Pharmacological exploration of the resting membrane potential reserve: Impact on atrial fibrillation

Author’s Accepted Manuscript Pharmacological exploration of the resting membrane potential reserve: Impact on atrial fibrillation Marcel A.G. van der Heyden, Thomas Jespersen www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(15)30363-0 http://dx.doi.org/10.1016/j.ejphar.2015.11.026 EJP70341

To appear in: European Journal of Pharmacology Received date: 28 August 2015 Revised date: 6 November 2015 Accepted date: 16 November 2015 Cite this article as: Marcel A.G. van der Heyden and Thomas Jespersen, Pharmacological exploration of the resting membrane potential reserve: Impact on atrial fibrillation, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.11.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

EJP-42564R2

Pharmacological exploration of the resting membrane potential reserve: impact on atrial fibrillation.

Marcel A.G. van der Heyden1,*, Thomas Jespersen2 1

Department of Medical Physiology, Division of Heart and Lungs, University Medical Center

Utrecht, Yalelaan 50, 3584 CM Utrecht, Netherlands. 2

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of

Copenhagen, Copenhagen, Denmark

Words 5062 (without references), Figures 2, Tables 1 Language: English (U.S.) Disclosures: MvdH: none; TJ: none

*corresponding author Dr. Marcel A.G. van der Heyden Dept. of Medical Physiology, Division of Heart & Lungs, Yalelaan 50, 3584 CM Utrecht, The Netherlands phone: ..31 30 2538901, fax: ..31 30 2539064, email: [email protected]

Abstract The cardiac action potential arises and spreads throughout the myocardium as a consequence of highly organized spatial and temporal expression of ion channels conducting Na+, Ca2+ or K+ currents. The cardiac Na+ current is responsible for the initiation and progression of the action potential. Altered Na+ current has been found implicated in a number of different arrhythmias, including atrial fibrillation. In the atrium, the resting membrane potential is more depolarized than in the ventricles, and as cardiac Na+ channels undergo voltage-dependent inactivation close to this potential, minor changes in the membrane potential have a relatively large impact on the atrial Na+ current. The atrial resting membrane potential is established following ionic currents through the inwardly rectifying K+ currents IK1, IK,ACh and IK,Ca and to a lesser extent by other ion channels as well as by exchangers and pumps. This review will focus on the relative and regulated contribution of IK1, IK,ACh and IK,Ca, and on pharmacological modification of the channels underlying these currents in respect to the resting membrane potential, Na+ channel availability and atrial electrophysiology in health and disease. Keywords: IK1; IK,ACh; IK,Ca; INa, resting membrane potential; atrial fibrillation

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1. Introduction Atrial arrhythmia, and in particular atrial fibrillation (AF), is an increasing health problem with the aging population (Lloyd-Jones et al., 2004; Krijthe et al., 2013). While the mainstay of current treatment of AF is rate and rhythm control pharmacotherapy, ablation of the atrial conduction pathway and anti-coagulation therapy (Camm et al., 2012), the present treatment regimes often are inadequate (Gillis et al., 2013). Therefore, development of novel pharmacological strategies based on mechanistic modulation of atrial excitability is of an imperative necessity as potential novel therapeutic options. The resting membrane potential (RMP), defined as the relatively stable potential between action potentials, also termed diastolic potential, plays a central role in controlling a number of electrophysiological parameters in the atrium and changes in this potential may prove pivotal in controlling excitability of the atrium and thereby define whether the atrium is prone to arrhythmia. Pharmacological modification of the ion channels underlying the RMP may therefore present new treatment options in AF. However, in cases with overlap in expression between atrium and ventricle it is possible that modulation of such a basic property as RMP may induce severe adverse effects in ventricular tissue. On the other hand, the RMP is generated due to activity of a series of ion channels and transporters, which may compensate each other their functions. The cardiac Na+ current, conducted through Nav1.5 voltage-gated channels, undergoes voltagedependent inactivation, a so-called state-dependent inactivation, at depolarizing potentials (Schneider et al., 1994; Petitprez et al., 2008). In non-diseased human atrium, RMP has been found to be approximately -75 mV, while the potential in atrial tissue from patients with chronic AF is around -79 mV (Christ et al., 2008). As these values are very close to the half inactivation of the cardiac Nav1.5 channel, and as it is the Na+ current that is strongly associated with the progression of action potentials, a minor change in RMP will have a large impact on the electrical impulse of the atrium (Schneider et al., 1994). Reduced Na+ current will both slow the

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conduction velocity of the action potential and prolong the refractory period between action potentials (Schneider et al. 1994).

