Desmin is essential for the structure and function of the sinoatrial node: implications for increased arrhythmogenesis.

Our objective was to investigate the effect of desmin depletion on the structure and function of the sinoatrial pacemaker complex (SANcl) and its implication in arrhythmogenesis. Analysis of mice and humans (SANcl) indicated that the sinoatrial node exhibits high amounts of desmin, desmoplakin, N-cadherin, and ß-catenin in structures we call "lateral intercalated disks" connecting myocytes side by side. Examination of the SANcl from an arrhythmogenic cardiomyopathy model, desmin-deficient (Des-/-) mouse, by immunofluorescence, ultrastructural, and Western blot analysis showed that the number of these lateral intercalated disks was diminished. Also, electrophysiological recordings of the isolated compact sinoatrial node revealed increased pacemaker systolic potential and higher diastolic depolarization rate compared with wild-type mice. Prolonged interatrial conduction expressed as a longer P wave duration was also observed in Des-/- mice. Upregulation of mRNA levels of both T-type Ca2+ current channels, Cav3.1 and Cav3.2, in the Des-/- myocardium (1.8- and 2.3-fold, respectively) and a 1.9-fold reduction of funny hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 could underlie these functional differences. To investigate arrhythmogenicity, electrocardiographic analysis of Des-deficient mice revealed a major increase in supraventricular and ventricular ectopic beats compared with wild-type mice. Heart rate variability analysis indicated a sympathetic predominance in Des-/- mice, which may further contribute to arrhythmogenicity. In conclusion, our results indicate that desmin elimination leads to structural and functional abnormalities of the SANcl. These alterations may be enhanced by the sympathetic component of the cardiac autonomic nervous system, which is predominant in the desmin-deficient heart, thus leading to increased arrhythmogenesis.NEW & NOTEWORTHY The sinoatrial node exhibits high amounts of desmin and desmoplakin in structures we call "lateral intercalated disks," connecting side-by-side adjacent cardiomyocytes. These structures are diminished in desmin-deficient mouse models. Misregulation of T-type Ca2+ current and hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 was proved along with prolonged interatrial conduction and cardiac autonomic nervous system dysfunction.

Telocytes in the human sinoatrial node.

The sinoatrial node (SAN) is composed mostly of pacemaker, transitional and Purkinje-like cells. Pacemaker cells, especially in the centre of the SAN, are surrounded by dense fibrous tissue and do not have any contact with transitional cells. We hypothesize that the SAN contains telocytes that have contacts with pacemaker cells and contractile myocardium. Immunohistochemistry using antibodies against HCN4 and antibody combinations against CD34 and HCN4 was carried out on 12 specimens. Confocal laser scanning microscopy (CLSM) with two mixtures of primary antibodies, namely CD34/S100 and vimentin/S100, was performed in three cases. In two cases, CLSM was carried out with CD117 antibody. Specimens for electron microscopy and immunocytochemistry with HCN4 immunogold labelling were taken from another three patients. In our study, we found cells with the immunophenotype of telocytes in the SAN. There were twice as many of these cells in the centre of the SAN as in the periphery (20.3 ± 4.8 versus 10.8 ± 4.4 per high-power field). They had close contact with pacemaker cells and contractile cardiomyocytes and expressed HCN4. The ultrastructural characteristics of these cells are identical to those of telocytes observed earlier in other organs. Our study provides evidence that telocytes are present in the SAN.

Biochemical and biomechanical properties of the pacemaking sinoatrial node extracellular matrix are distinct from contractile left ventricular matrix.

Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.

Desmosomal junctions are necessary for adult sinus node function.

