Mechanism of Pacemaking in I_K1-Downregulated Myocytes

Abstract

Biological pacemakers were recently created by genetic suppression of inward rectifier potassium current, I_K1, in guinea pig ventricular cells. We simulated these cells by adjusting I_K1 conductance in the Luo-Rudy model of the guinea pig ventricular myocyte. After 81% I_K1 suppression, the simulated cell reached steady state with pacemaker period of 594 ms. Pacemaking current is carried by the Na⁺-Ca²⁺ exchanger, I_NaCa, which depends on the intracellular calcium concentration [Ca²⁺]_i. This [Ca²⁺ ]_i dependence suggests responsiveness (increase in rate) to β-adrenergic stimulation (βAS), as observed experimentally. Simulations of βAS demonstrate such responsiveness, which depends on I_NaCa expression. However, a simultaneous βAS-mediated increase in the slow delayed rectifier, I_Ks, limits βAS sensitivity.

Recent experiments demonstrate that cardiac biological pacemakers (BPs) can be created by genetic suppression of inward rectifier potassium current (I_K1) in guinea pig ventricular myocytes.¹ A potential advantage of this approach, as a therapeutic alternative to electronic pacemaking, is possible responsiveness to regulatory inputs, eg, β-adrenergic stimulation (βAS).

To advance this technology, it is important to understand the BP pacemaking mechanism. In the present study, we demonstrate that Na⁺-Ca²⁺ exchanger (I_NaCa) is the pacemaker current and explore BP responsiveness to βAS.

Materials and Methods

The Luo-Rudy (LRd) guinea pig ventricular myocyte model² was used to investigate BP pacemaking. Two I_K1 suppression levels (81% and 100%) and I_NaCa expression levels (control² and 100% increase) were simulated. βAS effects were simulated³ based on experimental observations. Abbreviations are defined in the Figure legend.

Selected processes during spontaneous initiation (first two APs) and steady-state oscillations in BP cells. I_K1 suppressed by 81%. Paced AP (same CL) is shown for reference (framed right column). A, Membrane potential, V_m. B, Sodium current, I_Na. C, Na⁺-Ca²⁺ exchange current, I_NaCa. D, Intracellular calcium, [Ca²⁺]_i. E, L-type calcium current, I_Ca,L. F, I_Ca,L gating: activation gate (d), voltage-dependent inactivation (f), and calcium-dependent inactivation (f_Ca). G, Fast delayed rectifier K⁺ current, I_Kr, and slow delayed rectifier K⁺ current, I_Ks. H, Inward rectifier potassium current, I_K1.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

Results

After 81% I_K1 suppression, we observe spontaneous action potentials (APs) that, after a 16-second transition, settle into a stable oscillatory pattern (Figure, panel A). Activity is initiated by slow depolarization generated by sodium and calcium leakage (background currents) and I_NaCa that extrudes calcium to maintain homeostasis at rest.² In unmodified cells (intact I_K1), these inward currents are balanced by outward I_K1 and resting V_m is stable.⁴ In the BP cell, when V_m reaches −60 mV, I_Na activates and increases depolarization rate. Peak I_Na is two orders of magnitude smaller than that of a paced AP² due to inactivation during the slow depolarization (Figure, panel B). I_Na and initial activation of I_Ca,L continue depolarizing V_m as I_NaCa decreases (higher V_m reduces its driving force). T-type calcium current (I_Ca,T) does not contribute because of inactivation during the slow depolarization (in ventricular myocytes these channels are unavailable at potentials above −65 mV).⁵ Once I_Ca,L is fully activated, it supports the subsequent upstroke and plateau of the AP (Figure, panel E). dV_m/dt_max corresponds to peak I_Ca,L and is much smaller than that of I_Na-dependent paced APs (15 V/s versus 388 V/s).² As I_Kr and I_Ks repolarize V_m, I_NaCa driving force increases, causing larger inward current (Figure, panel C). At −67.8 mV (maximum diastolic potential, MDP, Figure, panel A), outward I_Kr, I_Ks, and the suppressed I_K1 (Figure, panel H) do not balance inward I_NaCa and background currents. This imbalance causes slow phase-4 depolarization (φ4d) that leads to generation of a subsequent AP and continuous pacemaking.