2. RMP reserve Three different non-voltage-gated cardiac K+ currents - IK1, IK,ACh, and IK,Ca - are important in controlling RMP (Fig. 1.). These currents partly overlap in temporal expression both during the later part of the action potential and during diastolic repolarization and do therefore constitute a RMP reserve, i.e. a decrease in one of the currents will have minor effect on the potential due to a buffering, or even increased activity, of the others. Still, an increased current from any of these three K+ channels will, modestly, hyperpolarize the RMP and thereby increase conduction velocity and decrease refractoriness, while a reduction will have the opposite effect. As the halfinactivation of cardiac Na+ channels is close to atrial RMP (Schneider et al., 1994; Petitprez et al., 2008), small changes in the atrial RMP will have a relatively profound effect on the Na+ channel availability and thereby be closely coupled to the genesis of AF. In the following molecular identity, electrophysiology and pharmacology of these three inwardly rectifying, non-voltage-gated K+ currents will be described. This will be followed by discussion of the extent to which pharmacological inhibition of these currents may alter RMP electrophysiology, and how this may be beneficial in the context of AF.

2.1. IK1 The IK1 channel is an inward rectifier which is fully open around the reversal potential of K+ (-90 mV) but non-conducting at depolarized potentials above -40 mV (Hibino et al., 2010; Tang et al., 2015) (Fig. 1.). This gives IK1 an important role during the late repolarisation of the cardiac action potential as well as in stabilizing the RMP. IK1 is present in all excitable cells, including

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cardiac, skeletal and smooth muscle (De Boer et al., 2010a). IK1 is conducted through tetrameric protein complexes composed of Kir2.1, Kir2.2, and Kir2.3 proteins, either as homomeric or heteromeric complexes (Preisig-Müller et al., 2002). Kir2.x proteins are highly conserved throughout evolution (Houtman et al., 2012 and 2014). Although Kir2.1 is most abundant in the human heart, the expression of the different subtypes differs between cell types and thereby biophysical properties of IK1 can be specifically adapted to the specific requirements of the tissue in question. In canine ventricular cardiomyocytes, IK1 is mainly dependent on Kir2.1, whereas in atrial myocytes Kir2.1, Kir2.2 and Kir2.3 are more equally expressed (Cordeiro et al., 2015). Similar findings have been made in other species (reviewed in De Boer et al., 2010a). The concept of RMP reserve can be appreciated from a vast amount of experimental evidence in which Ba2+ was used to inhibit IK1. At relatively low concentrations, e.g. 10 mM, Ba2+ is rather specific for the Kir2.x-carried IK1 and provides >70% blockade (Biliczki et al., 2002) (Table 1). For example, 10 mM BaCl2 applied to guinea-pig papillary muscle preparations did not change RMP (-82 mV vs -83 mV), whereas action potential duration was significant prolonged (action potential duration at 90% of repolarization (APD90) 187 vs 167 ms) (Wang et al., 1993). Similar findings were obtained in canine papillary muscle, in which again 10 mM BaCl2 prolonged APD90 by 9-11% without affecting RMP (Biliczki et al., 2002; Nagy et al., 2013). Interestingly, in human right ventricular muscle preparations neither APD90 nor RMP was significantly affected by 10 mM BaCl2 (Nagy et al., 2013). Finally, in Langendorff perfused rat hearts, a 10fold higher concentration of BaCl2 (100 mM) resulted in APD90 prolongation by almost 3-fold in left ventricular subepicardial cells compared to baseline, whereas RMP was depolarized by approximately 2 mV (Baiardi et al., 2003). BaCl2 concentrations from 50 mM and higher were able to induce RMP depolarization and automaticity in isolated guinea-pig ventricular cells and multicellular preparations from guinea pig and dog (Malécot et al., 1984; Hirano and Hiraoka, 1988; Shen et al., 1996). Using the specific IK1 inhibitor PA-6, no change in RMP of isolated

