Transcriptional response of the heart to vagus nerve stimulation.

Heart failure is a major clinical problem, with treatments involving medication, devices, and emerging neuromodulation therapies such as vagus nerve stimulation (VNS). Considering the ongoing interest in using VNS to treat cardiovascular disease, it is important to understand the genetic and molecular changes developing in the heart in response to this form of autonomic neuromodulation. This experimental animal (rat) study investigated the immediate transcriptional response of the ventricular myocardium to selective stimulation of vagal efferent activity using an optogenetic approach. Vagal preganglionic neurons in the dorsal motor nucleus of the vagus nerve were genetically targeted to express light-sensitive chimeric channelrhodopsin variant ChIEF and stimulated using light. RNA sequencing of the left ventricular myocardium identified 294 differentially expressed genes (false discovery rate < 0.05). Qiagen Ingenuity Pathway Analysis (IPA) highlighted 118 canonical pathways that were significantly modulated by vagal activity, of which 14 had a z score of ≥2/≤-2, including EIF-2, IL-2, integrin, and NFAT-regulated cardiac hypertrophy. IPA revealed the effect of efferent vagus stimulation on protein synthesis, autophagy, fibrosis, autonomic signaling, inflammation, and hypertrophy. IPA further predicted that the identified differentially expressed genes were the targets of 50 upstream regulators, including transcription factors (e.g., MYC and NRF1) and microRNAs (e.g., miR-335-3p and miR-338-3p). These data demonstrate that the vagus nerve has a major impact on the myocardial expression of genes involved in the regulation of key biological pathways. The transcriptional response of the ventricular myocardium induced by stimulation of vagal efferents is consistent with the beneficial effect of maintained/increased vagal activity on the heart.NEW & NOTEWORTHY This experimental animal study investigated the immediate transcriptional response of the ventricular myocardium to selective stimulation of vagal efferent activity. Vagal stimulation induced significant transcriptional changes in the heart involving the pathways controlling autonomic signaling, inflammation, fibrosis, and hypertrophy. This study provides the first direct evidence that myocardial gene expression is modulated by the activity of the autonomic nervous system.

Hypoxia Promotes Atrial Tachyarrhythmias via Opening of ATP-Sensitive Potassium Channels.

BACKGROUND: Hypoxia-ischemia predisposes to atrial arrhythmia. Atrial ATP-sensitive potassium channel (KATP) modulation during hypoxia has not been explored. We investigated the effects of hypoxia on atrial electrophysiology in mice with global deletion of KATP pore-forming subunits. METHODS: Whole heart KATP RNA expression was probed. Whole-cell KATP current and action potentials were recorded in isolated wild-type (WT), Kir6.1 global knockout (6.1-gKO), and Kir6.2 global knockout (6.2-gKO) murine atrial myocytes. Langendorff-perfused hearts were assessed for atrial effective refractory period (ERP), conduction velocity, wavefront path length (WFPL), and arrhymogenicity under normoxia/hypoxia using a microelectrode array and programmed electrical stimulation. Heart histology was assessed. RESULTS: Expression patterns were essentially identical for all KATP subunit RNA across human heart, whereas in mouse, Kir6.1 and SUR2 (sulphonylurea receptor subunit) were higher in ventricle than atrium, and Kir6.2 and SUR1 were higher in atrium. Compared with WT, 6.2-gKO atrial myocytes had reduced tolbutamide-sensitive current and action potentials were more depolarized with slower upstroke and reduced peak amplitude. Action potential duration was prolonged in 6.1-gKO atrial myocytes, absent of changes in other ion channel gene expression or atrial myocyte hypertrophy. In Langendorff-perfused hearts, baseline atrial ERP was prolonged and conduction velocity reduced in both KATP knockout mice compared with WT, without histological fibrosis. Compared with baseline, hypoxia led to conduction velocity slowing, stable ERP, and WFPL shortening in WT and 6.1-gKO hearts, whereas WFPL was stable in 6.2-gKO hearts due to ERP prolongation with conduction velocity slowing. Tolbutamide reversed hypoxia-induced WFPL shortening in WT and 6.1-gKO hearts through ERP prolongation. Atrial tachyarrhythmias inducible with programmed electrical stimulation during hypoxia in WT and 6.1-gKO mice correlated with WFPL shortening. Spontaneous arrhythmia was not seen. CONCLUSIONS: KATP block/absence leads to cellular and tissue level atrial electrophysiological modification. Kir6.2 global knockout prevents hypoxia-induced atrial WFPL shortening and atrial arrhythmogenicity to programmed electrical stimulation. This mechanism could be explored translationally to treat ischemically driven atrial arrhythmia.

