Pathology and function of conduction tissue in Fabry disease cardiomyopathy.

BACKGROUND: Cardiac arrhythmias are common in Fabry disease (FD) and may occur in prehypertrophic cardiomyopathy suggesting an early compromise of conduction tissue (CT). Therefore, FD X-linked and CT may be variously involved in male and female patients with FD cardiomyopathy, affecting CT function. METHODS AND RESULTS: Among 74 patients with endomyocardial biopsy diagnosis of FD cardiomyopathy, 13 (6 men; 7 women; mean age, 50.1±13.5 years; maximal wall thickness, 16.7±3.7 mm) had CT included in histological specimens and 6 also at electron microscopy. CT glycolipid infiltration was defined as focal, moderate, extensive, or massive, if involved ≤30%, ≤50%, >50%, or 100% of cells; identified as loosely arranged small myocytes positive to HCN4 immunostaining, supplied by a centrally placed thick-walled arteriole. CT involvement was correlated with age, sex, and α-Gal gene mutation. CT function was evaluated by electrophysiological study and arrhythmias at Holter registration. CT infiltration was focal/moderate in 4 women with no arrhythmias and normal electrophysiological study, extensive in 3 women with atrial or ventricular arrhythmias and short HV interval, and massive in 6 men with atrial fibrillation or ventricular arrhythmias and short HV. Short PR/AH with increased refractoriness was additionally found in 3 patients with extensive/massive CT infiltration. A male patient with the shortest HV presented infra-Hissian block during decremental atrial stimulation. There was no correlation with age, maximal wall thickness, and type of gene mutation. CONCLUSIONS: CT infiltration in FD cardiomyopathy is constant in men and variable in women because of skewed X-chromosome inactivation; its extensive/massive involvement causes accelerated conduction with prolonged refractoriness and electric instability.

High resolution imaging of the human cardiac conduction system using reflectance confocal microscopy.

Rhythmical contraction of the heart is controlled by the cardiac conduction system (CCS) that consists of the three main parts: the sino-atrial node, the atrioventricular node and the His-Purkinje system. A heartbeat signal, originated from CCS, spreads through its branches to the different parts of the heart, initiating depolarization of the ventricles. However, this highly important system could not be distinguished visually from the surrounding heart tissues: myocardium (MC) and connective tissue (CT). Thus, during surgical procedures, CCS could be easily damaged; namely, the reliable method for identification of CCS either in vivo or ex vivo does not exist. Accordingly, there is a definite need for developing a CCS imaging method. Reflection confocal microscopy (RCM) offers non-destructive imaging of the tissue at depths of up to 0.35 mm with the capability of identification of a single cell. During the visualization procedure, a given tissue is illuminated with infrared laser light and the image is obtained because of different reflections from the tissue structures. However, the reflective structures in the heart tissues are still not identified. In the present study, for the first time we investigated cardiac tissues by RCM. The resolution of the method allowed us to distinguish MC cells and CCS cells. The method also allowed us to distinguish the network-like structures that are main components of CT. The ability to visualize different tissue components indicates a great potential for RCM to be used in non-destructive cardiac investigations and for imaging CCS.

The structure of the atrioventricular node in the heart of the female laying ostrich (Struthio camelus).

The electrical impulse for cardiac contraction is generated in the Sinoatrial node (SA node), subsequently spreads to the Atrioventricular node (AV node) and continues in the Atrioventricular bundle (AV bundle). The AV node may not always be present in different avian species and seems to differ in location and contents between species. In this study, the anatomy and histology of the AV node were studied five female adult ostriches (Struthio camelus). Routine paraffin sectioning and transmission electron microscopic method were performed. The study showed that in the ostrich, the AV node is located in the endocardium of the atrial surface of the right atrioventricular valve adjacent to the fibrous ring. The parenchyma of the AV node is formed by small specialized muscle fibres that are spread within a loose connective tissue network. The AV node is not covered by a connective tissue sheath and some arterioles are present. Nerve fibres are seen related to the node. Ultrastructurally, they stain lighter and contain fewer organized myofibrils than usual myocardial cells. The myofibril bundles run parallel to one another and have interspersed mitochondria, which display distinct cristae. The cells have a large euchromatic nucleus with a clear perinuclear area, and they connected by desmosomes. The ostrich is, thus, one of the birds that have the AV node, whose position varies from the other birds.

