Calmodulin Kinase and L-Type Calcium Channels

Calmodulin Kinase and L-Type Calcium Channels: A Recipe for Arrhythmias? Mark E. Anderson* L-type Ca2+ channels (LTCCs) are the main portal for Ca2+ entry into cardiac myocytes. These ion channel proteins open in response to cell membrane depolarizations elicited by action potentials, and LTCC current (ICa) flows during the action potential plateau, to increase cellular Ca2+ (Ca2+i) and trigger myocardial contraction. ICa is also implicated in the genesis of cardiac arrhythmias under conditions such as heart failure and cardiac hypertrophy, in which the action potential plateau and QT interval are prolonged. This article reviews recent findings about the molecular regulation of LTCCs by the Ca2+-dependent signaling molecule, calmodulin kinase II (CaMKII), and compares this form of regulation with regulation by calmodulin-binding domains and b-adrenergic receptor agonists. LTCC dysregulation is discussed in the context of new results showing that CaMKII can be a proarrhythmic signal in disease conditions in which Ca2+i is disordered and cardiac repolarization is excessively prolonged. (Trends Cardiovasc Med 2004;14:152–161) n 2004, Elsevier Inc.

L-type Ca2+ channels (LTCCs) are the primary portal for Ca2+ entry into cardiac myocytes. LTCCs are a macromolecular complex of proteins that serve to interpret and integrate a wide array of regulatory signals—including cell membrane potential and phosphorylation by protein kinases—and transduce these signals into LTCC current (ICa) responses to meet cellular requirements for cytoplasmic Ca2+ (Ca2+i). LTCCs are the target for

Mark E. Anderson is at the Division of Cardiovascular Medicine, Departments of Medicine and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA. * Address correspondence to: Mark E. Anderson, MD, PhD, Vanderbilt University Medical Center, 2220 Pierce Avenue, 383 Preston Research Building, Nashville, TN 37232-6300, USA. Tel.: (+1) 615-936-1873; fax: (+1) 615-936-1872; e-mail: mark.anderson@ vanderbilt.edu. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter

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pharmacologic therapies by Ca2+ channel antagonist agents (mediated by direct binding to the pore-forming a subunit) (Clusin and Anderson 1999) and b-adrenergic receptor antagonists, which indirectly reduce ICa by suppressing protein kinase A (PKA) activity (Bunemann et al. 1999). ICa is an inward current that can influence the duration and stability of the cardiac action potential plateau, but Ca2+i is also an important cellular second messenger that binds to target proteins to actuate key physiologic processes, including contraction. Regulation of Ca2+i requires special diligence by cardiac myocytes because of the important role of Ca2+i for regulating cellular processes, and because the concentration of free Ca2+ in the extracellular space is approximately 105 times greater than the permissible concentration of free Ca2+ in the cell. Ca2+i regulates ICa, in part via binding to the ubiquitous Ca2+i -sensing protein calmodulin (CaM). Ca2+-bound CaM (Ca2+/ CaM) activates the multifunctional Ca2+/

CaM-dependent protein kinase II (CaMKII), which targets several critical Ca2+i homeostatic proteins, including LTCCs. Perhaps because of the complexity of these processes, dysregulation of LTCC activity can occur in disease (Schroder et al. 1998) and is linked to the genesis of arrhythmias (January and Riddle 1989, Wu et al. 2002, Zeng and Rudy 1995) and initiation of hypertrophic and signaling pathways (Cohn et al. 2001, Muth et al. 2001). This review focuses on the regulation of LTCCs by CaMKII, PKA, and CaM, and discusses evidence regarding the hypothesis that CaMKII is a proarrhythmic signaling molecule. 

