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The mitral early to late diastolic flow velocity ratio (E/A ratio) is age-dependent. It has been considered that its age dependency reflects the age-related lengthening of left ventricular (LV) relaxation; however, the change in E/A ratio is far larger than that expected from those in LV relaxation. We hypothesized that an age-related reduction of the parasympathetic activity increases left atrial (LA) contractility, and that this accounts for the age-related change in E/A ratio. (1) Exercise stress test was performed in 61 normal subjects (age range, 8-80 years, mean, 40 years) to assess heart rate (HR) recovery because slowed HR recovery indicates lowered parasympathetic activity. There were good interrelations among age, E/A ratio, and HR recovery. Among those aged ≤30 years, the age no longer correlated with E/A ratio or HR recovery, but there was a significant correlation between HR recovery and E/A ratio (r = 0.44, p < 0.05). (2) Pulsed Doppler and two-dimensional speckle tracking echocardiography (2DSTE) were performed before and after administration of parasympathetic blockade (atropine) in ten young healthy subjects. LA booster pump function was assessed with LA emptying index calculated by 2DSTE. LA emptying index was calculated from ([LA volume before the atrial contraction - minimal LA volume]/LA volume before the atrial contraction) × 100. Atropine increased mitral A velocity (p < 0.001) and LA emptying index (p < 0.05) along with a decrease in E/A ratio (p < 0.001). Parasympathetic withdrawal enhances LA contraction and increases mitral A velocity, which likely cause a reciprocal decrease in mitral E velocity and E/A ratio. Thus, parasympathetic deactivation with aging should be closely involved in the age-related change in mitral E/A ratio.

Aim: The functional significance of the autonomic nerves in the anterior mitral valve leaflet (AML) is unknown. We tested the hypothesis that remote stimulation of the vagus nerve (VNS) reduces AML stiffness in the beating heart., Methods: Forty-eight radiopaque-markers were implanted into eleven ovine hearts to delineate left ventricular and mitral anatomy, including an AML array. The anesthetized animals were then taken to the catheterization laboratory and 4-D marker coordinates obtained from biplane videofluoroscopy before and after VNS. Circumferential (E(circ)) and radial (E(rad)) stiffness values for three separate AML regions, Annulus, Belly and Edge, were obtained from inverse finite element analysis of AML displacements in response to trans-leaflet pressure changes during isovolumic contraction (IVC) and isovolumic relaxation (IVR)., Results: VNS reduced heart rate: 94±9 vs. 82±10min(-1), (mean±SD, p<0.001). Circumferential AML stiffness was significantly reduced in all three regions during IVC and IVR (all p<0.05). Radial AML stiffness was reduced from control in the annular and belly regions at both IVC and IVR (P<0.05), while the reduction did not reach significance at the AML edge., Conclusion: These observations suggest that one potential functional role for the parasympathetic nerves in the AML is to alter leaflet stiffness. Neural control of the contractile tissue in the AML could be part of a central control system capable of altering valve stiffness to adapt to changing hemodynamic demands., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The presence of nerves in heart valves was first depicted decades ago and identified into subpopulations: sympathetic, parasympathetic. So valves are expected to be greatly affected by the autonomic nerves. However, few studies have focused on the regulation of heart valves by the autonomic nervous system., Objective: We sought to identify the role of the autonomic nervous system in the regulation of the mechanical properties of porcine mitral valve tissues., Methods: Mechanical properties of porcine mitral valve leaflets were evaluated in response to norepinephrine (NE) and acetylcholine (ACH), the main neurotransmitters. At the same time, phentolamine (Phent), metoprolol (Metop), atropine (Atrop) and endothelial denudation were added to the reactive system., Results: Under physiological conditions, the stiffness was not affected by endothelial denudation (p > 0.05). NE elevated the valve stiffness significantly per 10-fold increase in concentration (10(-6) vs 10(-7), p < 0.05; 10(-5) vs 10(-6), p < 0.05). This response was mitigated by Phent, Metop or endothelial denudation (p < 0.05), however, it was still increased significantly when compared to Controls (p < 0.05). ACH caused a decrease in stiffness accompanied by an increase in its concentration (significant change in stiffness per 10-fold increase in ACH concentration, 10(-6) vs Control, p < 0.05; 10(-5) vs 10(-6), p < 0.05), which were reversed by endothelial denudation and Atrop (p > 0.05 vs Control)., Conclusion: These findings highlight the role of the autonomic nervous system in the regulation of the mechanical properties of porcine mitral valve cusps, which underline the importance of autonomic nervous status for optimal valve function.

