first week of culture employing a culture medium consisting of 90% minimum essential medium and 10% horse serum, which is similar to that used by other investigators [24, 47]. The more mitogenic culture medium stimulates proliferation of the cardiomyocytes but not differentiation, and results in modulation of the normal cardiomyocyte phenotype. When fetal cardiomyocytes are cultured in a mitogen-rich medium containing 20% FBS, 1% chick embryo extract, 50ng/ml FGF and 25ng/ml multiplication-stimulating activity factor, the cardiomyocytes proliferate in culture and can undergo 30 passages or more [46]. These cells will quickly lose their contractile apparatus. However, when cardiomyocytes are cultured in lower serum concentrations (e.g. 0-5% FBS), the cell number shows no or only a small increase, but the cells maintain their phenotype and contractile properties. For example, when fetal cardiomyocytes are cultured in a mitogen-poor medium (4% horse serum), the cells cease mitosis and undergo differentiation [46].

In addition to the concentration of serum, the medium itself may also be a crucial factor. We have tested Dulbecco' s-modified Eagle's medium (DMEM), medium 199, minimum essential medium, alpha-medium, and IMDM, all with 10% FBS. We found the greatest preservation of cultured cardiomyocyte morphology over time with IMDM. With the other media, cultured cardiomyocytes underwent marked phenotypic changes and became stellate or spider-shaped after several days in culture. Although the particular characteristics of IMDM that are responsible for its optimal preservation of cardiomyocyte morphology remain to be clarified, it is possible that particular components, such as the concentration of calcium or of particular vitamins, may be important.

5. APPLICATIONS OF A CARDIOMYOCYTE CULTURE SYSTEM

5.1 Cardiac Development

Studies of cardiomyocyte differentiation are an important step in understanding the development of the normal heart. A number of in vivo studies have identified specific morphological, biochemical and immunohistochemical changes in cardiomyocytes during heart development [25, 48, 49]. The regulation of gene expression in cardiac cells plays a fundamental role in heart development. It has not yet been possible to devise a model to evaluate gene expression in cardiomyocytes in vivo. Human cardiomyocyte cultures are therefore an excellent model with which to study development of the normal heart. These cell culture models allow for cell proliferation and differentiation. Cultured cells at different stages can be used to study changes in fetal gene expression [22, 50]. Using these models, expression of a number of fetal genes has been correlated with cell maturation and differentiation. In an analogous manner, the structural and biochemical changes occurring in cardiomyocytes during cell differentiation can also be studied in these cultured cells.

5.2 Cardiac Physiology

Since fetal and adult cardiomyocytes can contract in culture, physiological studies can be performed on single cells. Patch clamping can be used to record ion channel activity. Cell surface receptor expression, translocation and activity can be studied. The effect of transmembrane channel activators and blockers on cell physiology and metabolism can be evaluated. Multicellular cardiomyocyte preparations can also be used to study the effect of stretch on ion channels [51]. This simple in vitro model led to the discovery of stretch-activated channels in the heart. These findings may explain the occurrence of length-dependent changes in pacemaker activity and stretch-activated arrhythmias in the whole heart.

Cardiomyocyte cultures also provide a simple model with which to study cell-to-cell interaction. Eid et al. [52] demonstrated that adult cardiomyocytes undergo phenotypic changes during adaptation to primary culture. However, co-culture of cardiomyocytes with specific non-muscle cardiac cells slowed and could even reverse this process of adaptation.

5.3 Cardiac Pathology and Cellular Defenses

Isolated cardiomyocytes can be used to evaluate the effect of various pathological stimuli on the myocardium. For example, rod-shaped cardiomyocytes were isolated from the myocardium of patients with a variety of cardiac pathologies [53-55]. A whole-cell patch-clamp technique was used to measure the hyperpolarization-activated inward current, sodium current and outward potassium current, providing important information about the effects of specific cardiac diseases on transmembrane ion channels.

Cultured cardiomyocytes have also been employed to study the effect of various hypertrophic stimuli. In a cardiomyocyte culture system, individual stimuli including growth factors, hormones, cytokines, vasoactive substances and catecholamines can be administered individually without concern for the contaminating effects of whole organ or whole organism physiology. The intracellular effects of these stimuli, including alpha-skeletal and smooth muscle actin changes, and atrial natriuretic factor, can be evaluated. From these data, potential mechanisms by which myocardial hypertrophy occurs can be hypothesized and specific therapeutic interventions proposed. For example, Simpson has demonstrated, using this cell culture system, that adrenergic receptor stimulation induces cardiomyocyte hypertrophy [56].

Cardiomyocyte culture has been employed as a model to select factors which act at a cellular level to protect heart cells from injury. Using cultured human cardiomyocytes, Bowes et al. [57] demonstrated that inhibition ofpoly-ADP-ribose synthetase activity reduced the cell death caused by H2O2. To protect the myocardium from free radical injury, we tested various antioxidants and demonstrated a cell-type specific protective effect of various antioxidants, as well as a syngergistic effect of these antioxidants [58]. These in vitro findings were subsequently confirmed in an in vivo study [59]. Insulin was also identified as a important factor by which myocardial metabolism may be favorably altered during ischemia and reperfusion, leading to a clinical trial of insulin cardioplegia to improve post-operative ventricular function in patients undergoing urgent coronary artery bypass surgery [60].

6. CONCLUSIONS

Human heart cells can be isolated, purified and cultured. Depending on donor age, the isolated heart cells may vary in structure and function. Although the cells undergo phenotypic modulation in culture, they have many characteristics of normal cardiomyocytes in vivo. These cells can therefore be used as an in vitro model for cardiovascular research. The selection of fetal or adult cells is dependent on the aims of the specific study.

Cells isolated from fetal, pediatric and adult myocardium may be spherical. These spherical cells attach to a culture dish and grow in vitro. Fetal cells can contract in culture. This model can be used to study the development and function of the immature heart. Primary cultures of pediatric and adult cardiomyocytes are de-differentiated, but retain many characteristics of normal cardiomyocytes including contractile protein content and enzyme activity. These cells proliferate in vitro, but with time, the levels of contractile proteins and cardiac enzymes gradually fall.

Rod-shaped cardiomyocytes can be obtained by partial digestion of human adult myocardium. These cells are groups of cardiomyocytes linked by cardiac junctions and extracellular matrix. They contain organized sarcomeres and retain many characteristics of adult myocardial tissue. They can be regarded as miniature samples of myocardium, and can be used to evaluate physiological and pathological changes under defined conditions. These cells, however, cannot attach to a culture dish and grow in vitro, and are therefore unsuitable for long-term studies or studies requiring a large population of cells.

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