Articles in PresS. Am J Physiol Heart Circ Physiol (January 10, 2014). doi:10.1152/ajpheart.00760.2013
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Title: Mechanisms of Cardiac Conduction: A History of Revisions Authors: Rengasayee Veeraraghavan Ph.D,1 Robert Gourdie Ph.D,1,2 Steven Poelzing Ph.D1,2 Affiliations: 1 Virginia Tech Carilion Research Institute, and Center for Heart and Regenerative Medicine, Virginia Polytechnic University, Roanoke, VA 2 School of Biomedical Engineering and Sciences, Virginia Polytechnic University, Blacksburg, VA
Short Title: Cardiac Conduction
21 22 23 24
Address correspondence to: Steven Poelzing, Ph.D. Virginia Tech Carilion Research Institute 2 Riverside Circle Roanoke, VA 24016 TEL: (540) 526-2108 FAX: (540) 985-3373 e-mail:
[email protected]
Copyright © 2014 by the American Physiological Society.
2 Mechanisms of Cardiac Conduction: A History of Revisions
25 26 27
Abstract
28
Cardiac conduction is the process by which electrical excitation spreads
29
through the heart, triggering individual myocytes to contract in synchrony.
30
Defects in conduction disrupt synchronous activation and are associated with life-
31
threatening arrhythmias in many pathologies. Therefore, it is scarcely surprising
32
that this phenomenon continues to be the subject of active scientific inquiry. Here
33
we provide a brief review of how the conceptual understanding of conduction has
34
evolved over the last century and highlight recent, potentially paradigm-shifting
35
developments.
36 37
Background
38
Cardiac conduction is the process by which electrical activation is
39
communicated between myocytes, triggering their synchronous contraction.
40
Impulses originating in the sinoatrial node spread to the atria and via the
41
specialized His-Purkinje conduction system to the ventricles. The sequence of
42
activation thus achieved is key in translating the lengthwise contraction of
43
individual myocytes into the complex three-dimensional pumping motion of the
44
heart.
45
It is well established that aberrant ventricular conduction is associated with
46
a high risk of sudden cardiac death, presumably due to ventricular
47
arrhythmias.(67)
The
prevention
of
arrhythmias
caused
by
conduction
3 48
abnormalities remains a topic of intense research in part because there are many
49
factors that are thought to govern cardiac conduction. This review will focus on
50
canonical determinants of conduction -- cellular excitability, gap junctions and
51
tissue architecture in the context of the historical development of the theoretical
52
understanding of conduction in the ventricular myocardium. Additionally, long
53
theorized and new experimental evidence will be discussed concerning
54
alternative modes of action potential propagation from myocyte-to-myocyte.
55 56
The study of conduction
57
Dr. Theodor Wilhelm Engelmann is credited with determining in 1875 that
58
electrical activity in strips of frog atrial muscle spread through muscular
59
tissue.(27) It took 39 years before the first direct measurements of cardiac
60
conduction velocity were reported, when Dr. Thomas Lewis and his colleagues
61
first quantified the velocity of cardiac conduction from canine myocardium.(61)
62
Conduction velocity is still the primary metric for quantifying the spread of
63
electrical activity in cardiac muscle in part because of its conceptual simplicity
64
and the ease of measuring the time it takes for the electrical wavefront to travel a
65
known distance. More importantly, velocity as a metric of conduction has yielded
66
insights into the mechanisms of electrical activity spread, as conduction velocity
67
is not uniform in all directions from the point of stimulation. This issue of
68
direction-specific conduction spread has provided the foundation of scientific
69
inquiry and debate. But before researchers could even begin to understand the
70
biophysical mechanisms governing cardiac conduction velocity, they first had to
4 71
determine what caused the action potential, and to do that, the determinants of
72
tissue excitability had to be understood.
