Molecular identification and immunohistochemical localization of atrial natriuretic peptide in the heart of the dromedary camel (Camelus dromedarius)

Atrial and B-type natriuretic peptide (ANP and BNP) are cardiac hormones synthesized and secreted by the myoendocrine cells of the heart. They exert potent actions on body fluid balance. Since various body organs including the heart are under high physiological stress during water and food deprivation in the desert nomads, we intended to perform molecular biological and histological studies of ANP in the heart of the dromedary camel Camelus dromedarius. Initially, we isolated cDNAs encoding ANP from the atrium and BNP from the atrium and ventricle of the dromedary camel. Putative mature ANP, deduced from the cDNA sequence, was identical to that of human and pig ANP, but the putative mature BNP was more diverse and was most similar to pig BNP (94% identity). Thus, we used antisera raised against human ANP that did not cross-react with pig BNP in the subsequent immunohistochemical studies. The ANP-expressing myoendocrine cells are most concentrated in the right atrium, to a lesser extent in the left atrium, and almost absent in the left ventricle. The immuno-positive cells are scattered uniformly in each region and are characterized by the presence of immunoreactive granular deposits around the nucleus. The left atrium comprises some ramifications of conductive cells (Purkinje fibers), some of which also contained ANP-immunoreactive granules. At the electron microscopic level, myoendocrine cells possessed secretory granules primarily in the perinuclear zone and a well-developed Golgi apparatus. The present study is the first comprehensive report dealing with the molecular cloning and immunohistochemical localization of ANP in the heart of a desert dwelling mammal.     

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles

Summary A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.Supported by a Medical Research Council of Canada Group Grant to the Multidisciplinary Research Group on Hypertension, by the National Research Council of Canada, the Pfizer Company (England), Bio-Méga Inc. and the Canadian Heart Foundation     

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.     

Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression

Purkinje fibers of the cardiac conduction system differentiate from heart muscle cells during embryogenesis. In the avian heart, Purkinje fiber differentiation takes place along the endocardium and coronary arteries. To date, only the vascular cytokine endothelin (ET) has been demonstrated to induce embryonic cardiomyocytes to differentiate into Purkinje fibers. This ET-induced Purkinje fiber differentiation is mediated by binding of ET to its transmembrane receptors that are expressed by myocytes. Expression of ET converting enzyme 1, which produces a biologically active ET ligand, begins in cardiac endothelia, both arterial and endocardial, at initiation of conduction cell differentiation and continues throughout heart development. Yet, the ability of cardiomyocytes to convert their phenotype in response to ET declines as embryos mature. Therefore, the loss of responsiveness to the inductive signal appears not to be associated with the level of ET ligand in the heart. This study examines the role of ET receptors in this age-dependent loss of inductive responsiveness and the expression profiles of three different types of ET receptors, ET(A), ET(B) and ET(B2), in the embryonic chick heart. Whole-mount in situ hybridization analyses revealed that ET(A) was ubiquitously expressed in both ventricular and atrial myocardium during heart development, while ET(B) was predominantly expressed in the atrium and the left ventricle. ET(B2) expression was detected in valve leaflets but not in the myocardium. RNase protection assays showed that ventricular expression of ET(A) and ET(B) increased until Purkinje fiber differentiation began. Importantly, the levels of both receptor isotypes decreased after this time. Retrovirus-mediated overexpression of ET(A) in ventricular myocytes in which endogenous ET receptors had been downregulated, enhanced their responsiveness to ET, allowing them to differentiate into conduction cells. These results suggest that the developmentally regulated expression of ET receptors plays a crucial role in determining the competency of ventricular myocytes to respond to inductive ET signaling in the chick embryo.     

In vitro expression of natriuretic peptides in cardiomyocytes differentiated from monkey embryonic stem cells

Functional characterization of ES cell-derived cardiomyocytes is important for differentiation control and application to the cell therapy. One of the crucial functions of cardiomyocytes is a production of atrial and brain natriuretic peptides (ANP and BNP, respectively), which have important endocrine, autocrine, and paracrine functions. In this study, we focused on the functional aspect of the cardiomyocytes differentiated from monkey ES cells in vitro and investigated the expression of ANP and BNP. Spontaneously contracting cells showed nodal-like action potentials, and expression of ANP and BNP by RT-PCR and immunocytochemistry. Interestingly, ANP and BNP expressions were detected as immunoreactive granules in the perinuclear area and these signals appeared to co-localize with trans-Golgi network. These findings suggest that monkey ES cells were able to differentiate into cardiomyocytes with functional characteristics in vitro and therefore can be used as a useful model to study mechanisms and functions in early cardiogenesis.     

