Cellular Structure of the Heart

The myocardium is composed of cardiac myocytes, which are highly differentiated and specialized cells responsible for the conduction of the electrical impulse and the heart’s contractile behavior, and nonmyocytes, which serve a number of functions. Myocytes occupy two thirds of the structural space of the heart; however, they represent only one third of all cells. Nonmyocytes include fibroblasts responsible for the turnover of extracellular matrix that predominantly consists of fibrillar collagen types I and III. The collagen scaffolding provides for myocyte alignment and coordinated transmission of contractile force to the ventricular chamber. Other nonmyocyte cells include the endothelial and smooth muscle cells of the intramural vasculature, neuronal elements (such as ganglia) and, under some conditions, inflammatory cells. The gross anatomic features of the heart, the extracellular matrix, and the intramural vasculature create both macroanatomic and microanatomic barriers that are central to both the normal electrophysiology of the heart and clinically important arrhythmias.

Cardiac myocytes are a family of structurally distinct cells, with a design commensurate with their function. Pacemaking cells such as those in the sinoatrial (SA) and atrioventricular (AV) nodes underlie the spontaneous electrical activity of the heart and contain relatively few contractile elements. In contrast, muscle cells are packed with actin and myosin filaments, which serve the main function of the heart—propulsion of blood through the vasculature. Contractile myocytes are rod-shaped cells of approximately 100 × 20 µm. The myocyte is enveloped by the cell membrane, a lipid bilayer 80 Å to 100 Å in thickness. This insulating bilayer permits little to no transport of ions and maintains a separation of charge established by active transporters that reside in the cell membrane. Ion channels are transmembrane proteins that serve as a conductive pathway between the inside and outside of the cell, allowing the flow of ions and, thus, charge (i.e., current). Also, no current flow exists between myocytes. However, unlike skeletal muscle, cardiac tissue is not a true syncytium; rather, cells are connected to each other by low-resistance communications called gap junctions that contain intercellular ion channels.

Membrane Potential and Conduction

Ion concentration and charge gradients across the cell membrane are responsible for the membrane potential of the cardiac myocyte. Transmembrane ionic and electrical gradients are maintained by a series of energy-requiring ion pumps and exchangers that perform the following functions: (1) Concentrate K⁺ inside the cell, (2) keep the intracellular sodium (Na⁺) low (<10 mM), and (3) tightly regulate intracellular calcium (Ca²⁺) concentrations (100–200 nmol/L). The pump responsible for establishing and maintaining most of the monovalent cation gradients is the Na⁺-K⁺ ATPase (sodium-potassium adenotriphosphatase), although other pumps and exchangers that transport Na⁺, Ca²⁺, and hydrogen (H⁺) play significant roles in the genesis of ionic concentration gradients and the membrane potential. Ion channels are passive but selective conduits for the flow of ions along electrical and chemical gradients established by active ion transport systems. The physicochemical basis of the membrane potential depends (as described below) on the Nernst potential (E_x):

(1)

where R is the gas constant, T is temperature, F is Faraday’s constant, and z is the valence of the ionic species. If the resting cardiac myocyte is assumed to have an intracellular [K⁺] of ~150 mmol/L and an extracellular [K⁺] of 4 mmol/L, then the Nernst potential for K⁺ (E_K) is roughly –90 mV. The Nernst potential represents the voltage at which the osmotic tendency for K⁺ to flow across the membrane is exactly balanced by the electrical tendency to flow in the opposite direction, which results in a net zero ionic flux and thus no net current flow. Accordingly, the K⁺ Nernst potential is sometimes referred to as the zero current potential, at which no K⁺ current would flow through open K channels. By contrast, the Nernst potential for Na⁺ in the resting cell is approximately +60 mV, indicating that if a pathway for Na⁺ to enter the cell were present, Na⁺ would enter the cell to move the membrane voltage (mV) toward the Nernst potential for Na⁺. Indeed, this is precisely what happens when Na channels are open and initiate phase 0 of the action potential. The Nernst potential for potassium is, in fact, very close to the resting membrane potential of the ventricular myocyte, indicating that the resting heart cell membrane is highly permeable to K⁺. Actual resting potentials are less negative than E_K due to small conductances of other ionic species with less negative Nernst potentials. In the course of the cardiac cycle, the cell membrane becomes permeable to different ionic species, and these changes in permeability determine time-dependent changes in the membrane potential, with each ion striving to move the membrane voltage to its Nernst potential and inscribing regionally specific action potentials (Figure 3-1).

