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Cardiovascular Research 2007 73(4):631-640; doi:10.1016/j.cardiores.2006.11.005
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Copyright © 2006, European Society of Cardiology

Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation–contraction coupling in the heart

Lars S. Maier* and Donald M. Bers

Abt. Kardiologie & Pneumologie / Herzzentrum, Georg-August-Universität Göttingen, 37075 Göttingen, Germany
Department Physiology, Loyola University Chicago, Maywood, Il, USA

* Corresponding author. Abt. Kardiologie & Pneumologie / Herzzentrum, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. Tel.: +49 551 39 9481 or 8921; fax: +49 551 39 8941 or 14370. Email address: lmaier{at}med.uni-goettingen.de

Received 5 September 2006; revised 3 November 2006; accepted 6 November 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Structure of Ca2+/calmodulin...
 3. CaMKII and excitation...
 4. Outlook
 References
 
Calcium (Ca2+) is the central second messenger in the translation of electrical signals into mechanical activity of the heart. This highly coordinated process, termed excitation–contraction coupling or ECC, is based on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). In recent years it has become increasingly clear that several Ca2+-dependent proteins contribute to the fine tuning of ECC. One of these is the Ca2+/calmodulin-dependent protein kinase (CaMK) of which CaMKII is the predominant cardiac isoform. During ECC CaMKII phosphorylates several Ca2+ handling proteins with multiple functional consequences. CaMKII may also be co-localized to distinct target proteins. CaMKII expression as well as activity are reported to be increased in heart failure and CaMKII overexpression can exert distinct and novel effects on ECC in the heart and in isolated myocytes of animals. In the present review we summarize important aspects of the role of CaMKII in ECC with an emphasis on recent novel findings.

KEYWORDS Calcium; Calmodulin; CaM kinase; E-C coupling; Heart


   1. Introduction
Top
 Abstract
 1. Introduction
2. Structure of Ca2+/calmodulin...
 3. CaMKII and excitation...
 4. Outlook
 References
 
Intracellular Ca2+ is the central second messenger in the translation of electrical signals (i.e. action potentials) into mechanical activity of the heart (i.e. contractions). This highly coordinated process of excitation–contraction coupling (ECC) and a number of transporters, pumps, and ion channels on the sarcolemmal membrane as well as in the sarcoplasmic reticulum (SR) contribute to these Ca2+ fluxes on a beat-to-beat basis. Recently it has become increasingly clear that several Ca2+-dependent proteins themselves contribute to the fine tuning of ECC. One of these intracellular proteins is the Ca2+/calmodulin-dependent protein kinase (CaMK) of which CaMKII is the predominant isoform in the heart [1–3]. Relevant to ECC, CaMKII phosphorylates several Ca2+ handling proteins including SR Ca2+ release channels or ryanodine receptors (RyR), phospholamban (PLB), and L-type Ca2+ channels with multiple functional consequences [4,5]. Interestingly, CaMKII was found to be co-localized to multiple target proteins along with phosphatases, which dephosphorylate target proteins and function as an antagonist to CaMKII and other kinases. In addition, novel data suggest that non-Ca2+ transporters such as sarcolemmal Na+ and K+ channels may be regulated by CaMKII and thus be sensitive to Ca2+ handling properties and also influence them via electrophysiological effects.

In the myocardium of patients with heart failure CaMKII expression and activity correlate positively with the impaired ejection fraction and it was speculated that CaMKII (mainly through PLB phosphorylation thereby increasing SR Ca2+ reuptake) might be a compensatory mechanism to keep diseased hearts from complete contractile failure [6,7]. However, transgenic overexpression of the cytosolic isoform CaMKII{delta}C in mice causes severe contractile dysfunction and heart failure with altered intracellular Ca2+ handling and protein expression, leading to a reduced SR Ca2+ content, typical of heart failure [8]. Interestingly, the frequency of diastolic spontaneous SR Ca2+ release events (or opening of SR Ca2+ release channels or RyR) was greatly enhanced, demonstrating increased diastolic SR Ca2+ leak. This was attributed to increased CaMKII-dependent RyR phosphorylation resulting in increased and prolonged openings of RyR. This review focuses on acute and chronic effects of CaMKII in ECC and summarizes recent findings in the field.


