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Molecular Physiology of Cardiac Repolarization

Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri; and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York

ABSTRACT

I. INTRODUCTION

II. MYOCARDIAL ACTION POTENTIALS AND VOLTAGE-GATED INWARD SODIUM AND CALCIUM CURRENTS

A. Voltage-Gated Na+(Nav) Currents

B. Voltage-Gated Ca2+ (Cav) Currents

III. MYOCARDIAL ACTION POTENTIALS AND REPOLARIZING VOLTAGE-GATED POTASSIUM CURRENTS

A. Transient Outward Kv Currents

B. Delayed Rectifier Kv Currents

C. Regional Differences in Kv Current Expression and Properties

IV. OTHER MYOCARDIAL POTASSIUM CURRENTS CONTRIBUTING TO REPOLARIZATION

V. MOLECULAR COMPONENTS OF MYOCARDIAL NAV AND CAV CHANNELS

A. Nav Channel Pore-Forming {alpha}-Subunits

B. Nav Channel Accessory Subunits and Other Interacting Proteins

C. Cav Channel Pore-Forming {alpha}-Subunits

D. Cav Channel Accessory Subunits and Other Interacting Proteins

VI. MOLECULAR COMPONENTS OF MYOCARDIAL KV CHANNELS

A. Kv Channel Pore-Forming {alpha}-Subunits

B. Kv Channel Accessory Subunits

C. Molecular Correlates of Cardiac Transient Outward Kv Channels

D. Molecular Correlates of Cardiac Delayed Rectifier Kv Channels

VII. MOLECULAR COMPONENTS OF OTHER CARDIAC POTASSIUM CHANNELS

A. Inwardly Rectifying Cardiac K+ (Kir) Channel Pore-Forming {alpha}-Subunits

B. Two-Pore Domain K+ (K2P) Channel Pore-Forming {alpha}-Subunits

VIII. MYOCARDIAL POTASSIUM CHANNELS AND THE ACTIN CYTOSKELETON

IX. SUMMARY AND CONCLUSIONS

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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The normal mechanical (pump) functioning of the mammalian heart depends on proper electrical functioning (56, 173), reflected in the sequential activation of cells in specialized, "pacemaker" regions of the heart and the propagation of activity through the ventricles (Fig. 1). Myocardial electrical activity is attributed to the generation of action potentials in individual cardiac cells, and the normal coordinated electrical functioning of the whole heart is readily detected in surface electrocardiograms (Fig. 1). The propagation of activity and the coordination of the electromechanical functioning of the ventricles also depend on electrical coupling between cells, mediated by gap junctions (251, 435). The generation of myocardial action potentials reflects the sequential activation and inactivation of ion channels that conduct depolarizing, inward (Na⁺ and Ca²⁺), and repolarizing, outward (K⁺), currents (24, 375). The waveforms of action potentials in different regions of the heart are distinct (Fig. 1), owing to differences in the expression and/or the properties of the underlying ion channels (24, 374). These differences contribute to the normal unidirectional propagation of excitation through the myocardium and to the generation of normal cardiac rhythms (23, 24, 259, 374, 375). Changes in the properties or the functional expression of myocardial ion channels, resulting from inherited mutations in the genes encoding these channels (23, 36, 51, 102, 204, 243, 253) or from myocardial disease (34, 49, 67, 184, 365, 496, 501–503, 510), can lead to changes in action potential waveforms, synchronization, and/or propagation, thereby predisposing the heart to potentially life-threatening arrhythmias (13, 14, 16, 24, 127, 259, 436). There is, therefore, considerable interest in delineating the molecular, cellular, and systemic mechanisms contributing to the generation and maintenance of normal cardiac rhythms, as well as in understanding how these mechanisms are altered in the diseased myocardium.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Electrical activity in the myocardium. Top: schematic of a human heart with illustration of typical action potential waveforms recorded in different regions. Bottom: schematic of a surface electrocardiogram; three sequential beats are displayed.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Action potential waveforms and underlying ionic currents in adult human and ventricular (left) and atrial (right) myocytes. The time- and voltage-dependent properties of the voltage-gated inward Na+ (Nav) and Ca2+ (Cav) currents expressed in human atrial and ventricular myocytes are similar. In contrast, there are multiple types of K+ currents, particularly Kv currents, contributing to atrial and ventricular action potential repolarization. The properties of the various Kv currents are distinct, and in contrast to the inward currents, there are multiple Kv currents expressed in individual myocytes throughout the myocardium.

The driving force for K⁺ efflux is high during the plateau phase of the action potential in ventricular and atrial myocardium and, as the Cav channels inactivate, the outward K⁺ currents predominate, resulting in (phase 3) repolarization, bringing the membrane voltage back to the resting potential (Fig. 2). In contrast to Nav and Cav currents, however, there are multiple types of voltage-gated K⁺ (Kv) currents, as well as non-voltage-gated, inwardly rectifying K⁺ (Kir) currents (Table 1), that contribute to myocardial action potential repolarization. The greatest functional diversity is among Kv channels (Table 1). At least two types of transient outward currents, I_to,f and I_to,s, and several components of delayed rectification, including I_Kr [I_K(rapid)], I_Ks [I_K(slow)], and I_Kur [I_{K(ultrarapid)}], for example, have been distinguished (Table 1). The time- and voltage-dependent properties of the various Kv currents identified in myocytes isolated from different species and/or from different regions of the heart in the same species, however, are remarkably similar, suggesting that the same (or very similar) molecular entities contribute to the generation of each of the various types of Kv channels (Table 1) in different cells/species. The relative Kv channel expression levels vary in cardiac cells in different regions (i.e., atria, ventricles) of the heart, and this heterogeneity contributes importantly to the observed regional differences in action potential waveforms (24, 127, 373, 374). Changes in the properties or the functional expression of Kv channels, as occurs in a variety of myocardial diseases (34, 49, 67, 365, 496, 503, 510), can, therefore, have dramatic effects on action potential waveforms, propagation, and rhythmicity.