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canine ventricular cardiomyocytes was observed at concentrations that inhibited IK1 by >70% (Takanari et al., 2013). As indicated above, the molecular constituents of IK1 in atrial cells are somewhat different than that from ventricular cells (De Boer et al., 2010a). Nevertheless, 10 mM BaCl2 did not affect RMP in stretched isolated atrial cardiomyocytes from guinea pig (Qi et al, 2009). On the other hand, Escande and colleagues report a significant RMP depolarization (~10 mV) in human atrial appendage tissue upon treatment with 50 mM BaCl2 (Escande et al., 1986). However, it should be noted that 50 mM BaCl2 will not only block IK1 but also have a profound effect on IK,ACh and IK,Ca (Table 1), thereby reducing all three primary conductances in the atrial RMP reserve. In conclusion, specific inhibition of IK1 with 10 mM Ba2+ (>70% blockade) does not, or maybe to a very low degree, affects RMP in ventricular cardiomyocytes and preparations, whereas APD is significantly prolonged. Further, the scarce information available from atrial cells and tissue points in the same direction.

2.2. IK,ACh The G protein-coupled inward rectifier K+ current IK,ACh is, like IK1, a non-voltage-gated inward rectifier current (Hibino et al., 2010) (Fig. 1.). However, while IK1 does not produce any significant current at potentials more depolarized to -40 mV, IK,ACh also conducts a relatively large current at depolarized potentials (Tang et al., 2015). The primary constituents underlying cardiac IK,ACh are G-protein Inward Rectifier K+-channel protein 1 (GIRK1) and 4 (GIRK4), also named Kir3.1 and Kir3.4, respectively. These proteins are highly expressed in nodal and atrial tissues (Gaborit et al., 2007; Atkinson et al., 2013), but have also been reported in both rodent and human ventricular tissue (Yang et al., 2010; Liang et al., 2014). In human atrium, IK,ACh has a minor permanent conducting component (Dobrev et al., 2005). Following G protein-coupled receptor activation, the open probability of these channels is profoundly increased (Logothetis et al., 1987). Originally, IK,ACh was found to be activated by ACh following vagal stimulation

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(Koumi and Wasserstrom, 1984), but it has been shown that this current can be activated by several different Gi/o coupled receptors, including adenosine receptors (Belardinelli and Isenberg, 1983; Wang et al., 2013), S1P3 sphingolipid receptors (Bünemann et al., 1995 and 1996), endothelin-A receptors (Kim, 1991; Yamaguchi et al., 1997) and somatostatin receptors (Lewis and Clapham, 1989). Thus, despite this current being named according to its regulation through the vagal ACh-muscarinic pathway it is likely that other transmitter pathways may be important in regulating IK,ACh too. Tertiapin, TertiapinQ and NTC-801 are selective IK,ACh blockers in the low nanomolar range (Jin and Lu, 1998, 1999; Kitamura et al., 2000; Machida et al., 2011) (Table 1). NTC-801 did not have significant effects on the RMP in guinea-pig papillary muscle preparations in concentrations up to 30 mM, nor in guinea-pig atrial or ventricular cells at 100 nM (Machida et al., 2011). In canine ventricular endocardial, epicardial and Purkinje fiber preparations treated with ACh, 100 nM Tertiapin did not affect the RMP, in contrast to atrial preparations where Tertiapin induced a slight RMP depolarization (Calloe et al., 2013). Upon treatment of guinea-pig atrial cells with aconite, an activator of depolarizing Na+ current resulting in a RMP decrease from -78.6 to -70.9 mV, Tertiapin at 30 nM caused strong RMP depolarization to -40.2 mV, whereas in non aconite-treated cells, Tertiapin had no effect (Suzuki et al., 2014), emphasizing the role of IK,ACh in conditions in which a stable negative RMP is challenged. In rat papillary muscle preparations stimulated at 1 Hz, 20 nM TertiapinQ depolarized RMP (-76.3 mV vs -80.2 mV), whereas at more physiological frequencies (5 Hz) no significant effects were observed (Liang et al., 2014). In contrast, in rat right atrium paced at 5 Hz, 60 nM of TertiapinQ was able to depolarize RMP (-76.3 mV vs -83.2 mV) (Wang et al, 2013). Finally, in mouse right ventricle and rat right atrium, ACh-induced hyperpolarization could be reversed by TertiapinQ application (Liang et al., 2014). Overall, we can conclude that

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specific inhibition of IK,ACh tends to be associated with a mild (1-5 mV) RMP depolarization in both non-treated and ACh-treated preparations.

