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Heart Rhythm. Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Heart Rhythm. 2016 May ; 13(5): 1172–1181. doi:10.1016/j.hrthm.2016.01.011.
Cardiac Purkinje fibers and Arrhythmias; The GK Moe award Lecture 2015 Penelope A. Boyden, PhD, Wen Dun, MD, PhD, and Richard B. Robinson, PhD Department of Pharmacology, Center for Molecular Therapeutics, Columbia University, New York NY
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Abstract Purkinje fibers/cells continue to be a focus of arrhythmologists. Here we review several new ideas that have emerged in the literature and fold them into important new points. These points include some proteins in Purkinje cells that are specific to Purkinjes, pacemaker function in Purkinje may be similar to that of the sinus node cell, sink-source concerns about tracts/sheets of Purkinje fibers, role of Ito in arrhythmias and genetic lesions in Purkinjes and their high impact on cardiac rhythm. Although new ideas about the remodeled Purkinje cell are not the focus of this review, one can easily imagine how Purkinjes and their function may be altered in diseased hearts.
Keywords Purkinje; arrhythmias; pacemaker activity; calcium; ion channels
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Introduction
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Despite the careful descriptions of Purkinje fibers by the Czech Jan Purkinje (1787–1869) cardiac cellular Electrophysiology was still in its infancy in late 1960 s and early 1970 s. Fine tipped microelectrodes and cardiac preparations of Purkinje fibers were the most commonly used experimental approaches. In fact, C. Mendez and Gordon K. Moe published several papers about the electrical properties of canine Purkinjes within the Purkinje-Muscle Junction (eg.1). Several hundred miles south of the Utica NY Moe lab, BF Hoffman would suggest that enhanced depolarizations in Purkinje cells might be a significant factor in human arrhythmias 2. This led to an intense effort to develop the single cell preparation of canine Purkinje fibers dissected from free running bundles 3. After all the idea was simple, by using a single Purkinje cell with whole cell voltage clamp techniques, one could understand the ionic currents flowing during Purkinje pacemaker activity. The problem was that the single Purkinje cell from a normal heart did not show spontaneous impulse
Address for Correspondence: Dr. Penelope A. Boyden, Dept of Pharmacology, Columbia College of Physicians and Surgeons, 630 West 168th ST., New York New York 10032, 212-305-7907 (phone) There are no potential conflicts of interest. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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generation. This has been discussed in our previous review 4 and it with other aspects of Purkinjes will be included here. Macro/Micro anatomy; location, location, location
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During this time, the concept that Purkinje cells ran together in nice, neat bundles was confirmed over and over again as they were used often in the cell experiments. This is to provide rapid synchronized conduction to the ventricles to ensure precise timing of contraction. It was easy to understand that Purkinje fibers would be a source of an arrhythmia since their location is unique; the superior Purkinje fibers are activated before most ventricular muscle. However today we must consider that Purkinje fiber bundles have been identified morphologically in various compartments of the heart. Figure 1A,B show examples of such where the phenotypes of the major Purkinje bundles of the human proximal LV conduction system are compared to those Purkinje fiber bundles found in the Moderator Band(MB) of the RV 5. With modern day electromapping and catheter techniques, we now know that premature ventricular depolarizations can originate from the MB often inducing ventricular fibrillation 6. Further, we know that a thick connective tissue sheath surrounds bundles of Purkinje cells which track into and below that endocardial surface 7 (Figure 1C). In our laboratory, we study these subendocardial canine Purkinje cells since they are important initiators of severe ventricular arrhythmias post MI 8.
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But Purkinje cells are not identified just by location. Their identity must be confirmed at least by histologic and ultrastructural criteria. Histologically, Purkinje cells stain lightly, presumably due to the reduced, but still significant, myofibrillar content and enhanced glycogen. EM studies show that they lack t-tubuli and the all-important cell core dyad 4. Finally, recent immunostaining confirm distinguishable Purkinje cell-to-cell junctions and the existence of the Cx40 protein as an important Purkinje connexin isoform9. Phenotypically distinct Purkinje cells also have proteins that differ from those of ventricular cells (eg. EC coupling proteins 10). In fact, using differential transcriptional profiling techniques, Atkinson et al 11 report significant differences in mRNA abundance in rabbit Purkinje versus Ventricular myocardium. These findings are in keeping with those of Gaborit et al 12 for human Purkinje fibers. We have identified an important marker, contactin-2(CNTN-2), of the murine/canine conduction system 13. The Fishman lab has gone on to use CNTN-2 as a powerful tool to help identify other potentially critical proteins that are in Purkinje cells(PCs) and not in ventricular cells (eg. 14,15). Cellular Electrophysiology; Pacemaker function Differences between PCs and SAN cells. Right and Left PCs
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A brief review of well-known differences in Purkinje and ventricular myocardial action potentials (APs) and currents (eg. IK1) is given in our previous reviews 16,17 (see also 18). At that time, we discussed a fundamental difference between ventricular muscle and Purkinje, in that Purkinje fibers show spontaneous impulse initiation like the pacemaker activity of SAN cells. Involvement of intracellular Ca2+ cycling in Ca2+ wave formation and resultant DADs in Purkinje cells was also discussed 17 but the role of intracellular Ca2+ cycling in pacemaker function of both normal canine Purkinje and SAN cells remains uncertain. Is
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there an interdependence of If and Ca2+ cycling (so called membrane and Ca2+ clocks 19) in these pacemakers of the heart?