A large number of pore-forming () subunits, encoding Nav, Cav, Kv, and Kir channels, and a variety of channel accessory (, , and ) subunits have been identified (Tables 2–6), and considerable progress has been made in defining the expression patterns of these subunits in the heart and the roles of the individual subunits in the generation of functional cardiac (Nav, Cav, Kv, and Kir) channels (Tables 2–6). These studies have demonstrated that distinct molecular entities underlie the various cardiac ion channels/currents that have been distinguished electrophysiologically and shown to contribute to myocardial action potential repolarization. It also has now been shown that mutations in the genes encoding the subunits involved in the generation of functional cardiac Nav, Kv, Cav, and Kir channels underlie several inherited cardiac arrhythmias (23, 36, 51, 102, 204, 253, 479, 481). Although inherited rhythm disorders are rare, these mutations belong to an ever-increasing number of "channelopathies," i.e., diseases linked to genes encoding ion channels (35, 123, 204, 220, 234, 243, 267, 309, 359, 403, 442, 459). Based on the rapid progression of this field (249) and the growing molecular complexity of ion channels, it seems certain that the number of genes encoding ion channels or ion channel regulatory molecules linked to inherited and acquired disorders of the cardiovascular (and other) system will continue to increase, perhaps dramatically, in the future. The densities and the functional properties of myocardial Nav, Cav, Kv, and Kir currents also change in a number of acquired myocardial disease states (34, 49, 67, 365, 496, 503, 510), and these changes can lead to the generation of potentially life-threatening cardiac arrhythmias. At present, therefore, there is considerable interest in understanding the detailed molecular mechanisms controlling the properties and the functional cell surface expression of the various ion channels controlling myocardial action potential repolarization, as well as the impact of genetic and epigenetic factors, including cardiac and noncardiac disease, on the functioning of these channels.

View this table: [in this window] [in a new window]   TABLE 2. Diversity of voltage-gated Na+ (Nav) channel - and -subunits

	II. MYOCARDIAL ACTION POTENTIALS AND VOLTAGE-GATED INWARD SODIUM AND CALCIUM CURRENTS

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Voltage-gated cardiac Na⁺ (Nav) channels open rapidly on membrane depolarization (Fig. 2) and underlie the rapidly rising phases of the action potentials recorded in mammalian ventricular and atrial myocytes and in cardiac Purkinje fibers (93, 375). Although not evident in all cells, Nav channels with similar properties are also expressed in subsets of mammalian SAN and AVN cells, and differences in functional Nav channel expression likely contribute to action potential heterogeneity in pacemaker cells (262, 361, 579, 582). Although the properties of the Nav channels expressed in different cardiac cells are similar, the biophysical and pharmacological properties of these channels are distinct from Nav channels expressed in other excitable cells, such as neurons and skeletal muscle (93, 573). Cardiac Nav channels, for example, are remarkably insensitive to the Nav channel toxin tetrodotoxin (TTX), which binds with high (nM) affinity to neuronal and skeletal muscle Nav channels and blocks Na⁺ influx (93, 573). This observation was probably the first indication that the molecular identities of the Nav channels in cardiac myocytes, neurons, and skeletal muscle were distinct, and as detailed in section VA, this has now been demonstrated.

On membrane depolarization, cardiac Nav channels activate and inactivate rapidly (172, 174). The threshold for Nav channel activation is quite negative (approximately –55 mV), and the activation of these channels is steeply voltage dependent. Importantly, inactivation is also voltage dependent, and cardiac Nav channels can undergo voltage-dependent inactivation without ever opening (174). Nevertheless, persistent openings of cardiac Nav channels are occasionally observed, even at depolarized membrane potentials (437, 591). At potentials corresponding to the action potential plateau in ventricular myocytes, present estimates are that 99% of the Nav channels are in an inactivated, nonconducting state (423, 525). There is, therefore, a finite, albeit small (1%), probability of Nav channels being open at potentials corresponding to the action potential plateau (525). A slow component of Nav channel inactivation has indeed been described in normal human ventricular myocytes (323). This current is modulated by lysolipids (501) and appears to be upregulated in failing myocardium (501–503). Importantly, the inward (Na⁺) current through open Nav channels during the action potential plateau (phase 2) will counter the effects of the increased K⁺ efflux, thereby slowing or delaying repolarization and increasing action potential durations (23, 102). It follows, therefore, that changes in Nav channel open probability at voltages corresponding to the plateau (i.e., phase 2) could markedly affect action potential waveforms, particularly in ventricular cells.

The probability of Nav channel opening at depolarized potentials (i.e., during phase 2) is determined by the overlap of the curves describing the voltage dependences of channel activation and inactivation (30). At the molecular level, the fact that some channels are open over this voltage "window" implies that there is a finite probability that inactivation is reversible, i.e., that inactivated channels can reopen at depolarized potentials. Consistent with these predictions, electrophysiological studies reveal the presence of a sustained component of inward Nav current, i.e., a "persistent" Nav current, during prolonged membrane depolarizations. Although the persistent Nav channel ("window") current is small, this current could, in principle, contribute to determining action potential durations (438, 439). In this context, it is interesting to note that it has been reported that the expression level of the persistent Nav current component varies in different regions of the ventricles (438), differences that could contribute to the observed regional heterogeneities in ventricular action potential durations (24, 374). The concept that small inward (Na⁺) currents could profoundly affect action potential waveforms and excitability in the myocardium was suggested more than 50 years ago in the pioneering studies of Silvio Weidman (542). The impact of alterations in the "persistent" Nav channel window current on cardiac rhythms has now been definitively demonstrated with the identification of mutations in the gene, SCN5A, encoding the TTX-insensitive cardiac Nav channels (see sect. VA) in patients with an inherited form of long QT syndrome, LQT3 (52). A number of SCN5A mutations in different affected individuals/families have been identified (Fig. 3A) and linked to Brugada syndrome and to conduction defects, in addition to LQT3 (23, 36, 51, 60, 93, 102, 105, 204, 253, 422, 519, 525, 540). Interestingly, mutations in noncardiac Nav channel genes have also been linked to familial paroxysmal dysfunction in the skeletal and nervous systems (123, 204, 220, 359).