AIMS: Current mechanisms driving cardiac pacemaker function have focused on ion channel and gap junction channel function, which are essential for action potential generation and propagation between pacemaker cells. However, pacemaker cells also harbour desmosomes that structurally anchor pacemaker cells to each other in tissue, but their role in pacemaker function remains unknown. METHODS AND RESULTS: To determine the role of desmosomes in pacemaker function, we generated a novel mouse model harbouring cardiac conduction-specific ablation (csKO) of the central desmosomal protein, desmoplakin (DSP) using the Hcn4-Cre-ERT2 mouse line. Hcn4-Cre targets cells of the adult mouse sinoatrial node (SAN) and can ablate DSP expression in the adult DSP csKO SAN resulting in specific loss of desmosomal proteins and structures. Dysregulation of DSP via loss-of-function (adult DSP csKO mice) and mutation (clinical case of a patient harbouring a pathogenic DSP variant) in mice and man, respectively, revealed that desmosomal dysregulation is associated with a primary phenotype of increased sinus pauses/dysfunction in the absence of cardiomyopathy. Underlying defects in beat-to-beat regulation were also observed in DSP csKO mice in vivo and intact atria ex vivo. DSP csKO SAN exhibited migrating lead pacemaker sites associated with connexin 45 loss. In vitro studies exploiting ventricular cardiomyocytes that harbour DSP loss and concurrent early connexin loss phenocopied the loss of beat-to-beat regulation observed in DSP csKO mice and atria, extending the importance of DSP-associated mechanisms in driving beat-to-beat regulation of working cardiomyocytes. CONCLUSION: We provide evidence of a mechanism that implicates an essential role for desmosomes in cardiac pacemaker function, which has broad implications in better understanding mechanisms underlying beat-to-beat regulation as well as sinus node disease and dysfunction.

[Autonomous cardioneuropathy in patients with coronary heart disease].

This paper reports the results of an electron microscopic study of the sinoatrial node in deceased patients with CHD in whom high-resolution rhythmocardiography revealed autonomous cardioneuropathy. Non-specific morphological changes in pacemaker cells of the sinoatrial node (swelling and disintegration of mitochondria, incorporation of calcium and accumulation of lipofuscin in mitochondria) can be regarded as manifestations of ischemic insufficiency of cardiac tissue perfusion. The rhythmocardiograms of such patients showed signs of autonomous cardioneuropathy manifest as the sharply decreased amplitude of all vegetative waves. The spectral power pattern was dominated by inefficient humoral-metabolic regulation due to suppression of protective parasympathetic regulation.

Morphological study of the sinus node and its artery in yak.

The sinus node of yak has been studied by the histological methods and transmission electron microscopy. The sinus node artery of yak was also determined by the injection-corrosion casting technique, the angiography, and histological methods. The results showed that the sinus node of yak contained an extensive framework of collagen and two main type cells: pacemaker cells (P cells) and transitional cells (T cells). The P cells had a perinuclear clear zone, contained less myofibrils, and appeared smaller mitochondria than T cells. The T cells were longer and slender than P cells, and had a variety of shapes. At the periphery of sinus node there were many nerve fibers and ganglions. Gap junction did not reveal reaction with anti-connexin43, but it was detected by electron microscopy in the central part of sinus node of yak. The sinus node artery of yak originated from left coronary artery more frequently (98%) than by right (2%). The artery located at the periphery of sinus node. It had an internal elastic membrane throughout its course, and a large nerve bundle was found running in a longitudinal direction.

Protective effects of adenosine in rabbit sinoatrial node ischemia-reperfusion model in vivo: control of arrhythmia by hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channels.

Disturbance of cardiac rhythm is one of the consequences of myocardial ischemia/reperfusion injury. Many researchers have prompted considerable interests in developing therapeutic approaches for its control. In present study, we want to determine whether that adenosine pre- and postconditioning have protective effects on sinoatrial node ischemia/reperfusion injury on morphology, arrhythmia score, serological markers (CK-MB and cTnT), SOD activities, MDA levels and expression of HCN4 channels in SA node cells. According to the arrhythmia score recorded, whether adenosine used in terms of ischemia or reperfusion, the total number of arrhythmia was significantly reduced, as well as the number of its episodes was also markedly decreased. We have also shown a clear correlation between HCN4 channels expression and the dysfunction of SA node cells. HCN4 immunoreactivity decreased after adenosine pre- and postconditioning, but changes were significantly smaller in the cells of the SA node compared with cells of I/R group. The content of cTnT, CK-MB and MDA in adenosine pre- and postconditioning group reduced significantly; but the level of SOD increased significantly. Histological examination and electron microscopy observations found in adenosine pre- and postconditioning group sinoatrial node injury also mitigated. These findings suggested that adenosine pre- or postconditioning were to reduce the incidence of ischemia/reperfusion arrhythmias, reduce myocardial ischemia reperfusion injury. The mechanism was to stabilize the SA node cells membrane and one possible mechanism involves modulation of HCN4 channels in pacemaker cells of the sinoatrial node.

Advancement in the examination of the human cardiac sinus node: an unexpected architecture and a novel cell type could interest the forensic science.

