Identification of candidate genes for histiocytoid cardiomyopathy (HC) using whole genome expression analysis: analyzing material from the HC registry.

Histiocytoid cardiomyopathy (HC) is a rare but distinctive arrhythmogenic disorder characterized by incessant ventricular tachycardia, cardiomegaly, and often sudden death by age 2 years. The underlying genetic mechanism of HC has eluded researchers for decades. To further identify the potential molecular-genetic bases of HC, molecular analyses of HC hearts and hearts of age-matched controls were performed. Total RNA and genomic DNA were prepared from formalin-fixed, paraffin-embedded cardiac tissue from 12 cases of HC and 12 age-matched controls. To identify genes differentially expressed in HC, whole genome cDNA-mediated annealing, selection, extension, and ligation profiling was performed. TaqMan quantitative polymerase chain reaction confirmed changes in RNA expression. DNA copy number changes were measured by TaqMan copy number variant analysis. Analysis of differential gene expression in HC cases identified 2 significantly downregulated gene sets aligned sequentially along the genome. The 1st gene cluster consisted of genes S100A8 , S100A9 , and S100A12 at 1q21.3c, and the 2nd cluster consisted of genes IL1RL1 ( ST2 ), IL18R1 , and IL18RAP at 2q12.1a. Strong decreases in interleukin 33 expression were also observed. Decreases in copy number of the S100A genes were confirmed by TaqMan copy number variant assays. S100A genes are downstream of the p38-MAPK pathway that can be activated by interleukin 33 signaling. These data suggest a model in which the interleukin 33-IL1RL1/p38-MAPK/ S100A8-S100A9 axis is downregulated in HC cardiac tissue and provide several candidate genes on 1q21.3c and 2q12.1a for inherited mutations that may predispose individuals to HC.

Contribution of neuronal sodium channels to the cardiac fast sodium current INa is greater in dog heart Purkinje fibers than in ventricles.

OBJECTIVE: To determine the presence and the potential contribution of neuronal sodium channels to dog cardiac function. METHODS: We used a combination of electrophysiological (patch clamp), RT-PCR, biochemical and immunohistochemical techniques to identify and localize neuronal Na(+) channels in dog heart and determine their potential contribution to the fast sodium current. RESULTS: In all cardiac tissues investigated, Na(v)1.1, Na(v)1.2 and Na(v)1.3 transcripts were detected. In immunoblots, we found Na(v)1.1 and Na(v)1.2 proteins in the ventricle (V) and in Purkinje fibers (PF). Na(v)1.3 immunoblots suggested strong proteolytic activity against this isoform in the heart. Na(v)1.6 was not found in any of the tissues tested. Confocal immunofluorescence on cardiac myocytes showed that Na(v)1.1 was predominantly localized at the intercalated disks in V and PF and around the nucleus (V). Na(v)1.2 was only present at the Z lines (V). Consistent with the immunoblot data, an intense but diffuse intracellular staining was observed for Na(v)1.3. Na(v)1.6 fluorescence staining was faint and diffuse. Surprisingly, immunoblots indicated the presence of two Na(v)beta 2 variants: a 42-kDa protein that co-localized with Na(v)1.2 at the Z lines in V and a 34-kDa protein that co-localized with Na(v)1.1 at the intercalated disks in PF. In agreement with the biochemical data, electrophysiological results suggest that neuronal sodium channels generate 10+/-5% and 22+/-5% of the peak sodium current in dog ventricle and Purkinje fibers, respectively. CONCLUSIONS: Our results suggest that neuronal NaChs are more abundant in Purkinje fibers than in ventricles, and this suggests a role for them in cardiac conduction.

Expression of slow skeletal myosin heavy chain 2 gene in Purkinje fiber cells in chick heart.