73 74
Tissue excitability
75
The definition of the term excitability has evolved with our understanding
76
of the mechanisms underlying this phenomenon. For the purpose of this review,
77
it is convenient to think of excitability in terms of the probability that a propagated
78
action potential will be triggered in response to some quantity of charge entering
79
a cell over a period of time. In neurons, Drs. Wallace Fenn and Doris Cobb(28)
80
suggested in 1936 that a propagated action potential was caused by sodium ions
81
entering an excitable cell and depolarizing the membrane. Subsequently, and
82
perhaps more famously, Drs. Alan Lloyd Hodgkin, Andrew Huxley and Bernard
83
Katz published a series of manuscripts on the topic of neuronal excitation.(39-46)
84
Finally, Drs. Alan Lloyd Hodgkin and Paul Horowicz demonstrated that
85
excitability in cardiac myocytes is based on similar mechanisms of sodium
86
entry.(38) These early studies, demonstrated that sodium conductance through
87
the cell membrane is very low when the cell is electrically quiescent, but
88
increases dramatically during the depolarization phase of the action potential.
89
These and other findings prompted Hodgkin and Huxley to suggest that there
90
may be specialized “pores” or channels through which Na+ permeates the cell
91
membrane.(44) In 1964, Dr. Toshio Narahashi and colleagues reported on the
92
sodium current blocking properties of tetrodotoxin,(72) and the use of
93
pharmacological blockers to elucidate properties of the sodium current began in
5 94
earnest. This line of investigation culminated in the landmark 1976 paper by Drs.
95
Erwin Neher and Bert Sackmann’s, in which currents were recorded from single
96
ion channels,(73)
97
voltage-gated sodium current (Nav1.5)(30) responsible for depolarizing the
98
preponderance of cardiac myocytes. Overall, it is now well-accepted that sodium
99
channel availability is an important determinant of cellular excitability.(95)
and finally, the identification of the cardiac isoform of the
100 101
Potassium Channels and Excitability
102
Potassium channels provide outward current that acts to set the resting
103
membrane potential and repolarize the membrane when it is depolarized. Thus,
104
these channels shape the action potential after the upstroke and their relevance
105
to conduction has been thought to be limited to an influence on resting
106
membrane potential. Under pathological conditions such as following ischemia,
107
changes in resting membrane potential are determined by potassium currents,
108
particularly the inward-rectifier current IK1 and the ATP-sensitive potassium
109
current IKATP. Since the resting membrane potential is a key determinant of
110
sodium channel availability, potassium currents have a significant influence on
111
conduction under such conditions. However, in recent years it has been
112
demonstrated that modulating potassium currents can also affect conduction
113
velocity and its dependence on the sodium current, independent of changes in
114
resting membrane potential: Partial inhibition of IK1 can speed up conduction
115
under normal physiological conditions but not when sodium channel availability is
116
compromised.(119) On the other hand, pharmacological activation of IKATP and
6 117
the slow component of the delayed-rectifier potassium current IKs both slow
118
conduction under normal physiological conditions; however, only the latter slows
119
conduction when sodium channel availability is reduced.(118) Overall, these
120
findings suggest that modulating voltage-dependent K+ currents affects
121
conduction independent of Na+ channel availability, whereas modulating K+
122
currents that do not display voltage-dependent kinetics only affect conduction
123
when Na+ channel availability is not reduced.
124
Further, ongoing studies suggest a co-dependence between the
125
membrane expression levels of inward rectifier potassium channels (Kir2.1) and
126
sodium channels (Nav1.5).(68) An additional line of evidence suggesting an
127
interrelationship between the two channel types comes from the recently
128
identified long QT syndrome type 9, where mutations in the scaffolding protein
129
caveolin-3 was associated with alterations in the biophysical properties of both
130
Kir2.1 and Nav1.5 channels. It is therefore likely that potassium channels will
131
continue to be a significant area of research focus in coming years.
132 133
Early Revisions and Models of Conduction
134
Cable Theory
135
In 1952 Dr. Silvio Weidmann demonstrated that electrical communication
136
between cardiac myocytes could be described using a theory first developed in
137
the 19th century by William Thomson (aka Lord Kelvin) to account for the
138
transmission of electrical signals through a transatlantic telegraph cable.(113,
139
114) Importantly, the application of what is known as cable or the continuous
7 140
core conductor theory supposes that myocardial tissue is a syncytium coupled
141
through purely resistive pathways. While the theory was initially applied to
142
understand action potential propagation through nerves, which are readily
143
conceived as a core of conductive material surrounded by a non-conductive
144
sheath, Weidman extended these treatments to understand electrical impulse
145
propagation
146
structure.(124)
through
cardiac
Purkinje
fibers,
which
are
cable-like
in
147
Briefly, for a fiber of radius a with intracellular resistance per unit length ri
148
and extracellular resistance per unit length re bounded by a membrane with
149
resistance per unit length rm and capacitance per unit length cm, the
150
transmembrane potential vm is can be described by:
151 rm ∂ 2 v m ∂v − c m rm m − v m = 0 2 ri + re ∂x ∂t
152 153 154
By extension, it was demonstrated that conduction velocity is related to ri and re
155
by:
156 157
θ=
k ri + re
158 159
where k is a constant representing membrane properties.(101) In the heart, ri and
160
re are determined by various anatomical structures that compose the electric
161
current path inside, outside and between myocytes.