   2. Structure of Ca2+/calmodulin-dependent protein kinase II (CaMKII)
Top
 Abstract
 1. Introduction
 2. Structure of Ca2+/calmodulin...
3. CaMKII and excitation...
 4. Outlook
 References
 
CaMKII is a multifunctional serine/threonine protein kinase [1–3] which can phosphorylate many different proteins in response to increasing intracellular Ca2+ concentrations ([Ca2+]i) [5]. Initially, CaMKII was identified in the nervous system but later CaMKII expression was discovered in the heart, albeit at lower levels [1,2]. There are four different but closely related CaMKII genes ({alpha}, β, {gamma}, {delta}) with the {alpha} and β isoform expression restricted to nervous tissue whereas the {gamma} and {delta} isoforms are more ubiquitously expressed, and the {delta} isoform is predominant in the heart [3,4,8]. Distinct splice variants of the {delta} isoform have different intracellular localization: Subcellular localizations of CaMKII{delta} were found with {delta}B being specifically compartmentalized to the nucleus due to an eleven amino acid nuclear localization sequence (NLS) and with {delta}C being the cytosolic isoform without NLS [9].

In contrast to the monomeric kinases CaMKI and CaMKIV, the multimeric CaMKII holoenzyme consists of homo- or heteromultimers of 6–12 kinase subunits forming a wheel-like structure [3–5]. Each CaMKII monomer contains an amino-terminal catalytic domain, a central regulatory domain (containing partially overlapping autoinhibitory and CaM binding regions) and a carboxy-terminal association domain responsible for oligomerization [3,4]. The autoinhibitory region close to the active site of the catalytic domain sterically blocks access to substrates. When [Ca2+]i increases, as during systole, intracellular calmodulin (CaM) binds up to four Ca2+ ions [10] and the Ca2+/CaM complex binds to the regulatory domain of CaMKII and displaces the autoinhibitory domain on CaMKII thereby activating the enzyme with half maximal activation at [Ca2+]i of 500–1000 nM. After this Ca2+/CaM-dependent activation, CaMKII can lock itself into an activated state upon autophosphorylation of Thr287 on the autoinhibitory segment [3,8]. Autophosphorylation can maintain CaMKII active even after [Ca2+]i has declined, (e.g. during diastole) when Ca2+/CaM has dissociated from its binding region (the so-called autonomous state). It is important to appreciate that autophosphorylation is not essential for CaMKII activity (in contrast to CaMKIV activation by phosphorylation by an upstream CaMK kinase [8]), but it does have important consequences, i.e. by increasing the affinity of the Ca2+/CaM-kinase complex [11]. This effect traps Ca2+/CaM on the autophosphorylated subunit. At high [Ca2+]i, the affinity of Ca2+/CaM to CaMKII increases ~700 fold (Kd decreases from 45 nM to 60 pM) [3,11]. Even when [Ca2+]i declines to resting levels during diastole (i.e. ~100 nM), CaM is still trapped for several seconds. As a result, the kinase retains close to fully active as long as CaM is trapped, regardless of the [Ca2+]i level [11]. Interestingly, autophosphorylation significantly disrupts autoinhibition, such that even after Ca2+/CaM has dissociated from the autonomous state CaMKII remains partially active (20–80%) [12–14]. For complete inactivation to occur, autophosphorylated CaMKII can be dephosphorylated by protein phosphatases including PP1, PP2A, and PP2C [8].

Several CaMKII inhibitors have been widely used in cardiac myocytes, including the organic inhibitors KN-62 and KN-93 [4] which competitively inhibit CaM binding at the regulatory domain to CaMKII (Ki ~370 nM) and are quite selective. Unfortunately, some of these agents appear to have direct ion channel effects which may be independent of CaMKII actions [15,16]. In contrast, peptide inhibitors are not known to affect ion channels. Two useful peptides are autocamtide-2 related inhibitory peptide (AIP) with 13 amino acids [17], and autocamtide-2 inhibitory peptide (AC3-I) [18].


