Control Of Action Potential Duration Research Articles

Regulation of cardiac action potential duration by neurotransmitters and co-transmitters

The autonomic nervous system plays a critical role in regulating cardiac contractility, but it must do so while maintaining precise control of action potential duration (APD) in order to ensure orderly propagation of electrical impulses throughout the heart. This requires the orchestration of multiple ion channel responses, but exactly which ones are most important in sustaining this balancing act vary depending on the species. Many studies have addressed this question using the hearts of small animals, such as the mouse or rat, where the ion channels involved in regulating APD are very different from those found in human cardiac myocytes.

Role of an Intersubunit Ca2+ Bridge in Structure and Function of BK Channels

Large conductance Ca2+-activated K+ (BK) channels are found in a variety of tissues and serve a range of functions, including control of action potential duration and smooth muscle contractility. Ca2+-dependent gating of BK channels is controlled by conformational changes of the “gating ring,” formed by the C-terminal domains (CTD) of its four pore-forming subunits, each comprised of two regulators of conductance of K+ (RCK) domains, RCK1 and RCK2. Residues D895-D897 comprise a well-established high-affinity Ca2+ binding site within RCK2 known as the “Ca2+ bowl”, and the crystal structure of the Ca2+ bound gating ring suggests that Ca2+ coordination at this site may also involve formation of bridge to residue N449 in the RCK1 domain of the adjacent subunit. Here we implemented a combination electrophysiology, biochemistry, and X-ray crystallography to interrogate the role of the putative Ca2+ bridge in BK channel activation. In excised patch recordings from transfected HEK cells, N449A mutant BK channels displayed a +50 mV shift in V1/2 compared to wild-type, suggesting that the N449 side chain plays a role in gating. We then overexpressed the wild-type and mutant BK-CTD in sf9 cells and analyzed the Ca2+-dependence of tetramerization by size exclusion chromatography. The wild-type BK-CTD formed tetramers in Ca2+-containing solutions with a biphasic dose-response relation, defined by K1/2 values of 25 μM and 11.7 mM. The N449A mutation greatly reduced the high-affinity effect of Ca2+, whereas the low affinity effect of Ca2+ on tetramerization remained (K1/2 =12.6 mM). Preliminary X-ray crystallographic data suggests that the N449A mutation does not effect the overall structure of the CTD. Together, these experiments point to a functional role for an intersubunit Ca2+ bridge between N449 and the Ca2+ bowl in BK channel activation.

Real-time feedback based control of cardiac restitution using optical mapping.

Cardiac restitution is the shortening of the action potential duration with an increase in the heart rate. A shorter action potential duration enables a longer diastolic interval which ensures that the heart gets adequate time to refill with blood. At higher rates however, restitution becomes steep and thus, can lead to unstable electrical activity (alternans) in the heart, leading to fatal cardiac rhythms. It has been proposed that maintaining a shallow slope of cardiac restitution could have potentially anti-arrhythmic effects. Previous studies involved the control of action potential duration (APD) or diastolic interval (DI) in isolated tissue samples based on the feedback from single microelectrode recordings. This limited the spatial resolution of the feedback system. Here, we aimed to develop a real time feedback control system that enabled the detection of APDs from various single pixels based on optical mapping recordings. Stimuli were applied after a predefined fixed DI after detection of an APD. We validated our algorithm using optical mapping movies from an ex-vivo rabbit heart. Thus, we provide an optical mapping based approach for the control of cardiac restitution and a potential means to validate its anti-arrhythmic effects.

Antifibrillatory Agents and Potassium Channels in the Atria: Pore Block versus Channel Trafficking

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The preferred therapy for AF is sustained sinus rhythm control; however, the efficacy of currently used antiarrythmic drugs is limited by adverse side effects resulting from both a lack of ion channel selectivity and nonspecific ventricular activity. The role of the voltage-gated potassium channels in atrial myocyte repolarization and the subsequent control of action potential duration renders them attractive targets for antiarrhythmic drugs in the treatment of AF. Conventional antiarrhythmic drugs generally target the ion permeability of potassium channels. This review discusses the limitations of this traditional approach and introduces, as a novel paradigm for antiarrhythmic pharmacology, the decrease of ion channel cell surface density through the modulation of ion channel trafficking pathways.

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.