2.3. IK,Ca Small conductance Ca2+-activated K+ (SK) channels underlying IK,Ca, are unique among ion channels due to their ability to sense changes in intracellular Ca2+ concentration and couple this to cellular electrical activity (Köhler et al., 1996). SK channels are widely expressed in the body. Cardiac IK,Ca was first shown by Giles and Imaizumi who observed larger Ca2+-activated K+ currents in rabbit atrial than ventricular myocytes (Giles and Imaizumi, 1988), which was later confirmed in mouse and human myocytes (Xu et al., 2003). Today, functional atrial IK,Ca has been described in a number of species, including rat, guinea pig, rabbit, dog and horse (Diness et al., 2010; Qi et al., 2014; Haugaard et al., 2015). The tetrameric SK channel complex can be formed either as homomers or heteromers from SK1, SK2 and SK3, all of which are reported to be expressed in the heart (Xu et al., 2003; Tuteja et al., 2005; Ozgen et al., 2007; Skibsbye et al., 2014). The intracellular C-terminal domain of the SK proteins constitutively binds calmodulin, serving as the Ca2+ sensor (Fig. 1.). The three SK channel subtypes are extremely sensitive to rises in Ca2+ with similar Ca2+ activation sensitivities with a half maximum around 300 nM [Ca2+]i and a Hill coefficient of 4-5 (Xia et al., 1998). The activated SK current shows a low degree of inward rectification, resulting in a significant conductance through this channel, if activated, also at depolarized potentials (Fig. 1.). SK channels are selectively blocked by low concentrations of the bee venom toxin apamin (Blatz and Magleby, 1986). Apamin has SK channel subtype specificity ranging from 100 pM to 10 nM (Xia et al., 1998). Other toxins, including scyllatoxin and tamapin, also block IK,Ca (Stocker, 2004). Lei-Dab7, which is a synthetic derivative of leiurotoxin has been reported to selectively block SK2 channels (Shakkottai et al., 2001). The small molecule inhibitors UCL1684 and

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ICAGEN (N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine) are, like the neurotoxin, pore blockers (Dunn, 1999; Skibsbye et al., 2014). This is in contrast to the negative allosteric modulator NS8593 that decreases the Ca2+ sensitivity by shifting the activation curve for Ca2+, but only marginally affects maximal activated IK,Ca (Strøbaek et al., 2006). With 1 µM NS8593 and 1-10 µM ICAGEN, both of which are reported to be specific at these concentrations, a reduced IK,Ca was reported in isolated human atrial cells, while no effect was observed in isolated ventricular myocytes (Skibsbye et al., 2014). In the same study, action potential recordings in multicellular human atrial preparations also revealed an effect with 10 µM ICAGEN and 30 µM NS8593. With both compounds the action potential duration and effective refractory period were prolonged, and RMP depolarized by 2-4 mV. The electrophysiological parameters in isolated human ventricular cells and muscle strips were not reported altered following NS8593 and ICAGEN application. Although IK,Ca does not seem to play a significant role in non-diseased ventricular electrophysiology, a number of publications have recently described up-regulation of IK,Ca in heart failure (Chua et al., 2011; Chang et al., 2013; Lee et al., 2013). Pharmacological treatment of ventricular fibrillation in Langendorff rat hearts with the negative allosteric modulator NS8593 was able to revert VF in 53% of the hearts tested, and was able to prevent reinduction of VF (Diness et al., 2015). In contrast, the poreblocker ICAGEN was found less effective in preventing VF reinduction and it was unable to revert VF in this model. These findings suggest a role for IK,Ca during ventricular arrhythmia (Diness et al., 2015).

2.4. Potential contribution of additional ion channels and transporters to the RMP Additional ion currents and transporters may also contribute to the RMP and thus have a role in establishing the RMP reserve. Ouabain is a Na+/K+-ATPase inhibitor in the low nanomolar range for human a1, a2 and a3 subtypes (Crambert et al., 2000) as well as many other species, with