   3. CaMKII and excitation–contraction coupling (ECC)
Top
 Abstract
 1. Introduction
 2. Structure of Ca2+/calmodulin...
 3. CaMKII and excitation...
4. Outlook
 References
 
Ca2+ is the central regulator of ECC. During an action potential Ca2+ enters the cell mainly through voltage dependent L-type Ca2+ channels, which triggers Ca2+ release from the SR via RyR, a process termed Ca2+-induced Ca2+ release [19]. These processes increase [Ca2+]i, causing Ca2+ binding to troponin C which activates the myofilaments leading to contraction (systole). For diastolic relaxation to occur, Ca2+ must be removed from the cytoplasm. The SR Ca2+-ATPase (SERCA) and the Na+/Ca2+-exchanger (NCX) are the main mechanisms for Ca2+ removal [19]. Sarcolemmal Ca2+ pumps and mitochondrial Ca2+ uniporter contribute only minimal to this Ca2+ cycling on a beat-to-beat basis (Fig. 1).


Figure 1
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  		Fig. 1 Effects of CaMKII{delta}C on excitation–contraction coupling (ECC). CaMKII phosphorylates several Ca2+-handling proteins including phospholamban (PLB), possibly SR Ca2+ ATPase (SERCA), SR Ca2+ release channels (RyR), and L-type Ca2+ channels responsible for Ca2+ influx (ICa). In addition, Na+ channels and K+ channels may be also regulated by CaMKII. During the action potential (AP), Ca2+ enters the cell via L-type Ca2+ channels (ICa) and reverse-mode of the Na+/Ca2+-exchanger (NCX, left) thereby triggering Ca2+ release through RyR from the SR (filled with Ca2+, red dots). This Ca2+ then activates the myofilaments during systole leading to contraction. During diastole, Ca2+ is taken up back into the SR by SERCA and is transported outside the cells mainly by NCX, leading to relaxation. PLB inhibits SERCA when PLB is dephosphorylated. Mitochondria and sarcolemmal Ca2+ pumps only contribute by 1–2% to Ca2+ extrusion.

 
This highly coordinated process of global ECC occurring at ~60 times per minute in man and up to 500 times per minute in mouse during rest, is composed of many synchronized local events. About 25 L-type Ca2+ channel proteins and 100 RyR proteins are co-localized forming a local SR Ca2+ release unit called a junction or couplon [20]. This local functional unit can be monitored by confocal microscopy measuring elementary Ca2+ release events from the SR (Ca2+ sparks) occurring spontaneously in resting cardiac myocytes and summating during normal Ca2+ transients in ECC.

There is considerable evidence that fluctuations in [Ca2+]i are not only involved in the direct myofilament activation leading to contraction, but also indirectly modify the activity of ion channels and transporters via CaM and CaMKII [4]. The integrative responses of these downstream messengers of Ca2+ giving a feedback on the ion channels and transporters that regulate [Ca2+]i serve to fine tune ECC. For example, CaMKII can modulate ECC by phosphorylating several important Ca2+ regulatory proteins in the heart in response to Ca2+ signals, including RyR [21,22], PLB [23,24], and L-type Ca2+ channels [4] with multiple functional consequences (Fig. 1). These proteins are mainly involved in Ca2+ influx, Ca2+ release from the SR, as well as Ca2+ uptake into the SR and their specific role in ECC is discussed in the following paragraphs. In addition to these "classic" CaMKII-dependent effects on Ca2+ handling proteins novel findings of CaM- and CaMKII-dependent regulation of sarcolemmal Na+ and K+ channels are presented below.

3.1. Ca2+ influx into the myocyte
CaMKII modulates voltage-gated L-type Ca2+ channels and thereby Ca2+ current (ICa), and this is most clearly seen functionally as a positive staircase of ICa with repeated depolarization from –90 to 0 mV, a process termed Ca2+-dependent ICa facilitation [25,26]. ICa amplitude increases and inactivation is slowed progressively over a series of a few pulses after a rest interval. Several groups independently demonstrated that Ca2+-dependent ICa facilitation is mediated by CaMKII-dependent phosphorylation [27–29]. CaMKII tethers to the {alpha}1C subunit of the cardiac L-type Ca2+ channel (on the long C-terminal tail) and can phosphorylate the {alpha}1C subunit at both amino and carboxy tails [30]. CaMKII also appears to phosphorylate a site on the β2a-subunit of the L-type Ca2+ channel (Thr-498), which may be involved in the functional effect of ICa facilitation [31]. At the single channel level this CaMKII-dependent ICa facilitation is manifested as longer single channel openings [32]. This positive ICa staircase is Ca2+-dependent, very local at the mouth of the L-type Ca2+ channels, and still occurs in the absence of SR Ca2+ release, since it is still observed when the intracellular milieu of cells is heavily Ca2+ buffered with 10 mM EGTA. The physiological role of ICa facilitation is not entirely clear, but it may partly offset reduced L-type Ca2+ channel availability at high heart rates (caused by direct Ca2+-dependent inactivation). Using specific CaMKII inhibitors or Ba2+ as charge carrier, this facilitation can be abolished. By overexpressing CaMKII{delta}C in transgenic mouse myocytes as well as in adenovirus-mediated rabbit myocytes ICa amplitude was increased and inactivation was slowed [33,34]. ICa amplitude could be reduced back to control levels by blocking CaMKII with KN-93 [33] or AIP [34].

