RESUMO
[Figure: see text].
Assuntos
Átrios do Coração/inervação , Nó Sinoatrial/inervação , Neurônios Adrenérgicos/fisiologia , Animais , Nó Atrioventricular/inervação , Nó Atrioventricular/fisiologia , Sistema Nervoso Autônomo/anatomia & histologia , Sistema Nervoso Autônomo/fisiologia , Biomarcadores/análise , Neurônios Colinérgicos/fisiologia , Vasos Coronários/anatomia & histologia , Feminino , Gânglios Autônomos/anatomia & histologia , Humanos , Masculino , Ilustração Médica , Contração Miocárdica/fisiologia , Fenótipo , Nó Sinoatrial/fisiologia , Suínos , Porco Miniatura , Sinapses/fisiologia , Função Ventricular Esquerda/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/análiseRESUMO
Patients with type 2 diabetes mellitus (T2DM) have a greater risk of developing life-threatening cardiac arrhythmias. Because the underlying mechanisms and potential influence of diabetic autonomic neuropathy are not well understood, we aimed to assess the relevance of a dysregulation in cardiac autonomic tone. Ventricular arrhythmia susceptibility was increased in Langendorff-perfused hearts isolated from mice with T2DM (db/db). Membrane properties and synaptic transmission were similar at cardiac postganglionic parasympathetic neurons from diabetic and control mice; however, a greater asynchronous neurotransmitter release was present at sympathetic postganglionic neurons from the stellate ganglia of db/db mice. Western blot analysis showed a reduction of tyrosine hydroxylase (TH) from the ventricles of db/db mice, which was confirmed with confocal imaging as a heterogeneous loss of TH-immunoreactivity from the left ventricular wall but not the apex. In vivo stimulation of cardiac parasympathetic (vagus) or cardiac sympathetic (stellate ganglion) nerves induced similar changes in heart rate in control and db/db mice, and the kinetics of pacing-induced Ca2+ transients (recorded from isolated cardiomyocytes) were similar in control and db/db cells. Antagonism of cardiac muscarinic receptors did not affect the frequency or severity of arrhythmias in db/db mice, but sympathetic blockade with propranolol completely inhibited arrhythmogenicity. Collectively, these findings suggest that the increased ventricular arrhythmia susceptibility of type 2 diabetic mouse hearts is due to dysregulation of the sympathetic ventricular control.NEW & NOTEWORTHY Patients with type 2 diabetes mellitus have greater risk of suffering from sudden cardiac death. We found that the increased ventricular arrhythmia susceptibility in type 2 diabetic mouse hearts is due to cardiac sympathetic dysfunction. Sympathetic dysregulation is indicated by an increased asynchronous release at stellate ganglia, a heterogeneous loss of tyrosine hydroxylase from the ventricular wall but not apex, and inhibition of ventricular arrhythmias in db/db mice after ß-sympathetic blockade.
Assuntos
Arritmias Cardíacas/fisiopatologia , Cardiomiopatias Diabéticas/fisiopatologia , Fibras Simpáticas Pós-Ganglionares/fisiopatologia , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/etiologia , Sinalização do Cálcio , Cardiomiopatias Diabéticas/complicações , Frequência Cardíaca , Ventrículos do Coração/inervação , Ventrículos do Coração/fisiopatologia , Masculino , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Neurônios/metabolismo , Propranolol/farmacologia , Fibras Simpáticas Pós-Ganglionares/citologia , Fibras Simpáticas Pós-Ganglionares/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.
Assuntos
Endocitose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Coração/inervação , Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/agonistas , Quinases da Família src/antagonistas & inibidores , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática , Feminino , Cobaias , Humanos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neurônios/enzimologia , Fosforilação , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Quinases da Família src/metabolismoRESUMO
Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels (Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.
Assuntos
Adenilil Ciclases/metabolismo , Colforsina/administração & dosagem , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Neurônios/efeitos dos fármacos , Animais , Carbazóis/administração & dosagem , Clatrina/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Flavonoides/administração & dosagem , Cobaias , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miocárdio/patologia , Neurônios/metabolismo , Neurônios/patologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Pirróis/administração & dosagem , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismoRESUMO
Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides (Adcyap1) signaling at the selective PAC1 receptor (Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels (Ih) and another flowing through low-voltage-activated calcium channels (IT), which support the peptide-induced increase in excitability. Thus a PACAP- and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.