View larger version (46K): [in this window] [in a new window]   FIG. 3. Pore-forming () subunits of cardiac Nav (A) and Kv (B and C) channels linked to inherited arrhythmias. A: membrane topology of the four domains (I–IV) of SCN5A is illustrated with mutations linked to LQT3 and Brugada syndromes and to conduction disorders. Schematics illustrating the transmembrane topologies of KCNH2 (B) and KCNQ1 (C) subunits with the mutations linked to LQT2 and LQT1, respectively, depicted by the open and closed circles.

In contrast to skeletal muscle, it has long been recognized that Ca²⁺ entry from the extracellular space is required for excitation-contraction coupling in the mammalian myocardium (56, 57, 154, 173). The pathway for plasmalemmal Ca²⁺ entry was first revealed in voltage-clamp recordings from multicellular (frog) atrial preparations and was termed the "slow inward" current pathway (416–418, 429). Subsequent studies revealed that this "slow inward" current is carried by Ca²⁺ through a membrane conductance distinct from the voltage-dependent (Nav channel) pathway for Na⁺ movement (45, 332, 386, 416–418, 429). Further studies detailed the time- and voltage-dependent properties of voltage-gated cardiac Ca²⁺ (Cav) currents, first, in multicellular preparations and later, in isolated single cardiac cells (45, 57, 172, 416–418).

Although the presence of two functionally distinct types of Cav currents in single (starfish egg) cells was first reported in 1975 (201), it was not until the late 1980s that the import and the generality of these observations became clear. Two types of Cav currents/channels, for example, were clearly distinguished in (chick and rat) sensory neurons, based primarily on differences in the thresholds for channel activation (85, 86). These channels were termed high voltage-activated (HVA) and low voltage-activated (LVA) Cav channels. Cardiac HVA and LVA Cav channels were first described in isolated canine atrial cells (44). LVA Cav channels, also referred to as T-type Ca²⁺ channels (394), activate at relatively hyperpolarized membrane potentials, i.e., approximately –50 mV, and these channels activate and inactivate rapidly (85, 86, 382). HVA Cav channels, in contrast, open on depolarization to membrane potentials positive to approximately –20 mV, and these channels inactivate over a time course of several tens of milliseconds to seconds, depending on the preparation and the charge carrying ion (85, 326). Under physiological conditions, with Ca²⁺ as the charge carrier, HVA channels in most cells inactivate in <100 ms at depolarized voltages (44, 326).

The detailed kinetic, pharmacological, and voltage-dependent properties of HVA Cav channels in different cell types are distinct, suggesting that HVA Cav channels are heterogeneous, particularly compared with LVA Cav channels. Consistent with this view, multiple types of HVA channels have now been identified in different cell types, and these are referred to as L, N, P, Q, or R channels (276, 382, 394). Although all HVA Cav channels exhibit relatively large single-channel conductances (13–25 pS) and have similar permeation properties, the detailed biophysical properties and the pharmacological sensitivities of the various types of HVA Cav channels are distinct. In the mammalian heart, L-type HVA Cav currents appear to be ubiquitously expressed (44, 49, 326). In addition, the properties and the densities of L-type Cav channel currents in cells isolated from different regions of the myocardium, as well as in cardiac cells from different species, are quite similar, suggesting that the molecular compositions of the underlying channels and the molecular mechanisms controlling the functional expression of these channels are the same. Importantly, however, the time- and voltage-dependent properties of cardiac L-type HVA currents are distinct from the L-type HVA Cav currents expressed in skeletal muscle and in neurons (44, 326, 340), suggesting that, similar to the Nav channels, distinct molecular entities underlie the L-type HVA Cav channels in different tissues (see sect. VC).

The opening of cardiac L-type Cav channels in response to membrane depolarization is delayed relative to the Nav channels (Fig. 2), and these channels, therefore, contribute little to phase 0 depolarization in Purkinje, atrial and ventricular cells. Rather, the opening of HVA L-type Cav channels and the Ca²⁺ entry through these channels underlies the action potential plateau (phase 2), which is particularly prominent in ventricular and Purkinje cells (Fig. 2). In addition, Ca²⁺ influx through the L-type HVA Cav channels triggers Ca²⁺release from intracellular Ca²⁺stores and underlies excitation-contraction coupling in the working (ventricular) myocardium (56, 57, 154, 173). L-type HVA Cav channels are also expressed in SAN and AVN cells, where they play a role in action potential generation, as well as in regulating automaticity (49, 72, 262, 340, 361, 579). Cardiac L-type HVA Cav channels undergo rapid voltage- and Ca²⁺-dependent inactivation (166, 281, 326), processes that will also influence action potential waveforms (Fig. 2) by affecting the duration of the plateau (phase 2) and the time course of action potential repolarization.

In addition to the ubiquitously expressed HVA L-type cardiac Cav currents, LVA or T-type Cav channel currents have also been identified in voltage-clamp recordings from adult atrial myocytes and conducting tissues in several different species (44, 200, 340, 394). Although not evident in normal adult ventricular myocytes (394), T type Cav currents have also been recorded in neonatal rat and rabbit ventricular myocytes (547, 548). In addition, it has been demonstrated that T-type Cav currents are expressed in ventricular myocytes in several animal models of ventricular hypertrophy (328, 383), findings consistent with the view that substantial remodeling occurs in the hypertrophied myocardium, reflecting a reversion to a fetal/neonatal pattern of gene expression (486). It is certainly possible that similar remodeling occurs in the hypertrophied human heart (49). Nevertheless, it is important to note that, to date, T-type LVA Cav channels have not been detected in normal or diseased human myocardial cells (49, 394).

