GCH1 attenuates cardiac autonomic nervous remodeling in canines with atrial-tachypacing via tetrahydrobiopterin pathway regulated by microRNA-206.

BACKGROUND/AIMS: Cardiac autonomic nerve remodeling (ANR) is an important mechanism of atrial fibrillation (AF). GTP cyclohydrolase I, encoded by GCH1, is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide (NO) synthesis. Previous studies reported that increased BH4 and NO content negatively regulated nerve regeneration. This study investigated the effects of GCH1 on ANR via BH4 pathway, regulated by microRNA-206 (miR-206). METHODS AND RESULTS: In canines, atrial tachypacing (A-TP), together with miR-206 overexpression, increased PGP9.5 level and inhibited GCH1 expression by quantitative real-time polymerase chain reaction and western blot analysis. GCH1 was validated to be a direct target of miR-206 by luciferase assays. Meanwhile, miR-206 overexpression by lentiviruses infection into right superior pulmonary vein fat pad decreased GCH1 expression to ∼40% and further reduced BH4 and NO content compared with the control canines. After infection of GCH1 overexpression lentiviruses for two weeks, atrial effective refractory period was increased compared with the control group (105.8 ± 1.537 ms vs 99.17 ± 2.007 ms, P < 0.05). Moreover, GCH1 overexpression attenuated canines' atrial PGP9.5 level to ∼56% of the controls. In myocardial cells, transfection of GCH1 overexpression lentiviruses also decreased PGP9.5 expression to 26% of the control group. In patients, plasma was collected and miR-206 expression was upregulated in AF patients (n = 18) than the controls (n = 12). CONCLUSIONS: Our findings suggested that GCH1 downregulation exacerbated ANR by decreasing atrial BH4 and NO content modulated by miR-206 in A-TP canines. This indicates that GCH1 may prevent the initiation of AF through inhibiting ANR.

Acquired long QT syndrome and phosphoinositide 3-kinase.

While it is well known that mutation of several different ion channels can cause congenital long QT syndrome, block of IKr is widely thought to be responsible for most cases of drug-induced acquired long QT syndrome (aLQTS). In this article, we review evidence supporting another cause of aLQTS due to inhibition of phosphoinositide 3-kinase (PI3K) signaling. Inhibition of PI3K affects multiple plateau currents, reducing IKr, IKs, and ICaL while increasing the persistent sodium current (INaP). The effects of PI3K inhibitors develop slowly, requiring hours to days to reach steady state. Dofetilide and terfenadine, an antihistamine on which much of the original IKr hypothesis was based, are among the many drugs that inhibit the PI3K pathway. Reduced PI3K signaling may also play a role in aLQTS associated with diabetes. Drug safety testing to identify aLQTS risk may be improved by examining PI3K-dependent effects that develop over time.

Progressive age-associated activation of JNK associates with conduction disruption in the aged atrium.

Connexin43 (Cx43) is critical for maintaining electrical conduction across atrial muscle. During progressive ageing atrial conduction slows associating with increasing susceptibility to arrhythmias. Changes in Cx43 protein expression, or its phosphorylation status, can instigate changes in the conduction of the cardiac action potential. This study investigated whether increased levels of activated c-jun N-terminal kinase (JNK) is responsible for the decline of Cx43 during ageing. Right atria from guinea pigs aged between 1 day and 38 months of age were examined. The area of the intercalated disc increased with age concurrent with a 75% decline in C43 protein expression. An age-dependent increase in activated-JNK correlated with a rise in phosphorylated Cx43, but also slowing of action potential conduction velocity across the atria from 0.38±0.01 m/s at 1 month of age to 0.30±0.01 m/s at 38 months. The JNK activator anisomycin increased activated JNK in myocytes and reduced Cx43 protein expression simulating ageing. The JNK inhibitor SP600125, was found to eradicate almost all trace of Cx43 protein. We conclude that in vivo activation of JNK increases with age leading to the loss of Cx43 protein resulting in impaired conduction and contributing to the increasing risk of atrial arrhythmias with advancing age.