In avian, there are three slow skeletal myosin heavy chain (MHC) isoforms, slow skeletal MHC 1, 2, and 3. While slow skeletal MHC 3 has been characterized, slow skeletal MHC 1 and 2 are not yet fully studied. To determine the complete sequence of slow skeletal MHC 2, we isolated six overlapping cDNA clones, each encoding a portion of chick slow skeletal MHC 2, using the reverse transcription polymerase chain reaction (RT-PCR). The entire slow skeletal MHC 2 cDNA consisted of 5927 nucleotides including a 104 bp 3'-untranslated region and encoded 1941 amino acids. Using one of the cDNA clones, we made a probe for in situ hybridization. We also used immunohistochemistry to localize slow skeletal MHC 2 in skeletal and cardiac tissues. These studies showed that in addition to its expected expression in the adult chicken slow skeletal muscle, slow skeletal MHC 2 was expressed in the subendocardial cluster of cells and around the blood vessels within the ventricle of late embryos and adults. This isoform was not expressed in the myocardium throughout the life of the chicken. Based on morphological criteria as well as rich desmin expression, we concluded that the subendocardial cluster of cells were Purkinje cells. Although the physiological significance of the slow skeletal MHC expression remains elusive at this time, this MHC isoform may be used as a specific marker for Purkinje cells.

Spatiotemporal and tissue specific distribution of apoptosis in the developing chick heart.

To investigate spatial and temporal distributions of apoptosis in the embryonic chick heart and its relation to different tissue types, we examined apoptosis in the embryonic chick heart from Hamburger and Hamilton stage 17 through 3 days after hatching. MF20 antibody, alpha-smooth muscle actin (SMA) antibody and EAP-300 antibody were applied to delineate specific cell types. During early development of the embryonic chick heart, very few apoptotic cells were detected. The first distinctive zone of apoptosis was observed in the outflow tract at stage 25. This focus was most prominent during septation of the pulmonary artery from the aorta (i.e., between stages 28 and 29), and diminished to virtually background level by stage 32, except in the subconal regions. Subsequently, remarkable apoptosis appeared in the atrioventricular cushions by stage 26, peaked at stages 29-31, and dropped significantly thereafter. Characteristic distribution patterns of apoptotic cells were also detected in the cardiac conduction tissues, including the His bundle, the bundle branches, and the ventricular trabeculae. After stage 36, cell death dropped to background level, except in developing coronary vessels. MF20 and TUNEL double staining revealed that apoptosis in cardiomyocytes was limited to a few specific regions, much less than in cushion tissues. SMA and TUNEL double staining demonstrated that vascular structures were the major foci of apoptosis from stage 40 to 44, whereas adjacent perivascular Purkinje cells displayed significantly less cell death at these stages. The characteristic spatiotemporal locations of apoptosis parallel the morphologic changes and tissue differentiation during heart development, suggesting that apoptosis is crucial to the transformation of the heart from a simple tube to a complex multichambered pump.

Calcium-activated Cl(-) current contributes to delayed afterdepolarizations in single Purkinje and ventricular myocytes.

BACKGROUND: The ionic mechanism underlying the transient inward current (I(ti)), the current responsible for delayed afterdepolarizations (DADs), appears to be different in ventricular myocytes and Purkinje fibers. In ventricular myocytes, I(ti) was ascribed to a Na(+)-Ca(2+) exchange current, whereas in Purkinje fibers, it was additionally ascribed to a Cl(-) current and a nonselective cation current. If Cl(-) current contributes to I(ti) and thus to DADs, Cl(-) current blockade may be potentially antiarrhythmogenic. In this study, we investigated the ionic nature of I(ti) in single sheep Purkinje and ventricular myocytes and the effects of Cl(-) current blockade on DADs. METHODS AND RESULTS: In whole-cell patch-clamp experiments, I(ti) was induced by repetitive depolarizations from -93 to +37 mV in the presence of 1 micromol/L norepinephrine. In both Purkinje and ventricular myocytes, I(ti) was inward at negative potentials and outward at positive potentials. The anion blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) blocked outward I(ti) completely but inward I(ti) only slightly. The DIDS-sensitive component of I(ti) was outwardly rectifying, with a reversal close to the reversal potential of Cl(-) currents. Blockade of Na(+)-Ca(2+) exchange by substitution of extracellular Na(+) by equimolar Li(+) abolished the DIDS-insensitive component of I(ti). DIDS reduced both DAD amplitude and triggered activity based on DADs. Conclusions-In both Purkinje and ventricular myocytes, I(ti) consists of 2 ionic mechanisms: a Cl(-) current and a Na(+)-Ca(2+) exchange current. Blockade of the Cl(-) current may be potentially antiarrhythmogenic by lowering DAD amplitude and triggered activity based on DADs.