8 162 163
Gap junctions
164
Up until 1954, many theorized that cytoplasmic continuity between
165
myocytes, but then Drs. Fritiof S. Sjostrand and Ebba Andersson showed by use
166
of electron microscopy that myocytes are fully bounded by a membrane.(96)
167
Therefore, the physical nature of electrical connectivity between cells remained
168
speculative. In the 1960’s, Dr. Lloyd Barr demonstrated the existence of low
169
resistance pathways between cardiac myocytes(5, 21), which he termed the
170
nexus. These structures have come to be known more widely as gap junctions,
171
the name having been coined by Drs. Milton W. Brightman and Thomas S.
172
Reese in 1969.(10)
173
Since their discovery, gap junctions have been intensely studied, with over
174
14,000 publications in the literature as of 2013. Each gap junction channel
175
consists of two hexameric connexon hemichannels, each in turn composed of 6
176
molecules from the connexin protein family.(97)
177
connexins 40, 43 and 45 are expressed(8, 9, 53) (122) which have different
178
conductances, permeabilities and cardiac tissue-specific expression patterns(15,
179
35). Further, different connexin isoforms can combine to form heterotypic and
180
heteromultimeric gap junctions which demonstrate composition-dependent
181
properties.(25, 69) However, the situation in mammalian ventricular myocardium
182
is somewhat simplified by the fact that it predominantly expresses connexin 43
183
(Cx43). Moreover, in the ventricle of humans, and many other mammals, Cx43 is
In the heart, three isoforms
9 184
almost exclusively localized to the intercalated disks(34). Thus myocytes are
185
coupled by gap junction channels in primarily end-to-end fashion.(49)
186
Since the majority of early conduction measurements in myocardial tissue
187
were performed macroscopically on Purkinje fibers, which resemble a cable, it is
188
unsurprising that cable theory continued to fit the available data well. To
189
reconcile the discovery of gap junctions with what appeared to be continuous
190
conduction, many deemed gap junctional conductance to be sufficiently high as
191
to render the cytoplasms of coupled myocytes electrically contiguous.(123, 125)
192
Gap junctional conductance was therefore, often incorporated into the
193
intracellular resistance term ri.
194 195
Anisotropic conduction
196
Although thought to be a true syncitium through the first half of the 20th
197
century, ultrastructural studies in the 1950s revealed the cellular nature of
198
cardiac muscle.(78, 81, 96) Atrial and ventricular myocardium came to be seen
199
as brick-wall-like structures composed of myocytes 100-150µm long and 10-
200
20µm wide.(19) Therefore, cardiac tissue is anisotropic, with lengthwise
201
orientation of cardiac myocytes and predominantly end-to-end gap junctional
202
coupling(49). This tissue architecture suggests that cardiac conduction should be
203
different parallel to the long axis relative to the short axis of myocytes. Drs. J.
204
Walter Woodbury and Wayne E. Crill showed in the late 1950’s that myocardium
205
exhibited a direction-dependent spatial decay of current injected into a point of
206
myocardium, with the longest decay, or space-constant, occurring parallel to the
10 207
long axis of moycytes and the shortest decay occurring perpendicular to
208
myocytes.(16) Continuous cable theory was quickly updated to 2 and 3-
209
dimensional models of anisotropic tissue to incorporate the vectors of directional
210
resistivity both inside (ri) and outside (re) cells.(50, 74, 75) From here, these
211
models as a group came to be referred to as ‘bidomain models’ and predicted
212
that cardiac conduction velocity in 2- and 3- dimensional tissue should be
213
anisotropic. By assuming that cardiac myocardium is a continuous but
214
anisotropic medium, Dr. L. Clerc demonstrated that conduction velocity parallel
215
and transverse to fibers is predicted by an inverse square relationship to total
216
axial resistance, similar to what would be anticipated from cable theory.(12)
217
Microelectrode recordings of conduction velocity from multiple sites of myocardial
218
tissue agreed well with these mathematical treatments, and therefore, anisotropic
219
conduction was linked to fiber orientation.(22, 93) In this context, it is important to
220
note that the continuous anisotropic resistivity envisaged by bidomain models is
221
a theoretical approximation. In tissue, end-to-end contacts between myocytes
222
can mediate both longitudinal and transverse coupling patterns.
