University of Groningen. Atrial electrical remodeling from barn to bedside Tieleman, Robert George

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1 University of Groningen Atrial electrical remodeling from barn to bedside Tieleman, Robert George IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1999 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Tieleman, R. G. (1999). Atrial electrical remodeling from barn to bedside: experimental and clinical studies on tachycardia-induced changes in atrial electrophysiology. Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 DiscussionIVpart

3

4 General Discussion 10 chapter

5 introduction During recent years animal experimental models and experiments in humans have shown that rapid atrial pacing and atrial fibrillation (AF) can induce electrophysiological changes to the atria that increase the vulnerability for reinduction and perpetuation of AF. 1-3 This so-called atrial electrical remodeling explains the long known clinical fact that paroxysmal AF has the tendency to deteriorate into chronic AF 4,5 and that with a longer duration of AF it becomes more difficult to restore sinus rhythm using pharmacological 6-9 and electrical cardioversion and to maintain sinus rhythm thereafter Wijffels et al 2 published the first study in which part of the underlying electrophysiological changes explaining the progressive nature of AF were demonstrated. In chronically instrumented healthy goats they showed that repetitive induction of AF increased the duration of successive episodes of AF, until AF finally did not convert spontaneously any more, illustrating what Rosenbaum had called the domestication of AF. 2 They discovered that the increased tendency of the atria to fibrillate was paralleled by a progressive shortening of the atrial refractory period and loss of the physiologic rate adaptation of the refractory period which they termed atrial electrical remodeling. 2 Later observations by the same authors 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, but that the increase in atrial rate during AF was the main determinant for atrial electrical remodeling. 16 High heart rates increase the inflow of calcium 17 and sodium 18 and AF induces intracellular calcium- and sodiumoverload. 19 Furthermore, verapamil administered during short episodes of AF could reduce atrial contractile dysfunction after cessation of AF, whereas administration of the calcium agonist Bay-K8644 resulted in an increased duration and magnitude of the hypocontractile period post AF. 20 These results suggest that after cessation of AF, hypocontractility is related to tachycardia-induced calcium overload during AF. Therefore we hypothesized that tachycardia-induced calcium- and perhaps sodiumoverload was one of the mechanisms which also could explain the initiation of atrial electrical remodeling. Another potential cause of atrial electrical remodeling could be ischemia-induced hydrogen overload due to the increased metabolic demand during the high atrial fibrillatory depolarization frequencies. These 2 potential mechanisms and data from previous physiological studies served to construct a hypothetical model of the intracellular processes explaining tachycardia-induced electrical remodeling of the atria. Subsequently, we used various pharmacological probes during rapid atrial stimulation at 300 beats per minute in chronically instrumented goats to modulate atrial electrical remodeling, in order to test this hypothetical model and to clarify the underlying mechanisms of remodeling. After briefly discussing the normal physiological processes during depolarization and repolarization, the hypothetical model of atrial electrical remodeling and the 166 CHAPTER 10 General Discussion

6 results from verifying pharmacological studies modulating electrical remodeling are described. In the second part of this discussion the results from clinical studies are reviewed, analyzing how they compare to the experimental studies on atrial electrical remodeling. Finally, other forms of AF-induced atrial remodeling, such as contractile dysfunction and morphological remodeling are discussed. physiology of the heart and the initiation of electrical remodeling Sodium and Calcium Homeostasis During Sinus Rhythm During the normal action potential, depolarization occurs due to a fast but brief sodium influx through the Na-channels in combination with a more slowly developing but prolonged calcium inflow through the voltage-dependent L-type calcium Schematic illustration of the sequence of excitation-contraction coupling in an adult mammalian ventricular myocyte. The intracellular ion transport events are shown in the main figures; the corresponding changes in membrane potential, cytosolic calcium and contractile force (shortening) in an isolated adult rabbit ventricular myocyte loaded with the calcium-sensitive dye indo-1 stimulated at a frequency of 1 Hz at 36 degrees celcius are shown in the insets on the left. A: The process ongoing in the resting myocyte or at end diastole. Figure 1A Atrial Electrical Remodeling From Barn To Bedside 167

7 Figure 1B Influx of sodium and calcium through the sarcolemma. channels. 21 This quantitatively small influx of calcium ( trigger-calcium ) induces the sarcoplasmic reticulum ryanodine receptors to release large amounts of calcium (calcium-induced calcium release) 22 which will bind to the contractile proteins to induce myocardial cell shortening (figure 1 A-C). During repolarization, the efflux of especially potassium ions restores the negative resting transmembrane potential. 21 In order to maintain steady state conditions, the systolic inflow of calcium and sodium ions and the repolarizing potassium currents are counterbalanced by diastolic homeostatic mechanisms which restore the respective ionic concentrations before the next activation occurs (figure 1D). The 2 most important homeostatic ion-transporters are the Na-/K-ATPase pump and the Na-/Ca exchanger (figure 1D). 23 The energy-dependent Na-/K-ATPase pump transports 3 Na-ions out against 2 K-ions in, and is therefore an electrogenic pump, contributing to repolarization and hyperpolarization, especially under conditions of an increased intracellular sodium concentration. 24 The direction of the passive Na- /Ca-exchanger depends on the concentrations of sodium and calcium on either side of the sarcolemma. Since it exchanges 3 Na-ions against 1 Ca-ion, 25 its action also depends on the membrane potential, 26 ie it preferentially transports Na out and Ca in during the more positive membrane potentials early after depolarization (reversed mode). When during repolarization the transmembrane potential decreases below the reversal potential of the Na-/Ca-exchanger it changes direction transporting sodium in and calcium out of the cell (figure 1D; forward mode). During diastole the Na-/K-ATPase pump transports sodium out of the cell. The resulting decreased intracellular sodium 168 CHAPTER 10 General Discussion