Efficacy of B-Type Natriuretic Peptide Is Coupled to Phosphodiesterase 2A in Cardiac Sympathetic Neurons.

Elevated B-type natriuretic peptide (BNP) regulates cGMP-phosphodiesterase activity. Its elevation is regarded as an early compensatory response to cardiac failure where it can facilitate sympathovagal balance and cardiorenal homeostasis. However, recent reports suggest a paradoxical proadrenergic action of BNP. Because phosphodiesterase activity is altered in cardiovascular disease, we tested the hypothesis that BNP might lose its efficacy by minimizing the action of cGMP on downstream pathways coupled to neurotransmission. BNP decreased norepinephrine release from atrial preparations in response to field stimulation and also significantly reduced the heart rate responses to sympathetic nerve stimulation in vitro. Using electrophysiological recording and fluorescence imaging, BNP also reduced the depolarization evoked calcium current and intracellular calcium transient in isolated cardiac sympathetic neurons. Pharmacological manipulations suggested that the reduction in the calcium transient was regulated by a cGMP/protein kinase G pathway. Fluorescence resonance energy transfer measurements for cAMP, and an immunoassay for cGMP, showed that BNP increased cGMP, but not cAMP. In addition, overexpression of phosphodiesterase 2A after adenoviral gene transfer markedly decreased BNP stimulation of cGMP and abrogated the BNP responses to the calcium current, intracellular calcium transient, and neurotransmitter release. These effects were reversed on inhibition of phosphodiesterase 2A. Moreover, phosphodiesterase 2A activity was significantly elevated in stellate neurons from the prohypertensive rat compared with the normotensive control. Our data suggest that abnormally high levels of phosphodiesterase 2A may provide a brake against the inhibitory action of BNP on sympathetic transmission.

Activation of angiotensin-converting enzyme 2 improves cardiac electrical changes in ventricular repolarization in streptozotocin-induced hyperglycaemic rats.

AIMS: Diabetic patients present a high level of cardiac arrhythmias and risk of cardiac sudden death. The renin-angiotensin system (RAS) plays a key role in diabetes and cardiac diseases. The present study aimed to evaluate whether an angiotensin-converting enzyme 2 (ACE2) activator, diminazene aceturate (DIZE), could improve the streptozotocin (STZ)-induced electrical changes in ventricular repolarization in hyperglycaemic rats. METHODS AND RESULTS: Hyperglycaemia was induced in Wistar male rats with STZ (60 mg/kg/iv). After 4 weeks of STZ injection, rats were daily treated with saline (control) or DIZE (1 mg/kg/gavage) for four consecutive weeks. The cardiac electrical function was evaluated in vivo by electrocardiogram and in vitro by cardiac action potential records in different pacing frequencies. Treatment with DIZE was not able to reverse hyperglycaemia nor body weight loss. However, DIZE reversed hyperglycaemia-induced cardiac electrical changes in ventricular repolarization. Specifically, animals treated with DIZE showed shorter QT and QTc intervals. In addition, ACE2 activation was capable to shorten the cardiac action potential and also reverse the arrhythmic markers. Diminazene aceturate treatment did not induce arrhythmic events in normal, as well as in hyperglycaemic animals. CONCLUSION: Our data indicate that activation of ACE2 has a beneficial effect in hyperglycaemic rats, improving the cardiac electrical function. Thus, DIZE represents a promising new therapeutic agent to treat hyperglycaemia-induced cardiac electrical changes in ventricular repolarization.

Histoenzymological characteristics of the heart conduction system: comparative study with left or right ventricle afterload.