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In a recent study, the Robinson and Boyden labs combined energies to look at this question. Canine SAN cells show several phenotypes (Figure 2A). SAN cells from the superior node show both elongated and spider like cells, while those from the inferior node are predominately elongated cells. In addition, there are cell specific differences in the kinetics of the pacemaker current If (Figure 2B), suggesting that the mechanism of the underlying pacemaker activity may differ. Further isoproterenol (ISO) effects on SAN rate are due to effects on If and early diastolic depolarization (EDD) and not the take off potential (TOP) of the action potential (AP) (Figure 2C). Importantly in the presence of ISO SAN rate is sensitive to ryanodine which slows the rate by affecting the TOP 20. Bucchi et al also showed that ryanodine disrupts the beta adrenergic signaling in these cells but the effect was not due to change in If responsiveness to cAMP since ryanodine did not affect the ISO induced SAN rate in the presence of either CPTcAMP or RPcAMP (Figure S1), Bucchi et al therefore predicted that the Ca2+ dependent effect on SAN rate is more proximal, suggesting it is at a Ca2+ sensitive adenylate cyclase cAMP-If interface 21. When canine SAN cells were probed for AC1 (Ca2+ sensitive adenylyl cyclase type 1) and the pacemaker isoform HCN4, an overlap in immunosignal is seen (Figure 3A) suggesting a proximity of the HCN channel and the Ca2+ sensitive AC isoform which would respond to ryanodine by decreasing activity. This overlap was not seen when same was done with AC5/6, the cardiac Ca2+ insensitive Adenylate cyclase.
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In canine Purkinje fibers, there is also a difference in pacemaker rate depending on location of the fiber in heart. Left ventricular Purkinje fiber spontaneous rates are faster than those from the RV 22(Figure S2). Rates are ryanodine sensitive 23,24, and AC1 is present in the single Purkinje cell but does not overlap with HCN4. In fact, there is little immunopositive HCN4 protein in single canine Purkinje cells (Figure 3B). Other isoforms such as HCN1 and HCN2 do show signals in canine Purkinje cells. These two proteins cluster with the wellknown HCN beta subunit, MiRP1 (Figure 4) which differs depending on the chamber origin of single cell. Coexpression of HCN1/HCN2 with MiRP1 enhances and speeds the If current in expression studies25. These results are in agreement with mRNA differences between left and right Purkinje fibers of the rabbit 26 and earlier findings about MiRP1 in Purkinje fibers 27. Finally like SAN cells, some canine Purkinje cells show If and the rate is ivabradine sensitive (Figure 5) but these results must be interpreted carefully due to additional blocking effects of ivabradine 28.
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In sum, major isoforms of the HCN protein differ in mature Purkinje cells versus SAN cells from canine hearts. Second, Ca2+ cycling affects pacemaker activity in both canine SAN and Purkinje cells. In SAN cells, the ryanodine effect is proximal and a possible candidate is a Ca2+ sensitive activated adenylyl cyclase. AC1 is present in specific membrane microdomains and associates with HCN isoforms in both types of pacemaker cells. Further in vitro studies in a model system confirm that introduction of AC1 leads to Ca-dependence of adrenergic modulation of HCN when it is otherwise absent29. We suggest that AC1 as a
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Ca2+ sensitive cyclase could be one central control point of the interdependence of the Ca2+ and membrane clock in both types of pacemaking cells. Triggered Activity; DADs and EADs; role of Ito Triggered activity from delayed after depolarizations(DADs) and early afterdepolarizations(EADs) can both be initiated by traveling Ca2+ waves (see 4). However, not all EADs show this Ca2+ dependence. Some cells with EADs arise due to a change in the balance of inward and outward currents activated with the cell s depolarization 30. Such examples are the theory of Ca2+ current reactivation as suggested by the January lab in 1989 31 and observed by Nattel lab 32, and the upregulation of the L type Ca2+ current into mode 2 gating. Others have suggested that common HERG mutations could lead to membrane oscillations seen upon repolarization (EADs) 33. In these cases, the intracellular Ca2+ changes that occur would be secondary to the membrane oscillations.