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the exception of the rat a1 (Jewell and Lingrel, 1991; reviewed in Blanco and Mercer, 1998), which has an impact on the concentration gradient of Na+ and K+ across the membrane. Ouabain (0.2 mM, 23.5 min) significantly depolarized RMP in adult canine Purkinje fibers (75 mV vs 89.2 mV) (Rosen et al., 1975). Interestingly, the authors showed that RMP sensitivity for ouabain was lower in preparations from neonates and juvenile dogs, whereas senescence animals displayed even larger changes in RMP (Hewett et al., 1982). In guinea-pig papillary muscle, ouabain at 1 mM induced a mild RMP depolarization measured at 6 min (-82.5 mV vs -84.5 mV) (Yang et al., 1992). SEA0400 is a specific inhibitor of the Na+/Ca2+-exchanger NCX (IC50 ~30-90 nM) (Lee and Hryshko, 2004). NCX contributes to cytoplasmic Ca2+ extrusion following cardiac contraction. No effect on the RMP was observed following 1 mM SEA0400 application to isolated canine ventricular cardiomyocytes (Johnson et al., 2010; Bourgonje et al., 2013). The cardiac Na+-H+-proton exchanger (NHE-1) regulates intracellular pH and can be inhibited by cariporide (IC50 ~0.1-1 mM) (Dhein and Salameh, 1999). Guinea-pig cardiac muscle preparations treated with 1 mM cariporide for 10 min did not display any significant change in RMP (-76 vs -77 mV), whereas APD90 was prolonged (212 vs 196 ms) (Dhein et al., 1998). Hence, of the main cardiac ion transporters, only modulation of the Na+/K+- ATPase appears to change the RMP in a way that most likely results from slight alteration in intracellular ion concentrations. However, it should be noted that intracellular ion concentrations become only affected in the presence of Na+/K+-ATPase inhibitors after a significant period of action potential formation, which may explain the discrepancy between short and long term ouabain treatment on RMP. K2P3 channels (TASK) are two-pore domain K+ channels. These channel proteins combine two pore-forming domains in one single polypeptide and hence a protein dimer can form a functional channel. Pharmacological inhibition of the K2P3.1 channels by 200 nM of A293 (IC50 ~195-220

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nM; Putzke et al., 2007) did not affect RMP in human atrial cardiomyocytes isolated from patients in sinus rhythm or atrial fibrillation (Schmidt et al., 2015).

3. Atrial electrophysiology (RMP and Na+ channel function) In human atrial tissue obtained from hearts beating at normal sinus rhythm, RMP has been found to be around -75 mV, while atrial tissue suffering from chronic AF has a potential around -79 mV (Christ et al., 2008; Wettwer et al., 2004). As the steady-state inactivation of cardiac Na+ channels are approximately -70 mV, a 4 mV shift in the RMP will have a major impact on the availability of the cardiac Na+ channels (Schneider et al., 1994; Petitprez et al., 2008) (Fig. 2.). This means that a significant fraction of the Na+ channels are inactivated, and thereby nonconducting, in normal beating atria, while in AF the majority of the Na+ channels will be ready to conduct Na+ ions (released from inactivation) when the membrane is depolarized. Such a voltage-dependent inactivation is termed a state-dependent Na+ channel block. The Na+ current will thereby be expected to be much larger in AF than in non-diseased atria which may be expected to be pro-arrhythmic (Schneider 1994). It should be noted that neither the peak nor the late Na+ currents are, unlike many other atrial currents, changed in expression in AF (Poulet et al., 2015, Bosch et al., 1999), but voltage-dependent inactivation has been found to be shifted to more positive potentials in AF contributing to a larger Na+ current (Bosch et al., 1999). The proarrhythmogenicity of a large Na+ current is underlined by the fact that class I anti-arrhythmics, which are Na+ channel blockers, can be used to treat AF (Workman et al., 2011).

4. Atrial arrhythmia and RMP The most frequent cardiac arrhythmia seen in the clinic is AF. AF is associated with increased mortality and morbidity (Camm et al., 2012). The incidence of AF is increasing due to an increasing life expectancy. About 5% of 65 year olds and 10% of 75 year olds are expected to