223 224
Discontinuous Conduction
225
As technology improved to allow for electrical measurements at greater
226
spatial and temporal resolution, studies in the 1970’s revealed yet more
227
complications with the understanding of cardiac conduction. Were the
228
myocardium to behave as a syncytium, the time constant of slow depolarization
229
preceding sodium channel activation (τfoot) should depend on only axial and
11 230
membrane resistance and therefore, be independent of conduction velocity (θ).
231
The model also predicted that the maximal rate of rise of the transmembrane
232
potential (dV/dtmax) should depend only on sodium channel availability; therefore,
233
faster conduction should be associated with a larger dV/dtmax.(100) However,
234
ground-breaking microscopic measurements made by Dr. Madison Spach and
235
colleagues in both atrial and ventricular myocardium revealed a very different
236
picture: Longitudinal conduction, which is faster, was associated with a longer
237
τfoot and smaller dV/dtmax, relative to slower transverse conduction. (105, 106)
238
These experiments began to call into question the use of models based on
239
continuous cable theory to describe anisotropic conduction.
240
In order to explain these new discrepancies between measurements made
241
in tissue and those mathematically modeled, the concept of discontinuous
242
conduction was proposed since gap junctions may represent high resistance
243
pathways between myocytes, rather than the low resistance structures previously
244
assumed. In fact, direct measurements of gap junctional resistance revealed that
245
resistance at the intercalated disks is approximately equal to the axial resistance
246
of a myocyte.(87) Importantly, discontinuous conduction suggested that
247
conduction transverse to the myocyte axis might be slower and also more
248
discontinuous relative to conduction parallel to myocytes, and further
249
experimental evidence supported this assessment.(100, 106)
250
The development of discontinuous conduction theory needed to account
251
for a more complex type of axial resistance that included the following important
252
parameters. First, the mechanisms underlying the resistance of the intercalated
12 253
disks needed to be explored since the number and function of gap junctions can
254
dramatically affect gap junctional conductance. Second, a conduction wavefront
255
will encounter more discontinuities caused by gap junctions when it travels
256
transverse to myocytes relative to their longitudinal axis, as myocytes are shorter
257
than they are long. Since myocytes organize in a roughly brick-like structure in a
258
sheet or bundle, and the orientation of these sheets and bundles maintain a 3-
259
dimensional geometry that is optimized for the efficient propulsion of blood,
260
tissue geometry could not be treated as a simple 2 or 3-dimensional sheet. (101)
261
Case-in-point, myocyte orientation changes from the epicardium to the
262
endocardium,(60, 80, 110) and this complex rotational anisotropy has been
263
shown
264
conduction.(111)
to
produce
important
differences
when
measuring
cardiac
265 266
Contemporary Understanding of Anisotropic Conduction
267
Myocyte geometry
268
Cardiac myocytes do not maintain the same size and geometry over an
269
organism’s lifetime.(63, 103) Changes in cell size and composition can alter
270
cytoplasmic and extracellular resistances in direction-dependent manners
271
leading to altered conduction anisotropy(92, 106) (the ratio of longitudinal to
272
transverse conduction velocities). Once again, changes in myocyte geometry will
273
also alter the number of gap junctions encountered by the electrical wavefront
274
over a given distance.(58, 87, 89) Additionally, cellular hypertrophy over
275
postnatal life is accompanied by changes in GJ localization from uniform
13 276
distribution around the cell to predominantly intercalated disk localization(3, 36) -
277
further confounding the problem of understanding growth-related changes in
278
conduction.
279 280
It is noteworthy though that there remain open questions regarding the
281
relationship between cell size and conduction velocity. Some in silico studies
282
have suggested a positive correlation between cell size and conduction
283
velocity,(32, 103) while experiments in hypertrophied myocardium have
284
suggested a negative correlation between myocyte diameter and conduction
285
velocity.(65) A combined experimental and simulation study by Dr. Rob F.