Repolarizing K+ currents in rabbit heart Purkinje cells.

1. Electrophysiological experiments on single myocytes obtained from Purkinje fibres and ventricular tissue of adult rabbit hearts were done to compare the contributions of three potassium (K+) currents to the action potentials in these two tissues. 2. In Purkinje cells reductions in extracellular potassium, [K+]o, from normal (5.4 mM) to 2.0 mM resulted in a large hyperpolarization and marked lengthening of the action potential. In ventricular myocytes, these changes were much less pronounced. Voltage clamp measurements demonstrated that these differences were mainly due to a much smaller inward rectifier K+ current, IK1, in Purkinje cells than in ventricular myocytes. 3. Application of 4-aminopyridine (4-AP, 2 mM) showed that all Purkinje cells exhibited a very substantial Ca2+-independent transient K+ outward current, It. 4-AP significantly broadened the early, rapid repolarization phase of the action potential. 4. Selective inhibitors of the fast component, IK, r (MK-499, 200 nM) and the slow component IK,s (L-735821 (propenamide), 20 nM) of the delayed rectifier K+ currents both significantly lengthened the action potential, suggesting that these conductances are present, but very small (< 20 pA) in Purkinje cells. Attempts to identify time- and voltage-dependent delayed rectifier K+ current(s) in Purkinje cells failed, although a slow delayed rectifier was observed in ventricular myocytes. 5. These results demonstrate significant differences in action potential waveform, and underlying K+ currents in rabbit Purkinje and ventricular myocytes. Purkinje cells express a much smaller IK1, and a larger It than ventricular myocytes. These differences in current densities can explain some of the most important electrophysiological properties of these two tissues.

Natriuretic peptide immunoreactivity in nerve structures and Purkinje fibres of human, pig and sheep hearts.

Atrial natriuretic peptide is a well-described peptide in cardiac Purkinje fibres and has been shown to interfere with the autonomic regulation in the heart of various species, including man. Recently, we detected immunoreactivity for the peptide in intracardial ganglionic cells and nerve fibre varicosities of bovine hearts, by the use of a modified immunostaining technique that induced an improved detection of natriuretic peptides. These findings raised the question as to whether natriuretic peptides are detectable in these tissues in man and other species. The conduction system from human, pig and sheep hearts was dissected processed with antisera against atrial natriuretic peptide and the closely related brain natriuretic peptide. Immunostaining for the brain natriuretic peptide was detected in some Purkinje fibres in all of these species. Interestingly, in pig, sheep and human hearts, some ganglionic cells and nerve fibres showed atrial natriuretic peptide immunoreactivity, particularly in the soma of human ganglionic cells. This is the first study showing immunoreactivity for the atrial natriuretic peptide in nerve structures and for the brain natriuretic peptide in Purkinje fibres of the human heart. The results give a morphological correlate for the documented effects of atrial natriuretic peptide on the heart autonomic nervous system and for the presumable effects of brain natriuretic peptide in the conduction system of man.

Immunohistochemical delineation of the conduction system. II: The atrioventricular node and Purkinje fibers.

Using an antibody that reacts specifically with the myocytes of the conduction system of the bovine heart, we have studied the atrioventricular node and the spatial distribution of the Purkinje fibers in the bovine heart. This study was complemented by studying the distribution of the gap junction protein connexin43 in these areas in the bovine heart and in the human heart. The large Purkinje fibers in the bovine heart are arranged in a two-dimensional network underneath the endocardium. At discrete sites, these fibers branch to the Purkinje fibers situated between the muscle bundles of the ventricular mass. These intramural Purkinje fibers are arranged in sheets that form a complex three-dimensional network of lamellas. Contacts with the ventricular myocytes are found throughout the myocardial wall, with the exception of a subepicardial layer of 2-mm thickness, ie, 10% to 15% of the wall thickness. The spatial arrangement of the Purkinje fibers correlates well with data on electrophysiology. Connexin43 was not detected in the myocytes of the atrioventricular node, whereas in the Purkinje fibers of the atrioventricular bundle and of the bundle branches, abundant expression of connexin43 was found in both humans and cows. In the bovine Purkinje fibers, a remarkable subcellular distribution of connexin43 is found: it occupies the entire plasma membrane facing other Purkinje cells but not that facing the surrounding connective tissue. The structural differences in architecture of the ventricular conduction system in humans and cows seems not to result in substantial differences in conduction velocities. However, the Purkinje fiber network in the bovine heart may explain the efficient ventricular excitation, as reflected by the relatively short QRS complex compared with that in the human heart, where intramural Purkinje fibers are not found.

