SUMMARYAugmentation of vagal tone increases ventricular fibrillation threshold (VFT) under nonischemic and ischemic conditions and protects against spontaneous ventricular fibrillation during experimental myocardial infarction. The purpose of this study was to identify the anatomic pathways responsible for this cholinergically-mediated enhanced electrical stability and to determine whether or not these pathways are present in human hearts. Rich cholinergic innervation of the sinoatrial node, atrioventricular node, and atrial myocardium was confirmed in both canine and human hearts. Although sparse cholinergic innervation was present in ventricular myocardium, numerous cholinergic nerve fibers were present in ventricular conduction tissue of both canine and human hearts. To determine whether these cholinergic fibers mediate the protective effects of vagal stimulation, cholinergic fibers to the ventricular conducting system were ablated in dogs. The ablation procedures used resulted in histologic absence of cholinergic nerves in the ventricular conducting system; innervation of the atrium, however, was histologically intact. In these animals vagal stimulation no longer increased VFT but still caused slowing of the sinus rate. The effect of vagal stimulation on VFT was shown to be independent of adrenergic innervation in a group of catecholamine depleted animals (6-hydroxydopamine). We conclude that 1) the enhanced ventricular electrical stability produced by vagal stimulation in dogs is mediated by cholinergic nerve fibers which supply the ventricular conduction system, and 2) this anatomic pathway is present in human hearts. were 1) to determine definitely whether the beneficial effects of vagal stimulation in dogs are in fact due to cholinergic innervation of the ventricle and 2) to determine whether cholinergic pathways exist in the human heart that could provide a basis for possible beneficial effects of increased vagal tone on the genesis of arrhythmias in man.Methods Human tissue was obtained at autopsy within 12 hours of death from five patients without known cardiac disease. Tissue from the sinoatrial (SA) node region, the atrioventricular (A-V) node, the His bundle, the left bundle branch, and the free wall of the left ventricle was examined. To determine whether postmortem time was a factor in the disappearance of acetylcholinesterase (AChE) activity in human tissue, fresh samples of atrial myocardium (atrial appendage removed at the time of cannulation for extracorporeal circulation) and ventricular myocardium (removed at time of myectomy for asymmetric septal hypertrophy) were obtained at the time of operation, frozen on dry ice, and analyzed immediately.In canine experiments tissue blocks of the SA node, A-V node with bundle of His and proximal left bundle branch, and left ventricular myocardium were placed on specimen

The anterior leaflet of the mitral valve of the dog contains blood vessels, nerve fibers, and cardiac muscle in addition to elastic fibers and collagen. When studied in a myograph, the electrically stimulated mitral valve actively developed tension and shortened. Active tension was found to be a function of initial length of the valve and was increased by norepinephrine and decreased by acetylcholine. The presence of neuronally releasable norepinephrine stores in the valve was indicated by responsiveness to tyramine. The negative inotropic response of the mitral valve to acetylcholine was consistent with an atrial origin of the tissue. Possible functional roles for mitral valve muscle and the potential significance of its neural control are discussed.

The localization of adrenergic and cholinergic nerves in normal and denervated cat hearts was studied histochemically. The norepinephrine content of atria and ventricles was chemically determined by a spectrofluorometric method. In hearts denervated 9 to 42 days, little or no norepinephrine was detected. Histochemically, many catecholamine-containing fibers were present in the atria and ventricles of normal cats, whereas in denervated cats there were none in one and very few in four. There were many cholinergic nerves in the atria and a small to moderate number in the ventricles. The left atria of denervated hearts showed a marked reduction in cholinergic nerve fibers. It is concluded that cardiac denervation by mediastinal neural ablation is often incomplete. When norepinephrine is not detectable by chemical analysis, individual nen'e fibers not sectioned can still be histochemically identified.ADDITIONAL KEY WORDS atrium acetylcholinesterase ganglia ventricles interatrial septum papillary muscle aorta catecholamines interventricular septum norepinephrine• Since extensive cardiac denervation results in loss of much of the capacity of the heart to store norepinephrine (NE) (1), surgical preparations have been used to clarify the role of NE stores in the intrinsic contractile state of the heart (2) and in the response of the heart to drugs that may depend on the release of local NE stores for their cardiac action (3, 4). Physiologic (5), pharmacologic and chemical (6) studies have been relied upon for assessment of completeness of cardiac denervation. A morphologic study of the effects of cardiac denervation on the distribution of cateFrom the

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The ability of normal and transplanted dog hearts to make, bind, store, and metabolize norepinephrine was studied. Transplanted hearts were used in order to assess the effects of adrenergic denervation. Normal hearts bound large quantities of administered C 14 -dopamine and synthesized considerable quantities of norepinephrine in both the atria and ventricles. Isolated perfused normal hearts steadily removed about 56% of infused dl -H 3 -norepinephrine; a binding mechanism was the major means of inactivation of the amine. Uptake of radioactive norepinephrine was greater in the ventricles than in the atria because they received more of the amine, and the turnover rate of the labeled amine was also highest in the ventricles. Subcellular fractionation of the bound amine demonstrated that it was localized in particles of microsomal size; radioautographic studies demonstrated the presence of H 3 -norepinephrine only in association with nerves. About 57% of the H 3 -norepinephrine released from normal hearts was metabolized, primarily by O-methylation. After autotransplantation, and degeneration of postganglionic sympathetic nerves, the average catecholamine content of the hearts fell to 1.2% of normal, C 14 -dopamine uptake and formation of norepinephrine were reduced to a few per cent of normal, and the uptake and retention of H 3 -norepinephrine was 6.1% of normal under the conditions used. Binding of norepinephrine to unknown sites in the transplanted tissue served temporarily as an important mechanism of inactivation of the amine, but this norepinephrine was very rapidly released and nearly 85% metabolized, again predominantly by O-methylation. The fine structure of the transplanted tissues was barely distinguishable from that of normal hearts by electron microscopy. In vitro assays of catechol-O-methyltransferase and monoamine oxidase showed little change in activity upon adrenergic denervation. These results demonstrate the importance of the adrenergic innervation of the heart for the normal synthesis, storage and inactivation of norepinephrine.

A technique for complete extrinsic denervation of the heart by the ablation of neural structures in the mediastinum is described. The completeness of denervation was verified by direct electrical stimulation of the main vagal and sympathetic trunks. Total deple tion of myocardial catecholamines followed the procedure in chronic survivors. Animals prepared in this manner are useful in the study of the neurologically isolated heart and simulate this aspect of the problem of cardiac transplantation.

In order to assess the role played by endogenous norepinephrine (NE) stores in the intrinsic contractile state of cardiac muscle, the right ventricular papillary muscles from normal cats and cats with cardiac NE depletion produced by chronic cardiac denervation or reserpine pretreatment were studied. The contractile state of NE-depleted ventricular myocardium was found to be normal. The resting and active length-tension curves, the forcevelocity relations, and the augmentation of isometric tension achieved by paired electrical stimulation and by increasing frequency of contraction were not depressed in either group of NE-depleted muscles. Similarly, no changes in the absolute refractory period and electrical excitability were observed. It is concluded that cardiac stores of NE are not fundamental for maintaining the intrinsic contractile state of the myocardium. Further, release of endogenous NE from cardiac muscle does not appear to play an essential role in the mediation of the positive inotropic effects of increasing frequency of contraction or of sustained postextrasystolic potentiation.