Interestingly, CaMKIV transgenic mice that also showed increased activity of cytosolic CaMK, showed an increased propensity for ventricular arrhythmias [35]. This was attributed to triggered activity due to early afterdepolarizations resulting from an inward current during the late plateau phase of the action potential. Enhanced open probability of L-type Ca2+ channels due to increased activity of CaMKII was causally linked to this increased propensity for arrhythmias [35]. Additionally, in the presence of the CaMKII-inhibitor KN-93, both the increased Ca2+ channel open probability as well as the increased occurrence of arrhythmias were reversed to control values [35]. In summary, it is now well accepted that CaMKII modulates ICa, and that this modulation may be important under pathophysiological conditions (e.g. arrhythmias).

3.2. Ca2+ release from the SR
CaMKII also affects RyR activity. Witcher et al. [21] first reported direct phosphorylation of cardiac RyR at Ser-2809 by CaMKII activating the SR Ca2+ release channel. However, Rodriguez et al. [36] showed that there may be at least four additional CaMKII sites on RyR2. Other studies also showed that RyR are substrates of CaMKII [22,37], but the specific effects of phosphorylation reported in these studies remained controversial. That is, CaMKII either increases [21,22] or decreases RyR open probability [37]. Most of the work on CaMKII effects on RyR has been conducted using RyR in lipid bilayers or by measuring Ca2+ release from SR vesicles, with little data in more physiological environments. Li et al. [15] reported that in intact voltage clamped cardiac myocytes endogenous CaMKII increased the amount of SR Ca2+ release for a given SR Ca2+ content and ICa trigger. In that study, the effect of CaMKII on RyR was evaluated when both L-type Ca2+ current and SR Ca2+ load were constant under control conditions and in the presence of the CaMKII inhibitor KN-93. This conclusion is also consistent with observations that protein phosphatases (PP1 and PP2A) can reduce RyR activity for a given ICa and SR Ca2+ load, and conversely that phosphatase inhibitors enhance it [38]. However, Wu et al. [39] found opposite results suggesting that constitutively active CaMKII inhibited SR Ca2+ release, while CaMKII inhibition (by AC3-I) enhanced SR Ca2+ release. Since they did not measure SR Ca2+ content in the same protocols, the dichotomy raised is still not resolved.

Exciting reports from our and other labs over the past few years provided new evidence in isolated cardiac myocytes that CaMKII indeed is directly associated with RyR [40–42] and overexpression of CaMKII{delta}C increases fractional SR Ca2+ release during ECC as well as spontaneous SR Ca2+ release (or Ca2+ spark) frequency for a given SR Ca2+ load [33,34,43]. In addition to this increased frequency (which is indicative of RyR2-mediated diastolic spontaneous SR Ca2+ release events), width, and duration of Ca2+ sparks were enhanced, demonstrating increased diastolic SR Ca2+ leak. In contrast, when blocking CaMKII (using KN-93) Ca2+ spark frequency decreases dramatically [33]. These results in myocytes from CaMKII{delta}C transgenic mouse hearts were confirmed recently by acute CaMKII{delta}C overexpression by adenovirus-mediated gene transfer in isolated rabbit myocytes [34] (Fig. 2) as well as direct application of pre-activated CaMKII to permeabilized mouse myocytes [43]. Similarly, Currie et al. [42] showed that in rabbit hearts the CaMKII peptide inhibitor AIP (1 µM) depresses Ca2+ spark frequency and ryanodine binding to RyR2 (an assay of RyR2 activation), indicating that CaMKII activates RyR2 in myocytes. Wehrens et al. [41] also recently showed that CaMKII-dependent RyR phosphorylation increased RyR open probability using single channel measurements in lipid bilayers, and using site-directed mutagenesis, that CaMKII-dependent RyR2 phosphorylation may be at Ser-2815, rather than Ser-2809 (the initially identified CaMKII target). Whether CaMKII phosphorylates RyR2 at Ser-2809 is now controversial (as is the role of PKA-dependent phosphorylation and subsequent FKBP12.6 dissociation) [44].