Figure 3-1 Schematic of action potentials in different regions of the heart. The permeability of the cell membrane determines membrane voltage. At rest, cells in all regions of the heart are more permeable to potassium (K⁺) than to any other cation, hence the negative membrane potential, near the Nernst potential for K⁺. The characteristic shapes of action potentials are determined by the ionic currents that are active in each cell type during the cardiac cycle.

Passive Membrane Properties and Cable Theory

The cardiac cell membrane can be modeled as a circuit comprising variable resistors (ion channels) in parallel with a capacitor (lipid bilayer), an RC circuit (Figure 3-2). The flow of current across the membrane will alter the charge on the capacitor (and therefore the membrane potential) and change the membrane resistance. The flow of current occurs not only across but obviously along the inside and outside of the cell membrane from cell to cell as well, that is, current propagates.

Figure 3-2 Electrical and biologic representations of the cardiac cell membrane. The cardiac (or any excitable) membrane can be modeled as an RC circuit with variable resistors (ion channels) in parallel with a capacitor (cell membrane). The transmembrane voltage is established by a series of energy-requiring pumps that maintain ionic gradients across the membrane.

Cable theory, originally developed to understand current flow in trans-oceanic telegraphic cables, can be used to model passive current flow and propagation in a cardiac muscle fiber. In their simplest formulation, the cable equations define the distribution of voltage along a continuous, uniform cable of infinite length stimulated by a point source. The predictions of cable theory are that (1) a change in voltage exhibits a characteristic decay along the cable defined by the space constant (distance over which voltage decays to 1/e of the value at the site of injection) (Figure 3-3, A); and (2) there is an inverse relationship between resistance to current flow (both transmembrane and intercellular) and the cable diameter (Figure 3-3, B). That is, the space constant is directly proportional to the cable diameter, and thus, greater lengths of the cable are influenced by the same current injection into a thick, rather than a thin, cable.

Figure 3-3 Spread of current in an idealized cable. A, Injection of current into the cable at site I produces a transmembrane voltage change of smaller amplitude, and slower kinetics are recorded at more distal sites in the cable. In an excitable tissue such as the heart, a stimulus of sufficient amplitude elicits a regenerative response (i.e., an action potential) and can propagate along the length of the preparation. Transmission of the action potential is associated with current flow across and along the membrane. B, The characteristics of the cable determine the distance over which the voltage difference induced by the current injection decays (space constant). In a cable of larger diameter, the voltage difference falls over a larger distance; that is, the space constant (the distance over which the voltage difference falls to 1/e of the value at the site of injection) is proportional to the radius of the cable.

Substantial anatomic and biophysical limitations exist when applying the cable theory description of conduction to cardiac muscle. Anatomically, the shape of the heart is complex, and at any level of integration above a single muscle fiber, it does not resemble a cable. Conduction through the myocardium is not continuous; instead, myocardial cells are connected by gap junction channels that create a non-uniformity of intercellular resistance. Macroscopic discontinuities such as fibrous tissue and blood vessels also significantly perturb the cable view of conduction in the myocardium. Finally, the cardiac cell membrane does not just comprise RC circuits; instead, when stimulated to the threshold, it will generate action potentials (see Figure 3-3, A). Despite the limitations of cable theory, it serves as the foundation for several important concepts in impulse propagation and was used to demonstrate the electrical nature of conduction in the heart.¹

