© 2000 American Heart Association, Inc.
Editorials |
Cardiac Gap Junction Remodeling by Stretch
Is It a Good Thing?
From the Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY.
Correspondence to José Jalife, MD, Department of Pharmacology, SUNY Upstate Medical University, 766 Irving Ave, Syracuse, NY 13210. E-mail jalifej{at}upstate.edu
Key Words: mechanical forces • pulsatile stress • cell-to-cell communication • conduction velocity • optical mapping • immunohistochemistry
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Introduction |
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Cardiac cells contract and are also normally exposed to the mechanical events in their surroundings. It is now well established that both cardiac gene expression and protein synthesis are subject to regulation by mechanical forces, including stretch. For example, mechanical stretch is known to be one of the most important stimuli leading to cardiac hypertrophy,1 2 3 4 5 and recent studies indicate that cardiac myocyte hypertrophy is stimulated in vitro by specific directions and degrees of stretch.6 Similarly, certain stretch-sensitive sarcolemmal ion channels and exchangers have been found in cardiac myocytes7 and have been implicated in the mechanism of stretch-induced arrhythmias.8 However, whereas signal transduction induced by mechanical stretch involves activation of a wide variety of second messenger systems,9 it remains to be determined which molecules are directly affected by stretch and which are the processes whereby mechanical stimuli trigger intracellular signaling pathways to activate protein kinase cascades and produce changes in function.
The process of filling and ejecting blood subjects the cells of the heart to repetitive pulsatile stress. Our understanding of the basic electrophysiology underlying the cardiac action potential and its propagation across cells is largely on the basis of patch clamp data and isolated tissue experiments in the absence of mechanical stress. On the other hand, whole-heart electrophysiological mapping studies are often carried out in in situ functioning hearts. In either case, the role of mechanical stress in impulse initiation and propagation has not been adequately addressed. In addition, although stretch is thought to play an important role in cardiac remodeling that is associated with heart failure, very little is known about its role in normal electrical function. The study by Zhuang et al10 in this issue of Circulation Research, which is the result of a successful collaboration between two outstanding laboratories, sheds new light on this important subject by detailing some of the electrophysiological consequences of mechanical stretch.
The responses to uniform pulsatile and static stress were investigated using an innovative approach developed in the laboratory of Dr André Kléber. Previously, Dr Kléber and his colleagues used cell cultures grown in specific shapes to study the role of tissue geometry and cellular coupling on impulse propagation.11 12 These studies have led to a better understanding of the effects of source-sink mismatching during impulse propagation. In the present study, cultured rat neonatal myocytes were grown on a flexible foundation constructed of thin silicone rubber. Once the cells were firmly attached to this surface, the edges of the silicone were connected to a mechanical device that would deform the silicone base in a controlled fashion. Using this system, various stretch protocols were applied along one or two axes for a predetermined amount of time. This approach provided a reliable source of cells that have been subjected to similar and reproducible stretch protocols.
Multiple-site optical mapping of voltage-dependent fluorescence was performed to assess the effects of pulsatile stretch on propagation and upstroke velocity. The authors hypothesized that pulsatile stretch would produce an immediate adaptive response that would affect conduction parameters, and this is exactly what they found. Measurements of conduction velocity from cells subjected to pulsatile stress demonstrated a modest increase in propagation velocity compared with control cells. These effects were observed within the first hour of pulsatile stretch and continued over the next 6 hours. The upstroke velocity of the action potential did not change significantly at any of the time points measured, which suggested that pulsatile stretch did not affect the active membrane currents involved in cardiac excitation.