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have AF (Krijthe et al., 2013). Longer periods of AF will provoke significant cellular remodeling in the atrium, making it difficult to revert the arrhythmia – a phenomenon known as "AF begets AF" (Wijffels et al., 1995). AF is often associated with remodeling, which can be both structural, often in the form of fibrosis, and electrical, as changes in the expression and/or function of several of the ion currents underlying the normally highly fine-tuned atrial action potential (Iwasaki et al., 2011). Thus, the atrial action potential often becomes shorter, more triangular and with shorter refractory periods, which is a measure of the minimum time interval between two action potentials. A shorter effective refractory period (ERP) increases the risk of continuous reactivation of action potentials giving rise to self-sustained circular propagation of the electrical conduction. This phenomenon is recognized as re-entry-based AF resulting in an atrial beating frequency of 300 to 600 pulses/min. It follows from the etiology of re-entry based AF that prolongation of the ERP will per se be anti-arrhythmic in the atrium. AF can also originate from so-called spiral waves where the center of a wave has a very high pulsing frequency (Iwasaki et al., 2011). Na+ channel blockage has been suggested to abrogate such spiral waves by slowing the conduction velocity, which is primarily defined by INa, obstructing the formation of these fast waves. Most of the presently used pharmacological treatment modalities are based on either prolonging the refractory period, by partly blocking the repolarising K+ currents (class III antiarrhythmics), or by decreasing the conduction velocity, by partly blocking the Na+ channels (class I anti-arrhythmics) (Workman et al., 2011). AF induces remodeling of a number of ionic currents in the atrium (Burstein B, Nattel, 2008; Schotten et al., 2011). While several of the K+ channels participating in shaping the action potential are down-regulated, it has consistently been shown, in both animal models and humans, that both IK1 and IK,ACh are up-regulated (Bosch et al., 1999; Dobrev et al., 2005). This fits very nicely with the 4 mV RMP hyperpolarization in AF tissue, as larger K+ currents will drive the potential closer to the reversal potential of K+ around -90 mV. SK channel expression has also

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been reported to change following AF. During the early stages of AF, an increased IK,Ca in rabbit and dog has been observed (Ozgen et al., 2007; Qi et al., 2014), while in patients with long-term AF, a decreased expression has been reported (Yu et al., 2012; Ling et al., 2013; Skibsbye et al., 2014). This could suggest an increased activity of SK channels caused by altered Ca2+ handling early in the AF, followed by a down-regulation when the disease progresses (Neef et al., 2010).

5. Pharmacological modification of IK1, IK,ACh and IK,Ca and atrial fibrillation In a number of experimental models, the pharmacological blockage of any of the three currents has shown promising effects in the perspective of AF treatment.

5.1. IK1 inhibition and AF At present only Ba2+ is available for whole animal AF studies. As indicated above, Ba2+ rapidly looses its IK1 specificity at higher concentrations, which results in a multitude of cardiac and extracardiac effects (Bhoelan et al., 2014) and therefore Ba2+ is unsuited for studies of AF in animals. Chloroquine which demonstrates IK1, IK,ACh and IKATP inhibiting activity, was able to counteract cholinergic and stretch-induced AF in the isolated sheep heart (Noujaim et al, 2010; Filgueiras-Rama et al., 2012).

5.2. IK,ACh inhibition and AF Both NIP-151 and Tertiapin dose-dependently terminated AF in canine models of rapid atrial pacing combined with vagal nerve stimulation and aconite-induced AF (Hashimoto et al., 2006 and 2008). This was accompanied by a preferential prolongation of the atrial ERP, whereas conduction and ventricular repolarization was not affected (Hashimoto et al., 2006 and 2008). AVE1231, a combined IK,ACh and IKur/Ito blocker, prolonged the atrial ERP in goats after 72 h of atrial tachypacing, but its efficacy in cardioversion has not been addressed (Wirth et al., 2007).

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NTC-801 has been evaluated in three canine AF models (vagal nerve stimulation-induced AF, aconite-induced AF and left or right rapid atrial pacing-induced AF) (Machida et al., 2011; Yamamoto et al., 2014). The authors report effective anti-AF activity (decreased AF inducibility, dose-dependent cardioversion) combined with prolongation of atrial ERP. NTC-801 reached clinical phase II, but for unknown reasons further development was discontinued (Zhao and Xiang, 2015). Finally, AZD2927 and A7071 displayed strong prolongation of atrial ERP and profound conversion rates to sinus rhythm in dogs with rapid right atrial pacing-induced AF (Walfridsson et al., 2015). Upon application in patients with atrial flutter, however, no prolongation of the left atrial ERP was observed (Walfridsson et al., 2015) illustrating the problems when translating animal model system findings to human AF patients.