286
Wiegerinck and colleagues found increased longitudinal, but not transverse
287
conduction velocity in failing rabbit hearts; however, the degree of increase was
288
insufficient to compensate for the increased path length resulting from
289
hypertrophy resulting in an increased total activation time.(126) To complicate
290
matters further, a recent study by Dr. Thomas Seidel and colleagues suggests
291
that myocyte shape may be as important as myocyte size, if not more so, with
292
respect to modulating conduction velocity in a predictable manner.(94)
293
It continues to be a challenge to understand the role that heterogeneous
294
cell geometry has on conduction in diseases such as cardiac hypertrophic
295
cardiomyopathy,(121) because these conditions often also affect the expression
296
and localization of membrane proteins, especially connexins. Additionally,
297
myocyte geometry can also change in the acute time scale in response to
298
ischemia(115) or osmolarity for example.(18)
14 299 300
Non-Myocytes
301
In addition to cardiac myocytes, the heart also consists of fibroblasts and
302
other non-myocyte cells. Indeed, these cells outnumber the myocytes although
303
the latter account for the majority of the heart's volume.(128) It has been
304
proposed that fibroblasts could influence cardiac conduction by either acting as
305
passive obstacles, or by coupling to myocytes and/or each other and acting as
306
either a current sink, or even participating actively in conduction. Additionally,
307
under pathophysiological conditions, fibroblasts can transform into myofibroblasts
308
which participate in inflammatory signaling and have also been proposed to
309
modulate conduction.(6) However, the extent to which fibroblasts and
310
myofibroblasts
311
myocardium remains a topic of ongoing inquiry.(51, 91, 127) For more detailed
312
discussion of the role of non-myocytes in conduction, the reader is referred to
313
reviews by Drs. Peter Kohl (55, 56) and Heather Duffy(6, 23).
electrotonically
couple
with
myocytes
in
the
ventricular
314 315
In the normal heart, fibroblasts are thought to provide structural support
316
directly, as well as by laying down the extracellular matrix.(6) However, under
317
pathophysiological conditions, excessive deposition of extracellular collagen can
318
occur, electrically isolating myocytes from each other, forming barriers to
319
conduction. Indeed, in an elegant study in aging human atrial fiber bundles, Dr.
320
Madison Spach demonstrated how collagenous septa deposited between
321
myocytes
create
a
tortuous
electrical
path
and
facilitate
reentrant
15 322
arrhythmias.(104) Likewise, in the ventricles, fibrosis can create resistive barriers
323
to conduction and act as an arrhythmogenic substrate. While a certain degree of
324
fibrosis occurs in aging hearts, it can be greatly exacerbated in a variety of
325
pathological conditions.(11, 33, 48)
326 327
Gap junctions
328
Given the role gap junctions play in electrically coupling myocytes, and
329
because they are remodeled so frequently in cardiac disease, they have received
330
the preponderance of experimental attention. While slowed conduction is well
331
correlated with an increased risk of arrhythmias, there is disagreement in the
332
literature concerning the relationship between the degree of gap junctional
333
uncoupling and conduction velocity changes. It is well established that
334
pharmacologically uncoupling gap junctions slows cardiac conduction.(57, 87,
335
88) With this observation comes an important experimental caveat: Most
336
manuscripts only report data from doses of gap junction uncouplers that
337
measurably slow conduction.(120) This binary data representation therefore
338
excludes the linear correlation between the degree of gap junction uncoupling
339
and conduction slowing – as well as non-specific (i.e., non-gap junction-related)
340
effects of the drugs. Importantly, pharmacologic gap junction inhibition studies
341
have provided sufficient evidence that gap junctions at the ends of myocytes
342
constitute the primary source of delay during microscopic conduction.(58, 89)
343
Thus, modulating gap junctional coupling preferentially impacts transverse
344
conduction velocity leading to altered anisotropy. (90, 92, 106)
16 345
Contrast pharmacologic gap junction manipulation with genetically
346
manipulating gap junction functional expression. There are a variety of studies on
347
cardiac conduction utilizing the same transgenic mouse lineage expressing 50%
348
of the wild-type levels of connexin43, the primary ventricular gap junction protein.