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More recently there has been an emphasis on the role of Ito (the transient outward current) in the genesis of EADs in cardiac cells 34. In normal canine Purkinje cells, the transient outward current is very large, rate and age dependent and easily remodeled 35,36,37(Figure S3). It is sensitive to block with 4 aminopyridine which inhibits both the transient and sustained component of the current. This results in less of a Purkinje AP notch. This leads to less repolarizing current and thus the Purkinje cell s membrane voltage comes into a range where EAD oscillations can occur, the so called basin of attraction 34 (Figure 6). Presumably complete block of Ito would suppress these EADs. The Sink Source problem with Purkinje fibers as pacemakers
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The Gelband lab showed that Purkinje fibers run along the endocardial surface and are electrically isolated 38. Thus a source sink problem would exist if cells showed EADs or DADs. For example, it is possible that a cluster of Purkinje cells susceptible to EADs or DADs could be embedded in well coupled tissues. EADs would also be suppressed due to this coupling. How is it then that DADs(or EADs in Purkinje cells) propagate into tissues for extra beats and/or arrhythmia initiation? Recent theoretical studies have shed some light on how Purkinje cells can become initiators of abnormal rhythms 39. They explain that in normal tissues, a substantial number (nearly 700,000) of cells in 3D tissue must synchronize to trigger an extra AP. This number decreases if there is an overall reduced gap conductance(Ggap). (Figure 7). An increase in fibrosis also lowers the number of cells needed to trigger an AP. And finally they show that if an electrically remodeled substrate having reduced IK1 and ICaL exists, then there is a further reduction in the number of cells required for EAD or DAD- induced triggered action potential (Figure 7C). Thus factors surrounding clusters of Purkinje cell tracts must be considered when trying to predict triggerability of these cells from a specific location. Mutations of high impact in Purkinje Based Arrhythmia Combining their unique location and the existence of some proteins that differ from ventricular muscle, Purkinje cells with genetic lesions will have a large impact on cardiac rhythm. One example is seen in the Idiopathic Ventricular Fibrillation (IVF) literature. In one study 5 patients with IVF were studied as they all had short coupled premature beats Heart Rhythm. Author manuscript; available in PMC 2017 May 01.
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that initiated VF 36. During preablation mapping, early signals from Purkinje network were clear at the onset of VF. Ablation at this site was successful. A molecular follow up of these patients showed that a chromosomal haplotype producing overexpressed DDP6 (dipeptidyl peptidase-like protein 6) caused this form of familial VF. Further work suggested that DPP6 was rich in Purkinjes and co expression of DPP6 with Kv4.3 (the alpha subunit of Ito in Purkinje) enhanced the amplitude of the expressed current. Thus, a gain in function in Purkinje cell DDP6 mediates an electrical effect on Purkinje Ito to shorten repolarization setting the stage for arrhythmia.
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A more recent proband has been described with a similar arrhythmia 40 (Figure 8). Here a novel splice form of DPP6, DPP6-T, was identified and its abundance was enhanced in the human heart. This truncated form was found in proband as a variant (p.H332R) and this truncated protein level was elevated. In coexpression work, it was found that the DPP6- T variant increased Kv4.3 currents augmenting Ito(Figure 8). These investigators go on to show the effects of Ito based therapies on this patient s arrhythmia. Importantly, a combination of dalfampridine (4 aminopyridine) and cilostazol (used to increase PKA) reduced VF events >90 fold and ECG has returned to normal. As noted by these authors, the DPP6 variant was originally dismissed due its predicted location in intronic sequence and minor allele frequency (MAF 0.0047 in NHLBI ESP; 0.0010 in 1000 Genomes). These limitations are noted, and in fact the authors suggested that due to the diversity of phenotypes even within the immediate family (proband, mother, uncle) that the specific variant represents a modifier allele requiring additional genetic and/or environmental factors for disease. Further, this case highlights the impact of ‘incomplete penetrance’ for our understanding of cardiac channelopathies. Also noted by the authors, congenital LQTS shows ~40% penetrance while BrS is significantly lower at ~16%. Nevertheless with the knowledge of the specific cell type involved and the underlying genetic lesion, personalized Ito therapy proved to be anti-arrhythmic in this patient. In sum, Purkinje fibers and cells have been studied for years but there is still much to learn. Future work will be involved in devising a way to identify them during electrical mapping, perhaps by involving the proteins that are specific to Purkinje. Other work could be to understand if mutations in arrhythmic probands manifest differently depending on cell type (PC vs ventricular). We need to discover how these important cells remodel at the molecular level. Finally we need to learn how to dissect/study the human Purkinje cell. They may surprise us!
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Refer to Web version on PubMed Central for supplementary material.
Acknowledgments Supported by grant HL114383 from the National Heart Lung and Blood Institute Bethesda, Maryland
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ABBREVIATIONS
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MB
moderator Band
Cx40
Connexin 40
CNTN-2
contactin 2
PCs
Purkinje cells
SAN
sinus node cells
EADs
Early afterdepolarizations
DADs
Delayed afterdepolarizations
ISO
Isoproterenol
EDD
early diastolic depolarization
TOP
take off potential
AC1
Ca2+ sensitive adenylyl cyclase type1
HCN
hyperpolarization activated cyclic nucleotide-gated cation channel protein
MiRP1
mink related peptide
IVF
idiopathic ventricular fibrillation
DPP6
dipeptidyl peptidase-like protein 6
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A: Remarkably similar diagrams of the proximal left-sided conduction system as observed in 20 normal human hearts demonstrating the origin of the anterior, posterior and median fascicles from Syed et al. from [5]. B: Pig moderator band histology demonstrating conduction tissue (Purkinje fibers) lying within the band from Syed et al. [5]. C(a): A tissue block of India ink injected in the LV wall illustrating the most distal components of the conduction system. ED Endocardium, EP epicardium, PF intramyocardial and subendocardial Purkinje fibers. C(b): A tissue block of India ink injected left ventricular wall made transparent, showing the three dimensional (3D) intramyocardial network. Division = 1 mm; C(c): Detail of C(b); ED endocardium side, OF oblique intramyocardial communicating Purkinje fibers, VPF vertical intramyocardial Purkinje fibers. C(d): Histological section showing subendocardial fibers within India ink stained inside fibrous sheath. ED Endocardium, IK India ink, PF subendocardial Purkinje fibers. From De Almedia et al. [7]
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Figure 2.