Figure 2
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  		Fig. 2 CaMKII-dependent SR Ca2+ leak. A. Elementary Ca2+ release events (Ca2+ sparks) measured by confocal microscopy (line scan mode) as a measure of diastolic ryanodine receptor (RyR) activity (Ca2+ leak). B. In the presence of CaMKII overexpression, there is an increased SR Ca2+ leak which can be reversed by CaMKII inhibition using KN-93 or AIP (based on data in [34]). C. Schematic diagram of RyR phosphorylation by CaMKII and Ca2+ leak (Ca2+ ions, red dots) from the SR.

 
Ai et al. [45], recently showed that in heart failure there is increased CaMKII expression (as seen in human heart failure) [7], more CaMKII is autophosphorylated (as seen in human heart failure) [6] and more of this CaMKII is physically associated with the RyR. There was also less phosphatase associated with the RyR (as seen in other heart failure models) [46], and RyR2 was more heavily phosphorylated (at both Ser-2809 and 2815). Moreover, the enhanced diastolic leak previously measured in heart failure cells from this model [47] could be completely reversed by CaMKII inhibition (but not by PKA inhibition). This CaMKII-dependent enhancement of SR Ca2+ leak in heart failure may contribute to both the diminished SR Ca2+ content characteristic of this disease, and also diastolic SR Ca2+ release which can activate transient inward Na+/Ca2+ exchange current that causes delayed afterdepolarizations and triggered arrhythmias. Indeed, CaMKII inhibition greatly increases SR Ca2+ content in heart failure myocytes, but interestingly this results in only modest inotropy [45]. The explanation is that while blocking the CaMKII effects on RyR limits diastolic SR Ca2+ leak and enhances SR Ca2+ content, it also prevents CaMKII-dependent stimulation of ECC at the RyR, such that there is lower fractional release (i.e. a smaller fraction of a higher SR load is released). In conclusion, CaMKII can enhance RyR2 activation during both ECC (influencing the fractional SR Ca2+ release during systole) and during diastole, when it may both unload Ca2+ from the SR and also contribute to triggered arrhythmias in the setting of heart failure.

3.3. SR Ca2+ uptake
Phospholamban (PLB) is an endogenous inhibitor of SERCA in its unphosphorylated state [48]. Upon PLB phosphorylation SERCA activity and SR Ca2+ uptake are enhanced. PLB can be phosphorylated by PKA at Ser-16 and by CaMKII at Thr-17 [48,49] which produces a similar lowering of Km(Ca2+) of SERCA as Ser-16 phosphorylation by PKA. While less generally accepted, there are also reports that suggest an increase of Vmax due to CaMKII phosphorylation of PLB with less effect on Km (vs. PKA). Bassani et al. [50] initially showed that CaMKII enhances SR Ca2+ uptake, and speculated that CaMKII-dependent PLB phosphorylation might be responsible for the frequency-dependent acceleration of relaxation (FDAR) of twitches and SR Ca2+ uptake. Hagemann et al. [51] showed a frequency-dependent increase in PLB Thr-17 phosphorylation in rat myocytes (independent of Ser-16 phosphorylation), and that the level of CaMKII-dependent Thr-17 phosphorylation correlated with the rate of relaxation.

