Time for primary review 20 days.

1 Introduction

In order to study cardiac myocyte development different approaches were established during the last decades. The main purpose of these studies was the differentiation of cardiac precursor cells into specialized, differentiated cell types, as well as the development of functional properties such as Ca2+ handling, rhythm generation and excitation-contraction coupling of cardiomyocytes during development. Although considerable data exist about skeletal myogenesis [1–3], limited knowledge is available with regard to the origin of the commitment and differentiation of cardiac cells. A comprehensive, morphological study on the cytodifferentiation from mesenchymal cells into cardiac myocytes is described in the embryonic murine heart [4]: According to the authors, different stages of myofibrillogenesis are present during embryological myocardial development. Cells with no or only little myofibrillar arrangement develop to myocardial cells with orientated myofibrils [5, 6]. A number of morphological studies have investigated heart development on embryonic, neonatal and adult isolated cardiomyocytes also from different species [7–16].

Although the ultrastructure during cardiac development has been thoroughly investigated [17], still relatively little is known on the development of excitability of the mammalian heart, most importantly: (1): The relation between expression of cardio-specific genes (see review [23]), the formation of cardiac phenotypes and the functional expression of different types of ion channels; (2): The regulation and genetic control of expression of ion channels (e.g. by growth factors, hormones, extracellular matrix); (3): The development of the regulation of ion channels and morphological correlates. The progress in this field is hampered by the inability to study cardiomyocytes from early, embryonal hearts because of their very small size and because of the lack of cardiac cell lines that mimic various stages of cardiac development. The development of ion currents has been studied in cardiomyocytes prepared from mammalian embryos not earlier than shortly before birth [18–21], but see Ref. [22]. Cells of this developmental stage already exhibit action potentials of highly differentiated cardiomyocytes (e.g. of ventricular-like type, [19]) and thus can serve as a model for studying only late stages of prenatal heart development.

The focus of this review is on the development-dependent changes of morphology and electrical activity during cardiomyogenesis. Since the amount and orientation of myofibrils allows the classification into the different stages of myocyte development, the morphological part deals in detail with the myofibrillogenesis. The combination of the analysis of the electrical membrane properties and morphological criteria allows the correlation of morphological and functional phenotypes. These aspects of cardiac myogenesis are studied in a model based on the differentiation of pluripotent murine embryonic stem (ES) cells into cardiomyocytes in vitro.

2 Cell lines to study cardiomyocytes

Many studies deal with primary myocardial cultures of mammals demonstrating ultrastructural and functional features of the terminally differentiated heart (for review see [24]). The cell-isolation techniques used to obtain these primary cultures are, however, not suitable for prenatal, early cardiac developmental stages due to the small quantity of myocardial material and the difficulty to maintain the developmental stage within the cell culture procedure. Therefore a first attempt to generate permanent cell lines of early cardiac differentiation was undertaken in the late 70's:

  1. 1.

    A permanent cell line, H9c2 derived from embryonic rat heart [25], exhibited dihydropyridine sensitive L-type Ca2+ channels activated by isoproterenol, presumably through the Gs α-subunit and the cAMP-dependent pathway [26]. In addition, H9c2 cells express two distinct K+ channels and a nonspecific cation channel [27]. Furthermore, both single channel studies and RT-PCR analysis with α1 isoform-specific primers revealed an expression of skeletal L-type Ca2+ channels in addition to the cardiac type of the channel [28].

  2. 2.

    Immortal cardiac cell lines have been established from embryonal avian heart [29]and adult rat myocardium [RCVC, [30]]. Under appropriate cultivation conditions these cell lines expressed some muscle-specific markers (e.g. α-sarcomeric-actinin, α-actin, desmin). Moreover, the immortal cell line established by Caviedes and coworkers [30]exhibited inward currents that resemble T- and L-type Ca2+ currents. In spite of the recent progress, the usage of these permanent cell lines is limited because they retain only a few markers of differentiated heart cells and lack many other important characteristics such as contraction and generation of action potentials.

  3. 3.

    A new approach based on transgenic mice with myocardial tumors has been developed to establish cardiomyocyte cell lines (see review [31]). The cultured cardiomyocytes derived from tumors (e.g. AT-1) can be maintained in culture, but have to be passaged into mice for regeneration. These cells express some skeletal muscle- and cardiac-specific markers and retain a highly organized ultrastructure and exhibit contractile activity as well as action potentials similar to those generated by normal cultured atrial cells.

  4. 4.

    Proliferating cardiac cells obtained by transfection with SV-40 large T antigen [32]also retain certain differentiated properties including myosin light chain expression and assembly into organized myofibrils, spontaneous contractile activity and chronotropic responses to adrenergic agonists.

The disadvantage is, however, that the cardiomyocytes derived from myocardial tumors or by virus transfection with SV-40 large T antigen can be passaged only for a limited time. The lack of other surrounding cells forming the natural microenvironment for cardiomyocytes impairs the use of these cell lines to model the in vivo cardiomyogenesis. It is also known from the literature that cellular contacts [34, 35, 11]and the extracellular matrix [33, 10, 8]influence myofibril assembly and myocyte cytoarchitecture. The loss of real three-dimensional geometry and cellular matrix relation in culture may therefore induce significant changes of the cardiomyocyte phenotype.

3 Factors influencing cardiac myogenesis

In contrast to the relatively accurately described functions of growth factors in skeletal myogenesis [36]and of extracellular matrix in the cardiovascular system [37], their role in cardiomyogenesis is not well characterized (for review see [38]). The role of growth factors in cardiac development has been mostly investigated in embryological hearts: (i) tumor growth factor β1 (TGFβ1) stimulates cardiac mesoderm formation [39], (ii) β fibroblast growth factor (βFGF) is involved in the autoregulatory processes of cardiomyocyte proliferation and differentiation [40]and in processes of down-regulation of initial stages of the vertebrate cardiac development in vivo [39]. However, with these models a direct experimental approach to study the underlying signal transduction cascades is hampered because of the complexity of the whole embryo. Experiments with insulin growth factors (IGFs), which are found to bind to receptors in plasma membranes of embryonic chicken heart tissue [41], as well as studies demonstrating platelet derived growth factor (PDGF)-receptors during somatogenesis localized in the murine heart implicate the importance of tyrosine-kinase-receptors in heart development.

4 The embryonic stem (ES) cell model

A new approach to study cardiomyogenesis is provided by the use of pluripotent murine embryonic stem cells (ES cells, ESC) differentiated in vitro. The ES cells originally have been derived from undifferentiated cells of the inner cell mass of murine embryos at the blastocyst stage or from eight-cell embryos [42–44]. They are kept in permanent culture if grown on feeder layer cells (embryonal murine fibroblasts) or leukemia inhibitory factor (LIF) [45]. ES cells are capable to take part in the embryonic development in vivo after retransfer into blastocysts (e.g. [46]). In vitro, ES cells have been shown to differentiate spontaneously into derivatives of all three primary germ layers, endoderm, ectoderm and mesoderm [47–50].

The principle of differentiation is schematically illustrated in Fig. 1:

  1. 1.

    the cultivation of a definite number of cells (e.g. 400 cells) in ‘hanging drops’ (20 μl) as embryoid bodies (EBs) for 2 days,

    Fig. 1

    Cultivation protocol for the generation of ES-cell derived cardiomyocytes.

    Fig. 1

    Cultivation protocol for the generation of ES-cell derived cardiomyocytes.

  2. 2.

    cultivation in bacteriological dishes for 5 days,

  3. 3.

    plating of ‘7d’-EBs for further differentiation.

Similar to in vivo development EBs consist of cellular phenotypes derived from the three germ layers forming a complex multicellular arrangement. Among others, skeletal [51]and smooth muscle cells, neuronal cells [52, 53]forming networks with synaptic connections, glial cells (micro- and macroglial) [54], blood vessels (containing endothelial and smooth muscle cells) [55, 56], hematopoietic cells, glandular cells (Addicks, unpublished data) as well as epithelial cells [57]form organ-like structures. Beside these different mesenchymal cells, components of extracellular matrix like different types of fibre collagen, laminin, nidogen and fibronectin form the connective tissue in the EB [58, 59]like in vivo.

Within this multicellular arrangement in EB outgrowths (from 7+1d to 7+24d) cardiomyocytes appeared as spontaneously contracting cell clusters. They increased in size during further differentiation. The cluster can be investigated in total, or dispersed into single cells by aid of widely used isolation techniques. Because of the small number of cell layers forming the total cellular mass of the outgrowth, the EB is transparent for light and therefore can be permanently observed and controlled by aid of vital microscopy.

