Calcium-Activated Potassium Channels, Cardiogenesis of Pluripotent Stem Cells, and Enrichment of Pacemaker-Like Cells

Calcium-Activated Potassium Channels, Cardiogenesis of Pluripotent Stem Cells, and Enrichment of Pacemaker-Like Cells Alexander Kleger and Stefan Liebau*

Potassium channels represent the largest group of pore proteins regulating K⫹ efflux from the K⫹-rich inner cell to the extracellular compartment, thereby inducing changes in the membrane potential. Activity is regulated either by voltage or calcium concentrations, thus the nomenclature of voltage- and calcium-activated potassium channels. The critical role of potassium ion channels in developmental processes remains enigmatic, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, a series of studies delineated the precise function of calcium-activated potassium channels during cardiac, particularly pacemaker, cell development using human and mouse pluripotent stem cell models. It has become evident that this protein family not only regulates proliferation, apoptosis, and cell metabolism but also drives critical events during organ development such as the heart. This review summarizes the literature on calcium-activated potassium channels, their role in cardiac stem cell differentiation and development, and provides an outlook on how this process could be mechanistically regulated. (Trends Cardiovasc Med 2011;21:74-83) © 2011 Elsevier Inc. All rights reserved. • Introduction Ion channels represent a large group of pore proteins that play a variety of functions not only in the adult organism but also during embryonic development. Interestingly, the presence of excitable membranes responding to variations in their electrical membrane potential had been observed long before the first ion channel was described (Hodgkin and Huxley 1952). Even more surprising was Alexander Kleger is at the Department of Internal Medicine, Ulm University Hospital, 89081 Ulm, Germany. Stefan Liebau is at the Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany. *Address correspondence to: Stefan Liebau, MD, Institute for Anatomy and Cell Biology, Ulm University, Albert-Einstein Allee 11, 89081 Ulm, Germany. Tel.: (⫹49) 731 500 15580; e-mail: [email protected]. © 2011 Elsevier Inc. All rights reserved. 1050-1738/$-see front matter

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the finding that ion channels possessed a specificity and selectivity of the conducted ion through the membrane, based on their structure (MacKinnon 2004). Ion channels, unlike other pore proteins such as certain transporters, are passive and are mostly distinguished by their conducted ion, mechanism of activity, and conductance. The subgroup of potassium channels is expressed both in prokaryotes and in eukaryotes and forms the largest group of ion channels in the human genome. These channels are further divided by their gating mechanism, activated by either voltage or ligands (eg, Ca2⫹ and ATP) (Hille et al. 1999). The family of potassium-selective, calcium-activated ion channels comprises four members that either exhibit small (SK1, KCa2.1, Kcnn1; SK2, KCa2.2, Kcnn2; SK3, KCa2.3, Kcnn3) or

intermediate (SK4, IK, KCa3.1, Kcnn4) unitary conductance for K⫹ ions. The currents that are conducted by ion channels are described in picosiemens (pS) per single channel (or unit), whereas 1 S is equal to the reciprocal of 1 “Ohm.” For comparison only, large conductance potassium channels exhibit a unitary conductance of 100-220 pS, intermediate conductance channels 20-85 pS, and small conductance represents a current between 2 and 20 pS. The functional form of the ion pore is mediated by the combination of four subunits, respectively. Thereby, SK channels can be constructed by different subunits in vitro and in vivo. In addition, widely distributed functional splice variants of SK channels have been found throughout the organism (Shmukler et al. 2001, Strassmaier et al. 2005, Tomita et al. 2003). Intracellular Ca2⫹ is the only known physiological activator of SK channels. Ca2⫹ sensitivity is mediated by calmodulin, which constitutively binds to SK channels. Upon submicromolar elevation of the intracellular Ca2⫹ concentration, channel opening occurs within a few milliseconds (Xia et al. 1998). The functional ion channels possess the unique ability to transfer changes in the concentration of the intracellular signaling molecule calcium to membrane conductance of potassium and finally to the membrane potential (Stocker 2004). Many functional aspects have been revealed by the fact that all family members exhibit varying sensitivity to both activating and blocking molecules (Ishii et al. 1997a, Xia et al. 1998), presumably depending on homomeric or heteromeric assembly (Tuteja et al. 2010). Once activated, all SK channels can be kept in an open conformation by 1-ethyl2-benzimidazolinone (EBIO), its derivatives (eg, DC-EBIO), or structurally related compounds such as riluzole by increasing their apparent Ca2⫹ sensitivity, though with differing potency among the SK family members (Pedarzani and Stocker 2008). Whereas SK1SK3 can be selectively blocked by apamin and scyllatoxin (although with varying potency; Chicchi et al. 1988, Ishii et al. 1997b, Jäger and Grissmer 2004), SK4 is apamin insensitive and its activity can be inhibited by, for example, the compound clotrimazol (Pedarzani and Stocker 2008). TCM Vol. 21, No. 3, 2011