Objective: To map aspects of the innervation of the mitral valve complex and determine any association with the development or progression of myxomatous mitral valve disease (MMVD) in dogs., Sample Population: Septal mitral valve leaflets from 11 dogs aged 6 months to > 10 years., Procedures: Expression of protein gene product 9.5 (general neuronal marker), tyrosine hydroxylase (adrenergic innervation marker), vasoactive intestinal peptide (parasympathetic innervation marker), and calcitonin gene-related peptide (sensory innervation marker) was assessed by use of a standard immunohistochemical technique. Innervation was assessed qualitatively and semiquantitatively. Differences between valvular zones and between groups were analyzed statistically., Results: MMVD was present in leaflets of all dogs > or = 5 years of age. Innervation was confirmed in all leaflets but was markedly reduced in leaflets of dogs > 10 years of age. Innervation was most dense at the base of valves and mainly associated with the epimysial, perimysial, and endomysial layers of the muscle and blood vessels within the valve. Innervation was reduced within the middle zone of the valve and lacking at the free edge. Innervation was not identified at the tip of the leaflet, the free edge, or the chordae. Nerve fibers were mostly sympathetic, with the remainder being parasympathetic or sensory. Existence of MMVD did not alter the pattern or density of innervation., Conclusions and Clinical Relevance: Mitral valve leaflets in the study dogs were innervated, with most of the nerve fibers associated with the myocardium in the valve base. Development of MMVD appeared to precede the reduction of innervation associated with advancing age.

Introduction: Ablation of the mitral isthmus to achieve bidirectional conduction block is technically challenging, and incomplete block slows isthmus conduction and is often proarrhythmic. The presence of the blood pool in the coronary venous system may act as a heat-sink, thereby attenuating transmural RF lesion formation. This porcine study tested the hypothesis that elimination of this heat-sink effect by complete air occlusion of the coronary sinus (CS) would facilitate transmural endocardial ablation at the mitral isthmus., Methods: This study was performed in nine pigs using a 30 mm-long prototype linear CS balloon catheter able to occlude and displace the blood within the CS (the balloon was inflated with approximately 5 cc of air). Using a 3.5 mm irrigated catheter (35 W, 30 cc/min, 1 minute lesions), two sets of mitral isthmus ablation lines were placed per animal: one with the balloon deflated (CS open) and one inflated (CS Occluded). After ablation, gross pathological analysis of the linear lesions was performed., Results: A total of 17 ablation lines were placed: 7 with CS Occlusion, and 10 without occlusion. Despite similar biophysical characteristics of the individual lesions, lesion transmurality was consistently noted only when using the air-filled CS balloon., Conclusions: Temporary displacement of the venous blood pool using an air-filled CS balloon permits transmurality of mitral isthmus ablation; this may obviate the need for ablation within the CS to achieve bidirectional mitral isthmus conduction.