In addition to marked differences in biophysical properties, the physiological role(s) of cardiac T-type LVA Cav channels appears to be quite different from the L-type HVA Cav channels. As noted previously, for example, Ca²⁺ entry through L-type channels in cardiac cells results in Ca²⁺-induced Ca²⁺ release from intracellular (Ca²⁺) stores and is the main trigger for excitation-contraction coupling (57, 154). Although Ca²⁺ entry through T-type channels also triggers Ca²⁺ release from intracellular stores, the coupling is less efficient (589), and it seems unlikely that T channels contribute importantly to excitation-contraction coupling. This may simply reflect the fact that LVA channel densities are low and that, owing to rapid inactivation, very little Ca²⁺ actually enters cells on depolarization. Alternatively, these functional differences may reflect the fact that HVA and LVA cardiac Cav channels are differentially localized, i.e., L-type, but not T-type, Cav channels are highly localized in the t tubules near the storage sites for intracellular Ca²⁺ sequestration/release (394, 589). Further experiments will be necessary to determine the mechanistic basis for the distinct functional roles of L- and T-type Ca²⁺ channels in regulating excitation-contraction coupling.

The finding that T-type LVA Cav currents activate at relatively hyperpolarized potentials and that these channels are expressed preferentially in pacemaker and conducting cells in the heart suggests the interesting possibility that there is a role for LVA channels in pacemaking (200). Although some experimental support for this hypothesis has been provided, rigorous testing is complicated by the paucity of highly selective LVA Cav channel blockers (394). In addition, it has been reported that (rabbit) SAN cells express rapidly activating Na⁺-dependent inward currents that are blocked by Ca²⁺ channel blockers (582). These observations suggest that pacemaker cells may express additional novel inward currents that contribute to shaping action potential waveforms and to regulating normal cardiac rhythms. Additional studies, focused on further characterization of the properties of LVA Cav channels in SAN and AVN cells and determination of the functional roles of these channels in regulating pacemaking, will be needed to explore these possibilities directly.

	III. MYOCARDIAL ACTION POTENTIALS AND REPOLARIZING VOLTAGE-GATED POTASSIUM CURRENTS

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Voltage-gated K⁺ (Kv) channels are the primary determinants of action potential repolarization in the mammalian myocardium, and compared with cardiac Nav and Cav channels, there is considerable electrophysiological and functional cardiac Kv channel diversity (42, 373, 375). Based primarily on differences in time- and voltage-dependent properties and pharmacological sensitivities (42, 373, 375), two broad classes of repolarizing cardiac Kv currents have been distinguished (42): transient outward K⁺ currents (I_to) and delayed, outwardly rectifying K⁺ currents (I_K) (Table 1). The transient currents (I_to) activate and inactivate rapidly on membrane depolarizations to potentials positive to approximately –30 mV and underlie the early phase (phase 1) of repolarization in ventricular and atrial cells (Fig. 2). Cardiac delayed rectifiers (I_K) activate at similar membrane potentials and with variable kinetics, and these currents determine the latter phase (phase 3) of repolarization back to the diastolic potential (Fig. 2). Multiple types of myocardial I_to and I_K channels with distinct time- and voltage-dependent properties (Table 1), however, have been identified, and differences in the densities and the biophysical properties of these channels contribute to variations in the waveforms of action potentials recorded in different cardiac cell types (Fig. 1), as well as in different species (24, 374, 375, 436). The detailed pharmacological, time- and voltage-dependent properties of each of the various repolarizing Kv currents characterized in different cardiac cell types and species are quite similar, thereby allowing Kv channels to be classified based on these biophysical properties (Table 1). The observed similarities in Kv channel properties also suggest that the molecular correlates of the underlying Kv channels are also similar (42, 373), and considerable experimental evidence has now been provided to support this hypothesis (see sect. VI).

A. Transient Outward Kv Currents

Although cardiac transient outward currents were first described in (sheep) Purkinje fibers and thought to reflect Cl^– conductances (143, 175), subsequent work demonstrated the presence of two transient outward currents with distinct properties and referred to as I_to1 and I_to2 (112). Pharmacological studies revealed that I_to1 is blocked by 4-aminopyridine (4-AP) and unaffected by changes in extracellular Ca²⁺, whereas I_to2 is not blocked by 4-AP and is Ca²⁺ dependent (257, 256). In further studies, it was shown that the Ca²⁺-dependent I_to2 in Purkinje fibers and ventricular cells is a Cl^– (not a K⁺) current (593, 594). In contrast, the Ca²⁺-independent component, I_to1, was shown to be K⁺ selective (594), and transient outward K⁺ currents, referred to variably by different laboratories as I_to, I_to1, or I_t (42, 83, 436), have now been described in many cardiac cell types and in most species. Comparison of the detailed biophysical properties of the transient outward K⁺ currents described in various cell types/species, however, suggested there might actually be two types of transient outward K⁺ currents (42), and electrophysiological and pharmacological studies have now provided considerable support for this hypothesis. In adult mouse ventricular myocytes, for example, two transient K⁺ currents, termed I_to,fast (I_to,f) and I_to,slow (I_to,s), have been distinguished (562). On membrane depolarization, mouse ventricular I_to,f channels activate and inactivate rapidly, and on membrane repolarization, these (I_to,f) channels recover rapidly from steady-state inactivation (562). In the adult mouse, I_to,f channels contribute importantly to the rapid repolarization of action potentials (194, 562) that is likely necessary to maintain the very high resting heart rates (700 beats/min) in these animals. In humans and other larger mammals, I_to,f underlies the early phase (phase 1) of repolarization in ventricular and atrial cells (Fig. 2) and likely also contributes to determining the plateau (phase 2).