A developmentally controlled expression pattern of the cardiac-specific genes encoding α- and β-cardiac myosin heavy chain (α-, β-MHC), atrial natriuretic factor (ANF) and myosin light chain isoform 2V (MLC-2V) can be detected by RT-PCR of the total mRNA of EB's (see Fig. 2): While α- and β-MHC expression remains constant the latter two are upregulated during the cardiac specification stage. The first cardiac-specific transcripts of the gene coding for the α1 subunit of the L-type Ca2+ channel (α1 CaCh) are already detectable at day 3, two days before the first beating cells appear [58]. Skeletal muscle-specific genes encoding the myogenic regulatory factors myogenin and MyoD as well as the cell adhesion molecule M-cadherin and the skeletal muscle-specific α1-subunit of the L-type Ca2+ channel (α1CaChsm) are found to be expressed in a stage when the first myocytes appeared in the EB outgrowths [51].

Fig. 2

Expression of cardiac- and skeletal muscle-specific genes during in vitro differentiation of ES cell (line D3)-derived embryoid bodies plated at day 5 as analyzed by RT-PCR. A developmentally controlled expression pattern of the cardiac-specific genes encoding α- and β-cardiac myosin heavy chain, ANF, and myosin light chain isoform 2V was found with the latter two being upregulated during cardiac-specific specification stage. The first cardiac-specific transcripts of the gene coding for the α1-subunit of the L-type Ca2+ channel (α1CaCh) were already detected at day 3, two days before the first beating cells appeared. Skeletal muscle-specific genes encoding the skeletal muscle-specific α1-subunit of the L-type Ca2+ channel (α1CaChsm) were found to be expressed at a stage when the first myocytes appeared in the embryoid body outgrowths. Primers specific for the house keeping gene β-tubulin were used as an internal standard, and skeletal muscle (M) and heart (H) from a day 16 p.c. mouse embryo as positive controls.

Fig. 2

Expression of cardiac- and skeletal muscle-specific genes during in vitro differentiation of ES cell (line D3)-derived embryoid bodies plated at day 5 as analyzed by RT-PCR. A developmentally controlled expression pattern of the cardiac-specific genes encoding α- and β-cardiac myosin heavy chain, ANF, and myosin light chain isoform 2V was found with the latter two being upregulated during cardiac-specific specification stage. The first cardiac-specific transcripts of the gene coding for the α1-subunit of the L-type Ca2+ channel (α1CaCh) were already detected at day 3, two days before the first beating cells appeared. Skeletal muscle-specific genes encoding the skeletal muscle-specific α1-subunit of the L-type Ca2+ channel (α1CaChsm) were found to be expressed at a stage when the first myocytes appeared in the embryoid body outgrowths. Primers specific for the house keeping gene β-tubulin were used as an internal standard, and skeletal muscle (M) and heart (H) from a day 16 p.c. mouse embryo as positive controls.

5 Morphology of ES cell derived cardiomyocytes

The formation of developing cardiomyocytes in EBs represents an in vitro tool demonstrating a progredient differentiation of cardiomyocytes from pluripotent ES cells. In early stages of EB development (7+2d) the spontaneously contracting areas consist of small rounded early-stage myocytes, which are situated in round accumulations (Fig. 3a). With maturation of such cardiomyocyte formations, the overall appearance changes to strands of elongated cardiomyocytes with well developed myofibrils (Fig. 3b). The arrangements of cardiomyocytes within the EB are located between a covering epithelial layer and a basal layer of mesenchymal cells. The cardiomyocytes are surrounded by a discontinuous basal lamina and the myocytes are linked by cellular contacts (Fig. 3c). Hence, cardiomyocytes of the EB are always in a structural and functional relationship to other cells and extracellular matrix proteins.

Fig. 3

Cardiomyocytes in a and b are identified by α-sarcomeric-actinin labelling. (a) Early stage EB (7+2d): Small rounded early myocytes form round accumulations. (b) Late stage EB (7+12d): Elongated cardiomyocytes with well developed myofibrils are situated in strands. (c) The cardiomyocyte aggregates within the EB are located between a covering epithelial layer and a basal layer of mesenchymal cells. (d) Early stage EB (7+2d): Actin and myosin filaments form alternating bundles and are in a irregularly and disorientated manner distributed in the cytoplasm. Z-bodies (zb) are rare. The cross-section demonstrates a hexagonal array of actin and myosin filaments. (e) Early stage EB (7+4d): Z-bodies are aligned periodically and are associated with actin filaments. The Z-bodies are interrupted by bundles of myosin filaments that are located between two dense bodies. Further development of the myofibrillar arrangement shows lateral alignment of several primitive myofibrils and fusion of Z-bodies to Z-line precursors which remain irregularly shaped. There is still no clear distinction of I- and A-bands. (f) Late stage EB (7+12d): The diameter of the Z-line corresponds to the diameter of the myofibrillar arrangement. Z-line, I- and A-band are distinct, but in comparison to adult cardiomyocytes the M-band is still missing.

Fig. 3

Cardiomyocytes in a and b are identified by α-sarcomeric-actinin labelling. (a) Early stage EB (7+2d): Small rounded early myocytes form round accumulations. (b) Late stage EB (7+12d): Elongated cardiomyocytes with well developed myofibrils are situated in strands. (c) The cardiomyocyte aggregates within the EB are located between a covering epithelial layer and a basal layer of mesenchymal cells. (d) Early stage EB (7+2d): Actin and myosin filaments form alternating bundles and are in a irregularly and disorientated manner distributed in the cytoplasm. Z-bodies (zb) are rare. The cross-section demonstrates a hexagonal array of actin and myosin filaments. (e) Early stage EB (7+4d): Z-bodies are aligned periodically and are associated with actin filaments. The Z-bodies are interrupted by bundles of myosin filaments that are located between two dense bodies. Further development of the myofibrillar arrangement shows lateral alignment of several primitive myofibrils and fusion of Z-bodies to Z-line precursors which remain irregularly shaped. There is still no clear distinction of I- and A-bands. (f) Late stage EB (7+12d): The diameter of the Z-line corresponds to the diameter of the myofibrillar arrangement. Z-line, I- and A-band are distinct, but in comparison to adult cardiomyocytes the M-band is still missing.

Although in the mature heart atrial granules (atrial natriuretic factor, ANF) containing cells are preferentially found in the atria [60], most of the cardiomyocytes developed within EBs contain these atrial granules [61], suggesting an important role of ANF in early cardiac development. Like in the developing heart, T-tubuli are not prominent in all stages of cardiomyogenesis within EBs. It has been shown that the T-tubular system only develops postnatally [62, 63]. Adjacent cardiomyocytes show different degrees of myofibrillar organization. Also, within the same cell, different stages of myofibrillar assembly, leading to the definite sarcomeric architecture, can coexist.

Corresponding to the cell shape, the α-actinin positive sarcomeric structures differentiate from spots in roundish cells (early and pacemaker-like cells) to complete sarcomeric structures and oriented myofibrils in elongated cells (differentiated atrial- and ventricular-like cells). In respect to the arrangement of filamentous elements and the appearance of an amorphous material (comparable to the Z-line material because of the electron density), the following patterns of myofibrillar assembly in cardiomyocytes can be distinguished (Fig. 3a–f) [61]:

  1. 1.

    Spots of Z-line material (Z-bodies) are dispersed in the cytoplasm.

  2. 2.

    Actin- and myosin filaments form alternating bundles which are irregularly and in a disoriented manner distributed in the cytoplasm. The cross-section demonstrates a hexagonal array of actin- and myosin filaments. The Z-bodies are seldom.

  3. 3.

    The Z-bodies are aligned periodically and associated with actin filaments. The Z-bodies are interrupted by bundles of myosin filaments which are located between two dense bodies; these have a greater diameter than the Z-bodies.

  4. 4.

    Further development of the myofibrillar arrangement shows lateral alignment of several primitive myofibrils and fusion of Z-bodies to Z-line-precursors which remain irregularly shaped. There is still no clear distinction of I- and A-bands.

  5. 5.

    The diameter of the Z-line corresponds to the diameter of the myofibrillar arrangement, the spacing between two Z-lines varies between 1.7 and 2.5 μm. The Z-line, the I- and the A-band are distinct, but in comparison to adult cardiomyocytes the M-band is still missing. The M-band formation is considered the endpoint of myofibrillar maturation [64, 65]and is presumably related to the mechanical requirements in the heart (pressure load). Similar mechanical functions are not required in the EBs and may explain the lack of the typical M-band formation.

6 Diversity of cardiac cell types in the EB

As shown also by the electrophysiological measurements (see below) the heterogeneous population of cardiomyocytes undergoes a shift from early stage cardiomyocytes with pacemaker activity to terminally differentiated atrial-/ventricular-like cells. This is from the morphological point of view due to the shift from small rounded cells with low myofibrillar content to elongated cardiomyocytes with high content of organized myofibrils.