We have investigated the morphology of the sinus node of the human cardiac conduction system. Until today the sinus node (SN) is described as a heterogeneous system composed of 2 types of cells, namely, P or pale and T or transitional cells which are immersed in the matrix around the sinus nodal artery. T cells are said to share characteristics of P cells and of peripheral working atrial myocardial cells. This study was carried out on autoptic and explanted specimens using histochemical, immunohistochemical, and electron microscopic methods.Our investigations show that SN tissue has a quite different cellular composition, ie, spherical and/or star-shaped cells organized in clusters with long cytoplasmic processes (type P), transitional cells, similar to myocytes but with a reduced number of sarcomeres (type T) and, finally, as yet not described in the literature, fibroblast-like cells with long bi-tripolar extensions contacting cells. Interestingly, SN is squared by connective and elastic fibers geometrically arranged. Immunohistochemistry shows that the 3 cell types of the SN node express mesenchymal markers revelatory of their embryological origin. Innervation appears to be more complex than previously thought; we identified a system of synaptophysin-positive cholinergic vesicles dependent on the sympathetic system and parasympathetic fibers expressing S100 protein.Overall results indicate that the SN has an unexpected, systematic architecture.

[Analysis of cardiac rhythm variability in patients with sinoatrial node dysfunction].

The study was designed to elucidate the wave structure of heart rhythm variability (HRV) for the assessment of vegetative regulation of pacemaker activity of the sinoatrial node in 362 patients with coronary heart disease. The results were compared with the data of routine cardiologic examination. Sinus node dysfunction and weakness were shown to be accompanied by differential autonomous dysregulations that may be used as additional symptoms for the diagnosis of arrhythmias. Precision analysis of HRV permits to evaluate the arrhythmogenic events behind vegetative regulation of the sinus node, hemodynamic significance of arrhythmic episodes, and dependence of sinoatrial blockade on the degree of autonomous dysregulation. Autonomous cardioneuropathy syndrome is distinguished, its association with necrobiotic changes in the sinus node is demonstrated by electron microscopy.

[Experimental study on influence of ischemia-reperfusion on expression of hyperpolarization activated cyclic nucleotide gated cation channel 4 of sinoatrial node cells in rabbits in vivo].

OBJECTIVE: To study the influence of ischemia-reperfusion on the expression of the hyperpolarization activated cyclic nucleotide gated cation channel 4 (HCN4) and to discuss the mechanism of functional disturbance of sinoatrial node tissue (SANT) after ischemia reperfusion injury (IRI). METHODS: Eighty five healthy adult rabbits, weighing 2-3 kg, were randomly divided into 3 groups: control group [a suture passed under the root section of right coronary artery (RCA) without ligation, n=5], experimental group A (occluding the root section of RCA for 30 minutes, then loosening the root 2, 4, 8 and 16 hours, n=10), experimental group B (occluding the root section of RCA for 1 hour, then loosening the root 2, 4, 8 and 16 hours, n=10). At the end of the reperfusion, the SANT was cut off to do histopathological, transmission electron microscopical and immunohistochemical examinations and semi-quantitative analysis. RESULTS: The result of HE staining showed that patho-injure of sinoatrial node cell (SANC) happened in experimental groups A and B after 2 hours of reperfusion, the longer the reperfusion time was, the more serious patho-injure of SANC was after 4 and 8 hours of reperfusion, SANC reached peak of damage after 8 to 16 hours of reperfusion; patho-injure of SANC was more serious in experimental group B than in experimental group A at the same reperfusion time. Immunohistochemical staining showed that the expression of HCN4 located in cellular membrane and cytoplasm in the central area of SANC and gradually decreased from the center to borderline. The integral absorbance values of HCN4 expression in the control group (397.40 +/- 34.11) was significantly higher than those in the experimental group A (306.20 +/- 35.77, 216.60 +/- 18.59, 155.40 +/- 19.11 and 135.00 +/- 12.30) and in the experimental group B (253.70 +/- 35.66, 138.70 +/- 13.28, 79.10 +/- 9.60 and 69.20 +/- 8.42) after 2, 4, 8 and 16 hours of reperfusion (P < 0.05). With reperfusion time, the expression of HCN4 of SANC decreased, which was lowest after 8 hours of reperfusion; showing significant difference among 2, 4 and 8 hours after reperfusion (P < 0.05) and no significant difference between 8 and 16 hours after reperfusion (P > 0.05). At the same reperfusion time, the expression of HCN4 was higher in the experimental group A than in the experimental group B. The result of transmission electron microscope showed that ultramicrostructure of SANC was damaged after reperfusion in experimental groups A and B. The longer the reperfusion time was, the more serious ultramicrostructure damage of SANC was, and reached the peak of damage after 8 hours of reperfusion. Ultramicrostructure of SANC was not different between 8 and 16 hours of reperfusion. At the same reperfusion time, the ultramicrostructure damage of SANC was more serious in experimental group B than in experimental group A. CONCLUSION: IRI is harmful to the morphous and structure of SANC, and effects the expression of HCN4 of SANC, which is concerned with functional disturbance and arrhythmia.