Initially, SK channels were found to mediate the afterhyperpolarization, influencing the intrinsic excitability of a variety of neurons, and to contribute to the refractory period (Stocker 2004). Thereby, the function of distinct SK channel subtypes in different neurons often results from their specific coupling to different calcium sources. More precisely, negative feedback systems exist in which Ca2⫹ entering the cell activates SK channels, which in turn close the sources of Ca2⫹ entry (Hallworth et al. 2003). This largely affects the threshold for the induction of long-term potentiation (LTP) and facilitates hippocampusdependent learning and memory processes (Behnisch and Reymann 1998). Furthermore, SK channels partake in a calcium-mediated feedback loop with NMDA receptors, controlling the threshold for induction of hippocampal LTP (Ngo-Anh et al. 2005). On the other hand, SK channels play important roles in multiple other cellular functions, namely in both cerebral and peripheral blood vessel smooth muscle cells. It has been shown that a vasodilatory mechanism that leads to smooth muscle hyperpolarization and subsequent vasodilatation is dependent on an intact endothelium, extracellular Ca2⫹, and activation of Ca2⫹-dependent K⫹ channels (Félétou and Vanhoutte 2006). In addition to the modulation of the membrane potential in various tissues and cell populations, these ion channels have been found to be involved in a number of biological processes, such as proliferation, cell differentiation, and cell morphology. Because these mechanisms are present in stem or progenitor cells of different origin and potency, a role in developmental processes can be hypothesized (Jäger et al. 2004, Liebau et al. 2007, Tharp et al. 2006, Wang et al. 2008). Furthermore, it has been shown for the Xenopus embryo that both the membrane potential and voltage gradients affect cell differentiation. Specifically, Pai et al. (2012) showed that bioelectrical communication specifies the type of new organ to be created at a particular location within a vertebrate organism. On the other hand, non-electrogenic roles have been described during heart development at least for cardiac sodium channels and L-type calcium channels (Chopra et al. 2010, Rottbauer et al. 2001). FurtherTCM Vol. 21, No. 3, 2011

more, it is well accepted that cell differentiation and maturation can affect the expression patterns of ion channels (Cingolani et al. 2002), but the role of ion channels, particularly SK channels, in developmental processes remains enigmatic in many aspects. • SK Channels and Cardiogenesis in Vitro It is well accepted that pluripotent stem cells represent a particularly attractive source for ex vivo generation of cardiomyocytes (Kolossov et al. 2006, Nelson et al. 2009). Based on extensive research on signaling pathways and by using techniques involving genetic engineering, cardiogenesis from murine and human pluripotent stem cells has been increased, and today fairly pure populations of cardiomyocytes can be generated. The signals involved comprise, for example, members of the Wnt pathway (Gessert and Kühl 2010) or the BMP pathway (Yuasa et al. 2005). Still, several major obstacles exist with regard to therapeutic or pharmaco-toxicological use of these generated cardiomyocytes, such as (1) avoiding the use of genetically modified stem cells, (2) immunotolerance in a transplantational setting, (3) efficiency, and (4) unguided subtype specification. Until we initiated our studies aiming to dissect the impact of SK channel activity on stem cell development and differentiation, little was known about the direct role of this channel family on developmental processes (Jäger et al. 2004, Wang et al. 2008). Using embryonic stem cells and induced pluripotent stem cells of mice (Kleger et al. 2010, Liebau et al. 2011b) and humans (Muller et al. 2011) as a developmental model system, we identified the SK channel family as a critical player for cardiomyocyte fate determination. Briefly, we increased SK channel activity using several chemical compounds (eg, EBIO) in a time-restricted regimen in pluripotent stem cells. This led to (1) rapid remodeling of the actin cytoskeleton, (2) inhibition of proliferation, (3) induction of differentiation, and (4) virtual elimination of any residual pluripotent cells. Further dissection of this phenomenon in a series of functional testings revealed the induction of cardiac mesoderm and commitment to the cardiac lineage. In addition, the differentiation into cardiomyocytes was mod-

