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Molecular adaptations in human atrial fibrillation
Brundel, Bianca Johanna Josephina Maria
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Brundel, B. J. J. M. (2000). Molecular adaptations in human atrial fibrillation: mechanisms of protein
remodeling s.n.
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Introduction
Introduction
Atrial Fibrillation, clinical aspects
Atrial fibrillation (AF) is currently the most common sustained clinical arrhythmia
and is responsible for a substantial proportion of hospital costs incurred in the treatment of
cardiac rhythm disorders.1 AF becomes increasingly common with age, having an incidence
averaging <0.5% in patients <40 years of age and reaching >5% in patients >65.2 Thus AF
is likely to become increasingly important with the ageing of the population. The arrhythmia
is defined by a very rapid atrial rate (generally >400/min in humans) along with irregular
atrial activation and lack of repetitive pattern of co-ordinated atrial activity. AF is associated
with a variety of complications, including thromboemboli resulting from coagulation in
the relatively static atrial blood pool, a loss of the fine adjustment of ventricular rate to the
body’s precise metabolic needs, potential impairment of cardiac function and subjective
symptoms like palpitations, dizziness, breathlessness and chest pain.
AF can occur in paroxysms of a duration shorter than 24 hours (but longer lasting
paroxysms are not unusual) with intermittent sinus rhythm. Paroxysmal AF either converts
spontaneously or is terminated with an intravenously administered antiarrhythmic drug.3,4
In contrast, during persistent AF, the arrhythmia is continuously present until the moment
of investigation, i.e. at least two consecutive electrocardiograms of AF more than 24 hours
apart and without intercurrent sinus rhythm. Persistent AF does not convert spontaneously.3,4
AF has the tendency to become more persistent over time. This is illustrated by the
fact that about 30% of patients with paroxysmal AF eventually will develop persistent
AF.5 Also pharmacological and electrical cardioversion and maintenance of sinus rhythm
thereafter become more difficult the longer the arrhythmia exists.6 The cumulative
percentage of patients who maintained sinus rhythm after serial cardioversion treatment
was not more than 42% after 1 year and 27% after 4 years.6 This relates to progression of
the underlying disease and possibly also to electrical remodeling of the atria.7
Mechanism: multiple-wavelet reentry
In 1959 Moe & Abildskov8 showed that AF could be produced by experimental
paradigms of both multiple circuit reentry and rapid activity and they suggested that either
type of mechanism might cause clinical AF. Moe put forward the ‘multiple wavelet
hypothesis’ of AF in 1962.9 This concept described the propagation of reentry waves as
involving multiple independent wavelets circulating around functionally refractory tissue.
The maintenance of AF then depends on the probability that electrical activity can be
sustained by a sufficient number of active wavelets at any time. Experimental support for
Moe’s ideas was obtained subsequently with the use of computerized mapping of AF
maintained in the presence of acetylcholine in dog hearts.10 It was demonstrated that during
9
Chapter 1
Figure 1
Schematic representation of inward and outward ionic currents involved in the ventricular action potential, resting
membrane potential and cytoplasmic Ca2+ transients.
The numbers 0 through 4 indicate the different phases of the action potential: 0, upstroke; 1, fast early repolarization
phase; 2, plateau phase; 3, repolarization phase; 4, resting membrane potential. Adapted from The Sicilian Gambit.47
INa, sodium current encoded by the gene SNC5A; ICaT, calcium current encoded by T-type Calcium channel; ICaL,
calcium current L-type Calcium channel; ICl(Ca), calcium dependent transient outward current encoded by the
gene Kv4.2; INaCa, sodium calcium exchanger; ITo, transient outward current encoded by Kv4.3; IK1 , inward rectifier
K+ current encoded by Kir2.1; I Ks, slow delayed rectifier K+ current encoded by minK and KvLQT; IKr, rapid
delayed rectifier K+ current encoded by HERG, IKACh, acetylcholine dependent potassium current encoded by
Kir3.1 and Kir3.4; INa-K, sodium potassium pump.