A ZASP missense mutation, S196L, leads to cytoskeletal and electrical abnormalities in a mouse model of cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a primary disease of the heart muscle associated with sudden cardiac death secondary to ventricular tachyarrhythmias and asystole. However, the molecular pathways linking DCM to arrhythmias and sudden cardiac death are unknown. We previously identified a S196L mutation in exon 4 of LBD3-encoded ZASP in a family with DCM and sudden cardiac death. These findings led us to hypothesize that this mutation may precipitate both cytoskeletal and conduction abnormalities in vivo. Therefore, we investigated the role of the ZASP4 mutation S196L in cardiac cytoarchitecture and ion channel biology. METHODS AND RESULTS: We generated and analyzed transgenic mice with cardiac-restricted expression of the S196L mutation. We also performed cellular electrophysiological analysis on isolated S196L cardiomyocytes and protein-protein interaction studies. Ten month-old S196L mice developed hemodynamic dysfunction consistent with DCM, whereas 3-month-old S196L mice presented with cardiac conduction defects and atrioventricular block. Electrophysiological analysis on isolated S196L cardiomyocytes demonstrated that the L-type Ca(2+) currents and Na(+) currents were altered. The pull-down assay demonstrated that ZASP4 complexes with both calcium (Ca(v)1.2) and sodium (Na(v)1.5) channels. CONCLUSIONS: Our findings provide new insight into the mechanisms by which mutations of a structural/cytoskeletal protein, such as ZASP, lead to cardiac functional and electric abnormalities. This work represents a novel framework to understand the development of conduction defects and arrhythmias in subjects with cardiomyopathies, including DCM.

The area composita of adhering junctions connecting heart muscle cells of vertebrates. VII. The different types of lateral junctions between the special cardiomyocytes of the conduction system of ovine and bovine hearts.

Postnatal development of mammalian cardiomyocytes in the working myocardium is characterized by a near-complete translocation of both kinds of adhering junctions (AJs), i.e. desmosomes and fasciae adhaerentes (FAs), to the polar intercalated disk (ID) regions where they cluster, fuse and molecularly amalgamate to extended hybrid intercellular junction structures, the area composita (composite junction; AC). Using immunofluorescence and immunoelectron microscopy we now report that the AJ structures of the conduction system, in particular those of the Purkinje fiber cells of cows and sheep are fundamentally different. Here the numerous AJs remain in lateral connections with other conductive cells. Desmosomal or desmosome-like junctions can still be distinguished from FA junctions, and a third type of AJs can be identified which shows colocalization of desmosomal and FA proteins, i.e. an AC character. These results, together with demonstrations of other cell type cytoskeletal markers such as alpha-cardiac actin and desmin, support the concept that conductive cells are derived from embryonal cardiomyocytes and are arrested at an early stage of differentiation. We also show that the conductive cells have extended plasma membrane regions characterized by an exceptionally high proportion of junctions with desmosomal character and proteins, amounting to 50% and more, resulting in the highest desmosome protein packing so far described in non-epithelial cells. The relevance of these junctions for the formation, maintenance and functions of the conductive system is discussed, together with the conclusion that the desmosome-rich regions of conductive cells are among the most vulnerable sites for functional disorders caused by desmosomal protein mutations.

Increased interstitial loading reduces the effect of microstructural variations in cardiac tissue.