Immediate and sustained increases in the activity of vagal preganglionic neurons during exercise and after exercise training.

AIMS: The brain controls the heart by dynamic recruitment and withdrawal of cardiac parasympathetic (vagal) and sympathetic activity. Autonomic control is essential for the development of cardiovascular responses during exercise, however, the patterns of changes in the activity of the two autonomic limbs, and their functional interactions in orchestrating physiological responses during exercise, are not fully understood. The aim of this study was to characterize changes in vagal parasympathetic drive in response to exercise and exercise training by directly recording the electrical activity of vagal preganglionic neurons in experimental animals (rats). METHODS AND RESULTS: Single unit recordings were made using carbon-fibre microelectrodes from the populations of vagal preganglionic neurons of the nucleus ambiguus (NA) and the dorsal vagal motor nucleus of the brainstem. It was found that (i) vagal preganglionic neurons of the NA and the dorsal vagal motor nucleus are strongly activated during bouts of acute exercise, and (ii) exercise training markedly increases the resting activity of both populations of vagal preganglionic neurons and augments the excitatory responses of NA neurons during exercise. CONCLUSIONS: These data show that central vagal drive increases during exercise and provide the first direct neurophysiological evidence that exercise training increases vagal tone. The data argue against the notion of exercise-induced central vagal withdrawal during exercise. We propose that robust increases in the activity of vagal preganglionic neurons during bouts of exercise underlie activity-dependent plasticity, leading to higher resting vagal tone that confers multiple health benefits associated with regular exercise.

Omega-3 polyunsaturated fatty acid-induced vasodilation in mouse aorta and mesenteric arteries is not mediated by ATP-sensitive potassium channels.

There is strong evidence that the omega-3 polyunsaturated fatty acids (n-3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have cardioprotective effects. n-3 PUFAs cause vasodilation in hypertensive patients, in part controlled by increased membrane conductance to potassium. As KATP channels play a major role in vascular tone regulation and are involved in hypertension, we aimed to verify whether n-3 PUFA-mediated vasodilation involved the opening of KATP channels. We used a murine model in which the KATP channel pore subunit, Kir6.1, is deleted in vascular smooth muscle. The vasomotor response of preconstricted arteries to physiologically relevant concentrations of DHA and EPA was measured using wire myography, using the channel blocker PNU-37883A. The effect of n-3 PUFAs on potassium currents in wild-type native smooth muscle cells was investigated using whole-cell patch clamping. DHA and EPA induced vasodilation in mouse aorta and mesenteric arteries; relaxations in the aorta were sensitive to KATP blockade with PNU-37883A. Endothelium removal didn't affect relaxation to EPA and caused a small but significant inhibition of relaxation to DHA. In the knock-out model, relaxations to DHA and EPA were unaffected by channel knockdown but were still inhibited by PNU-37883A, indicating that the action of PNU-37883A on relaxation may not reflect inhibition of KATP. In native aortic smooth muscle cells DHA failed to activate KATP currents. We conclude that DHA and EPA cause vasodilation in mouse aorta and mesenteric arteries. Relaxations in blocker-treated arteries from knock-out mice demonstrate that KATP channels are not involved in the n-3 PUFA-induced relaxation.

Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models.

BACKGROUND: Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. METHODS: In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. RESULTS: Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. CONCLUSION: Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

The Pharmacology of ATP-Sensitive K+ Channels (KATP).

ATP-sensitive K+ channels (KATP) are inwardly-rectifying potassium channels, broadly expressed throughout the body. KATP is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels thus playing an important physiological role by coupling cellular metabolism with membrane excitability. The hetero-octameric channel complex is formed of 4 pore-forming inward rectifier Kir6.x subunits (Kir6.1 or Kir6.2) and 4 regulatory sulfonylurea receptor subunits (SUR1, SUR2A, or SUR2B). These subunits can associate in various tissue-specific combinations to form functional KATP channels with distinct electrophysiological and pharmacological properties. KATP channels play many important physiological roles and mutations in channel subunits can result in diseases such as disorders of insulin handling, cardiac arrhythmia, cardiomyopathy, and neurological abnormalities. The tissue-specific expression of KATP channel subunits coupled with their rich and diverse pharmacology makes KATP channels attractive therapeutic targets in the treatment of endocrine and cardiovascular diseases.

Vascular KATP channels protect from cardiac dysfunction and preserve cardiac metabolism during endotoxemia.