ulated in a qualitative manner, resulting in a strong enrichment of pacemakerlike cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamberspecific myocardium (Kleger et al. 2010, Muller et al. 2011). Interestingly, both overexpression of SK4 (Liebau et al. 2011b) and elevated activity of SK channels resulted in the generation of highly cardiac-enriched cultures with a majority of cardiomyocytes exhibiting all hallmarks of functional cardiac pacemaker cells (Kleger et al. 2010, Muller et al. 2011). Timing and Dosing the Channels In our recent studies, we investigated the dose dependency of EBIO on the impact of initiating differentiation in pluripotent cells toward mesoderm and the cardiac lineage. There, the half-maximal effect was obtained at 0.1 mM, corresponding to its described EC50 value on SKCa activation (Pedarzani and Stocker 2008). Maximal effects were obtained at a concentration of 1 mM (Kleger et al. 2010). Different culture models have been used in our studies to differentiate pluripotent stem cells from mice and humans toward either mesoderm or functional cardiomyocytes: Pluripotent stem cells were differentiated in a twodimensional layer (referred to as the monolayer) upon withdrawal of pluripotency factors from the culture medium, whereas differentiation was initiated by the formation of three-dimensional aggregates, so-called embryoid bodies resembling the early embryo until gastrulation (Kleger et al. 2012, Linta et al. 2011, Stockmann et al. 2011, Wobus et al. 2001). Using these model systems, we found that when SK channel activity was increased immediately after cell seeding, every pluripotent cell reacted by stopping self-renewal and immediate differentiation toward a cardiac-prone cell type. Interestingly, despite strong differentiation, no beating clusters were detected even upon prolonged SK channel activation in this setting (Figure 1A). This holds true in both EB- and monolayer-based assays and points to a very strong mechanism directly interfering with the pluripotency machinery, such as the Erk signaling cascade. An appropriate balance between inductive and 75

preventive differential signals represents a hallmark of directed differentiation from pluripotent cells (Kattman et al. 2011, Ying et al. 2008). This seems to be particularly important for the cardiac system, for which precise timing and concentrations of each signal transducer are very importance (Kattman et al. 2011). Thus, we assume that a similar mechanism seems to be present for the expression and activity of SK channels. Several lines of evidence suggest that a pluripotent cell has to acquire an as yet undefined differentiation state to allow an optimal response for SK channel activation-induced cardiac/pacemaker-like cell commitment (Figures 1B and C): When starting with SK channel modulation after several days of monolayer differentiation, most probably allowing the formation of proper cardiac mesoderm, we found marginally increased cardiogenesis at the end of the experiment (Figure 1B). The setup to allow the generation of mesendoderm, mesoderm, and cardiac mesoderm is the embryoid body culture (Tran et al. 2009). However, the EB-based differentiation assays have some disadvantages compared to monolayer differentiation assays, such as variability in EB size and higher concentrations of morphogens/growth factors in culture required for the supplements to reach the innermost layers of EBs and for the applied substances to build a gradient throughout the EBs (Dhara and Stice 2008, Messana et al. 2008, Sachlos and Auguste 2008, Schulz et al. 2003). The EB favors the generation of the respective germ layers in particular mesendodermal monolayer derivatives that are dependent on precisely regulated intercellular signaling events (Tran et al. 2009). Those conditions are more likely to be present in EBs than in monolayer cultures because EBs have been shown to resemble the in vivo situation more precisely (Tran et al. 2009). Although we found that monolayer culture conditions and SK channel activation using mouse embryonic stem cells were able to govern differentiation toward the cardiac lineage, EB-based assays were still superior in efficiency and reproducibility. Using day 4 EBs to start SK channel activation, we found the most pure populations of cardiomyocytes and pacemaker cells compared to those without any genetic modulation (Figure 1C). 76

Thus, it remains to be determined whether this precise differentiation stage represents a particular cardiac progenitor population arising in a certain time window of development to drive optimal SK channel response. This time window seems to be between days 3 and 5 of spontaneous differentiation. Of note, we found that activation on day 4 was superior to that on day 5 in terms of efficiency, and lower EBIO concentrations were more efficient for cardiac commitment in human iPS cells compared to murine cells. This was mostly due to the fact that EB outgrowth after plating was hindered by EBIO concentrations higher than 0.75 m M. We observed that higher concentrations of EBIO restricted cell division in human cultures more than in murine cultures. We therefore hypothesize that the initial proliferation of progenitor cells in these cultures is important for a viable culture during later cell differentiation. Certainly, we could not exclude that the human pluripotent cells express other subforms or splice variants of SK channels that might be more sensitive to compounds such as EBIO. Direct SK channel activation in pluripotent cells (as in Figure 1A) could lead to premature signaling events being optimal for mesodermal but not yet for cardiac specification. This is in line with the recent finding that minimal alterations in cytokine timing completely abolished cardiac differentiation (Kattman et al. 2011). Previously, we found that SK4 is the predominant subtype for cardiac specification in murine ES cells and the knock down of this channel leads to a lack of beating cardiomyocytes (Kleger et al. 2010). On the other hand, a continuous rise in SK4 expression levels using an SK4 knockin ES cell line led to an increased cardiac commitment (Liebau et al. 2011b). Thus, several lines of evidence indicate that precise regulation of SK channel activity levels, dosage, and timing along with SK expression levels can lead to optimal efficiency for cardiac commitment. SK Channel–Derived Pacemaker Cells Dressing for Clinics? Modulation of ion channel activity is a new noninvasive strategy to enhance cardiac differentiation. Indeed, our

