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Shift of leading pacemaker site during reflex vagal stimulation and altered electrical source-to-sink balance.
KEY POINTS: Vagal reflexes slow heart rate and can change where the heartbeat originates within the sinoatrial node (SAN). The mechanisms responsible for this process - termed leading pacemaker (LP) shift - have not been investigated fully. We used optical mapping to measure the effects of baroreflex, chemoreflex and carbachol on pacemaker entrainment and electrical conduction across the SAN. All methods of stimulation triggered shifts in LP site from the central SAN to one or two caudal pacemaker regions. These shifts were associated with reduced current generation capacity centrally and increased electrical load caudally. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. However, our findings indicate the LP region is defined by both pacemaker rate and capacity to drive activation. Shifts in LP site provide an important homeostatic mechanism for rapid switches in heart rate. ABSTRACT: Reflex vagal activity causes abrupt heart rate slowing with concomitant caudal shifts of the leading pacemaker (LP) site within the sinoatrial node (SAN). However, neither the mechanisms responsible nor their dynamics have been investigated fully. Therefore, the objective of this study was to elucidate the mechanisms driving cholinergic LP shift. Optical maps of right atrial activation were acquired in a rat working heart-brainstem preparation during baroreflex and chemoreflex stimulation or with carbachol. All methods of stimulation triggered shifts in LP site from the central SAN to caudal pacemaker regions, which were positive for HCN4 and received uniform cholinergic innervation. During baroreflex onset, the capacity of the central region to drive activation declined with a decrease in amplitude and gradient of optical action potentials (OAPs) in the surrounding myocardium. Accompanying this decline, there was altered entrainment in the caudal SAN as shown by decreased conduction velocity, OAP amplitude, gradient and activation time. Atropine abolished these responses. Chemoreflex stimulation produced similar effects but central capacity to drive activation was preserved before the LP shift. In contrast, carbachol produced a prolonged period of reduced capacity to drive and altered entrainment. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. Our findings indicate that cholinergic LP shifts are also determined by altered electrical source-to-sink balance in the SAN. We conclude that the LP region is defined by both rate and capacity to drive atrial activation.
The autonomic nervous system and cardiac arrhythmias: current concepts and emerging therapies.
Research into cardiac autonomic control has received great interest in the past 20 years, and we are now at a critical juncture with regard to the clinical translation of the experimental findings. A rush to develop clinical interventions and implant a range of devices aimed at cardiac neuromodulation therapy has occurred. This interest has been driven by research, superimposed on commercial opportunities and perhaps the more relaxed regulatory framework governing implantable devices and interventions compared with that for pharmacotherapy. However, many of the results of the clinical trials into these therapies have been disappointing or conflicting. This lack of positive results is partly attributable to a scramble to find simple solutions for complex problems that we do not yet fully understand. Are there reasons to be optimistic? In this Review, we highlight areas in the field of cardiac autonomic control that we feel show the most promise for clinical translation and areas in which our current range of blunt tools need to be refined to bring about long-term success in treating arrhythmias.
FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons.
Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.
A governing equation for rotor and wavelet number in human clinical ventricular fibrillation: Implications for sudden cardiac death.
BACKGROUND: Ventricular fibrillation (VF) is characterized by multiple wavelets and rotors. No equation to predict the number of rotors and wavelets observed during fibrillation has been validated in human VF. OBJECTIVE: The purpose of this study was to test the hypothesis that a single equation derived from a Markov M/M/∞ birth-death process could predict the number of rotors and wavelets occurring in human clinical VF. METHODS: Epicardial induced VF (256-electrode) recordings obtained from patients undergoing cardiac surgery were studied (12 patients; 62 epochs). Rate constants for phase singularity (PS) (which occur at the pivot points of rotors) and wavefront (WF) formation and destruction were derived by fitting distributions to PS and WF interformation and lifetimes. These rate constants were combined in an M/M/∞ governing equation to predict the number of PS and WF in VF episodes. Observed distributions were compared to those predicted by the M/M/∞ equation. RESULTS: The M/M/∞ equation accurately predicted average PS and WF number and population distribution, demonstrated in all epochs. Self-terminating episodes of VF were distinguished from VF episodes requiring termination by a trend toward slower PS destruction, slower rates of PS formation, and a slower mixing rate of the VF process, indicated by larger values of the second largest eigenvalue modulus of the M/M/∞ birth-death matrix. The longest-lasting PS (associated with rotors) had shorter interactivation time intervals compared to shorter-lasting PS lasting <150 ms (∼1 PS rotation in human VF). CONCLUSION: The M/M/∞ equation explains the number of wavelets and rotors observed, supporting a paradigm of VF based on statistical fibrillatory dynamics.