LTCCs and Cardiac Physiology

The cardiac action potential has a distinct morphology that includes a long plateau, which serves as a cell membrane potential substrate to support Ca2+ entry through voltage-gated LTCCs. LTCCs participate in a wide range of functions, but their central role in myocardium is to provide the Ca2+ trigger for excitation – contraction coupling (Figure 1). Most of the Ca2+ for cross-bridge formation and myocyte contraction comes from the sarcoplasmic reticulum (SR) intracellular Ca2+ store, and ICa serves to trigger release of this Ca2+i by activating the ryanodine receptor (RyR) (Fabiato and Fabiato 1975), an intracellular Ca2+ channel that is positioned across a narrow cleft from the cytoplasmic face of the LTCC (Sun et al. 1995). This restricted cytoplasmic space between LTCCs and RyRs is a Ca2+i microdomain that can focus Ca2+i-dependent signals on T-tubular membrane proteins important for regulating electric responses, such as Ca2+ channels (the focus of this review), Na + channels (Tan et al. 2002), K+ channels (Clark et al. 2001), and the Na+/Ca2+ exchanger (Ritter et al. 2003). LTCCs also play specialized roles in the conduction system. In the sinoatrial node, LTCCs contribute to impulse formation (Kodama et al., 1997), but other Ca2+ channel types (namely, a primarily neuronal LTCC, Cav1.3) are also important (Platzer et al. 2000). LTCCs are also a critical source of inward current for generating action potentials in the atrioventricular nodal tissue (Hancox and Levi 1994), where many cells may lack a Na + current (Petrecca et al. TCM Vol. 14, No. 4, 2004

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time Figure 1. L-type Ca2+ channels (LTCCs), ryanodine receptors (RyR), and calmodulin kinase II (CaMKII) colocalize in a subcellular microdomain (Wu et al., 1999a) and provide the ultrastructural framework for cardiac excitation – contraction coupling. Most LTCCs are concentrated in sarcolemmal invaginations called T tubules. These LTCCs face off across a cytoplasmic space with intracellular channels called RyR that control Ca2+ release from the sarcoplasmic reticulum (SR) (Flucher and Franzini-Armstrong 1996). (a) Peak LTCC current (ICa) occurs early in the action potential plateau and provides the Ca2+ trigger for (b) RyR opening and release of SR Ca2+, a process called Ca2+-induced Ca2+ release (Fabiato and Fabiato 1975). SR Ca2+ drives myofilament contraction and activates CaMKII. CaMKII is persistently activated by a mechanism involving intersubunit (intraholoenzyme) autophosphorylation (Lisman et al. 2002), and is bound to RyRs (Hain et al. 1995) and LTCCs (Dzhura et al. 2002) to regulate excitation – contraction coupling (Li et al. 1997, Wu et al. 2001a).

1997) that initiates the rapid depolarization phase of action potentials in contractile myocardium. Ca2+ is both a charge carrier and a second messenger. LTCCs provide Ca2+ that is critical for activating Ca2+i-dependent cardiomyopathic transcriptional signaling pathways (Muth et al. 2001). Precedence for the concept that LTCCs constitute a privileged pathway to supply Ca2+i for critical cellular functions comes in part from studies in neurons (Dolmetsch et al. 2001) and adrenal glomerulosa cells (Artalejo et al. 1994), where LTCCs serve as high efficiency Ca2+ entry sites for activating the transcription factor CRE binding protein and for secretion. The second messenger function of Ca2+i also serves to recruit molecular determinants of structural disease that can create the tissue substrate for arrhythmias and sudden cardiac death (Molkentin et al. 1998, Wu et al. TCM Vol. 14, No. 4, 2004

2002, Zhang et al. 2003, Zhang et al. 2002a). On the other hand, Ca2+ acting as a charge carrier can also contribute to the physiology of action potentials and to electrical instability and arrhythmias (see below), particularly in heart disease in which action potentials are prolonged (Beuckelmann et al. 1993). This dual nature of Ca2+ participation in electrical instability and pathological transcription may be an important factor underlying the clinical association between structural heart disease and arrhythmic sudden death. 

LTCC Structure and Function

L type refers to ‘‘long lasting,’’ a historic nomenclature that antedated molecular identification of the determinants of ICa. ICa was also known as the slow inward current, to contrast with the far more rapid kinetics of the Na+ current. The

pore-forming a subunit of LTCCs (Figures 2a and b) is now referred to as Cav1.2, to indicate that it is a member of the voltage-gated (v) Ca2+ channel family. Cav1.2 is structurally homologous to voltage-gated Na+ channels, and belongs to a family of six membranespanning, voltage-gated, ion channels. Voltage gating indicates that LTCCs open in response to a depolarizing change in cell membrane potential (Figure 2e), as occurs during the cardiac action potential. There is only one gene encoding the Cav1.2, but multiple isoforms are reported from alternative splicing. The cytoplasmic C terminus is a region richly endowed with motifs and consensus sites for cell membrane targeting (Gao et al. 2000), phosphorylation, and Ca 2 + i -dependent signal transduction. Alternative splicing among the 12 exons that encode the C terminus results in a mixture of channel isoforms