On the single cell level the cardiomyocytes derived from EBs can be classified as early pacemaker-like, Purkinje-like and atrial-/ventricular-like cells in regard to their overall shape and myofibrillar organization stage [61]. The cells can be clearly classified in these cell types, even though morphological intermittent stages are observed (Fig. 4):

  • Early cells (Fig. 4e) with no or rarely developed sarcomeres are predominantly small and round (35 μm diameter).

    Fig. 4

    At the single cell level EB derived cardiomyocytes can be classified based on overall shape and myofibrillar organization into early, pacemaker-like (see a, e, f, and j), Purkinje-like (b, g, and k), atrial-like (c, h, and l) and ventricular-like (see d, i, and m) cells. (a) Early cells (Fig. 4e) with no or rarely developed sarcomeres are relatively small (35 μm diameter) and display a round to branched shape. (b) Purkinje-like cells (see Fig. 4b, g) are large with one or two round nuclei and show a highly branched shape. The peripherally placed myofibrils are aligned in the direction of the tension forces. In contrast to freshly dissociated ventricular myocytes, the typical parallel orientation of the myofibrils is not present. (c) The atrial-/ventricular-like cells are elongated with an oval nucleus and well organized tension-orientated myofibrils, spanning the long axis of the cells. (e–i) The characteristic morphological pattern of EB derived cardiomyocytes can also be distinguished by vital microscopy into early pacemaker-like cells (e, f), pacemaker-like cells, Purkinje-like cells (g), atrial- (h) and ventricular-like (i) cells. (k–n) Representative action potential recordings from spontaneously beating, single cardiomyocytes in the current-clamp mode: (k) Relatively depolarized cardiomyocyte with short lasting AP of small amplitude characteristic for an early, pacemaker-like cell. (l) Cell with a relatively stable resting potential, notch and plateau phase characteristic for a Purkinje-like cell. (m) Cell with a relatively stable resting potential and short lasting AP typical for an atrial-like cell. (n) Cell with relatively stable, negative resting potential and long plateau phase typical for a ventricular-like cell.

    Fig. 4

    At the single cell level EB derived cardiomyocytes can be classified based on overall shape and myofibrillar organization into early, pacemaker-like (see a, e, f, and j), Purkinje-like (b, g, and k), atrial-like (c, h, and l) and ventricular-like (see d, i, and m) cells. (a) Early cells (Fig. 4e) with no or rarely developed sarcomeres are relatively small (35 μm diameter) and display a round to branched shape. (b) Purkinje-like cells (see Fig. 4b, g) are large with one or two round nuclei and show a highly branched shape. The peripherally placed myofibrils are aligned in the direction of the tension forces. In contrast to freshly dissociated ventricular myocytes, the typical parallel orientation of the myofibrils is not present. (c) The atrial-/ventricular-like cells are elongated with an oval nucleus and well organized tension-orientated myofibrils, spanning the long axis of the cells. (e–i) The characteristic morphological pattern of EB derived cardiomyocytes can also be distinguished by vital microscopy into early pacemaker-like cells (e, f), pacemaker-like cells, Purkinje-like cells (g), atrial- (h) and ventricular-like (i) cells. (k–n) Representative action potential recordings from spontaneously beating, single cardiomyocytes in the current-clamp mode: (k) Relatively depolarized cardiomyocyte with short lasting AP of small amplitude characteristic for an early, pacemaker-like cell. (l) Cell with a relatively stable resting potential, notch and plateau phase characteristic for a Purkinje-like cell. (m) Cell with a relatively stable resting potential and short lasting AP typical for an atrial-like cell. (n) Cell with relatively stable, negative resting potential and long plateau phase typical for a ventricular-like cell.

  • Pacemaker like-cells (Fig. 4a,f) vary in size from the smallest to the largest cells in the cardiomyocyte arrangements and also in shape from round to branched cells. The typical pacemaker-like cell shows rare myofibrillar content; the few myofibrils are mainly organized rudimentary and the nucleus is round and prominent.

  • Purkinje-like cells (Fig. 4b,g) are classified into three different groups [66–68]. Structurally only one of these can be distinguished from atrial- and ventricular cardiomyocytes. This type of Purkinje-like cell is larger than atrial-/ventricular-like myocytes, possesses one or two round nuclei and shows a highly branched shape. The peripherally located myofibrils are aligned in the direction of tension forces. The Purkinje-like cells exhibit a gradual increase in the density of myofibrils and a decrease in cell size in transition to the atrial-/ventricular-like cells.

  • The atrial-(Fig. 4c,h)/ventricular-(Fig. 4d,i) like cells are elongated (72–110 μm length), have an oval nucleus and well organized tension-orientated myofibrils, spanning the long axis of the cells.

Although all cardiac phenotypes simultaneously occur at each developmental stage in the EB, the percentage of the different cell types changes (from Ref. [61]): Early, pacemaker-like cardiomyocytes predominate in early EBs (7+4d, i.e. 11d in culture) while a higher percentage of atrial-/ventricular like cells is found in older EBs (17/21 days).

This morphological pattern of cardiomyocytes from EBs can also be distinguished by vital microscopy in early stage cells, pacemaker-like cells, atrial- and ventricular-like cells as exemplified in Fig. 4e–i. These cells have been recorded on videotape, prior to patch clamp recordings and characterized electrophysiologically (Fig. 4h, m, picture and action potential are not from the same cell). By this approach it is possible to correlate cellular morphology strongly with electrophysiological features of the different cardiac phenotypes as shown in Fig. 4k–n. Action potentials and ionic currents are measured on spontaneously beating cardiomyocytes using the patch clamp technique. While the atrial-like, Purkinje-like and ventricular-like cell types display a more stable and hyperpolarized resting potential (Fig. 4l, m, n), the early pacemaker-like cell is characterized by a relatively depolarized resting membrane potential (Fig. 4k). The independent phenotype classification using morphological and electrophysiological criteria yielded nearly identical results. Thus developing cardiomyocytes can be selected by vital microscopy before the electrophysiological measurements.

7 Development of electrophysiological characteristics of ES cell derived cardiomyocytes

ES cell-derived cardiomyocytes express action potentials of sinusnodal, atrial and ventricular types: Differentiation of ES cell derived EB's allows for the first time to study in vitro all consequent stages of action potential development in mammalian cardiomyocytes (Fig. 5) [69, 70]. At any given time during development cardiomyocytes with the different types of action potentials (early/pacemaker-, AV-, Purkinje-, atrial- and ventricular-like) can be found within the same EB. While cardiomyocytes of an early differentiation stage (‘7+2 d’ to ‘7+4 d’) mostly reveal pacemaker-like action potentials, mainly three major types of action potentials can be found in cardiomyocytes of the terminal differentiation stage (‘7+9 d’ to ‘7+12 d’). Cells of atrial and ventricular phenotypes (elongated cells with orientated myofibrils) can be characterized by a stable resting potential of about −75 mV and by action potentials of high amplitude and upstroke velocity. Similar as described for adult myocardium [71, 72], atrial action potentials differ from ventricular action potentials by a less pronounced plateau and by an acetylcholine-induced hyperpolarization. In cardiomyocytes of both phenotypes, tetrodotoxine (TTX), a selective blocker of voltage-dependent Na+ channels, strongly reduces the upstroke velocity of action potentials. The 1,4-dihydropyridine-derived opener and blocker of L-type Ca2+ channels, BayK 8644 and isradipine, increases and decreases the plateau phase of action potentials, respectively. The third type of action potential measured in ES cell-derived cardiomyocytes (small, roundish cells with rare and disoriented myofibrils) shows all characteristics of sinusnodal pacemaker cells including the typical shape and the hormonal regulation (Fig. 5) (see also [73]). Chronotropic measurements demonstrated functional expression of adrenoceptors, cholinoceptors and L-type Ca2+ channels [44].

Fig. 5

Typical action potentials recorded in embryonic stem cell derived cardiomyocytes at different developmental stages in relation to ionic currents expressed in the cells. Both atrial and ventricular types of action potentials were elicited in current clamp mode by current pulse stimulation (from Ref. [70]).

Fig. 5

Typical action potentials recorded in embryonic stem cell derived cardiomyocytes at different developmental stages in relation to ionic currents expressed in the cells. Both atrial and ventricular types of action potentials were elicited in current clamp mode by current pulse stimulation (from Ref. [70]).

