Specialized Excitable Cells Tightly Regulate Cardiac Depolarization

The highly coordinated and efficient propagation of electrical activity through the heart is maintained by the combined activities of a diverse set of specialized excitable cardiac cells, each with its own structural, electrical, and molecular signature. The cardiac sinoatrial (SA) node, a small group of spontaneously active cells in the right atria, is the primary initiation site of cardiac electrical activity because of its relatively positive threshold potential.¹ Once generated by the sinus node, the cardiac action potential propagates through the atria to the atrioventricular (AV) node (the maximum diastolic potential of the AV node is only slightly more negative than that of the SA node), a second small but critical group of specialized cells that display slow conduction properties preventing inappropriate depolarization of the ventricles. In fact, the slow conduction of the AV node is a critical safeguard against the development of ventricular arrhythmias from pathologic atrial pacing defects (i.e., atrial flutter/fibrillation). After the AV node, the cardiac action potential propagates through the cardiac conduction system comprising the AV bundle (bundle of His), the left and right bundle branches, and the cardiac Purkinje system. Interestingly, this conduction system, particularly the cardiac Purkinje fibers, has evolved to rapidly propagate cardiac electrical activity at up to 2 to 4 m/s for the nearly instantaneous spread of depolarization through the sub-endocardium of the left and right ventricles.² In comparison with the rapid conduction pathways of the Purkinje system, left ventricular tissue conduction velocity is significantly slower (0.3 to 1 m/s).² Importantly, Purkinje fibers communicate with the ventricular mass at well-defined discrete loci (Purkinje-muscle junctions).

Form Fits Function: The Ventricular Cardiomyocyte and Excitation-Contraction Coupling

As is discussed in detail in Chapter 3, the electrical activity of cardiac cells (the action potential) is primarily modulated by the coordinated movement of sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) across the external plasma membrane (sarcolemma) and the internal sarcoplasmic reticulum (SR) membrane. The specialized function of each cardiac cell type is the result of the evolution of specific molecular and structural components that regulate ion flux across the membrane and dictate specific cell properties.³ In this section, the primary structural and molecular components of different cardiac cells is discussed in relation to cell type–specific action potentials. Because of its central role in cardiac excitability, the ventricular cardiomyocyte will be used as the central point of comparison for other excitable cardiac cell types.

The ventricular action potential is notable for its hyperpolarized resting membrane potential, rapid upstroke, and prolonged plateau (Figure 2-1). The resting membrane potential of the ventricular cardiomyocyte (held at ~–90 mV, roughly 30 mV more negative than the human sinus node) is the most negative of all excitable cell types (hence the final cell type to depolarize), primarily because of a large inwardly rectifying K⁺ current, I_K1, prominently expressed in these cells.⁴ The rapid upstroke, because of the presence of rapidly activating voltage-gated Na⁺ channels, allows for rapid propagation of the electrical signal through the ventricles, which is required for synchronized muscle contraction. Finally, the extended plateau allows sufficient time for Ca to enter the cell and signal contraction.³

Figure 2-1 Key ion currents responsible for the phases of the cardiac ventricular action potential. A mathematical model of the ventricular cardiac myocyte was used to generate representative ventricular action potential with rapid phase 0 (upstroke), characteristic phase 1 repolarization (“spike”), prominent phase 2 plateau (“dome”), delayed phase 3 repolarization, and stable rest potential (phase 4, ~–90 mV).

(From Hund TJ, Rudy Y: Rate dependence and regulation of action potential and calcium transient in a canine cardiac ventricular cell model, Circulation 110[20]:3168–3174, 2004; Hund TJ, Decker KF, Kanter E, et al: Role of activated CaMKII in abnormal calcium homeostasis and I(Na) remodeling after myocardial infarction: insights from mathematical modeling, J Mol Cell Cardiol 45[3]:420–428, 2008.)