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A: A variety of phenotypes of canine single SAN cells: elongated (left), spider (right) and spindle (middle). Percentages of different phenotype cells from superior and inferior nodes are summarized below. E/S: elongated or spindle cell. B: Different characteristics of If in elongated/spindle (E/S) versus spider cells. Left: Activation kinetics differ significantly at indicated membrane potentials (*; Bonferoni). Right: Steady-state activation relations differ (ANOVA). C: Modification of cell aggregates of SAN AP parameters by isoprenaline 1 μM (Iso) emphasizing the effects on rate by the effects on EDD. C(a); Dot-plots of time course of action potential parameters during application of Iso (bar). C(b); Sample traces recorded in control and following perfusion with Iso, as indicated (arrows); corresponding takeoff potential (TOP) values plotted (filled squares, control; open squares, Iso). C(c); Cycle-bycycle TOP vs rate plot (upper) and end diastolic depolarization(EDD)vs rate plot (lower) as from corresponding panels in A. From Bucchi et al. [21]
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Figure 3.
Immunofluorescent images of canine SAN(A) and Purkinje cells (B) co-stained with AC1 (Green) and HCN4 (Red). There was intense colocalization of AC1 and HCN4 in single SAN cells, however there was little immuno positive HCN4 protein in Purkinje cells.
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A: Immunofluorescent images of canine Purkinje single cells co-stained with MiRP1 (Green) and HCN2 (Red), demonstrating that MiRP1 colocalizes with HCN2, the major isoform of the pacemaker channel in Purkinje cells B: Immunofluorescent images of the right and left ventricular Purkinje cells staining HCN1 (Left images) and MiRP1 (Right images). The staining of both proteins showed membrane immunofluorescence intensity, suggesting that HCN1 and MiRP1 colocalized on the membrane.
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Figure 5.
Representative If recorded from single canine SAN cells (L. Protas) (A) and Purkinje cells (B). Ivabradine significantly reduced If in SAN cells and decreased rate in both SAN and Purkinje cells, demonstrating that like SAN cells, the canine Purkinje fibers have ivabradinesensitive rate effect (E. Sosunov, E. Anyukhovsky).
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Figure 6.
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Role of Ito in EAD genesis in different species. A: The EAD distribution in the ICa,L and Ito conductance (GCa and Gto) space. The inactivation time constant of the Ito is the intermediate value (th0= 200 ms). EAD occurrence region is marked in grey. B: Single cell APs recorded in different species as indicated (a–d). The black and colored symbols mark the putative locations(including canine Purkinjes) of the APs in the distribution diagram (A). C: Summarized bar graphs showing the incidence of EADs within 20 Aps.. D: The same as (A), except that the inactivation time constant of the Ito is fast (th0= 20 ms), which is similar to that of canine ventricular myocytes. E: APs recorded from canine ventricular myocytes. The symbols mark the putative locations of the APs in the distribution diagram (C). From Zhao et al. [34].
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Figure 7.
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Computational study showing that the number of cells exhibiting EAD or DAD matters. A(a):Schematic of 1D tissue, with central region of EAD/DAD susceptible cells demarcated in red, and unsusceptible cells in blue. A(b): Schematic of 2D tissue, with central elliptical region of EAD-/DAD-susceptible cells demarcated in red. A(c): Schematic of bricklike 2D tissue with fibroblasts (F) randomly interspersed at the ends (I) or sides (II) of the myocytes (M). B(a): Selected AP traces along a 1D cable, with the red traces indicating the EADsusceptible cells in the central region, and the normal unsusceptible cells in black. The EAD failed to propagate with 69 susceptible cells in the central region (left), but propagated successfully with 70 susceptible cells (right). B(b): Same as for B(a), but with the central region exhibiting DAD-susceptible cells. The DAD failed to trigger an AP with 79 susceptible cells in the central region (left), but did so successfully with 80 susceptible cells (right). C: Number of contiguous susceptible myocytes required to trigger an EAD- or DADmediated PVC in simulated 2D tissue with lateral fibrosis. Fibroblasts were randomly interspersed exclusively along the sides of myocytes throughout the entire tissue, with EADor DAD-susceptible myocytes only in the central region. As the fibroblast/myocyte (FM) ratio increased, the required size of the central region (and hence the number of susceptible cells in the central region) progressively decreased, approaching the 1D case at the maximum FM ratio of 5, above which transverse propagation failed. From Xie et al. [39]
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Abnormal repolarization and ventricular fibrillation. A: Presenting ECG of proband with right bundle branch block; J point elevation (arrows)
Heart Rhythm. Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Heart Rhythm. 2016 May ; 13(5): 1172–1181. doi:10.1016/j.hrthm.2016.01.011.