Electrical propagation in diseased and aging hearts is strongly influenced by structural changes that occur in both the intracellular and interstitial spaces of cardiac tissue; however, very few studies have investigated how interactions between the two spaces affect propagation at the microscale. In this study, we used one-dimensional microstructural computer models of interconnected ventricular myocytes to systematically investigate how increasing the effective interstitial resistivity (rho(oeff)) influences action potential propagation in fibers with variations in intracellular properties such as cell coupling and cell length. Changes in rho(oeff) were incorporated into a monodomain model using a correction to the intracellular properties that was based on bidomain simulations. The results showed that increasing rho(oeff) in poorly coupled one-dimensional fibers alters the distribution of electrical load at the microscale and causes propagation to become more continuous. In the poorly coupled fiber, this continuous state is characterized by decreased gap junction delay, sustained conduction velocity, increased sodium current, reduced maximum upstroke velocity, and increased safety factor. Long, poorly coupled cells experience greater loading effects than short cells and show the greatest initial response to changes in rho(oeff). In inhomogeneous fibers with adjacent well-coupled and poorly coupled regions, increasing rho(oeff) in the poorly coupled region also reduces source-load mismatch, which delays the onset of conduction block and reduces the dispersion of repolarization at the transition between the two regions. Increasing the rho(oeff) minimizes the effect of cell-to-cell variations and may influence the pattern of activation in critical regimes characterized by low intercellular coupling, microstructural heterogeneity, and reduced or abnormal membrane excitability.

Spectroscopic studies of the human heart conduction system ex vivo: implication for optical visualization.

Fluorescence excitation and emission spectra of the heart tissues specimens have been measured ex vivo with the aim of finding out the optical differences characteristic for the human heart conduction system (the His bundle) and ventricular myocardium. The optimal conditions enhancing the spectral differences between the His bundle and myocardium were found by recording the fluorescence signal in the range from 420 nm to 465 nm under the excitation at wavelengths starting from 320 nm to 370 nm. In addition, the spectral differences between the His bundle and the connective tissue, which is often present in the heart, could be displayed by comparing the ratios of fluorescence intensities being measured at above 460 nm under the preferred excitation of elastin and collagen. The left and right branches of the His bundle were visualized ex vivo in the interventricular septum of the human heart under illumination at 366 nm.

Extracting intramural wavefront orientation from optical upstroke shapes in whole hearts.

Information about intramural propagation of electrical excitation is crucial to understanding arrhythmia mechanisms in thick ventricular muscle. There is currently a controversy over whether it is possible to extract such information from the shape of the upstroke in optical mapping recordings. We show that even in the complex geometry of a whole guinea pig heart, optical upstroke morphology reveals the 3D wavefront orientation near the surface. To characterize the upstroke morphology, we use V(F)(*), the fractional level at which voltage-sensitive fluorescence, V(F), has maximal time derivative. Low values of V(F)(*)( approximately 0.2) indicate a wavefront moving away from the surface, high values of V(F)(*) ( approximately 0.6) a wavefront moving toward the surface, and intermediate values of V(F)(*) ( approximately 0.4) a wavefront moving parallel to the surface. We further performed computer simulations using Luo-Rudy II electrophysiology and a simplified 3D geometry. The simulated V(F)(*) maps for free wall and apical stimulations as well as for sinus rhythm are in good quantitative agreement with the averaged experimental results. Furthermore, computer simulations show that the effect of the curvature of the heart on wave propagation is negligible.

Mechanism of origin of conduction disturbances in aging human atrial bundles: experimental and model study.