Assuntos
Potenciais de Ação/fisiologia , Coração/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Transdução de Sinais/fisiologia , Animais , Canais de Cálcio/metabolismo , Feminino , Cobaias , Masculino , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Canais de Potássio Shal/metabolismoRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Cav3.1, Cav3.2, and Cav3.3 T-type isoforms and R-type Cav2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization-induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents (Ih). The T-type calcium channel inhibitors, nickel (Ni(2+)) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2-4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni(2+), a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni(2+) also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni(2+)-sensitive current contributes to PACAP-induced modulation of neuronal excitability.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Níquel/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Feminino , Expressão Gênica , Cobaias , Masculino , Mibefradil/farmacologia , Microeletrodos , Miocárdio/citologia , Miocárdio/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Análise de Célula ÚnicaRESUMO
Parasympathetic dysfunction after chronic myocardial infarction (MI) is known to predispose ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation [VT/VF]). VT/VF after MI is more common in males than females. The mechanisms underlying the decreased vagal tone and the associated sex difference in the occurrence of VT/VF after MI remain elusive. In this study, using optogenetic approaches, we found that responses of glutamatergic vagal afferent neurons were impaired following chronic MI in male mice, leading to reduced reflex efferent parasympathetic function. Molecular analyses of vagal ganglia demonstrated reduced glutamate levels, accompanied by decreased mitochondrial function and impaired redox status in infarcted males versus sham animals. Interestingly, infarcted females demonstrated reduced vagal sensory impairment, associated with greater vagal ganglia glutamate levels and decreased vagal mitochondrial dysfunction and oxidative stress compared with infarcted males. Treatment with 17ß-estradiol mitigated this pathological remodeling and improved vagal neurotransmission in infarcted male mice. These data suggest that a decrease in efferent vagal tone following MI results from reduced glutamatergic afferent vagal signaling that may be due to impaired redox homeostasis in the vagal ganglia, which subsequently leads to pathological remodeling in a sex-dependent manner. Importantly, estrogen prevents pathological remodeling and improves parasympathetic function following MI.
Assuntos
Estradiol , Ácido Glutâmico , Infarto do Miocárdio , Transmissão Sináptica , Nervo Vago , Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Masculino , Feminino , Camundongos , Estradiol/farmacologia , Estradiol/metabolismo , Nervo Vago/efeitos dos fármacos , Nervo Vago/metabolismo , Nervo Vago/fisiopatologia , Transmissão Sináptica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Fatores Sexuais , Modelos Animais de Doenças , Estresse Oxidativo/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Intrinsic cardiac neurons (ICNs) play a crucial role in the proper functioning of the heart; yet a paucity of data pertaining to human ICNs exists. We took a multidisciplinary approach to complete a detailed cellular comparison of the structure and function of ICNs from mice, pigs, and humans. Immunohistochemistry of whole and sectioned ganglia, transmission electron microscopy, intracellular microelectrode recording and dye filling for quantitative morphometry were used to define the neurophysiology, histochemistry, and ultrastructure of these cells across species. The densely packed, smaller ICNs of mouse lacked dendrites, formed axosomatic connections, and had high synaptic efficacy constituting an obligatory synapse. At Pig ICNs, a convergence of subthreshold cholinergic inputs onto extensive dendritic arbors supported greater summation and integration of synaptic input. Human ICNs were tonically firing, with synaptic stimulation evoking large suprathreshold excitatory postsynaptic potentials like mouse, and subthreshold potentials like pig. Ultrastructural examination of synaptic terminals revealed conserved architecture, yet small clear vesicles (SCVs) were larger in pigs and humans. The presence and localization of ganglionic neuropeptides was distinct, with abundant VIP observed in human but not pig or mouse ganglia, and little SP or CGRP in pig ganglia. Action potential waveforms were similar, but human ICNs had larger after-hyperpolarizations. Intrinsic excitability differed; 93% of human cells were tonic, all pig neurons were phasic, and both phasic and tonic phenotypes were observed in mouse. In combination, this publicly accessible, multimodal atlas of ICNs from mice, pigs, and humans identifies similarities and differences in the evolution of ICNs.
