Phase 4: The Resting Membrane Potential

The E _m of resting atrial and ventricular cardiomyocytes remains steady throughout diastole. The resting E _m is caused by the differences in ionic concentrations across the membrane and the selective membrane permeability (conductance) to various ions. Large concentration gradients of Na ⁺ , K ⁺ , Ca ²⁺ , and Cl ⁻ across the cell membrane are maintained by the ion pumps and exchangers ( Table 1.2 ).

Under normal conditions, the resting membrane is most permeable to K ⁺ and relatively impermeable to other ions. K ⁺ has the largest resting membrane conductance (g _K is 100 times greater than g _Na ) because of the abundance of open K ⁺ channels at rest, whereas Na ⁺ and Ca ²⁺ channels are generally closed. Thus K ⁺ exerts the largest influence on the resting E _m . As a consequence, the resulting E _m is almost always close to the K ⁺ reversal potential (E _m approximates E _K ). The actual resting E _m is slightly less negative than E _K because the cell membrane is slightly permeable to other ions.

The inwardly rectifying K ⁺ (Kir) channels underlie an outward K ⁺ current (I _K1 ) responsible for maintaining the resting potential near the E _K in atrial, His-Purkinje, and ventricular cells, under normal conditions. Kir channels preferentially allow currents of K ⁺ ions to flow into the cell with a strongly voltage-dependent decline of K ⁺ efflux (i.e., reduction of outward current) on membrane depolarization. As such, I _K1 is a strong rectifier that passes K ⁺ currents over a limited range of E _m . At a negative E _m , I _K1 conductance is much larger than that of any other current; thus it clamps the resting E _m close to the reversal potential for K ⁺ (E _K ) (see Chapter 2 for detailed discussion on the concept of rectification). I _K1 density is much higher in ventricular than in atrial myocytes, a feature that largely prevents the ventricular cell from having diastolic membrane depolarization and pacemaker activity. By contrast, I _K1 is almost absent in sinus and AV nodal cells, thus allowing for relatively more depolarized resting diastolic potentials compared with atrial and ventricular myocytes ( Table 1.3 ). The effect of outward K ⁺ current to resist membrane depolarization (keeping voltage fixed) is sometimes referred to as a voltage clamping effect.

A unique property of Kir currents is the unusual dependence of rectification on extracellular K ⁺ concentration. Specifically, with an increase in extracellular K ⁺ , the I _K1 current-voltage relationship shifts nearly in parallel with the E _K and leads to a crossover phenomenon. One important consequence of such behavior is that at potentials positive to the crossover, K ⁺ conductance increases rather than decreases, against an expectation based on a reduced driving force for K ⁺ ions as a result of elevated extracellular K ⁺ concentration.

The resting E _m is also powered by the Na ⁺ -K ⁺ adenosine triphosphatase (ATPase) (the Na ⁺ -K ⁺ pump), which helps to establish concentration gradients of Na ⁺ and K ⁺ across the cell membrane. Under physiological conditions, the Na ⁺ -K ⁺ pump transports two K ⁺ ions into the cell against its chemical gradient and three Na ⁺ ions outside against its electrochemical gradient at the expense of one ATP molecule. Because the stoichiometry of ion movement is not 1 : 1, the Na ⁺ -K ⁺ pump is electrogenic and generates a net outward movement of positive charges (i.e., an outward current). At faster heart rates, the rate of Na ⁺ -K ⁺ pumping increases to maintain the same ionic gradients, thus counteracting the intracellular gain of Na ⁺ and loss of K ⁺ with each depolarization.

Ca ²⁺ does not contribute directly to the resting E _m because the voltage-activated Ca ²⁺ channels are closed at the hyperpolarized resting E _m . However, changes in intracellular free Ca ²⁺ concentration can affect other membrane conductance values. Increases in intracellular Ca ²⁺ levels can stimulate the Na ⁺ -Ca ²⁺ exchanger (I _Na-Ca ), which exchanges three Na ⁺ ions for one Ca ²⁺ ion; the direction depends on the Na ⁺ and Ca ²⁺ concentrations on the two sides of the membrane and the E _m difference. At resting E _m and during a spontaneous sarcoplasmic reticulum Ca ²⁺ release event, this exchanger would generate a net Na ⁺ influx, possibly causing transient membrane depolarizations.