Cardiovascular autonomic responses in patients with Parkinson disease to pedunculopontine deep brain stimulation.
PURPOSE: Dysautonomia can be a debilitating feature of Parkinson disease (PD). Pedunculopontine nucleus (PPN) stimulation may improve gait disorders in PD, and may also result in changes in autonomic performance. METHODS: To determine whether pedunculopontine nucleus stimulation improves cardiovascular responses to autonomic challenges of postural tilt and Valsalva manoeuver, eight patients with pedunculopontine nucleus deep brain stimulation were recruited to the study; two were excluded for technical reasons during testing. Participants underwent head up tilt and Valsalva manoeuver with stimulation turned ON and OFF. Continuous blood pressure and ECG waveforms were recorded during these tests. In a single patient, local field potential activity was recorded from the implanted electrode during tilt. RESULTS: The fall in systolic blood pressure after tilt was significantly smaller with stimulation ON (mean - 8.3% versus - 17.2%, p = 0.044). Valsalva ratio increased with stimulation from median 1.15 OFF to 1.20 ON (p = 0.028). Baroreflex sensitivity increased during Valsalva compared to rest with stimulation ON versus OFF (p = 0.028). The increase in baroreflex sensitivity correlated significantly with the mean depth of PPN stimulating electrode contacts. This accounted for 89% of its variance (r = 0.943, p = 0.005). CONCLUSION: PPN stimulation can modulate the cardiovascular system in patients with PD. In this study, it reduced the postural fall in systolic blood pressure during head-up tilt and improved the cardiovascular response during Valsalva, presumably by altering the neural control of baroreflex activation.
Anti-arrhythmic targeting of sympathetic stellate ganglion P2X3 receptors.
Sympathetic hyperactivity associated with many primary cardiovascular diseases is pro-arrhythmic and a key contributor to sudden cardiac death. Surgically removing the sympathetic stellate ganglion has demonstrated therapeutic potential in minimising cardiac arrhythmia, but there are off-target risks prompting the need for more site-specific non-invasive approaches. Here we hypothesised that P2X3 purinergic receptors (P2X3R) may be a therapeutic target and play a role in the excessive sympathetic drive to the heart in cardiovascular disease. We investigated the role of P2X3R in the sympathetic stellate ganglion of Wistar and spontaneously hypertensive rats, and human-induced pluripotent stem cell (hiPSC)-derived sympathetic neurons, using a combination of immunohistochemistry, qRT-PCR, and in vitro calcium fluorescence imaging. Responses to ATP and P2X3R inhibition were investigated in intact stellate ganglion-heart preparations in situ. We confirm expression of P2X3R in sympathetic neurons of rat stellate ganglia at mRNA and protein levels. P2X3R mRNA was upregulated in SHR stellate ganglia compared to Wistar. Activation of P2X3R increased [Ca2+]i in both isolated post-ganglionic sympathetic stellate neurons and hiPSC-derived sympathetic neurons, whereas microinjection of αβ-methylene ATP directly into right stellate ganglia induced tachycardia. Attenuation of these responses by a selective antagonist (AF353) indicated the tachycardia was P2X3R mediated. Inhibition of P2X3R rapidly restored the normal heart rhythm from sympathetic-induced cardiac arrhythmia. We show that stellate ganglion P2X3R contribute to sympathetically mediated cardiac chronotropic regulation. Our findings highlight the potential for inhibition of P2X3R located in the sympathetic stellate ganglion as a promising novel therapeutic for sympathetic nervous system-induced cardiac arrhythmias. KEY POINTS: Sympathetic nerve overactivity is pro-arrhythmic and a key contributor to left ventricular tachycardia and sudden cardiac death. P2X3 purinergic receptors are upregulated in stellate ganglia from prehypertensive rats. Stellate ganglion P2X3 receptors mediate increases in neuronal intracellular calcium and heart rate. Inhibiting P2X3 receptors rapidly and profoundly recovers normal heart rhythm from arrhythmia, highlighting the potential for inhibition of P2X3 receptors as a novel anti-arrhythmic.