Depolarization of cardiac muscle results in the generation of an action potential at the site of excitation and, in doing so, sets up a voltage gradient between the excited cells and their nearest neighbors. The current generated by the action potential serves as an excitatory current for neighboring cells (source), which at their resting membrane potential (sink) are activated by the source. Impulse propagation depends on the balance between the magnitude of the currents in the source and the sink and the resistance along the fiber. Failure of conduction may result from alterations in the source current; examples include reduction of the source by drug blockade of Na (atrial or ventricular muscle) or Ca⁺ channels (nodal cells) or from changes in the characteristics of the sink, such as ischemia and activation of ATP-dependent potassium channels (I_K-ATP). In the latter case, propagation fails because the tissue with greatly increased repolarizing K current, due to activated I_K-ATP, acts as an infinite sink and cannot be sufficiently depolarized to reach the threshold for the generation of an action potential. Myocardial ischemia will produce changes in the intracellular environment such as decreased pH and increased intracellular Ca²⁺ that will serve to reduce gap junctional conductance and functionally uncouple myocardial cells, altering the relationship between the source and the sink and hindering impulse propagation.

The safety factor for conduction is the magnitude of the current provided by the source that is in excess of that required to activate the sink. The main factors influencing source current are the rate of rise of the upstroke and the amplitude of the action potential, which are the metrics that reflect the magnitude of inward currents. The factors that influence the current requirements of the sink are the membrane resistance and the difference between the resting and threshold potentials. One major reason for the mismatch between the source and the sink is an abrupt anatomic change, such as that which occurs at the Purkinje fiber–ventricular muscle junction. Orthodromic conduction over the Purkinje system results in the activation of a broad band of ventricular muscle by narrow strands of Purkinje fibers. Such an abrupt transition from an anatomically narrow source to a massive sink makes propagation tenuous such that small changes in the characteristics of the source or the sink are likely to produce failure of conduction. Antidromic conduction from ventricular muscle to Purkinje fibers produces just the opposite source-sink relationship and thus a higher safety factor for conduction.

While this discussion implicitly treats the activation wave in one dimension, the behavior of propagating waves in the heart is more complex. The complexity can be appreciated if one considers propagation in two dimensions. In this circumstance, the shape of the wavefront is a major determinant of the efficiency of propagation. A convex wavefront, as might be observed after point stimulation, creates a large sink around a smaller activating source. This mismatch reduces conduction velocity and the safety factor for propagation. Conversely, a concave activation front produces a source-sink mismatch that favors the source; this results in a high safety factor and more rapid impulse transmission. Thus, not only do source-sink characteristics influence propagation, they also influence the curvature of the wave front (see Figure 3-4).

Figure 3-4 Relationship of current sources and sinks. A, Compared with the steady-state conduction velocity for the planar wavefront in a homogeneous medium (θ₀), the conduction velocity of a convex wavefront is slower, and a concave wavefront is faster with lower and higher safety margins for conduction, respectively. In situations of structural inhomogeneity, as when a Purkinje fiber activates ventricular muscle (B), the conduction velocity is slower, with a lower safety margin at the structural discontinuity. C, In the case of a larger mass of tissue activating a smaller mass, the conduction velocity is fast, with a high safety margin.

Directionally different conduction velocity is a characteristic feature of cardiac muscle known as anisotropic conduction. Anisotropic conduction has its basis in the structure of the myocyte and cardiac tissue; myocytes are rod shaped and are organized in bundles that are oriented along the long axis of the cell. Transmurally, the axes of these bundles undergo significant changes in orientation through the ventricular wall (~120 degrees maximal deviation).² Communication among myocytes occurs via gap junction channels that are non-uniformly distributed over the surface of the heart cell, with larger numbers of channel poised to propagate the impulse longitudinally, rather than transversely, to the long axis of the muscle fiber.³ The implications of anisotropy for conduction in the longitudinal and transverse direction under pathologic conditions are controversial. In the context of uniform depression of conduction, as might exist with antiarrhythmic drug treatment, propagation in the transverse direction is preserved compared with conduction in the longitudinal direction. However, when cellular uncoupling occurs, such as in ischemia, longitudinal propagation may exhibit a higher safety factor than does transverse conduction.