Once the excitation reaches the ventricles through the cardiac conduction system (see above), the electrical signal passes from cell to cell as a flux of ions through specialized intercellular ion channels called gap junctions (discussed in detail below). This flow of ions into the cell from neighboring activated cells depolarizes the membrane potential. If the membrane reaches a threshold potential (~–60 mV), a large population of voltage-gated Na⁺ channels (primarily Na_v1.5, encoded by SCN5A) is activated, which results in a large inward influx of Na⁺ across the membrane into the myocyte. Na⁺ channels are functionally well suited to this task as they undergo rapid activation (activation time constant <0.5 ms) in response to membrane depolarization. Importantly, these channels also experience rapid voltage-dependent inactivation, which prevents reactivation until the membrane has returned to rest. The inward flux of Na⁺ ions carried by voltage-gated Na⁺ channels produces the initial rapid spike of the ventricular action potential (phase 0; see Figure 2-1).³ Depolarization of the cardiac membrane by rapidly activating voltage-gated Na⁺ channels activates higher threshold, more slowly activating voltage-gated Ca²⁺ channels (primarily CACNA1C-encoded Ca_v1.2 in the ventricle; see Figure 2-1).² Ca²⁺ influx through voltage-gated Ca²⁺ channels serves two major purposes: (1) maintaining the action potential plateau (important for controlling heart rhythm), and (2) triggering Ca²⁺ release from internal stores for the purpose of triggering the mechanical contraction of the heart (excitation-contraction coupling).² During excitation-contraction (EC) coupling, a small inward Ca²⁺ current across the plasma membrane is sensed by functional SR ryanodine receptor (RyR2 in the ventricle, encoded by RYR2) clusters, which subsequently release large quantities of Ca²⁺ from the internal SR stores into the cytosol, giving rise to a dramatic increase (order of magnitude) in intracellular Ca²⁺ (the Ca²⁺ transient).² In this process, termed Ca²⁺-induced Ca²⁺ release, a small increase in local Ca²⁺ via Ca_v1.2 produces a relatively large release of SR Ca²⁺ (high gain function).

While voltage-gated Na⁺ and Ca²⁺ channels are responsible for myocyte depolarization and contraction, a host of plasma membrane–associated K⁺ channels regulate ventricular cardiomyocyte repolarization during phases 1, 2, and 3, as well as the rest potential.⁴ Specifically, the characteristic repolarization “notch” of phase 1 is modulated by the outward flux of K⁺ carried by the transient outward K⁺ current, I_to (primarily K_v4.2/K_v4.3 channels; see Figure 2-1). The duration of the action potential plateau (phase 2) is determined by a delicate balance between the inward Ca²⁺ current and the outward delayed rectifier K⁺ currents I_Kr and I_Ks (erg1/MiRP1 and KvLQT1/MinK channels encoded by KCNH2/KCNE2 and KCNQ1/KCNE1, respectively; see Figure 2-1).⁴ As the action potential proceeds, the Ca²⁺ current decreases because of deactivation as well as inactivation, and the K⁺ currents I_Kr and I_Ks increase, ultimately tilting the balance in favor of the late repolarization phase (phase 3; see Figure 2-1).⁴ The cell eventually returns to a resting potential maintained primarily by the time-independent inward rectifier current I_K1 (see Figure 2-1). Other currents such as I_KATP, while not central to the healthy control action potential, may have key roles in disease. Finally, the Na⁺-K⁺ adenotriphosphatase (ATPase) (which uses ATP to remove 3 Na⁺ from the cell and bring in 2 K⁺) is a key feature for generating and maintaining the myocyte electrochemical gradient.

