doi: 10.1161/01.RES.0000091364.90121.0C
© 2003 American Heart Association, Inc.
Editorials |
Heterogeneous Cell Coupling in the Heart
An Electrophysiological Role for Fibroblasts
From the University Lab of Physiology, Oxford, UK.
Correspondence to Peter Kohl, MD, PhD, University Lab of Physiology, Parks Road, Oxford OX1 3PT, UK. E-mail peter.kohl{at}physiol.ox.ac.uk
Key Words: cardiomyocytes • cardiac fibroblasts • gap junctions • in vitro • in vivo
The heart is a muscle. Muscles are made up of myocytes. These may differ in form and function, but—in essence—they are the cells in which we are interested when we consider the structural makeup of the heart. Likewise, when we assess electrical coupling of cardiac cells by connexins, we are often content to assume that gap junctions occur exclusively—or at least in the overwhelming majority of cases—between homologous cell types.
It is sobering, in this context, to reflect on the fact that cardiac myocytes form a minority of cells in the heart, insofar as cell numbers are concerned (which, for cell coupling, is more relevant than total volume occupied by a cell population). A meticulous study by Adler et al1 demonstrated that myocyte and connective tissue cell numbers increase at a similar rate in early human development, from about 0.5x109 at 28 weeks of fetal development to 2 to 3x109 several weeks postpartum. Thereafter, myocyte cell numbers remain stable, while the connective tissue cell count increases with cardiac weight to 
7x109 at 2 months of age.
This mitotic potential of cardiac fibroblasts is maintained after cell isolation and is the key reason for which fibroblasts are omnipresent in primary cardiac cell cultures. This is not for lack of effort to eliminate nonmyocytes. Measures to enrich myocyte content in cardiac cell culture include addition of mitotic inhibitors, substrate restrictions, and, most prominently, preplating steps (occasionally in the presence of antibodies against muscle cell surface adhesion factors).2 Nonetheless, cardiac cell cultures are essentially always cocultures of myocytes and (proliferating) nonmyocytes. This insight is reflected in some of the earliest cardiac cell culture work from the 1950s, where mitosis was observed—but not in pulsating cells (ie, in cells other than myocytes).3
A contribution of nonmyocytes to in vitro impulse conduction was first established in 1966 by Mark and Strasser4 who identified two main cell populations in trypsin-digested neonatal rat heart cell cultures: myocytes and "endothelioid" cells (whose detailed description and photographic presentation allows classification of them as what nowadays is referred to as fibroblasts). Using time-lapse and real-time video microscopy, they discovered that synchronization of spontaneous contractile activity in individual cardiomyocytes required cell contact, which could be either direct or indirect via nonmuscle cells (see Figures 6 through 8 in Reference 4). This line of investigation was followed by Goshima et al,5–7 who combined cinematographic, histological, and electrophysiological methods to confirm that a whole range of heterocellular constructs can conduct excitation between cultured cardiac myocytes. Goshima and Tonomura6 also described effective synchronization of distant myocytes (
150 µm) via conduction pathways involving multiple nonmyocytes (see Figure).
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The practical relevance of these observations was intensely debated in the late 1980s/early 1990s when, on the one hand, homogeneous and heterogeneous gap junctions between cardiac myocytes and fibroblasts in vitro were characterized in great detail (down to single-channel conductances),8–10 while, on the other hand, in vivo investigations largely drew blanks on the underlying histological substrate.11 This issue of Circulation Research contains a report by Gaudesius et al12 that, in all likelihood, will rekindle this debate on an elevated level.
In their thorough study, Gaudesius et al12 describe a novel cardiac coculture system in which myocyte strands (80 µm wide) are interrupted by inserts of variable length (50 to 800 µm) that are filled with heterogeneous cell types, including cardiac fibroblasts, wild-type HeLa cells, and HeLa cells transfected with connexin43 (HeLa-Cx43). Using a combination of time-lapse and real-time video microscopy, immunocytochemistry, and optical measurement of impulse propagation, they show that cardiac fibroblasts can transmit electrical excitation, bridging gaps of up to 300 µm. They further show that heterotypic cell inserts can replace cardiac fibroblasts only if they express suitable connexins (here Cx43). This highlights the requirement for formation of gap junctional coupling between nonmyocytes, and between myocytes and nonmyocytes, for successful impulse transmission via the latter. It also begs the question as to whether highly sophisticated in vitro systems can become suitable models of in vivo cardiac structure and function, including gap junctional coupling patterns.