Major Breakthroughs: Voltage Clamp, Molecular Cloning

A stimulus of sufficient magnitude applied to a myocyte (or any excitable cell) elicits a typical change in membrane potential known as the action potential. The ionic current basis of the action potential was confirmed and quantitatively studied using the voltage clamp developed in the middle of the twentieth century.⁴ Voltage clamping is a technique whereby the experimenter controls the transmembrane voltage and measures the current at that defined voltage. Much of what we know about ionic currents in myocytes comes from voltage clamp experiments and a more recently developed type of voltage clamp called the patch clamp.⁵ A variant of the patch clamp technique permits the measurement of ionic currents through single ion channels.

Typically, in a voltage clamp experiment, the membrane voltage (V) is held near the resting membrane potential, (approximately −80 mV for ventricular myocytes) and then stepped to more positive voltages. This voltage step induces two components of the membrane current (I_M) flow; at the instant of the voltage change, ions (I_C) flow to charge the membrane capacitance (C_M), after which the current reflects the movement of ions through the ion channels (I_i).

(2)

Capacitive current is generally small and transient and can usually be electronically compensated, so the voltage clamp provides a robust measure of current flow through ion channels, thereby permitting the study of the detailed biophysics and pharmacology of ionic currents and channels. The major limitation of such experiments in myocytes is the existence of many currents that are simultaneously active in response to the voltage step. Experimental conditions can be altered to isolate a current of interest; however, this often requires the presence of drugs, toxins, or highly nonphysiologic conditions. An alternative to the study of ionic currents in native cells was afforded with the molecular cloning and heterologous expression of ion channel genes. Expression of an ion channel gene in a non-excitable cell without other overlapping currents permits the study of the ionic current of interest under more physiologic conditions. The fundamental limitation of heterologous expression is that the ion channel is removed from its native cellular background, which may change the behavior of the channel.

The combination of highly sensitive electrophysiological methods, such as patch-clamp recording, and deoxyribonucleic acid (DNA) cloning, heralded the era of understanding of the molecular basis of cardiac excitability.

Ion Channels and Transporters: Molecular Building Blocks of the Action Potential

Currents that underlie the action potential are carried by complex, multi-subunit transmembrane glycoproteins called ion channels (Table 3-1). These channels open and close in response to a number of biologic stimuli, including a change in voltage, ligand binding (directly to the channel or to a G-protein–coupled receptor), and mechanical deformation. Other ion-motive transmembrane proteins such as exchangers and transporters make important contributions to cellular excitability in the heart. Ion pumps establish and maintain the ionic gradients across the cell membrane that permit current flow through ion channels. If pumps, transporters, or exchangers are not electrically neutral (e.g., 3 Na⁺ for 1 Ca²⁺), they are termed electrogenic and can further influence electrical signaling in the heart.

The most abundant superfamily of ion channels expressed in the heart consists of voltage-gated ion channels. Various structural themes are common to all voltage-dependent ion channels. First, the architecture is modular, consisting either of four homologous subunits (in K channels, see Figures 3-7 and 3-8) or of four internally homologous domains (in Na and Ca channels) (see Figures 3-5 and 3-6). Second, proteins wrap around a central pore (see Figure 3-8). The pore-lining (P segment) regions exhibit exquisite conservation within a given channel family of like selectivity (e.g., jellyfish, eel, fruit fly, and human Na channels have very similar P segments), but not among families with different selectivities. Third, the general strategy for activation gating (opening and closing in response to changes in the membrane voltage) is highly conserved: The fourth transmembrane segment (S4), typically studded with positively charged residues, lies within the membrane field and moves in response to depolarization, thus opening the channel. Fourth, most ion channel complexes include not only the pore-forming proteins (α-subunits) but also auxiliary subunits (e.g., β-subunits) that modify channel function.