Similar to I_to,f, mouse ventricular I_to,s channels activate and inactivate rapidly (562). In contrast to I_to,f, however, I_to,s channels recover very slowly (time constants of seconds) from (steady-state) inactivation and are functionally distinct from I_to,f channels (196, 562). In addition, I_to,f is readily distinguished from other Kv currents, including I_to,s,(562), using the spider K⁺ channel toxins Heteropoda toxin-2 or -3 (449). The distinct properties of I_to,f and I_to,s suggested that these currents reflect the functioning of two molecularly distinct Kv channels, and considerable evidence has now been provided to support this hypothesis (see sect. VIC). Detailed comparisons of the properties of the transient outward K⁺ currents expressed in other species, whether termed I_to, I_to1, or I_t, suggest that, in each case, these currents could also be classified as I_to,s or I_to,f, based on the kinetics of current inactivation and recovery from steady-state inactivation, as well as by the differential sensitivities of the channels to the Heteropoda toxins. Although the properties of the transient K⁺ currents in different cell types and species are similar and are amenable to classification as either I_to,f or I_to,s (Table 1), there are differences in the detailed biophysical properties of the (I_to,f and I_to,s) currents in different cells/species (15). These observations suggest that there may well be subtle, albeit potentially important, molecular heterogeneity among I_to,f and I_to,s channels in different cell types and/or in different species (see sect. VIC).

Although originally identified in Purkinje fibers, I_to,f is a prominent repolarizing current in atrial and ventricular myocytes in most species (26, 58, 65, 69, 75, 79, 160, 178, 264, 294, 297, 500, 530, 545, 546, 578), including humans. Nevertheless, there are exceptions. In guinea pig ventricular cells, for example, I_to,f has not been detected except when extracellular Ca²⁺ is removed (224). In addition, I_to,f is not detected in rabbit atrial or ventricular cells (156, 160, 183, 530). Nevertheless, there are transient Kv currents in rabbit myocytes (typically referred to as I_t), which inactivate slowly and recover from (steady-state) inactivation very slowly (160, 530). The properties of these currents, therefore, more closely resemble mouse ventricular I_to,s than I_to,f (562). Similar to the mouse, however, two distinct transient outward Kv currents have been described in the ventricles of other mammals (77, 178, 294, 500), including humans (225, 264, 545, 546), and the properties of these currents are quite similar to those of mouse ventricular I_to,f and I_to,s (79, 196, 562), permitting their classification as such (Table 1). In ferret, the rates of inactivation and recovery (from steady-state inactivation) of the transient Kv currents in myocytes isolated from the left ventricular (LV) endocardium are significantly slower than the currents in cells from the epicardial surface of the LV, suggesting the presence of two distinct transient Kv currents that are differentially expressed (77). Examination of the reported biophysical properties (77) suggests that the endocardial and epicardial LV currents can be classified as I_to,s and I_to,f, respectively (Table 1). The distinct transient Kv currents in the epicardial, midmyocardial, and endocardial layers of canine (294, 500) and human (264, 545, 546) ventricles can also be appropriately referred to as I_to,f or I_to,s (Table 1).

Transient Kv currents that can be classified as I_to,f (Table 1) have also been shown to be expressed in (rabbit) SAN cells, although, similar to Nav currents, I_to,f densities vary markedly among (SAN) cells (213, 283). I_to,f densities are higher, for example, in the larger cells isolated from the periphery, compared with the smaller cells in the center, of the SAN (213, 283). In addition, when expressed, I_to,f appears to play a role in shaping action potential waveforms and in regulating automaticity in SAN cells (72, 213, 283). Cells isolated from the (rabbit) AVN also express I_to,f (349, 361, 371), and detailed kinetic analysis of the currents reveals the presence of two components with distinct rates of inactivation and recovery (349). It is unclear whether these findings reflect differences in the kinetic properties of a single type of I_to,f channel or if two distinct types of I_to channels are expressed in (rabbit) AVN cells. Similar to the (rabbit) SAN, there is considerable heterogeneity in I_to densities among (rabbit) AVN cells (349). In contrast to SAN cells (213, 283), however, the differences in I_to,f densities are not correlated with cell size in AVN cells (349). Interestingly, and similar to findings in guinea pig atrial and ventricular cells, I_to,f is not detected in guinea pig AVN cells (579). It is presently unclear, however, whether currents with properties similar to ventricular I_to,s are expressed in conducting tissues in guinea pig heart. Owing to the marked differences in inactivation and recovery kinetics of I_to,f and I_to,s channels, however, the differential expression of these two channel types would be expected to have profound functional effects on the regulation of rhythmicity in the normal heart, effects that will be augmented in the diseased myocardium.

B. Delayed Rectifier Kv Currents

Myocardial delayed rectifier Kv currents, I_K, also first described in (sheep) Purkinje fibers (379), have been characterized in atrial and ventricular myocytes, as well as in pacemaker cells, isolated from a variety of different species, and, in most cases, multiple components of I_K are coexpressed (Table 1). In guinea pig ventricular and atrial myocytes, for example, two prominent components of I_K, I_Kr (I_K,rapid) and I_Ks (I_K,slow), were first distinguished, based on marked differences in time- and voltage-dependent properties (216, 445, 446). Both I_Kr and I_Ks are also coexpressed in guinea pig AVN cells (579). Although I_Kr activates rapidly, inactivates very rapidly, and displays marked inward rectification, no inward rectification is evident for the slowly activating I_Ks (445, 446). These channels can also be distinguished at the microscopic level, as well as by their unique pharmacological profiles (37, 524). Similar to guinea pig, I_Kr and I_Ks are also reportedly coexpressed in human atrial and ventricular myocytes (290, 514, 532, 533), as well as in canine (296, 297, 513, 522, 578) and rabbit (440, 518) ventricles and in canine Purkinje fibers (513) and, in each case, are prominent repolarizing currents. In adult rodent ventricles, in contrast, I_Kr and I_Ks densities are very low (104) or the currents are undetectable (562).