8 Calcium-induced calcium release through the ryanodine receptor (SR-Ca2+ release channel). Na/Caexchanger shifts to forward mode. Calcium binds to contractile elements to start contraction. Figure 1C concentration in combination with the high cytosolic calcium concentration released from the sarcoplasmic reticulum will further stimulate the forward mode of the Na- /Ca exchanger to transport calcium out of the cell. 27 In addition, diastolic re-uptake of cytosolic calcium by the sarcoplasmic reticulum is warranted by the sarcoplasmic reticulum Ca-ATPase (SERCA; figure 1D). 28 Regulation of SERCA occurs primarily by phosphorylation of pospholamban. Pospholamban is an inhibitory protein that binds to and inhibits sarcoplasmatic calcium re-uptake by SERCA. When pospholamban is phosphorylated by camp-dependent protein kinase, eg during sympathetic stimulation, the inhibition is removed and the activity of SERCA increases. 29 A third route by which calcium is extruded from the cytosol is through the sarcolemmal calcium ATPase. 30 However, over the range of physiologic calcium concentrations the rate of calcium extrusion by this transporter is estimated to be about 1/10 of that of the Na-/Ca exchanger. 31 Finally, intracellular storage sites such as mitochondria 32,33 and calcium-binding proteins 34 can buffer short-term increases in calcium. The diastolic Na-/Ca exchange, sarcoplasmic reticulum calcium reuptake and the extrusion by sarcolemmal calcium ATPase will lower the cytosolic calcium concentration, which will subsequently dissolve calcium from the contractile elements resulting in relaxation (figure 1 D). Of note, these diastolic homeostatic processes such as the outward sodium transport by the Na-/K-ATPase pump, the sarcoplasmic reuptake of calcium by SERCA, and the sarcolemmal calcium ATPase, and therefore myocardial relaxation, are energy-dependent processes. Atrial Electrical Remodeling From Barn To Bedside 169

9 Figure 1D The events associated with a fall in cytosolic calcium during repolarization leading to relaxation. See text for discussion. (1A-D Adapted from Barry et al, 23 Circulation 1993) Increases in heart rate during sinus rhythm cause an increase in calcium 17 and sodium inflow. 18 Sodium is rapidly exchanged against calcium through the Na-/Ca-exchanger, adding to the increase in cytosolic calcium. 35,36 This will induce more calcium-induced calcium release from the sarcoplasmic reticulum, 22 which explains the increased contractile force exhibited during increased frequencies (positive staircase). 17,37 In order to maintain this enhanced contractility, diastolic calcium removal from the cytosol must also increase. During physiologic conditions of an increased heart rate (eg sympathetic stimulation) this is achieved by shortening of the refractory period 38,39 which favors the forward mode of the Na-/Ca exchanger 26 and by camp-dependent inactivation of pospholamban and subsequent activation of the sarcoplasmic reticulum Ca-ATPase, which provides re-uptake and calcium loading of the sarcoplasmic reticulum. 29 Of note, most of the above-mentioned processes have only been investigated in ventricular cells and it is not known to what extent these processes apply to atrial tissue. Hypothesis on Increased Ion-fluxes During the first hours of Pathological Tachycardia During rapid atrial pacing or AF, the pathological increase in depolarization frequency ranging from 300 to 600 bpm will also lead to increased calcium and sodium inflow per unit of time (figure 2). Extrapolating the normal physiology to the pathological situation during AF, we hypothesized that this increased influx will be too high for the normal homeostatic 170 CHAPTER 10 General Discussion

10 counter transport mechanisms, leading to a pathological increase of the intracytosolic sodium and calcium concentrations. As a result of the increased sodium concentration and the subsequent fall in driving force for sodium entry, one would expect pacing thresholds to increase, or conduction to slow down, 24 neither of which occurred in our experiments during 24 hours of rapid atrial pacing. This suggests that, parallel to the situation during digitalis therapy in sinus rhythm patients, 40,41 the excess sodium which is not pumped out by the Na-/K-ATPase pump will be exchanged by the concentrationdependent Na-/Ca exchanger (figure 2). This will further increase calcium influx, adding to intracellular calcium overload. When in a later stage further intracellular calcium overload favors the forward mode of the Na-/Ca-exchanger, intracellular sodium may also increase. 42 Indeed, Leistad and co-workers 19 demonstrated increased levels of intracellular sodium but especially calcium in the atria of pigs after only 15 minutes of AF. Furthermore, Gaspo et al 43,44 demonstrated that shortening of the atrial refractory period and loss of rate-adaptation (due to calcium overload and a subsequent decrease in calcium-current, see below) occurred early during pacinginduced electrophysiological remodeling, with near-maximal changes after 1 week of pacing, whereas a decrease in sodium current and subsequent decrease of the conduction velocity occurred at a slower pace and was maximal after 6 weeks of pacing. Schematic representation of the hypothesized ionic events that take place during pathological tachycardia which initiates atrial electrical remodeling. See text for discussion. Figure 2 Atrial Electrical Remodeling From Barn To Bedside 171