studies identified for the first time the functional relevance of a specific ion channel for the differentiation of ES cells and iPS cells into cardiac pacemaker-like cells. This differentiation system represents an interesting tool for applications in biomedical engineering and even potential therapeutic applications. To date, the highest cardiac differentiation efficiencies lacking a purification step have been reported using a serumfree differentiation system from the Keller laboratory. In this report, a precise sequence of inductive and repressive stimuli of BMP4 and activin A signaling led to a yield of ⬎60% pure cardiomyocytes from mouse pluripotent stem cells and approximately 50% from the human counterparts (Kattman et al. 2011). This study established a new standard of purity in the field. Nevertheless, there are certain limitations in the study that are bypassed using our differentiation approach, at least in the case of the mouse system: (1) The arising cardiomyocytes have not been characterized for their specific subtype, namely atrium, ventricle, or conduction system, and (2) the protocol requires tight control of the stage-specific addition of certain cytokines. In contrast, our system offers (1) high purity of the cell system (⬎60% cardiomyocytes), (2) specifically differentiated cell subtypes (⬃60% of the cardiomyocytes are pacemaker-like cells), (3) ease of handling and robustness in terms of different cell types, and (4) applicability under various culture conditions (Kleger et al. 2010, Muller et al. 2011). Still, regarding human pluripotent stem cell differentiation, our protocols need further optimization in terms of functionality and efficiency because the cardiac differentiation of our keratinocyte-derived hiPSCs was relatively poor, possibly related to the somatic memory of our hiPSCs (Bar-Nur et al. 2011, Pfaff et al. 2011, Polo et al. 2010). Thus, in the future, these assays could be important for correcting malfunctions of cardiac pacemaker cells that lead to rhythm generation disorders. In this respect, the replacement of pacemaking cells and tissue in the failing heart has been a focus in the cardiovascular field of research for a long time. Traditional treatments require pharmacological intervention and/or implantation of electronic pacemakers. Although such therapy is effective, it is also assoTCM Vol. 21, No. 3, 2011

A) SKCa activation Day 0 - Harvest

d i Mo ffe n re ola nt ye ia r tio n

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ES/iPS cell Cardiac progenitor cell Mesodermal progenitor cell Cardiomyocyte

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Figure 1. Modeling the optimal time window for SK channel activity to drive cardiogenesis from pluripotent stem cells. (A) Pluripotent cells were held under monolayer differentiation conditions and increased SK channel activation started at day 0 until harvest on day 13. (B) Pluripotent cells were held under monolayer differentiation conditions and increased SK channel activation started at day 4 until harvest on day 13. (C) Pluripotent cells were subjected to embryoid body formation and increased SK channel activation started at day 4 until harvest on day 13.

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Erk-/- ES-cells Figure 2. Proposed signaling mechanism downstream of SK channels to mediate cardiogenesis from pluripotent cells. (A) Selected genes on a microarray involved in the proposed pathway depicted in panel B. (B) Based on the microarray data from murine embryonic stem cells that were harvested under pluripotency conditions for 3 days with and without increased SK channel activity. (C) ERK knockout cells (kindly provided by Sylvain Meloche) were differentiated with and without increased SK channel activity under monolayer conditions for 3 days showing no differentiation under both conditions. Immunocytochemical staining of the pluripotency factor Oct3/4 (red) and nuclei were stained by DAPI. Scale bars ⫽ 10 ␮M.

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ciated with significant risks (for example infection, hemorrhage, lung collapse, and death) and expense. Other disadvantages include limited battery life (replaced every 5-10 years), permanent implantation of catheters, and lack of autonomic neurohumoral responses (Kong et al. 2010). The electric pacemaker has been used for a long time, but the development of biological pacemakers has increased in recent years. Several techniques have been investigated, including the transfer of pacemaking, bioengineered ion channels of the HCN family (Tse et al. 2006). In addition, the transplantation of pluripotent stem cell– derived pacemaker cells has been extensively investigated, with success. Several studies showed the functional integration of stem cell– derived pacemaker cells into the existing organ, leading to benefits of pacemaking in the heart (Kehat et al. 2004, Menasché 2004, Shiba et al. 2009). This was also true for ectopic pacemaking activity guiding the graft. Nevertheless, also resulting from the detection of the induced pluripotent stem cell, individual and biological pacemakers consisting of iPS cell– derived pacemaker cells are considered a valuable medical tool in cardiac cell-based therapies (Yoshida and Yamanaka 2010). Concerning the feasibility of cell therapy, major obstacles need to be overcome, such as improvement of contractile heart function, prevention of arrhythmias caused by insufficient graft guidance, potential teratoma formation (Henkel et al. 2006), and immunotolerance. Another safety concern in relation to the use of stem cells pertains to the potential proarrhythmic effects of transplanted stem cells in the host heart (Liao et al. 2010). Our system provides a cardiac differentiation system exclusive of genetic manipulation and lineage selection and thus could meet most of these criteria. However, functional tests using pacemaker cells generated upon SK channel activation, followed by transplantation into a mouse model of myocardial infarction, have to unequivocally test this hypothesis. • SK Channels and the Heart In Vivo Whereas SK1 and SK2 are predominantly expressed in the neuronal system, SK3 is widely distributed along the nervous system and endothelial and smooth 78