KATP channels in the vasculature composed of Kir6.1 regulate vascular tone and may contribute to the pathogenesis of endotoxemia. We used mice with cell-specific deletion of Kir6.1 in smooth muscle (smKO) and endothelium (eKO) to investigate this question. We found that smKO mice had a significant survival disadvantage compared with their littermate controls when treated with a sub-lethal dose of lipopolysaccharide (LPS). All cohorts of mice became hypotensive following bacterial LPS administration; however, mean arterial pressure in WT mice recovered to normal levels, whereas smKO struggled to overcome LPS-induced hypotension. In vivo and ex vivo investigations revealed pronounced cardiac dysfunction in LPS-treated smKO, but not in eKO mice. Similar results were observed in a cecal slurry injection model. Metabolomic profiling of hearts revealed significantly reduced levels of metabolites involved in redox/energetics, TCA cycle, lipid/fatty acid and amino acid metabolism. Vascular smooth muscle-localised KATP channels have a critical role in the response to systemic infection by normalising cardiac function and haemodynamics through metabolic homeostasis. KEY MESSAGES: • Mice lacking vascular KATP channels are more susceptible to death from infection. • Absence of smooth muscle KATP channels depresses cardiac function during infection. • Cardiac dysfunction is accompanied by profound changes in cellular metabolites. • Findings from this study suggest a protective role for vascular KATP channels in response to systemic infection.

Endothelial ATP-Sensitive Potassium Channel Protects Against the Development of Hypertension and Atherosclerosis.

In the endothelium, ATP-sensitive potassium (KATP) channels are thought to couple cellular metabolism with membrane excitability, calcium entry, and endothelial mediator release. We hypothesized that endothelial KATP channels have a broad role protecting against high blood pressure and atherosclerosis. Endothelial-specific Kir6.1 KO mice (eKO) and eKO mice on an apolipoprotein E KO background were generated (A-eKO) to investigate the role of KATP channels in the endothelium. Basal blood pressure was not elevated in eKO mice. However, when challenged with a high-salt diet and the eNOS inhibitor L-NAME, eKO mice became more hypertensive than their littermate controls. In aorta, NO release at least partly contributes to the endothelium-dependent vasorelaxation induced by pinacidil. In A-eKO mice atherosclerotic plaque density was significantly greater than in their littermate controls when challenged with a high-fat diet, particularly in the aortic arch region. Levels of endothelial dysfunction markers were higher in eKO compared with WT mice; however, these were not significant for A-eKO mice compared with their littermate controls. Furthermore, decreased vascular reactivity was observed in the mesenteric arteries of A-eKO mice, but not in aorta when on a high-fat diet. Our data support a role for endothelial Kir6.1-containing KATP channels in the endothelial protection against environmental stressors: the maintenance of blood pressure homeostasis in response to high salt and endothelial integrity when challenged with a high-fat diet.

Genetic variants in TRPM7 associated with unexplained stillbirth modify ion channel function.

Stillbirth is the loss of a fetus after 22 weeks of gestation, of which almost half go completely unexplained despite post-mortem. We recently sequenced 35 arrhythmia-associated genes from 70 unexplained stillbirth cases. Our hypothesis was that deleterious mutations in channelopathy genes may have a functional effect in utero that may be pro-arrhythmic in the developing fetus. We observed four heterozygous, nonsynonymous variants in transient receptor potential melastatin 7 (TRPM7), a ubiquitously expressed ion channel known to regulate cardiac development and repolarization in mice. We used site-directed mutagenesis and single-cell patch-clamp to analyze the functional effect of the four stillbirth mutants on TRPM7 ion channel function in heterologous cells. We also used cardiomyocytes derived from human pluripotent stem cells to model the contribution of TRPM7 to action potential morphology. Our results show that two TRPM7 variants, p.G179V and p.T860M, lead to a marked reduction in ion channel conductance. This observation was underpinned by a lack of measurable TRPM7 protein expression, which in the case of p.T860M was due to rapid proteasomal degradation. We also report that human hiPSC-derived cardiomyocytes possess measurable TRPM7 currents; however, siRNA knockdown did not directly affect action potential morphology. TRPM7 variants found in the unexplained stillbirth population adversely affect ion channel function and this may precipitate fatal arrhythmia in utero.

Acute Isolation of Cells from Murine Sino-atrial Node.