11 Sodium and Calcium Overload and Shortening of the Action Potential Sodium overload is known to result in shortening of the action potential duration by activation of the electrogenic outward Na-/K-ATPase current and the reversed mode of the Na-/Ca-exchange current, 24,45 and could thereby add to shortening of the atrial refractory period during electrical remodeling. Calcium overload will cause inactivation of the L-type calcium channels (calcium-induced inactivation) which in combination with the decreased transsarcolemmal concentration gradient will reduce the magnitude and duration of calcium inflow and the plateau phase, one of the mechanisms normally responsible for the physiological rate adaptation of the refractory period Furthermore, increased levels of intracellular calcium are known to activate calcium-dependent potassium channels such as the delayed rectifier potassium channel 51,52 and the calcium-dependent transient outward current I to2, 53 which will further shorten atrial refractoriness. Focussing on intracellular calcium homeostasis, it is conceivable that it is favorable for the rapidly activated cells to shorten their plateau phase. In case the action potential is shortened by activation of I to2 or calcium-dependent potassium-channels, the voltage-dependent L-type calcium channels will close earlier, decreasing transsarcolemmal calcium inflow. 54 Furthermore, early repolarization to values below the reversal potential will favor the Na/Ca-exchanger to switch to its forward mode, extruding calcium from the cytosol. In this way, the short action potential duration helps to achieve a new homeostatic balance between calcium-entry and efflux. However, during the pacing or AF-induced pathological increase in atrial rate these physiological processes may fall short in preventing calcium overload. This may induce structural adaptations to the pathological conditions as described below. Furthermore, the adaptive mechanisms which prevent calcium overload occur at the cost of a decreased refractory period which perpetuates reentrant arrhythmias such as atrial flutter and AF. Metabolic Demand and Energy Supply During Tachycardia Apart from the increased inflow of sodium and calcium per unit of time during onset of rapid atrial pacing and AF, it is conceivable that the increased frequency of contraction will lead to an increased metabolic demand with the potential risk of energy depletion and metabolic derangements (figure 2). When the Na-/K-ATPase pump comes short in restoring pre-activation ionic concentrations, the extracellular potassium concentration will increase, which will lead to a hypopolarization of the resting membrane potential with conduction slowing and shortening of the refractory period. Recent findings indeed indicate an increased extracellular potassium concentration after 30 minutes of pacing-induced AF. 55 Furthermore, Leistad et al 20 demonstrated decreased levels of creatine phosphate following 5-minute periods of experimental AF, indicating an increased metabolic demand, whereas the lactate and ATP content remained unchanged. White et al 56 demonstrated that 15 minutes of pacing-induced AF in anesthetized dogs caused the 172 CHAPTER 10 General Discussion

12 atrial oxygen consumption to increase more than threefold, with a 2-3 fold increase in atrial myocardial perfusion, but a reduction in flow reserve. This increased blood flow, possibly due to metabolic feedback mechanisms, indicates the increased energy consumption during tachycardia. In case the flow is too low to meet the increased demand during AF, the cytosol will become acidified, and the increased hydrogen concentration may be exchanged by sodium through activation of the Na-/Hexchanger. 57 This in turn will lead to increased levels of intracellular sodium, which as explained above will induce calcium overload. Apart from an early increase in metabolic demand, energy production may be impaired, since calcium overload has been shown to impair mitochondrial functioning with depressed ATP production 58 which will further compromise the energy demandsupply balance. An adaptation to this may be excitation-contraction uncoupling 59 and the proteolysis of contractile proteins 60 which has been described during intracellular calcium overload in ventricular myocytes. In case of atrial remodeling, this will decrease atrial contractility and therefore reduce the metabolic demand preventing cell necrosis, and explains the transient contractile dysfunction after AF Finally, in case tachycardia reduces ATP production, the sodium efflux by the Na-/K- ATPase pump, calcium extrusion by the sarcolemmal calcium ATPase and the diastolic re-uptake of calcium by the sarcoplasmic reticulum Ca-ATPase (SERCA) will also be reduced (figure 2). This will further increase the calcium concentration, which again adds to atrial remodeling. Furthermore, in case of a reduced SERCA activity the sarcoplasmic reticulum becomes depleted from calcium which decreases contractility during subsequent activations. All proposed mechanisms of remodeling (tachycardia-induced increased ion flow/unit of time and ischemia-induced metabolic derangements) have cytosolic calcium overload as a final common pathway. Therefore, calcium handling and overload play a crucial role in our hypothetical model of the initiation of electrical remodeling. atrial electrical remodeling in experimental models of AF Testing the Calcium Overload Hypothesis: Pharmacological Modulation of Atrial Electrical Remodeling The fact that verapamil and ryanodine 68 could reduce pacing-induced electrical remodeling suggests an important role for intracellular calcium overload in the shortening of the atrial refractory period during electrical remodeling. By blocking calcium inflow through the L-type calcium channels (verapamil) or inhibition of calcium-induced calcium release from the sarcoplasmic reticulum (ryanodine), homeostasis will be Atrial Electrical Remodeling From Barn To Bedside 173