Proper functioning of ion channels is required for normal cardiac physiology, as channel dysfunction has been linked to both congenital and acquired forms of heart disease and arrhythmias. For example, genetic mutations in Na⁺, K⁺, and Ca²⁺ channels and channel subunits have been associated with lethal cardiac arrhythmia syndromes, including congenital long QT syndrome (mutations in KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and KCNJ2 genes), short QT syndrome (mutations in KCNH2, KCNQ1, and KCNJ2 genes), Brugada syndrome (mutations in the SCN5A gene), and Timothy syndrome (mutations in the CACNA1C gene). Ion channel defects arising from electrical remodeling in the setting of acquired heart disease have also been linked to arrhythmia. Specifically, Na⁺ channel changes after myocardial infarction have been linked to slowed conduction, and changes in K⁺ and Ca²⁺ channels have been linked to action potential prolongation in failing hearts.

Cell Membrane Architecture Defines Myocyte Local Electrical Activity

Over the past 15 years, the use of high-resolution imaging techniques in the field of molecular cardiology has revolutionized the understanding of cardiac cell biology and electrical function. Specifically, unlike the first plant cell imaged by Hooke in the mid-1600s, it is now known that the vertebrate myocyte is not simply a large pool of cytosol surrounded by a simple membrane. Rather, the metazoan myocyte has evolved complex membrane structures to facilitate efficient electrical activity and signaling to regulate cardiac physiology. Not surprisingly, specific cell types in the heart possess a distinct set of membrane structures based on their unique function.

The ventricular cardiomyocyte plasma membrane, or sarcolemma, is divided into multiple and unique membrane structures (Figure 2-2). In addition to the external sarcolemma (resident proteins include the Na⁺-Ca²⁺ exchanger (NCX1) and plasma membrane Ca²⁺-ATPase [PMCA1]), the ventricular cardiomyocyte contains a large array of regularly spaced (~1.8 μm) plasma membrane invaginations, termed transverse tubules, or T-tubules. This membrane system, instrumental in myocyte EC coupling, evolved to facilitate coordinated EC coupling in the relatively large ventricular cardiomyocyte (system not present in smaller atrial and sinoatrial node cells). T-tubule–resident proteins include the L-type Ca²⁺ channel Ca_v1.2, the Na⁺-Ca²⁺ exchanger, and Na⁺-K⁺-ATPase.² Finally, a highly specialized domain is present where the ventricular cardiomyocyte plasma membrane lies in close apposition to the plasma membrane of a neighboring cell. This complex membrane system, termed the intercalated disc, is required for myocyte cell-cell adhesion as well as intercellular action potential propagation and comprises three subdomains: (1) the gap junction, (2) the adherens junction, and (3) the desmosome (Figure 2-3).² The gap junction comprises hundreds of hemi-channels (connexons) that span the lipid bilayer and allow electrical and metabolic coupling when docked with hemi-channels from a neighboring cell. At least four different connexin proteins (functional units of the connexon) with distinct biophysical properties are expressed throughout the heart. Gap junctions in ventricular tissue consist primarily of connexin43, which forms large conductance channels to allow rapid conduction. In contrast, gap junctions in the sinus node contain mostly connexin45, which forms lower conductance channels ideal for slow but safe conduction. The adherens junction is maintained by the function of a cadherin-catenin complex that provides a stabilizing link from the intercalated disc to the actin cytoskeleton (see Figure 2-3). Finally, the desmosome also supports cell adhesion through a complex, including plakoglobin (γ-catenin, also found in the adherens junction), desmoplakin, and plakophilin, that interacts with intermediate filaments (see Figure 2-3). Interestingly, defects in desmosomal proteins have been linked to cardiomyopathies and arrhythmias, including arrhythmogenic right ventricular cardiomyopathy (ARVC). In fact, loss of plakoglobin immunostaining in human heart biopsies has been recently developed into a diagnostic marker for Naxos disease, a cardiocutaneous syndrome characterized by wooly hair, palmoplantar keratoderma, and severe cardiomyopathy.⁵

Figure 2-2 Local membrane architecture, which is critical for vertebrate cardiomyocyte electrical activity and physiology. Image of ventricular cardiomyocyte with denoted membrane structures, including the intercalated disc (yellow), transverse tubules overlying the cellular Z-line (red, stained with α-actinin antibody), sarcoplasmic reticulum (green), nucleus (silver), and nuclear envelope (violet).