Pacemaking mechanism remains similar during steady state. While removing residual Ca²⁺ from calcium-induced calcium release (CICR) of the previous AP, I_NaCa generates inward current that, in absence of balancing I_K1, depolarizes V_m to AP threshold. During sustained oscillations, there is higher [Ca²⁺]_i due to loading (Figure, panel D). Increased [Ca²⁺]_i affects rate by augmenting forward-mode I_NaCa (inward current) during φ4d (Figure, panel C), which accelerates depolarization (Figure, panel A). At the end of φ4d, as I_NaCa decreases, I_Na transiently increases and depolarizes V_m to threshold for I_Ca,L activation, which generates the AP upstroke. At the end of the AP (beginning of φ4d), I_Ks is still partially activated (Figure, panel G) and is important in determining MDP and rate of early φ4d. I_K1 expression also affects the rate of φ4d; 81% I_K1 suppression results in oscillations at cycle length (CL) of 594 ms (Figure, panel A), and complete I_K1 suppression leads to much faster rate (CL = 366 ms, not shown).

Changes in pacemaker rate under βAS are investigated by modifying AP currents according to their experimental response to β-agonists (see Reference ³ in the online data supplement). Enhanced I_up (Ca²⁺ uptake by the sarcoplasmic reticulum, SR), I_Ca,L, or I_NaK (the Na⁺-K⁺ pump) accelerates rate. Increased I_Ks or negative shift of I_Na inactivation decreases rate. The Table provides data for individual protocols and their combined effect. Increasing I_up (110%) in the control BP cell (81% I_K1 suppression) results in a 24% rate increase. This increase corresponds to SR loading and increased [Ca²⁺]_i that augments forward I_NaCa. Similarly, increasing I_Ca,L (300%) increases rate (11%) by augmenting [Ca²⁺]_i and I_NaCa. This increase occurs despite I_Ca,L-mediated increase in AP duration (APD), which decreases rate. Contrary to expectation, increasing I_NaK (an outward current) also increases rate. Augmenting I_NaK increases the sodium gradient, which increases I_Na and I_NaCa by increasing their driving force, accelerating φ4d. Because of I_Na participation during φ4d, negative shift of its inactivation decreases rate. This effect abolishes I_Na in BP cells, in contrast to cells with intact I_K1 where I_Na is only reduced by 14%. I_Ks increase by βAS decreases APD, which accelerates rate. However, it also hyperpolarizes V_m to a more negative MDP, which prolongs φ4d to the next AP threshold. The net effect is slowing of rate (13%), indicating that effects on MDP dominate APD changes. The overall effect of βAS on rate (only 4% increase) is very small, indicating low BP sensitivity to βAS. However, the simulated control BP cell is epicardial,² which expresses relatively low I_NaCa density (average midmyocardial is 50% higher).⁶ When I_NaCa density is increased 100% (estimated upper limit), βAS causes a 24% rate increase (Table). Note that all other model parameters were kept constant, to study the isolated effect of I_NaCa expression. We conclude that the βAS sensitivity of BP cells depends strongly on I_NaCa expression levels.

BP cells show increased [Ca²⁺]_i at steady state compared with paced cells at the same CL (1.22 and 0.94 μmol/L, respectively, Figure, panel D). [Ca²⁺]_i is further increased by βAS and by rate increases, which could cause calcium overload. We test this possibility by applying βAS to a rapidly paced cell (I_K1 fully suppressed; control I_NaCa) and comparing [Ca²⁺]_i to that of a slower BP cell (81% I_K1 suppression; control I_NaCa) without βAS, finding 85% increase in peak [Ca²⁺]_i (from 1.22 to 2.25 μmol/L). This result suggests that, from this perspective, increasing I_NaCa expression would be a preferred method of increasing βAS sensitivity, because of enhanced calcium removal capacity and protection against calcium overload.

The modulatory role of calcium in pacemaking suggests that I_Ca,L antagonists may overly suppress pacemaking in BP cells. We simulate this effect by 50% I_Ca,L block and observe 18.7% decrease in CL, in the range observed for similar block in sinoatrial node (SAN) cells.⁷

Discussion

In a recent study,¹ viral gene transfer was used to convert quiescent myocardial cells into pacemaker cells. With ~80% of I_K1 channels suppressed, these cells generated a rhythmic excitation at an intrinsic CL of 600 ms. The spontaneous APs were initiated by slow φ4d from MDP of −60.7 ± 2.1 mV.