The unique time- and voltage-dependent properties of I_Kr and I_Ks suggest that these currents play prominent roles in action potential repolarization, particularly in ventricular myocytes and Purkinje fibers. Nevertheless, in some cardiac cells, only I_Kr or I_Ks appears to be expressed. In isolated human (225), feline (171), and rat (404) ventricular myocytes and in rat atrial (404), mouse SAN (108), rabbit AVN and SAN (218, 233, 467) cells, for example, only I_Kr is detected. It has also been reported, however, that both I_Kr and I_Ks are coexpressed in rabbit SAN cells (284). In this study, the measured densities of I_Kr and I_Ks (in rabbit SAN cells) were quite variable, although, in general, I_Kr and I_Ks densities are highest in the larger cells found at the periphery of the node (284). It may be that the densities of I_Kr and/or I_Ks in some cells are too low to be resolved reliably or, alternatively, that the properties of I_Ks and I_Kr in each of these cell types are distinct from guinea pig ventricular and atrial I_Ks and I_Kr. The apparent absence of I_Kr and I_Ks in some cells, as well as the observation that I_Kr and I_Ks expression is heterogeneous and variable, might also reflect the fact that functional cardiac Kv channel expression is labile and might well be affected by the isolation methods, which typically involve the use of enzymes (578). Detailed studies focused on current characterizations in intact preparations, as well as on examining the effects of specific enzymes and cell isolation methods on Kv current densities and properties, will be needed to explore these various possibilities further.

Although I_Kr and I_Ks are not prominent repolarizing Kv currents in rodent atria or ventricles, there are other components of delayed rectification with time- and voltage-dependent properties distinct from I_Ksand I_Kr(Table 1) in myocytes from these (and other) species. In rat ventricular myocytes, for example, there are multiple delayed rectifier Kv currents that are coexpressed, and these are referred to as I_K, I_Klate, and I_ss (26, 210, 561). In adult mouse ventricular myocytes, three distinct delayed rectifier Kv currents have also been separated and characterized (168, 196, 263, 291, 303, 560, 562, 586, 587), and these are referred to as I_K,slow1, I_K,slow2, and I_ss (Table 1). Multiple components of delayed rectification have also been described in rodent atrial myocytes (68, 69, 74, 75, 497). In both rat and mouse, it has been demonstrated that all the various delayed rectifier Kv current components contribute, together with I_to,f channels, to (ventricular and atrial) action potential repolarization (291, 303, 560, 586, 587). It is interesting to note that a steady-state, noninactivating K⁺ current, which resembles I_ss in rodent atria and ventricles, has also been described in human atrial myocytes (58).

In rat (74, 75), canine (577), and human (531–533) atrial myocytes, a novel, very rapidly activating, and largely noninactivating, outward Kv current, now typically referred to as I_Kultrarapid or I_Kur (479), has been described (Table 1). In most species, I_Kur is not detected in ventricular cells, and it seems likely that the expression and the properties of I_Kur, together with I_to,f, contribute to determining the more rapid repolarization evident in atrial, compared with ventricular, myocytes (Fig. 2). Importantly, as in most other species, I_Kur is not expressed in human ventricular myocytes or in Purkinje fibers, suggesting that I_Kur channels might represent a therapeutic target for the treatment of atrial arrhythmias without complicating effects on impulse propagation, ventricular functioning, or cardiac output (444). The potential of this pharmacological strategy, however, will have to be determined by the atrial specificity/selectivity of the drugs that are developed. In contrast to rat, canine, and human, Kv currents have been described in guinea pig (576) and mouse (168, 291, 587) ventricular myocytes that have biophysical properties very similar to human (rat or canine) atrial I_Kur. Indeed, the properties of the rapidly activating, I_Kur-like, current in guinea pig ventricular myocytes, referred to as I_Kp (576), and the micromolar 4-AP-sensitive component of mouse ventricular I_K,slow, referred to as I_K,slow1 (69, 291, 302, 587), are indistinguishable from human (canine and rat) atrial I_Kur. These currents should, therefore, probably be renamed I_Kur (Table 1) to reflect the similarities in properties, as well as molecular identities of the channels underlying I_Kp and I_K,slow1 and I_Kur (see sect. VID).

C. Regional Differences in Kv Current Expression and Properties

Although the properties of I_to,f in different cardiac cells are similar (Table 1), there are marked regional differences in current densities. In humans (160, 367, 527, 542, 543) and in rats (26, 74, 75), for example, I_to,f densities are significantly higher in atrial, compared with ventricular, myocytes. Similarly, in the rabbit, I_to,s densities are higher in atrial myocytes and Purkinje fibers than in ventricular cells (75, 514). In the mouse, however, I_to,fdensity is significantly higher in ventricular (79, 562), than in atrial (69), myocytes. The density of I_to,f is also quite variable in sheep Purkinje fibers (520) and in different regions of the ventricles in canine (294, 297, 522), cat (178), ferret (77), human (367, 545, 546), mouse (79, 196, 562), and rat (107, 545) hearts. In canine (522) and mouse (79, 196, 562) heart, for example, I_to,f density is higher in the right ventricle (RV), compared with the left ventricle (LV), and I_to,f densities are lower in the base of the LV than in the LV apex (79). In addition, in canine and in human heart, I_to,f density varies throughout the thickness of the ventricular walls, being severalfold higher in the epicardial and midmyocardial, than in the endocardial, layers (297, 546). In large mammals, the regional and cellular heterogeneities in I_to,f densities are directly reflected in the differences in action potential waveforms in Purkinje, ventricular, and atrial cells (75, 107, 366, 520, 551). Within the ventricles, for example, the differences in I_to,f densities are revealed by the presence and the appearance and depth of the "notch" in the initial phase (phase 1) of action potential repolarization (24, 364, 374; see Fig. 2).

There are also marked regional differences in the expression/distribution of I_to,s in adult rat, mouse, human, and canine ventricles (77, 79, 196, 366, 367, 551, 562). In mouse RV and LV, for example, I_to,s is undetectable, whereas cells in the interventricular septum express only I_to,s or both I_to,f and I_to,s (79, 196, 562). Even when expressed, however, I_to,f density is significantly lower in septum, compared with ventricular (or atrial), cells (79, 562). The densities of the delayed rectifier Kv currents, I_K,slow1, I_K,slow2, and I_ss, in contrast, are similar throughout adult mouse ventricles (79, 196, 562). The main determinant of action potential heterogeneity in the mouse, therefore, appears to be the differential expression of I_to,f (79, 196).