5.3. IK,Ca inhibition and AF In human lone AF, genetic variations in the gene encoding SK3 have been associated with AF (Ellinor et al., 2010). Nattel and colleagues found that NS8593 had anti-AF properties in dogs with atrial remodeling induced by 7-day atrial tachypacing (Qi et al., 2014) and likewise, acutely induced AF in horses was also reverted with NS8593 (Haugaard et al., 2015). In rodents, IK,Ca blockage by UCL-1684, NS8593 and ICAGEN can protect against, and abrogate acutely induced AF (Diness et al., 2010 and 2011). In freshly isolated human cardiac tissue, a prolongation of both action potential duration and ERP in both AF and non-AF tissue following ICAGEN and NS8593 blockage of IK,Ca have been found (Skibsbye et al., 2014). This supports the notion of SK channel blockage also being anti-arrhythmic in humans. In both rats and humans IK,Ca inhibition induces a 2-3 mV depolarization of the resting membrane potential (Skibsbye et al., 2014 and 2015). Though promising, the beneficial results of pharmacological intervention as indicated above in cell and animal models still need to be corroborated in clinical trials, an enterprise that not

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always leads to satisfying results, and in which many promising preclinical compounds become “stuck in translation” (Rongen and Wever, 2015).

6. Mechanistic considerations about AF treatment and IK1, IK,ACh and IK,Ca inhibition Although intensively studied, there is a lack in mechanistic understanding on how blockers of these three currents act anti-arrhythmic. As an RMP hyperpolarization in AF seems to be a common denominator, it is worth investigating whether restoring the RMP may protect atria against AF. Also, it is important to keep in mind that the minimal diastolic potential, which in this context maybe more appropriately should be named the off-set potential (i.e. of the activation of Na+ channels), is drastically depolarized during atrial arrhythmia. This was first shown, but not quantified, in isolated dog atria with atrial flutter (Burashnikov et al., 2014) and later in rat atria with atrial fibrillation/flutter reported to be around -60 mV (Skibsbye et al., 2015). At this potential, only a very small fraction of the cardiac Na+ channels would be available for activation, while the rest will be inactivated. However, at approximately -60 mV the fraction of available Na+ channels is presumably sufficient to support at new action potential. The increased activity of IK1, IK,ACh and IK,Ca reported in AF will lead to a faster repolarization, whereby potentials around – 60 mV are reached faster, whereby the tissue is faster released from conduction block (shorter refractory period) and a new action potential can progress through the tissue. Hence, the increase in K+ channel function associated with the RMP reserve will support shortening of time between subsequent action potentials. As a consequence, pharmacological inhibition of either of these channels may lead to reduced repolarization and thereby longer refractoriness, serving as an anti-arrhythmic mechanism. Further, for IK,Ca an additional factor can be proposed to play a role. During AF, the Ca2+ handling is compromised suggested to provide higher diastolic [Ca2+]i (Neef et al., 2010). As SK channels are activated by submicromolar concentrations of Ca2+, it is possible that IK,Ca is more

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or less constitutively active during AF. This will result in an increased repolarizing current, also at the peak of the action potential, as SK channels have a low degree of rectification (Tang et al., 2015). Such an increased repolarization will lead to a short refractory period, which may support an increased fibrillatory frequency. From this follows that if IK,Ca is inhibited, the action potential will be less depressed and longer, which will be an additional anti-arrhythmic mechanism.

7. Conclusions IK1, IK,ACh and IK,Ca contribute to the RMP reserve. Inhibition of only one of these currents at a time, has only very limited effects on the ventricular RMP and has not been associated with ventricular proarrhythmia. The atrial RMP, especially under disease conditions, appears more sensitive to blockade of each of these three currents. In general, blocking effects are more outspoken with respect to changes in APD and ERP lengthening than RMP. In AF, increased functional expression of these currents is likely associated with a mild hyperpolarization, which in turn increases Na+ channel availability. An increased Na+ current increases conduction velocity, shortens APD and ERP and thus promotes AF. Specific pharmacological inhibition of each of the currents contributing to the RMP, or maybe in combination, demonstrated anti-AF properties in which lengthening of the ERP is a common denominator.

Acknowledgements This work was supported by The Danish Council for Independent Research to TJ (DFF-133100313B).