Human-derived cardiac-neural microtissues reveal catecholaminergic polymorphic ventricular tachycardia is also a disease of the sympathetic neuron.
Sudden cardiac death in young individuals with structurally normal hearts represents a critical unresolved clinical challenge and typically occurs in patients with inherited arrhythmia syndromes due to cardiac channelopathies. Catecholaminergic polymorphic ventricular tachycardia (CPVT) can cause fatal arrhythmias triggered by adrenergic stimulation. Therapeutic interventions primarily target cardiac myocytes (CMs) despite robust clinical evidence demonstrating the life-saving efficacy of cardiac sympathetic denervation. To understand this therapeutic paradox, we developed human induced pluripotent stem cell (hiPSC)-derived CMs and sympathetic neurons (SNs) from healthy individuals and CPVT patients to investigate neurocardiac interactions using two- and three-dimensional microtissue models. We tested the hypothesis that CPVT is also a disease of the autonomic nervous system and observed that CPVT hiPSC-derived SNs had enhanced calcium transients, elevated cyclic adenosine monophosphate levels, and hyperexcitability, similar to diseased cardiomyocytes. Critically, co-culturing diseased neurons with healthy CMs induced arrhythmogenic activity, establishing that neuronal dysfunction directly triggers cardiac arrhythmias. Multielectrode array recordings, optical mapping and single-cell RNA sequencing revealed dysregulated neurotransmitter pathways and identified druggable molecular targets within SNs. These findings may explain why surgically interrupting sympathetic nerves helps CPVT patients and identify the nervous system as a therapeutic target. They further suggest that CPVT is more than a disease of the CM and should be re-defined as a neuro-cardiac disorder that paves the way for neuromodulation therapy. KEY POINTS: Sympathetic nerve overactivity is pro-arrhythmic and a key contributor to ventricular tachycardia and sudden cardiac death in patients with cardiac channelopathies. Catecholaminergic polymorphic ventricular tachycardia (CPVT) sympathetic neurons (SNs) exhibit enhanced calcium transients, elevated cAMP levels, and hyperexcitability that directly trigger arrhythmias in healthy cardiomyocytes. Novel human induced pluripotent stem cell-derived cardiac-neural microtissue models reveal CPVT is also a neurological disorder involving dysfunctional neurocardiac interactions. Single-cell RNA sequencing identifies dysregulated neurotransmitter pathways in SNs, providing new therapeutic targets for neuromodulation therapy.
Multimodal, device-based therapeutic targeting of the cardiovascular autonomic nervous system.
The miniaturization of implantable sensors and actuators, combined with advances in interactive modelling and high-resolution imaging, is propelling the use of medical devices for counteracting impaired neural control of the cardiovascular system. In this Review, we discuss the current effectiveness of this technology for modulating autonomic activity in numerous cardiovascular conditions, including high blood pressure, heart failure and cardiac arrhythmias. We advocate for smarter closed-loop bionic devices fitted with feedback from multiple sensors to allow adaptive, state-dependent control, and discuss how the adoption of artificial intelligence technology would facilitate auto-personalization to meet the needs of patients. We also describe how transcriptomics of autonomic circuits can guide device-based approaches. Finally, the use of stem cell therapies to target sympathetic circuits more precisely will help to optimize the therapeutic effects of autonomic modulation for the treatment of arrhythmia. For bioelectronic medicine to achieve clinical utility in neurocardiology, these innovations must demonstrate improved efficacy beyond that offered by contemporary interventions.
Uncovering Anti-Arrhythmic Potential of Stellate Ganglion Purinergic Receptors.