Autonomic Control of Cardiac Action Potentials

I(Ks), the slowly activating component of the delayed rectifier current, plays a major role in repolarization of the cardiac action potential (AP). Genetic mutations in the alpha- (KCNQ1) and beta- (KCNE1) subunits of I(Ks) underlie Long QT Syndrome type 1 and 5 (LQT-1 and LQT-5), respectively, and predispose carriers to the development of polymorphic ventricular arrhythmias and sudden cardiac death. beta-adrenergic stimulation increases I(Ks) and results in rate dependent AP shortening, a control system that can be disrupted by some mutations linked to LQT-1 and LQT-5. The mechanisms by which I(Ks) regulates action potential duration (APD) during beta-adrenergic stimulation at different heart rates are not known, nor are the consequences of mutation induced disruption of this regulation. Here we develop a complementary experimental and theoretical approach to address these questions. We reconstituted I(Ks) in CHO cells (ie, KCNQ1 coexpressed with KCNE1 and the adaptator protein Yotiao) and quantitatively examined the effects of beta-adrenergic stimulation on channel kinetics. We then developed theoretical models of I(Ks) in the absence and presence of beta-adrenergic stimulation. We simulated the effects of sympathetic stimulation on channel activation (speeding) and deactivation (slowing) kinetics on the whole cell action potential under different pacing conditions. The model suggests these kinetic effects are critically important in rate-dependent control of action potential duration. We also investigate the effects of two LQT-5 mutations that alter kinetics and impair sympathetic stimulation of I(Ks) and show the likely mechanism by which they lead to tachyarrhythmias and indicate a distinct role of I(KS) kinetics in this electrical dysfunction. The full text of this article is available online at http://circres.ahajournals.org.

Ca2+ regulation of a large conductance K+ channel in cultured rat cerebellar Purkinje neurons.

The calcium sensitivity of a large conductance voltage-sensitive potassium channel found in cultured rat cerebellar Purkinje neurons was studied in membrane patches from the somatic region of the cultured Purkinje neurons using single-channel recording techniques. The potassium channel had a conductance of approximately 94 picosiemens (pS) under physiologic ionic conditions and was active at depolarized membrane potentials. Activity due to this channel type was not observed when the saline at the internal surface of the membrane was calcium free. Low intracellular calcium concentration (10 nM) triggered channel activity at depolarized membrane potentials. Channel activity increased further with increasing intracellular calcium concentrations and was evident at more negative membrane potentials. The high sensitivity of this potassium channel type to intracellular calcium and its abundance in the Purkinje neuron membrane may reflect a prominent role in the control of action potential duration and interspike interval when the neurons are firing in a rapid, repetitive mode.

Regulation of Transient Outward K + Current in Human Atria Involves G Proteins

The role of G protein in regulating the transient outward K + current (I to) was examined by using isolated human atrial myocytes. When a nonhydrolyzable analogue of guanosine 5′-triphosphate (GTPγs) was introduced intracellularly, I to was irreversibly decreased and reached a new steady-state within 3 minutes. These results suggest that regulation of I to is similar to that of other K + current in the heart and involves guanine nucleotide-binding proteins. This has important implications with respect to autonomic control of action potential duration in the human heart.

Autonomic regulation of delayed rectifier K+ current in mammalian heart involves G proteins

The role of adrenergic and cholinergic stimulation in regulating the delayed outward K+ current (IK) was examined by using isolated guinea pig ventricular myocytes. Isoproterenol (ISO) stimulated IK in a reversible manner. Similar effects were seen by directly stimulating adenylate cyclase with forskolin (FSK). The responses to ISO and FSK were reversed by concurrent application of acetylcholine (ACh), but ACh alone did not affect IK. When a nonhydrolyzable analogue of guanosine 5'-triphosphate was introduced intracellularly, in the presence of extracellular ISO, IK was irreversibly stimulated. In cells pretreated with pertussis toxin the ACh response was blocked. These results suggest that autonomic regulation of IK is similar to that of the Ca2+ current and involves guanine nucleotide-binding proteins. This has important implications with respect to autonomic control of action potential duration and pacemaker activity in the heart.

Effects of moderate hypoxia on premature action potentials of rabbit papillary muscle.

Experiments were performed to study the effects of hypoxia on the characteristics of premature action potentials of rabbit papillary muscles. At normal resting potential, the duration of the premature action potential at the shortest coupling intervals was always greater than that of the control response. As the coupling interval was increased beyond 150 ms, the duration of the premature action potential regained control values. In cells depolarized to -70 mV by KCl, early lengthening of the premature response was attenuated. After 60 min of hypoxia, recovery of action potential duration at normal and reduced resting potentials was accelerated. The maximum rate of depolarization and its reactivation time constant were not affected by 60 min of hypoxia. It is suggested that intracellular free Ca is important in the control of action potential duration via the outward background potassium current.

Control of action potential duration by calcium ions in cardiac Purkinje fibers.

It is well known that cardiac action potentials are shortened by increasing the external calcium concentration (Cao). The shortening is puzzling since Ca ions are thought to carry inward current during the plateau. We therefore studied the effects of Cao on action potentials and membrane currents in short Purkinje fiber preparations. Two factors favor the earlier repolarization. First, calcium-rich solutions generally raise the plateau voltage; in turn, the higher plateau level accelerates time- and voltage-dependent current changes which trigger repolarization. Increases in plateau height imposed by depolarizing current consistently produced shortening of the action potential. The second factor in the action of Ca ions involves iK1, the background K current (inward rectifier). Raising Cao enhances iK1 and thus favors faster repolarization. The Ca-sensitive current change was identified as an increase in iK1 by virtue of its dependence on membrane potential and Ko. A possible third factor was considered and ruled out: unlike epinephrine, calcium-rich solutions do not enhance slow outward plateau current, ikappa. These results are surprising in showing that calcium ions and epinephrine act quite differently on repolarizing currents, even though they share similar effects on the height and duration of the action potential.