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Beat number Figure 2. L-type Ca2+ channel (LTCC) current (ICa) contributes to early afterdepolarizations and is increased by calmodulin kinase II (CaMKII). (a) A schematic depiction of the LTCC with numerals labeling the four homologous domains of the pore-forming a subunit. The a subunit C terminus is large and cytoplasmic and is a site for phosphorylation by protein kinase A (PKA) (indicated by a circled S) and contains at least 2 calmodulin-binding domains (IQ and CBD). The h subunit binds the a subunit at the cytoplasmic linker region between homologous domains I and II, and contains two PKA phosphorylation sites (each indicated by a circled S) that can increase ICa. (b) An en face projection of the LTCC a subunit with ‘‘Ca2+’’ marking the location of the pore region. (c) Voltage clamp configurations that can be used to measure single LTCC currents (left panel) or all LTCC currents (i.e., ICa) on a cardiomyocyte (right panel). LTCCs are depicted as paired rectangles. (d) The relationship between ventricular action potentials (top) and the QT interval (bottom). An early afterdepolarization (arrow) arises in the setting of action potential prolongation (right top panel) and is associated with a U wave (arrow at right bottom panel). (e) An action potential waveform with an early afterdepolarization (arrow at top panel) was digitized and used repetitively as a voltage clamp command to elicit ICa that increases from the first to the fifth ‘‘beat,’’ by a process termed facilitation. There is a secondary increase in ICa associated with the early afterdepolarization (arrow at lower panel). (f) ICa facilitation requires CaMKII activity, because the normal pattern of ICa facilitation is prevented by dialysis of a CaMKII inhibitory peptide (modeled after the pseudosubstrate region on CaMKII; see Figure 3a), but not by an inactive control peptide. (Data from panels e and f are modified from Wu et al. 1999a.)

with different functional characteristics. For example, PKA is a serine/threonine kinase that catalyzes transfer of a phosphate from adenosine triphophate to a serine or threonine residue. A single PKA phosphorylation consensus site on the C terminus—Ser 1928—is critical for transduction of b-adrenergic receptor agonist increases in I Ca (Gao et al. 1997), but this phosphorylation site is absent from at least one truncated LTCC isoform (Gerhardstein et al. 2000). Kinases are typically ‘‘paired’’ with phosphatases—enzymes that dephosphorylate specific amino acids to dynamically regulate protein function. Although much of this review is dedicated to the action of protein kinases, it is important to consider that the balance of kinase

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signaling actions also depends on phosphatase activity. In addition to the pore-forming a subunit, auxiliary subunit proteins associate with the a subunit to modulate a-subunit responses to voltage and phosphorylation. b subunits bind to a subunits through well-defined interaction domains (Figure 2a) (De Waard et al. 1994), and serve as chaperones to enhance a-subunit expression at the cell membrane by binding and masking a retention signal that would otherwise limit LTCC expression (Bichet et al. 2000). b subunits increase LTCC openings (Dzhura and Neely 2003) and regulate the kinetics of ICa inactivation (Wei et al. 2000). This versatile subunit is also a substrate for serine/threonine protein

kinases, and has two PKA consensus sites important for b-adrenergic, receptor-mediated increases in I Ca (Bunemann et al. 1999). There are at least four genes encoding b subunits, and the mixture of b subunits in heart is a matter of active investigation (Wei et al. 2000). However, functionally significant PKA consensus sites and the a – b interaction domains are conserved across isoforms. The a2d subunit is heavily glycosylated and predominantly extracellular (Brickley et al. 1995), and serves to facilitate functional a – b subunit interactions and to regulate the voltage dependence of a-subunit gating charge (Shirokov et al. 1998). Although skeletal and neuronal LTCCs also associate with a c subunit, these are not known to be TCM Vol. 14, No. 4, 2004

present in heart. Thus, LTCCs are a macromolecular complex, built around the pore-forming a subunit, capable of responding to diverse stimuli. 