349
Some groups have reported significantly slowed conduction in mice with a 50%
350
reduction in Cx43 expression relative to wild-type mice,(26, 37) while others
351
could not measure a difference between them. (7, 17, 71, 109, 112, 116, 117)
352
These very basic studies are critically important to understanding cardiac
353
arrhythmias because gap junction remodeling is a hallmark of cardiac disease.
354
To make matters worse, in disease, the relationship between gap junction
355
remodeling and conduction is even less clear. For example, a reduction of total
356
Cx43 expression in a canine pacing induced heart failure model was associated
357
with aberrant ventricular conduction and increased arrhythmogenesis.(79) In a
358
similar model, gap junction remodeling (relocation) and conduction slowing
359
preceded loss of Cx43 expression.(2)
360
The phrase, “gap junctional remodeling” is a catchall term to describe
361
connexin redistribution at the cellular level, post-translational modification, and
362
total protein expression changes. While it has been demonstrated that cellular
363
redistribution of connexins to the lateral membrane is associated with altered
364
conduction, in hypertrophy for example, lateralization occurs concomitantly with
365
changes in myocyte geometry.(102) Whether or not lateralized Cx43 in the
366
pathological myocardium forms functional gap junctions remains an important
367
and complex question.(24) The connexin life-cycle is also dynamically regulated
17 368
by
a
variety
of
biochemical
pathways
including
phosphorylation
and
369
dephosphorylation of specific amino acid residues of the channel.(4, 82, 98, 99)
370
Therefore, gap junctions do not exist in isolation and are part of larger
371
macromolecular complexes and biological pathways.
372 373
Dynamic Determinants of Conduction:
374
In addition to the structural substrate, conduction is also modulated by
375
dynamic functional changes. Primarily, these dynamics result from the interplay
376
between the strength of the excitatory impulse - the source - and the electrical
377
load represented by the tissue it must excite - the sink. In the adult canine
378
ventricular myocardium, each myocyte is coupled to an average of 11±3 other
379
myocytes.(92) As an activation wavefront spreads through the myocardium, the
380
amount of source available per unit mass of tissue is determined by its
381
excitability whereas the balance between source and sink is determined by the
382
curvature of the wavefront and its interaction with the architecture of the
383
myocardium - intercellular coupling, fiber orientation, rotational anisotropy,
384
branching tissue geometry etc.(58, 88, 92, 106) Since local excitability in tissue is
385
dynamically modulated by changes in the shape and duration of action
386
potentials, mismatch between source and sink can arise locally and dynamically,
387
creating
388
Pathophysiological gap junction remodeling and fibrosis can exacerbate source-
389
sink mismatch and thereby the propensity for arrhythmias. For a more detailed
a
functional
substrate
for
arrhythmogenic
conduction
defects.
18 390
discussion of source-sink mismatch, the reader is referred to the in depth review
391
of electrotonic conduction by Drs. Andre Kleber and Yoram Rudy.(54)
392 393
Ion Channels at the Intercalated Disk: Functional Implications
394
The major impetus for reassessing our understanding of conduction arises
395
from structural insights into the subcellular localization of ion channels. In 1996,
396
Dr. Sidney A. Cohen published the first immunofluorescence images of rat TTX-
397
resistant sodium channels (rH1) demonstrating their strong localization at the
398
intercalated disk of cardiac myocytes.(13) While the importance of ion channels
399
at the intercalated disk has been long postulated as a mechanism of non-gap
400
junction mediated coupling, this work was mostly the domain of mathematical
401
models.(14, 59, 66, 70, 107, 108, 129) These models envision intercellular
402
coupling as occurring thusly: A depolarized myocyte withdraws sodium ions from
403
the restricted junctional cleft via its intercalated disk-localized Nav1.5 channels
404
(figure 1A). The resulting depletion of positive charge from the junctional cleft
405
would render the local extracellular electrical potential more negative.
406
Consequently, the transmembrane potential across the apposed membrane of
407
the neighboring myocyte becomes more positive, causing the activation of
408
Nav1.5 channels (figure 1B). Thus electrical activation is communicated from one
409
cell to another without the direct transfer of ions between them (figure 1C).
410
Dr. Nicholas Sperelakis is perhaps best known for championing these
411
mechanisms which he summarized in a 2002 review.(108) Later that year, Drs.