Figure 3-5 Models of the sodium (Na) channel. Top, Topographic illustration of the Na channel α-subunit with four pseudohomologous domains (I to IV). One or more transmembrane β-subunits coassemble with the α-subunit to form the intact channel. The fourth membrane-spanning repeat (S4) is charged and serves as the voltage sensor for channel activation. The segments or linkers between S5 and S6 in each domain, called permeation segments, or P segments, form the outer pore mouth and the selectivity filter. The linker between the third and fourth domains underlies fast inactivation and contains one of the mutations that underlies the chromosome 3–linked form of long QT syndrome. The S6 segment of the fourth domain contains residues critical for the binding of the local anesthetic to the channel. Bottom, Illustration of the caniliculus through which the S4 segment slides during channel activation. An outward movement of the S4 during channel activation is proposed.

Figure 3-6 Top, Subunit structure of the cardiac calcium (Ca) channel. The α₁1.2 (α_1C) subunit forms the pore and contains drug-binding sites. β₂ and α₂– to δ-subunits coassemble with the α₁ subunit. The intact cardiac L-type Ca channel containing the α₁1.2-subunit is referred to as Ca_V1.2. Bottom, The transmembrane topology of the Ca channel is similar to the sodium (Na) channel with four homologous domains, each containing six membrane-spanning repeats. The S4 segments (dark gray) are generally conserved, but the molecular basis of inactivation is distinct from Na channels. The Ca channel exhibits both voltage-dependent inactivation and Ca²⁺-dependent inactivation. Calmodulin (CaM), which is required for Ca²⁺-induced inactivation of the channel, is tethered to the channel in the carboxyl terminus; with binding of Ca²⁺, a conformation change occurs in this region of the channel that requires CaM, the CaM binding motif (IQ), and the EF hand motif, and together these constitute the Ca²⁺ inactivation region of the channel.

Sodium Channels

Sodium (Na) channels have been highly conserved through evolution and exist in all species, from the jellyfish to humans; they are nature’s solution to the conundrum of coordination and communication within large organisms, particularly when speed is of the essence. Thus, Na channels are richly concentrated in axons and muscle, where they are often the most plentiful ion channels. A mammalian heart cell, for example, typically expresses more than 100,000 Na channels but only 20,000 or so L-type (large and long-lasting) Ca channels and fewer copies of each family of voltage-dependent K channels.

Na channels were the first ion channels to be cloned and have their sequence determined.⁷ In humans, more than 10 distinct Na channel genes have been cloned from excitable tissues, with striking homology to the complementary DNA (cDNA) cloned from eel electroplax. The cardiac Na channel gene (SCN5A) resides on the short arm of chromosome 3 (3p21) (see Table 3-1). The Na channel complex is composed of several subunits, but only the α-subunit is required for function. Figure 3-5 shows that the α-subunit consists of four internally homologous domains (labeled I to IV), each of which contains six transmembrane segments. The four domains fold together so as to create a central pore, whose structural constituents determine the selectivity and conductance properties of the Na channel.

Peptide linkers between the fifth (S5) and sixth (S6) membrane-spanning repeats in each domain, referred to as the P segments, come together to form the pore. The primary structure of the S5-S6 linkers of Na channels in each domain is unique. Thus, the structural basis of the permeation of Na channels differs fundamentally from that of K channels, in which four identical P segments can come together to form a K⁺-selective pore (see below).