Ca2+i Regulates LTCCs

It has been known for some time that Ca2+i can reduce (by increasing inactivation) (Brehm and Eckert 1978) or increase (by a process termed facilitation) (Marban and Tsien 1982) the magnitude of various Ca2+ currents, and relatively recently the Ca2+i -sensing protein CaM was implicated in both of these processes (Zuhlke et al. 1999). Several groups

identified CaM-binding motifs on the asubunit C terminus (Pate et al. 2000, Qin et al. 1999, Zuhlke et al. 1999) and established that mutant forms of CaM that do not bind Ca2+ normally are incapable of supporting Ca2+i-dependent regulation of ICa in heterologous cells (Peterson et al. 1999) and in cardiomyocytes (Alseikhan et al. 2002). A single mutation (I1654A) in one of these C-terminus CaM binding domains resulted in an exaggerated pattern of ICa increases with repetitive stimulation (Zuhlke et al. 1999), suggesting the possibility that CaM binding domains themselves are signaling molecules, in addition to bind-

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ing CaM, by acting as ‘‘autofacilitation’’ ligands to increase ICa when Ca2+i is low. This concept is supported by recent findings (Dzhura et al. 2002 and 2003, Wu et al. 2001b) that peptides modeled after two separate, but adjacent, CaM binding domains (Figure 1a) increase the probability of LTCC openings by inducing LTCCs to enter a gating state characterized by prolonged channel openings. Thus, Ca2+-bound CaM (Ca2+/CaM) can reduce ICa by binding to the a-C terminus and these C-terminus CaM-binding sites can increase ICa facilitation under conditions of low Ca2+/CaM activity. The physiologic and pathologic implications

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Figure 3. Calmodulin kinase II (CaMKII) activity increases with action potential prolongation and afterdepolarizations to increase L-type Ca2+ channel (LTCC) opening probability. (a) Each CaMKII monomer consists of an N-terminus catalytic domain, a regulatory domain, and a Cterminus autoassociation domain that assembles the multimeric holoenzyme. Under basal conditions of intracellular Ca2+ (Ca2+i), CaMKII is inactive. Elevations in Ca2+i increase the amount of Ca2+-bound calmodulin (Ca2+/CaM) that binds to a CaM-binding sequence within the regulatory domain of CaMKII. This Ca2+/CaM binding activates CaMKII by inducing a conformational change that exposes the catalytic domain (marked by a star). High-frequency stimulation or prolonged Ca2+i pulses (of a duration relevant to action potential prolongation in heart) favor intersubunit phosphorylation of a Thr286 (or 285) (De Koninck and Schulman 1998) that is within the regulatory domain. The 286 phosphorylation causes persistent CaMKII activation, even after Ca2+i returns to basal levels (Lisman et al. 2002). (b) Action-potential prolongation and afterdepolarizations result in increased Ca2+-independent CaMKII activity (Anderson et al. 1998). (c) A cell-free, excised cell membrane voltage clamp configuration (lower panel) can measure single LTCC currents under conditions in which macromolecules can be added directly to the cytoplasmic face of the cell membrane. (d) A single LTCC current (channel openings are seen as downward deflections from baseline) is elicited by repetitive depolarizing voltage clamp steps and reveals infrequent, brief openings under basal conditions. Both protein kinase A (PKA) (e) and CaMKII (f) cause frequent and prolonged LTCC openings compared with baseline. PKA and CaMKII effects were nonadditive under these experimental conditions. The probability of LTCC opening during a depolarizing voltage clamp step is dramatically increased upon addition of PKA or CaMKII, compared with basal conditions (lower panels). (Data from panels d and f were modified from Dzhura et al. 2002.)

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of I Ca increases by CaM-binding domains remains uncertain, but the CaM inhibitory drug W-7 effectively suppresses Torsade de Pointes, a type of polymorphic ventricular tachycardia favored by excessive action potential prolongation (Gbadebo et al. 2002, Mazur et al. 1999), suggesting that CaM itself does participate in arrhythmogenic cellular signaling. 