The concept that the mitral valve of the heart is a passive flap that opens and closes like a barn door has been emphasized for decades by medical and biology professors to their students. But experimental findings, which are outlined in this report, support the theory of an active valve. We hypothesize that the two leaflets of the mitral valve are actively contractile; that physical forces generated in the valve itself may stabilize and add precision to the sum of forces that regulate valve movement. This precision could be of critical significance both in the moments preceding, and during, valve opening and closing. Evidence supporting our active valve hypothesis includes the profuse innervation of motor and sensory nerves that are present in the mitral valves of all animals studied. In addition, multiple contractile cell types have been found in the mitral valve, including cardiac muscle cells, smooth muscle cells, and cardiac valvular interstitial cells. In vitro work in our laboratories using the rat mitral valve shows that not only are the valves capable of contraction and relaxation, but that the contractions and relaxations are nerve-mediated. We theorize that the rich innervation and contractile cells in the mitral valve work together to modulate fine-tuning of valve movements and tone, thereby ensuring the integrity of the valve seal. Other investigators have reported that the mitral valve demonstrates contractile activity and that denervation localized to the mitral valve affects valve competence. The evidence for an active mitral valve presented by these and other experimental studies warrant a reexamination of the validity of the passive valve concept. An accurate and full understanding of the precise movements of the valve leaflets and the mechanisms that regulate these movements is likely to provide the information needed to understand and develop treatments for many different cardiac valve problems, including mitral valve diseases such as prolapse and myxomatous degeneration. In view of the available experimental evidence, the concept that the mitral valve functions only as a passive structure is challenged by numerous anomalies. A reinterpretation of the concept of valve function that incorporates active as well as passive roles for the valve leaflets and other components of the valve apparatus would have significant implications both for the directions taken in research involving the cardiac valves and for the approaches to treatment.

Several previous studies have described the distribution of neuropeptide Y (NPY)-like and calcitonin gene related peptide (CGRP)-like immunoreactive nerve fibres in the atrioventricular valves of humans and various animals. It has been suggested that peptide-containing nerve fibres might have motor or sensory roles in valvular function. Although there is evidence that diabetic changes occur in the sympathetic (preganglionic and postganglionic), parasympathetic (vagal) and peptidergic nerves of rats, the changes of peptide-containing nerve fibres in the atrioventricular valves of the diabetic rat have not been studied. The distribution, relative density and staining intensity of NPY-like and CGRP-like immunoreactive nerve fibres in the mitral and tricuspid valves were studied in whole mount preparations using confocal microscopy with a computer-assisted image analysis system. Streptozotocin-induced diabetic and control rats were sacrificed at 12 and 24 months. The nerve staining intensity within the tricuspid valve was greater than the mitral valve in both control (P < 0.01) and diabetic (P < 0.001) rats. Nerve density in the anterior leaflet was greater than the posterior leaflet of the mitral valve. However, the anterior leaflet of the mitral and tricuspid valves showed a decreased number of nerve fibres, followed by drastic reduction in the staining intensities for both the peptides studied (P < 0.001) in the long-term diabetic rat. The decrease in the number of nerve fibres that follow the mechanical interruption of nerves raises the possibility that cycles of degeneration may occur. It is suggested that these peptide-containing nerve fibres in the atrioventricular valves may be involved in valvular dysfunction in the diabetic state., (Copyright 2000 Wiley-Liss, Inc.)

Previous reports demonstrated that mammalian atrioventricular (AV) valves possess a dense nerve plexus, consisting of nerve subpopulations which differ from each other in densities and patterns of distribution in the valves, and which may have sensory or motor roles in valve function. Although there is extensive evidence that age-related changes occur in autonomic nerves of animals and humans (Daly et al. J. Pharm. Exp. Ther., 1988;245(3):798-803; Ingall et al. Aust. NZ J. Med., 1990;20:570-577; Tumer et al. Exp. Gerontol., 1992;27:301-307), and that these changes contribute to changes in cardiac function (Klausner and Schwartz Clin. Geriat. Med., 1985;1(1):119-114), there is little information about age-related changes in heart valve innervation. In this study, we used acetylcholinesterase (AChE) histochemistry to localize and compare qualitative and quantitative changes in the innervation of the mitral valves in young adult and aged animals of three species. Young adult and aged guinea pigs, mice, and Wistar and Fischer 344 rats were anesthetized with Nembutal, the hearts removed, and the mitral valves dissected out and processed for AChE localization. Camera lucida drawings of the AChE-positive nerves in representative segments of valve cusps were made directly from slides; these drawings were digitized and subjected to computer-assisted image analysis to obtain quantitative information about nerve plexus density in the valves. All three animal species showed profuse AChE-positive innervation in the mitral valves of young adult animals, and decreases in the density of this innervation in aged animals. The most striking loss of innervation, compared to the young adult, occurred in the mitral valves of aged Fischer 344 rats, in which large regions of the valves appeared virtually devoid of nerves. Further studies are needed to investigate whether and to what extent age-related losses in heart valve innervation affect valvular structure and function.