The cardiac conduction system allows the synchronized propagation of electrical activity through heart muscle. This is initiated by the spontaneous activity of the specialized pacemaker cells of the sino-atrial node (SAN). The SAN region underlies automaticity in mammals and therefore has a crucial role in the pathogenesis of cardiac disorders such as arrhythmia. Isolation of SAN tissue and SAN cells is critical to advance our understanding of SAN structure and function in health and disease. Initially, isolation of SAN tissue and SAN cells was carried out in the rabbit owing to its larger size and similar electrical properties to human. This protocol was optimized by Mangoni and Nargeot (2001) for use in mice to take advantage of advancements in transgenic models. Here, we provide a step-by-step guide to dissecting the SAN tissue and isolating pacemaker cardiomyocytes from mouse hearts using an enzyme digestion approach.

Whole-cell and Perforated Patch-clamp Recordings from Acutely-isolated Murine Sino-atrial Node Cells.

Cardiac pacemaker cells of the sino-atrial node are responsible for the initiation of the heart beat and express an array of ion channels. The patch-clamp technique is the gold standard method for investigating the function of ion channels expressed in electrically active cells. Conventional whole-cell and perforated patch-clamp techniques can be used to investigate ionic currents in the voltage-clamp mode and changes in membrane potential (e.g., action potential) in the current-clamp mode. Here, we provide details of protocols used to measure spontaneous and triggered action potentials and whole-cell funny current If (HCN4) in single cardiomyocytes isolated from the mouse sino-atrial node (SAN).

A critical role for the ATP-sensitive potassium channel subunit KIR6.1 in the control of cerebral blood flow.

KIR6.1 (KCNJ8) is a subunit of ATP sensitive potassium channel (KATP) that plays an important role in the control of peripheral vascular tone and is highly expressed in brain contractile cells (vascular smooth muscle cells and pericytes). This study determined the effect of global deletion of the KIR6.1 subunit on cerebral blood flow, neurovascular coupling and cerebral oxygenation in mice. In KIR6.1 deficient mice resting cerebral blood flow and brain parenchymal partial pressure of oxygen (PO2) were found to be markedly lower compared to that in their wildtype littermates. However, cortical blood oxygen level dependent responses triggered by visual stimuli were not affected in conditions of KIR6.1 deficiency. These data suggest that KATP channels containing KIR6.1 subunit are critically important for the maintenance of normal cerebral perfusion and parenchymal PO2 but play no significant role in the mechanisms underlying functional changes in brain blood flow.

Modulation of Cardiac Ventricular Excitability by GLP-1 (Glucagon-Like Peptide-1).

BACKGROUND: Glucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular outcomes in patients with type 2 diabetes mellitus. However, systemic actions of these agents cause sympathetic activation, which is generally considered to be detrimental in cardiovascular disease. Despite significant research interest in cardiovascular biology of GLP-1, the presence of GLP-1R in ventricular cardiomyocytes remains a controversial issue, and the effects of this peptide on the electrical properties of intact ventricular myocardium are unknown. We sought to determine the effects of GLP-1R agonist exendin-4 (Ex4) on ventricular action potential duration (APD) and susceptibility to ventricular arrhythmia in the rat heart in vivo and ex vivo. METHODS: Ventricular monophasic action potentials were recorded in anaesthetized (urethane) rats in vivo and isolated perfused rat hearts during sinus rhythm and ventricular pacing. RESULTS: In vivo, systemic administration of Ex4 (5 µg/kg intravenously) increased heart rate, and this effect was abolished by ß-adrenoceptor blockade. Despite causing sympathetic activation, Ex4 increased APD at 90% repolarization during ventricular pacing by 7% ( P=0.044; n=6) and reversed the effect of ß-adrenoceptor agonist dobutamine on APD at 90% repolarization. In isolated perfused hearts, Ex4 (3 nmol/L) increased APD at 90% repolarization by 14% ( P=0.015; n=6) with no effect on heart rate. Ex4 also reduced ventricular arrhythmia inducibility in conditions of ß-adrenoceptor stimulation with isoproterenol. Ex4 effects on APD and ventricular arrhythmia susceptibility were prevented in conditions of muscarinic receptor blockade or inhibition of nitric oxide synthase. CONCLUSIONS: These data demonstrate that GLP-1R activation effectively opposes the effects of ß-adrenoceptor stimulation on cardiac ventricular excitability and reduces ventricular arrhythmic potential. The effect of GLP-1R activation on the ventricular myocardium is indirect, mediated by acetylcholine and nitric oxide and, therefore, can be explained by stimulation of cardiac parasympathetic (vagal) neurons.

Potassium channels in the sinoatrial node and their role in heart rate control.

Potassium currents determine the resting membrane potential and govern repolarisation in cardiac myocytes. Here, we review the various currents in the sinoatrial node focussing on their molecular and cellular properties and their role in pacemaking and heart rate control. We also describe how our recent finding of a novel ATP-sensitive potassium channel population in these cells fits into this picture.