Phase 0: The Upstroke—Rapid Depolarization

On excitation of an atrial, ventricular, or Purkinje cardiomyocyte by electrical stimuli from adjacent cells, its resting E _m (approximately −85 mV) depolarizes, leading to opening (activation) of Na ⁺ channels from its resting (closed) state and enabling a large and rapid influx of Na ⁺ ions (inward I _Na ) into the cell down their electrochemical gradient. As a consequence of increased Na ⁺ conductance, the excited membrane no longer behaves like a K ⁺ electrode (i.e., exclusively permeable to K ⁺ ) but more closely approximates an Na ⁺ electrode, and the E _m moves toward the E _Na (see Table 1.2 ). Once an excitatory stimulus depolarizes the E _m beyond the threshold for activation of Na ⁺ channels (approximately −65 mV), the activated I _Na is regenerative and no longer depends on the initial depolarizing stimulus. As a consequence, the influx of Na ⁺ ions further depolarizes the membrane and thereby increases conductance to Na ⁺ more, which allows more Na ⁺ to enter the cell (thus “regenerative”).

Normally, activation of Na ⁺ channels is transient; fast inactivation (closing of the channel pore) starts simultaneously with activation, but because inactivation is slightly delayed relative to activation, the channels remain transiently (less than 1 millisecond) open to conduct I _Na during phase 0 of the action potential before it closes. In addition, the influx of Na ⁺ into the cell increases the positive intracellular charges and reduces the driving force for Na ⁺ . When the E _Na is reached, no further Na ⁺ ions enter the cell.

The rate at which depolarization occurs during phase 0 (i.e., the maximum rate of change of voltage over time [dV/dt _max ]) is a reasonable approximation of the rate and magnitude of Na ⁺ entry into the cell and a determinant of conduction velocity for the propagated action potential (see later).

The threshold for activation of I _CaL is approximately −30 to −40 mV. Although I _CaL is normally activated during phase 0 by the regenerative depolarization caused by the fast I _Na , I _CaL is much smaller than the peak I _Na . Furthermore, the amplitude of I _CaL is not maximal near the action potential peak because of the time-dependent nature of I _CaL activation, as well as the low driving force (E _m − E _Ca ) for I _CaL . Therefore I _CaL contributes little to the action potential until the fast I _Na is inactivated, after completion of phase 0. As a result, I _CaL affects mainly the plateau of action potentials recorded in atrial and ventricular muscle and His-Purkinje fibers. On the other hand, I _CaL plays a prominent role in the upstroke of slow response action potentials in partially depolarized cells in which the fast Na ⁺ channels have been inactivated.

Phase 1: Early Repolarization

Phase 0 is followed by phase 1 (early repolarization), during which the membrane repolarizes rapidly and transiently to almost 0 mV (early notch), partly because of the inactivation of I _Na and concomitant activation of several outward currents. The transient outward K ⁺ current (I _to ) is mainly responsible for phase 1 of the action potential. I _to rapidly activates (with time constants less than 10 milliseconds) by depolarization and then rapidly inactivates (25 to 80 milliseconds for the fast component of I _to [I _to,f ], and 80 to 200 milliseconds for the slow component of I _to [I _to,s ]). The influx of K ⁺ ions via I _to channels partially repolarizes the membrane, thus shaping the rapid repolarization (phase 1) of the action potential and setting the height of the initial plateau (phase 2) (see Fig. 1.1 ). In addition, an Na ⁺ outward current through the Na ⁺ -Ca ²⁺ exchanger operating in reverse mode likely contributes to this early phase of repolarization.

Phase 2: The Plateau

Phase 2 (plateau) represents a delicate balance between the depolarizing inward currents (I _CaL and a small residual component of inward I _Na ) and the repolarizing outward currents (ultrarapidly [I _Kur ], rapidly [I _Kr ], and slowly [I _Ks ] activating delayed outward rectifying currents) (see Fig. 1.1 ). Phase 2 is the longest phase of the action potential, lasting tens (atrium) to hundreds of milliseconds (His-Purkinje system and ventricle). The plateau phase is unique among excitable cells and marks the phase of Ca ²⁺ entry into the cell. It is the phase that most clearly distinguishes the cardiac action potential from the brief action potentials of skeletal muscle and nerve.

I _CaL is activated by membrane depolarization, is largely responsible for the action potential plateau, and is a major determinant of the duration of the plateau phase. I _CaL also links membrane depolarization to myocardial contraction. L-type Ca ²⁺ channels activate on membrane depolarization to potentials positive to −40 mV. I _CaL peaks at an E _m of 0 to +10 mV and tends to reverse at +60 to +70 mV, following a bell-shaped current-voltage relationship.

