The role of the autonomic nervous system in the genesis of life-threatening arrhythmias has been the subject of intense investigation yet remains incomplete and fragmented. Sympathetic imbalance has been implicated as a trigger of ventricular arrhythmias in the long QT syndrome (LQTS) by enhancing spatial heterogeneities of i) action potential durations (APDs), ii) dispersion of repolarization (DOR) and iii) perhaps conduction. The project will address fundamental questions regarding the neuromodulation of cardiac function by autonomic activity, the effects of intra-cardiac reflex responses and their role in LQT-related arrhythmias. Rabbit hearts will be isolated with bilateral innervation of sympathetic and parasympathetic branches, will be perfused, stained with voltage and Ca 2+ - sensitive dyes to simultaneously map action potentials (APs) and intracellular Ca 2v(Cai) transients from 256 sites at high spatial and temporal resolution. The sympathetic system will be stimulated bilaterally with electrodes inserted in the vertebral column and the parasympathetic system with electrodes on the right and left vagus nerves. Innervated hearts will be perfused with inhibitors of IKs (HMR 1556), IKr (E4031) or INa inactivation kinetics (Anthopleurin A) (e.g. models of LQTS types 1-3) to elucidate the role of autonomic activity on Torsade de Pointes (TdP).
The specific aims are: 1) To test the hypothesis that autonomic activity to the heart modulates APDs, DOR and to determine the mechanisms underlying this neuromodulation by mapping simultaneously cardiac APs and Cai from 256 sites of innervated, Langendorff rabbit hearts. Sympathetic and parasympathetic modulation of heart rate, conduction of the specialized conduction system and ventricular myocardium, AP upstroke velocity, APDs, DOR, and Cai transients will be analyzed during various autonomic nerve stimulation paradigms. Stimulation nerve paradigms will be developed to obtain a spectrum of cardiac responses. Pharmacological interventions will be used to identify the receptors mediating the cardiac responses (131,132,cq-adrenergic receptors: AR; muscarinic cholinergic; peptidergic and puronergic) and the contribution of efferent and afferent fibers involved in this neuromodulation by blocking ganglionic transmission with hexamethonium. 2) To test the hypothesis that intra-cardiac reflexes via afferent and efferent neurons and ganglia in the heart muscle regulate electrical and contractile properties. We will apply a focal sensory stimulus (mechanical or chemical) at a site on the heart (i.e. the apex of the left ventricle) while recording changes in electrical and contractile parameters mediated by cardiac reflex responses in other regions of the heart. Pharmacological agents will then be per'fused to block specific neural pathways to identify the underlying neuronal mechanisms. 3) The synergistic effects of right and left sympathetic or right-left vagus nerves activation and the cross-interactions between the sympathetic and parasympathetic branches are central to our understanding of the neuromodulation of the heart. We will compare the changes in APs and Ca_ at a constant heart rate during i) bilateral versus unilateral (right or left) vagal stimulation; ii) vertebral column stimulation (bilateral sympathetic activation) and bilateral versus unilateral vagus stimulation; iii) vertebral column stimulation with right or left stellectomy. Sympathetic inputs to the heart are fractionated and emanate from different thoracic segments that target different regions of the heart. We will selectively stimulate a single sympathetic branch without activating the others inputs (up to 4) to identify the targets on the heart of each input. The convergence or divergence of sympathetic inputs to the heart may be important for normal cardiac function and enhance QT dispersion and TdP in the LQTS. 4) The role of 'autonomic imbalance' on the genesis of LQT-related arrhythmias will be determined in rabbit heart with LQTS type 1,2 or 3 by measuring changes in APDs, DOR, the propensity to fire early afterdepolarizations (EADs) and the initiation of TdP before and during various nerve stimulation paradigms (determined in aim 1). Stimulation paradigms that i) enhance DOR or ii) elicit a bradycardia followed by a tachycardia are more likely to increase the incidence of EADs and TdP.
The specific aims are: 1) To test the hypothesis that autonomic activity to the heart modulates APDs, DOR and to determine the mechanisms underlying this neuromodulation by mapping simultaneously cardiac APs and CaI from 256 sites of innervated, Langendorff rabbit hearts. Sympathetic and parasympathetic modulation of heart rate, conduction of the specialized conduction system and ventricular myocardium, AP upstroke velocity, APDs, DOR, and CaI transients will be analyzed during various autonomic nerve stimulation paradigms. Stimulation nerve paradigms will be developed to obtain a spectrum of cardiac responses. Pharmacological interventions will be used to identify the receptors mediating the cardiac responses (beta1, beta2, alpha1-adrenergic receptors: AR; muscarinic cholinergic; peptidergic and puronergic) and the contribution of efferent and afferent fibers involved in this neuromodulation by blocking ganglionic transmission with hexamethonium. 2) To test the hypothesis that intra-cardiac reflexes via afferent and efferent neurons and ganglia in the heart muscle regulate electrical and contractile properties. We will apply a focal sensory stimulus (mechanical or chemical) at a site on the heart (i.e. the apex of the left ventricle) while recording changes in electrical and contractile parameters mediated by cardiac reflex responses in other regions of the heart. Pharmacological agents will then be perfused to block specific neural pathways to identify the underlying neuronal mechanisms. 3) The synergistic effects of right and left sympathetic or right-left vagus nerves activation and the cross-interactions between the sympathetic and parasympathetic branches are central to our understanding of the neuromodulation of the heart. We will compare the changes in APs and Cai at a constant heart rate during i) bilateral versus unilateral vagus stimulation; ii) vertebral column stimulation (bilateral sympathetic activation) and bilateral versus unilateral vagus stimulation; iii) vertebral column stimulation with right or left stellectomy. Sympathetic inputs to the heart are fractionated and emanate from different thoracic segments that target different regions of the heart. We will selectively stimulate a single sympathetic branch without activating the others inputs (up to 4) to identify the targets on the heart of each input. The convergence or divergence of sympathetic inputs to the heart may be important for normal cardiac function and enhance QT dispersion and TdP in the LQTS. 4) The role of 'autonomic imbalance' on the genesis of LQT-related arrhythmias will be determined in rabbit heart with LQTS type 1, 2, or 3 by measuring changes in APDs, DOR, the propensity to fire early afterdepolarizations (EADs) and the initiation of TdP before and during various nerve stimulation paradigms (determined in aim 1). Stimulation paradigms that i) enhance DOR or ii) elicit a bradycardia followed by a tachycardia are more likely to increase the incidence of EADs or TdP.
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