Cardiac Purkinje fibers and Arrhythmias; The GK Moe award Lecture 2015 Penelope A. Boyden, PhD, Wen Dun, MD, PhD, and Richard B. Robinson, PhD Department of Pharmacology, Center for Molecular Therapeutics, Columbia University, New York NY
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Abstract Purkinje fibers/cells continue to be a focus of arrhythmologists. Here we review several new ideas that have emerged in the literature and fold them into important new points. These points include some proteins in Purkinje cells that are specific to Purkinjes, pacemaker function in Purkinje may be similar to that of the sinus node cell, sink-source concerns about tracts/sheets of Purkinje fibers, role of Ito in arrhythmias and genetic lesions in Purkinjes and their high impact on cardiac rhythm. Although new ideas about the remodeled Purkinje cell are not the focus of this review, one can easily imagine how Purkinjes and their function may be altered in diseased hearts.
Keywords Purkinje; arrhythmias; pacemaker activity; calcium; ion channels
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Introduction
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Despite the careful descriptions of Purkinje fibers by the Czech Jan Purkinje (1787–1869) cardiac cellular Electrophysiology was still in its infancy in late 1960 s and early 1970 s. Fine tipped microelectrodes and cardiac preparations of Purkinje fibers were the most commonly used experimental approaches. In fact, C. Mendez and Gordon K. Moe published several papers about the electrical properties of canine Purkinjes within the Purkinje-Muscle Junction (eg.1). Several hundred miles south of the Utica NY Moe lab, BF Hoffman would suggest that enhanced depolarizations in Purkinje cells might be a significant factor in human arrhythmias 2. This led to an intense effort to develop the single cell preparation of canine Purkinje fibers dissected from free running bundles 3. After all the idea was simple, by using a single Purkinje cell with whole cell voltage clamp techniques, one could understand the ionic currents flowing during Purkinje pacemaker activity. The problem was that the single Purkinje cell from a normal heart did not show spontaneous impulse
Address for Correspondence: Dr. Penelope A. Boyden, Dept of Pharmacology, Columbia College of Physicians and Surgeons, 630 West 168th ST., New York New York 10032, 212-305-7907 (phone) There are no potential conflicts of interest. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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generation. This has been discussed in our previous review 4 and it with other aspects of Purkinjes will be included here. Macro/Micro anatomy; location, location, location
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During this time, the concept that Purkinje cells ran together in nice, neat bundles was confirmed over and over again as they were used often in the cell experiments. This is to provide rapid synchronized conduction to the ventricles to ensure precise timing of contraction. It was easy to understand that Purkinje fibers would be a source of an arrhythmia since their location is unique; the superior Purkinje fibers are activated before most ventricular muscle. However today we must consider that Purkinje fiber bundles have been identified morphologically in various compartments of the heart. Figure 1A,B show examples of such where the phenotypes of the major Purkinje bundles of the human proximal LV conduction system are compared to those Purkinje fiber bundles found in the Moderator Band(MB) of the RV 5. With modern day electromapping and catheter techniques, we now know that premature ventricular depolarizations can originate from the MB often inducing ventricular fibrillation 6. Further, we know that a thick connective tissue sheath surrounds bundles of Purkinje cells which track into and below that endocardial surface 7 (Figure 1C). In our laboratory, we study these subendocardial canine Purkinje cells since they are important initiators of severe ventricular arrhythmias post MI 8.
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But Purkinje cells are not identified just by location. Their identity must be confirmed at least by histologic and ultrastructural criteria. Histologically, Purkinje cells stain lightly, presumably due to the reduced, but still significant, myofibrillar content and enhanced glycogen. EM studies show that they lack t-tubuli and the all-important cell core dyad 4. Finally, recent immunostaining confirm distinguishable Purkinje cell-to-cell junctions and the existence of the Cx40 protein as an important Purkinje connexin isoform9. Phenotypically distinct Purkinje cells also have proteins that differ from those of ventricular cells (eg. EC coupling proteins 10). In fact, using differential transcriptional profiling techniques, Atkinson et al 11 report significant differences in mRNA abundance in rabbit Purkinje versus Ventricular myocardium. These findings are in keeping with those of Gaborit et al 12 for human Purkinje fibers. We have identified an important marker, contactin-2(CNTN-2), of the murine/canine conduction system 13. The Fishman lab has gone on to use CNTN-2 as a powerful tool to help identify other potentially critical proteins that are in Purkinje cells(PCs) and not in ventricular cells (eg. 14,15). Cellular Electrophysiology; Pacemaker function Differences between PCs and SAN cells. Right and Left PCs
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A brief review of well-known differences in Purkinje and ventricular myocardial action potentials (APs) and currents (eg. IK1) is given in our previous reviews 16,17 (see also 18). At that time, we discussed a fundamental difference between ventricular muscle and Purkinje, in that Purkinje fibers show spontaneous impulse initiation like the pacemaker activity of SAN cells. Involvement of intracellular Ca2+ cycling in Ca2+ wave formation and resultant DADs in Purkinje cells was also discussed 17 but the role of intracellular Ca2+ cycling in pacemaker function of both normal canine Purkinje and SAN cells remains uncertain. Is
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there an interdependence of If and Ca2+ cycling (so called membrane and Ca2+ clocks 19) in these pacemakers of the heart?