In larger mammals, including humans, the differential expression of I_to,f is also a primary determinant of action potential heterogeneity (24, 374). In human heart, however, it is clear that differences in the expression levels of the various delayed rectifier Kv currents, as well as the persistent component of the Nav current (see sect. IIA), also play important roles in regulating action potential heterogeneity (24, 374). In canine heart, for example, I_Ks density is higher in cells in the RV, compared with the LV, whereas I_Kr densities are similar in both chambers (24, 522). I_Ks density is also higher in canine LV epicardial and endocardial cells than in M cells (24, 296). In guinea pig heart, I_Kr and I_Ks densities are approximately twofold higher in atrial, than in ventricular, myocytes (37, 216, 442, 524). There are also regional differences in functional I_Kr and I_Ks expression within the ventricles (80, 316). In cells isolated from the (guinea pig) LV free wall, for example, I_Kr density is higher in subepicardial, than in either midmyocardial or subendocardial, myocytes (316). At the base of the LV, however, the densities of both I_Kr and I_Ks are significantly lower in endocardial, than in either epicardial or midmyocardial, cells (80). These differences clearly contribute to the marked differences in action potential waveforms and frequency-dependent properties in cells through the thickness of the ventricular wall (24). In addition to having a major impact on action potential repolarization, it is now very clear that differences in functional I_Kr and I_Ks densities are also expected to influence the maintenance of normal cardiac rhythms and the susceptibility to rhythm disturbances (24).

	IV. OTHER MYOCARDIAL POTASSIUM CURRENTS CONTRIBUTING TO REPOLARIZATION

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In addition to the depolarization-activated Kv currents, non-voltage-gated inwardly rectifying K⁺(Kir) currents, through I_K1 channels, also contribute to myocardial action potential repolarization, particularly in ventricular cells (232, 306, 377, 380). There are also other types of Kir channels that are expressed and are important in the normal functioning of the heart, although these do not seem to play important roles in action potential repolarization under normal physiological conditions (306, 377). One example of a functionally important class of myocardial Kir channels is the I_KATP channels, which are inhibited by intracellular ATP, activated by nucleotide diphosphates, and thought to provide a link between cellular metabolism and membrane potential (232, 380). In the ventricular myocardium, the opening of I_KATP channels is thought to be important under conditions of metabolic stress, as occurs during ischemia or hypoxia, and to result in shortening action potential durations and minimizing K⁺ efflux (165, 232). The opening of I_KATP channels has also been suggested to contribute to the cardioprotection resulting from ischemic preconditioning (141, 188). Although I_KATP channels appear to be distributed uniformly in the RV and LV and through the thickness of the ventricular wall, these channels are expressed at much higher density than other sarcolemmal K⁺ channels, suggesting that action potentials could be shortened markedly when only very small numbers of I_KATP channels are activated (463).

Another important cardiac Kir channel type is the I_K(ACh) channels, which are gated through a G protein-coupled mechanism mediated by muscarinic acetylcholine receptor activation (275, 564). Physiologically, I_K(ACh) channels are activated by the binding of G protein -subunits in response to the acetylcholine released on vagal stimulation (414). Although I_K(ACh) channels are expressed in AVN, SAN, atrial, and Purkinje cells, and are activated by acetylcholine released on vagal stimulation, these channels are not thought to contribute appreciably to action potential repolarization under normal physiological conditions. Consistent with this hypothesis, targeted deletion of one of the Kir subunits (Table 6) encoding I_K(ACh) channels, Kir3.4, does not measurably affect resting heart rates (552). Interestingly, however, atrial fibrillation is not evident in Kir3.4 null mice exposed to the acetylcholine receptor agonist carbachol, suggesting that activation of I_K(ACh) channels is involved in the cholinergic induction of atrial fibrillation (269).

View this table: [in this window] [in a new window]   TABLE 6. Diversity of inwardly rectifying K+ (Kir) and two-pore domain K+ (K2P) channel -subunits

The expression of I_K1 is clearly reflected in the negative slope region (between approximately –50 and –10 mV) of the (total steady-state) myocyte conductance-voltage relation, which is prominent in ventricular myocytes, but is small or undetectable in atrial cells (183). The fact that the strongly inwardly rectifying I_K1channels conduct at negative membrane potentials suggests that these channels will play a role in establishing the resting membrane potentials of Purkinje fibers, as well as of atrial and ventricular myocytes. Direct experimental support for this hypothesis was provided with the demonstration that ventricular membrane potentials are depolarized in the presence of Ba²⁺ (377), which blocks I_K1 channels. In addition, action potentials are prolonged, and phase 3 repolarization is slowed in the presence of extracellular Ba²⁺(306), suggesting that I_K1 channels also contribute to repolarization, particularly in the ventricular myocardium. The voltage-dependent properties of I_K1 channels (306, 377), however, are such that the conductance is low at potentials positive to approximately –40 mV. Nevertheless, because the driving force on K⁺ is markedly increased at depolarized potentials, these channels should contribute outward K⁺current during the phase 2 plateau and during phase 3 repolarization (Fig. 2). In contrast to atrial, ventricular, and Purkinje cells, I_K1 density is low or undetectable in SAN and AVN cells (244, 381, 468). These observations, as well as the fact that pacemaker currents are expressed and functional in SAN and AVN cells, likely explain the findings that resting membrane potentials in these (SAN/AVN) cells are depolarized (significantly) and that the rising phases of the action potentials in these cells are less steep, relative to resting membrane potentials/action potentials in atrial and ventricular cells (Fig. 1).