412
Jan P. Kucera, Stephan Rohr, and Yoram Rudy confirmed the presence of both
19 413
sodium channels and connexins at the intercalated disc.(59) More importantly,
414
they revised mathematical models of cardiac conduction in a one-dimensional
415
strand demonstrating that the high density of sodium channels at the intercalated
416
disk could impact cardiac conduction in previously un-appreciated ways.
417
Specifically, they concluded that gap junctions are still likely the principal
418
mechanism of electrical transmission between cells, but sodium channels at the
419
intercalated disk could modulate the conduction velocity - gap junction
420
relationship particularly if the space between the myocytes were very small and
421
densely packed with sodium channels.
422
Since then much evidence has been uncovered to support the existence
423
at the intercalated disk of a macromolecular complex containing the gap junction
424
protein Cx43, as well as cardiac sodium channels (Nav1.5). Indeed, Cx43 and
425
Nav1.5 were found to co-immunoprecipitate from mouse heart lysates(64) and
426
more recently to colocalize at the intercalated disk(76). Work from the group of
427
Dr. Mario Delmar has demonstrated that mechanical adhesion proteins first
428
localize to sites of cell-cell contact followed by recruitment of Cx43 gap junctions
429
and ankyrin-G, a sub-membrane adapter protein involved in localizing cardiac
430
sodium channels (Nav1.5) in the membrane.(31) Recent results from the
431
laboratory of Dr. Mario Delmar demonstrate a loss of Nav1.5 from the membrane
432
in conditional Cx43 knockout mice(52) and even suggest that Cx43 is important
433
for the recruitment of Nav1.5 channels into the membrane at the intercalated
434
disk.(1, 20)
20 435
In addition to sodium channels, evidence has also emerged placing
436
various potassium channel isoforms at the intercalated disk - specifically the
437
inward rectifier potassium channel (Kir2.1)(68), the ATP-sensitive potassium
438
channel (Kir6.2)(47), the delayed rectifier potassium channel (KvLQT1 encoded
439
by KCNQ1)(83) and the 'rapid' delayed rectifier potassium channel (Kv11.1
440
encoded by hERG)(130). As previously discussed, potassium channels can have
441
significant effects on conduction. Additionally, being localized at the intercalated
442
disk, they could also play a role in intercellular coupling via a potassium-
443
mediated ephaptic mechanism. Briefly, potassium efflux from a depolarized
444
myocyte could lead to a transient accumulation of potassium in the narrow
445
junctional cleft, causing the membrane of the neighboring myocyte to depolarize
446
via an inward potassium current.(108)
447
Although the presence of ion channels at the intercalated disk is
448
suggestive, the ephaptic coupling hypothesis has yet to be experimentally tested
449
in the heart. A key missing element has been the identification of a well-defined
450
structure that could serve as a functional unit of ephaptic coupling, an ephapse.
451
The localization of ion channels at the intercalated disk could have important
452
implications in this regard given the necessity of close apposition between
453
membranes of adjacent cells for ephaptic coupling.(62, 70) Taking the
454
experimental evidence together with predictions made by the models,
455
intermembrane spacing could be a key variable in identifying specific
456
microdomains within the intercalated disk that could function as an ephapse.
457
21 458
Interstitial Volume
459
As with any electrical circuit, one must not only consider electrical
460
conduction “forward” through the circuit, but also how the circuit is completed by
461
a current return path. In tissue, the return path can be the interstitial space
462
between the myocytes. At this point, it is useful to re-visit cable theory for a
463
moment, as there is no other mathematical theory that has been applied so
464
rigorously to cardiac conduction. Since myocyte geometry is anisotropic, the
465
interstitial space outside the cells is also anisotropic and can affect axial
466
resistance and thereby conduction in an anisotropic manner.(77, 101) Cable
467
theory-based models predict a direct proportionality between the volume of the
468
extracellular space and conduction velocity,32,81 and this has been experimentally
469
supported in the cable-like papillary muscle.(29)
470
However, one of our recent manuscripts provides evidence that
471
modulating the extracellular volume in a heart changes epicardial conduction in a
472
manner inconsistent with bidomain models of cardiac conduction that lack
473
ephaptic coupling.(120) Additionally, we demonstrated that small degrees of gap
474
junction uncoupling that did not alter conduction normally, significantly slowed
475
conduction when the interstitial space was increased. This study suggested that
476
the effects of small degrees of gap junctional uncoupling on cardiac conduction
477
can be unmasked by increasing the interstitial volume. What remains unknown
478
though is how increasing interstitial volume produces changes in cardiac
479
conduction inconsistent with bi-domain models.