muscle cells (Bond et al. 2005, Luján et al. 2009, Pedarzani and Stocker 2008, Stocker 2004). For a long time, it was considered that SK4 was more or less restricted to non-neuronal cells such as muscle, epithelia, and blood cells (Berkefeld et al. 2010). During cardiac development, both activity and expression of SK channels change throughout the varying cardiac subtypes (Davies et al. 1996). During the past few years, SK4 not only was found to be modulating the cardiac action potential (Nouchi et al. 2008) but also was found to be expressed in the developing heart (Horsthuis et al. 2009). During postnatal development and cellular function, SK channels are widely distributed across the heart and vessels and also contribute to the action potential of different cardiac regions (Zhang et al. 2008). In particular, SK4 has been described in atrial stretch–induced atrial natriuretic factor secretion (Ogawa et al. 2009), and it was found to be the only SK subtype upregulated during regeneration/remodeling in a rodent model of myocardial infarction (Saito et al. 2002). However, only a few studies have investigated the functional role of SK channels in the heart in vivo. In this respect, their role in the repolarization/ afterhyperpolarization in the heart and their contribution to the AP are still extensively discussed (Nagy et al. 2009). A major point in these discrepancies was the differing effect of blocking reagents on repolarization, hyperpolarization, and AP length. One hypothesis for these discrepancies between the different studies was given by Diness et al. (2010), who stated that different mammalian species express variable forms of SK1SK3. This in turn can lead to differing binding capacities of the channels to apamin, for example (Dale et al. 2002). This might explain the varying results from studies using apamin as a blocking reagent (Diness et al. 2010). Interestingly, SK channels form certain heterodimers in the heart consisting of SK1-SK3, which might explain the controversy in detection and function of these channels throughout the organism and also between species (Tuteja et al. 2005, Tuteja et al. 2010, Xu et al. 2003). Lu et al. (2007) found a molecular coupling of the SK subtype SK2 to the L-type Ca2⫹ channel with the cardiac structure protein ␣-actinin as a bridging

cytoskeletal element. The contribution of SK channels to the afterhyperpolarization has been discussed for a long time. Still, several in vivo studies confirm the contribution of SK channels to the cardiac action potential. Li et al. (2009) demonstrated the functional abundance of SK2 in the cardiac system with a predominant expression in the atria. SK Channels and Pacemaking In Vivo SK channels have been found to be predominantly expressed in the atrium as well as in the atrioventricular node, representing one of the most crucial pacemaking centers in the heart besides the sinoatrial node (Tuteja et al. 2010, Zhang et al. 2008). Recently, a Tbx3EGFP BAC transgenic mouse line was used as reporter for the developing conduction system to perform subsequent whole transcriptome analysis. Intriguingly, data have shown that even the SK channel subtypes are differentially expressed in the conduction system, namely a ninefold higher SK4 expression compared to SK1-SK3 in the Tbx3GFP–positive cells (Horsthuis et al. 2009). Nevertheless, SK channel null mice do not display conduction system malformations or developmental defects (Begenisich et al. 2004, Romanenko et al. 2007, Zhang et al. 2008); instead, functional abnormalities such as atrial arrhythmias have been frequently reported (Zhang et al. 2008). This is particularly the case for SK2 but also for SK3, in which common genetic variants have been reported to be associated with lone atrial fibrillation (Ellinor et al. 2010). Specifically, gain-of-function analysis for SK2 resulted in prolonged action potentials in the atrioventricular node, whereas loss of function reacted in the opposite manner (Zhang et al. 2008). Moreover, atrial extrastimuli led to the most common atrial arrhythmia, namely atrial fibrillation, in SK2 null mice (Li et al. 2009). The fibrillations seen in the SK2 knockout model were considered as nonpacemaking driven due to the fact that the late phase of the cardiac action potential is highly susceptible to aberrant excitation. This also reflects the role of these channel subtypes during development and their expression in the pacemaking tissues (Zhang et al. 2008). Whereas a series of TCM Vol. 21, No. 3, 2011