RESUMO
Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, ß4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, ß4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, ß4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.
Assuntos
Gânglios Autônomos/metabolismo , Guanilato Quinases/metabolismo , Plexo Hipogástrico/metabolismo , Proteínas de Membrana/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , RNA Mensageiro/metabolismo , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Acetilcolina/metabolismo , Animais , Modelos Animais de Doenças , Regulação para Baixo , Galanina/genética , Galanina/metabolismo , Guanilato Quinases/genética , Plexo Hipogástrico/lesões , Masculino , Potenciais da Membrana , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Traumatismos dos Nervos Periféricos/genética , Receptores Nicotínicos/genética , Transmissão Sináptica , Fatores de Tempo , Técnicas de Cultura de Tecidos , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Bladder and erectile dysfunction are common urologic complications of diabetes and are associated with reduced parasympathetic autonomic control. To determine whether disruption of ganglionic neurotransmission contributes to the loss of function, we investigated synaptic transmission at parasympathetic, major pelvic ganglion (MPG) neurons in control and chronically (20 wk) diabetic mice. In contrast to what has been reported for sympathetic neurons, diabetes did not cause an interruption of synaptic transmission at parasympathetic MPG neurons from streptozotocin-treated C57BL/6J (STZ) or db/db mice. Cholinergically mediated excitatory postsynaptic potentials (EPSPs) were suprathreshold during 5-s trains of 5-, 10-, and 20-Hz stimuli. Asynchronous neurotransmitter release, observed as miniature EPSPs (mEPSPs) during and after stimulation, permitted quantitative assessment of postganglionic, cholinergic receptor sensitivity. mEPSP amplitude following tetanic stimulation (recorded at -60 mV) was reduced in STZ (4.95 ± 0.4 vs. 3.71 ± 0.3 mV, P = 0.03), but not db/db mice. The number of posttetanic mEPSPs was significantly greater in db/db mice at all frequencies tested. Assessment of basic electrophysiological properties revealed that parasympathetic MPG neurons from db/db mice had less negative membrane potentials, lower input resistances, and shorter afterhyperpolarizations relative to their control. MPG neurons from STZ had longer afterhyperpolarizations but were otherwise similar to controls. Membrane excitability, measured by the membrane responsiveness to long-duration (1 s), suprathreshold depolarizing pulses, was unchanged in either model. The present study indicates that, while parasympathetic neurotransmission at the MPG is intact in chronically diabetic mice, obese, type 2 diabetic animals exhibit an altered presynaptic regulation of neurotransmitter release.
Assuntos
Neurônios Colinérgicos/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Potenciais Pós-Sinápticos Excitadores , Gânglios Parassimpáticos/fisiopatologia , Pelve/inervação , Potenciais de Ação , Animais , Gânglios Parassimpáticos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Potenciais Pós-Sinápticos em MiniaturaRESUMO
The cell bodies of postganglionic sympathetic neurons innervating the heart primarily reside in the stellate ganglion (SG), alongside neurons innervating other organs and tissues. Whether cardiac-innervating stellate ganglionic neurons (SGNs) exhibit diversity and distinction from those innervating other tissues is not known. To identify and resolve the transcriptomic profiles of SGNs innervating the heart, we leveraged retrograde tracing techniques using adeno-associated virus (AAV) expressing fluorescent proteins (GFP or Td-tomato) with single cell RNA sequencing. We investigated electrophysiologic, morphologic, and physiologic roles for subsets of cardiac-specific neurons and found that three of five adrenergic SGN subtypes innervate the heart. These three subtypes stratify into two subpopulations; high (NA1a) and low (NA1b and NA1c) neuropeptide-Y (NPY) -expressing cells, exhibit distinct morphological, neurochemical, and electrophysiologic characteristics. In physiologic studies in transgenic mouse models modulating NPY signaling, we identified differential control of cardiac responses by these two subpopulations to high and low stress states. These findings provide novel insights into the unique properties of neurons responsible for cardiac sympathetic regulation, with implications for novel strategies to target specific neuronal subtypes for sympathetic blockade in cardiac disease.