Na ⁺ channels also make a contribution, although minor, to the plateau phase. After phase 0 of the action potential, some Na ⁺ channels occasionally fail to inactivate or exhibit prolonged opening or reopening repetitively for hundreds of milliseconds after variable and prolonged latencies, resulting in a small inward I _Na (with a magnitude of less than 1% of the peak I _Na ). This persistent or “late” I _Na (I _NaL ), along with I _CaL , helps to maintain the action potential plateau.

I _Kr and I _Ks are activated at depolarized membrane potentials. I _Kr activates relatively fast (in the order of tens of milliseconds) on membrane depolarization, thus allowing outward diffusion of K ⁺ ions in accordance with its electrochemical gradient, but voltage-dependent inactivation thereafter is very fast. Hence only limited numbers of channels remain in the open state, whereas a considerable fraction resides in the nonconducting inactivated state. The fast voltage-dependent inactivation limits outward current through the channel at positive voltages and thus helps to maintain the action potential plateau phase that controls contraction and prevents premature excitation. However, as the voltage becomes less positive at the end of the plateau phase of repolarization, the channels recover rapidly from inactivation; this process leads to a progressive increase in I _Kr amplitudes during action potential phases 2 and 3, with maximal outward current occurring before the final rapid declining phase of the action potential.

I _Ks , which is approximately 10 times larger than I _Kr , also contributes to the plateau phase. I _Ks activates in response to membrane depolarization to potentials positive to −30 mV and gradually increases during the plateau phase because its time course of activation is extremely slow, slower than any other known K ⁺ current. In fact, steady-state amplitude of I _Ks is achieved only with extremely long membrane depolarization. Hence the contribution of I _Ks to the net repolarizing current is greatest late in the plateau phase, particularly during action potentials of long duration. Importantly, although I _Ks activates slowly compared with action potential duration, it is also slowly inactivated. As heart rate increases, I _Ks increases because channel deactivation is slow and incomplete during the shortened diastole. This allows I _Ks channels to accumulate in the open state during rapid successive action potentials and mediate the faster rate of repolarization. Hence I _Ks plays an important role in determining the rate-dependent shortening of the cardiac action potential.

I _Kur is detected only in human atria but not in the ventricles, such that it is the predominant delayed rectifier current responsible for atrial repolarization and is a basis for the much shorter duration of the action potential in the atrium. I _Kur activates rapidly on depolarization in the plateau range and displays outward rectification, but it inactivates slowly during the time course of the action potential.

The Na ⁺ -Ca ²⁺ exchanger operating in forward mode (three Na ⁺ ions in for one Ca ²⁺ ion out) and the Na ⁺ -K ⁺ pump provide minor current components during phase 2.

Importantly, during the plateau phase, membrane conductance to all ions falls to rather low values. Thus less change in current is required near plateau levels than near resting potential levels to produce the same changes in E _m . In particular, K ⁺ conductance falls during the plateau phase as a result of inward rectification of I _K1 (i.e., voltage-dependent decline of K ⁺ efflux and hence reduction of outward current) on membrane depolarization, in spite of the large electrochemical driving force on K ⁺ ions during the positive phase of the action potential (phases 0, 1, and 2). This property allows membrane depolarization following Na ⁺ channel activation, slows membrane repolarization, and helps to maintain a more prolonged cardiac action potential. This also confers energetic efficiency in the generation of the action potential.

Phase 3: Final Rapid Repolarization

Phase 3 is the phase of rapid repolarization that restores the E _m to its resting value. Phase 3 is mediated by the increasing conductance of the delayed outward rectifying currents (I _Kr and I _Ks ), the inwardly rectifying K ⁺ currents (I _K1 and acetylcholine-activated K ⁺ current [I _KACh ]), and time-dependent inactivation of I _CaL (see Fig. 1.1 ). Final repolarization during phase 3 results from K ⁺ efflux through the I _K1 channels, which open at potentials negative to −20 mV.

Phase 4: Restoration of Resting Membrane Potential

During the action potential, Na ⁺ and Ca ²⁺ ions enter the cell and depolarize the E _m . Although the E _m is quickly repolarized by the efflux of K ⁺ ions, restoration of transmembrane ionic concentration gradients to the baseline resting state is necessary. This is achieved by the Na ⁺ -K ⁺ ATPase (Na ⁺ -K ⁺ pump, which exchanges two K ⁺ ions inside and three Na ⁺ ions outside) and by the Na ⁺ -Ca ²⁺ exchanger (I _Na-Ca , which exchanges three Na ⁺ ions for one Ca ²⁺ ion).