One of the seminal contributions of Hodgkin and Huxley was the notion that Na channels occupy several “states” (which are now viewed as different conformations of the protein) in the process of opening (activation); yet another set of conformations is entered when the channels close during maintained depolarization (inactivation).⁴ The m gates that underlie activation and the h gate that mediates inactivation were postulated to have intrinsic voltage dependence and to function independently.⁸ While some of the implicit structural predictions of that formulation have withstood the test of time, others have not. For example, the four S4 segments are now widely acknowledged to serve as activation voltage “sensors.” In the process of activation, several charged residues in each S4 segment physically traverse the membrane (see Figure 3-5, bottom panel). The contributions of each S4 segment to activation are markedly asymmetrical; some of the charged residues play a much more prominent role than do others in “homologous” positions.⁹ Other studies have revealed that activation is coupled with inactivation. Indeed, the time course of current decay during maintained depolarization predominantly reflects the voltage dependence of activation, although single-channel inactivation itself does vary with voltage (particularly in cardiac Na channels). If the S4s are the sensors, where are the activation “gates” themselves? This crucial question still remains unresolved. However, according to experimental evidence, S6 is the leading contender for the physical activation gate.

Inactivation of Na channels is as arcane a process as activation. Not only is there loose coupling to activation, but there are multiple inactivation processes. One common approach to distinguishing inactivated states is to determine the rate at which they recover the ability to activate: Repriming from the traditional “fast” inactivation occurs over tens of milliseconds, while recovery from “slow” inactivation may need tens of seconds or longer. Fast inactivation is at least partly mediated by the cytoplasmic linker between domains III and IV (the crucial residues are labeled IFM in Figure 3-5), which may function as a hinged lid, docking onto a receptor formed by amino acids in the S4-S5 linkers of domains III and IV. This notion is consistent with observations that fast inactivation can be disrupted by internal proteases. Nevertheless, it is increasingly clear that mutations scattered widely throughout the channel affect inactivation gating. The structural determinants of slow inactivation are less well localized than those of fast inactivation. Mutations in the P region of domain I affect both activation gating and slow inactivation, while various widely scattered disease mutations identified in paramyotonia congenita and other skeletal myopathies suppress slow inactivation of the Na channel.

The S6 segment of domain IV has been proposed to contain the receptor for local anesthetics that block Na channels in a voltage-dependent manner. The homologous domains on Ca and K channels are also loci for drug binding. Na current blockade is enhanced at depolarized potentials and/or with repetitive pulsing. These observations are consistent with the idea that local anesthetics act as allosteric effectors of the inactivation gating mechanism: When they bind to the channel, they facilitate inactivation. It is clear that gating interacts with local anesthetic blockade so profoundly that it is difficult to interpret the localization of a “receptor” to S6. Mutations in S6 at putative receptor sites alter gating, independent of superimposed drug effects. Further, mutations in distant parts of the molecule can also dramatically alter the phenotype of local anesthetic blockade. Despite these caveats, the S6 segments appear to play a special role in the effects of drugs in all of the voltage-gated ion channels.

Pharmacologic competition studies and mutagenesis have defined a number of neurotoxin binding sites on the Na channel. Among these, tetrodotoxin (TTX)—a guanidinium-containing blocker—has contributed the most to our understanding of Na channel structure and function. Externally applied TTX blocks neural and skeletal muscle Na channel isoforms potently (in the nM range) but blockade of cardiac channels requires much higher concentrations (~10⁻⁵ M). The identity of one particular residue in the P region of domain I accounts for most of the isoform-specific TTX sensitivity: An aromatic residue at this position (373 in the human heart Na channel) confers high affinity, while its absence renders the channel TTX resistant. Many other residues in the outer mouth of the channel contribute to the binding of TTX and the related divalent guanidinium toxin saxitoxin (STX), suggesting that the toxin has a large footprint on the external surface of the channel.