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In a recent study, the Robinson and Boyden labs combined energies to look at this question. Canine SAN cells show several phenotypes (Figure 2A). SAN cells from the superior node show both elongated and spider like cells, while those from the inferior node are predominately elongated cells. In addition, there are cell specific differences in the kinetics of the pacemaker current If (Figure 2B), suggesting that the mechanism of the underlying pacemaker activity may differ. Further isoproterenol (ISO) effects on SAN rate are due to effects on If and early diastolic depolarization (EDD) and not the take off potential (TOP) of the action potential (AP) (Figure 2C). Importantly in the presence of ISO SAN rate is sensitive to ryanodine which slows the rate by affecting the TOP 20. Bucchi et al also showed that ryanodine disrupts the beta adrenergic signaling in these cells but the effect was not due to change in If responsiveness to cAMP since ryanodine did not affect the ISO induced SAN rate in the presence of either CPTcAMP or RPcAMP (Figure S1), Bucchi et al therefore predicted that the Ca2+ dependent effect on SAN rate is more proximal, suggesting it is at a Ca2+ sensitive adenylate cyclase cAMP-If interface 21. When canine SAN cells were probed for AC1 (Ca2+ sensitive adenylyl cyclase type 1) and the pacemaker isoform HCN4, an overlap in immunosignal is seen (Figure 3A) suggesting a proximity of the HCN channel and the Ca2+ sensitive AC isoform which would respond to ryanodine by decreasing activity. This overlap was not seen when same was done with AC5/6, the cardiac Ca2+ insensitive Adenylate cyclase.
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In canine Purkinje fibers, there is also a difference in pacemaker rate depending on location of the fiber in heart. Left ventricular Purkinje fiber spontaneous rates are faster than those from the RV 22(Figure S2). Rates are ryanodine sensitive 23,24, and AC1 is present in the single Purkinje cell but does not overlap with HCN4. In fact, there is little immunopositive HCN4 protein in single canine Purkinje cells (Figure 3B). Other isoforms such as HCN1 and HCN2 do show signals in canine Purkinje cells. These two proteins cluster with the wellknown HCN beta subunit, MiRP1 (Figure 4) which differs depending on the chamber origin of single cell. Coexpression of HCN1/HCN2 with MiRP1 enhances and speeds the If current in expression studies25. These results are in agreement with mRNA differences between left and right Purkinje fibers of the rabbit 26 and earlier findings about MiRP1 in Purkinje fibers 27. Finally like SAN cells, some canine Purkinje cells show If and the rate is ivabradine sensitive (Figure 5) but these results must be interpreted carefully due to additional blocking effects of ivabradine 28.
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In sum, major isoforms of the HCN protein differ in mature Purkinje cells versus SAN cells from canine hearts. Second, Ca2+ cycling affects pacemaker activity in both canine SAN and Purkinje cells. In SAN cells, the ryanodine effect is proximal and a possible candidate is a Ca2+ sensitive activated adenylyl cyclase. AC1 is present in specific membrane microdomains and associates with HCN isoforms in both types of pacemaker cells. Further in vitro studies in a model system confirm that introduction of AC1 leads to Ca-dependence of adrenergic modulation of HCN when it is otherwise absent29. We suggest that AC1 as a
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Ca2+ sensitive cyclase could be one central control point of the interdependence of the Ca2+ and membrane clock in both types of pacemaking cells. Triggered Activity; DADs and EADs; role of Ito Triggered activity from delayed after depolarizations(DADs) and early afterdepolarizations(EADs) can both be initiated by traveling Ca2+ waves (see 4). However, not all EADs show this Ca2+ dependence. Some cells with EADs arise due to a change in the balance of inward and outward currents activated with the cell s depolarization 30. Such examples are the theory of Ca2+ current reactivation as suggested by the January lab in 1989 31 and observed by Nattel lab 32, and the upregulation of the L type Ca2+ current into mode 2 gating. Others have suggested that common HERG mutations could lead to membrane oscillations seen upon repolarization (EADs) 33. In these cases, the intracellular Ca2+ changes that occur would be secondary to the membrane oscillations.
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More recently there has been an emphasis on the role of Ito (the transient outward current) in the genesis of EADs in cardiac cells 34. In normal canine Purkinje cells, the transient outward current is very large, rate and age dependent and easily remodeled 35,36,37(Figure S3). It is sensitive to block with 4 aminopyridine which inhibits both the transient and sustained component of the current. This results in less of a Purkinje AP notch. This leads to less repolarizing current and thus the Purkinje cell s membrane voltage comes into a range where EAD oscillations can occur, the so called basin of attraction 34 (Figure 6). Presumably complete block of Ito would suppress these EADs. The Sink Source problem with Purkinje fibers as pacemakers
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The Gelband lab showed that Purkinje fibers run along the endocardial surface and are electrically isolated 38. Thus a source sink problem would exist if cells showed EADs or DADs. For example, it is possible that a cluster of Purkinje cells susceptible to EADs or DADs could be embedded in well coupled tissues. EADs would also be suppressed due to this coupling. How is it then that DADs(or EADs in Purkinje cells) propagate into tissues for extra beats and/or arrhythmia initiation? Recent theoretical studies have shed some light on how Purkinje cells can become initiators of abnormal rhythms 39. They explain that in normal tissues, a substantial number (nearly 700,000) of cells in 3D tissue must synchronize to trigger an extra AP. This number decreases if there is an overall reduced gap conductance(Ggap). (Figure 7). An increase in fibrosis also lowers the number of cells needed to trigger an AP. And finally they show that if an electrically remodeled substrate having reduced IK1 and ICaL exists, then there is a further reduction in the number of cells required for EAD or DAD- induced triggered action potential (Figure 7C). Thus factors surrounding clusters of Purkinje cell tracts must be considered when trying to predict triggerability of these cells from a specific location. Mutations of high impact in Purkinje Based Arrhythmia Combining their unique location and the existence of some proteins that differ from ventricular muscle, Purkinje cells with genetic lesions will have a large impact on cardiac rhythm. One example is seen in the Idiopathic Ventricular Fibrillation (IVF) literature. In one study 5 patients with IVF were studied as they all had short coupled premature beats Heart Rhythm. Author manuscript; available in PMC 2017 May 01.