Histoenzymological changes, indicating inhibition of the main metabolic processes, were found in the conduction cardiomyocytes of the left ventricle and ventricular septum in experimental stenosis of the aorta. The histoenzymological changes in the conduction system of both ventricles and ventricular septum were similar in experimental stenosis of the pulmonary artery and indicated primarily activation of glycolysis. The histoenzymological profile of conduction cardiomyocytes differed little in cases when the increase of the pressure load was complicated or not complicated by the development of heart failure, particularly in pulmonary artery stenosis. The histoenzymological changes in the conduction system in response to increased afterload differed significantly from those in the contractile myocardium and correlated with the level of cellular functional activity and sensitivity to the regulatory and alterative exposure. These data attest to minor role of metabolic shifts in conduction cell injuries with increasing afterload, primarily, of the right ventricle.

Cardiovascular safety of tyrosine kinase inhibitors: with a special focus on cardiac repolarisation (QT interval).

The development of tyrosine kinase inhibitors (TKI) represents a major milestone in oncology. However, their use has been found to be associated with serious toxicities that impinge on various vital organs including the heart. Sixteen TKIs have been approved for use in oncology as of 30 September 2012, and a large number of others are in development or under regulatory review. Cardiovascular safety of medicinal products is a major public health issue that has concerned all the stakeholders. This review focuses on three specific cardiovascular safety aspects of TKIs, namely their propensity to induce QT interval prolongation, left ventricular (LV) dysfunction and hypertension (both systemic and pulmonary). Analyses of information in drug labels, the data submitted to the regulatory authorities and the published literature show that a number of TKIs are associated with these undesirable effects. Whereas LV dysfunction and systemic hypertension are on-target effects related to the inhibition of ligand-related signalling pathways, QT interval prolongation appears to be an off-target class III electrophysiologic effect, possibly related to the presence of a fluorine-based pharmacophore. If not adequately managed, these cardiovascular effects significantly increase the morbidity and mortality in a population already at high risk. Hitherto, the QT effect of most QT-prolonging TKIs (except lapatinib, nilotinib, sunitinib and vandetanib) is relatively mild at clinical doses and has not led to appreciable morbidity clinically. In contrast, LV dysfunction and untreated hypertension have resulted in significant morbidity. Inevitably, dilemmas arise in determining the risk/benefit of a TKI therapy in an individual patient who develops any of these effects following the treatment of the TKI-sensitive cancer. QT interval prolongation, hypertension and LV dysfunction can be managed effectively by using reliable methods of measurement and careful monitoring of patients whose clinical management requires optimisation by a close collaboration between an oncologist and a cardiologist, an evolving subspecialty referred to as cardio-oncology. Despite their potential adverse clinical impact, the effects of TKIs on hypertension and LV function are generally inadequately characterised during their development. As has been the case with QT liability of drugs, there is now a persuasive case for a regulatory requirement to study TKIs systematically for these effects. Furthermore, since most of these novel drugs are studied in trials with relatively small sample sizes and approved on an expedited basis, there is also a compelling case for their effective pharmacovigilance and on-going reassessment of their risk/benefit after approval.

Mesenchymal stem cells improve cardiac conduction by upregulation of connexin 43 through paracrine signaling.

Mesenchymal stem cells (MSCs) were shown to improve cell survival and alleviate cardiac arrhythmias when transplanted into cardiac tissue; however, little is known about the mechanism by which MSCs modify the electrophysiological properties of cardiac tissue. We aimed to distinguish the influence of cell-cell coupling between myocytes and MSCs from that of MSC-derived paracrine factors on the spontaneous activity and conduction velocity (θ) of multicellular cardiomyocyte preparations. HL-1 cells were plated on microelectrode arrays and their spontaneous activity and θ was determined from field potential recordings. In heterocellular cultures of MSCs and HL-1 cells the beating frequency was attenuated (t(0h): 2.26 ± 0.18 Hz; t(4h): 1.98 ± 0.26 Hz; P < 0.01) concomitant to the intercellular coupling between MSCs and cardiomyocytes. In HL-1 monolayers supplemented with MSC conditioned media (ConM) or tyrode (ConT) θ significantly increased in a time-dependent manner (ConT: t(0h): 2.4 cm/s ± 0.2; t(4h): 3.1 ± 0.4 cm/s), whereas the beating frequency remained constant. Connexin (Cx)43 mRNA and protein expression levels also increased after ConM or ConT treatment over the same time period. Enhanced low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation after ConT treatment implicates the Wnt signaling pathway. Suppression of Wnt secretion from MSCs (IWP-2; 5 µmol/l) reduced the efficacy of ConT to induce phospho-LRP6 and to increase θ. Inhibition of ß-catenin (cardamonin; 10 µmol/l) or GSK3-α/ß (LiCl; 5 mmol/l) also suppressed changes in θ, further supporting the hypothesis that MSC-mediated Cx43 upregulation occurs in part through secreted Wnt ligands and activation of the canonical Wnt signaling pathway.