Experimental models for the study of cardiovascular function must always be a compromise between relevance (the only true model here would be humans), reproducibility (where lower levels of functional integration with fewer degrees of freedom yield less variability), and cost, as expertly explained by Hearse and Sutherland.13 Cell culture models offer a very attractive compromise, as they are about midscale on all three parameters. Extensive efforts have been made to improve the properties of cardiac tissue culture. Microstructuring of the cell adhesion matrix, for example, allows one to predetermine areas for cell attachment,14 and this has been found to improve characteristic cell morphology and alignment, particularly if cells are grown on lines that 
40 µm wide.15 Further improvements in cell culture quality can be achieved by application of controlled stretch to deformable cell culture substrates, which affects gene expression, protein synthesis, autocrine and paracrine signaling, and connexin expression in cocultures of cardiomyocytes and fibroblasts.16–19 Recently, the advantages of microstructuring and mechanical control have been combined in a single cell culture system.15
The issue of mechanical effects on cellular interactions is relevant even for cultures grown on solid substrates, since the contractile activity of cardiomyocytes affects adjacent cells. Gaudesius et al12 neatly reconfirm the role of gap junctional coupling for impulse conduction via nonmyocytes by showing that replacement of HeLa-Cx43 with wild-type cells lacking the electrotonic conduction pathway afforded by Cx43 prevents impulse transmission. While this finding provides convincing evidence in favor of a role for gap junctional coupling, it should be reconfirmed in the absence of streptomycin in the cell culture medium, since this aminoglycoside is a potent blocker of cardiac stretch-activated ion channels20 and could cause false-negative findings insofar as possible stretch-mediated effects are concerned.
A vital remaining question is that of the applicability of in vitro findings on (homogeneous and) heterogeneous cell coupling to the in vivo setting. Gaudesius et al12 report that myocytes and fibroblasts express Cx43—the main connexin to which cultured cells tend to revert—and Cx45, and that small punctate labeling for both connexins can be found at points of contact between both homogeneous and heterogeneous cell types. Cx40 label was not observed.
How representative of the in vivo setting are in vitro model findings? As the authors point out, at present there is little hard evidence on fibroblast-myocyte coupling in native cardiac tissue. Functional studies are hampered by the fact that cardiac fibroblasts have a very high membrane resistance (G
range). This may be an advantage for electrotonic impulse transmission, but it means that—if fibroblasts are well-coupled to cardiac myocytes—they mimic the intracellular membrane potential dynamics of the latter (albeit with reduced upstroke velocities, as observed in rat atrium21 and isolated cell pairs9), which makes it difficult to identify them electrophysiologically in situ. If fibroblasts are not coupled to neighboring cardiac muscle cells, they display a resting membrane potential of between 0 and -50 mV and are of little interest in the given context.
A painstaking transmission electron microscopy study by De Mazière et al11 revealed only "one tiny gap junction-like structure" in a sinoatrial node tissue volume containing an estimated 104 homologous nexus contacts. Instead, abundant heterogeneous cell approximations were reported, where fibroblast membranes anchor directly into the basal membrane of cardiomyocytes. Whether these membrane approximations accommodate dispersed gap junction channels that are not clustered densely enough to form a sufficiently electron-dense substrate for recognition by electron microscopy is not known. The present communication by Gaudesius et al12 reconfirms, however, that heterogeneous gap junctions between cardiac myocytes and fibroblasts are of much smaller dimension than those between myocyte pairs.
More recently, sinoatrial node fibroblasts have been reported to express punctate Cx40 and Cx45 label (but not Cx43) in vivo, and Cx45 was found at the point of contact of cardiac fibroblasts and myocytes in native sinoatrial node tissue.22 There are also first indications of functional heterogeneous coupling, as witnessed by dye transfer studies in rabbit sinoatrial node.23 These findings highlight the desirability of cell-type identification in immunohistochemical studies of connexin distribution in cardiac tissue, as it is inappropriate to assume that all label will be located between homologous cells.
What would be the functional relevance of electrical coupling between cardiac myocytes and fibroblasts? First of all, there can be no doubt that connective tissue can form barriers that interfere with the orderly conduction of excitation. The report by Gaudesius et al12 supports the possibility that fibroblasts may also act as a substrate for electrical coupling. This opens up a number of scenarios. Fibroblasts could act as a current sink, thereby contributing to the formation of unidirectional block of conduction.24 They could, via short-range interaction, contribute to the smoothing of propagation, in particular in the sinoatrial node (where pacemaker cells form islands embedded in connective tissue) and in the cross-sheet direction of ventricular tissue (which otherwise would give rise to fragmented conduction patterns).25 As long-distance communication lines, fibroblasts could bridge posttransplantation (or other) scar tissue with beneficial or detrimental effects on organ function, or explain the delay in atrioventricular conduction, as discussed by the authors.12 Furthermore, the inherent mechanosensitivity of cardiac fibroblasts could allow them to play a sensory role and affect cardiac electrophysiology in the context of mechano-electric feedback.26
The dynamics of the development of the idea of cardiac heterogeneous cell coupling bears striking similarities to that of neuron-glia interaction: early reports on in vivo heterogeneous structural and functional coupling27,28 were reassessed in the 1990s, largely rejected,29 and are now being reconsidered as potentially of functional relevance.30 As in the case of the nervous system, further advancement of our insight into the issue of direct electrical coupling of "main" and "bystander" cells of the heart will be technically challenging, yet conceptually rewarding.
The heart is a muscle, and muscles are structurally and functionally complex, highly integrated heterocellular constructs.
Acknowledgments
This work was supported by the British Heart Foundation and the Biotechnology and Biological Sciences Research Council. P.K. is a Royal Society Research Fellow. The author thanks Patricia J. Cooper for her help with this manuscript.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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