Ventricular Microanatomy, Arrhythmias, and the Electrochemical Driving Force for Na ⁺

HomeCirculation: Arrhythmia and ElectrophysiologyVol. 10, No. 2Ventricular Microanatomy, Arrhythmias, and the Electrochemical Driving Force for Na+ Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBVentricular Microanatomy, Arrhythmias, and the Electrochemical Driving Force for Na+Is There a Need for Flipped Learning? Luiz Belardinelli, MD, Mary M. Maleckar, PhD and Wayne R. Giles, PhD Luiz BelardinelliLuiz Belardinelli From the InCarda Therapeutics, Inc, Clinical Research and Development, Brisbane, CA (L.B.); Allen Institute for Cell Science, Seattle, WA (M.M.M.); and Faculties of Kinesiology and Medicine (W.R.G.), The University of Calgary, Alberta, Canada. , Mary M. MaleckarMary M. Maleckar From the InCarda Therapeutics, Inc, Clinical Research and Development, Brisbane, CA (L.B.); Allen Institute for Cell Science, Seattle, WA (M.M.M.); and Faculties of Kinesiology and Medicine (W.R.G.), The University of Calgary, Alberta, Canada. and Wayne R. GilesWayne R. Giles From the InCarda Therapeutics, Inc, Clinical Research and Development, Brisbane, CA (L.B.); Allen Institute for Cell Science, Seattle, WA (M.M.M.); and Faculties of Kinesiology and Medicine (W.R.G.), The University of Calgary, Alberta, Canada. Originally published17 Feb 2017https://doi.org/10.1161/CIRCEP.117.004955Circulation: Arrhythmia and Electrophysiology. 2017;10:e004955is corrected byCorrection to: Ventricular Microanatomy, Arrhythmias, and the Electrochemical Driving Force for Na+: Is There a Need for Flipped Learning?A well-known feature of the electrophysiological activity of the mammalian heart is that it can be regulated by tissue microanatomy. This occurs both on a cell-to-cell basis and regionally, that is, within defined structures in the cardiac syncytium.1,2 The initiation and conduction of the action potential and its ability to trigger and regulate contraction are modulated by connexin-mediated intercellular communication at well defined gap junctions.3 Recent results strongly suggest that the expression of some of the ion channels responsible for the cardiac action potential is not uniform on the surface membrane or sarcolemma of each myocyte. For example, Na+ channels seem to be preferentially expressed and in fact concentrated at the intercalated disc regions between ventricular myocytes, where connexins are also located.4,5 These features (Figure), when combined with the fact that in the adult myocardium, myocytes are often arranged in trabeculae (tightly packed cylindrical structures) can also result in activity- or rate-dependent changes in the electrolyte composition of the immediately adjacent restricted extracellular space or "clefts".3 A well-known example is the extracellular potassium accumulation that has been studied in detail in the ventricle6 and the Purkinje conduction system.7Download figureDownload PowerPointFigure. Diagram illustrating the 2-dimensional structure of ventricular myocytes within the cardiac syncytium. A, Three rows of idealized myocytes. The hatched lines in this panel draw attention to 3 specialized regions of intercellular communication. One such gap junction is illustrated in B with the connexin-mediated cell-to-cell conduits denoted as the 3 cylinders. Such extremely narrow intercellular spaces are denoted the "perinexus" in this article and include highly concentrated (up to 90% of the total) expression of cardiac Na+ channels. Adapted from Lin and Keener10 with permission of the publisher. Copyright © 2014, Biophysical Society. Authorization for this adaptation has been obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.See Article by Greer-Short et alBackgroundAn interesting and forward looking article8 in this issue of Circulation: Arrhythmia and Electrophysiology draws on this background while testing a novel hypothesis for underlying causes of the proarrhythmia associated with the marked action potential lengthening denoted long-QT syndrome. This experimental and theoretical work invokes a fundamental role for what is termed ephaptic conduction9 in the mammalian ventricular myocardium.10–12 These authors build on their previous data13,14 from similar experiments on guinea pig ventricle. They now propose that changes in highly localized intercellular electric fields (the corner stone of the principle of ephaptic transmission) arise because of activity-dependent changes in extracellular sodium, [Na+]o.In this article, Greer-Short et al8 attempt to identify and document plausible ways in which the principles of ephaptic conduction may contribute to the genesis of some aspects of the long-QT syndrome.9 Specifically, they have studied the consequences of 2 genetic variants of the long-QT type 3 (LQT3) syndrome based on known changes in the mammalian cardiac Na+ channel-mediated current, Nav 1.5 or INa. Measurements of the action potential waveforms and changes in conduction velocity, based on elegant voltage-sensitive dye experiments, are presented and analyzed. These findings are complemented with electron microscopic measurements of "gap width" within the intercalated disc regions denoted the perinexus. In addition, a mathematical model15 for impulse conduction in the guinea pig heart that includes preferentially increased Na+ channel expression within the intercalated disc has been used. This model can account for the changes in Na+ current dynamics that contribute importantly to the action potential lengthening that is characteristic of the LQT3 syndrome phenotypes.16 Interestingly, these simulations reveal conditions under which