8 ES cell-derived cardiomyocytes express all major cardiac-specific ion channels

Undifferentiated stem cells exhibit no electrical activity. They are unable to generate action potentials and revealed only slight linear current voltage relations [74]. The various shapes of action potentials in ES cell-derived cardiomyocytes of different developmental stages are well correlated with the expression of specialized types of ion channels [70]. The expression of ionic currents and the corresponding action potentials measured in cardiomyocytes from different developmental stages are summarized in Fig. 5. While in cardiomyocytes of an early differentiation stage the primitive pacemaker action potentials are generated by only two main types of ion channels, i.e. voltage-dependent L-type Ca2+ channels (ICa) and transient K+ channels (IK,to), terminal differentiated cardiomyocytes express various additional types, including voltage-dependent Na+ channels (INa), delayed outward rectifying K+ channels (IK), inward rectifying K+ channels (IK1), muscarinic acetylcholine-activated K+ channels (IK,Ach) and hyperpolarization-activated pacemaker channels (If). Ventricular- and atrial-like cardiomyocytes express INa and IK1 underlying the high upstroke velocity and the stable resting potentials, respectively [71, 75]. Acetylcholine-induced hyperpolarization of atrial-like cells corresponds to the selective expression of IK,ACh. Sinusnodal-like cells preferentially exhibit neither INa nor IK1, but ion channels regulated by cardiotropic hormones, i.e., If, IK,ACh and ICa (see [73]). Most biophysical and pharmacological properties of the ionic currents of ES cell-derived cardiomyocytes are similar to those previously described for adult [24]or perinatal cardiomyocytes [76, 19, 77]. There are however some differences, which will be discussed below.

9 Ca2+ channels

The most prominent current component found throughout the entire differentiation period of ES-cell derived cardiomyocytes is the voltage-dependent ICa. The voltage dependence of ICa as well as parameters of steady-state inactivation are similar in cardiomyocytes of all differentiation stages. The whole-cell ICa exhibits all major biophysical properties and pharmacological characteristics inherent to the L-type Ca2+ current of the heart [72, 78, 79]. It is characterized by blockage with isradipine, gallopamil and Cd2+. A specific antagonist of N-type Ca2+ current, ω-conotoxin, blocked ICa insignificantly. As reported for early stage embryonic, murine cardiomyocytes [22]T-type Ca2+ channels could not be detected in the ES-cell derived early stage cardiomyocytes. In some cardiomyocytes of intermediate and late stage T-type like Ca2+ currents with (i) negative threshold of activation, (ii) insensitivity to dihydropiridines, (iii) complete block by 50 μM Ni2+ were detected [70]; T-type Ca2+ currents were described in adult rat ventricle cardiomyocytes [123]and also the embryonic chick heart [124]. Bay K 8644, a specific opener of L-type Ca2+ channels, strongly stimulated the current (from 2 to 3 times) and shifted the maximum of the current–voltage curve to more negative potentials [70]. ICa was also stimulated by adrenaline or forskolin, a direct activator of adenylyl cyclase [80]suggesting a cAMP-dependent regulation for ICa. The regulation of ICa appears to differ between embryonal cardiomyocytes and adult ventricular myocytes. The development of signal transduction pathways for the regulation of ion channels preferentially has been studied at postnatal stages. In contrast to adult cardiomyocytes differences in the expression pattern of G-proteins and the activity of cAMP and cGMP can be found in postnatal rabbit and rat cardiomyocytes [81]. Moreover, a postnatal increase of ICa upon β-adrenergic stimulation was demonstrated [82], while there seems to be a postnatal decrease in the muscarinergic modulation of ICa[83]. Recent electrophysiological studies on embryonal murine cardiomyocytes and on ES-cell derived cardiomyocytes of early developmental stage provide a more detailed insight in developmental changes of the β-adrenergic and muscarinergic modulation of ICa. Ligand binding experiments have already proven expression of β-adrenoceptors as early as day 13 of murine gestation [84], but patch-clamp experiments on day 12–14 old murine embryonal cardiomyocytes have demonstrated the lack of ICa stimulation [85]upon isoproterenol application. Moreover, the cAMP analogue 8-bromo-cAMP did not stimulate ICa, indicating a limiting step in the β-adrenergic modulation of ICa downstream from the receptor and the G-protein. Cellular dialysis with the catalytic subunit of the cAMP-dependent protein kinase A (PKA) stimulated ICa and upon dialysis with the holoenzyme of PKA, ICa could be stimulated again by cAMP or forskolin. However, these cells still lacked the β-adrenergic modulation of ICa[85]. These and biochemical studies [86]support the idea of a reduced PKA activity in the embryonal heart. The insensitivity of ICa towards β-adrenergic modulation after dialysis with the holoenzyme of PKA points towards an additional defect in the signal transduction pathway. Hence, data from embryonal rats [87]show a lack of the functional coupling between G-protein and β-receptors in early gestation, suggesting a deficient receptor-G protein coupling. Studies in our laboratory have confirmed the lack of the β-adrenergic modulation of ICa for the young stage (7+2-4d) of ES cell-derived cardiomyocytes. In contrast, muscarinergic receptor stimulation strongly depresses basal ICa in the early stage cells. The muscarinergic depression of ICa is G-protein mediated, but not by a direct inhibition of the adenylylcyclase, as described for adult ventricular myocytes [88–90]. Preliminary results point towards an activation of the cGMP dependent phosphodiesterase II and a concomitant reduction in cAMP activity [91]. Further studies are needed to investigate the modulation of ICa in late stage cardiomyocytes.

The density of ICa significantly increases during cardiomyocyte differentiation in EBs up to 30 pA/pF [70]. This value is slightly higher than the reported current density of guinea pig and rat ventricular myocytes (26.5 pA/pF and 17–25 pA/pF, respectively) but lower than that of bovine ventricle (34–42 pA/pF) [79]. In chick embryonic cardiomyocytes a reduced or constant Ca2+ channel density has been reported [92, 20]suggesting species dependent differences in the development.

10 Na+ channels of low TTX sensitivity

INa could not be detected at the earliest differentiation stage (‘7+2 d’ to ‘7+3 d’) but appears during further development [70]. While INa has been detected in rabbit sinus-node cells [125], ES cell derived sinus-node like cardiomyocytes do not express INa. Almost all terminally differentiated, ES derived cardiomyocytes express Na+ currents. INa is maximally expressed in cells exhibiting atrial- and ventricular-like action potentials at the terminal differentiation stage [70]. A low TTX sensitivity of INa is a characteristic for the mammalian myocardium. TTX-block of INa in mouse cardiomyocytes was described by 1:1 binding with a Kd value of 1.4 μM [93]. The Kd value ranges from 1.5 to 2.5 μM for different mammalian cardiac preparations [94]. The whole-cell INa was further characterized by blockage with external Cd2+ (IC50 of near 0.5 mM). The Cd2+ sensitivity is an additional specific property of cardiac Na+ channels [95, 96]. INa reverses closely to the calculated Na+ equilibrium potential ENa (46.6 mV). As previously described, positive to ENa a slight inward-going rectification can be observed [97, 98]. Major biophysical characteristics of INa including parameters of steady-state activation and inactivation prove to be similar to neonatal or adult mammalian heart cells [70].

The normalized conductance of INa at the intermediate differentiation stage is smaller than that of adult cardiac tissue. However, the density of INa apparently increases during cardiomyocyte differentiation from intermediate to terminal stage. From indirect estimations (by measurement of partially inactivated INa) the density of the current is more than 5 μS/μF. Interestingly, in some precursor cells of the intermediate stage the Na+ channels are electrically inactivated due to the depolarized membrane potential of −40 mV to −60 mV (see also [99]). Thus the coexpression of inwardly rectifying K+ channels (see below) leads to a lowering of the resting potential, to an increased availability of Na+ channels and ultimately to the INa-triggered fast upstroke velocity of action potentials.

11 ES cell-derived cardiomyocytes express basic cardiac-specific K+ currents

Three major K+ currents, Ito, IK and IK1 are present in ES cell-derived cardiomyocytes. Ito was expressed throughout the whole differentiation period in the spontaneously beating cells [70], similar to findings in murine embryonic cardiomyocytes [22]. Depolarization-activated Ito attains its maximum within about 10 ms and inactivates thereafter during 100–200 ms. The amplitude of Ito depends on the holding potential. The voltage of half inactivation varies in a range from −60 mV to −30 mV for cardiomyocytes of different ages with more negative values for cells of terminal differentiation as compared to those of early stages. The current density of Ito increases considerably in terminally differentiated cardiomyocytes as compared to the early differentiation stage. An augmentation in the density was reported for rat ventricular myocytes during postnatal development [76]. This results in shortening of ventricular action potentials, which is important for high beating rates of the heart. As demonstrated by the prolongation of action potential duration by 4-AP (about twice by 2 mM 4-AP), Ito plays a prominent role in the determination of the action potential duration in ES cell-derived cardiomyocytes.