BACKGROUND: Aging is associated with a significant increase in atrial tachyarrhythmias, especially atrial fibrillation. A macroscopic repolarization gradient created artificially by a stimulus at one site before a premature stimulus from a second site is widely considered to be part of the experimental protocol necessary for the initiation of such arrhythmias in the laboratory. How such gradients occur naturally in aging atrial tissue is unknown. OBJECTIVE: The objective of this study was to determine if the pattern of cellular connectivity in aging human atrial bundles produces a mechanism for variable early premature responses. METHODS: Extracellular and intracellular potentials were recorded after control and premature stimuli at a single site in aging human atrial bundles. We also measured cellular geometry, the distribution of connexins, and the distribution of collagenous septa. A model of the atrial bundles was constructed based on the morphological results. Action potential propagation and the sodium current were analyzed after premature stimuli in the model. RESULTS: Similar extracellular potential waveform responses occurred after early premature stimuli in the aging bundles and in the model. Variable premature conduction patterns were accounted for by the single model of aging atrial structure. A major feature of the model results was that the conduction events and the magnitude of the sodium current at multiple sites were very sensitive to small changes in the location and the timing of premature stimuli. CONCLUSION: In aging human atrial bundles stimulated from only a single site, premature stimuli induce variable arrhythmogenic conduction responses. The generation of these responses is greatly enhanced by remodeling of cellular connectivity during aging. The results provide insight into sodium current structural interactions as a general mechanism of arrhythmogenic atrial responses to premature stimuli.

Effects of carvedilol on M2 receptors and cholinesterase-positive nerves in adriamycin-induced rat failing heart.

Heart failure is correlated with attenuation of parasympathetic nervous function and enhanced sympathetic activity. Carvedilol, a third-generation beta-blocker, may improve the prognosis of heart failure better than selective beta(1)-blockers. Not all of its effects, however, can be explained by direct actions on the sympathetic nervous system. This study was therefore performed to investigate the possible alterations of muscarinic cholinergic (M)(2) receptors and cholinesterase-positive nerves in different regions of the adriamycin-induced failing rat heart, and the potential effects of carvedilol on these M(2) receptors and cholinesterase-positive nerves. Karnovsky-Roots histochemical staining combined with point counting methods, and immunochemical streptavidin-biotin complex staining and image analysis were used to test the distribution of cholinesterase-positive nerves and the expression of M(2) receptors, respectively. Our results show that the cholinesterase-positive nerve system was downregulated in the adriamycin-induced failing heart group, while the density of M(2) receptors was increased in the carvedilol 3- and 10-mg/kg body weight groups, especially in the endocardial tissues of the left-ventricular free wall. It is concluded that upregulation of M(2) receptors may be one of the potential mechanisms by which carvedilol exert its action on heart failure.

Cardiomyocyte bridging between hearts and bioengineered myocardial tissues with mesenchymal transition of mesothelial cells.

BACKGROUND: For the reconstruction of 3-dimensional (3D) tissues, we exploited an original method of tissue engineering that layers individual cell sheets harvested from temperature-responsive culture dishes. Stacked cardiomyocyte sheets demonstrated electrical and morphologic communication, resulting in synchronously beating myocardial tissue. When these bioengineered 3D tissue grafts are transplanted onto damaged hearts, gap junction communication between graft and host is likely critical for synchronized beating and functional improvement. In this study, these graft-to-heart morphologic communications were examined. METHODS: Neonatal rat cardiomyocyte sheets were harvested from temperature-responsive culture dishes and layered to create 3D tissues. These constructs were then transplanted onto infarcted rat hearts. Histologic analyses and transmission electron microscopy (TEM) were performed to examine morphologic communications. The passage of small molecules through functional gap junctions was also detected using a dye-transfer assay. RESULTS: Transplanted cardiomyocytes bridged between the grafts and hearts in intact areas. Connexin-43 staining and TEM revealed the existence of gap junctions and intercalated disks between the bridging cardiomyocytes. Furthermore, it was confirmed that a low-molecule fluorescent dye, calcein, was transferred from the grafts to the hearts via the bridging cardiomyocytes. Immunohistochemistry with anti-intercellular adhesion molecule-1 antibodies revealed that mesothelial cells in the epicardium scattered and transdifferentiated into mesenchymal cells between the graft and host. CONCLUSIONS: The direct attachment of layered cardiomyocyte sheets on the heart surface promotes mesothelial cell transdifferentiation and cardiomyocyte bridging, leading to functional communication via gap junctions. These results indicate that these bioengineered myocardial tissues may improve damaged heart function via synchronized beating.

Emerin-lacking mice show minimal motor and cardiac dysfunctions with nuclear-associated vacuoles.