Cardiovascular disease affects over 30% of people worldwide and is one of the leading causes of death each year. Elevated sympathetic nerve activity is a common feature of cardiovascular disease, contributing to end-organ damage, morbidity and mortality. Recent findings indicate that short-circuiting sympathetic nerve overactivity by removal of the stellate ganglion can eradicate arrhythmias, emphasising the need for novel therapeutic targets to correct signalling non-invasively. We have revealed upregulation of the P2X3 purinergic receptor in stellate ganglia of Spontaneously Hypertensive (SHR) compared to Wistar rats (16 week-old), via RNASeq transcriptional profiling. We hypothesise that these purinergic receptors within cardiac stellate ganglia play a role in the excessive sympathetic drive to the heart in cardiovascular disease and can initiate cardiac arrhythmias. Thus, we have investigated the functional role of purinergic receptors in the stellate ganglion. Administration of ATP to acutely isolated post-ganglionic sympathetic neurones from the stellate ganglia of Wistar rats (4-6 week-old) evokes a significant increase (Median; 0.21) in [Ca2+ ]i as measured by Fura-2AM imaging (n=15, Wilcoxon matched-pairs test; p<0.0001). This ATP-induced calcium transient was inhibited by specific P2X3 receptor antagonism with either NF-10 or AF-130. Cardiac effects of stellate ganglion purinergic stimulation were investigated in the working heart-brainstem preparation of Wistar rats (4-6 week-old). Increasing doses of ATP (50-250 µg) delivered via microinjection (50-250 µL) directly to the right stellate ganglion produced either a tachycardia (18 ± 6 bpm, n=6) or bradycardia (-24 ± 12 bpm, n=5); these responses were typically found at distinct sites within the ganglion. Preliminary data from SHR suggest that higher doses of ATP (≥250µg) may be arrhythmogenic (n=3). P2X3 purinergic receptors are present in the sympathetic stellate ganglion and contribute to calcium ion flux and cardiac chronotropic regulation. Overexpression of P2X3 receptors is likely to contribute to excessive cardiac sympathetic activity and the development of hypertension and cardiac arrhythmias, making them a promising novel therapeutic target.
Macrophages Can Drive Sympathetic Excitability in the Early Stages of Hypertension.
Hypertension is a major health burden worldwide with many cases resistant to current treatments. Hyperactivity of the sympathetic nervous contributes to the etiology and progression of the disease, where emerging evidence suggests that inflammation may underpin the development of sympathetic dysautonomia. This study examined whether macrophages could drive the sympathetic phenotype in Spontaneously Hypertensive Rats (SHR) before animals develop high pressure. Stellate neurons from wild-type control Wistar rats and SHRs were co-cultured with blood leukocytes from their own strain, and also crossed cultured between strains. The calcium transient response to nicotinic stimulation was recorded using Fura-2 calcium imaging, where SHR neurons had a greater calcium transient compared with Wistar neurons. However, when co-cultured with leukocytes, Wistar neurons began to phenocopy the SHR sympathetic hyperactivity, while the SHR neurons themselves were unaltered. Resident leukocyte populations of the SHR and Wistar stellate ganglia were then compared using flow cytometry, where there was a shift in monocyte-macrophage subset proportions. While classical monocyte-macrophages were predominant in the Wistar, there were relatively more of the non-classical subset in the SHR, which have been implicated in pro-inflammatory roles in a number of diseases. When bone marrow-derived macrophages (BMDMs) were co-cultured with stellate neurons, they made Wistar neurons recapitulate the SHR nicotinic stimulated calcium transient. Wistar BMDMs however, had no effect on SHR neurons, even though SHR BMDMs increased SHR neuron responsiveness further above their hyper-responsive state. Taken together, these findings show that macrophages can be potent enhancers of sympathetic neuronal calcium responsiveness, and thus could conceivably play a role in peripheral sympathetic hyperactivity observed in the early stages of hypertension.
The cardiac sympathetic co-transmitter neuropeptide Y is pro-arrhythmic following ST-elevation myocardial infarction despite beta-blockade.