Role of Pyk2 in cardiac arrhythmogenesis.

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein kinase regulated by intracellular Ca(2+), CaMK, and PKC and can be activated by different stress signals involved in heart failure. However, Pyk2 has not been investigated in the human heart, and the functional role of Pyk2 signaling at the whole heart level has not been elucidated. We hypothesize that Ca(2+)-dependent activation of Pyk2 is involved in cardiac electrophysiology. We examined the expression of Pyk2 in nonfailing versus ischemic and nonischemic failing human hearts (n = 6 hearts/group). To investigate Pyk2 function, we optically mapped perfused hearts from wild-type (WT; n = 7) and knockout (Pyk2(-/-); n = 8) mice during autonomic stimulation. Experiments were done in control mice and after 1 wk of transverse aortic constriction. We used the Illumina beadarray approach for transcriptional profiling of WT and Pyk2(-/-) mouse ventricles. Western blot analysis revealed a doubling of Pyk2 activation in nonischemic failing versus nonfailing human hearts. In mouse hearts, we observed a much higher probability of ventricular tachyarrhythmia during ACh perfusion in Pyk2(-/-) versus WT mice. Parasympathetic stimulation resulted in a dose-dependent decrease of atrial action potential duration (APD) in both WT and Pyk2(-/-) mice, whereas in ventricles it induced APD shortening in Pyk2(-/-) mice but not in WT mice. Deficiency of Pyk2 abolished ACh-induced prolongation of atrioventricular delay in Pyk2(-/-) mouse hearts but did not affect heart rate. Lower mRNA and protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and higher mRNA levels of Na(+)/Ca(2+) exchanger 1 were detected in Pyk2(-/-) hearts compared with WT hearts. The transverse aortic constriction protocol did not change the phenotype. In conclusion, our results indicate a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to Ca(2+) handling. We hypothesize that activation of Pyk2 in the human heart during heart failure may contribute to protection against arrhythmia.

Pharmacological inhibition of leukotriene biosynthesis: effects on the heart conductance.

Leukotrienes are lipid mediators produced via 5-lipooxygenase pathway of arachidonic acid. At least two cysteinyl-leukotrienes receptors are highly expressed in the heart, including the conduction system. Coronary angiography or angioplasty is accompanied by release of cysteinyl leukotrienes into coronary circulation and into urine. We tested the hypothesis that inhibition of leukotrienes biosynthesis would affect the conductance system function. In a double-blind placebo controlled study, patients with stable angina undergoing elective coronary catheterization or angioplasty were randomly assigned to 48 hrs treatment with a 5-lipoxgenase inhibitor (n=54) or placebo (n=49). ECG Holter recording was carried out for 24 hrs before and after the procedure and urinary leukotriene E(4) measurements were done. Inhibition of 5-lipoxygenase caused 26% reduction of urinary leukotriene E(4), associated with: 1) decrease in heart rate by about 7%, 2) enhanced heart rate variability; 3) protection against depressions in atrioventricular conductance and ventricular repolarization induced by the procedure. No effects on either arrhythmias, or ECG patterns of ischemia were noted. We conclude that pharmacological inhibition of 5-lipoxygenase, shortly before percutaneous coronary intervention, reveals specific actions of leukotrienes on the heart rhythm. Inhibitors of 5-lipoxygenase might be of interest as a novel class of cardiac drugs affecting the conductive system.