Ca2+ and CaM Activate CaMKII

Ca2+-bound CaM (Ca2+/CaM) activates the multifunctional CaMKII. CaMKII is a serine/threonine kinase that is abundant in heart, where it exists in multiple splice variants of two distinct gene products (isoforms d and c) (Hoch et al. 1999). All significant regulatory and catalytic domain sequences are conserved across isoforms, except for a nuclear localization sequence that is present in dB (Zhang et al. 2002a), but not dC (Zhang et al. 2003). The CaMKII holoenzyme is a multimeric protein consisting of 8 to 12 monomers (Kolodziej et al. 2000), and this higher-order structure has significant implications for enzyme function (De Koninck and Schulman 1998). Under basal conditions of Ca2+i (dictated largely by heart rate and action potential duration) most CaMKII is inactive, because the N-terminus catalytic domain is constrained through an interaction with a pseudosubstrate region within the regulatory domain (Figures 3a and b). CaMKII is activated when Ca2+i is increased and Ca2+/CaM binds to a region within the regulatory domain that induces a conformational change, freeing the catalytic domain to phosphorylate protein substrates, favored by the consensus sequence X 1 X 2 RX 3 X 4 S/T, where X1 is a hydrophobic amino acid and X 3 is a nonbasic amino acid (White et al. 1998). CaMKII is itself a substrate for CaMKII, and phosphorylation of Thr 286 (numbered for the neuronal a isoform) alters the requirement for activating Ca2+i by inducing a dramatic increase in Ca2+/CaM binding avidity (from nanomolar to picomolar) (Meyer et al. 1992). CaMKII autophosphorylation at Thr 286 also preserves a significant fraction of CaMKII activity in the absence of Ca2+/CaM binding (Figure 3a). CaMKII autophosphorylation at Thr 286 is very sensitive to both

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the frequency and pulse duration of the Ca2+i stimulus (De Koninck and Schulman 1998). Action potential durations vary according to disease states and between species and importantly determine the Ca2+i transient duration. Thus, differences in Ca2+i stimulus duration likely explain how CaMKII is dynamically activated over heart rates from mouse (short action potentials and fast rates) to human (longer action potentials and slower rates). The multimeric nature of CaMKII, combined with the dynamic features of CaMKII activity that are driven by autophosphorylation, make CaMKII ideal for integrating Ca2+i responses in heart. 

CaMKII Stimulates LTCCs

In cardiomyocytes, CaMKII activity results in a dynamic pattern of I Ca increases and slowed inactivation that are collectively termed facilitation (Figures 2e and f ) (Anderson et al. 1994, Yuan and Bers 1994). At the single LTCC level, CaMKII increases Ca2+ entry by inducing Ca2+ channels to enter into a behavior (called a gating mode) with markedly prolonged openings (Figures 3c – f) that likely underlie ICa facilitation (Dzhura et al. 2000). PKA and CaMKII produce similar effects on LTCCs (Figures 3d – f), because PKA also augments peak ICa and slows inactivation by inducing a gating mode (mode 2) characterized by long channel openings (Dzhura et al. 2002). PKA catalyzes phosphorylation of amino acid Ser 1928 on the a-subunit C terminus (Gao et al. 1997) and two amino acids on the b subunit (Gerhardstein et al. 1999) that cause increased channel openings. CaMKII can also phosphorylate an LTCC a subunit (Jahn et al. 1988) but, in contrast to PKA, the identity of critical amino acid determinants for CaMKII action is as yet unknown. CaMKII appears to be anchored near to cardiac LTCCs by a cytoskeletal-dependent mechanism that positions CaMKII for activation by Ca2+i outside of the channel pore microdomain (Dzhura et al. 2002). The location of CaMKII outside of the LTCC pore fits with the finding that CaMKII is primarily activated by Ca2+ released from intracellular (SR) Ca2+ stores, rather than by ICa (Bartel et al. 2000) (Figure 1). ICa facilitation does not occur when Cav1.2 is

expressed in noncardiac cell types (Zuhlke et al. 1999), or when cardiomyocytes are treated with drugs (e.g., ryanodine) that eliminate SR Ca2+ release (Wu et al. 1999a). This requirement of normal ICa function for the ultrastructural context of a cardiomyocyte may be due to the fact that nonheart cells lack SR Ca2+ release that is essential for activation of CaMKII. CaMKII activity increases are steeply graded over a range of stimulation rates and Ca2+i transient durations present in heart (see Figure 4 in De Koninck and Schulman 1998), and CaMKII is most effective for increasing ICa over a time course and at cell membrane potentials that occur during the cardiomyocyte action potential plateau (Wu et al. 2004). Taken together, these findings strongly suggest that CaMKII is functionally poised to increase LTCC openings and, during excessive activity, has the potential to trigger arrhythmias by further enhancing LTCC reopenings during the action potential plateau (see below).