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Figure legends

Fig. 1. Topology of single protein subunits constituting IK1, IK,ACh and IK,Ca currents. Kir2.x and Kir3.x proteins contain 2 transmembrane regions, an extracellular pore-loop and intracellularly located N- and C-termini. SK proteins cross the membrane six times and carry an extracellular pore loop between transmembrane region 5 and 6. Calmodulin binding occurs at the C-terminus. Strong rectification of IK1 is achieved by polyamine/Mg2+ pore block upon depolarisation, whereas less stringent rectification is observed for IK,ACh and IK,Ca channels (B).

Fig. 2. Voltage-dependent inactivation of cardiac Na+ channels. Topology of the Nav1.5 Na+ channel displaying a 4-subunit conformation of the pore forming a-subunit that interacts with modulating b-subunits (A). Na+ current traces transferred into current-voltage relationship, indicating maximal current flow around -30 mV (B). The inactivation profile of Nav1.5 channels (C). The approximate RMP of an atrium in sinus rhythm (sino-atrial nodal rhythm, SANR) and in atrial fibrillation (AF) is indicated by dashed lines. At a ventricular RMP of -85 mV (not illustrated) most of the Na+ channels will be released from inactivation and thereby ready to open when the next action potential arrives. In contrast, in AF atria a minor fraction of the channels will be inactivated while in non-diseased (SANR) atria a larger proportion of the channels will not be available for activation when an action potential arrives (Petitprez et al., 2008). A hyperpolarizing prepotential (-90 mV) results in a subsequent larger Na+ current than with a more depolarized (-65 mM) prepotential (D).

36

TABLE Table 1 Inhibitors of cardiac ion channel associated with RMP

Channel

Compound

other cardiac targets (IC50)

reference

IK1

Ba2+

mM), IKATP (1 mM)

IC50

Affinity to

0.15-40 mM.

IKAch (10-92

Hibino et al., 2010

Coetzee et al., 1999 Pentamidine1

170 nM2

IKr (252 mM)

De Boer et al., 2010b 14 nM2

PA-6

Takanari et al., 2013

Ba2+

IKACh mM), IKATP (1 mM)

10-92 mM

IKAch (10-92

Hibino et al., 2010

Coetzee et al., 1999 Tertiapin

8 nM Jin and Lu, 1998, 1999

Kitamura et al., 2000

37

TertiapinQ

13 nM Jin and Lu, 1999

NTC-801 Ito (11.6 mM), IKATP (7.8 mM)

5.7 nM

INa (8.3 mM),

Machida et al., 2011

NIP-151

1.6 nM

IKr (57.6 mM)

Hashimoto et al., 2008

AVE1231 IKur (3.6 mM)

8.4 mM

Ito (5.9 mM),

Wirth et al., 2007

A7071

590 nM

IKr (>33 mM), IKur, Ito, IKs, INa and ICa,L

Walfridsson et al., 2015

(all > 100 mM)

AZD2927 INa (100 mM), IKr, Ito, IKs

350 nM

IKur (>33 mM),

Walfridsson et al., 2015

and ICa,L (all > 100 mM)

IK,Ca

ICAGEN

0.3-0.5 mM

Ito (21mM), INa (~30 mM)3

Gentles et al., 2008

Skibsb ye et al., 2014

38

NS8593 INa (5 mM), IKr (12 mM),

0.4-0.7 mM

ICa,L (2.5 mM ),

Strøbaek et al., 2006

IKACh (24 mM)

Skibsbye et al., 2014

UCL-1684

3-30 nM

IKAch (6 nM)

Dunn, 1999

Apamin

0.1-10 nM

IKAch (> 1 mM)

Xia et al., 1998

1

affects Kv11.1 maturation and plasmamembrane expression in chronic treatment; 2measured in inside/out

conditions (neglecting membrane penetrance); 3depending on frequency (Skibsbye et al., 2015).

39

Figure 1

Figure 1

0

B)

A)

IK1

-40

-20

0

20

IK,ACh

0

IK,Ca

Figure 2

A)

Topology

Na

Na+

+

Na

B) Na

+

Current-voltage relationship

+

Vm (mV) -60

ß Nav1.5 ß

-40

-20

0 -2

I (nA)

1 ms

-4 -6 -8

Na+

Voltage-dependent inactivation

Normalized current

C)

D)

INa as function of prepotential

RMP AF RMP SANR

1

Minimal diastolic potential during AF

0.5

-65 mV in prepotential

-100

-80

-60

Vm (mV)

-40

-90 mV in prepotential

20

40