Cardiac Rac1 overexpression in mice creates a substrate for atrial arrhythmias characterized by structural remodelling.

AIMS: The small GTPase Rac1 seems to play a role in the pathogenesis of atrial fibrillation (AF). The aim of the present study was to characterize the effects of Rac1 overexpression on atrial electrophysiology. METHODS AND RESULTS: In mice with cardiac overexpression of constitutively active Rac1 (RacET), statin-treated RacET, and wild-type controls (age 6 months), conduction in the right and left atrium (RA and LA) was mapped epicardially. The atrial effective refractory period (AERP) was determined and inducibility of atrial arrhythmias was tested. Action potentials were recorded in isolated cells. Left ventricular function was measured by pressure-volume analysis. Five of 11 RacET hearts showed spontaneous or inducible atrial tachyarrhythmias vs. 0 of 9 controls (P < 0.05). In RacET, the P-wave duration was significantly longer (26.8 +/- 2.1 vs. 16.7 +/- 1.1 ms, P = 0.001) as was total atrial activation time (RA: 13.6 +/- 4.4 vs. 3.2 +/- 0.5 ms; LA: 7.1 +/- 1.2 vs. 2.2 +/- 0.3 ms, P < 0.01). Prolonged local conduction times occurred more often in RacET (RA: 24.4 +/- 3.8 vs. 2.7 +/- 2.1%; LA: 19.1 +/- 6.3 vs. 1.2 +/- 0.7%, P < 0.01). The AERP and action potential duration did not differ significantly between both groups. RacET demonstrated significant atrial fibrosis but only moderate systolic heart failure. RacET and statin-treated RacET were not significantly different regarding atrial electrophysiology. CONCLUSION: The substrate for atrial arrhythmias in mice with Rac1 overexpression is characterized by conduction disturbances and atrial fibrosis. Electrical remodelling (i.e. a shortening of AERP) does not play a role. Statin treatment cannot prevent the structural and electrophysiological effects of pronounced Rac1 overexpression in this model.

Lamin A/C haploinsufficiency causes dilated cardiomyopathy and apoptosis-triggered cardiac conduction system disease.

Mutations in the lamin A/C (LMNA) gene, which encodes nuclear membrane proteins, cause a variety of human conditions including dilated cardiomyopathy (DCM) with associated cardiac conduction system disease. To investigate mechanisms responsible for electrophysiologic and myocardial phenotypes caused by dominant human LMNA mutations, we performed longitudinal evaluations in heterozygous Lmna(+/-) mice. Despite one normal allele, Lmna(+/-) mice had 50% of normal cardiac lamin A/C levels and developed cardiac abnormalities. Conduction system function was normal in neonatal Lmna(+/-) mice but, by 4 weeks of age, atrioventricular (AV) nodal myocytes had abnormally shaped nuclei and active apoptosis. Telemetric and in vivo electrophysiologic studies in 10-week-old Lmna(+/-) mice showed AV conduction defects and both atrial and ventricular arrhythmias, analogous to those observed in humans with heterozygous LMNA mutations. Isolated myocytes from 12-month-old Lmna(+/-) mice exhibited impaired contractility. In vivo cardiac studies of aged Lmna(+/-) mice revealed DCM; in some mice this occurred without overt conduction system disease. However, neither histopathology nor serum CK levels indicated skeletal muscle pathology. These data demonstrate cardiac pathology due to heterozygous Lmna mutations reflecting a 50% reduction in lamin protein levels. Lamin haploinsufficiency caused early-onset programmed cell death of AV nodal myocytes and progressive electrophysiologic disease. While lamin haploinsufficiency was better tolerated by non-conducting myocytes, ultimately, these too succumbed to diminished lamin levels leading to dilated cardiomyopathy, which presumably arose independently from conduction system disease.