13 achieved at a lower level of intracellular calcium, reducing calcium-induced shortening of the refractory period. Another possibility is that the latter occurs due to the negative inotropic action of these drugs which prevent metabolic derangements with hydrogen overload. The subsequent reduction in Na/H-exchange and Na/Ca-exchange will also attenuate calcium overload. The class 1C anti-arrhythmic drug flecainide also attenuated atrial electrical remodeling (chapter 5), which may be explained by a reduction of sodium inflow during tachycardia which will lead to a lower sodium load and therefore less activation of the repolarizing Na-/K-ATPase pump. Furthermore, reduction of sodium entry may lead to less Na-/Ca- exchange which also results in a reduction of intracellular calcium overload. In contrast, the study by Yu et al 67 and Daoud et al 66 showed no reduction of electrical remodeling by the sodium channel blocker procainamide. This could be explained by the fact that flecainide exhibits a more use-dependent action than procainamide which may be especially important in blocking sodium inflow during tachycardia-induced electrical remodeling. On the other hand, since only short-term adaptation to several minutes of AF or rapid pacing was studied in the procainamide studies 66,67, it remains questionable what the effect of procainamide will be during longer periods of atrial tachycardia. The Na-/K-ATPase inhibitor digoxin administered during rapid atrial pacing slightly but significantly accelerated electrical remodeling, which is in agreement with the abovementioned principles. 69 However, a more pronounced feature of digoxin was that it significantly delayed the recovery from electrical remodeling after cessation of pacing. 69 Recovery from electrical remodeling may relate to wash-out of the excess cytosolic calcium. Inhibition of the Na-/K-ATPase pump by digoxin increases intracellular sodium, which will favor the reversed (Na out, Ca in) mode of the Na- /Ca-exchanger, slowing down calcium extrusion, which explains the delayed recovery from electrical remodeling. Similarly, Goette et al 64 showed that during hypercalcemia, recovery from electrical remodeling was also delayed. In this case the high extracellular calcium concentration might have had a similar effect on the Na-/Ca-exchanger, inhibiting efficient calcium extrusion. Whether ischemia also plays a significant role during atrial electrical remodeling remains controversial. Administration of the Na-/H-exchange-inhibitor HOE642 (cariporide) has been shown to reduce pacing-induced atrial electrical remodeling, 70,71 which suggests an important role for intracellular acidosis during atrial electrical remodeling. On the other hand, blockade of the ATP-dependent K-channels by glibenclamide during rapid atrial pacing 64 did not prevent shortening of the atrial refractory period. However, as explained above, shortening of the atrial refractory periods due to tachycardia-induced ischemia may be the result from calcium overload, and not only the opening of ATP-dependent K-channels. Recently, Jayachandran et al 72 demonstrated a reduced atrial myocardial blood flow in dogs with 6 weeks of pacing-induced AF using a sophisticated mini positronemission-tomograph. They interpreted this finding as proof for the hypothesis that ischemia induced atrial electrical remodeling. However, the reduction in blood flow 174 CHAPTER 10 General Discussion

14 during AF may also be the consequence of electrical and contractile remodeling, expressing a reduced metabolic state as an adaptation to the high frequency of stimulation (see below). Therefore, the findings by Jayachandran et al 72 do not prove the presence of ischemia during AF. The Autonomic Nervous System and Electrical Remodeling The autonomic tone, and especially the vagal limb, is a factor long recognized as increasing the susceptibility to AF 73,74 by shortening the atrial refractory period and increasing the dispersion of refractoriness. 78,79 On the other hand, sympathetic stimulation also shortens the atrial refractory period 75,77,80,81 due to camp dependent shortening of the plateau phase, 38,39 although in the experimental setting sympathetic activation had little effect on the inducibility of AF. 82 Nevertheless, Coumel described patients with a typical vagal or adrenergic form of paroxysmal AF. 83 We have demonstrated that the autonomic nervous system may be important in modulating recovery from electrical remodeling 84,85 (chapter 6). Using parameters of heart rate variability, we demonstrated that in goats with a high vagal tone after cessation of 24 hours of rapid atrial pacing recovery from electrical remodeling was significantly reduced, compared with goats with a low vagal tone, while the autonomic status did not seem to influence the time course of electrical remodeling during the first 24 hours of rapid atrial pacing. During high vagal tone, activation of the acetylcholine-dependent potassium channel I kach will result in shortening of the atrial refractory period 86 and probably slow down recovery from electrical remodeling. Furthermore, vagal activation inhibits camp production which leads to dephosphorylation of pospholamban, reducing the activity of SERCA. This would decrease the diastolic re-uptake of calcium by the sarcoplasmic reticulum, prolonging cytosolic calcium overload. Likewise, other neurohormones such as catecholamines, natriuretic peptides and the reninaldosterone-angiotensin system, but also nitric oxide affect intracellular calcium handling and therefore may modulate atrial electrical remodeling. An indication that neurohormonal modulation has an effect on the rate of atrial remodeling during atrial tachycardia and the recovery from atrial electrical remodeling after cessation of tachycardia can be derived from the comparison of different studies performed with or without autonomic blockade. First Goette et al 64 induced electrical remodeling during 7 hours of rapid atrial pacing at 800 beats per minute (bpm) in dogs during total autonomic blockade. In their study verapamil completely blocked electrical remodeling, while in our goat study verapamil only reduced electrical remodeling during rapid atrial pacing at 300 bpm but in the absence of autonomic blockade. 65 Similarly, after cessation of rapid atrial pacing (780 bpm for 8 weeks) in dogs Lee et al 87 studied recovery from electrical remodeling during 48 hours of autonomic blockade. A full recovery of normal refractory periods occurred within 28 hours. By contrast, Elvan et al 88 studied recovery from electrical remodeling in dogs after 2-6 weeks of rapid atrial pacing-induced AF. They only temporarily applied autonomic blockade during Atrial Electrical Remodeling From Barn To Bedside 175