Four different β-subunits (Na_Vβ₁ to 4, SCN1B-SCN4B) have been isolated, and all appear to be single membrane–spanning domain (type I topology) proteins with a large extracellular V-shaped immunoglobulin (Ig) fold often found in cell adhesion molecules and small carboxyl terminal cytoplasmic domain.¹⁰ The effects of the particular β-subunit on the kinetics and voltage dependence of the α-subunit vary depending on the particular β-subunit and the cell expression system. Despite the expression of β₁ mRNA, using subtype-specific antisera, the functional role of β-subunits in cardiac Na currents is still being debated. Several pieces of evidence are consistent with a role for β-subunit(s) in heart cells. First, β-subunits are found in cardiac myocytes, although no Na_Vβ1 is found in association with the α-subunit protein from the rat heart. Second, Na_Vβ1 variably modulates the function of Na_V1.5 in heterologous expression systems, including the sensitivity of the channel to blockade by antiarrhythmic drugs and free fatty acids. Finally, mutations in β-subunits have been directly or indirectly implicated in heritable cardiac arrhythmias.

Regulation of the Na channel by serine/threonine phosphorylation is a complex process. Isoforms of the Na channel α-subunit fall into one of two groups, long (neuronal and cardiac) and short (skeletal muscle and eel). Neuronal isoforms have a substantially larger intracellular linker between domains I and II, which contains five consensus sites for cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA) phosphorylation. In fact, PKA modulates the function of expressed neuronal and cardiac Na channels. The cardiac Na channel has eight candidate consensus PKA phosphorylation sites in the I-II linker, all of which are distinct from neuronal channels. In vitro studies of the expressed cardiac Na channel demonstrate cAMP-dependent phosphorylation on only two of these serines. Interestingly, when the cardiac channel is phosphorylated by PKA, whole-cell conductance increases, suggesting that the specific pattern of phosphorylation is responsible for the functional effect and may involve changes in the trafficking of the channel to the cell membrane.

In contrast to PKA, protein kinase C (PKC) alters the function of all of the mammalian Na channel isoforms. The PKC effect is largely attributable to phosphorylation of a highly conserved serine in the III-IV linker (see Figure 3-5). Conventional PKC isoforms reduce the maximal conductance of the channels and alter gating in an isoform-specific fashion. The macroscopic current decay of neuronal channels is uniformly slowed by PKC, which suggests destabilization of the inactivated state. Cardiac channels exhibit a hyperpolarizing shift in the steady-state availability curve consistent with an enhancement of inactivation from closed states.

Overexpression of calmodulin kinase (CaMKIIδC) in transgenic murine heart alters cardiac I_Na function, stabilizing inactivation and increasing persistent current; however, this finding is complicated by the presence of heart failure in this murine model. Subacute expression after adenoviral infection of adult ventricular myocytes has a similar effect on I_Na. In contrast, in acutely isolated guinea pig ventricular myocytes, enhanced CaMKII activity destabilizes inactivation gating and increases the persistent or late current, thus prolonging the action potential duration.

Alteration of ion channel function is an important pathophysiologic mechanism of various familial diseases of muscle and brain and of inherited arrhythmias. The Na channelopathies were among the first molecularly characterized human ion channel diseases.¹¹ Rare allelic variants in SCN5A have been linked to inherited ventricular arrhythmias,¹² conduction system disease, and sudden infant death syndrome. Complex electrophysiological phenotypes have been associated with mutations in both α- and β-subunits of Na channels. Rare variants or mutations have been associated with sudden death in women (in a population-based study) and with atrial fibrillation. More common acquired forms of long QT syndrome (LQTS), generally associated with drug ingestion, have been linked to common variants of SCN5A. Finally, disease-causing mutations in the Na channel have been associated with alterations in drug blockade. The highly variant phenotypes of these arrhythmic syndromes are, in part, explained by the variable effects of the mutations on channel subunit function or expression.