highly concentrated Na+ channel expression (70% to 90% of the total Na+ channel population) at the intercalated disc, when combined with the extremely narrow extracellular spaces between the myocytes at the perinexus can give rise to significant localized decreases in [Na+]o, thus reducing the electrochemical driving force for Na+, ENa. This causes a temporary, but significant, reduction in INa current measured in terms of both its peak amplitude and the slowly inactivating or "late" component of INa.17 This is perhaps the key tenet of this study. The authors refer to this phenomenon as "self-attenuation" of INa.Because late INa provides an inward current that tends to prolong the action potential and promote the occurrence of early afterdepolarizations, the authors relate the pattern of changes illustrated by their computations to a phenomenon sometimes denoted "concealed conduction" in the ventricular myocardium. Specifically, they write "…that self-attenuation can "mask" the LQT3 phenotype by reducing the driving force and late sodium current that produces early afterdepolarizations (EADs)." This supposition is novel and intriguing, but it deserves additional analysis. This includes consideration of the specific and essential assumptions governing the mathematical model and recollection of previously published data that may be relevant to the overall interpretation of this intriguing study.Experimental and Computational ResultsA strong element of this article8 is the microanatomical evidence that demonstrates the required, exceptionally narrow extracellular spaces near the connexins that are within the intercalated disc regions between myocytes. These features of the "perinexus" are supported by recent results obtained using "super resolution microscopy".18 As mentioned, previous publications have provided evidence for preferential binding of antibodies directed toward selected epitopes of the cardiac Na+ channel5 at the intercalated disc. In combination, these 2 key data sets provide necessary starting points for the possibility of electric field-mediated or ephaptic intercellular communication/conduction in the mammalian ventricular myocardium.Electron micrographs shown in this article illustrate an increase in the width of the perinexus caused by an experimental maneuver (addition of mannitol to the perfusing solution) that mimics some aspects of the tissue edema observed after ventricular injury. In fact, this procedure provided an important, but unexpected, pattern of results. After mannitol treatment the action potential duration in the guinea pig ventricular preparations exhibited much less variation than would otherwise be the case in the chosen model of LQT3 proarrhythmia. In this model, very low levels of sea anenome toxin-III enhance the late INa and result in an LQT3 phenotype. However, in the setting of tissue edema, it is likely that other important properties of the myocyte such as its input resistance could change. This is known to strongly modulate "cell-to-cell interactions during both early and late repolarization".19None of the essential localized electrophysiological changes or required alterations in electrochemical driving force for Na+ can be directly monitored within the microanatomical spaces of interest. Accordingly, Greer-Short et al8 explore the basis for their experimental findings using a mathematical model as outlined above. These computations reveal that, even at the relatively low heart rates that typically unmask the prominent action potential lengthening that is characteristic of LQT3, there are significant changes (decreases) in the [Na+]o concentration within the small extracellular spaces at the intercalated disc regions. As shown in their Figure 3D and supplemental data (Figures S3 and S4) the main consequence of this is a marked decrease or (movement in the hyperpolarizing direction) of ENa. According to these calculations, ENa decreases substantially (as much as 30 mV) from its control value. This reduces peak INa somewhat (not shown) and also decreases late INa. As late INa is a prominent proarrhythmic factor in the ventricular myocardium, its reduction can have significant stabilizing or antiarrhythmic effects.15–18 Greer-Short et al,8 argue that under baseline conditions, the normal pattern of electric activity is sufficient to produce cyclic changes in [Na+]o in the perinexus regions. If so, this would consistently reduce the magnitude of late INa, thus giving this component of Na+ influx only a minimal role in early afterdepolarization generation. In this sense, the authors' working hypothesis can perhaps account for the combination of microanatomy and ephaptic transmission concealing the appearance of the marked action potential lengthening that is a hallmark feature of both the genetic and the acquired variants of LQT3. These authors also postulate that this "self-attenuation mechanism… can operate independent of specific (Na+ channel) mutations". Finally, they suggest that "future therapies could target intercellular cleft separation" as an antiarrhythmic maneuver.The likelihood of this mechanism contributing importantly to the proarrhythmia characteristic of the LQT3 syndrome should be further evaluated after considering the specific assumptions on which these interesting sets of simulations are based. As part of this consideration, some previous experimental findings that are relevant to any working hypothesis that requires significant changes in the electrochemical driving force for Na+ in the mammalian myocardium are presented.Additional ConsiderationsChanges in the Electrochemical Gradient