Increased LTCC Activity Is Proarrhythmic

Increased LTCC openings can contribute to an ICa ‘‘window current’’ (Figure 2e) that destabilizes the cardiac action potential plateau (Hirano et al. 1992) to cause arrhythmia-initiating afterdepolarizations (January and Riddle 1989, Zeng and Rudy 1995). Early afterdepolarizations (EADs) occur during action potential repolarization (Figures 2d and e), whereas delayed afterdepolarizations (DADs) follow completion of action potential repolarization (Luo and Rudy 1994), and both EADs and DADs are thought to trigger polymorphic ventricular tachycardia, including Torsade de Pointes. EADs are linked to LTCC openings (January and Riddle 1989), and can be initiated by the LTCC agonist drug BAYK8644 (January and Riddle 1989). On the other hand, DADs represent the response of the electrogenic Na+/Ca2+ exchanger to Ca2+ overload that can follow excessive Ca 2+ entry through LTCCs (Wu et al. 1999b). DADs are linked to ‘‘waves’’ of Ca2+i (Boyden et al. 2000), raising the possibility that local Ca2+i elevations may create proarrhythmic microdomains of TCM Vol. 14, No. 4, 2004

is not fully understood, it suggests that successful therapies for cardiac arrhythmias in the setting of structural heart disease may need to target molecules with multiple effects on Ca2+i homeostasis. b-adrenergic receptor antagonists are also indirect PKA inhibitors, and PKA and CaMKII both phosphorylate LTCCs (Armstrong et al. 1991, Dzhura et al. 2000 and 2002), RyR (Lokuta et al. 1995, Reiken et al. 2003), and phospholamban (Tada et al. 1974)—molecules critical for Ca2+i homeostasis—suggesting that CaMKII inhibition could mimic favorable effects of b-adrenergic receptor antagonists.

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Figure 4. Calmodulin kinase II (CaMKII) activity links afterdepolarizations and arrhythmias in a CaMKIV transgenic (TG) mouse model of cardiac hypertrophy. (a) Multiple early afterdepolarizations (EADs) are seen as oscillations of the action potential plateau. (b) EADs were present in TG mice, but not wild-type (WT) littermate controls, and (c) were completely suppressed by intracellular dialysis of a CaMKII inhibitory peptide. (d) The EAD-initiated arrhythmia Torsade de Pointes is recorded from an unanesthetized and unrestrained electrocardiogram-telemetered TG mouse (asterisks mark initiation of arrhythmia salvos). TG mice had higher arrhythmia scores at baseline (e) and after isoproterenol (f), compared with WT mice, and the arrhythmia scores were significantly reduced by injection of the CaMKII inhibitory drug KN-93, but not by the inactive drug KN-92 (g). (Data are modified from Wu et al. 2002.)

increased CaMKII activity. Both EADs and DADs can be seen in isolated cardiomyocytes after exposure to b-adrenergic receptor agonists (De Ferrari et al. 1995, Priori and Corr 1990), suggesting a possible mechanism for the reduction of sudden cardiac death in patients treated with b-adrenergic receptor antagonist drugs (Anonymous 1981 and 1999). b adrenergic receptor stimulation increases Ca2+i, in part by increasing the activity of PKA, which in turn increases TCM Vol. 14, No. 4, 2004

LTCC activity (Dzhura et al. 2002). Thus, conditions that prolong cardiac repolarization increase LTCC activity and disorder Ca 2+ i to favor arrhythmia initiation. It is interesting to contrast the demonstrated efficacy of b-adrenergic receptor antagonist drugs in reducing sudden death mortality with the disappointing results from LTCC antagonist agents (Clusin and Anderson 1999). Although the biologic basis for this discrepancy