Emery-Dreifuss muscular dystrophy is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures, and cardiomyopathy with conduction block. The X-linked recessive form is caused by mutation in the EMD gene encoding an integral protein of the inner nuclear membrane, emerin. In this study, mutant mice lacking emerin were produced by insertion of a neomycin resistance gene into exon 6 of the coding gene. Tissues taken from mutant mice lacked emerin. The mutant mice displayed a normal growth rate indistinguishable from their littermates and were fertile. No marked muscle weakness or joint abnormalities were observed; however, rotarod test revealed altered motor coordination. Electrocardiography showed mild prolongation of atrioventricular conduction time in emerin-lacking male mice older than 40 weeks of age. Electron microscopic analysis of skeletal and cardiac muscles from emerin-lacking mice revealed small vacuoles, which mostly bordered the myonuclei. Our results suggest that emerin deficiency causes minimal motor and cardiac dysfunctions in mice with a structural fragility of myonuclei.

Cytoarchitecture and intercalated disks of the working myocardium and the conduction system in the mammalian heart.

Working and specialized cardiac myocytes and their intercalated disks (ID) in the mammalian heart were examined by transmission and scanning electron microscopy. The NaOH/ultrasonication treatment of cardiac tissues resulted in the digestion of collagen fibers and separation of intercellular junctions. Auricular and ventricular myocytes were cylindrical in shape, bifurcated, and connected end-to-end at the ID. The ID in the working myocardium showed a stair-like profile, consisting of steps (plicate segments) and corresponding risers (interplicate segments). The ventricular myocytes had many steps and risers. The steps were filled with numerous finger-like microprojections, including desmosomes, fasciae adherentes, and small gap junctions. The risers showed the smooth surface, including desmosomes and large gap junctions. The cell strands of the sinoatrial node were oriented linearly, while those of the atrioventricular node formed a reticular network. The ID in both nodal cells was underdeveloped, having few microprojections. Myocytes in the His bundle and its branches were arranged in parallel, and Purkinje cell strands formed reticular networks. The ID in the His-Purkinje system was irregular in appearance, and the microprojections were larger in size and smaller in number than those of working myocytes. There were few microprojections in the sheep Purkinje cells. The gap junctions in the conduction system were few or small in size in the nodal tissue, but large in the His-Purkinje system.

High resolution magnetic images of planar wave fronts reveal bidomain properties of cardiac tissue.

We magnetically imaged the magnetic action field and optically imaged the transmembrane potentials generated by planar wavefronts on the surface of the left ventricular wall of Langendorff-perfused isolated rabbit hearts. The magnetic action field images were used to produce a time series of two-dimensional action current maps. Overlaying epifluorescent images allowed us to identify a net current along the wavefront and perpendicular to gradients in the transmembrane potential. This is in contrast to a traditional uniform double-layer model where the net current flows along the gradient in the transmembrane potential. Our findings are supported by numerical simulations that treat cardiac tissue as a bidomain with unequal anisotropies in the intra- and extracellular spaces. Our measurements reveal the anisotropic bidomain nature of cardiac tissue during plane wave propagation. These bidomain effects play an important role in the generation of the whole-heart magnetocardiogram and cannot be ignored.

Significant damage of the conduction system during cardioplegic arrest is due to necrosis not apoptosis.