functional data have been reported on SK2 null mice, there are few data on the other SK channels. Particularly, in the case of SK4, we showed using mouse ES cells that this subtype is critically involved in cardiac development and the generation of functional pacemaker cells. Although SK4 null mice have not been reported to exhibit developmental or obvious functional disorders of the cardiac conduction system, detailed functional analysis is pending (Begenisich et al. 2004). We hypothesize that compensatory mechanisms might apply differently in the in vivo situation. Thus, we believe that based on the observations from single SK channel knockout mice, it is difficult to predict the precise function of one channel because multiple deletions have been shown to result in distinct effects, for example, in noncardiac systems (Brähler et al. 2009). Further studies including precise functional analysis of the conduction system using combined SK channel knockout mice may shed more light on these issues. SK Channels as Therapeutic Targets of Cardiac Dysfunction The aforementioned animal models establish SK channels as potential targets of arrhythmic treatment. Further support for this hypothesis is given by drugbased modulation of SK channel function. In this respect, SK channels have been shown to play a role in the development of cardiac fibrillations, whereas in human chronic atrial fibrillations the expression of SK channels is reduced (Chua et al. 2011, Li et al. 2009, Skibsbye et al. 2011, Yu et al. 2011). Several studies have used varying substances modulating SK channel activity, thereby decreasing the risk of ventricular proarrhythmic effects and preventing fibrillations in an atrial-specific manner (Diness et al. 2011, Li et al. 2009, Skibsbye et al. 2011). Specifically, class III antiarrhythmic drugs are restricted to sotalol and amiodaron-like drugs, mostly delaying potassium outflux and thereby inducing AP delay (Milberg et al. 2004). Several studies have added SK channel inhibitors such as NS8593 to this class. Briefly, NS8593 prolongs the atrial effective refractory period without affecting QT interval and prevents and thereby terminates atrial fibrillation (Diness et TCM Vol. 21, No. 3, 2011

al. 2010). This has been further extended to pacing-induced atrial fibrillation, which can be reduced in vivo by the inhibition of SK channels (Skibsbye et al. 2011). A superficial look suggests that loss of SK channel function could, on the one hand, be used to treat atrial arrhythmias but, on the other hand, potentially contribute to relevant proarrhythmic events. However, a precise view of the inhibitor kinetics reveals that NS8593 inhibits not only SK2 but also SK1 and SK3, thus virtually abolishing SK channel signaling (Strobaek et al. 2006). This points to the hypothesis that disturbing the balance of SK channel activity leads to rather distinct phenotypes depending on the dosage and type of loss of function. Another important point in this respect is made by Tuteja et al. (2010), who showed that SK channels form distinct heteromultimeric complexes among different SK channel subunits in atrial myocytes, differing from ventricular myocytes. They propose the use of specific ligands of the different isoforms of SK channel subunits, offering a unique therapeutic opportunity to directly modify atrial cells without interfering with ventricular myocytes. This could complicate the development of drugs because the heteromultimeric complexes may also differ among several individuals. In summary, SK channel inhibitors may become important new members of class III anti-arrhythmias, but major obstacles have to be overcome to understand and control the SK channel subtype effects of the inhibitors that have hampered their clinical use to date (Grunnet et al. 2011, Nagy et al. 2011). • Signaling and Mechanism SK channel gating is tightly controlled by intracellular calcium levels (Stocker 2004), leading to opening of the channel with subsequent potassium efflux and hyperpolarization. This electrical function is particularly important for the devolution of an action potential in excitable cells such as neurons or cardiomyocytes, but it also triggers signaling events downstream of the membrane potential such as the ERK1/2 pathway mediated by calmodulin (CaM) and CaM-dependent kinases (Schmitt et al. 2005). The most important trigger in this respect is the intracellular calcium levels, tightly regulated by SK channel ac-

tivity, influencing various downstream signaling pathways (Berkefeld et al. 2010, Fakler and Adelman 2008). In addition to these electrogenic triggers of downstream signaling, our group has reported a protein-protein interactionbased mechanism of SK channel signaling (Liebau et al. 2007, Liebau et al. 2011a, Proepper et al. 2007). Further evidence for the presence of nonelectrogenic mechanisms in ion channel signaling is given by recent work on sodium channels in the developing zebra fish heart (Chopra et al. 2010). However, further details on the downstream pathways of these electrogenic and nonelectrogenic events upon SK channel activation remain underdeveloped so far. Membrane potential hyperpolarization of endothelial cells, for example, elevates the driving force for calcium entry, possibly through transient receptor potential channels (Barrett et al. 1982). In cardiomyocytes, SK channels have been shown to be critical in determining the duration of the cardiac action potential, especially promoting the repolarization. They functionally integrate changes in intracellular Ca2⫹ concentration with changes in potassium conductance and membrane potential (Stocker 2004). Xia and colleagues (1998) showed that Ca2⫹ sensitivity is conferred by the interaction of CaM with SK channel subunits, thereby governing the open/close stage of the channel pore (Xia et al. 1998). Subtle changes in the membrane potential can lead to a feedforward loop of calcium release from internal stores, thus affecting different signaling pathways and gene expression pattern. Therefore, calcium serves as a second messenger depending on kinetics, amplitude, and subcellular localization (Carrasco et al. 2004). Our previous work has shed light on a potential mechanism mediating SK channel– driven cardiogenesis from pluripotent stem cells, namely the downstream activation of the ERK signaling cascade (Kleger et al. 2010). However, it remains to be investigated whether electrogenic or nonelectrogenic mechanisms govern ERK activation. In addition, the pathway is certainly not complete because other downstream or interacting mediators are likely to be involved in this process. One example is the previously mentioned direct proteinprotein interaction of SK channels with 79