Effects of enzyme replacement therapy on the cardiomyopathy of Anderson-Fabry disease: a randomised, double-blind, placebo-controlled clinical trial of agalsidase alfa.

BACKGROUND: Anderson-Fabry disease is an X-linked glycosphingolipid storage disorder caused by deficient activity of the lysosomal enzyme alpha-galactosidase A. This leads to a progressive accumulation of globotriaosylceramide (Gb(3)) in the lysosomes of cells throughout the body that ultimately results in premature death from renal, cardiac or cerebrovascular complications. Until recently, there was no effective therapy available for this disease. The present study was designed to assess the safety and efficacy of enzyme replacement therapy with agalsidase alfa on the cardiac manifestations of Anderson-Fabry disease. METHOD: The effects of therapy with agalsidase alfa on cardiac structure and function were assessed in a randomised, double-blind, placebo-controlled study of 15 adult male patients with Anderson-Fabry disease. The following parameters were measured at baseline and 6 months: left ventricular mass, QRS duration and levels of Gb(3) in cardiac tissue, urine sediment and plasma. After 6 months of the randomised trial patients were enrolled in a 2-year open-label extension study. RESULTS: Left ventricular mass, as measured by MRI, was significantly reduced following 6 months of treatment with agalsidase alfa compared with placebo (p = 0.041). A mean 20% reduction in myocardial Gb(3) content as assessed by serial transvenous endomyocardial biopsies was demonstrated over the 6 months of enzyme replacement compared to a mean 10% increase in patients receiving placebo (p = 0.42) CONCLUSION: Enzyme replacement therapy with agalsidase alfa resulted in regression of the hypertrophic cardiomyopathy associated with Anderson-Fabry disease.

Regulation of Ca2+ and electrical alternans in cardiac myocytes: role of CAMKII and repolarizing currents.

Alternans of cardiac repolarization is associated with arrhythmias and sudden death. At the cellular level, alternans involves beat-to-beat oscillation of the action potential (AP) and possibly Ca(2+) transient (CaT). Because of experimental difficulty in independently controlling the Ca(2+) and electrical subsystems, mathematical modeling provides additional insights into mechanisms and causality. Pacing protocols were conducted in a canine ventricular myocyte model with the following results: 1) CaT alternans results from refractoriness of the sarcoplasmic reticulum Ca(2+) release system; alternation of the L-type calcium current has a negligible effect; 2) CaT-AP coupling during late AP occurs through the sodium-calcium exchanger and underlies AP duration (APD) alternans; 3) increased Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity extends the range of CaT and APD alternans to slower frequencies and increases alternans magnitude; its decrease suppresses CaT and APD alternans, exerting an antiarrhythmic effect; and 4) increase of the rapid delayed rectifier current (I(Kr)) also suppresses APD alternans but without suppressing CaT alternans. Thus CaMKII inhibition eliminates APD alternans by eliminating its cause (CaT alternans) while I(Kr) enhancement does so by weakening CaT-APD coupling. The simulations identify combined CaMKII inhibition and I(Kr) enhancement as a possible antiarrhythmic intervention.

Noradrenergic neuron-specific overexpression of nNOS in cardiac sympathetic nerves decreases neurotransmission.