Figure 2-3 Structures of the intercalated disc. The adherens junction contains a catenin-cadherin complex that anchors the disc to actin. The desmosome provides a link to intermediate filaments and contains primarily desmoplakin, plakoglobin (γ-catenin), plakophilin-2, and the desmosomal cadherins (desmoglein-2 and desmocollin-2). Finally, the gap junction establishes electrical and metabolic communication between neighboring cells. Six individual connexin proteins (primarily Cx45 in the SA node, Cx40 to Cx45 in the atria, and Cx43 in the ventricle) combine to form heteromeric connexon hemi-channels that dock with apposing connexons from the adjoining cell.

In addition to the external plasma membrane, the cardiac myocyte (as well as many other excitable cells, including neurons and skeletal muscle) contains SR, the specialized endoplasmic reticulum that has key roles in the regulation of intracellular Ca²⁺.² The cardiac SR is an extensive network of tubules linking key signaling networks, including the plasma membrane, nuclear envelope, nucleus, and mitochondria, to mediate a host of diverse functions. The pre-eminent role of the cardiac SR is to regulate myocyte EC coupling. Specifically, the vertebrate SR sequesters a large pool of releasable Ca²⁺ (1 mM inside SR vs. 100 nM in cytosol²) that serves as the primary source of Ca²⁺ for troponin-C (TnC) activation and muscle contraction (discussed in detail below).² The majority of this Ca²⁺ is internally buffered by the Ca²⁺-binding protein calsequestrin, which forms a critical RyR2 regulatory complex with triadin and junctin. However, Ca²⁺ entry into the cell during the action potential plateau through voltage-gated Ca²⁺ channels activates SR ryanodine receptor Ca²⁺ channels. These RyR2 channels then rapidly release sequestered SR Ca²⁺ into the cytosol (see below) to signal contraction. During diastole, SR Ca²⁺-ATPase (SERCA2) and its regulatory protein phospholamban play central roles in the reuptake of released Ca²⁺ from the myocyte cytosol into the SR. Interestingly, inappropriate regulation of SR Ca²⁺ because of defects in either Ca²⁺ buffering (i.e., human calsequestrin-2 gene mutations) or Ca²⁺ release (human RyR2 gene mutations) has been linked with potentially fatal human arrhythmia (catecholaminergic polymorphic ventricular tachyarrhythmia). SR membrane–resident proteins also include inositol 1,4,5 trisphosphate (InsP₃) receptors that have been linked to cardiac hypertrophy and arrhythmia.⁶

During the ventricular action potential, the rise in cytosolic Ca²⁺ via the SR membrane–associated RyR2 is rapidly translated into mechanical activity between the thick myosin filaments and the thin actin filaments through the regulatory functions of the troponin-tropomyosin complex.² This complex consists of four subunits. Tropomyosin is a double-stranded α-helical molecule, which, under basal conditions (low Ca²⁺), covers myosin-binding sites along a span of seven actin monomers.² Troponin T (TnT; tropomyosin-binding subunit) connects tropomyosin to the two remaining subunits TnC (Ca²⁺-binding) and troponin I (TnI; inhibitory subunit). The cardiac isoform of TnC has two high-affinity binding sites for Ca²⁺ or Mg²⁺ in the C-terminal domain and a low-affinity regulatory binding site that is Ca²⁺ specific in the N-terminal domain. During diastole, the C-terminal domain of TnI interacts with actin. During systole, Ca²⁺ binds to a low-affinity binding site in the TnC N-terminal domain, causing an increased affinity between this domain and the TnI N-terminal domain. As a result, the TnI-actin interaction is destabilized, which ultimately leads to a conformational change in the troponin-tropomyosin complex.²