Physiologically, FDAR may be an important intrinsic mechanism to allow faster relaxation (and diastolic filling) when heart rate is increased. FDAR is also manifest as slowing of twitch relaxation as time between beats is prolonged (i.e. at post-rest contractions) [52]. An attractive hypothesis (as above) was that FDAR might be due to enhanced SR Ca2+ uptake via PLB phosphorylation by CaMKII, activated by the cyclic increase in [Ca2+]i during ECC (with rest allowing PLB dephosphorylation). However, we found that FDAR is still quite prominent in PLB deficient (PLB-KO) mice and still sensitive to CaMKII inhibition by KN-93 and AIP [53]. Also, the time course of FDAR development is much faster during changes in frequency than that of PLB phosphorylation [54,55]. Thus, while PLB might contribute to FDAR, it cannot be the sole mechanism. There have been reports of direct CaMKII-dependent phosphorylation of cardiac SERCA, and increased Vmax for Ca2+ transport [56,57]. While this might fit with FDAR results above, critical studies have shown that CaMKII does not directly phosphorylate SERCA2 [58,59]. Thus, the identity of the CaMKII target involved in accelerating SR Ca2+ transport during FDAR is not yet clearly identified. While we and some others have shown that FDAR can be strongly suppressed by CaMKII inhibitors (KN-93, KN-62, AIP), [50,51,53,60] some reports could not detect FDAR inhibition with the organic inhibitors KN-93 or KN-62 [54,61–63]. Thus, CaMKII may be critical in mediating FDAR, but further study is required.

Pathophysiologically, during acidosis, there is a progressive increase in {delta}[Ca2+]i causing a partial recovery of contractions [64]. Interestingly, the recovery of Ca2+ transients can be prevented with the CaMKII inhibitor KN-93 and it was proposed that CaMKII-dependent PLB phosphorylation may be responsible for the faster [Ca2+]i decline and recovery of contractions that partially overcomes the direct inhibitory effect of acidosis [64–66]. We confirmed that PLB and CaMKII were both required for recovery of Ca2+ transients and contraction during acidosis in mouse myocytes [67]. Indeed, recovery was prevented in isolated myocytes from PLB knockout (PLB-KO) vs. wild-type (WT) mice. In addition, inhibition of CaMKII abolished recovery in WT mice, but was without effect in PLB-KO mice [67]. Moreover, our preliminary results show that acute overexpression of CaMKII in rabbit cardiac myocytes even improves recovery during late acidosis, with increased twitch shortening, [Ca2+]i transient amplitude and decline as well as accelerated relaxation parameters, however these preliminary data need to proven [68]. Thus CaMKII-dependent enhancement of SR Ca2+ uptake may be important in both FDAR and maintenance of contractile function during acidosis.

3.4. Na+ channels, [Na+]i, and K+ channels
In addition to Ca2+ channels, some reports show that CaMKII may also target cardiac Na+ channels [69] and K+ channels [70]. Tan et al. first reported a CaM-dependent regulation of cardiac voltage-gated Na+ channels [71]. They showed that Ca2+/CaM can bind to an IQ motif (isoleucine, glutamine; amino acids 1908–1909 plus subsequent ~10 amino acids) at the carboxy-tail of the Na+ channel {alpha} subunit. This interaction specifically altered Na+ channel gating properties. Accumulation of intermediate inactivation was enhanced consistent with a reduced channel function. The inhibition of Na+ channel function by Ca2+/CaM displays some similarities to the CaM-dependent regulation of L-type Ca2+ channels. The Ca2+-dependent regulation of Na+ current (INa) may thus modulate excitability as a feedback mechanism during ECC.

However, the molecular mechanisms involved in the altered Na+ channel gating are not fully understood. CaM modulates the interaction of the III–IV cytoplasmic linker with the C-terminus that is required for fast inactivation to occur [72], and enhanced fast INa inactivation in the presence of activated CaM had been observed by Tan et al. [71]. However, since a peptide antagonist of Ca2+-dependent CaM binding exerted the same enhancement of fast inactivation, it was suggested not to be specifically CaM-dependent [71]. In this respect, even direct binding of Ca2+ ions to a paired EF hand motif has also been recently proposed as an alternative mode of Ca2+ regulation [73] (although direct binding of Ca2+ to the C-terminus has been questioned [72]).

Interestingly, mutations within or near the Na+ channel IQ motif are arrhythmogenic and can result either in Brugada syndrome or may predispose to drug-induced LQTS [74]. Disrupted interaction of either Ca2+ alone or CaM may be responsible for the altered gating resulting in the clinical phenotype.