Calcium Channels: L-type

The pore-forming subunit (α₁) of the calcium (Ca) channel is built on the same structural framework as the Na channel.¹³ As is the case with the Na channel, a number of genes encode surface membrane Ca channel α₁-subunits. The predominant sarcolemmal Ca channels in the heart are the L-type (large and long-lasting) and T-type (tiny and transient) Ca channels (Table 3-2). The cardiac L-type Ca channel (Ca_V1.2) is a multi-subunit transmembrane protein composed of α_1C (α₁1.2) (165 kDa), β (55 kDa), and α₂ (130 kDa) to δ (32 kDa) subunits. Three genes are known to encode L-type Ca channel α₁-subunits (Ca_Vα₁1.x to α₁3.x), and the Ca_Vα₁1.2 is the gene expressed in the heart (see Table 3-1). Distinct splice variants of the Ca_Vα₁1.2 gene have been described, and these contribute to the diversity of the cardiac L-type Ca channel function. Similar to the α-subunit of the Na channel, the S5-S6 linkers (P segments) of the α₁-subunit of the Ca channel form the ion-selective pore (Figure 3-6). However, unlike Na channels, each P segment contributes a glutamic acid to a cluster that serve to bind Ca²⁺ in the channel pore. The β-subunit is completely cytoplasmic and noncovalently binds to the α_1C subunit, modifying its function and contributing to appropriate membrane trafficking of the channel complex. Although β_2a has been proposed to be the major L-type Ca channel β-subunit, splice variants of all β-subunits β₁ to β₄ are expressed in the mammalian heart in a complex spatial and temporal pattern. As many as five genes have been suggested to encode the α₂-δ subunit: These gene products undergo post-translational processing to produce the mature extracellular α₂-subunit linked by a disulfide bond to the transmembrane δ-subunit. In heterologous expression systems, α₂– to δ-subunits enhance expression of Ca channels and hasten current activation and deactivation in the presence of α₁– and β-subunits.

Table 3-2 Properties of Calcium Channels

Ba, Barium; Cd, cadmium; Ni, nickel.

The α₁-subunit of the Ca channel contains a highly basic S4 transmembrane segment in each homologous domain, which, by analogy, is thought to be the voltage sensor for channel activation. Activation of skeletal muscle and cardiac L-type Ca channels differ; skeletal muscle channels activate much more slowly than do their cardiac counterparts. Based on the properties of chimeric channels constructed from cardiac (Ca_Vα₁1.2) and skeletal muscle (Ca_Vα₁1.1) α₁-subunits, the difference in activation gating resides in the first homologous domain; however, it is unclear if the S4 membrane-spanning segment is the crucial structural motif.

Ca channels inactivate by both Ca²⁺-dependent inactivation (CDI) and voltage-dependent inactivation (VDI) processes. CDI and VDI are regulated by channel phosphorylation and β-subunits, which suggests shared structural mechanisms that perhaps involve the I-II domain linker. The C-terminus contains peptide sequences that bind calmodulin (CaM) and Ca²⁺; these are an IQ motif named for the signature amino acids (isoleucine and glutamine) in the sequence and a helix-loop-helix structural domain, referred to as an EF hand, that mediate CDI. CaM is permanently tethered to the channel complex and serves as a Ca²⁺ sensor for the L-type channel. The Ca²⁺-CaM complex facilitates the interaction of CaM with the IQ motif resulting in the occlusion of the inner mouth of the Ca channel pore and terminating the inward Ca²⁺ flux despite continued depolarization. As cytoplasmic Ca²⁺ concentration falls, calmodulin unbinds Ca²⁺ and the IQ motif, thus relieving Ca²⁺-dependent inactivation. Spatial discrimination of Ca²⁺ concentration by CaM may involve regions in the N-terminus of the channel. A Ca²⁺-binding EF hand motif in the carboxyl terminus of α₁ appears to be necessary to confer Ca²⁺-dependent inactivation to the Ca_Vα₁1.2-subunit, although not through a direct binding of Ca²⁺ (see Figure 3-6, bottom