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that initiated VF 36. During preablation mapping, early signals from Purkinje network were clear at the onset of VF. Ablation at this site was successful. A molecular follow up of these patients showed that a chromosomal haplotype producing overexpressed DDP6 (dipeptidyl peptidase-like protein 6) caused this form of familial VF. Further work suggested that DPP6 was rich in Purkinjes and co expression of DPP6 with Kv4.3 (the alpha subunit of Ito in Purkinje) enhanced the amplitude of the expressed current. Thus, a gain in function in Purkinje cell DDP6 mediates an electrical effect on Purkinje Ito to shorten repolarization setting the stage for arrhythmia.
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A more recent proband has been described with a similar arrhythmia 40 (Figure 8). Here a novel splice form of DPP6, DPP6-T, was identified and its abundance was enhanced in the human heart. This truncated form was found in proband as a variant (p.H332R) and this truncated protein level was elevated. In coexpression work, it was found that the DPP6- T variant increased Kv4.3 currents augmenting Ito(Figure 8). These investigators go on to show the effects of Ito based therapies on this patient s arrhythmia. Importantly, a combination of dalfampridine (4 aminopyridine) and cilostazol (used to increase PKA) reduced VF events >90 fold and ECG has returned to normal. As noted by these authors, the DPP6 variant was originally dismissed due its predicted location in intronic sequence and minor allele frequency (MAF 0.0047 in NHLBI ESP; 0.0010 in 1000 Genomes). These limitations are noted, and in fact the authors suggested that due to the diversity of phenotypes even within the immediate family (proband, mother, uncle) that the specific variant represents a modifier allele requiring additional genetic and/or environmental factors for disease. Further, this case highlights the impact of ‘incomplete penetrance’ for our understanding of cardiac channelopathies. Also noted by the authors, congenital LQTS shows ~40% penetrance while BrS is significantly lower at ~16%. Nevertheless with the knowledge of the specific cell type involved and the underlying genetic lesion, personalized Ito therapy proved to be anti-arrhythmic in this patient. In sum, Purkinje fibers and cells have been studied for years but there is still much to learn. Future work will be involved in devising a way to identify them during electrical mapping, perhaps by involving the proteins that are specific to Purkinje. Other work could be to understand if mutations in arrhythmic probands manifest differently depending on cell type (PC vs ventricular). We need to discover how these important cells remodel at the molecular level. Finally we need to learn how to dissect/study the human Purkinje cell. They may surprise us!
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Refer to Web version on PubMed Central for supplementary material.
Acknowledgments Supported by grant HL114383 from the National Heart Lung and Blood Institute Bethesda, Maryland
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ABBREVIATIONS
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MB
moderator Band
Cx40
Connexin 40
CNTN-2
contactin 2
PCs
Purkinje cells
SAN
sinus node cells
EADs
Early afterdepolarizations
DADs
Delayed afterdepolarizations
ISO
Isoproterenol
EDD
early diastolic depolarization
TOP
take off potential
AC1
Ca2+ sensitive adenylyl cyclase type1
HCN
hyperpolarization activated cyclic nucleotide-gated cation channel protein
MiRP1
mink related peptide
IVF
idiopathic ventricular fibrillation
DPP6
dipeptidyl peptidase-like protein 6
Reference List Author Manuscript Author Manuscript
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A: Remarkably similar diagrams of the proximal left-sided conduction system as observed in 20 normal human hearts demonstrating the origin of the anterior, posterior and median fascicles from Syed et al. from [5]. B: Pig moderator band histology demonstrating conduction tissue (Purkinje fibers) lying within the band from Syed et al. [5]. C(a): A tissue block of India ink injected in the LV wall illustrating the most distal components of the conduction system. ED Endocardium, EP epicardium, PF intramyocardial and subendocardial Purkinje fibers. C(b): A tissue block of India ink injected left ventricular wall made transparent, showing the three dimensional (3D) intramyocardial network. Division = 1 mm; C(c): Detail of C(b); ED endocardium side, OF oblique intramyocardial communicating Purkinje fibers, VPF vertical intramyocardial Purkinje fibers. C(d): Histological section showing subendocardial fibers within India ink stained inside fibrous sheath. ED Endocardium, IK India ink, PF subendocardial Purkinje fibers. From De Almedia et al. [7]
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Figure 2.