[Complex morpho-functional characteristic of adrenal medulla and cardiac sino-auricular area in dogs after 30 day hypokinesia].

The aim of this study was to characterize tissue components of adrenal medulla and cardiac sino-auricular area in dogs exposed to hypokinesia for 30 days. The material was studied using the methods of light and electron microscopy. The quantitative and qualitative morphological features indicative of high functional activity of both structures were detected at tissue, cellular and subcellular level. Cardiomyocytes in the sino-auricular area demonstrated the reactive changes of energy-producing apparatus and membrane systems, responsible for ion exchange, while myofibrils remained intact. It is concluded that after 30-day hypokinesia adrenomedullary secretory activity was markedly increased, which in association with an increased chronotropic function of the heart may cause negative changes in contractile and conducting cardiomyocytes of sino-auricular area.

Angiotensin II may mediate apoptosis via AT1-receptors in the rat cardiac conduction system.

INTRODUCTION: Apoptosis and angiotensin II (Ang II) have been suggested as possible causes of arrhythmias. In addition, Ang II via Ang II type I (AT(1)-) receptors, has been demonstrated to induce cardiomyocyte apoptosis. The transgenic m(Ren-2)27 (TG) rat carries the additional Ren-2 gene, the expression of which results in an increase in cardiac Ang II, thus potentially affecting the cell growth/death equilibrium. In this study we have investigated the effect of Ang II, via AT(1)-receptors, on mediating apoptosis in a cardiac conduction system (SA node and AV nodes). MATERIALS AND METHODS: Heart sections from male two-day, one-week and two-week TG and Sprague-Dawley (SD) rats were stained with Masson Trichrome to localise the SA and AV nodes. The sections containing SA or AV nodes were processed for quantitation of apoptotic nuclei and AT(1)-receptors. RESULTS: The number of apoptotic nuclei/mm(2) in the SA and AV nodes were found to decrease from two days to two weeks in both the TG and the SD rats, and the number of apoptotic nuclei/mm(2) in the TG groups was significantly higher than that of the SD groups for all ages (p<0.05). The number of AT(1)-receptors/mm(2) in the SA node were found to decrease with increasing age, whereas the number of AT(1)-receptors/mm(2) in the AV node was increased in both TG and SD rats and the number of AT(1)-receptors/mm(2) in the three TG groups was significantly more than that of the three SD groups (p<0.05). DISCUSSION AND CONCLUSION: As a consequence of the additional renin gene in the TG rats, which results in the alteration of the local renin-angiotensin system, the numbers of AT(1)-receptors/mm(2) and apoptotic nuclei/mm(2) are increased. The number of apoptotic nuclei/mm(2) and AT(1)-receptors/mm(2) in the SA node decrease with maturation, whereas, the number of AT(1)-receptors in the AV node increase. Thus, there may be a correlation between Ang II and apoptosis in the SA node, which does not appear to be present in the AV node.

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.

Primary culture and identification of sinoatrial node cells from newborn rat.

OBJECTIVE: To establish a reliable approach to primary culture and identification of sinoatrial node (SAN) cells. METHODS: The SAN cells were cultured from SAN tissue removed from neonatal Wistar rats and purified with differential attachment and 5'-bromodeoxyuridine (BrdU) treatment. The obtained cells were morphologically observed with inverted microscopy and transmission electron microscopy. Its action potential was recorded using electrophysiological methods. RESULTS: Three distinctly different cells were observed in the cultured SAN cells: spindle, triangle and irregular. Of these, the spindle cells comprised the greatest proportion, with their shape, structure and electrophysiological characteristics consistent with those of the pacemaker cells of SAN. The triangle cells were similar in features to the similarly shaped myocytes located in the atrial myocardium. CONCLUSIONS: The culture method of differential attachment combined with BrdU treatment is a reliable approach to growing SAN cells. Of the cells cultured from SAN, the spindle cells appear to function as pacemaker cells.