IK can be measured by membrane depolarization from a holding voltage of −45 mV where Ito is almost completely inactivated. IK is stimulated by adrenaline and forskolin, suggesting a modulation by adrenoceptors through a cAMP-dependent pathway [100–102]. IK can be mostly detected in cells of the intermediate and terminal differentiation stage.

The most prominent expression of the inwardly rectifying K+ current (IK1) is observed in cells with stable resting potential, i.e. in atrial- and ventricular cardiomyocytes. This current is evoked by hyperpolarizing voltage pulses and reveals a time-dependence which is more prominent at more negative potentials. IK1 exhibits the same properties as described for embryonal [19, 103], perinatal [104, 105]and adult [106, 107]ventricular cardiomyocytes. IK1 is highly sensitive to external Ba2+ with 2 mM Ba2+ almost completely blocking the current. ES cell-derived cardiomyocytes reveal lower values of IK1 (usually less than 0.5 nA, by voltage pulse to −100 mV) as compared with those reported for adult cardiomyocytes (2–3 nA). An additional large increase of IK1 expression in mammals is found. This increase is suggested to be due to the appearance of the adult type of IK1-channel of 42 pS, in addition to the embryonal type of 30 pS [104, 105]or alternatively by a change in the open probability of the embryonal channel [126].

Furthermore, in cardiomyocytes of terminal differentiation the appearance of another type of K+ current can be detected which progressively increases during prolonged (40–60 min) recordings of membrane currents in the whole-cell configuration. The K+ selectivity of the current is confirmed by the measurement of its reversal potential which is close to the calculated EK (−87 mV). The assumption that this current is due to openings of ATP-dependent K+ channels is corroborated by the finding that its development is much faster at the onset (5–10 min) when the patch pipette solution is depleted of ATP. These results suggest the expression of ATP-dependent K+ current (IK,ATP) in ES cell-derived cardiomyocytes [108, 109]. Recent experiments in our laboratory have demonstrated the activation of IK,ATP by the mitochondrial uncoupler 2,4-DNP in early and late stage cardiomyocytes (S. Viatchenko-Karpinski and B.K. Fleischmann, unpublished data). As also reported for murine, embryonic cardiomyocytes [22], the current density was similar in the early and late stage cells. This may indicate an important function of this channel during embryonic cardiac development.

At the terminal differentiation stage, ES cell-derived cardiomyocytes (about 30% of all cells assayed) of the atrial and sinusnodal types selectively express inward rectifying K+-currents (IK,Ach) activated by the cholinoceptor agonist carbachol. Under carbachol, IK,Ach is seen as an inward rectifying current component depending on extracellular K+ concentration. After its activation, IK,Ach is almost completely suppressed by subsequent addition of 20 μM atropin, suggesting the involvement of muscarinic receptors [110]. Under conditions of equimolar K+ solutions we have recently detected IK,Ach in more than 50% of early stage cells (S. Viatchenko-Karpinski and B.K. Fleischmann, unpublished data). The current density is low and the functional importance is questionable, because application of the muscarinergic agonist carbachol causes only in about 20% of the early stage cells a hyperpolarization of the resting membrane potential (Q. Ji and B.K. Fleischmann, unpublished data).

12 Pacemaker (If)-current

Another current component important for hormonal regulation of pacemaker action potentials is the hyperpolarization-activated current (If) current [111, 112]. Expression of If could be detected in early-, pacemaker- and Purkinje-like cells, but not in atrial- and ventricular-like cells [70]. This is in contrast to findings in chick atrial and ventricular cardiomyocytes [127]and to a recent report, where If was also found in adult canine ventricular cardiomyocytes at very negative potentials [128]. The amplitude of If varied in individual ES-cell derived cardiomyocytes between 50 and 500 pA (measured at hyperpolarizing voltage steps from −35 mV to −100 mV). In the terminally differentiated pacemaker-like cells, current densities up to 2 nA were observed. All cells expressing If generate spontaneous pacemaker action potentials.

The shape of the hyperpolarization-activated If and its steady-state activation curve in cardiomyocytes of terminally differentiated pacemaker like cells proves to be similar to those previously found in sinusnodal cells or Purkinje fibres from adult hearts (for review see [113], for embryonic chick heart see [114, 115]). The If activation curve measured from a holding potential of −35 mV extends over the range from −40 mV to −100 mV [70]. The reversal potential obtained from the current–voltage relationship can be found at about −27 mV. The current almost completely disappears in ‘cation free’-medium suggesting that If channels are predominantly permeable for cations. If can be blocked by 2 mM Cs+. Adrenaline and carbachol stimulate and inhibit If shifting the current activation curve to positive and negative potentials, respectively. In addition, forskolin modifies If similar as adrenaline suggesting a cAMP-dependent regulation of the current [70].

13 Limitations of the ES-cell preparation

While in the late stage cells, cardiomyocytes can be recognized by their typical myofibrillar content under the light microscope prior to measurements by the patch clamp technique, cells of the earliest stage cannot easily be distinguished from other cell types by morphological criteria (small, roundish shape). For this reason, electrophysiological measurements on early stage cells have to be exclusively performed on spontaneously beating cells. This type of cell selection bears the risk of overlooking an early cardiomyocyte population, which does not yet contract. Moreover, as described above, the M-band and T-tubulus-formation is not finalized, indicating that cardiomyogenesis only reaches a perinatal-like stage. Differentiation protocols have to be standardized in order to exclude clonal diversity. Some differences of ion channel expression and biophysical characteristics between the ES cell derived and fetal, mammalian cardiomyocytes have been also noticed (see chapters above).

14 Conclusions and future perspectives

Taken together, the data reviewed suggest that pluripotent embryonic stem cells cultivated within EBs reproduce cardiomyocyte development from primitive precursor cells to highly specialized phenotypes of the cardiac tissue. The differentiation of cardiomyocytes in EBs represents a process of developmentally controlled gene expression [116–118], increasing myofibrillar organization, changing cellular shape and size as well as electrophysiological properties. It is demonstrated that most cardiac-specific ion currents, L-type ICa, INa, Ito, IK, IK1, IK,Ach, IK,ATP and If are expressed in cardiomyocytes developed in vitro from pluripotent ES cells. Most of the biophysical and pharmacological properties of ion currents were similar to those previously described for adult cardiomyocytes or neonatal mammalian heart cells [19, 21, 76, 105, 104, 119, 120].

The ES-cell derived cardiomyocyte differentiation system has the following advantages: (i) cardiomyocytes develop among cells of all 3 germlayers in contrast to monocultures; (ii) nearly two-dimensional growth allows microscopical observation during development; (iii) morphological and electrophysiological characterization of the same cell; (iv) easy access to very early stage cardiomyocytes.

Furthermore, this cell model is valuable for more detailed studies on commitment and differentiation of cardiomyocytes, on the role of growth factors, extracellular matrix components and connexins, on cardiac myogenesis, as well as on pharmacological and toxicological effects on morphology, gene expression, cardiac-specific ionic currents and action potentials. It may further provide a unique model to analyze the quantitative expression of ion channels and the corresponding structural changes during the cardiac development which may reveal new insights into inborn heart diseases (see review, [121]). A first attempt for studying the role of the extracellular matrix (β1-integrins) has been made already [58], finding that the lack of integrins significantly influence cardiac development, in particular the expression of ion channels as well as myofibrillar proteins. The absence of integrins leads to a retarded differentiation of cardiomyocytes.

Simultaneous quantitative analysis of channel expression [122]and ultrastructure in ES cell-derived cardiomyocytes as well as temporally and spatially controlled gene expression may offer for the first time the possibility to study pathophysiological phenomena. For example, an abnormal development of ionic channels may lead to an electrical instability of the cardiomyocyte and consequently to arrhythmias. A limitation for many studies is the identification of cardiomyocytes, especially of the early stage. This appears particularly critical for molecular biological and biochemical studies, where a pure population of cardiomyocytes is needed. This may help to address such critical questions as time point of differentiation into the cardiac lineage and regulation of gene expression. Recently Metzger et al. have established an ES-cell line, where lacZ expression was under the control of the cardiac specific promoter human cardiac α-actin [129]. This allowed also a vital stain approach, in order to investigate early stage cardiomyocytes functionally. In the future, this approach may be further improved by the establishment of stably transfected ES-cell lines, where instead of lacZ in vivo reporter genes are under control of very early, cardiac specific promoters.

In the future the ES-cell differentiation model may prove also helpful for the investigation of the development of other cell types.