Reduction of cytosolic Ca ²⁺ concentration during diastole is achieved by the reuptake Ca ²⁺ by the sarcoplasmic reticulum via activation of the sarco/endoplasmic reticulum calcium-ATPase calcium pump (SERCA), in addition to extrusion across the sarcolemma via the Na ⁺ -Ca ²⁺ exchanger. In the human heart under resting conditions, the time required for cardiac myocyte depolarization, contraction, relaxation, and recovery is approximately 600 milliseconds.

Regional Heterogeneity of the Action Potential

Substantial differences in expression levels of ion channels underlie the considerable heterogeneity in action potential duration and configuration between cardiomyocytes located in different regions of the heart. The characteristics of the action potential differ in atrial versus ventricular myocardium, as well as across the ventricular myocardial wall from endocardium, midmyocardium, to epicardium (see Fig. 1.2 ).

Atrioventricular Heterogeneity of the Action Potential

Compared with the atrium, ventricular myocytes maintain a slightly more hyperpolarized resting E _m (approximately −85 mV vs. −80 mV). In addition, the action potential duration is longer, the plateau phase reaches a more depolarized E _m (approximately +20 mV), and phase 3 repolarization curve is steeper in ventricular myocytes as compared with the atrial action potential (see Table 1.3 ).

The differences in action potential configuration between atria and ventricles are mainly related to differences in ionic current densities and ion channel expression (especially K ⁺ channels) between ventricular and atrial myocytes. Although I _Kr and I _Ks densities are similar in atrial and ventricular myocytes, I _Kur is detected only in human atria and not in the ventricles. In fact, I _Kur is the predominant delayed rectifier current responsible for human atrial repolarization, with only small contribution of I _Kr and I _Ks .

Furthermore, the density of I _to is twofold higher in the atria compared with ventricular myocytes. In addition, I _to subtypes (I _to,f and I _to,s ) are differentially expressed in the heart. I _to,f is the principal subtype expressed in human atrium. Conversely, I _to,s is larger and I _to,f is smaller in the ventricles compared with atrial tissue.

The markedly higher densities of I _to,f , together with the expression of I _Kur , accelerate the early phase of repolarization and lead to lower plateau potentials and shorter action potential durations in atrial as compared with ventricular cells.

I _K1 density is much higher in ventricular than in atrial myocytes, and this explains the steep repolarization phase in the ventricles (where the more abundant I _K1 plays a larger role in accelerating the terminal portion of repolarization) and the shallower phase in the atria. Furthermore, the higher I _K1 channel expression underlies the hyperpolarized resting E _m in ventricular myocytes, and prevents the ventricular cell from exhibiting pacemaker activity.

Several other K ⁺ channels are atrial selective and potentially contribute significantly to the atrial, but not ventricular, action potential. These include I _KACh , two-pore K ⁺ channels (K _2P ), and small-conductance Ca ²⁺ -activated K ⁺ (SK) channels.

Ventricular Regional Heterogeneity of the Action Potential

Action potential differences exist among the different layers across the ventricular wall, between the left and right ventricles, and from the apical region to the base.

Three distinct action potential waveforms have been distinguished from three predominant cell types contributing to the transmural heterogeneity of ventricular repolarization: the epicardial, midmyocardial, and endocardial cardiomyocytes. The most notable differences among these three layers are the prominent phase 1 notch and the spike and dome morphology of epicardial and midmyocardial action potentials compared with endocardium. The action potential duration of epicardial myocytes is shorter than that of endocardial myocytes. The duration of the action potential is longest in midmyocardial myocytes.

The distinct notch phase in the action potential waveform of epicardial myocytes has mainly been attributed to the regional differences in I _to density across the myocardial wall. In human ventricles, I _to densities are much higher in the epicardium and midmyocardium than in the endocardium. Furthermore, although both I _to,f and I _to,s are expressed in the ventricle, I _to,f is more prominent in the epicardium and midmyocardium than in the endocardium, whereas I _to,s is mainly present in the endocardium and Purkinje cells. A prominent I _to -mediated action potential notch in ventricular epicardium but not endocardium produces a transmural voltage gradient during early ventricular repolarization that registers as a J wave or J point elevation on the electrocardiogram (ECG).

Some experimental studies in wedge preparations strongly suggest the presence of a subpopulation of cells in the midmyocardium (referred to as the M cells) that exhibits distinct electrophysiological (EP) properties, although the presence of M cells has not been consistently confirmed by intact heart experiments. The putative M cells appear to have the longest action potential duration across the myocardial wall, largely attributed to their weaker I _Ks current but stronger late I _Na and Na ⁺ -Ca ²⁺ exchanger currents. Hence the M cells have been proposed to underlie the EP basis for transmural ventricular dispersion of repolarization and the T wave on the surface ECG, with the peak of the T wave (in wedge preparations) coinciding with the end of epicardial repolarization and the end of the T wave coinciding with the end of repolarization of the M cells. Although the role of M cells under physiological conditions remains controversial, these cells appear to have a significant role in arrhythmogenesis under a variety of pathological conditions, such as the long QT and Brugada syndromes, secondary to exaggeration of transmural repolarization gradients.