Similar to the Kv channels, I_K1 densities and the detailed biophysical properties of the currents do vary in different myocardial cell types. In human heart, for example, I_K1 density is more than twofold higher in ventricular, than in atrial, cells (514). In guinea pig, the properties of the atrial and ventricular I_K1 currents are also distinct in that ventricular I_K1 inactivates during maintained depolarizations, whereas atrial I_K1 does not (133, 223). In addition, changes in extracellular K⁺ modulate the magnitude of ventricular I_K1, but have little effect on atrial I_K1 (223). At the microscopic level, (guinea pig) atrial and ventricular I_K1 channels are also distinct. Mean channel open times of ventricular I_K1 channels, for example, are approximately five times longer than those of atrial I_K1 channels, whereas the single atrial and ventricular I_K1 channel conductances are indistinguishable (223). Taken together, these observations suggested the interesting possibility that distinct molecular entities underlie ventricular and atrial I_K1 channels, and experimental support for this hypothesis has now been provided (133).

	V. MOLECULAR COMPONENTS OF MYOCARDIAL NAV AND CAV CHANNELS

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A. Nav Channel Pore-Forming -Subunits

Voltage-gated Na⁺ (Nav) channel pore-forming () subunits (Fig. 3A) belong to the "S4" superfamily of voltage-gated ion channel genes (93, 172, 174, 573). Nav -subunits have four homologous domains (I to IV), each of which contains six transmembrane-spanning regions (S1-S6), and these four domains come together to form the Na⁺-selective pore. Structure-function studies have revealed many of the important features of voltage-dependent Nav channel gating (91, 93). The cytoplasmic linker between domains III and IV, for example, has been shown to play a pivotal role in voltage-dependent Nav channel inactivation (392), and a critical isoleucine, phenylalanine, methionine (IFM) motif within this linker (91, 444) has been identified as an important molecular component of the inactivation gate (516, 517, 544). Voltage-dependent inactivation of Nav channels is attributed to the rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV that occurs within milliseconds of membrane depolarization (483). Consistent with the functional electrophysiological data, solution NMR analysis of this cytoplasmic linker peptide revealed a rigid helical structure positioned to block the pore (427).

Although there are a number of homologous Nav -subunits (Table 2), Nav1.5 (SCN5A) is the prominent Nav -subunit expressed in the mammalian myocardium, and this subunit encodes the rapidly activating and inactivating, tetrodotoxin (TTX)-insensitive Nav channels that underlie rapid (phase 0) depolarization in atrial and ventricular myocytes and in Purkinje fibers (Fig. 1). Nevertheless, several studies have demonstrated that mRNAs encoding other Nav -subunits, notably Nav1.1, Nav1.3 (120, 426, 471), and Nav1.4 (590), which are typically considered the Nav -subunits encoding brain and skeletal muscle Nav channels, respectively, are also expressed in the myocardium. In contrast to the Nav channels formed by Nav1.5, however, Nav1.1-, Nav1.3- and Nav1.4-encoded Nav channels are blocked by nanomolar concentrations of TTX (120, 426, 471, 590). In addition, although cardiac Nav currents are generally considered relatively TTX insensitive (174, 573), application of nanomolar concentrations of TTX has been reported to shorten canine Purkinje fiber action potential durations (113). These findings suggest a possible role for TTX-sensitive Nav channels in the generation of the persistent component of cardiac Nav currents, at least in canine Purkinje fibers. Nevertheless, there have been very few reports documenting the presence of TTX-sensitive inward Nav current components in cardiac cells, raising some concern about the functional significance of the expression data, in spite of the fact that the (message) expression levels of Nav1.1, Nav1.3, and Nav1.4 in the myocardium appear to be quite high (120, 426, 471, 590).

It has also been reported that there are several Nav1 -subunit proteins in addition to Nav1.5 in adult (mouse) myocardium (314). These include Nav1.1, Nav1.3, and Nav1.6 (314). The immunolocalization data also suggest that the Nav1.1, Nav1.3, and Nav1.6 -subunits are localized in the t tubules in adult mouse ventricles (314), whereas Nav1.5 appears to be localized preferentially to intercalated disks in mouse, as well as in rabbit and rat, hearts (270, 315, 400). The subcellular localization of Nav1.5-encoded myocardial Nav channels at the intercalated disks has been interpreted as suggesting that these (Nav) channels play a major role in regulating conduction (270). Although the functional role(s) of t-tubular Nav channels in cardiac functioning has not been established, voltage-clamp studies have clearly demonstrated that TTX-sensitive Nav currents can be measured in whole cell recordings from adult mouse ventricular myocytes treated with -scorpion toxin, which shifts the voltage dependence of activation of brain Nav channels, but does not affect cardiac (i.e., SCN5A-encoded) Nav channels (314). These observations have been interpreted as suggesting a distinct role for the neuronal Nav channels localized to the t tubules, i.e., linking depolarization of the sarcolemmal membrane with the t tubules, thereby coupling depolarization with excitation-contraction coupling (314). This hypothesis has important functional implications and certainly warrants further direct experimental testing.

Immunohistochemical studies have also provided evidence suggesting that Nav1.1 and Nav1.3, but not Nav1.5, are expressed in rat and mouse SAN (314). These findings suggest a substantive molecular difference between the SAN and the remainder of the myocardium in terms of Nav channel expression. Given the primary role of the SAN in regulating heart rate, it would seem certain that modulating the (TTX-sensitive) Na⁺ current in the SAN should impact heart rate. Nevertheless, exposure to TTX reportedly has no effect on heart rate in the mouse (314). In mice in which one copy of the SCN5A gene has been disrupted, however, conduction defects, as well as ventricular dysfunction, are evident (389), suggesting that SCN5A encodes most, if not all, of the cardiac Nav current. It seems reasonable to suggest, therefore, that additional studies focused on exploring the functioning of Nav1.1-, Nav1.3- and Nav1.6-encoded channels in the heart are warranted.

During the plateau phase of the action potential in human ventricular myocytes, 99% of the Nav channels are in an inactivated, nonconducting state with the inactivation gate occluding the inner mouth of the conducting pore through specific interactions with sites on either the S6 segment (345) or the S4-S5 loop (346) of domain IV. Mutations in the linker between domains III and IV in SCN5A, linked to the LQT3 syndrome (Fig. 3A), disrupt Nav channel inactivation (52, 346). These "gain of function" mutations lead to an increase in the amplitude of the sustained component of the Na⁺ current