CaMKII Is Proarrhythmic

CaMKII activity and expression are increased in patients (Hoch et al. 1999, Kirchhefer et al. 1999, Tessier et al. 1999) and in a variety of animal models of structural heart disease (Colomer et al. 2003, Currie and Smith 1999, Hagemann et al. 2001, Wu et al. 2002), conditions generally marked by action potential (Beuckelmann et al. 1993) and QT interval prolongation (Boccalandro et al. 2003, Brooksby et al. 1999, Vrtovec et al. 2003), disordered Ca2+i (Beuckelmann et al. 1992, Gwathmey et al. 1987), and an increased risk of sudden death. CaMKII is activated during drug-induced action potential prolongation (Anderson et al. 1998) (Figure 3b), and in ischemia-induced ventricular fibrillation (Billman et al. 1991). EADs (Wu et al. 1999a and 2002) and the transient inward current underlying DADs (Wu et al. 1999b) are both suppressed by CaMKII inhibition in isolated cardiomyocytes, and the Ca2+/CaM antagonist agent W-7 effectively suppresses the afterdepolarization-initiated arrhythmia Torsade de Pointes in a rabbit model of pharmacologic QT interval prolongation (Gbadebo et al. 2002, Mazur et al. 1999). LTCCs are likely involved in Torsade de Pointes initiation because this arrhythmia was evoked by the LTCC agonist BAYK8644 (Mazur et al. 1999), whereas BAYK8644 also enhanced inducibility of ventricular fibrillation in ischemic canine myocardium (Billman et al. 1991). Suppression of Torsade de Pointes occurred without shortening the QT interval (a possible antiarrhythmic effect) (Fish et al. 1990), but rather by preventing development of oscillations (U waves)

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Structural heart disease Action potential prolongation

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Arrhythmias & sudden death Figure 5. The hypothesized mechanism for linking structural heart disease phenotypes to increased arrhythmias and sudden death. Ca2+i, cellular Ca2+; CaMKII, calmodulin kinase II; LTCC, L-type Ca2+ channel.

The cardiac Ca2+ channel Cav1.2 is central to physiologic and pathologic events in myocardium and is a central target for second messenger regulation by b-adrenergic receptor stimulation that generates PKA. More recently, it has become clear that CaMKII also regulates ICa and that under disease conditions marked by a high risk for arrhythmic sudden death, CaMKII activity and Ca2+ channel openings are increased. Experimental studies in animals implicate CaMKII as a proarrhythmic signaling molecule and suggest the possibility that CaMKII inhibition may be a novel and effective antiarrhythmic therapy.



in the QT segment that reliably heralded arrhythmia onset (Gbadebo et al. 2002). EADs are a cellular correlate of U waves (Antzelevitch and Sicouri 1994); thus, this finding suggests that W-7 may inhibit arrhythmias by preventing CaMKII-triggered EADs. In support of this concept, the more specific CaMKII inhibitory agent KN-93 also effectively suppressed EADs in isolated hearts (again, without shortening the action potential duration) (Anderson et al. 1998), further implicating CaMKII as a critical Ca2+/CaM-activated proarrhythmic target. More recently, we used mice with cardiac overexpression of a type of CaMK not endogenous to heart (CaMKIV) that develop cardiac hypertrophy, marked QT interval and action potential prolongation, arrhythmias, and increased CaMKII expression and activity to further dissect this hypothesized pathway between CaMKII and arrhythmias (Wu et al, 2002). Cardiomyocytes from these mice had LTCCs with increased opening probability, as occurs in failing human cardiomyocytes (Schroder et al. 1998), and action potentials with spontaneous EADs (Figure 4a) (Wu et al. 2002). CaMKII inhibition normalized the LTCC opening probability and eliminated EADs (Figures 4b and c), indicating that CaMKII was responsible for these electric phenotypes. We used implanted electrocardiogram telemeters and a scoring system to quantify ar-

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rhythmia burden in unanesthetized and unrestrained mice. Mice with cardiac hypertrophy had more arrhythmias at baseline and after the b-adrenergic receptor agonist isoproterenol (Figures 4d – f). KN-93—a CaMK inhibitor that selectively targeted CaMKII in these hearts—reduced high arrhythmia scores seen in response to isoproterenol (Figure 4g), suggesting that PKA and CAMKII may converge on a common set of arrhythmia-activating cellular targets. To date, the cardiac CaMKII isoforms (d and c) (Hoch et al. 1999) have not been knocked out. Mice with overexpression of the predominant cardiac d isoform develop dilated cardiomyopathy and die prematurely (Zhang et al. 2003), although arrhythmias have not been studied in these mice. Our laboratory recently developed a genetic model of cardiac CaMKII inhibition and these mice have a phenotype marked by reduced LTCC opening at baseline and after isoproterenol (Zhang et al. 2002b) and resistance to chamber dilation after myocardial infarction (Khoo et al. 2003). Taken together, a wide range of in vivo and in vitro studies link action potential prolongation, disordered Ca2+i, neurohumoral activation, and structural heart disease to increased CaMKII activity, increased LTCC openings, afterdepolarizations, and arrhythmias and raise the question whether CaMKII will be a useful new therapeutic target in heart disease (Figure 5).