22 480
As mentioned earlier, non-gap junction mediated coupling or "ephaptic"
481
coupling had been proposed by mathematical models, and the distance between
482
cells at the intercalated disk might be an important factor for mediating this type
483
of coupling. A candidate structure that could serve as a functional ephaptic unit
484
as definitive as a synapse or gap junction emerges from recent work by an
485
author of this review together with his colleague Dr. J Matthew Rhett. In these
486
studies a new feature of cardiac ultrastructure was described: The perinexus - a
487
juxta-gap
488
hemichannels wherein interaction between connexin43 and the cardiac sodium
489
channel (Nav1.5) occurs. Given the close (0-20 nm) apposition of membranes
490
from adjacent myocytes in the vicinity of the gap junction, the perinexus emerges
491
as a strong candidate structure for the cardiac ephapse.(84-86)
junction
membrane
microdomain
rich
in
undocked
connexin
492
As it stands, there is evidence from both experiments and mathematical
493
models to suggest that ephaptic coupling could be important to cardiac
494
conduction. While previously viewed as a possible alternative to electrotonic
495
coupling, ephaptic coupling has since come to be viewed as operating in tandem
496
with gap junctions, helping sustain conduction when gap junctional coupling is
497
compromised. To fully appreciate the role of ephaptic coupling, a multi-pronged
498
strategy will be necessary: a) Further whole-heart experiments will need to be
499
performed to generate additional examples of complex conduction; b) The
500
biochemical and functional ultrastructure of the ephaptic machinery will need to
501
be dissected by high resolution microscopic, molecular and physiological
23 502
methods and c) Multi-dimensional mathematical models will need to be revised to
503
incorporate ephaptic coupling and tested against the new experimental data.
504 505
Conduction: A new multi-factorial understanding
506
Like all science, the understanding of cardiac conduction has undergone
507
revisions and refinements over the last 130 years, and it appears that discoveries
508
will only continue to accelerate. The picture that is emerging though is that
509
cardiac conduction is not a simple phenomenon mechanistically determined by a
510
few independent biophysical parameters. Rather, cellular excitability, gap
511
junctional conductance, cell size, gap junction localization, sub-cellular
512
architecture, and ion channel localization are important and inter-related
513
determinants of the conduction phenomenon. While these factors were initially
514
studied vis-à-vis conduction in isolation using a reductionist approach, we are
515
beginning to appreciate that changes in one determinant can potentiate the
516
effects of altering another. This type of higher-level understanding of conduction
517
is critical for determining why certain therapies developed to treat conduction
518
defects are sometimes ineffective or even produce deleterious effects. While it
519
could be argued the inter-related nature and complexity of biological processes
520
means that we will never completely prevent conduction-related arrhythmias, we
521
would suggest that understanding the myriad factors underpinning conduction
522
could eventually provide individualized therapeutic targets that might provide for
523
improved treatment of patients suffering from disease of the heart.
524
24 525
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Figure legend
897
Figure 1: Schematic cartoon illustrating the mechanism of ephaptic coupling. A) Sodium
898
channels (shown in red) on the depolarized (green) myocyte's membrane activate and
899
withdraw sodium ions (Na+) from the restricted extracellular cleft at the intercalated disk.
900
As a result, the transmembrane potential (Vm,1) of the first myocyte is elevated. B) The
901
concomitant depletion of positive charge from the extracellular cleft lowers the local
902
extracellular potential (Φe). This leads to an increase in the second, resting (red)
903
myocyte's transmembrane potential (Vm,2) - defined as the difference between its
904
intracellular potential and the extracellular potential (Φe). In turn, sodium channels
905
located at or near the intercalated disk of the second myocyte activate. C) Sodium
906
enters the second myocyte via these channels further depolarizing it and triggering an
907
action potential. Thus activation is communicated 'ephaptically' from cell-to-cell without
908
the direct transfer of ions between them.
909
A) + +
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Vm,2
Vm,1 B)
+
+ +
+ +
+ +
+
+
+
+
Vm,2
Vm,1 C) + +
Vm,1
+ +
+ +
+ +
+
+
+
+
Vm,2