References

1
Buckingham
M.
Houzelstein
D.
Lyons
G.
Ontell
M.
Ott
M.O.
Sassoon
D.
Expression of muscle genes in the mouse embryo
Symp Soc Exp Biol
 
1992
46
203
217
2
Olson
E.N.
Brennan
T.J.
Chakraborty
T.
Cheng
T.C.
Cserjesi
P.
Edmondson
D.
James
G.
Li
L.
Molecular control of myogenesis: antagonism between growth and differentiation
Mol Cell Biochem
 
1991
104
7
13
3
Weintraub
H.
Davis
R.
Tapscott
S.
Thayer
M.
Krause
M.
Benezra
R.
Blackwell
T.K.
Turner
D.
Rupp
R.
Hollenberg
S.
et al
The myoD gene family: nodal point during specification of the muscle cell lineage
Science
 
1991
251
761
766
4
Viragh
S.
Challice
C.E.
Origin and differentiation of cardiac muscle cells in the mouse
J Ultrastruct Res
 
1973
42
1
24
5
Reedy
M.C.
Beall
C.
Ultrastructure of developing flight muscle in Drosophila. II Formation of the myotendon junction
Dev Biol
 
1993
160
466
479
6
Peng
H.B.
Wolosewick
J.J.
Cheng
P.C.
The development of myofibrils in cultured muscle cells: a whole-mount and thin-section electron microscopic study
Dev Biol
 
1981
88
121
136
7
Sanger
J.W.
Mittal
B.
Sanger
J.M.
Formation of myofibrils in spreading chick cardiac myocytes
Cell Motil
 
1984
4
405
416
8
Terai
M.
Komiyama
M.
Shimada
Y.
Myofibril assembly is linked with vinculin, alpha-actinin, and cell-substrate contacts in embryonic cardiac myocytes in vitro
Cell Motil Cytoskeleton
 
1989
12
185
194
9
Schultheiss
T.
Lin
Z.X.
Lu
M.H.
Murray
J.
Fischman
D.A.
Weber
K.
Masaki
T.
Imamura
M.
Holtzer
H.
Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils
J Cell Biol
 
1990
110
1159
1172
10
Hilenski
L.L.
Ma
X.H.
Vinson
N.
Terracio
L.
Borg
T.K.
The role of beta 1 integrin in spreading and myofibrillogenesis in neonatal rat cardiomyocytes in vitro
Cell Motil Cytoskeleton
 
1992
21
87
100
11
Simpson
D.G.
Decker
M.L.
Clark
W.A.
Decker
R.S.
Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes
J Cell Biol
 
1993
123
323
336
12
Manasek
F.J.
Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo
J Morphol
 
1968
125
329
365
13
Tokuyasu
K.T.
Maher
P.A.
Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. I. Presence of immunofluorescent titin spots in premyofibril stages
J Cell Biol
 
1987
105
2781
2793
14
Tokuyasu
K.T.
Maher
P.A.
Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation
J Cell Biol
 
1987
105
2795
2801
15

Tokuyasu KT. Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. III. Generation of fasciae adherentes and costameres. J Cell Biol, 1989;108:43-.

16

Challice CE, Edwards GA. The micromorphology of the developing ventricular muscle. In: Paes de Carvalho A, Carlos de Mello W. Hoffmann B, editors. The Specialized Tissues of the Heart. Amsterdam: Elsevier, 1961:44-75.

17
Hiruma
T.
Hirakow
R.
An ultrastructural topographical study on myofibrillogenesis in the heart of the chick embryo during pulsation onset period
Anat Embryol Berl
 
1985
172
325
329
18
Kojima
M.
Ishima
T.
Taniguchi
N.
Kimura
K.
Sada
H.
Sperelakis
N.
Developmental changes in beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in rat ventricular muscles
Br J Pharmacol
 
1990
99
334
339
19
Huynh
T.V.
Chen
F.
Wetzel
G.T.
Friedman
W.F.
Klitzner
T.S.
Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal, and adult rabbit ventricular myocytes
Circ Res
 
1992
70
508
515
20
Tohse
N.
Masuda
H.
Sperelakis
N.
Novel isoform of Ca2+ channel in rat fetal cardiomyocytes
J Physiol Lond
 
1992
451
295
306
21
Conforti
L.
Tohse
N.
Sperelakis
N.
Tetrodotoxin-sensitive sodium current in rat fetal ventricular myocytes — contribution to the plateau phase of action potential
J Mol Cell Cardiol
 
1993
25
159
173
22
Davies
M.P.
An
R.H.
Doevendans
P.
Kubalak
S.
Chien
K.R.
Kass
R.S.
Developmental changes in ionic channel activity in the embryonic murine heart
Circ Res
 
1996
78
15
25
23
Hunter
J.J.
Zhu
H.
Lee
K.J.
Kubalak
S.
Chien
K.R.
Targeting gene expression to specific cardiovascular cell types in transgenic mice
Hypertension
 
1993
22
608
617
24

Piper HM, Isenberg G, editors. Electrophysiology and contractile function. Boca Raton: CRC Press, 1989: vol 2.

25
Kimes
B.W.
Brandt
B.L.
Properties of a clonal muscle cell line from rat heart
Exp Cell Res
 
1976
98
367
381
26
Hescheler
J.
Meyer
R.
Plant
S.
Krautwurst
D.
Rosenthal
W.
Schultz
G.
Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart
Circ Res
 
1991
69
1476
1486
27
Sipido
K.R.
Marban
E.
Jr.
L-type calcium channels, potassium channels, and novel nonspecific cation channels in a clonal muscle cell line derived from embryonic rat ventricle
Circ Res
 
1991
69
1487
1499
28
Mejia Alvarez
R.
Tomaselli
G.F.
Marban
E.
Simultaneous expression of cardiac and skeletal muscle isoforms of the L-type Ca2+ channel in a rat heart muscle cell line
J Physiol Lond
 
1994
478
315
329
29
Jaffredo
T.
Chestier
A.
Bachnou
N.
Dieterlen Lievre
F.
MC29-immortalized clonal avian heart cell lines can partially differentiate in vitro
Exp Cell Res
 
1991
192
481
491
30
Caviedes
P.
Olivares
E.
Salas
K.
Caviedes
R.
Jaimovich
E.
Calcium fluxes, ion currents and dihydropyridine receptors in a new immortal cell line from rat heart muscle
J Mol Cell Cardiol
 
1993
25
829
845
31
Field
L.J.
Transgenic mice in cardiovascular research
Annu Rev Physiol
 
1993
55
97
114
32
Sen
A.
Dunnmon
P.
Henderson
S.A.
Gerard
R.D.
Chien
K.R.
Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen
J Biol Chem
 
1988
263
19132
19136
33
Minkoff
R.
Rundus
V.R.
Parker
S.B.
Beyer
E.C.
Hertzberg
E.L.
Connexin expression in the developing avian cardiovascular system
Circ Res
 
1993
73
71
78
34
Atherton
B.T.
Meyer
D.M.
Simpson
D.G.
Assembly and remodelling of myofibrils and intercalated discs in cultured neonatal rat heart cells
J Cell Sci
 
1986
86
233
248
35
Borg
T.K.
Rubin
K.
Lundgren
E.
Borg
K.
Obrink
B.
Recognition of extracellular matrix components by neonatal and adult cardiac myocytes
Dev Biol
 
1984
104
86
96
36
Florini
J.R.
Ewton
D.Z.
Magri
K.A.
Hormones, growth factors, and myogenic differentiation
Annu Rev Physiol
 
1991
53
201
216
37
Carey
D.J.
Control of growth and differentiation of vascular cells by extracellular matrix proteins
Annu Rev Physiol
 
1991
53
161
177
38
Parker
T.G.
Schneider
M.D.
Growth factors, proto-oncogenes, and plasticity of the cardiac phenotype
Annu Rev Physiol
 
1991
53
179
200
39
Muslin
A.J.
Williams
L.T.
Well-defined growth factors promote cardiac development in axolotl mesodermal explants
Development
 
1991
112
1095
1101
40
Consigli
S.A.
Joseph Silverstein
J.
Immunolocalization of basic fibroblast growth factor during chicken cardiac development
J Cell Physiol
 
1991
146
379
385
41
Bassas
L.
Lesniak
M.A.
Serrano
J.
Roth
J.
de Pablo
F.
Developmental regulation of insulin and type I insulin-like growth factor receptors and absence of type II receptors in chicken embryo tissues
Diabetes
 
1988
37
637
644
42
Doetschman
T.C.
Eistetter
H.
Katz
M.
Schmidt
W.
Kemler
R.
The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium
J Embryol Exp Morphol
 
1985
87
27
45
43
Robbins
J.
Gulick
J.O.
Sanchez
A.
Howles
P.
Doetschman
T.
Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro
J Biol Chem
 
1990
265
11905
11909
44
Wobus
A.M.
Wallukat
G.
Hescheler
J.
Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers
Differentiation
 
1991
48
173
182
45
Williams
R.L.
Hilton
D.J.
Pease
S.
Willson
T.A.
Stewart
C.L.
Gearing
D.P.
Wagner
E.F.
Metcalf
D.
Nicola
N.A.
Gough
N.M.
Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells
Nature
 