The distribution of nerves immunoreactive to protein gene product 9.5 (PGP 9.5), tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP) and calcitonin gene related peptide (CGRP) antisera was investigated in the atrioventricular valves of the Sprague-Dawley rat and the Dunkin-Hartley guinea pig using confocal and epifluoresence microscopy. No major differences were noted between the innervation of the mitral and tricuspid valves in either species. For all antisera the staining was more extensive in the guinea pig valves. Two distinct nerve plexuses separated by a 'nearly nerve free' zone were identified in both species with each antiserum tested. This was most apparent on the anterior cusp of the mitral valve. The major nerve plexus extends from the atrioventricular ring through the basal, intermediate and distal zones of the valves towards the free edge of the valve cusp. These nerve bundles, arranged as primary, secondary and tertiary components, ramify to the free edge of the valve and extend to the attachment of the chordae. They do not contribute to the innervation of the chordae tendineae. The second, minor chordal plexus, runs from the papillary muscles through the chordae tendineae and passes parallel to the free edge of the cusp. The nerves of this minor plexus are interchordal, branching to terminate mainly in the distal zone, free edge of the valve cusp and adjacent chordae tendineae. Some interchordal nerve fibres loop from a papillary muscle up through a chorda, along the free edge and pass down an adjacent chorda into another papillary muscle. The nerve fibres of the major and minor plexuses intermingle although no evidence was found for interconnectivity between them. In the distal zone between the major plexus which extends from the base of the valve and the minor chordal plexus there is a zone completely free of nerves staining with antisera to TH and NPY. Occasional nerves which stained positive for PGP 9.5, SP and CGRP immunoreactivities crossed this 'nearly nerve free zone' passing either from the chordal/free edge nerves to the intermediate and basal zones or vice versa. An additional small nerve plexus which displayed immunoreactivity to CGRP antiserum extended from the atrioventricular ring into the basal zone of the valve cusp. Not all chordae tendineae displayed immunoreactive nerve fibres. It is concluded that the innervation patterns of the sensory and sympathetic neurotransmitters and neuropeptides examined in the atrioventricular valves of the rat and guinea pig are ubiquitous in nature. The complexity of the terminal innervation network of the mammalian atrioventricular valves and chordae tendineae may contribute to the complex functioning of these valves in the cardiac cycle.