In addition to the transmural action potential gradient that exists across the three layers of myocardium in the left and right ventricles, the right ventricular (RV) action potential duration overall is shorter and the spike and dome morphology is more pronounced compared with that of the left ventricle (LV). These differences have been attributed to higher I _to densities in the right than in the left ventricular myocytes.

Evidence also suggests an apicobasal ventricular action potential heterogeneity. Action potential duration appears to be shorter in LV base compared with the apex. Larger I _to and I _Ks in apical compared with basal myocytes likely underlie those observations.

Phase 4: Diastolic Depolarization

The sinus and AV nodal cells lack the inward rectifier K ⁺ current (I _K1 ), which acts to stabilize the resting E _m in the normal working atrial and ventricular myocardium and Purkinje fibers. Sinus and AV nodal excitable cells exhibit a spontaneous, slow, and progressive decline in the E _m during diastole (spontaneous diastolic depolarization or phase 4 depolarization) that underlies normal automaticity and pacemaking function. Once this spontaneous depolarization reaches threshold (approximately −40 mV), a new action potential is generated.

The ionic mechanisms responsible for diastolic depolarization and normal pacemaker activity in the sinus node are still controversial. Originally, a major role was attributed to the decay of the delayed K ⁺ conductance (an outward current) activated during the preceding action potential (the I _K -decay theory). This model of pacemaker depolarization lost favor upon the discovery of the “funny” current (I _f ), sometimes referred to as the pacemaker current. I _f is a hyperpolarization-activated inward current that is carried largely by Na ⁺ and, to a lesser extent, K ⁺ ions. Once activated, I _f depolarizes the membrane to a level where the Ca ²⁺ current activates to initiate an action potential.

Other ionic currents gated by membrane depolarization (i.e., I _CaL and T-type Ca ²⁺ current [I _CaT ]), nongated and nonspecific background leak currents, and a current generated by the Na ⁺ -Ca ²⁺ exchanger have also been proposed to be involved in pacemaking. The “membrane clock” (also referred to as the “voltage clock” or “ion channel clock”) refers to the time- and voltage-dependent membrane ion channels underlying pacemaking activity, including the decay of the outward rectifier K ⁺ current and the activation of several inward currents (I _f , I _CaL , I _CaT , and I _Na ).

Newer evidence suggests that the sarcoplasmic reticulum, a major Ca ²⁺ store in sinus nodal cells, can function as a physiological clock (the so-called calcium clock) within the cardiac pacemaker cells and has a substantial impact on late diastolic depolarization.

There remains some degree of uncertainty about the relative role of I _f versus that of intracellular Ca ²⁺ cycling in controlling the normal pacemaker cell automaticity. Furthermore, the interactions between the membrane clock and the intracellular calcium clock and cellular mechanisms underlying this internal Ca ²⁺ clock are not completely elucidated. A further debate has arisen around their individual (or mutual) relevance in mediating the positive and negative chronotropic effects of neurotransmitters. Nevertheless, these interactions are of fundamental importance for understanding the integration of pacemaker mechanisms at the cellular level (see Chapter 3 for detailed discussion on the mechanisms of automaticity and pacemaker activity).

Phase 0: The Upstroke—Slow Depolarization

I _K1 is almost absent in sinus and AV nodal cells, thus allowing for relatively more depolarized resting diastolic potentials (−50 to −65 mV) compared with atrial and ventricular myocytes and facilitating diastolic depolarization mediated by the inward currents (e.g., I _f ). At the depolarized level of the maximum diastolic potential of pacemaker cells, most Na ⁺ channels are inactivated and unavailable for phase 0 depolarization. Consequently, action potential upstroke is mainly achieved by I _CaL .

L-type Ca ²⁺ channels activate on depolarization to potentials positive to −40 mV, and I _CaL peaks at 0 to +10 mV. The peak amplitude I _CaL is less than 10% that of I _Na , and the time required for activation and inactivation of I _CaL is approximately an order of magnitude slower than that for I _Na . As a consequence, the rate of depolarization in phase 0 (dV/dt) is much slower and the peak amplitude of the action potential is less than that in the working myocardial cells.