Inositol 1,4,5-trisphosphate receptor in heart: evidence for its concentration in Purkinje myocytes of the conduction system.

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers capable of releasing Ca2+ from sarcoplasmic reticulum/ER subcompartments. The mRNA encoding the intracellular IP3 receptor (Ca2+ channel) has been detected in low amounts in the heart of various species by Northern blot analysis. The myocardium, however, is a heterogeneous tissue composed of working myocytes and conduction system cells, i.e., myocytes specialized for the beat generation and stimulus propagation. In the present study, the cellular distribution of the heart IP3 receptor has been investigated. [3H]IP3 binding experiments, Western blot analysis and immunofluorescence, with anti-peptide antibodies specific for the IP3 receptor, indicated that the majority of Purkinje myocytes (the ventricular conduction system) express much higher IP3 receptor levels than atrial and ventricular myocardium. Heterogeneous distribution of IP3 receptor immunoreactivity was detected both at the cellular and subcellular levels. In situ hybridization to a riboprobe generated from the brain type 1 IP3 receptor cDNA, showed increased accumulation of IP3 receptor mRNA in the heart conduction system. Evidence for IP3-sensitive Ca2+ stores in Purkinje myocytes was obtained by double immunolabeling experiments for IP3 receptor and cardiac calsequestrin, the sarcoplasmic reticulum intralumenal calcium binding protein. The present findings provide a molecular basis for the hypothesis that Ca2+ release from IP3-sensitive Ca2+ stores evoked by alpha 1-adrenergic stimulation is responsible for the increase in automaticity of Purkinje myocytes (del Balzo, U., M. R. Rosen, G. Malfatto, L. M. Kaplan, and S. F. Steinberg. 1990. Circ. Res. 67:1535-1551), and open new perspectives in the hormonal modulation of chronotropism, and generation of arrhythmias.

Immunoelectron microscopic visualization of the gap junction protein connexin 40 in the mammalian heart.

In mammalian myocardium, myocytes are electrically coupled by gap junctions, which are collections of membrane channels spanning the intercellular gap. Each channel is composed of twelve protein molecules--six in each membrane--enclosing a pore of 2 nm diameter. In addition to the main cardiac gap junction protein connexin 43 (cx43), two other constituents, connexin 40 (cx40) and connexin 45 (cx45), recently have been identified. In this study, the ultrastructural distribution of cx40 has been traced in several mammalian cardiac tissues, using a specific antibody and immunogold labeling. In rabbit, cx40 was found in gap junctions between myocytes of the right atrium as well as in the false tendons of the left ventricle. Labeling within the free ventricular wall could be obtained only with higher primary antibody concentrations. In rat atrial and ventricular working myocardium a similar distribution of label was observed. Double labeling of cx40 and cx43 in rat atrium, with 5 nm and 15 nm gold particles respectively, demonstrates limited patches of cx40 in gap junctions otherwise homogeneously labeled for cx43. Since cx40 has a much higher single channel conductance than cx43, topographical differences in their relative distribution might contribute to regional differences in conduction velocity of the cardiac impulse.

Dystrophin-glycoprotein complex and laminin colocalize to the sarcolemma and transverse tubules of cardiac muscle.