Using confocal fluorescence microscopy we studied, in whole mounts of heart mitral valves of young adult and aged Fischer 344 rats, the distribution of nerves containing the catecholamine marker tyrosine hydroxylase (TH) or the synthetic enzyme marker for nitric oxide, nitric oxide synthase (NOS). TH-IR was localized in two separate nerve plexuses which do not intermingle. The 'major' plexus arose from the annulus region, traversed the basal zone of the valve, and ramified in the intermediate zone to form a dense network of fine fibers. The 'minor' plexus was restricted to the distal zone and originated from bundles that ascended the chordae tendineae to enter the valve cusp. A concentric zone located between the major and minor plexuses was devoid of TH-IR nerve fibers. Both plexuses demonstrated (i) nerves that contained numerous varicosities along the length of each fiber, (ii) many terminal axons and (iii) different shaped terminal axon endings. With age, the density of TH-IR innervation in the mitral valve was markedly reduced; and nerve fibers of the minor plexus were limited to the chordae tendinae, without extending into the valve cusp itself. NOS-IR fibers in the mitral valve formed a loose network that extended from the annulus to more than halfway down the cusp. The varicose beads of the terminal NOS-IR axons appeared to become progressively smaller and less intensely fluorescent until they disappeared at the terminal endings, which showed no specializations. No NOS-IR fibers were observed in the distal zone of the valve leaflet or in the chordae. In the aged mitral valve, the density of NOS-IR nerves was decreased, as compared with NOS-IR innervation in the young adult valve. The existence of TH and NOS as well as other signal molecule markers in heart valve nerves and the disparate patterns of their distribution and localization provide evidence supporting the theory that heart valve nerves form a complex reflexogenic control system in the mitral heart valve. In summary, two distinct neural architectures are described for TH-IR and NOS-IR valve nerves, respectively. The former are believed to be axons dedicated to sympathetic motor functions. The NOS-IR valve nerves may have sensory and/or postganglionic parasympathetic motor functions. An implication of these findings is that different, but perhaps related, valve functions may be mediated by separate, dedicated circuits.

The distribution of vasoactive intestinal polypeptide-, neuropeptide Y-, and calcitonin gene-related peptide-immunoreactive nerve fibres was investigated in the atrioventricular valves of the rat. These nerve fibres were visualized by immunostaining of whole-mount preparations by the avidin-biotin-peroxidase complex method. Vasoactive intestinal polypeptide-immunoreactive nerve fibres were observed mainly in the anterior cusp of the mitral valve and, to a lesser extent, in the medial cusp of the tricuspid valve. Numerous neuropeptide Y-immunoreactive nerve fibres were found covering all of the cusps. Both types of peptidergic nerve fibre formed dense networks that consisted of interlacing and anastomosing nerve fibres. Calcitonin gene-related peptide-immunoreactive nerve fibres were seen in every cusp, but did not form a fine network. These results provide detailed anatomical information for evaluation of the possible roles of each type of peptide-containing nerve fibre in the function of atrioventricular valves.

We evaluated the distribution of the nerves in valve tissue of humans to clarify the relationship between mitral valve prolapse and autonomic nerve dysfunction. We studied 15 autopsy specimens of normal mitral valve, 10 prolapsed mitral valves, five each of normal tricuspid, aortic, and pulmonary valves, and three prolapsed mitral valves obtained at cardiac surgery. Immunohistochemical studies utilized the avidinbiotin peroxidase complex (ABC) method and several nerve-related antigens: 1) S-100 protein, glial fibrillary acidic protein (GFAP), and neurofilament protein (NFP) as markers of glial and Schwann cells of the nervous system; 2) choline acetyltransferase (ChAT) to identify cholinergic nerve endings; 3) neuropeptide Y (NPY), a neuropeptide that is distributed in accordance with sympathetic nerves; and 4) calcitonin gene-related peptide (CGRP), a neuropeptide that is distributed in accordance with afferent nerves. Distribution of adrenergic nerve fibers was also examined by fluorescence method. Morphology of nerve endings of the normal mitral valve was studied by electron microscopy. In normal valves, distributions of S-100 protein, GFAP, and NFP immunoreactivities were clearly visible along the subendocardial site on the coaptation aspect of the base-to-body portion of each valve, regardless of the kind of valve. In contrast, there was only a scanty distribution of these reactivities on the physiologic coaptation area of the tip. In prolapsed mitral valves, there was no distribution of S-100-positive protein or other nerve-related antigens in areas of the valve with myxomatous degeneration. Distribution of CGRP, ChAT, and NPY immunoreactivities, and adrenergic fluorescence, were the same as those of the nerve-related antigens in both normal and prolapsed mitral valves. Electron microscopic study of the atrial aspect of normal mitral valves revealed numerous small axons with aggregations of small clear vesicles, indicating cholinergic features. The results suggest that the subendocardial site on the atrial aspect at the middle portion of the mitral valve is rich in nerve endings, including the afferent nerves, and that mechanical stimuli from this area caused by abnormal coaptation in mitral valve prolapse may produce an improper circuit in autonomic nerve function between the central and mitral valve nervous systems.