In vivo induction of cardiac Purkinje fiber differentiation by coexpression of preproendothelin-1 and endothelin converting enzyme-1

The rhythmic heart beat is coordinated by electrical impulses transmitted from Purkinje fibers of the cardiac conduction system. During embryogenesis, the impulse-conducting cells differentiate from cardiac myocytes in direct association with the developing endocardium and coronary arteries, but not with the venous system. This conversion of myocytes into Purkinje fibers requires a paracrine interaction with blood vessels in vivo, and can be induced in vitro by exposing embryonic myocytes to endothelin-1 (ET-1), an endothelial cell-associated paracrine factor. These results suggest that an endothelial cell-derived signal is capable of inducing juxtaposed myocytes to differentiate into Purkinje fibers. It remains unexplained how Purkinje fiber recruitment is restricted to subendocardial and periarterial sites but not those juxtaposed to veins. Here we show that while the ET-receptor is expressed throughout the embryonic myocardium, introduction of the ET-1 precursor (preproET-1) in the embryonic myocardium is not sufficient to induce myocytes to differentiate into conducting cells. ET converting enzyme-1 (ECE-1), however, is expressed preferentially in endothelial cells of the endocardium and coronary arteries where Purkinje fiber recruitment takes place. Retroviral-mediated coexpression of both preproET-1 and ECE-1 in the embryonic myocardium induces myocytes to express Purkinje fiber markers ectopically and precociously. These results suggest that expression of ECE-1 plays a key role in defining an active site of ET signaling in the heart, thereby determining the timing and location of Purkinje fiber differentiation within the embryonic myocardium.     

ANP and BNP Responses to Dehydration in the One-Humped Camel and Effects of Blocking the Renin-Angiotensin System

The objectives of this study were to investigate and compare the responses of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in the circulation of hydrated, dehydrated, and dehydrated losartan - treated camels; and to document the cardiac storage form of B-type natriuretic peptide in the camel heart. Eighteen male camels were used in the study: control or hydrated camels (n = 6), dehydrated camels (n = 6) and dehydrated losartan-treated camels (n = 6) which were dehydrated and received the angiotensin II (Ang II) AT-1 receptor blocker, losartan, at a dose of 5 mg/kg body weight intravenously for 20 days. Control animals were supplied with feed and water ad-libitum while both dehydrated and dehydrated-losartan treated groups were supplied with feed ad-libitum but no water for 20 days. Compared with time-matched controls, dehydrated camels exhibited a significant decrease in plasma levels of both ANP and BNP. Losartan-treated camels also exhibited a significant decline in ANP and BNP levels across 20 days of dehydration but the changes were not different from those seen with dehydration alone. Size exclusion high performance liquid chromatography of extracts of camel heart indicated that proB-type natriuretic peptide is the storage form of the peptide.We conclude first, that dehydration in the camel induces vigorous decrements in circulating levels of ANP and BNP; second, blockade of the renin-angiotensin system has little or no modulatory effect on the ANP and BNP responses to dehydration; third, proB-type natriuretic peptide is the storage form of this hormone in the heart of the one-humped camel.     

Remodeling of the peripheral cardiac conduction system in response to pressure overload

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.     