Assuntos
Neurônios , Gânglio Estrelado , Camundongos , Animais , Neurônios/metabolismo , Gânglio Estrelado/metabolismo , Coração , Neuropeptídeo Y/metabolismo , Perfilação da Expressão GênicaRESUMO
Imbalances in the opposing actions of sympathetic and parasympathetic nerves controlling the heart enhance risk for arrhythmia and sudden cardiac death after myocardial infarction (MI). Plasticity in peripheral neuron function may underlie the observed changes in cardiomotor nerve activity. We studied vagal control of the heart in pigs after chronic infarction of the left ventricle. Stimulation of the cervical vagus nerve produced greater bradycardic responses 8-weeks after MI. Recordings of epicardial electrocardiograms demonstrate increased severity and duration of atrioventricular (AV) block in MI-pigs during 20 Hz vagal stimulation. Intracellular voltage recordings from isolated neurons of the inferior vena cava-inferior left atrium (IVC-ILA) ganglionated plexus, a cluster of epicardial neurons receiving innervation from the vagus known to regulate the AV node, were used to assess plasticity of membrane and synaptic physiology of intrinsic cardiac neurons (ICNs) after MI. Changes to both passive and active membrane properties were observed, including more negative resting membrane potentials and greater input resistances in MI-pig ICNs, concomitant with a depression of neuronal excitability. Immunoreactivity to pituitary adenylate cyclase-activating polypeptide (PACAP), a cardiotropic peptide known to modulate cardiac neuron excitability, was localized to perineuronal varicosities surrounding pig IVC-ILA neurons. Exogenous application of PACAP increased excitability of control but not MI-ICNs. Stimulation (20 Hz) of interganglionic nerves in the ex vivo whole-mount preparations elicited slow excitatory postsynaptic potentials (sEPSPs) which persisted in hexamethonium (500 µM), but were blocked by atropine (1 µM), indicating muscarinic receptor-mediated inhibition of M-current. Extracellular application of 1 mM BaCl2 to inhibit M-current increased neuronal excitability. The muscarine-sensitive sEPSPs were observed more frequently and were of larger amplitude in IVC-ILA neurons from MI animals. In conclusion, we suggest the increased probability of muscarinic sEPSPs play a role in the potentiation of the vagus nerve mediated-slowing of AV nodal conduction following chronic MI. We identify both a novel role of a muscarinic sensitive current in the regulation of synaptic strength at ICNs projecting to the AV node, and demonstrate changes to both intrinsic plasticity and synaptic plasticity of IVC-ILA neurons which may contribute to greater risk for heart block and sudden cardiac death after MI.
RESUMO
The cardiac autonomic nervous system (ANS) plays an integral role in normal cardiac physiology as well as in disease states that cause cardiac arrhythmias. The cardiac ANS, comprised of a complex neural hierarchy in a nested series of interacting feedback loops, regulates atrial electrophysiology and is itself susceptible to remodelling by atrial rhythm. In light of the challenges of treating atrial fibrillation (AF) with conventional pharmacologic and myoablative techniques, increasingly interest has begun to focus on targeting the cardiac neuraxis for AF. Strong evidence from animal models and clinical patients demonstrates that parasympathetic and sympathetic activity within this neuraxis may trigger AF, and the ANS may either induce atrial remodelling or undergo remodelling itself to serve as a substrate for AF. Multiple nexus points within the cardiac neuraxis are therapeutic targets, and neuroablative and neuromodulatory therapies for AF include ganglionated plexus ablation, epicardial botulinum toxin injection, vagal nerve (tragus) stimulation, renal denervation, stellate ganglion block/resection, baroreceptor activation therapy, and spinal cord stimulation. Pre-clinical and clinical studies on these modalities have had promising results and are reviewed here.