OBJECTIVES: Ventricular conduction disturbances following cardioplegic arrest remains a serious, yet unsolved problem. In the present study we examined whether myocardial conduction cells (MCC, Purkinje fibers) are more vulnerable to ischemia/reperfusion injury than working myocardial cells and whether the damage is due to necrosis or apoptosis. METHODS: Mini-pigs were subjected to 60 min of crystalloid (St Thomas; n = 15 group I) or blood (Buckberg; n = 6 group II) cardioplegic arrest followed by 3 h of reperfusion. Animals not subjected to either procedures served as controls (n = 5). Ventricular myocardial specimens were investigated by hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining and immunohistochemical labeling of apoptosis-associated proteins (Bax, Bcl-2, Caspase-3). DNA-breaks were visualized by in situ end labeling (terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling, TUNEL). Electron microscopy confirmed apoptosis or necrosis. RESULTS: MCC of control hearts intrinsically expressed Bax, Bcl-2, and Caspase-3 without signs of either apoptotic or necrotic damage. Subendocardial Purkinje fibers of groups I and II hearts exhibited focal damage, with reduced labeling of apoptosis-associated proteins, glycogen loss, karyopycnosis and increased eosinophilia (15/21 hearts). The majority of damaged MCC displayed nuclear TUNEL-positivity (2.8+/-2.5% of MCC), whereas the average TUNEL-rate of the adjacent working myocardium was low (<0.1%). Electron microscopy demonstrated ischemic changes in MCC consistent with cellular necrosis. CONCLUSIONS: Ischemia/reperfusion injury due to cardioplegic arrest inflicts significant damage on subendocardial MCC, but not on working myocardium. Ultrastructural and light-microscopic findings are consistent with coagulation necrosis, rather than apoptosis.

Alterations of cardiac innervation in evolving myocardial infarction and transplanted hearts. An anatomo-clinical reappraisal.

BACKGROUND: Debate regarding the alterations of the cardiac innervation in an evolving myocardial infarction and transplanted hearts is still raging and most studies are based on radionuclide uptake of neurotransmitters or on the evaluation of the cardiorespiratory reflex. METHODS: The present investigation, upon human autoptic specimens of 57 infarcts and 8 cardiac transplants, was carried out with traditional neuropathology and modern molecular biology techniques. The specimens were selected for the identification of neurons, nerve fibers and their sheaths. RESULTS: First of all, these techniques confirmed the gross difference in the vulnerability of infarcted myocytes if compared with the local innervation, the metabolism of which is infinitely less oxygen-dependent than that of working myocardium (approximate quantitation below). Delicate technicalities of the traditional silver impregnation for nerves usually yield a large incidence of artifacts. Thereby, only perfect results (20% of cases), corroborated by parallel nerve sheath immunostaining (70% of cases), were retained and documented herein. In the meantime, acidosis and free radicals increase, while catabolite accumulation supervenes. These three factors threaten myocardial viability. Thereby, nervelets can be seen to survive the hyperacute phase of ischemia, but may be in part damaged by the successive granulocytic-macrophage inflammation enzyme lysis of the infarcted muscle. The delayed and incomplete anatomical neural damage is confirmed by the observation of preserved nerve sheaths and neural filaments surviving in postinfarction scars, almost devoid of myocardiocytes. CONCLUSIONS: The rich sympatho-vagal cardiac network might further provide alternative bypasses for post-infarct reinnervation. The functional implications of this process remain unclear.

Evidence of specialized conduction cells in human pulmonary veins of patients with atrial fibrillation.

UNLABELLED: Specialized Conducting Cells in Human PV. INTRODUCTION: Depolarizations similar to those from the sinus node have been documented from the pulmonary veins after isolation procedures. We assessed the hypothesis that sinus node-like tissue is present in the pulmonary veins of humans. METHODS AND RESULTS: Pulmonary vein tissue was obtained from five autopsies (four individuals with a history of atrial fibrillation and one without a history of atrial arrhythmias) and five transplant heart donors. Autopsy veins were fixed in formaldehyde and processed for light microscopy to identify areas having possible conductive-like tissue. Areas requiring additional study were extracted from paraffin blocks and reprocessed for electron microscopy. Donor specimens were fixed in formaldehyde for histologic sections and glutaraldehyde for electron microscopy. Myocardial cells with pale cytoplasm were identified by light microscopy in 4 of the 5 autopsy subjects. Electron microscopy confirmed the presence of P cells, transitional cells, and Purkinje cells in the pulmonary veins of these cases. CONCLUSION: Our report is the first to show the presence of P cells, transitional cells, and Purkinje cells in human pulmonary veins. Whether these cells are relevant in the genesis of atrial fibrillation requires further study.