1988
336
684
687
46
Bradley
A.
Evans
M.
Kaufman
M.H.
Robertson
E.
Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines
Nature
 
1984
309
255
256
47
Evans
M.J.
Kaufman
M.H.
Establishment in culture of pluripotential cells from mouse embryos
Nature
 
1981
292
154
156
48
Martin
G.R.
Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells
Proc Natl Acad Sci USA
 
1981
78
7634
7638
49
Wobus
A.M.
Holzhausen
H.
Jakel
P.
Schoneich
J.
Characterization of a pluripotent stem cell line derived from a mouse embryo
Exp Cell Res
 
1984
152
212
219
50
Wobus
A.M.
Grosse
R.
Schoneich
J.
Specific effects of nerve growth factor on the differentiation pattern of mouse embryonic stem cells in vitro
Biomed Biochim Acta
 
1988
47
965
973
51
Rohwedel
J.
Maltsev
V.
Bober
E.
Arnold
H.H.
Hescheler
J.
Wobus
A.M.
Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents
Dev Biol
 
1994
164
87
101
52
Strubing
C.
Ahnert Hilger
G.
Shan
J.
Wiedenmann
B.
Hescheler
J.
Wobus
A.M.
Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons
Mech Dev
 
1995
53
275
287
53
Bain
G.
Kitchens
D.
Yao
M.
Huettner
J.E.
Gottlieb
D.I.
Embryonic stem cells express neuronal properties in vitro
Dev Biol
 
1995
168
342
357
54
Fraichard
A.
Chassande
O.
Bilbaut
G.
Dehay
C.
Savatier
P.
Samarut
J.
In vitro differentiation of embryonic stem cells into glial cells and functional neurons
J Cell Sci
 
1995
108
3181
3188
55
Risau
W.
Sariola
H.
Zerwes
H.G.
Sasse
J.
Ekblom
P.
Kemler
R.
Doetschman
T.
Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies
Development
 
1988
102
471
478
56

Drab M, Haller H, Bychkow R, Erdmann B, Lindschau C, Haase H, Luft FC, Wobus AM. Embryonic stem cells differentiate into spontaneously contracting vascular smooth-muscle-like cells characterized by vascular-specific gene expression and pharmacological functions (submitted).

57
Bagutti
C.
Wobus
A.M.
Faessler
R.
Watt
F.M.
Differentiation of embryonal stem cells into keratinocytes: Comparison of wild-type and 1integrin-deficient cells
Dev Biol
 
1996
179
184
196
58

Fassler R, Rohwedel J, Maltsev V, Bloch W, Lentini S, Kaomei G, Gullberg D, Hescheler J, Addicks K, Wobus AM. Differentiation and integrity of cardiac muscle cells are impaired in the absence of 1integrin. J Cell Sci 1996;109:2989–2999.

59
Fassler
R.
Sasaki
T.
Timpl
R.
Chu
M.L.
Werner
S.
Differential regulation of fibulin, tenascin-c, and nidogen expression during wound healing of normal and glucocorticoid-treated mice
Exp Cell Res
 
1996
222
111
116
60

Forssmann WG, Nokihara K, Gagelmann M, Hock D, Feller S, Schulz Knappe P, Herbst F. The heart is the center of a new endocrine, paracrine, and neuroendocrine system. Arch Histol Cytol, 1989;52 Suppl:293-315.

61

Lentini S, Wellner MC, Ji G, Pereverzev A, Wobus AM, Richter K, Bloch W, Fleischmann BK, Hescheler J, Addicks K. Myofibrillar pattern formation and elctrophysiological determination during in vitro cardiomyogenesis. (manuscript in preparation).

62
Viragh
S.
Challice
C.E.
Variations in filamentous and fibrillar organization, and associated sarcolemmal structures, in cells of the normal mammalian heart
J Ultrastruct Res
 
1969
28
321
334
63
Ishikawa
H.
Fukuda
Y.
Yamada
E.
Freeze-replica observations on frog sartorius muscle. I. Sarcolemmal specialization
J Electron Microsc Tokyo
 
1975
24
97
107
64
Forsgren
S.
Strehler
E.
Thornell
L.E.
Differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in the bovine heart: an immuno- and enzyme histochemical study
Histochem J
 
1982
14
929
942
65
Forsgren
S.
Carlsson
E.
Strehler
E.
Thornell
L.E.
Ultrastructural identification of human fetal Purkinje fibres — a comparative immunocytochemical and electron microscopic study of composition and structure of myofibrillar M-regions
J Mol Cell Cardiol
 
1982
14
437
449
66
Challice
C.E.
Viragh
S.
Origin and early differentiation of the sinus node in the mouse embryo heart
Adv Myocardiol
 
1980
1
267
277
67
Viragh
S.
Challice
C.E.
The development of the conduction system in the mouse embryo heart
Dev Biol
 
1980
80
28
45
68
Viragh
S.
Challice
C.E.
The development of the conduction system in the mouse embryo heart
Dev Biol
 
1982
89
25
40
69
Maltsev
V.A.
Rohwedel
J.
Hescheler
J.
Wobus
A.M.
Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types
Mech Dev
 
1993
44
41
50
70
Maltsev
V.A.
Wobus
A.M.
Rohwedel
J.
Bader
M.
Hescheler
J.
Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents
Circ Res
 
1994
75
233
244
71

Irisawa H. The action potentials of cardiomyocytes. In: Piper HM, Isenberg G, editors. Electrophysiology and contractile function. Boca Raton: CRC Press, 1989: vol 2. 1-12.

72
Trautwein
W.
Hescheler
J.
Regulation of cardiac L-type calcium current by phosphorylation and G proteins
Annu Rev Physiol
 
1990
52
257
274
73
DiFrancesco
D.
Pacemaker mechanisms in cardiac tissue
Annu Rev Physiol
 
1993
55
455
472
74
Kleppisch
T.
Wobus
A.M.
Strubing
C.
Hescheler
J.
Voltage-dependent L-type Ca channels and a novel type of non-selective cation channel activated by cAMP-dependent phosphorylation in mesoderm-like (MES-1) cells
Cell Signal
 
1993
5
727
734
75

Trube G. Potassium currents in isolated adult cardiac myocytes. In: Piper HM, Isenberg G, editors. Electrophysiology and contractile function., vol 2. Boca Raton: CRC Press, 1989:75–95.

76
Kilborn
M.J.
Fedida
D.
A study of the developmental changes in outward currents of rat ventricular myocytes
J Physiol Lond
 
1990
430
37
60
77
Sakmann
B.
Noma
A.
Trautwein
W.
Acetylcholine activation of single muscarinic K+ channels in isolated pacemaker cells of the mammalian heart
Nature
 
1983
303
250
253
78
Schultz
G.
Rosenthal
W.
Hescheler
J.
Trautwein
W.
Role of G proteins in calcium channel modulation
Annu Rev Physiol
 
1990
52
275
292
79

Pelzer D, Cavalie A, McDonald TF, Trautwein W. Calcium channels in single heart cells. In: Piper HM, Isenberg G, editors. Electrophysiology and contractile function., vol 2. Boca Raton: CRC Press, 1989:29–73.

80
Seamon
K.B.
Daly
J.W.
Forskolin: its biological and chemical properties
Adv Cyclic Nucleotide Protein Phosphorylation Res
 
1986
20
1
150
81
Kumar
R.
Joyner
R.W.
Hartzell
H.C.
Ellingsen
D.
Rishi
F.
Eaton
D.C.
Lu
C.
Akita
T.
Postnatal changes in the G-proteins, cyclic nucleotides and adenylyl cyclase activity in rabbit heart cells
J Mol Cell Cardiol
 
1994
26
1537
1550
82
Osaka
T.
Joyner
R.W.
Developmental changes in the beta-adrenergic modulation of calcium currents in rabbit ventricular cells
Circ Res
 
1992
70
104
115
83
Osaka
T.
Joyner
R.W.
Kumar
R.
Postnatal decrease in muscarinic cholinergic influence on Ca2+ currents of rabbit ventricular cells
Am J Physiol
 
1993
264
H1916
1925
84
Chen
F.M.
Yamamura
H.I.
Roeske
W.R.
Ontogeny of mammalian myocardial beta-adrenergic receptors
Eur J Pharmacol
 
1979
58
255
264
85
An
R.H.
Davies
M.P.
Doevendans
P.A.
Kubalak
S.W.
Bangalore
R.
Chien
K.R.
Kass
R.S.
Developmental changes in beta-adrenergic modulation of L-type Ca2+ channels in embryonic mouse heart
Circ Res
 
1996
78
371
378
86
Haddox
M.K.
Roeske
W.R.
Russell
D.H.
Independent expression of cardiac type I and II cyclic AMP-dependent protein kinase during murine embryogenesis and postnatal development
Biochim Biophys Acta
 
1979
585
527
534
87
Slotkin
T.A.
Lau
C.
Seidler
F.J.
Beta-adrenergic receptor overexpression in the fetal rat: distribution, receptor subtypes, and coupling to adenylate cyclase activity via G-proteins
Toxicol Appl Pharmacol
 
1994
129
223
234
88
Reuter
H.
Calcium channel modulation by neurotransmitters, enzymes and drugs
Nature
 
1983
301
569
574
89
Hescheler
J.
Kameyama
M.
Trautwein
W.
On the mechanism of muscarinic inhibition of the cardiac Ca current
Pflugers Arch
 
1986
407
182
189
90
Kameyama
M.
Hescheler
J.
Hofmann
F.
Trautwein
W.
Modulation of Ca current during the phosphorylation cycle in the guinea pig heart
Pflugers Arch
 
1986
407
123
128
91

Ji GJ, Wellner MC, Wobus AM, Fleischmann BK, Hescheler J. Muscarinic agonists modulate voltage dependent calcium channels in cardiomyocytes differentiated in vitro from embryonic stem cells. Pflugers Arch, 1996; R46 (Abstract).