Immunoreactive atrial and brain natriuretic peptides are co-localized in Purkinje fibres but not in the innervation of the bovine heart conduction system

Summary Recently, we observed that atrial natriuretic peptide (ANP) immunoreactivity was present in Purkinje fibres and nerve fibre varicosities in the conduction system of the bovine heart. In order to elucidate further the morphological correlation between natriuretic peptides and the conduction system, the distribution of brain natriuretic peptide (BNP) was examined. The different parts of the conduction system in the bovine heart were dissected out and processed for immunohistochemistry with antisera against BNP and ANP. BNP immunoreactivity was frequently observed in Purkinje fibres of the atrioventricular bundle, whereas only a few Purkinje fibres in the ventricular part of the conduction system showed immunoreaction. BNP immunoreactivity was detected in regions of the Purkinje fibres which also showed ANP immunoreactivity. BNP immunoreactivity was not observed in nerve fibre varicosities. Methodologically, a larger number of small BNP immunofluorescent granular structures was observed by using an elution-restaining technique instead of conventional immunohistochemistry. The present study shows that BNP and ANP immunoreactivities frequently occur in the atrioventricular bundle and that they are co-localized in Purkinje fibres, but not in nerve fibre varicosities, in the conduction system. As previously has been proposed for ANP, the present observations suggest that also BNP may act in an autocrine and/or paracrine way in the conduction cells.     

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP = 0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.     

Alpha actin isoforms expression in human and rat adult cardiac conduction system

In the adult heart, cardiac muscle comprises the working myocardium and the conduction system (CS). The latter includes the sinoatrial node (SAN), the internodal tract or bundle (IB), the atrioventricular node (AVN), the atrioventricular bundle (AVB), the bundle branches (BB) and the peripheral Purkinje fibers (PF). Most of the information concerning the phenotypic features of CS tissue derives from the characterization of avian and rodent developing hearts; data concerning the expression of actin isoforms in adult CS cardiomyocytes are scarce. Using specific antibodies, we investigated the distribution of α-skeletal (α-SKA), α-cardiac (α-CA), α-smooth muscle (α-SMA) actin isoforms and other muscle-typical proteins in the CS of human and rat hearts at different ages. SAN and IB cardiomyocytes were characterized by the presence of α-SMA, α-CA, calponin and caldesmon, whereas α-SKA and vimentin were absent. Double immunofluorescence demonstrated the co-localisation of α-SMA and α-CA in I-bands of SAN cardiomyocytes. AVN, AVB, BB and PF cardiomyocytes were α-SMA, calponin, caldesmon and vimentin negative, and α-CA and α-SKA positive. No substantial differences in actin isoform distribution were observed in human and rat hearts, except for the presence of isolated subendocardial α-SMA positive cardiomyocytes co-expressing α-CA in the ventricular septum of the rat. Aging did not influence CS cardiomyocyte actin isoform expression profile. These findings support the concept that cardiomyocytes of SAN retain the phenotype of a developing myogenic cell throughout the entire life span.     

Chick heart peptidergic innervation: localization and development

Localization and development of chick heart peptidergic innervation (Substance P, VIP and Somatostatin) were investigated by means of immunofluorescence technique. The peptidergic component of the heart innervation was observed, for the first time, in older than 11 day chick embryos, i.e., subsequently to the appearance of the cholinergic component. The peptidergic structures achieve nearly full development in about 16-17 day embryos. Substance P is the most represented of the three peptides. It is localized both in nerve bundle fibers and in isolated fibers within the myocardium, the pericardium, the vessel walls; it is also present in fibers of some heart base ganglia. VIP is mostly contained in some thick single fibers travelling along the vessel walls of the heart base, the myocardium and the pericardium. Some VIP immunoreactive cells were also observed in the base ganglia. Somatostatin is mostly contained in some ganglia cells, whilst thin Somatostatin-immunoreactive fibers form a rich plexus among the atrial and ventricular myofibers, without contacting the vessel walls.     

Immunolocalization of the substances P- and FMRFamide in the atrium of the snail <Emphasis Type="Italic">Achatina fulica</Emphasis>

The methods of immunohystochemistry and electron microscopic immunocytochemistry were applied for the investigation of the localization of the substances P (SP)- and FMRFamide in the auricle of the mollusc Achatina fulica. Nerve fibers that innervate a snail auricle are in close contact with the granular cells (GC) disposed among muscular and endocardial cells, forming the neuroendocrinal complexes. Both neuromediators were detected in the cells of these auricular neuroendocrinal complexes. The method of immunoperoxidase histochemistry has shown the localization of SP- and FMRFamide immunoreactive material in granules of auricular GCs. Electron microscopic immunocytochemistry has confirmed the presence of SP- and FMRFamide-immunoreactive material in GC granules; moreover, it has also shown its presence in the neurosecretory granules of nerve fibers, both located in neuroendocrinal complexes in contact with cardiomyocytes.     

