Laurine Marger

Bradycardia and Slowing of the Atrioventricular Conduction in Mice Lacking CaV3.1/α1G T-Type Calcium Channels

The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for Cav3.1/&agr;1G T-type calcium channels (cacna1g) abolishes T-type calcium current (ICa,T) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (ICa,L). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no ICa,T was detected in right atrium myocytes in both wild-type and Cav3.1−/− mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that Cav3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.

Control of heart rate by cAMP sensitivity of HCN channels

“Pacemaker” f-channels mediating the hyperpolarization-activated nonselective cation current If are directly regulated by cAMP. Accordingly, the activity of f-channels increases when cellular cAMP levels are elevated (e.g., during sympathetic stimulation) and decreases when they are reduced (e.g., during vagal stimulation). Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation by the autonomic nervous system, the exact contribution of the major If-mediating cardiac isoforms HCN2 and HCN4 to sinoatrial node (SAN) function remains highly controversial. To directly investigate the role of cAMP-dependent regulation of hyperpolarization activated cyclic nucleotide activated (HCN) channels in SAN activity, we generated mice with heart-specific and inducible expression of a human HCN4 mutation (573X) that abolishes the cAMP-dependent regulation of HCN channels. We found that hHCN4–573X expression causes elimination of the cAMP sensitivity of If and decreases the maximum firing rates of SAN pacemaker cells. In conscious mice, hHCN4–573X expression leads to a marked reduction in heart rate at rest and during exercise. Despite the complete loss of cAMP sensitivity of If, the relative extent of SAN cell frequency and heart rate regulation are preserved. Our data demonstrate that cAMP-mediated regulation of If determines basal and maximal heart rates but does not play an indispensable role in heart rate adaptation during physical activity. Our data also reveal the pathophysiologic mechanism of hHCN4–573X–linked SAN dysfunction in humans.

Functional roles of Ca v 1.3, Ca v 3.1 and HCN channels in automaticity of mouse atrioventricular cells Insights into the atrioventricular pacemaker mechanism

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Cav1.3, Cav3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Cav3.1 (Cav3.1-/-), Cav1.3 (Cav1.3-/-), channels or both (Cav1.3-/-/Cav3.1-/-). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Cav3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Cav3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Cav1.3 channels were required for spontaneous automaticity, because Cav1.3-/- and Cav1.3-/-/Cav3.1-/- AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Cav1.3 channels are required for automaticity, Cav3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.

The G-protein–gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.

Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of INa by tetrodotoxin (TTX) or of ICa,L by isradipine abolished AVNCs pacemaker activity. TTX-resistant (INar) and TTX-sensitive (INas) Na+ currents were recorded in mouse AVNCs, as well as T- (ICa,T) and L-type (ICa,L) Ca2+ currents ICa,L density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (If) and that of the fast component of the delayed rectifier current (IKr) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of If by 3 µM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for If in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K+ current (Ito). In contrast, no detectable slow component of the delayed rectifier current (IKs) could be recorded in AVNCs. The lower densities of If and ICa,L, as well as higher expression of IKr in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.

G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block

Significance The “sick sinus” syndrome (SSS) is characterized by abnormal formation and/or propagation of the cardiac impulse. SSS is responsible for about half of the total implantations of electronic pacemakers, which constitute the only currently available therapy for this disorder. We show that genetic ablation or pharmacological inhibition of the muscarinic-gated K+ channel (IKACh) prevents SSS and abolishes atrioventricular block in model mice without affecting the relative degree of heart rate regulation. We propose that “compensatory” genetic or pharmacological targeting of IKACh channels may constitute a new paradigm for restoring defects in the balance between inward and outward currents in pacemaker cells. Our study may thus open a new therapeutic perspective to manage dysfunction of formation and conduction of the cardiac impulse. Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies “sick sinus” syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca2+ channels (Cav1.3−/−) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K+ channel (IKACh) could rescue SSS and heart block in Cav1.3−/− mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3−/− mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3−/− mice or following selective inhibition of Cav1.3-mediated L-type Ca2+ (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.

0252: Bradycardia and arrhythmia caused by cardiac-specific suppression of the “funny” (If) current are rescued by Girk

The spontaneous activity of pacemaker myocytes controls the heartbeat. Automaticity is due to the presence of the slow diastolic depolarization phase, which leads the membrane potential from the end of the repolarization phase to the threshold of the following action potential. f- (HCN) channels underlying the hyperpolarization-activated “funny” current (If) are thought to play a key role in the generation and autonomic regulation of the diastolic depolarization and heart rate, but their role is still subject of controversy. Here we show that conditional and time-controlled expression of a dominant-negative non-conductive human HCN4 channel subunit (hHCN4-AYA) in the mouse heart leads to virtually complete silencing of If current (>95%) in the diastolic depolarization range in the sino-atrial node and in the conduction system. Heart-specific If silencing induced sino-atrial bradycardia, sinus pauses, severe dysfunction of atrioventricular conduction and ventricular arrhythmias. In comparison to control myocytes, the basal automaticity of hHCN4-AYA SAN myocytes was reduced by 76% and by 67% in myocytes of the conduction system. However, the relative maximal positive chronotropic effect of badrenergic activation on in vivo heart rate, isolated atria or pacemaking of individual SAN and conduction myocytes was preserved showing that If does not play an exclusive role in heart rate regulation. Unexpectedly, crossing hHCN4-AYA mutant mice with mice lacking the cardiac muscarinic G-protein-activated channel Girk4 (Girk4-/-) eliminated atrioventricular blocks and ventricular arrhythmias without preventing the autonomic regulation of heart rate. Our study shows, for the first time, the functional consequences of If silencing on heart rate and rhythm and indicates the possibility of managing cardiac disease related to HCN loss-of-function in humans by pharmacologic or genetic inhibition Girk4 channels.