15 Figure 3 Relation between the duration of AF or rapid atrial pacing and the time needed to return to normal refractory periods as reported by several studies in animals ( ) and humans ( ) the measurements and normalization of the refractory period took more than 1 week. This suggests that total autonomic blockade may speed up recovery from electrical remodeling. Electrical Remodeling and Conduction Velocity and Dispersion of Refractoriness Apart from shortening of the refractory period which was described in all animal models of tachycardia-induced atrial electrical remodeling, studies examining other parameters that are expected to be of importance for the induction and perpetuation of AF, such as dispersion of refractoriness and the conduction velocity, showed conflicting results. In Wijffels goats the maximal spatial dispersion in fibrillation intervals did not significantly increase after 24 hours or 2 weeks of artificially maintained AF, 2 while Gaspo et al 43 showed a significant increase in AF cycle length variability after 1 day and 1 and 6 weeks of rapid atrial pacing in dogs. Furthermore, while Fareh et al 89 demonstrated a tachycardia-induced increase in the spatial dispersion of atrial refractory periods after 24 hours of rapid atrial pacing, we found no change in dispersion of refractoriness during 24 hours of atrial remodeling in goats while dispersion increased after cessation of rapid atrial pacing, ie during recovery from electrical remodeling 85,90 (chapter 6). Similar to our results, regional differences in the time course of recovery from electrical remodeling after cessation of 8 weeks of rapid atrial pacing in dogs also induced a temporary increase in the 176 CHAPTER 10 General Discussion

16 dispersion of refractoriness during the first 24 hours following cessation of pacing, which was related to an increased inducibility and duration of secondary episodes of AF during that time. 87 Likewise, some groups reported slowing of conduction velocity after longer periods of atrial tachycardia or AF, 43,88 while in other studies this could not be demonstrated. 2,65 On the other hand, molecular biologic studies revealed a tachycardia-induced reduction in connexin 43 in dogs 91 and an increased dispersion of connexin 40 expression in the atria of Allessie s fibrillating goats. 92 Therefore, it is still questionable to what extent changes in conduction velocity and dispersion of refractoriness add to the concept of electrical remodeling. Nevertheless, from clinical studies it is clear that both an increased dispersion of refractoriness 93,94 as well as conduction slowing are associated with AF. However, it remains controversial whether these arrhythmogenic factors not only cause AF, but also could result from AF, as is the case with the shortened refractory period. Time Dependence of Tachycardia-induced Electrophysiological Changes So far, none of the results from pharmacological studies disprove the hypothesized model for the functional ionic mechanisms which could be responsible for the initiation of electrical remodeling during the first minutes to 24 hours of AF or rapid atrial pacing. Findings from histological and molecular biological studies investigating arrhythmia-induced morphological changes and alterations in channel expression and function after longer periods of tachycardia indicate subsequent long-term structural changes, which are described below. Figure 3 exhibits the relation between the duration of tachycardia and the duration till normalization of the refractory period after restoration of sinus rhythm. It shows that with episodes of atrial tachycardia ranging from 7 minutes to 7 hours, recovery to normal refractory periods occurs within minutes, 3,64,66,67,98 while in case the atrial tachycardia has lasted for 24 hours or more (up to 6 years!), normalization of the refractory period occurs within 1 to 4 days. 2,65,87,88,99 This suggests that after longer periods of AF, structural rather than functional or metabolic changes come into play. Therefore, Allessie proposed a classification of atrial adaptation to heart rate depending on the duration of the arrhythmia: 100 a. Short-term adaptation (metabolic; seconds-minutes); depending on ion concentrations, ion-pump activities and phosphorylation of ion channels. b. Moderate-term adaptation (electrical remodeling; hours-days); depending on altered gene expression and changes in protein synthesis, assembly and degradation. c. Long-term adaptation (contractile remodeling; weeks); degenerative changes of normal subcellular structures, resembling hibernation. d. Very long-term adaptation (anatomical remodeling; months-years); fibrosis, fatty degeneration. Atrial Electrical Remodeling From Barn To Bedside 177

17 Figure 4 A-C: Action potentials recorded at 0.1 ( ) and 2 Hz ( ) in cells from a sham operated dog (A), from a dog after 42 days of rapid atrial pacing (B), and from a sham operated dog after exposure to nifedipine (C). Note that in panel B the action potential shortened and that the normal adaptation of the action potential to rate disappeared. These long-term adaptations of the action potential to 42 days of atrial tachycardia could be mimicked by blocking the L-type calcium channels (C). D: Action potentials from a 42 days paced dog before and after administration of BayK8644 E and F: Action potentials recorded at 0.1 Hz before (control; Ctl ) and after administration of 4 aminopyridine (4AP) in a low ( ) and high ( ) dose in a non-paced cell without (E) and with (F) the continuous presence of nifedipine. (Adapted from Yue et al, 103 Circulation 1997) 178 CHAPTER 10 General Discussion