10
Introduction
AF, multiple independent wavelets activate the atria in a random reentrant way. Individual
wavelets could brake-up, fuse or collide with each other and wavelets would extinct when
they reached the border of the atria or met refractory tissue. From time to time a varying
number of wavelets was present in the atria and the duration of each individual wavelet
lasted only several hundreds of a millisecond. Further it was shown that the number of
wavelets that fit into the atria determined the perpetuation of AF. Below a critical number
of wavelets (between 3 and 6), there was a considerable chance for the wavelets to die out
all at the same time. When more than 6 independent wavelets were present, the arrhythmia
would not convert spontaneously anymore. The number of wavelets that fit into the atria
depends on the atrial refractory period, conduction velocity and atrial mass.8,9 During the
last decade, mapping studies in humans with AF have further confirmed the multiple wavelet
theory.11
Electrophysiology of the atria
The processes that signal the heart to contract (excitation-contraction coupling) begin
when an action potential depolarizes the plasma membrane surrounding the myocardial
cell. This electrical signal is generated by the passage of ions through ion channels in the
plasma membrane that changes the electrical potential of the interior of the cardiac cell
relative to the extracellular space. These ion fluxes include two major inward currents that
depolarize the heart. The phase 0 depolarization is initiated by a rapid inflow of sodium
ions through the voltage-gated Na-channels and later by calcium ions via the L-type calcium
channel (ICaL) (Figure 1).12,13 A large transient outward current has been held responsible
for the phase 1 repolarization. This current is composed of a 4-aminopyridine-sensitive
component (ITo1) and a Ca2+ activated and verapamil blocked Cl- current (ITo2 or ICl(Ca)).14-16
During the plateau phase (phase 2), there is a delicate balance of inward and outward
currents. Inward currents can be carried through the Na+ channel and the Ca2+ channels.
During reverse-mode, the Na+/Ca2+ exchanger will transport three Na+ ions outside and
exchanged for one Ca2+ resulting in a net movement of charge across the plasma membrane
from inside to outside. The influx of calcium through the L-type calcium channels is
responsible for the plateau phase during repolarization and initiates calcium release from
the sarcoplasmic reticulum that binds to the contractile filaments of the myocardial cell,
causing contraction (see calcium homeostasis in normal cardiac cells).17 Outward currents
during the plateau are carried by a number of K+ channels (IKs, IKr, IKACh ) and the Na+-K+
pump. For the initiation of phase 3 the contributions of rapidly and slowly activating
delayed rectifier K+ currents are of key importance. Here the outward potassium current
IK1 is activated and the action potential returns to its transmembrane resting potential,
which remains at that level during phase 4 of the action potential until the cell becomes reactivated.
11
Chapter 1
The duration of the repolarization phase is different between atrial and ventricular
myocardial cells. This phase is shorter in the atrium compared to ventricle.18 An other
difference is the distribution and magnitude of the ionic currents responsible for the resting membrane potential. The inward rectifier current, which is responsible for maintenance of the resting membrane potential, IKs is smaller in atrial cells compared with ventricular cells.19,20 Secondly, the acetylcholine-dependent potassium current IKACh is very
important in maintaining and hyperpolarizing the resting membrane potential in atrial
cells, while less active in ventricular cells.21 It is this current which is responsible for
hyperpolarization and shortening of the atrial action potential during parasympathic stimulation. Vagal stimulation is indeed one of the oldest models to induce sustained atrial
fibrillation by applying vagal stimulation. The resulted increased parasympathetic tone
will shorten the atrial refractory period through opening of these acetylcholine-dependent
potassium channels. Furthermore, inhomogeneous distribution of vagal nerve endings will
increase the spatial dispersion in refractoriness.22 In this way, atrial fibrillation will persist
after induction with premature stimuli or atrial burst pacing as long as the parasympathetic
system is stimulated, either by vagal nerve stimulation or by acetylcholine application.
Figure 2
Schematic representation of the calcium triggered calcium release in the myocardial cell. Small amount of
calcium enters the cell via the Na+/Ca2+ exchanger, but mainly through voltage gated L-type calcium channels,
which open in response to the action potential. This causes a much larger amount of calcium to be released by
the ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). Calcium will bind to the troponin C
resulting in contraction. The calcium pump of the SR (SR Ca2+ ATPase) has a high affinity for calcium and
reduces cytosolic Ca2+ concentrations to levels low enough to dissociate this cation from its binding sites on
troponin C. The SR Ca2+ ATPase is inhibited by phospholamban (PLB). When PLB is phosphorylated this
inhibition is reversed.
