Pacemaker Channels and the Chronotropic Response in Health and Disease.

Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease-including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating ß-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate-sensitive hyperpolarization-activated current (If) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate-dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond.

E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation.

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel KV1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.

Characterization of Endo-Lysosomal Cation Channels Using Calcium Imaging.

Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca2+-permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca2+ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca2+ measurements, (2) peri-endo-lysosomal Ca2+ imaging using genetically encoded Ca2+ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca2+ imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca2+ imaging by directing Ca2+ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca2+ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca2+ imaging.

Paradigm shift: new concepts for HCN4 function in cardiac pacemaking.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the If current and are critically involved in controlling neuronal excitability and the autonomous rhythm of the heart. The HCN4 isoform is the main HCN channel subtype expressed in the sinoatrial node (SAN), a tissue composed of specialized pacemaker cells responsible for generating the intrinsic heartbeat. More than 40 years ago, the If current was first discovered in rabbit SAN tissue. Along with this discovery, a theory was proposed that cyclic adenosine monophosphate-dependent modulation of If mediates heart rate regulation by the autonomic nervous system-a process called chronotropic effect. However, up to the present day, this classical theory could not be reliably validated. Recently, new concepts emerged confirming that HCN4 channels indeed play an important role in heart rate regulation. However, the cellular mechanism by which HCN4 controls heart rate turned out to be completely different than originally postulated. Here, we review the latest findings regarding the physiological role of HCN4 in the SAN. We describe a newly discovered mechanism underlying heart rate regulation by HCN4 at the tissue and single cell levels, and we discuss these observations in the context of results from previously studied HCN4 mouse models.

In vivo and ex vivo electrophysiological study of the mouse heart to characterize the cardiac conduction system, including atrial and ventricular vulnerability.

The mouse is a common and cost-effective animal model for basic research, and the number of genetically engineered mouse models with cardiac phenotype is increasing. In vivo electrophysiological study in mice is similar to that performed in humans. It is indispensable for acquiring intracardiac electrocardiogram recordings and determining baseline cardiac cycle intervals. Furthermore, the use of programmed electrical stimulation enables determination of parameters such as sinoatrial conduction time, sinus node recovery time, atrioventricular-nodal conduction properties, Wenckebach periodicity, refractory periods and arrhythmia vulnerability. This protocol describes specific procedures for determining these parameters that were adapted from analogous human protocols for use in mice. We include details of ex vivo electrophysiological study, which provides detailed insights into intrinsic cardiac electrophysiology without external influences from humoral and neural factors. In addition, we describe a heart preparation with intact innervation by the vagus nerve that can be used as an ex vivo model for vagal control of the cardiac conduction system. Data acquisition for in vivo and ex vivo electrophysiological study takes ~1 h per mouse, depending on the number of stimulation protocols applied during the procedure. The technique yields highly reliable results and can be used for phenotyping of cardiac disease models, elucidating disease mechanisms and confirming functional improvements in gene therapy approaches as well as for drug and toxicity testing.

MicroRNA-365 regulates human cardiac action potential duration.

Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.

Speeding Up the Heart? Traditional and New Perspectives on HCN4 Function.

The sinoatrial node (SAN) is the primary pacemaker of the heart and is responsible for generating the intrinsic heartbeat. Within the SAN, spontaneously active pacemaker cells initiate the electrical activity that causes the contraction of all cardiomyocytes. The firing rate of pacemaker cells depends on the slow diastolic depolarization (SDD) and determines the intrinsic heart rate (HR). To adapt cardiac output to varying physical demands, HR is regulated by the autonomic nervous system (ANS). The sympathetic and parasympathetic branches of the ANS innervate the SAN and regulate the firing rate of pacemaker cells by accelerating or decelerating SDD-a process well-known as the chronotropic effect. Although this process is of fundamental physiological relevance, it is still incompletely understood how it is mediated at the subcellular level. Over the past 20 years, most of the work to resolve the underlying cellular mechanisms has made use of genetically engineered mouse models. In this review, we focus on the findings from these mouse studies regarding the cellular mechanisms involved in the generation and regulation of the heartbeat, with particular focus on the highly debated role of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 in mediating the chronotropic effect. By focusing on experimental data obtained in mice and humans, but not in other species, we outline how findings obtained in mice relate to human physiology and pathophysiology and provide specific information on how dysfunction or loss of HCN4 channels leads to human SAN disease.

Beyond pacemaking: HCN channels in sinoatrial node function.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key proteins involved in the initiation and regulation of the heartbeat. Pacemaker cells within the sinoatrial node generate the electrical impulse that underlies the contraction of all atrial and ventricular cardiomyocytes. To generate a stable heart rhythm, it is necessary that the spontaneous activity of pacemaker cells is synchronized. Entrainment processes in the sinoatrial node create synchrony and also mediate heart rate regulation. In the past years it has become clear that the role of HCN channels goes beyond just pacemaking and that the channels play pivotal roles in these entrainment processes that coordinate and balance sinoatrial node network activity. Here, we review the role of HCN channels in the central pacemaker process and highlight new aspects of the contribution of HCN channels to stabilizing the electrical activity of the sinoatrial node network, especially during heart rate regulation by the autonomic nervous system.

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice.

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR. Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.

Role of the C-terminus of SUR in the differential regulation of ß-cell and cardiac KATP channels by MgADP and metabolism.

KEY POINTS: ß-Cell KATP channels are partially open in the absence of metabolic substrates, whereas cardiac KATP channels are closed. Using cloned channels heterologously expressed in Xenopus oocytes we measured the effect of MgADP on the MgATP concentration-inhibition curve immediately after patch excision. MgADP caused a far more striking reduction in ATP inhibition of Kir6.2/SUR1 channels than Kir6.2/SUR2A channels; this effect declined rapidly after patch excision. Exchanging the final 42 amino acids of SUR was sufficient to switch the Mg-nucleotide regulation of Kir6.2/SUR1 and Kir6.2/SUR2A channels, and partially switch their sensitivity to metabolic inhibition. Deletion of the C-terminal 42 residues of SUR abolished MgADP activation of both Kir6.2/SUR1 and Kir6.2/SUR2A channels. We conclude that the different metabolic sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channels is at least partially due to their different regulation by Mg-nucleotides, which is determined by the final 42 amino acids. ABSTRACT: ATP-sensitive potassium (KATP ) channels couple the metabolic state of a cell to its electrical activity and play important physiological roles in many tissues. In contrast to ß-cell (Kir6.2/SUR1) channels, which open when extracellular glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. This is due to differences in the SUR subunit rather than cell metabolism. As ATP inhibition and MgADP activation are similar for both types of channels, we investigated channel inhibition by MgATP in the presence of 100 µm MgADP immediately after patch excision [when the channel open probability (PO ) is near maximal]. The results were strikingly different: 100 µm MgADP substantially reduced MgATP inhibition of Kir6.2/SUR1, but had no effect on MgATP inhibition of Kir6.2/SUR2A. Exchanging the final 42 residues of SUR2A with that of SUR1 switched the channel phenotype (and vice versa), and deleting this region abolished Mg-nucleotide activation. This suggests the C-terminal 42 residues are important for the ability of MgADP to influence ATP inhibition at Kir6.2. This region was also necessary, but not sufficient, for activation of the KATP channel in intact cells by metabolic inhibition (azide). We conclude that the ability of MgADP to impair ATP inhibition at Kir6.2 accounts, in part, for the differential metabolic sensitivities of ß-cell and cardiac KATP channels.