ATP-Sensitive Potassium Channels and Their Physiological and Pathophysiological Roles.

ATP sensitive potassium channels (KATP ) are so named because they open as cellular ATP levels fall. This leads to membrane hyperpolarization and thus links cellular metabolism to membrane excitability. They also respond to MgADP and are regulated by a number of cell signaling pathways. They have a rich and diverse pharmacology with a number of agents acting as specific inhibitors and activators. KATP channels are formed of pore-forming subunits, Kir6.1 and Kir6.2, and a large auxiliary subunit, the sulfonylurea receptor (SUR1, SUR2A, and SUR2B). The Kir6.0 subunits are a member of the inwardly rectifying family of potassium channels and the sulfonylurea receptor is part of the ATP-binding cassette family of proteins. Four SURs and four Kir6.x form an octameric channel complex and the association of a particular SUR with a specific Kir6.x subunit constitutes the KATP current in a particular tissue. A combination of mutagenesis work combined with structural studies has identified how these channels work as molecular machines. They have a variety of physiological roles including controlling the release of insulin from pancreatic ß cells and regulating blood vessel tone and blood pressure. Furthermore, mutations in the genes underlie human diseases such as congenital diabetes and hyperinsulinism. Additionally, opening of these channels is protective in a number of pathological conditions such as myocardial ischemia and stroke. © 2018 American Physiological Society. Compr Physiol 8:1463-1511, 2018.

Postmortem Genetic Testing for Cardiac Ion Channelopathies in Stillbirths.

BACKGROUND: Although stillbirth is a significant health problem worldwide, the definitive cause of death remains elusive in many cases, despite detailed autopsy. In this study of partly explained and unexplained stillbirths, we used next-generation sequencing to examine an extended panel of 35 candidate genes known to be associated with ion channel disorders and sudden cardiac death. METHODS AND RESULTS: We examined tissue from 242 stillbirths (≥22 weeks), including those where no definite cause of death could be confirmed after a full autopsy. We obtained high-quality DNA from 70 cases, which were then sequenced for a custom panel of 35 genes, 12 for inherited long- and short-QT syndrome genes (LQT1-LQT12 and SQT1-3), and 23 additional candidate genes derived from genome-wide association studies. We examined the functional significance of a selected variant by patch-clamp electrophysiological recording. No predicted damaging variants were identified in KCNQ1 (LQT1) or KCNH2 (LQT2). A rare putative pathogenic variant was found in KCNJ2(LQT7) in 1 case, and several novel variants of uncertain significance were observed. The KCNJ2 variant (p. R40Q), when assessed by whole-cell patch clamp, affected the function of the channel. There was no significant evidence of enrichment of rare predicted damaging variants within any of the candidate genes. CONCLUSIONS: Although a causative link is unclear, 1 putative pathogenic and variants of uncertain significance variant resulting in cardiac channelopathies was identified in some cases of otherwise unexplained stillbirth, and these variants may have a role in fetal demise. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01120886.

ATP-sensitive potassium channels in the sinoatrial node contribute to heart rate control and adaptation to hypoxia.

ATP-sensitive potassium channels (KATP) contribute to membrane currents in many tissues, are responsive to intracellular metabolism, and open as ATP falls and ADP rises. KATP channels are widely distributed in tissues and are prominently expressed in the heart. They have generally been observed in ventricular tissue, but they are also expressed in the atria and conduction tissues. In this study, we focused on the contribution and role of the inwardly rectifying KATP channel subunit, Kir6.1, in the sinoatrial node (SAN). To develop a murine, conduction-specific Kir6.1 KO model, we selectively deleted Kir6.1 in the conduction system in adult mice (cKO). Electrophysiological data in single SAN cells indicated that Kir6.1 underlies a KATP current in a significant proportion of cells and influences early repolarization during pacemaking, resulting in prolonged cycle length. Implanted telemetry probes to measure heart rate and electrocardiographic characteristics revealed that the cKO mice have a slow heart rate, with episodes of sinus arrest in some mice. The PR interval (time between the onset of the P wave to the beginning of QRS complex) was increased, suggesting effects on the atrioventricular node. Ex vivo studies of whole heart or dissected heart regions disclosed impaired adaptive responses of the SAN to hypoxia, and this may have had long-term pathological consequences in the cKO mice. In conclusion, Kir6.1-containing KATP channels in the SAN have a role in excitability, heart rate control, and the electrophysiological adaptation of the SAN to hypoxia.