The expression and subcellular distribution of the dystrophin-glycoprotein complex and laminin were examined in cardiac muscle by immunoblot and immunofluorescence analysis of rabbit and sheep papillary muscle. The five dystrophin-associated proteins (DAPs), 156-DAG, 59-DAP, 50-DAG, 43-DAG, and 35-DAG, were identified in rabbit ventricular muscle and found to codistribute with dystrophin in both papillary myofibers and Purkinje fibers. The DAPs and dystrophin codistributed not only in the free surface sarcolemma but also in interior regions of the myofibers where T tubules are present. Neither the DAPs nor dystrophin were detected in intercalated discs, a specialized region of cardiac sarcolemma where neighboring myocardial cells are physically joined by cell-cell junctions. Similarly, in bundles of Purkinje fibers, which lack T tubules, DAPs and dystrophin were also found to codistribute at the free surface sarcolemma but were not detected either in the region of surface sarcolemma closely apposed to a neighboring Purkinje fiber or in interior regions of these myofibers. Comparison between the distribution of the dystrophin-glycoprotein complex and laminin showed that laminin codistributes with the components of this complex in both papillary myofibers and Purkinje fibers. These results are consistent with previous findings demonstrating that the extracellularly exposed 156-DAG (dystroglycan) of the skeletal muscle dystrophin-glycoprotein complex binds laminin, a component of the basement membrane. Although we demonstrate that DAPs, dystrophin, and laminin colocalize to the sarcolemma in rabbit and sheep papillary myofibers as they do in skeletal myofibers, the most striking difference between the subcellular distribution of these proteins in cardiac and skeletal muscle is that the dystrophin-glycoprotein complex and laminin also localize to regions of the fibers where T tubules are distributed in cardiac but not in skeletal muscle. These results imply that the protein composition and thus possibly some functions of T tubules in cardiac muscle are distinct from those of skeletal muscle.

Cardiac expression of polysialylated NCAM in the chicken embryo: correlation with the ventricular conduction system.

The neural cell adhesion molecule (NCAM) and its polysialic acid moeity (PSA) affect cellular interactions during the development of the nervous system and skeletal muscle. NCAM has also been identified in the embryonic heart of various species including humans. However, knowledge regarding the role of NCAM and its function-modulating PSA in cardiogenesis is limited. The distribution of NCAM and its PSA in the ventricular myocardium of chicken embryos was determined by indirect immunofluorescence staining. The NCAM polypeptide was found throughout the cardiac myocardium. In contrast PSA was located in discrete regions in stage 20 to 44 embryos (during and after septation). Myocardium at the subendocardial regions of the atrioventricular canal and ventricular trabeculae were PSA positive by stage 20. At later stages, transverse sections of the postseptation heart just below the level of the atrioventricular interface revealed a PSA-positive bundle of myocardium in the septum. This bundle was continuous with two branches at a more apical level which in turn were continuous with the PSA-positive subendocardial myocardium lining the left and right ventricles. This pattern of PSA in the myocardium was similar to that of the ventricular conduction system configuration defined in the adult heart. Electron micrographs of the subendocardium of the ventricular septum revealed PSA positivity on myofibril-containing cells with the ultrastructural location of Purkinje fibers. At later stages (35-44) a subset of cells within PSA-positive regions was stained by an antibody against an isoform of the myosin heavy chain found in adult Purkinje fibers. These cells and surrounding tissue lacked PSA in the adult heart. Thus polysialylated NCAM may be modulating cell-cell interactions during the development of the ventricular conduction system.

Demonstration of connective tissue sheaths surrounding working myocardial cells and Purkinje cells of the sheep moderator band.

Morphological studies were carried out to delineate the characteristics of connective tissue sheaths surrounding working myocardial cells and Purkinje cells in the moderator band of adult sheep hearts, using a series of techniques including silver staining, immunohistochemistry, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For SEM, tissue blocks were treated with 2N NaOH at room temperature to digest cellular elements. Individual working myocardial cells were ensheathed by thin argyrophil fibers (reticular fibers), while fascicles of 4-8 Purkinje cells (Purkinje strands) were encircled by rather thick reticular fibers. Some collagen fibers were located between masses of myocardial cells as well as between the Purkinje strands. Immunohistochemical analyses indicated that reticular fibers directly surrounding both myocardial cells and Purkinje strands showed moderately positive reactions for anti-type I collagen and intensely positive reactions for anti-type III collagen. Deserves particular note is that the three-dimensional architecture of the reticular sheaths varied widely at different places. The thin reticular sheaths surrounding each myocardial cell consisted of fibrils which were arranged in a coarse network and directed circularly along the long axis of cells. By contrast, the sheaths enclosing Purkinje strands were thicker, and their reticular fibrils were woven into a compact "felt-like" texture. The functional significance of these connective tissue sheaths is also discussed.

In situ localization of transforming growth factor beta 1 in porcine heart: enhanced expression after chronic coronary artery constriction.