Next, the authors hypothesized that the electrophysiological changes were the result of an alteration in the expression of proteins integrally involved in propagation of the electrical impulse. One of the important parameters involved in determining how fast action potentials propagate through cardiac tissue is intercellular coupling. The cells in the heart are electrically and metabolically coupled through gap junction channels. These channels provide a low-resistance pathway, which allows the excitation current to spread throughout the heart. Over the last 12 years, the laboratory of Dr Jeffery Saffitz has pioneered the study of connexins in normal and diseased heart tissues using immunohistochemistry.13 14 15 With this approach, the effects of pulsatile stretch on the expression of the major ventricular gap junction protein, connexin43 (Cx43), as well as the fascia adherens junction protein N-cadherin, were determined. Previously, Wang et al16 reported that 4 hours of pulsatile stretch of cultured rat myocytes caused a 3-fold increase in Cx43 levels. In the study by Zhuang et al,10 stretch also caused a significant increase in the amount of Cx43 immunoreactive signal. However, this change occurred after 1 hour of pulsatile stretch and continued for up to 6 hours. The authors attributed this increase in immunofluorescence to a greater number of individual gap junction plaques and not an overall increase in their mean size. Similar results were found for N-cadherin. It is important to note here that Zhuang et al10 demonstrated a temporal correlation between the increases in the Cx43 protein levels and conduction velocity. On the other hand, in the study by Wang et al,16 there was a discrepancy between the onset of the protein level changes and the electrophysiological response. Interestingly, Wang et al16 found that stretch induced an increase in intracellular pH, which may have led to an increase in gap junctional coupling17 and could have been a contributing factor to the increase in conduction velocity demonstrated by Zhuang et al.10 In fact, the changes in conduction velocity observed experimentally by these authors seem higher than those expected on the basis of Cx43 upregulation alone (32% instead of 39%). Thus, other factors, including increases in intracellular pH,16 cannot be ruled out.
As previously discussed in work from the Saffitz laboratory,13 when interpreting immunoreactive signals, it is important to remember that there is a complex nonlinear relation between the amount of antibody bound to an epitope and the intensity of secondary detection signals. Therefore, the number and size of those signals do not necessarily represent the number and dimension of the immunoreactive target. It is possible that the immunofluorescent signal that is emitted from several closely spaced gap junction plaques would appear as a single spot. This would artefactually lower the number of target structures while raising their apparent size. In addition, it is also possible that small gap junctions below the resolution of the detection system would be missed. On the other hand, an overestimation of the number of gap junctions could occur as a result of nonuniform staining of individual gap junction plaques. Careful calibration requires that the sensitivity and minimal detectable size of immunofluorescent signal be determined. Another important consideration in interpreting changes in immunoreactive signals is the determination of the linear range of the fluorescence measurements. Despite these limitations, which Zhuang et al10 clearly recognize, it is likely that changes in the total immunoreactive signal parallel the changes in the total Cx43 content of the preparation.
The study by Zhuang et al10 represents an important contribution to our understanding of the relationship between cardiac electrophysiology and mechanical stress. It also raises several interesting questions. For example, does the magnitude of stretch cause a change in the adaptive response? The heart undergoes pulsatile stretch as a normal part of its function. It is not clear whether greater levels of stretch would cause an additional increase in Cx43 content, resulting in greater conduction velocities. As gap junctional conductance increases, it is no longer the limiting resistor in the electrical circuit of propagation. Recently, Jongsma and Wilders18 demonstrated, using computer modeling, that relatively large changes in gap junctional conductance are required to affect conduction velocity. Those data were obtained without considering the effects of pulsatile stretch. In this regard, the results of Zhuang et al10 indicate that pulsatile stretch shifts the range of gap junctional conductance to levels where conduction velocity would no longer be sensitive to moderate changes in intercellular coupling. This suggests that the early stages of gap junction remodeling associated with heart failure may not cause large changes in the electrophysiological behavior of the heart.
It would be important to determine whether the changes observed in the present study are part of a response associated with normal cardiac function or whether they are part of the pathophysiology of cardiac hypertrophy and failure. Another interesting question is whether this experimental model can reproduce the changes associated with the latter stages of clinical heart failure, namely, a reduction in cell-cell coupling.19 20 21 Subjecting cell cultures to stretch over a prolonged period of time may reproduce these findings. Other questions include whether intercellular coupling is the only electrophysiological parameter that pulsatile stretch affects. Clearly, the adaptive changes responsible for the alterations observed during heart failure involve multiple signaling pathways. Mechanical stretch has been associated with the activation of several signaling mechanisms, including tyrosine kinases, Ras/mitogen-activated protein kinase, and protein kinase C phospholipases C and D.1 3 4 These pathways are known to regulate other membrane ion channels, which would contribute to the electrophysiological changes associated with stretch. On the other hand, given the dramatic upregulation of protein levels induced by stretch and the relatively modest changes in conduction velocity, it seems reasonable to speculate that perhaps the most important consequences of Cx43 overexpression may have to do more with metabolic than electrical events.
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Acknowledgments |
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This work was supported in part by grant PO1 HL39707 from the National Heart, Lung, and Blood Institute, National Institutes of Health. We thank Dr Mario Delmar for reading the manuscript and Dhananjay Vaidya for helpful discussions.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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