AIMS: ST-elevation myocardial infarction is associated with high levels of cardiac sympathetic drive and release of the co-transmitter neuropeptide Y (NPY). We hypothesized that despite beta-blockade, NPY promotes arrhythmogenesis via ventricular myocyte receptors. METHODS AND RESULTS: In 78 patients treated with primary percutaneous coronary intervention, sustained ventricular tachycardia (VT) or fibrillation (VF) occurred in 6 (7.7%) within 48 h. These patients had significantly (P
Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1.
AIMS: When activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear. METHODS AND RESULTS: NHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium prepulse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by low [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yl)ation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling. CONCLUSIONS: Physiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Na+-driven cellular Ca2+ loading. These responses are positively inotropic but also increase the likelihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive.
Healthy cardiac myocytes can decrease sympathetic hyperexcitability in the early stages of hypertension.
Sympathetic neurons are powerful drivers of cardiac excitability. In the early stages of hypertension, sympathetic hyperactivity is underpinned by down regulation of M current and increased activity of Cav2.2 that is associated with greater intracellular calcium transients and enhanced neurotransmission. Emerging evidence suggests that retrograde signaling from the myocyte itself can modulate synaptic plasticity. Here we tested the hypothesis that cross culturing healthy myocytes onto diseased stellate neurons could influence sympathetic excitability. We employed neuronal mono-cultures, co-cultures of neonatal ventricular myocytes and sympathetic stellate neurons, and mono-cultures of sympathetic neurons with media conditioned by myocytes from normal (Wistar) and pre-hypertensive (SHR) rats, which have heightened sympathetic responsiveness. Neuronal firing properties were measured by current-clamp as a proxy for neuronal excitability. SHR neurons had a maximum higher firing rate, and reduced rheobase compared to Wistar neurons. There was no difference in firing rate or other biophysical properties in Wistar neurons when they were co-cultured with healthy myocytes. However, the firing rate decreased, phenocopying the Wistar response when either healthy myocytes or media in which healthy myocytes were grown was cross-cultured with SHR neurons. This supports the idea of a paracrine signaling pathway from the healthy myocyte to the diseased neuron, which can act as a modulator of sympathetic excitability.
Electrophysiological and Proarrhythmic Effects of Hydroxychloroquine Challenge in Guinea-Pig Hearts.
Hydroxychloroquine (HCQ), clinically established in antimalarial and autoimmune therapy, recently raised cardiac arrhythmogenic concerns when used alone or with azithromycin (HCQ+AZM) in Covid-19. We report complementary, experimental, studies of its electrophysiological effects. In patch clamped HEK293 cells expressing human cardiac ion channels, HCQ inhibited IKr and IK1 at a therapeutic concentrations (IC50s: 10 ± 0.6 and 34 ± 5.0 μM). INa and ICaL showed higher IC50s; Ito and IKs were unaffected. AZM slightly inhibited INa, ICaL, IKs, and IKr, sparing IK1 and Ito. (HCQ+AZM) inhibited IKr and IK1 (IC50s: 7.7 ± 0.8 and 30.4 ± 3.0 μM), sparing INa, ICaL, and Ito. Molecular induced-fit docking modeling confirmed potential HCQ-hERG but weak AZM-hERG binding. Effects of μM-HCQ were studied in isolated perfused guinea-pig hearts by multielectrode, optical RH237 voltage, and Rhod-2 mapping. These revealed reversibly reduced left atrial and ventricular action potential (AP) conduction velocities increasing their heterogeneities, increased AP durations (APDs), and increased durations and dispersions of intracellular [Ca2+] transients, respectively. Hearts also became bradycardic with increased electrocardiographic PR and QRS durations. The (HCQ+AZM) combination accentuated these effects. Contrastingly, (HCQ+AZM) and not HCQ alone disrupted AP propagation, inducing alternans and torsadogenic-like episodes on voltage mapping during forced pacing. O'Hara-Rudy modeling showed that the observed IKr and IK1 effects explained the APD alterations and the consequently prolonged Ca2+ transients. The latter might then downregulate INa, reducing AP conduction velocity through recently reported INa downregulation by cytosolic [Ca2+] in a novel scheme for drug action. The findings may thus prompt future investigations of HCQ's cardiac safety under particular, chronic and acute, clinical situations.
FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons.
Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.