18 However, one should keep in mind that the long-term changes may be the result of a short-term metabolic adaptation, i.e. the structural changes occur in reaction to (the stimulus that induced) the functional adaptations. Therefore, it should be emphasized that the classes are arbitrarily chosen and that in fact adaptation to an increased heart rate consists of a continuum with on one end the metabolic changes and on the other end the irreversible structural changes. Short-term and moderate-term adaptations to atrial tachycardia The tachycardia-induced increases in intracellular sodium and calcium concentrations all are expected to occur within minutes of AF or rapid atrial pacing. Therefore, only the experiments by Olsson et al, 98 Daoud et al, 3,66 and Yu et al 67 presumably truly represent these early pathophysiological processes. The experiments of moderate duration by Goette et al 64 (7 hours) and Jayachandran et al 70 (6 hours) were probably too short to induce structural alterations (recovery from electrical remodeling in 30 minutes) 64. In contrast, after 24 hours of rapid atrial pacing in our goats, 65,69,84,101 genetic reprogramming of the cell might have changed the expression of ion-channels and proteins in the cell membrane and sarcoplasmic reticulum in order to adapt to the tachycardia. This would explain why after cessation of pacing it takes more than 1 day before the atrial refractory periods return to their baseline values. 65,69,84,101 However, verapamil reduced the shortening of the refractory period in a large variety of different experiments ranging from minutes 20,66,67 to hours of tachycardia. 64,65 This illustrates that reduction of calcium inflow not only reduces the early functional changes but also might prevent genetic reprogramming, possibly by removing its common trigger, which could well be calcium overload. Long-term Electrical Remodeling in experimental models of AF Like the original studies by Morillo et al 1 and Wijffels et al, 2 the extensive set of experiments by the group of Nattel also examined moderate to long-term changes in atrial electrophysiology due to rapid atrial pacing for 1, 7 and 42 days in dogs. The changes described after this long duration of tachycardia indicate further adaptation of the cells to the changes in atrial rate and also point to changes in calcium handling. First, Gaspo et al 43 demonstrated that tachycardia-induced atrial electrical remodeling is associated with an increase in the number of circulating wavelets, due to a decrease of the atrial refractory period and the conduction velocity. The decrease in conduction velocity occurred somewhat later than the changes in refractoriness and was suggested to account for the additional increases in AF sustenance after shortening of the refractory period had become maximal. 43 In a subsequent study Gaspo described a progressive reduction in the sodium current (I Na ) after 7 and 42 days of tachycardia, which paralleled the decrease in conduction velocity, and accounted for the increase in AF vulnerability. 44 As was shown previously, this decrease in I Na could be the result of intracellular calcium overload, 102 although it may also be an adaptation to the increased sodium load during tachycardia. Atrial Electrical Remodeling From Barn To Bedside 179

19 In the same animal model, Yue et al 103 demonstrated that after 1, 7 and 42 days of rapid atrial pacing there was a significant and progressive reduction of the L-type calcium current density (I Ca ) and transient outward current (I to ) density, without concomitant changes in kinetics or voltage dependence, suggesting a decrease in the number of functional I Ca and I to channels. Other important ionic currents such as the inward and delayed rectifier currents, the T-type Ca current and the Ca-dependent chloride current (I to2 ) remained unchanged. Then they performed action potential voltage clamp studies to investigate the effects of pharmacological blockers of I to and I Ca on the action potential and the ionic currents flowing during the action potential (figure 4). It was demonstrated that blocking the L-type calcium current by nifedipine in unpaced atrial cells had similar effects on the action potential morphology and action potential duration adaptation to rate as occurring in atrial cells after 42 days of rapid atrial pacing (figure 4A-C). Furthermore, administration of the I Ca agonist Bay K 8644 was able to restore the plateau phase of rapidly paced atrial cells, although this usually did not completely normalize the action potential duration (figure 4D). These results suggest an important role of a reduced I Ca in the action potential abnormalities after prolonged periods of rapid atrial pacing. Experiments in which Ito was blocked using 4-aminopyridine revealed that the reduction of I to in the presence of strongly reduced I Ca was unlikely to have contributed importantly to the changes in the action potential morphology (figure 4 E and F). 103 The results from long-term experiments all point in the direction of changes in calcium handling. Apart from the functional effects of calcium overload (calciuminduced inactivation, opening of calcium activated potassium channels, etc) it may also cause a downregulation of the calcium and sodium channels. This will reduce the influx of calcium and sodium even long after the tachycardia has terminated and therefore would explain the 1 to 4 days period needed to recover from electrical remodeling (figure 3). A recent report from Nattel s group using the same dog model as described above showed a downregulation of mrna of the 1subunit of the L-type Ca-channel, the 1subunit of the Na-channel and of Kv4.3 (I to -channel), with the same time course as the reduction in the respective currents. 104 This suggests that downregulation of gene expression of important ionic channels may play a crucial role in moderate- to long-term tachycardia-induced atrial electrical remodeling. This transcriptional downregulation may have been caused by the initial increase in cytosolic calcium and sodium, and could be a way of the cell to protect itself against a potentially lethal calcium or sodium overload. Of note, Garratt and co-workers 105 recently investigated whether the AF-induced changes were subjected to cardiac memory, ie whether repeated 5 day episodes of AFinterrupted by 2 days of sinus rhythm altered the time course of the subsequent episodes of AF. The results of this study clearly demonstrated that atrial electrical remodeling during the different episodes was not influenced by the earlier episodes. Therefore, 2 days of sinus rhythm make forget 5 days of AF CHAPTER 10 General Discussion