We investigated the expression of transforming growth factor beta 1 (TGF-beta 1), a polypeptide differentiation factor probably associated with angiogenic properties in chronically hypoperfused heart tissue. A slowly swelling ameroid constrictor was implanted around the coronary circumflex artery (CX) of young domestic pigs. Two to three weeks after, significant CX stenosis of more than 90% and coronary collateralization could be demonstrated angiographically. The CX dependent experimental myocardial tissue (E) was investigated, with the LAD dependent area of the same pig serving as a control (C). We found significantly enhanced TGF-beta 1 mRNA expression by northern blot hybridization in the experimental myocardium (E) of those pigs with demonstrable coronary collaterals in the absence of a major myocardial infarction. The presence of TGF-beta 1 protein could be demonstrated quantitatively in extracts of the experimental and the control area by immunoblot analysis. By in situ techniques, TGF-beta 1 mRNA and protein could be localized predominantly in cardiac myocytes. We conclude that one adaptive mechanism of the pig heart in chronic coronary artery constriction is the enhanced expression of TGF-beta 1. Cardiac myocytes are a major source of TGF-beta 1. The observed coronary collateralization could be mediated-at least in part-by the angiogenic properties of TGF-beta 1.

Persistence of an embryonic intermediate filament-associated protein in the smooth muscle cells of elastic arteries and in Purkinje fibres.

During differentiation of most myogenic tissues, vimentin is transiently expressed as the intermediate filament (IF) protein subunit and is progressively replaced by desmin. However, smooth muscle cells of mature vascular tissue contain variable amounts of vimentin whose cellular content decreases as function of the distance from the heart. IFAPa-400 is a developmentally regulated IF crosslinker protein whose expression appears to parallel that of vimentin during chick myogenesis. Immunohistological and immunoblot techniques were employed to study the expression of this protein in cardiac and vascular smooth muscle cells of the adult chicken. As observed for vimentin, the expression of IFAPa-400 persists in the mature smooth muscle cells of the large arteries as they leave the heart. However, both of these proteins are down-regulated according to a proximo-distal gradient with respect to the distance from the heart. Conversely, desmin is much more abundant in the distal segments of the aorta. Thus, the co-ordinate expression of vimentin with IFAPa-400 may be a characteristic feature of elastic vascular tissue in which it could meet mechanical requirements close to those of the embryonic cells expressing them. This hypothesis is supported by the observation that the single cell type which continues to express the vimentin-IFAPa-400 combination in the mature heart is the Purkinje fibres, which are also subjected to high mechanical tensions but in which myofibrils are generally sparse compared to working myocytes.

Brain-associated glycoproteins expressed in bovine heart Purkinje fibres.

The Purkinje fibres of bovine heart were investigated immunohistochemically by use of monoclonal antibodies with specificity against the glycoproteins Thy-1 and Gp120, expressed in human brain. The existence and expression in bovine tissues (brain and thymus) of antigens displaying similar properties and immunochemical crossreactivity with monoclonal antibodies against the human antigens were confirmed. Both these antigens, as identified by use of anti Thy-1 and anti-Gp120 monoclonal antibodies were found to be associated with the membranes of the impulse conduction system. The presence of the antigens was seen in areas facing other conduction cells. No parts of the cells facing the basal membrane of the fibres were stained. The continuous staining between the cells was different from that of desmosome related proteins. These findings may have physiological and functional implications and are interesting in relation to recent evidences suggesting that the conduction tissue might be a derivative of the neural crest.

Fine structure of subendocardial (Purkinje) cells of the insect-eating bat (Pipistrellus pipistrellus).

Subendocardial cells of the right ventricular myocardium of the West African bat, Pipistrellus pipistrellus, were investigated at the ultrastructural level. The prominent features of these cells include a well-developed T-tubule system and numerous mitochondria with closely packed cristae. Additionally, the cells display large stores of lipid bodies. These unusual features confirm that Purkinje cells are heterogeneous in structural detail. From the paucity and poor structure of myofibrils of these cells, it is likely that the T-tubules may have a primary nutritional role in the regulation of electrolytes in an animal in which the cardiac cycle is particularly rapid. The well-developed mitochondria and the large stores of lipid bodies are appropriate for such active cells in which metabolism is probably of the aerobic type.