Background: A rich supply of nerves to the atrioventricular valve has been demonstrated. The role of the valvular nerves is still controversial because the target sites of the nerves have not been confirmed., Methods: The innervation of the atrioventricular valves of the Japanese monkey (Macaca fuscata) was examined by acetylcholinesterase staining and electron microscopy. Immunoreactivity for neuropeptide Y (NPY) was also investigated by a post-embedding immunogold method., Results: The valvular nerve elements were clearly concentrated between the endothelium and interstitial cells on the atrial side of cusps. Naked axon terminals were observed to make direct contact (20-nm gaps) with interstitial cells and also to be in close proximity (approximately 200-nm cleft) to the endothelium. NPY immunoreactivity was clearly detected on the large granular vesicles in some terminals that were in close proximity to interstitial cells and/or the endothelium., Conclusion: The present study suggests that the extensive innervation of the atrioventricular valve, which includes NPY-containing nerves, might affect valvular function via interstitial cells and/or the endothelium.

Dense distribution of varicose fibres containing neuropeptide Y-like immunoreactivity (NPY-LI) was found in the atrioventricular valves of the Japanese monkey, and moderately in the rat. The immunoelectron microscopy using immunogolds resulted in the localization of NPY-LI within the dense-cored vesicles which existed with the small clear vesicles in the unmyelinated axons near the endocardium. These NPY-LI-containing fibres may participate in regulation of vasomotor role or other functions of the atrioventricular valves.

To demonstrate innervation in human atrioventricular valves, we examined the tricuspid and mitral valves of apparently normal autopsied hearts of four men (ages ranging from 50 to 74 years). Whole valve tissues were stained for acetylcholinesterase by a histochemical method. Acetylcholinesterase-positive nerve fibers with a diameter of 2 to 5 microns were distributed widely in the deep atrialis of the atrioventricular valves and partly in the fibrosa. The nerve fibers formed a network or plexus from the base to the anatomical edge of the valves. Meshes of the nerve fiber network were more dense towards the base and at the commissure than either towards the edge or at the body. Thicker nerve fibers, which were interspersed coarsely in the leaflets, were intercalated by special varicose apparatuses at a few sites in their long running course. On the contrary, thinner nerve fibers which were distributed abundantly, ended, as a rule, in small dotor brush-like formations. Approximately half of the chordae tendineae were innervated by the nerve fibers. The mode of vagal innervation suggests that the nerve system may assist valve movement by moderating myocyte contraction in the valve base and change valve structure by sensing a stress in the valves.

Innervation of the human atrioventricular (AV) valves is microscopically studied by histopathological methods. The tricuspid and mitral valves of 4 autopsied hearts of adult men (age range from 50 to 74 years old) without any cardiovascular diseases were stained for acetylcholine-esterase by histochemical method in the medium containing acetylthiocholine iodide. Acetylcholine-esterase positive nerve fibers of 2 to 50 microns in diameter were widely distributed in the subepicardial space of the atrial of the AV valve. They formed a coarse network of the nerve elements from the valve base to the anatomical edge. The nerve network was more dense at the valve ring and base, as well as at the commissure, than at the edge and body. Some thick nerve fibers ran in the chordae tendineae. The thick fibers were intercalated with varicose-like special structures at several places in the leaflets, which seemed to be a kind of sensory apparatus. The thin nerve fibers ended, as usual, at small dot or brush-like apparatus. It is widely accepted that the acetylcholine-esterase positive nerve fibers are identical with vagal nerves which are insisted on participating in development of mitral valve prolapse syndrome. We suggest that the vagal innervation in the AV valves could play an important role for valvular function.