20 Pharmacological Modulation of Long-term Electrical Remodeling As stated above, verapamil both reduced short-term contractile dysfunction 20,63 and adaptation of the refractory period to the increase in atrial rate 66,67 and moderate-term electrical remodeling. 64,65 It still remains to be investigated whether verapamil can reduce electrical and anatomical remodeling and contractile dysfunction after longer periods of AF. However, it is well possible that the different stadia of electrical and anatomical remodeling all result from the initial ionic changes and that prevention of these functional changes will also reduce long-term electrical and anatomic remodeling. Another question that remains to be answered is whether during late start of verapamil, ie after AF has been present for a longer period of time (eg several weeks), blockade of calcium entry still can reduce electrical remodeling. Analogous to the situation where it is of no use and potentially harmful to buckle-up after a car has crashed, it is conceivable that in the setting of a downregulated number of L-type calcium channels after a long period of AF, verapamil completely abolishes the plateau phase of the action potential, resulting in a further shortening of the refractory period. Preliminary results from experimental studies indeed show shortening of the AF intervals after administration of verapamil during AF. 106,107 However, the resulting decreased calcium entry may lower the cytosolic calcium load and in turn upregulate the L-type calcium channels. Eventually this will restore a plateau phase, while on the other hand the cytosolic calcium concentration may decrease and shortening of the refractory period due to other calcium activated processes may be reduced. Preliminary data from Olsson et al 108 point in this direction. Administration of verapamil in patients with chronic AF resulted in progressive lengthening of the atrial fibrillatory intervals in the course of several days to weeks. Furthermore, it remains to be investigated whether reduction of sodium overload by flecainide eventually also can prevent long-term tachycardia-induced downregulation of the Na-channels and slowing of the conduction velocity. 44 electrical remodeling in humans with AF Evidence Derived from the Clinical Course of AF To date, although there is no direct proof of the existence of electrical remodeling in humans, indirect evidence supporting the animal experimental findings is building up. Of course, atrial electrical remodeling would explain the progressive nature of AF and the decreasing efficacy of anti-arrhythmic treatments the longer the arrhythmia exists Furthermore, it explains why patients with rapid atrial activation due to other arrhythmias, such as atrial flutter or supraventricular tachycardia in the setting of the Wolf-Parkinson-White-Syndrome, are prone to develop AF In our study Atrial Electrical Remodeling From Barn To Bedside 181

21 evaluating the daily recurrence rate of AF after successful cardioversion we found an increased incidence of relapses of AF during the first week after cardioversion, with a sharp decline thereafter. This could be due to a vulnerable atrial substrate for the reinduction of AF, eg due to incomplete or asynchronous recovery from electrical remodeling during the first days after restoration of sinus rhythm, 113 similar to the situation after cessation of rapid pacing in instrumented goats 90 or dogs. 87 Furthermore, patients using calcium-lowering drugs during AF experienced significantly fewer recurrences of AF than patients who did not use these drugs, and who were mostly on monotherapy with digoxin. This is in agreement with the animal experimental findings evaluating pharmacological modulation of electrical remodeling ,69 Likewise, in 3 other studies concomitant treatment with verapamil during oral amiodarone 114,115 or quinidine 116 therapy increased the likelihood for cardioversion, possibly due to a reduction of electrical remodeling during AF. On the other hand, although many patients with paroxysmal AF eventually develop persistent AF, 4,5 it is a clinical fact that some patients with paroxysmal AF never deteriorate into persistent AF, while other patients with long-standing AF spontaneously convert into sinus rhythm, although usually within 7 days after AF onset. Furthermore, by definition patients with paroxysmal AF experience self-terminating episodes of AF,a phenomenon which is also not in agreement with the concept of electrical remodeling. Capucci and colleagues 117 indeed showed prolongation of the fibrillation intervals during AF, indicating an increase of the functional atrial refractory period, just before spontaneous conversion of AF. In patients with paroxysmal AF in whom AF persisted, the fibrillation intervals shortened. This suggests a multifactorial cause of the dynamic behavior of the atrial electrophysiologic characteristics in patients with AF, one of them being atrial electrical remodeling. Electrophysiologic Studies in Patients with AF Most findings from studies examining the electrophysiologic characteristics of the atria in patients with AF are in agreement with the animal experimental results. Already in 1972, long before electrical remodeling had been described, Olsson et al 118,119 demonstrated short right atrial monophasic action potentials in AF patients immediately after cardioversion. Furthermore, they found a positive correlation between the duration of the monophasic action potential and the tendency of the arrhythmia to recur. 118,119 One decade later, Attuel and co-workers 120,121 described a reduced rate adaptation of the atrial refractory period in patients susceptible to atrial arrhythmias, while Boutjdir and Le Heuzey et al 122,123 found short transmembrane action potentials and short atrial refractory periods without adaptation to rate in atrial appendages from patients with chronic AF and rheumatic heart disease undergoing surgery. But also in AF patients without an underlying heart disease the atrial refractory period was short compared with control patients in sinus rhythm, and the conduction velocity was decreased. 124 Till that time all investigators interpreted their findings as the electrophysiologic anomalies playing a crucial role in the cause of AF. 182 CHAPTER 10 General Discussion