Summary

Acknowledgments

This work was possible due to the collaborative efforts of a number of gifted scientists. Special thanks are due to Drs. Andy Braun, Roger Colbran, Igor Dzhura, Dan Roden, Howard Schulman, Yuejin Wu, and Rong Zhang. Ms. Linda Selfridge provided excellent secretarial assistance. M.E.A. is an established investigator of the American Heart Association and receives funding from the National Institutes of Health (HL62494, HL70250, and HL46681).

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PII S1050-1738(04)00028-3

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Modulation of Atherogenesis by Chemokines William A. Boisvert*

Migration of leukocytes into the vasculature—which involves the concerted effort of many molecules, including chemokines—is a requisite step for atherogenesis. The three chemokines that have been implicated most strongly in atherogenesis are monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8)/growth-regulated oncogene a (Gro-a), and fractalkine. Although all three chemokines appear to impact atherogenesis by attenuating monocyte/macrophage accumulation in the lesion, the precise mechanism of action of each of the chemokines, as well as their interactive role in atherosclerosis, have not been elucidated. This review focuses on the latest findings that describe the individual roles of MCP-1, IL-8/Gro-a, and fractalkine on macrophage recruitment in atherosclerosis. Furthermore, based on present knowledge of the participation of these three chemokines and their receptors in monocyte/macrophage recruitment, a possible interactive role of these chemokines in atherogenesis is explored. (Trends Cardiovasc Med 2004;14:161–161) n 2004, Elsevier Inc. William A. Boisvert is at the Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts, USA. * Address correspondence to: William A. Boisvert, Vascular Medicine Research, Brigham and Women’s Hospital, Harvard Medical School, 65 Landsdowne Street, Room 286, Cambridge, MA 02139, USA. Tel.: (+1) 617768-8415; fax: (+1) 617-768-8421; e-mail: [email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter

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Chemokines (chemotactic cytokines) are a family of small molecules (8 to 10 kDa) that are classically known to play an essential role in trafficking of leukocytes. In recent years, an expanded role of chemokines has been identified, including activation of leukocytes and mediation of inflammation (Reape and Groot 1999, Terkeltaub et al. 1998). Chemokines can be categorized into several classes according to the position of the first four conserved cysteine residues.

The chemokines of C-X-C (e.g., interleukin 8 [IL-8]/growth-regulated oncogene a [Gro-a], and epithelial-derived neutrophil attractant-78 [ENA-78]) and C-C (e.g., monocyte chemoattractant protein [MCP] 1 to 5, regulated on activation, normal T cell expressed and secreted [RANTES] and macrophage inflammatory protein-1 [MIP-1]) families constitute by far the majority of these molecules, with only a few chemokines belonging to CX3C (fractalkine) and C (lymphotactin) families (Baggiolini 1997, Luster 1998, Schall 1997). The receptors for these chemokines are comprised of a family of heterotrimeric, 7-transmembrane, Gprotein-coupled receptors, and can often bind multiple chemokines within the same family. Atherosclerosis is widely recognized as a chronic inflammatory disease in which leukocytes—especially monocytes/macrophages—play a pivotal role (Glass and Witztum 2001, Hansson 2001, Ross 1999). Leukocyte interaction with the vasculature requires that these cells be able to migrate to the atherosclerosis-prone vessel wall, a process that involves the concerted effort of several different classes of molecules such as selectins and integrins and their receptors. Chemokines also are an important class of molecules that mediate chemotaxis of leukocytes into the vasculature. To learn more about the general features of chemokines, the readers are referred to several excellent review articles devoted to the biology of chemokines and their receptors (Baggiolini 1997, Luster 1998, Ono et al. 2003). The focus of this review is to summarize some of the most recent literature on the role of three main chemokines, each belonging to a different family, that have been studied most extensively as to their role in the pathogenesis of atherosclerosis: MCP-1, IL-8/Gro-a, and fractalkine. 

MCP-1 in Atherosclerosis

As the name implies, MCP-1 is a key mediator of monocyte trafficking. MCP1 is made by multiple cell types—including endothelial cells, smooth muscle cells, and macrophages—all of which are important players in atherogenesis (Glass and Witztum 2001, Hansson 2001). Because macrophage recruitment to the vasculature is a key event in its pathology and because MCP-1 is a key

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