The bicuspid (mitral) valves were obtained from male albino New Zealand rabbits. The noradrenaline (NA) content (12.93 +/- 1.14 nmol/g) of the valve tissue was determined by high pressure liquid chromatography (HPLC) combined with electrochemical detection. After incubation with tritiated NA for 45 min, the tissues were mounted in perfusion baths and superfused with Krebs solution at a constant perfusion rate. After a 90 min washing period, the tissues were stimulated three times (S1; S2; S3) at a frequency of 1 or 10 Hz, and the release of NA was expressed as the stimulus-induced overflow of radioactivity. Using a constant number of impulses, the release of NA was significantly higher when the frequency applied was 10 Hz than in the case of 1 Hz. The release of NA was inhibited by stimulating the presynaptic alpha 2-adrenoceptors with xylazine or by stimulating the presynaptic muscarinic receptors with oxotremorine. Yohimbine (1 microM) not only overcame the effect of the alpha 2-adrenoceptor stimulation caused by xylazine, but increased it over the control level, whereas atropine blocked the inhibitory effect of oxotremorine. It is concluded that the adrenergic nerves in the valve tissue release NA in a frequency-dependent fashion, and the release of NA can be modulated through presynaptic alpha 2- and muscarinic receptors. This is the first case that neurochemical evidence was obtained showing that NA is released from the mitral valve and is subject to presynaptic modulation.

In this series of studies, the innervation patterns of whole-mount preparations of bicuspid and tricuspid valves were studied by light microscopy in the mouse, rat, guinea pig, and opossum. The acetylcholinesterase-positive networks of nerve fibers showed many similarities in the basic patterns of valve innervation in all of the species studied, but several interspecies variations were observed. The basal zone of the valve adjacent to the fibromuscular atrioventricular ring displayed the most dense plexus of nerves, with acetylcholinesterase-positive fibers being seen across the width of the valve. In the intermediate zone of the valve, less dense plexuses of nerve fibers were found; and these were more numerous in the cuspal areas and less numerous in the intervening commissural areas. In the distal portions of the valve, nerve networks arborized extensively, with some of their nerve fibers extending toward the chordae tendineae and the free edges of the valve cusps. Only in the guinea pig and opossum did these fibers reach the free margin of the valve cusp, where they either ended directly as free nerve endings or lay parallel to the free edge of the cusp, often running between adjacent chordae tendineae. Although the patterns of innervation were similar in both bicuspid and tricuspid valves, the innervation density of the bicuspid valve was greater than that of the tricuspid valve for each species examined. A distinguishing feature of guinea pig and opossum tricuspid valves was that their chordae tendineae were relatively more prominent and more densely innervated than the bicuspid chordae tendineae. Free nerve endings with no light microscopic evidence of specialization were present throughout the bicuspid and tricuspid valves of all species studied. Some nerve endings in the opossum showed evidence of specialization, with brush-like arborizations leading to presumed free terminals seen chiefly in the distal zone of the valve cusps. Although some general tendencies were apparent, we have demonstrated that interspecies heterogeneity exists in the terminal networks of the atrioventricular valves of mouse, rat, guinea pig, and opossum.

Method of silver nitrate impregnation was used in order to study 50 preparations of not-changed atrioventricular valves of the heart of domestic bulls and 30 preparations of the same valves of adult humans. It has been shown that in heart valves there are certain relationships between striated muscle fibres, blood vessels and nerve elements. The nerve structures of the valves are represented by nerve bundles of different thickness. In their composition there are comparatively thin non-myelinated and thicker myelinated fibres. Towards the free edge of cusps the nerve bundles become thinner and the nerve trunks give off separate thin nerve fibres disposed along the vessels of a capillary type and in some places getting around them. In certain portions of cusps the nerve bundles, some of which have zigzag sinuosity, cross blood vessels in different directions. In man the major mass of blood vessels and nerve elements are disposed near the base of the valve cusps, accompanying the muscle fibre bundles penetrating from the base side. In the bull heart valves an amount of blood vessels and nerve elements is found in considerable portions of the cusps not connected with muscle fibres.