12
Introduction
Calcium homeostasis in normal cardiac cells
When an action potential depolarizes the plasma membrane surrounding the
myocardial cell, the processes that signal the heart to contract begins. Calcium transients
underlying this excitation-contraction coupling in cardiac cells result mainly from calcium
release from the sarcoplasmic reticulum triggered by calcium entry during the action
potential. This process is called calcium-induced calcium release (Figure 2). Under normal
circumstances, calcium entry into cardiac myocytes is carried primarily via IcaL, whereas
additional fractions can enter via on reverse-mode Na+/Ca2+ exchange (one calcium ion is
transported inside the cell against three sodium ions outside) and ICaT. All three pathways
are capable of triggering sarcoplasmic reticulum (SR) calcium release and contraction,
but the relative contribution and efficiency is largest for ICaL.23,24 Upon depolarization,
influx of calcium through discrete clusters of L-type calcium channels in the plasma
membrane triggers the opening of ryanodine receptors in the sarcoplasmic reticulum
membrane, resulting in the major release of calcium into the cytoplasm which in turn
triggers myofibril contraction by binding of troponin C. Following contraction, the SR
calcium ATPase enzyme in the network of sarcoplasmic reticulum surrounding the
myofibrils, rapidly pumps the calcium back into the SR lumen, causing the myofibrils to
relax. The SR calcium ATPase is regulated by phospholamban, a small protein and when
phosphorylated it will activate SR calcium ATPase and the calcium content of the SR will
increase.25
Modern concepts of excitation-contraction coupling rely on a ‘local-control theory’ a
close association between L-type calcium channels and ryanodine receptors and
subsequently received experimental support documenting local functional interaction
between these channels.26 Changes in the close association between the L-type calcium
channels and ryanodine receptors result in a reduction of calcium release from the
sarcoplasmic reticulum and reduced contractility of the cardiac myocyte.26
The membrane machinery allows each myocyte to function as an autonomous
contractile unit. To produce a heart beat, the contractile capabilities of myocytes that
make up the heart have to work in a highly synchronous fashion. This requires both an
orderly spread of the wave of electrical activation and effective transmission of contractile
force from one cell to the next, throughout the heart.
Electrical remodeling of the atria
Over the past several years, AF-induced remodeling and its underlying mechanisms
have been studied in substantial detail. Wijffels and coworkers published the first study in
1995 which part of the underlying electrophysiological changes explaining the progressive
nature of AF was demonstrated.7 In healthy goats they showed that repetitive induction of
AF increased the duration of successive episodes of AF, until AF finally did not convert
13
Chapter 1
spontaneously any more. They discovered that the increased tendency of the atria to fibrillate
was paralleled by a progressive shortening of the atrial effective refractory period (AERP)
and loss of the physiological rate adaptation of the refractory period which they termed
atrial electrical remodeling.7 After cardioversion of atrial fibrillation that had been present
for two to four weeks, this so-called atrial electrical remodeling appeared to be completely
reversible within one week after restoration of sinus rhythm.
In 1995 Morillo et al. 27 also showed that rapid atrial pacing (400 bpm) strongly
promotes the ability to maintain AF in dogs, with changes quite similar to those observed
by Wijffels et al.7 Later observations by Wijffels et al. suggested that acute volume loading,
opening of the ATP-dependent potassium channels, neurohumoral activation or an increase
in ANP were not responsible for the altered electrophysiological characteristics in this
experimental model. This supports the idea that AF-induced remodeling is primarily due
to the rapid atrial activation rates caused by AF.28 Consequentially, many investigators
have used rapid atrial pacing in experimental models to study the electrophysiological
changes caused by sustained atrial tachycardia, hoping to gain insights into the atrial
electrophysiological changes caused by AF in man.
Experimental and clinical studies have shown that sustained AF decreases the atrial
effective refractory period (AERP).7,29-32 AERP changes occur over a period of days to
weeks7,27,33,34, but AF can decrease AERP over a time interval as short as several minutes.32
Although the AERP reduction caused by AF favours arrhythmia maintenance, it cannot be
the only factor involved because AF-induced AERP alterations become maximal well before
AF-promoting effects stabilize.7,34 One of the AF-promoting effects is tachycardia induced
atrial conduction slowing.27,33,34 It has a slower time course than AERP changes, probably
due to structural changes and could account for at least a part of the continued development
of AF promotion after AERP changes have stabilized.
Tieleman and coworkers found that the AERP shortened with loss of the normal rate
adaptation in response to 24 hours of rapid atrial pacing in goats.35 Here the tendency of
the atria to fibrillate increased. With resumption of sinus rhythm after cessation of pacing,
the refractory period normalized over a period of slightly more than 24 hours. This electrical
remodeling could be modulated using several pharmacological agents. First, the calcium
channel blocker verapamil reduced atrial electrical remodeling; suggesting that tachycardiainduced calcium overload might trigger the shortening of the refractory period.35 By contrast,
digoxin delayed the recovery from electrical remodeling of the atria.36 This could be due
to the effect of digoxin on calcium handling, preventing effective wash-out of calcium
after cessation of pacing.