Similar behavior is observed in the computer simulations; when I_K1 is suppressed by 81%, stable oscillatory behavior is attained. Slow φ4d from MDP of −67.3 mV sustains rhythmic excitation at a CL of 594 ms. Complete I_K1 suppression increases the rate to a CL of 366 ms, implying that altering I_K1 expression levels could be used to set intrinsic BP cell pacemaker rate.

The simulations identify I_NaCa as the regulated membrane process responsible for φ4d and pacemaking. Large I_K1 conductance determines resting V_m, which is close to K⁺ reversal potential. When I_K1 is suppressed, the steady-state balance between inward and outward currents shifts in the inward direction. The most important currents at this phase are involved with [Ca²⁺]_i homeostasis: calcium leakage that brings calcium into the cell and I_NaCa that extrudes calcium. These inward currents depolarize V_m to initiate a spontaneous AP. After this AP, residual [Ca²⁺]_i from CICR determines the magnitude of I_NaCa and consequently the rate of diastolic depolarization. At steady state, CICR (triggered by I_Ca,L) generates similar [Ca²⁺]_i transients every beat, resulting in a similar φ4d rate between APs and stable pacemaking at constant rate. This mechanism differs from spontaneous activity where spontaneous SR calcium release, an irregular process, underlies AP generation.⁸ This distinction is essential to the regular rhythm generated by BP cells, a prerequisite for any functional pacemaker.

An important determinant of I_NaCa is [Ca²⁺]_i, which enhances its forward mode. This property links BP rhythm to βAS and may explain why it accelerates with isoprenaline.¹ However, because of simultaneous βAS-mediated I_Ks increase and I_Na reduction, our simulations suggest that BP responsiveness to βAS is very limited (quantitative data are not provided in Reference ¹) and strongly depends on I_NaCa expression. For simulated high I_NaCa density, βAS increases the rate by 24%. For comparison, 115% increase is observed in isolated SAN cells,⁹ indicating much greater responsiveness to βAS.

The role of [Ca²⁺]_i in modulating CL suggests further experimental investigation. βAS can cause excessive Ca²⁺ SR loading and spontaneous release during φ4d, interrupting the regular rhythm. At fast rates with strong βAS, simulated peak [Ca²⁺]_i increases 85% compared with only 50% for SAN cells.⁹ Therefore, experiments examining a range of βAS are required to determine [Ca²⁺]_i overload levels and likelihood of arrhythmic APs. Ca²⁺ overload is also likely to induce long-term electrophysiological remodeling, which should be considered. Additionally, the model prediction that I_Ca,L antagonists will have similar effects in BP and SAN cells should be confirmed.

There are other mechanistic differences between BP and SAN cells. BP cells rely on a single dominant membrane process, I_NaCa, as the carrier of pacemaker current causing φ4d. Nodal cells rely on several depolarizing currents for φ4d and pacemaking. These include I_Ca,T, I_f (the hyperpolarization-activated current), I_NaCa, I_Ca,L, and possibly I_st (a sustained inward current, see review¹⁰). This multiplicity provides many control points for pacemaking regulation by various (neural and other) inputs. As suggested,¹⁰ this multiplicity underlies spatial heterogeneity within the SAN structure, which may be important for its function. In addition, I_K,Ach, an acetylcholine-sensitive current not detected in ventricular myocytes, provides vagal control of SAN rate. Finally, SAN ability to drive the heart depends on its architecture (gap-junction distribution; branching fibers), which facilitates optimization of its electrical loading by the surrounding atrial tissue. Therefore, it should be recognized that the engineering of single BP cells is only a first step toward creation of functional BP complexes.

Relationship Between Pacemaking Rate and Currents Affected by βAS

	CL, ms	Frequency, bpm	Percent Change
Control (81% IK1 suppression)	594	101	0
Iup increase (110%)	480	125	24
ICa,L increase (300%)	548	109	8
INaK pump increase (20%)	549	109	8
IKs increase (60%)	682	88	−13
INa shift of inactivation (33.4 mV)	636	94	−7
Total βAS (all of the above)	570	104	4
INaCa increase (100%)	567	106	5
βAS with INaCa increase (100%)	481	125	24

Results of modulating each individual process are shown followed by their cumulative effect (total βAS). Total βAS is also simulated with increased I_NaCa expression (bottom row), showing dramatic increase in sensitivity. Changes are relative to control.