To evaluate the relationship between mitral valve prolapse (MVP) and autonomic nerve dysfunction, clinical and immunohistochemical studies were performed. I. Clinical study Autonomic function tests and supine bicycle exercise were performed in 60 patients with MVP, 41 under 35 years of age and 19 of 35 years and older, and the results were compared with those of 31 normal controls, 19 under 35 years of age and 12 of 35 years and older. Case with positive response on postural stress and cold pressor tests were more frequent in both MVP groups than those of controls. Plasma nor-adrenaline levels were higher in the MVP groups than in the control groups during exercise. Our clinical observation suggested that autonomic dysfunction, particularly sympathetic hyperactivity, probably is present in MVP irrespective of age. II. Immunohistochemical study In 23 mitral valves (15 of normal controls and eight of MVP), three normal tricuspid, three normal aortic and three normal pulmonic valves, immunohistochemical localizations of S-100 protein, glial fibrillary acidic protein (GFAP) and neurofilament protein (NFP) were examined to detect the distribution of all nerve endings, choline acetyltransferase (ChAT) to detect the distribution of cholinergic nerve fibers, and neuropeptide Y (NPY) and calcitonin gene related peptide (CGRP) to detect the distribution of afferent nerve fibers, using the avidin-biotin peroxidase complex (ABC) method. The distribution of adrenergic nerve fibers was examined by fluorescence with the glyoxylic acid method. In normal valves, S-100 protein was mainly demonstrated in the base and body of the valve cusp. It was very scanty in the tip, and was only found in the coaptation zone that is, the layer of the atrialis or along the boundary between the atrialis and the spongiosa in the atrioventricular valves. The distribution of S-100 protein in the prolapsed mitral valve was the same as that of the normal valve except for the lack of S-100 protein in the degenerated portion. The distribution of GFAP, NFP, ChAT, NPY, CGRP and adrenergic fluorescence were the same as that of S-100 protein in both the normal and prolapsed mitral valves. It is suggested that the above-mentioned anatomical distribution of the nerve in the mitral valve is closely related to the dysfunction of the autonomic nerve system in the condition of mitral valve prolapse, because the body of the mitral valve is rich in nerve endings and this area is most easily influenced by the mechanical stimulation due to abnormal coaptation.

The development of the adrenergic and cholinergic innervation of the rat atrioventricular valves was studied in whole mount stretch preparations. Specimens obtained from rats ageing 2-30 days were processed for the histofluorescence and enzyme histochemical demonstration of monoamines and acetylcholinesterase (AChE) activity, respectively. Adrenergic and AChE-positive nerve fibres could be detected from the 9-10th postnatal days onwards. Fluorescence microscopy showed the presence of many brightly fluorescent mast cells in the close vicinity of the ingrowing terminals. The presence of mast cells during early stages of development of the ground plexus may be related either to the preneural state of the tissue or the structural and/or functional maturation of the autonomic nerve terminals.

The present paper describes peculiar lesions in papillary muscles and mitral valves produced by vagal manipulations. In the 109 rabbits killed one week after clipping or crushing of their cervical vagi, papillary muscle and mitral valve lesions were found in 53 (48.6%) and 52 (47.7%) hearts respectively. The lesions were identified by visible deposits of colloidal carbon which had been injected intravenously during the first 3 postoperative days. The left ventricular free wall, interventricular septum and right ventricle were free of carbon deposits. The papillary muscles involved were characterized by swelling and increased stiffness which corresponded to degeneration of the myocardial cells and interstitial fibrosis on microscopic observation. Hydroxyproline content of the papillary muscles involved was 1.4 times more than that of normal hearts, while there was no significant difference in hydroxyproline content of either interventricular septum or left ventricular free wall between the manipulated and the normal preparations. Heart murmurs were heard in 23 animals studied. Phonocardiograms revealed mid to late systolic murmurs with or without midsystolic click. These results indicate that the vagus nerve plays an important role in the pathogenesis of some forms of papillary muscle and mitral valve lesions.