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A: A variety of phenotypes of canine single SAN cells: elongated (left), spider (right) and spindle (middle). Percentages of different phenotype cells from superior and inferior nodes are summarized below. E/S: elongated or spindle cell. B: Different characteristics of If in elongated/spindle (E/S) versus spider cells. Left: Activation kinetics differ significantly at indicated membrane potentials (*; Bonferoni). Right: Steady-state activation relations differ (ANOVA). C: Modification of cell aggregates of SAN AP parameters by isoprenaline 1 μM (Iso) emphasizing the effects on rate by the effects on EDD. C(a); Dot-plots of time course of action potential parameters during application of Iso (bar). C(b); Sample traces recorded in control and following perfusion with Iso, as indicated (arrows); corresponding takeoff potential (TOP) values plotted (filled squares, control; open squares, Iso). C(c); Cycle-bycycle TOP vs rate plot (upper) and end diastolic depolarization(EDD)vs rate plot (lower) as from corresponding panels in A. From Bucchi et al. [21]
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Figure 3.
Immunofluorescent images of canine SAN(A) and Purkinje cells (B) co-stained with AC1 (Green) and HCN4 (Red). There was intense colocalization of AC1 and HCN4 in single SAN cells, however there was little immuno positive HCN4 protein in Purkinje cells.
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A: Immunofluorescent images of canine Purkinje single cells co-stained with MiRP1 (Green) and HCN2 (Red), demonstrating that MiRP1 colocalizes with HCN2, the major isoform of the pacemaker channel in Purkinje cells B: Immunofluorescent images of the right and left ventricular Purkinje cells staining HCN1 (Left images) and MiRP1 (Right images). The staining of both proteins showed membrane immunofluorescence intensity, suggesting that HCN1 and MiRP1 colocalized on the membrane.
Author Manuscript Heart Rhythm. Author manuscript; available in PMC 2017 May 01.
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Figure 5.
Representative If recorded from single canine SAN cells (L. Protas) (A) and Purkinje cells (B). Ivabradine significantly reduced If in SAN cells and decreased rate in both SAN and Purkinje cells, demonstrating that like SAN cells, the canine Purkinje fibers have ivabradinesensitive rate effect (E. Sosunov, E. Anyukhovsky).
Author Manuscript Author Manuscript Heart Rhythm. Author manuscript; available in PMC 2017 May 01.
Boyden et al.
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Figure 6.
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Role of Ito in EAD genesis in different species. A: The EAD distribution in the ICa,L and Ito conductance (GCa and Gto) space. The inactivation time constant of the Ito is the intermediate value (th0= 200 ms). EAD occurrence region is marked in grey. B: Single cell APs recorded in different species as indicated (a–d). The black and colored symbols mark the putative locations(including canine Purkinjes) of the APs in the distribution diagram (A). C: Summarized bar graphs showing the incidence of EADs within 20 Aps.. D: The same as (A), except that the inactivation time constant of the Ito is fast (th0= 20 ms), which is similar to that of canine ventricular myocytes. E: APs recorded from canine ventricular myocytes. The symbols mark the putative locations of the APs in the distribution diagram (C). From Zhao et al. [34].
Author Manuscript Heart Rhythm. Author manuscript; available in PMC 2017 May 01.
Boyden et al.
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Figure 7.
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Computational study showing that the number of cells exhibiting EAD or DAD matters. A(a):Schematic of 1D tissue, with central region of EAD/DAD susceptible cells demarcated in red, and unsusceptible cells in blue. A(b): Schematic of 2D tissue, with central elliptical region of EAD-/DAD-susceptible cells demarcated in red. A(c): Schematic of bricklike 2D tissue with fibroblasts (F) randomly interspersed at the ends (I) or sides (II) of the myocytes (M). B(a): Selected AP traces along a 1D cable, with the red traces indicating the EADsusceptible cells in the central region, and the normal unsusceptible cells in black. The EAD failed to propagate with 69 susceptible cells in the central region (left), but propagated successfully with 70 susceptible cells (right). B(b): Same as for B(a), but with the central region exhibiting DAD-susceptible cells. The DAD failed to trigger an AP with 79 susceptible cells in the central region (left), but did so successfully with 80 susceptible cells (right). C: Number of contiguous susceptible myocytes required to trigger an EAD- or DADmediated PVC in simulated 2D tissue with lateral fibrosis. Fibroblasts were randomly interspersed exclusively along the sides of myocytes throughout the entire tissue, with EADor DAD-susceptible myocytes only in the central region. As the fibroblast/myocyte (FM) ratio increased, the required size of the central region (and hence the number of susceptible cells in the central region) progressively decreased, approaching the 1D case at the maximum FM ratio of 5, above which transverse propagation failed. From Xie et al. [39]
Author Manuscript Heart Rhythm. Author manuscript; available in PMC 2017 May 01.
Boyden et al.
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Author Manuscript Author Manuscript Figure 8.
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Abnormal repolarization and ventricular fibrillation. A: Presenting ECG of proband with right bundle branch block; J point elevation (arrows)