Assuntos
Antiarrítmicos/uso terapêutico , Fibrilação Atrial/terapia , Denervação Autônoma , Sistema Nervoso Autônomo/fisiopatologia , Terapia por Estimulação Elétrica , Coração/inervação , Neurotransmissores/uso terapêutico , Potenciais de Ação , Animais , Antiarrítmicos/efeitos adversos , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/fisiopatologia , Remodelamento Atrial , Denervação Autônoma/efeitos adversos , Terapia por Estimulação Elétrica/efeitos adversos , Frequência Cardíaca , Humanos , Neurotransmissores/efeitos adversos , Estimulação da Medula Espinal , Resultado do Tratamento , Estimulação do Nervo VagoRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) evokes tachycardia followed by a larger cholinergic bradycardia in isolated guinea pig hearts. We used the selective PAC1 receptor agonist maxadilan and vasoactive intestinal polypeptide (VIP) to test the hypothesis that PACAP27-evoked tachycardia and bradycardia are mediated by VPAC and PAC1 receptors, respectively. Chronotropic actions of these peptides were evaluated in isolated perfused hearts. Direct neuronal actions were determined by intracellular voltage recordings from cholinergic neurons in atrial ganglion whole mounts. Administration of 1 nmol of PACAP27 to isolated hearts evoked typical biphasic rate responses, whereas 1 nmol of maxadilan caused only a minor rate decrease. Desensitization with VIP eliminated the positive chronotropic effect of PACAP27 selectively. Local application of PACAP27 to cardiac neurons frequently evoked slow depolarization and caused prolonged increase of neuronal excitability. Maxadilan rarely affected membrane potential but consistently increased excitability. VIP had no effect on excitability and evoked depolarization in only a few neurons. Because maxadilan increased neuronal excitability but did not trigger action potentials as PACAP often does, we evaluated the interaction of maxadilan with substance P (SP) in isolated hearts. SP depolarizes cardiac neurons more consistently than PACAP, often triggers neuronal action potentials, and causes bradycardia but does not increase neuronal excitability. Maxadilan had a persistent effect to augment negative chronotropic responses to SP. These findings support our hypothesis that PACAP evokes tachycardia and bradycardia through VPAC and PAC1 receptors, respectively. They also suggest that maxadilan and PACAP27 differ in activating PAC1 receptors on cardiac neurons and/or stimulating downstream signaling mechanisms.
Assuntos
Sistema de Condução Cardíaco/fisiologia , Proteínas de Insetos/fisiologia , Neurônios/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/agonistas , Potenciais de Ação/fisiologia , Animais , Células Cultivadas , Feminino , Gânglios Autônomos/fisiologia , Cobaias , Masculino , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Proteínas e Peptídeos Salivares/fisiologiaRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated by PACAP-selective PAC(1) receptors. In dissociated guinea pig cardiac neurons, PACAP causes a positive shift of the voltage dependence of activation of the hyperpolarization-activated nonselective cation current (I(h)). This observation suggested that an enhancement of I(h) contributed to the increase in excitability in neurons within whole-mount cardiac ganglia preparations. To evaluate the role of I(h) in the PACAP-induced increase in excitability, we compared the increase in action potentials generated by 10 nM PACAP in control neurons and in neurons treated with ZD7288 (10 or 100 muM) or CsCl (2 or 2.5 mM), drugs known to inhibit I(h). In control cells exposed to PACAP, 1-s depolarizing current pulses elicited multiple action potential firing in 79% of the neurons. In ZD7288- or CsCl-containing solutions, the 10 nM PACAP-induced increase in excitability was markedly suppressed, with 7% and 21% of the neurons generating multiple action potentials, respectively. Prior results indicated that PACAP initiates depolarization by activating an inward current, which is separate from its enhancement of I(h). Here, we show that a PACAP-induced depolarization was comparable in control neurons and neurons bathed in a CsCl-containing solution, an observation indicating that CsCl did not interfere with activation of the PAC(1) receptor by PACAP. Additional experiments indicated that pretreatment with the putative M current (I(M)) inhibitor 1 mM BaCl(2), but not 10 microM XE991, initiated multiple firing in a majority of neurons, with resting potentials maintained at approximately -60 mV. Furthermore, in Ba(2+)-treated cells, 10 nM PACAP increased the number of action potentials generated. Our results indicate that PACAP enhancement of I(h), rather than inhibition of I(M) and other 1 mM Ba(2+)-sensitive K(+) currents, is a key ionic mechanism contributing to the peptide-induced increase in excitability for neurons within whole-mount cardiac ganglia preparations.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Coração/inervação , Neurônios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Potenciais de Ação , Animais , Compostos de Bário/farmacologia , Césio/farmacologia , Cloretos/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Estimulação Elétrica , Feminino , Cobaias , Técnicas In Vitro , Masculino , Neurônios/efeitos dos fármacos , Potássio/metabolismo , Canais de Potássio/metabolismo , Pirimidinas/farmacologia , Fatores de TempoRESUMO
We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.
Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/química , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Dependovirus/metabolismo , Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Genes Reporter , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Injeções Intravenosas , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Miocárdio/citologia , Miocárdio/metabolismo , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/metabolismo , Transgenes , Proteína Vermelha FluorescenteRESUMO
During the production process, the authors of this paper supplied revised versions of Figs. 2-5, Supplementary Tables 1-4, and Supplementary Videos 1-3, but because of publisher error, these revised items were not included in the final published version of the protocol. The figures have been updated in the PDF and HTML versions of the paper, and the revised Supplementary Information files are now available online. We note that the figures have been revised to improve their resolution only; the content of the figures and the data reflected remain unchanged. Also, print requirements impose some limits on figure resolution, but the authors have made very high-resolution versions of Figs. 2-5 available at as Source data.
RESUMO
Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.
Assuntos
Frequência Cardíaca/fisiologia , Neurônios Motores/fisiologia , Animais , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Eletrofisiologia , Feminino , Masculino , Camundongos , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/fisiologia , Nervo Vago/metabolismo , Nervo Vago/fisiologiaRESUMO
The pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated through activation of PAC1 receptors. The signaling cascades that couple activation of the PAC1 receptor to alterations in membrane ionic conductances responsible for the PACAP effect are unknown. Intracellular recordings were made from neurons in kinase inhibitor-treated cardiac ganglia preparations to determine which of the intracellular cascades activated by PAC1 receptor stimulation mediate the PACAP effect. In control cells, long depolarizing-current steps elicited one to three action potentials. In contrast, during the application of 10 nM PACAP, depolarizing-current pulses elicited multiple action potential firing (greater than or equal to five action potentials) in 79% of the neurons. Pretreatment with an adenylyl cyclase inhibitor, SQ 22536 (100 microM), suppressed the PACAP-induced increase in excitability, whereas the presence of U-73122 (10 microM), a potent phospholipase C (PLC) inhibitor, had no effect. Thus, the activation of adenylyl cyclase, but not PLC, was a critical step mediating the PACAP effect. Pretreatment with H-89 (1 microM), a protein kinase A inhibitor, and PD 98059 (50 microM), a MEK kinase inhibitor, also significantly blunted the PACAP-induced increase in excitability. Furthermore, treatment with forskolin (5 microM), an activator of adenylyl cyclase, or exposure to the cell-permeable cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (1 mM), partially recapitulated the effect of PACAP on excitability. We conclude that the activation of signaling cascades downstream of cAMP mediate the PACAP-induced increase in cardiac neuron excitability.
Assuntos
Miocárdio/citologia , Neurônios/efeitos dos fármacos , Fibras Parassimpáticas Pós-Ganglionares/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/metabolismo , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/metabolismo , Estrenos/metabolismo , Flavonoides/metabolismo , Cobaias , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Fibras Parassimpáticas Pós-Ganglionares/citologia , Fibras Parassimpáticas Pós-Ganglionares/fisiologia , Pirrolidinonas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) effects on intracellular calcium ([Ca2+]i) and excitability have been studied in adult guinea pig intracardiac neurons. PACAP increased excitability, but did not elicit Ca2+ release from intracellular stores. Exposure to a Ca2+-deficient solution did not deplete [Ca2+]i stores but did eliminate the PACAP-induced increase in excitability. We postulate that Ca2+ influx is required for the PACAP-induced increase in excitability.