Na+ channel activity may also be regulated by CaMKII, as these channels may also be multiprotein regulatory complexes [75]. The first indirect evidence for CaMK-dependent regulation of cardiac Na+ channels was by Deschênes et al. [76]. They showed that for the cardiac Na+ channel isoform the CaMK-inhibitor KN-93 slowed current decay, consistent with an inhibition of fast inactivation. Additionally, the steady-state voltage dependence of inactivation was shifted in the depolarizing direction resulting in an increased channel availability at a given membrane voltage. Entry into the intermediate inactivated state was also slowed, while the recovery from inactivation was hastened. This was consistent with a CaMK-dependent inhibition of Na+ channel activity similar to the above CaM-dependent effects on Na+ channel gating. However, KN-92 (the inactive analog of the CaMKII inhibitor KN-93) also had effects on Na+ channel gating, and the specific CaMKII-inhibitor AIP did not appear to affect Na+ current. Therefore, the authors concluded that a CaM-dependent kinase other than CaMKII might modulate Na+ channels (suggesting CaMKIV). However, the expression levels of this kinase in the heart are very low [77]. Furthermore, since CaMKIV localizes to the nucleus [35], it would seem an unlikely candidate for acute modulation of sarcolemmal Na+ channels.

We recently examined Na+ channel gating in rabbit ventricular myocytes specifically overexpressing CaMKII{delta}C using adenovirus-mediated gene transfer [69] (Fig. 3). CaMKII{delta}C overexpression resulted in a significant leftward-shift in the steady-state voltage dependence of inactivation which could be reversed by CaMKII inhibition. The development of intermediate inactivation was enhanced and recovery from inactivation was prolonged. Both effects were completely reversible with CaMK-inhibition using KN-93 or AIP [69]. These effects of specific CaMKII overexpression argue for specific CaMKII-(vs. CaMKIV-) dependent modulation of Na+ channels. However, more information is needed to clarify how CaMKII regulates INa under physiological conditions (without CaMKII{delta}C overexpression). Thus, some effects previously attributed to CaM may actually be mediated by CaMKII{delta}C. In this respect, we also found evidence for a direct association of CaMKII with the Na+ channel and phosphorylation of Na+ channels by CaMKII [69].


Figure 3
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  		Fig. 3 Modulation of Na+ current gating in mutant Na+ channels (1795InsD) and upon CaMKII{delta}C overexpression. The curves are theoretical, but indicate qualitatively the common INa gating alterations reported by Veldkamp et al. [80] for mutant vs. wild type Nav1.5 (1795InsD vs. WT) expressed in HEK293 cells and reported by Wagner et al. [69] in adult rabbit ventricular myocytes overexpressing CaMKII{delta}C (vs. control β-galactosidase, Ctl) via adenovirus (where shifts could be reversed by CaMKII inhibitors). With either 1795InsD or CaMKII{delta}C, INa availability (or steady state inactivation) is shifted to more negative potentials, while activation voltage dependence is not (A), more INa accumulates in intermediate inactivation at –20 mV (i.e. taking more than 10 ms to recover at –120 ms; B). INa recovers more slowly from inactivation (C) and there is more persistent or slowly inactivating INa (D).

 
This may be especially important since upregulation of CaMKII activity and expression seem to be typical of cardiomyopathy from diverse causes in patients [6,7] and animal models [33,45,78]. Furthermore, transgenic mice overexpressing cytosolic CaMKII{delta}C develop heart failure and die early [40], and CaMKII inhibition can prevent myocardial remodeling after myocardial infarction or excessive β-adrenergic stimulation [79].

Several human cardiac Na+ channel mutations have been linked to either long-QT (LQT3) and Brugada syndromes with consequent life-threatening arrhythmias [74]. One such human mutation (Asp insertion at 1795 in the C-terminus, 1795InsD), shows simultaneous LQT3-like and Brugada-like phenotypes in the same patients [80]. Remarkably, Na+ channels bearing this mutation expressed in mammalian cells exhibit the same phenotype that we found for CaMKII-modified normal Na+ channel above. That is, there was a hyperpolarizing shift in steady-state INa availability, unaltered activation (Fig. 3A), slowed fast inactivation with more intermediate inactivation (Fig. 3B), slowed recovery from inactivation (Fig. 3C and enhanced persistent INa (Fig. 3D) [80,81]. At low frequencies, the impaired inactivation and persistent INa can cause AP prolongation consistent with LQT3 syndrome. However, at higher heart rates, incomplete INa recovery and limited INa availability, further shorten action potential duration, slow propagation and increase dispersion of repolarization. The intriguing thing with respect to CaMKII is that CaMKII-dependent INa modulation due to upregulated CaMKII in heart failure could constitute a common acquired form of arrhythmia (combined LQT3 and Brugada syndrome), in otherwise normal Na+ channels. Such an acquired Na+ channel dysfunction may contribute to arrhythmias under conditions when CaMKII effects are enhanced, as in heart failure. Interestingly, CaMKII has already been linked causally to ventricular arrhythmias in a mouse model of cardiac hypertrophy and failure by Anderson's group [35,79].