Contractile dysfunction of the atrium
Besides the electrical remodeling observed in experimental studies, clinical studies
14
Introduction
demonstrated atrial contractile dysfunction after AF.37,38 Leistad and co-workers investigated
the contractile dysfunction in an experimental model for AF.39 They demonstrated that the
atrial contractile dysfunction after acute atrial fibrillation is reduced by the calcium channel
antagonist verapamil, which suggests that transsarcolemmal calcium influx contributed to
this dysfunction. The calcium agonist BAY K8644 increased postfibrillation atrial contractile
dysfunction. A remarkable finding was that atrial contractility increased in the first seconds
after atrial fibrillation before a longer period of reduced atrial contractility ensued. On the
basis of these observations they hypothesised that cytosolic calcium overload due to rapid
depolarization during the preceding fibrillation might be responsible for the atrial contractile
dysfunction. This hypothesis was also suggested by Shapiro et al. in 1988.40It should be
noted that in the pig model of Leistad and coworkers39 atrial contractile dysfunction was
observed after only five minutes of AF, indicating that contractile dysfunction, like electrical
remodeling in the goat7, starts early.39
Structural remodeling
Until now, research focused on electrical and contractile remodeling. AF is also
associated with structural changes.27,31,41 Ausma and coworkers described and quantified
the structural remodeling in atrial myocardium due to sustained atrial fibrillation in the
goat.41 They maintained atrial fibrillation in normal goats for a prolonged period of time.
After 9 to 23 weeks of sustained atrial fibrillation, several areas of the right and left atria
were examined by light and electron microscopy. They found that a substantial proportion
of the atrial myocytes (up to 92%) revealed marked changes in their cellular substructures,
such as loss of myofibrils, accumulation of glycogen, changes in mitochondrial shape and
size, fragmentation of sarcoplasmic reticulum and dispersion of nuclear chromatin. They
pointed out that these changes are the same as observed in chronically hibernating
myocardium. This is a condition that occurs in patients as a result of low flow ischemia
caused by stenosis of one or more coronary arteries.41 The clinical condition is defined as
the ability of the myocardium to adapt to chronic ischemia by down-regulating its contractile
function, thereby maintaining cell viability for a prolonged period of time. Furthermore, it
was found that these hibernating myocytes resembled a form of dedifferentiation as a
result of chronic atrial fibrillation.42 Some of the atrial cardiomyocytes acquired a
dedifferentiated phenotype, as deduced from the re-expression of α-smooth muscle actin,
the disappearance of cardiotin and the staining patterns of titin, which resembled those of
embryonic cardiomyocytes.
Considerations for the thesis
Thus, experimentally it has been shown that contractile dysfunction starts accutely
after AF onset and was reduced by verapamil but increased by BAY K8644.39 These results
15
Chapter 1
strongly indicate that changes in the calcium homeostasis triggered by tachycardia induced
intracellular calcium overload 43-45, play a pivotal role in the induction of atrial contractile
dysfunction.
Shortening of the atrial effective refractory period was an other important factor contributing
to the maintenance of AF 7,46. This shortening could also be mediated by verapamil and
gave a reduction of the tachycardia induced electrical remodeling of the atria.35 This finding
also suggests that electrical remodeling could be due to changes in the calcium homeostasis.
Aim of the thesis
The main goal was to study the molecular remodeling in human atrial fibrillation. We
focussed on gene expression of proteins which influence the calcium homeostasis and
action potential duration in human AF. The impact of modulating systems like the natriuretic
peptide system and the endothelin system were also studied.
For the purpose of the thesis, right and left atrial appendages were collected during
four years from different groups of patients undergoing cardiac surgery. The patients clinical
characteristics (underlying heart disease, type of AF, electrocardiograms, medication and
exercise tolerance) were assessed. The result was a unique human atrial appendage collection
of around 150 individual patients. Because of this amount of different atrial appendages it
was possible to match AF patients with control patients in sinus rhythm for age, sex,
underlying heart disease, left ventricular function and as far as possible for medication
use. In this way, dissecting the effect of AF was optimized.
During the progression of the study a discrepancy between alterations in mRNA and
protein expression of several ion-channels in patients with paroxysmal AF was observed.
No alterations in mRNA expression of ion-channels compared to important reductions on
the protein level were found in human paroxysmal AF. This new finding prompted us to
explore an adaptive mechanism unknown to occur in AF. We hypothesized reduction in
protein channels due to calcium activated neutral protease calpain. To test this the calpain
activity, protein expression and localization were determined in tissue of patients with
atrial fibrillation. Finally the role of calpain activity in ion-channel protein, structural and
electrophysiological remodeling was studied.
16
Introduction
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18