Atrial and brain natriuretic peptide in right atrium myocytes in the postreperfusion period in rat

We have studied the accumulation and excretion of atrial (ANP) and brain (BNP) natriuretic peptides in the early and late postreperfusion period (60 min and 60 days) in the myocardium of the right atrium in rats. The model of total ischemia proposed by Korpachev et al. (1982) was used. Immunocytochemical localization of peptides in cardiomyocytes was performed on ultrathin sections using the polyclonal antibodies. The intensities of accumulation (excretion) of ANP and BNP were analyzed by counting the immunolabeled granules (types A and B) with a transmission electron microscope. At 60 min and 60 days of the postreperfusion period, an increase in the synthesis and release of ANP and BNP was found. A more pronounced BNP reaction could be explained by the fact that, under normal conditions, the main hormone of the natriuretic peptide system regulating blood pressure is ANP, while BNP regulates blood pressure in cardiovascular pathology.     

Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia. This work was supported by NIH grant HL65481 (R.L.P.) and HL54633 (D.B.H.). Use of the DeltaVision Restoration microscope was provided through the Imaging/Physiology Core supported by NIH Grant P20 RR16435 from the COBRE program of the National Center for Research Resources     

Changes in the myocardium of fetuses and newborn infants as a result of hypoxia

Myocardium has been investigated in 68 human fetuses and newborns suffered from intrauterine acute and prolonged hypoxia, occurred as a result of various maternal, fetal and placental diseases. Under acute hypoxia only reactive changes of the contractile myocardium appear. They are demonstrated as degeneration of muscle fibers and appearance of scattered foci of necrosis in cardiomyocytes. Under a prolonged hypoxia, besides analogous reactive changes, compensatory hypertrophy of some part of cardiomyocytes is noted; this is manifestation of a compensatory-adaptive reaction of the myocardium.     

突触体素、S-100蛋白、NSE免疫反应神经纤维在人淋巴结的分布

探讨突触体素、S－100蛋白、NSE免疫反应神经纤维在人淋巴结的分布,为淋巴结的神经免疫相互作用提供形态学资料。应用免疫组织化学ABC法观察人类腹股沟、腋窝、肠系膜、肺等淋巴结40例,10％福尔马林固定,石蜡包埋组织切片。结果显示：突触体素、S－100蛋白、NSE免疫反应神经纤维呈细丝状沿被膜和门部结缔组织小梁及血管进入皮质后主要分布于副皮质区,环境淋巴小结,进一步分支到达髓质。同时在淋巴小结发生中心及副皮质区有S－100蛋白免疫反应阳性细胞。在髓质髓窦内有NSE免疫反应阳性细胞。结论;淋巴结内有突触体素、S－100蛋白、NSE免疫反应神经纤维的支配、并有S－100蛋白、NSE免疫反应阳性细胞,为淋巴结的神经免疫相互作用提供形态学资料。     

Xenogenic cardiomyocytes transplantation for the treatment of curing acute myocardial infarction

The autogenic cardiomyocytes transplantation presents numerous challenges in clinical application, such as the difficulty to obtain the autogenic cells, etc. Therefore, it is necessary to investigate allogenic or xenogenic cardiomyocytes transplantation. In this study, the experimental rabbits with acute infarcted myocardium were randomly divided into 3 groups: the 7-day cultured cardiomyocytes group, the 2-day cultured cardiomyocytes group and the control group. Neonate rat cardiomyocytes were labeled by DAPI and then injected into the acute infarcted myocardium of rabbits. After transplantation, results showed that, compared to the control group, the survival number of grafted cardiomyocytes in the cultured group is significantly larger (P < 0.05), with the implanted cardiomyocytes parallel to the host myocardium in an aligning direction. However, compared to the control group, the ventricular wall of the two experimental groups is thicker and the condition of myocardial fibrosis is better, especially to 7-day cultured cardiomyocytes group. These results suggested that the transplantation of xenogenic cardiomyocytes into curing acute ischemic heart of animal model is possible.