for Na+An essential tenet of this article8 is that there is a substantial change in [Na+]o within the perinexus or very narrow cleft space. This change occurs at baseline and also at the very low heart rates that typically are associated with the manifestation of the LQT3 syndrome phenotype. Certainly, the simulations illustrate these changes clearly. An important question therefore arises: is there a commensurate marked increase in [Na+]i, resulting from the same Na+ influx that "depletes" [Na+]o?Rate-dependent alterations in [Na+]i arising from Na+ fluxes into small, [Na+]i domains that exhibit restricted diffusion, (the so-called fuzzy space) have previously been studied20 including their relationship to the Na+ influx produced by late INa.21 An increase in [Na+]i would activate the electrogenic [Na+/K+] pump22 and, therefore, hyperpolarize the target cells. Hyperpolarization would remove inactivation from Na+ channels during diastole and consequently alter excitability. However, this hyperpolarization may not be detected with the same voltage sensitive dye mapping that is optimized for recording the action potential. In addition, any significant change in the electrochemical gradient for [Na+] would be expected to alter the [Na+/Ca2+] exchange activity and [Na+/H+] regulatory mechanisms.23 The resulting changes in [Ca2+]i, pHi may alter INa,24 or connexin-mediated communication25 as well as a number of other time- and voltage-dependent currents.Possibility of Potassium [K+]o AccumulationPrevious studies of mammalian hearts and the possibility of rate-dependent alterations in the electrochemical gradients for plasma electrolytes have focused mainly on changes in [K+]o. These occur within small spaces immediately adjacent to the myocytes. This [K+]o accumulation can alter both the resting potential and a number of the K+ currents that are needed for repolarization of the action potential.6,7 In this article,8 the very low heart or stimulus rates would make it likely that [K+]o accumulation would not be significant. However, although heart rate is low, the experiments aimed at mimicking the LQT3 syndrome phenotype necessarily involve inducing long action potentials. This sea anemone toxin–induced prolongation of the action potential may also give rise to significant K+ accumulation because of relatively long-lasting activation of intrinsic K+ currents.17A Purkinje Fiber "Thought Experiment"Additional experimental work to further evaluate and perhaps validate important aspects of the novel working hypothesis developed by Greer-Short et al8 could perhaps be done using conventional microelectrode recordings from isolated Purkinje fibers. In this preparation, the initial depolarization phase of the action potential is distinct because of the prominent early repolarization to a plateau level of approximately −20 mV. The membrane potential at the peak overshoot of the action potential closely approximates the theoretical value for ENa. In addition, in most multicellular Purkinje fibers, the intercellular spaces are small.7 This microanatomical feature, combined with the exceptionally high density of expression for INa in this specialized conduction tissue, should yield conditions under which the consequences of the proposed change in the perinexus or cleft [Na+]o could be monitored by detailed recordings of the peak height of the action potential. Certianly, with respect to a key result revealed by the computations in this article,8 if the changes in ENa are as large as 20 to 30 mV, they would be detected. Recently, a somewhat similar approach for estimating putative changes of [Na+]i in ventricular myocytes has been described by Hegyi et al.26 We note, however, that both our proposed experiment and this recent work need to carefully consider the fact that near the ENa, Na+ channels (somewhat akin to Ca2+ channels) are not perfectly selective. Both allow a significant [K+] efflux at strongly depolarized membrane potentials and therefore do not behave as ideal Na+ or Ca2+- sensitive "electrodes."The authors are to be commended for their clever multidisciplinary study. This novel and provocative article clearly sets out a fundamental mechanism that could contribute to the development of a proarrhythmic substrate in the mammalian ventricle. Key elements of the working hypothesis will continue to be elucidated by ongoing studies of the molecular features of the "Na+ channel complex."27 Additional data on the microanatomy of the heart and intercellular conduction after myocardial ischemia/infarction28 will be relevant. Recent experimental results regarding new ways in which changes in [Ca2+]o can alter INa29 are also worth considering. Finally, when myocardial substrates characterized by abnormally long action potentials are being studied, electromechanical effects may also be important.30AcknowledgmentsDr Maleckar of the Allen Institute for Cell Science wishes to thank the AICS founder, Paul G. Allen, for his vision, encouragement, and support. Dr Giles is supported by the Alberta Innovates Health Solutions Foundation and the Canadian Institutes for Health Research.DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Wayne R. Giles, PhD, Faculty of Kinesiology, University of Calgary, 2500 University Dr, NW, Calgary, Alberta T2N 1N4, Canada. E-mail [email protected]References1. Kléber AG, Rudy Y. Basic mechanisms of cardiac impulse propagation and associated arrhythmias.Physiol Rev. 2004; 84:431–488. doi: 10.1152/physrev.00025.2003.CrossrefMedlineGoogle Scholar2. Aronsen JM, Swift F, Sejersted OM. 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