proteins associated with the cytoskeleton and signaling cascades. We showed in initial studies that SK channel activity modulates the cytoskeleton in neural precursor cells. In this respect, we found the SK3 subtype to be linked to the Abelson interactor protein 1 (Abi-1), which in turn is involved in the cytoskeletal machinery and can also translocate to the nucleus, where it modulates the activity of the MAX/myc complex, for example (Liebau et al. 2007, Liebau et al. 2011a, Proepper et al. 2007). Activation of SK channels in pluripotent stem cells stops the self-renewal machinery and drives the channels toward a primitive, cardiac-prone mesodermal cell type (Kleger et al. 2010). We extensively characterized the arising cell type by whole genome expression profiling in previous work (Kleger et al. 2010). To analyze the SK channel– driven signaling network in more depth, we took advantage of published transcriptome data to model a potential downstream scenario upon SK channel activation (Figures 2A and B). As previously mentioned, the only known physiological SK channel activators are tightly regulated fluctuations of intracellular Ca2⫹ levels. Nevertheless, SK channel activation cannot be explained by Ca2⫹ binding directly to the channel because there is no Ca2⫹ binding motif in the primary structure of the SK channel subunits (Stocker 2004). However, the calcium binding protein CaM has been shown to transmit calcium fluctuations by direct, constitutive interaction with the calmodulin binding site of SK channel subunits, whereas each SK channel complex has up to four CaM molecules (Xia et al. 1998). Of note, calmodulin signaling has been shown to be a critical parameter for cardiomyocyte integrity and development (Backs et al. 2006, Gangopadhyay and Ikemoto 2010). Specifically, SK channels sense Ca2⫹ concentrations via the associated Ca2⫹ binding protein CaM (Xia et al. 1998), and Ca2⫹ binding to the so-called EF hands (helix-loophelix structural domain) of CaM constitutively associated with channel subunits leads to rapid opening of the SK channel pore (Maylie et al. 2004). In addition, CaM is known to be involved in trafficking of SK channels (Barrett et al. 1982). CaM is known to activate downstream Ca2⫹/calmodulin-dependent protein kinase II (CaMKII), a kinase simi80

larly critical for heart development (Zhang et al. 2003). Interestingly, we found both CaM and CaMKII upregulated upon SK channel activation in our transcriptome data (Figure 2A). The upregulation of the CaM mRNA in this respect might be explained by the increased utilization and probable trafficking of CaM during early cardiac differentiation. Second, in previous studies, we observed an upregulation of SK channels during early cardiac differentiation steps (represented by the previously mentioned RNA arrays), thus explaining the increased requirement for CaM. A series of biochemical experiments noted activation of Ras and ERK1/2 upon SK channel activation (Kleger et al. 2010). Interestingly, inhibition of this pathway virtually abolished cardiac pacemaker cell induction followed by SK channel activation (Kleger et al. 2010). These findings are entirely mirrored in our transcriptome data model (Figures 2A and B). Of note, ERK⫺/⫺ ES cells lacked the differential response toward SK channel activity (Figure 2C). Based on these findings, we tried to generate a pathway model that is adapted from “KEGG pathway” (a collection of manually drawn pathway maps; http://www.genome.jp/kegg/pathway.html), “long-term potentiation” (mmu04720), and “MAPK signaling pathway” (mmu04010): We propose that in SK channel activity exposed embryonic stem cells, SK channels bind to CaM, probably to Calm1 and Calm2. The recruitment of CaM leads to activation of the Ras/ Raf/MEK/ERK pathway. In addition, CaMKII (CamkIIg or CamkIId) has an indirect effect on ERK. MKP could have a Ca2⫹-dependent negative effect on ERK1 as a kind of fine adjustment (Agell et al. 2002). Therefore, the sustained phase of ERK1 activation could lead to ES cell differentiation toward the cardiac lineage. • Outlook In recent years, ion channels and especially potassium channels have emerged from a status of “merely” being involved in the action potential of excitable cells to proteins playing multiple roles in developmental and cell biological events. In addition, there is accumulating evidence that these proteins act not only via the regulation of