22 Na-channel L-type Calcium to-current other repolarizing currents I f channel (mrna unless stated otherwise) I Na mrna I ca mrna prot I to1 mrna I kur Kv 1.5 I kach I kach I kach I katp I ksus I k1 mrna prot cur prot cur cur v Wagoner 126,127 v Gelder 128 Brundel 129,130 - Lai 131,132 Bosch Ohkusa 134 Na-channel, sodium channel; Ito1, 4-aminopyridine sensitive transient outward current; I kr, delayed rectifier current; I kur, ultra rapid delayed rectifier current; I kach, acetylcholine dependent potassium current; I katp, ATP dependent potassium current; I ksus, sustained outward current; I k1, inward rectifier current; I f, funny current; prot, protein ; cur, current. Atrial expression of channels, currents and proteins that are important for the electrophysiologic characteristics of patients with chronic AF. Table 1 After publication of the original articles by Wijffels 2 and Morillo, 1 Franz et al 125 were the first to demonstrate similar short atrial refractory periods and a reduced rate adaptation in patients with AF and atrial flutter when compared with sinus rhythm. However, they did not find a correlation between the duration of the arrhythmia and the duration of the refractory period or the action potential, which might be due to the long AF duration (3 weeks to 3 years). 125 The same was true for our study of the atrial refractory period in AF patients undergoing cardiac surgery (chapter 9). However, patients with paroxysmal AF had less shortening of the refractory period than patients with chronic AF and in the paroxysmal AF patients the refractory period was correlated with the duration of sinus rhythm after the last episode of AF (figure 2, chapter 9). This is suggestive for recovery from electrical remodeling, but also may represent the increased tendency of the atria to fibrillate in patients with a short atrial refractory period. In an attempt to study recovery from electrical remodeling after restoration of sinus rhythm we repeatedly measured the atrial refractory periods during the first week following surgery, using programmed electrical stimulation on temporary pacing electrodes that were left behind on the right atrium. Compared with the refractory periods during surgery, all patients with chronic AF had longer refractory periods during post-operative day 1, while sinus rhythm or paroxysmal AF patients showed more variation in their response. During the subsequent days the refractory period did not change significantly any more in any of the groups, which means that in case the prolongation of the refractory period indeed represents recovery from electrical remodeling, this process is completed within 24 hours after cardioversion of AF. These results are compatible with a previous study which demonstrated prolongation of the right atrial refractory periods when remeasured 4 weeks after cardioversion of lone AF. 94 Recently, results from a study by Yu et al, 99 who measured the atrial refractory Atrial Electrical Remodeling From Barn To Bedside 183

23 L-type Calcium SERCA PL RyR calseq Na/Ca-X channel I ca mrna prot mrna prot mrna mrna prot mrna mrna Van Wagoner 126,127 Van Gelder Brundel 129, Lai 131, Bosch 133 Ohkusa 134 SERCA, sarcoplasmic reticulum calcium ATPase; PL, pospholamban; RyR, ryanodine receptor; Calseq, calsequestrin; Na/Ca-X, sodium/ calcium-exchanger; prot, protein ; cur, current. Table 2 Atrial expression of channels, currents and proteins that are important for calcium handling in patients with chronic AF. periods with repeated cardiac catheterization during 1 week following electrical cardioversion, demonstrated complete recovery from electrical remodeling within 3 days after cardioversion. Molecular Biologic Studies of Human Atrial Tissue As in the animal studies, the altered electrophysiology in patients with long-lasting AF could be the result of changes in the sarcolemmal ion-channel expression. Therefore, several groups have investigated the ionic currents and expression of ionic channels and proteins that are important in the intracellular calcium handling and determination of the atrial refractory period in patients with AF (table 1). Van Wagoner et al 126 demonstrated that in atrial appendages obtained from patients with chronic AF undergoing cardiac surgery, there was a decrease in the calcium current density and the outward potassium current densities I to and I Ksus with a reduced mrna content of the delayed rectifier protein Kv In patients with persistent AF, and mostly NYHA III symptomatic valvular or coronary artery disease, Van Gelder et al 128 demonstrated an inverse relation between the duration of AF and the L-type calcium channel α1-subunit mrna content. Patients with AF < 6 months had a non-significant decrease of 26% (p=0.2) of the L-type calcium channel mrna content versus a decrease of 49% in patients with AF > 6 months (p=0.01). In this study, sarcoplasmic reticulum calcium ATPase, pospholamban and the Na-/Ca-exchanger mrna contents were not affected by AF. In a second study by our group, the gene expression of calcium-handling proteins in patients with paroxysmal AF and persistent AF (median duration 18, range 8-64 months) was compared with sinus rhythm patients. 129 Of note, most of these patients were in NYHA class I for exercise tolerance and had lone AF and therefore differed substantially from the study patients reported by Van Gelder. 128 In this study Brundel et al 129 demonstrated a reduction in both mrna and protein contents of the L-type 184 CHAPTER 10 General Discussion