CaMKII may also regulate transient outward K+ current (Ito) in human atrial myocytes from hearts with chronic fibrillation as shown by Tessier et al. [70]. These authors showed that CaMKII regulates Ito and CaMKII inhibition results in faster Ito inactivation. It was even speculated that K+ channels or associated regulatory proteins must be in a certain state of phosphorylation in order to be available for cumulative inactivation [82] resulting in a CaMKII-dependent "memory effect". Similarly, CaMKII was shown to regulate Kv4.2 [83] and Kv4.3 [84] in neuronal cells, and these two channels are known to contribute to cardiac Ito. There is even evidence suggesting that CaMKII directly phosphorylates Kv4.3 at Ser-550 [84]. CaMKII-dependent regulation of Ito is interesting in so far that in heart failure, Ito,fast mainly mediated by Kv4.2/3 is functionally reduced and expression of the channel proteins is lower [52]. In preliminary studies in ventricular cardiac myocytes acutely overexpressing CaMKII{delta}c, we find CaMKII-dependent enhancement of Ito consistent with increased Kv1.4 function, and consequent APD shortening [85]. Further studies need to be performed to elucidate the role of CaMK-dependent K+ channel regulation under physiological and pathophysiological conditions.

CaM itself is also known to regulate other K+ channels, and CaM is an essential subunit responsible for the Ca2+-dependent activation of small conductance Ca2+-activated K+ channels (prominent in many cell types, but not ventricular myocytes) [86]. The amino terminal lobe of CaM serves as the activating switch in this case, and strong binding of the Ca2+-free carboxy lobe of CaM to the channel seems to distort the Ca2+ binding sites, thereby preventing Ca2+ binding. Ca2+ is known to modulate the cardiac delayed rectifier K+ current IKs (the channel that is the most common cause of LQTS) [87]. Recent studies by two groups [88,89] have shown that CaM binds to the carboxy terminal of KCNQ1 as part of the IKs complex (KCNQ1+KCNE1+CaM), is required for channel assembly and delivery to the cell surface, and mediates Ca2+-dependent regulation of IKs. Moreover, if CaM interaction is disrupted the current shows prominent inactivation, which would interfere with the normal function of IKs in cardiac repolarization. Thus CaM is an important modulator of numerous ion channels that directly and indirectly impact ECC.


   4. Outlook
Top
 Abstract
 1. Introduction
 2. Structure of Ca2+/calmodulin...
 3. CaMKII and excitation...
 4. Outlook
References
 
CaMKII in heart has gained attention during the last few years. Its involvement at multiple levels in ECC and ion channel regulation indicates that it is an important modulator of both electrophysiological and contractile properties in the heart. Moreover, since CaMKII expression and activation may be elevated in important pathophysiological situations (e.g. heart failure), understanding CaMKII regulation in heart more completely will help to understand the normal physiology of the heart, how it changes during diseases, and may identify new modalities of treatment. We are just starting to understand the important subcellular mechanisms in which CaMKII is involved. The next few years should bring more insight into these mechanisms and possibly novel therapeutic approaches.


   Acknowledgements
 
Dr. Maier is funded by the Deutsche Forschungsgemeinschaft (DFG) through an Emmy Noether-grant (MA 1982/1-4 and 1-5), and by a grant through the DFG Klinische Forschergruppe (MA 1982/2-1). Dr. Bers is supported by National Institutes of Health Grants (HL30077, HL64724 and HL80101).


   Notes
 
Time for primary review 33 days


   References
Top
 Abstract
 1. Introduction
 2. Structure of Ca2+/calmodulin...
 3. CaMKII and excitation...
 4. Outlook
 References
 

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