the membrane potential but also via direct protein-protein interactions. In particular, our studies have shed light on the distinct roles of SK channels in different stem cell compartments and fate determination toward different lineages. Nevertheless, further studies are required to investigate this promising class of proteins for therapeutic purposes but even more for developmental biology. • Acknowledgments We thank Ralf Köhntop and Sabine Seltenheim for excellent technical assistance enabling research conducted in our laboratories. We thank all members of the Kleger and Liebau lab for discussions and critically reading the manuscript. The research that enabled this review was funded by a fellowship by the Medical Faculty of Ulm University (Bausteinprogramm, L. SBR.0011) and by the Deutsche Forschungsgemeinschaft (KL 2544/1-1, SL BO1718/4-1), the German Foundation for Heart Research (F/34/11; to AK and SL), and the Else-Kröner-Fresenius-Stiftung (2011_ A200; to AK and SL). We apologize to authors whose work has not been covered or directly cited due to space limitations. We thank Sylvain Meloche for providing the ERK knockout cells.

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PII S1050-1738(12)00040-0

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Bioengineered Vascular Grafts: Can We Make Them Off-the-Shelf?夡 Shannon L. M. Dahl*, Juliana L. Blum, and Laura E. Niklason

Surgical treatments for vascular disease have progressed during the past century from autologous bypass conduits to synthetic materials, animal-derived tissues, cryopreserved grafts, and, finally, bioengineered conduits. In all cases, alternative vascular grafting materials have been developed with the goal of treating patients who have severe vascular disease requiring bypass but who have no suitable autologous conduit. Synthetic vascular grafts, animal-derived tissues, and cryopreserved grafts all have drawbacks in terms of availability and functionality that have limited their routine clinical adoption. Although bioengineered vascular graft technologies remain early and highly investigational, they have the potential to revolutionize the way in which severe vascular disease is treated. However, before they can have a clinical impact, bioengineered grafts must be available immediately and “off-the-shelf.” (Trends Cardiovasc Med 2011;21: 83-89) © 2011 Elsevier Inc. All rights reserved. • Introduction Regenerative medicine therapies have the potential to repair, replace, or regenShannon L. M. Dahl and Juliana L. Blum are at Humacyte, Inc., Research Triangle Park, NC 27709, USA. Laura E. Niklason is at Humacyte, Inc., Research Triangle Park, NC 27709, USA; and Departments of Anesthesiology and Biomedical Engineering, Yale University, New Haven, CT 06520, USA. 夡 The authors are all founders of Humacyte, Inc. All authors have a financial interest in

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erate human cells, tissues, or organs in order to restore or establish normal function (Greenwood et al. 2006, Mason Humacyte, which is developing decellularized bioengineered vascular grafts. *Address correspondence to: Shannon L. M. Dahl, PhD, Humacyte, Inc., P.O. Box 12695, 7020 Kit Creek Road, Research Triangle Park, NC 27709, USA. Tel.: (⫹1) 919 313 9633, extension 231; fax: (⫹1) 919 313 9634; e-mail: [email protected]. © 2011 Elsevier Inc. All rights reserved. 1050-1738/$-see front matter

and Dunnill 2008). In particular, advances in recent years have allowed researchers and companies to produce readily available, mechanically functional, biological vascular grafts for treating arterial and venous disease. Each year, cardiovascular disease contributes to the death of an estimated 17.3 million people worldwide. In the United States alone, cardiovascular disease accounts for approximately 38% of all deaths (World Health Organization 2011). Cardiac surgeons worldwide perform more than 800,000 coronary artery bypass grafting operations annually (Cleveland Clinic 2011), and in the United States more than 300,000 dialysis patients require vascular grafts to provide access to their bloodstream (United States Renal Data System 2008). The overall demand for vascular grafts and other medical devices is growing substantially. In the United States, demand for all types of medical devices will increase by 8.3% annually through 2014 (Freedonia Group 2011). Autologous vascular grafts (artery and vein), and synthetic graft options such as polytetrafluoroethylene (PTFE) and Dacron, were the bypass conduits of choice during the 20th century. The first documented placement of an autologous vascular graft occurred in 1902, and the placement of the first synthetic graft occurred in 1952 (Ku and Allen 2000). However, the drawbacks of synthetic graft materials are multiple, including thrombogenicity, pseudointimal hyperplasia, risk of infection, and exuberant foreign body reactions. These drawbacks have led investigators, since the 1980s, to attempt development of tissuebased, bioengineered arterial grafts (Niklason et al. 1999, Weinberg and Bell 1986). However, it was not until very recently that a clinically viable type of vascular graft product has emerged—a bioengineered biological conduit that is mechanically robust and is available offthe-shelf, with no waiting time for graft production (Dahl et al. 2011). In this review, we focus on biological vascular grafts that are commercially available or in late-stage preclinical development and that offer the patient and the surgeon minimal waiting times prior to implantation. We discuss the challenges facing these types of products and strategies for overcoming these challenges. 83