Gene transfer of neuronal nitric oxide synthase (nNOS) with nonspecific adenoviral vectors can cause promiscuous transduction. We provide direct evidence that nNOS targeted only to cardiac sympathetic neurons inhibits sympathetic neurotransmission. An adenovirus constructed with a noradrenergic neuron-specific promoter (PRSx8), driving nNOS or enhanced green fluorescence protein (eGFP) gene expression caused exclusive expression in tyrosine hydroxylase (TH) positive rat cardiac sympathetic neurons. There was no detectable leakage of transgene expression in other cell types in the preparation nor did the transgene express in choline acetyltransferase (CHAT)-positive intracardiac cholinergic ganglia. Functionally, Ad.PRS-nNOS gene transfer increased nNOS activity and significantly reduced norephinephrine release evoked by field stimulation of isolated right atria. These effects were reversed by the NOS inhibitor N(omega)-Nitro-L-arginine. Our results demonstrate that noradrenergic cell-specific gene transfer with nNOS can inhibit cardiac sympathetic neurotransmission. This targeted technique may provide a novel method for reducing presynaptic sympathetic hyperactivity.

Comparative quantitative study of the intrinsic cardiac ganglia and neurons in the rat, guinea pig, dog and human as revealed by histochemical staining for acetylcholinesterase.

This study was conducted to determine the overall number of intrinsic neurons distributed through-out the entire heart, in which most neurons are located inside of intramural ganglia and are hidden to observers. For this reason, we attempted to ascertain: (1) how the number of neurons located inside of intrinsic cardiac ganglion is related to its area, and (2) whether this relationship is dependent on age and species of animals. Hearts of rats, guinea pigs, dogs and humans were used to examine intramural ganglia stained histochemically for acetylcholinesterase (AChE). The number and parameters of neurons located inside of 104 ganglia were estimated in serial sections. Although the revealed intrinsic cardiac ganglia varied extremely in shape and size, two different types were identified: the globular and plain ones. In the plain ganglia, perikarya of side by side situated neurons were always intensely stained for AChE and, being clearly discernible, they could be reliably counted in any plain ganglia on total heart preparations using a contact microscope. Contrarily, neuron somata in the globular ganglia were densely packed above one another and their perikarya were almost indiscernible for the observer. Counting of neurons located inside of globular ganglia was possible in serial sections only. The largest cardiac ganglia were revealed in dogs, in which some globular ganglia containing up to 2000 neurons occupied more than 1 mm2. In spite of evident species-dependent differences with respect to frequency of large ganglia, the majority of intrinsic cardiac ganglia both in humans and animals were comparatively small, involved approximately 100-200 nerve cells and occupied an area ranging from 0.01 to 0.17 mm2. Overall, the number of neurons located inside of globular ganglion was related to its area (correlation coefficient = 0.82). However, the correlation coefficients between the globular ganglion area and its neuron number were unequal in different species (0.92 in guinea pig; 0.80 in dog; 0.72 in human; and 0.44 in rat) as well as dependent on (1) ganglion size (0.8 for ganglia equal to or larger than 0.17 mm2 and 0.6 for ganglia smaller than 0.17 mm2) and (2) age of specimens (respectively, 0.98 for juvenile and 0.87 for adult dogs; 0.71 for infants and 0.54 for aged human). In all examined animals and humans, the mean measurements of neuron perikarya were similar (on average, 23 microm in width, 32 microm in length, and 615 microm2 in area) and differences between them were statistically insignificant. However, neuron perikarya of adult dogs and aged humans were significantly larger than those revealed in the juvenile dogs and infants, respectively. Based on the data of this study, we concluded that the number of intrinsic cardiac neurons may be approximated in the total heart preparation via counting and measuring of intramural ganglia, contours of which are well-discernible following a histochemical reaction for AChE.

The intrinsic origin of nitric oxide synthase immunoreactive nerve fibers in the right atrium of the guinea pig.

We previously reported three kinds of nitric oxide synthase-immunoreactive (NOS-ir) axons in the guinea pig heart: the sparse fiber network covering the right atrium, the basket-like endings around intracardiac neurons, and the axons in the septal region. The sparse NOS-ir nerve fiber network in the right atrium remained after vagotomy and has been suggested to be originated from intrinsic cardiac ganglia. Using Chorera toxin B as a retrograde tracer, we determined a part of them were derived from cardiac ganglionic neurons located in the area near the vena cavae.