92
Kawano
S.
DeHaan
R.L.
Developmental changes in the calcium currents in embryonic chick ventricular myocytes
J Membr Biol
 
1991
120
17
28
93
Benndorf
K.
Boldt
W.
Nilius
B.
Sodium current in single myocardial mouse cells
Pflugers Arch
 
1985
404
190
196
94
Satin
J.
Kyle
J.W.
Chen
M.
Rogart
R.B.
Fozzard
H.A.
The cloned cardiac Na channel alpha-subunit expressed in Xenopus oocytes show gating and blocking properties of native channels
J Membr Biol
 
1992
130
11
22
95
DiFrancesco
D.
Ferroni
A.
Visentin
S.
Zaza
T.
Cadmium induced blockage of the cardiac fast sodium channels in calf Purkinje fibres
Proc. R Soc London
 
1985
223
475
481
96
Backx
P.H.
Yue
D.T.
Lawrence
J.H.
Marban
E.
Tomaselli
G.F.
Molecular localization of an ion-binding site within the pore of mammalian sodium channels
Science
 
1992
257
248
251
97
Mitsuiye
T.
Noma
A.
A new oil-gap method for internal perfusion and voltage clamp of single cardiac cells
Pflugers Arch
 
1987
410
7
14
98
Brown
A.M.
Lee
K.S.
Powell
T.
Sodium current in single rat heart muscle cells
J Physiol Lond
 
1981
318
479
500
99
Fujii
S.
Ayer
R.K.
Jr.
DeHaan
R.L.
Development of the fast sodium current in early embryonic chick heart cells
J Membr Biol
 
1988
101
209
223
100
Walsh
K.B.
Begenisich
T.B.
Kass
R.S.
Beta-adrenergic modulation in the heart. Independent regulation of K and Ca channels
Pflugers Arch
 
1988
411
232
234
101
Walsh
K.B.
Kass
R.S.
Regulation of a heart potassium channel by protein kinase A and C
Science
 
1988
242
67
69
102
Yazawa
K.
Kameyama
M.
Mechanism of receptor-mediated modulation of the delayed outward potassium current in guinea-pig ventricular myocytes
J Physiol Lond
 
1990
421
135
150
103
Josephson
I.R.
Sperelakis
N.
Developmental increases in the inwardly-rectifying K+ current of embryonic chick ventricular myocytes
Biochim Biophys Acta
 
1990
1052
123
127
104
Chen
F.
Wetzel
G.T.
Friedman
W.F.
Klitzner
T.S.
Single-channel recording of inwardly rectifying potassium currents in developing myocardium
J Mol Cell Cardiol
 
1991
23
259
267
105
Wahler
G.M.
Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes
Am J Physiol
 
1992
262
C1266
1272
106
Sakmann
B.
Trube
G.
Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart
J Physiol Lond
 
1984
347
641
657
107
Biermans
G.
Vereecke
J.
Carmeliet
E.
The mechanism of the inactivation of the inward-rectifying K current during hyperpolarizing steps in guinea-pig ventricular myocytes
Pflugers Arch
 
1987
410
604
613
108
Belles
B.
Hescheler
J.
Trube
G.
Changes of membrane currents in cardiac cells induced by long whole-cell recordings and tolbutamide
Pflugers Arch
 
1987
409
582
588
109
Knoll
A.
Maltsev
V.A.
Wobus
A.M.
Hescheler
J.
Adenosine triphosphate-sensitive and inwardly rectifying potassium channels expressed in cardiomyocytes differentiated from embryonic stem cells
Heart and Vessels
 
1995
9
216
220
110

Belardinelli L, Kloeckner U, Iseberg G. Modulation of potassium currents and calcium currents in atrial and nodal cells. In: Piper HM, Isenberg G, editors. Electrophysiology and contractile function., vol 2. Boca Raton: CRC Press, 1989:155–180.

111
DiFrancesco
D.
Ohba
M.
Ojeda
C.
Measurement and significance of the reversal potential for the pace-maker current (iK2) in sheep Purkinje fibres
J Physiol Lond
 
1979
297
135
162
112
DiFrancesco
D.
Ojeda
C.
Properties of the current if in the sino-atrial node of the rabbit compared with those of the current iK, in Purkinje fibres
J Physiol Lond
 
1980
308
353
367
113
DiFrancesco
D.
The onset and autonomic regulation of cardiac pacemaker activity: relevance of the f current
Cardiovasc Res
 
1995
29
449
456
114
Satoh
H.
Sperelakis
N.
Identification of the hyperpolarization-activated inward current in young embryonic chick heart myocytes
J Dev Physiol
 
1991
15
247
252
115
Satoh
H.
Sperelakis
N.
Hyperpolarization-activated inward current in embryonic chick cardiac myocytes: developmental changes and modulation by isoproterenol and carbachol
Eur J Pharmacol
 
1993
240
283
290
116
Harvey
R.P.
NK-2 homeobox genes and heart development
Dev Biol
 
1996
178
203
216
117
Edmondson
D.G.
Lyons
G.E.
Martin
J.F.
Olson
E.N.
Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis
Development
 
1994
120
1251
1263
118
Srivastava
D.
Cserjesi
P.
Olson
E.N.
A subclass of bHLH proteins required for cardiac morphogenesis
Science
 
1995
270
1995
1999
119
Cohen
N.M.
Lederer
W.J.
Changes in the calcium current of rat heart ventricular myocytes during development
J Physiol Lond
 
1988
406
115
146
120
Osaka
T.
Joyner
R.W.
Developmental changes in calcium currents of rabbit ventricular cells
Circ Res
 
1991
68
788
796
121
Robbins
J.
Gene targeting. The precise manipulation of the mammalian genome
Circ Res
 
1993
73
3
9
122
Eberwine
J.
Yeh
H.
Miyashiro
K.
Cao
Y.
Nair
S.
Finnell
R.
Zettel
M.
Coleman
P.
Analysis of gene expression in single live neurons
Proc Natl Acad Sci USA
 
1992
89
3010
3014
123
Gomez
J.P.
Potreau
D.
Branka
J.E.
Raymond
G.
Developmental changes in Ca2+ currents from newborn rat cardiomyocytes in primary culture
Pflugers Arch.
 
1994
428
3-4
241
249
124
Kawano
S.
DeHaan
R.L.
Developmental changes in the calcium currents in embryonic chick ventricular myocytes
J. Membr. Biol.
 
1991
120
1
17
28
125

Muramatsu H, Zou AR, Berkowitz GA, Nathan RD. Characterization of a TTX-sensitive Na+ current in pacemaker cells isolated from rabbit sinoatrial node. Am. J. Physiol. 1996;270(6 Pt 2):H2108–H2119.

126
Masuda
H.
Sperelakis
N.
Inwardly rectifying potassium current in rat fetal and neonatal ventricular cardiomyocytes
Am. J. Physiol.
 
1993
265
4 Pt 2
H1107
1111
127
Brochu
R.M.
Clay
J.R.
Shrier
A.
Pacemaker current in single cells and in aggregates of cells dissociated from the embryonic chick heart
J. Physiol. Lond.
 
1992
454
503
515
128
Yu
H.
Chang
F.
Cohen
I.S.
Pacemaker current i(f) in adult canine cardiac ventricular myocytes
J. Physiol. Lond.
 
1995
485
Pt 2
469
483
129
Metzger
J.M.
Lin
W.I.
Samelson
L.C.
Vital staining of cardiac myocytes during embryonic stem cell cardiogenesis in vitro
Circ Res
 
1996
78
547
552