Role of pacemaking current in cardiac nodes: insights from a comparative study of sinoatrial node and atrioventricular node

Cardiac pacemaking in the sinoatrial (SA) node and atrioventricular (AV) node is generated by an interplay of many ionic currents, one of which is the funny pacemaker current (I_f). To understand the functional role of I_f in two different pacemakers, comparative studies of spontaneous activity and expression of the HCN channel in mouse SA node and AV node were performed. The intrinsic cycle length (CL) is 179±2.7 ms (n=5) in SA node and 258±18.7 ms (n=5) in AV node. Blocking of I_f current by 1 μmol/L ZD7288 increased the CL to 258±18.7 ms (n=5) and 447±92.4 ms (n=5) in SA node and AV node, respectively. However, the major HCN channel, HCN4 expressed at low level in the AV node compared to the SA node. To clarify the discrepancy between the functional importance of I_f and expression level of HCN4 channel, a SA node cell model was used. Increasing the I_f conductance resulted in decreasing in the CL in the model, which explains the high pacemaking rate and high expression of HCN channel in the SA node. Resistance to the blocking of I_f in the SA node might result from compensating effects from other currents (especially voltage sensitive currents) involved in pacemaking. The computer simulation shows that the difference in the intrinsic CL could explain the difference in response to I_f blocking in these two cardiac nodes.     

Characterization of the effects of ryanodine, TTX, E-4031 and 4-AP on the sinoatrial and atrioventricular nodes

AIMS: To characterize the effects of inhibition of Ryanodine receptor (RyR), TTX-sensitive neuronal Na+ current (iNa), "rapidly activating" delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) on the pacemaker activity of the sinoatrial node (SAN) and the atrioventricular node (AVN) in the mouse. METHODS: The structure of mouse AVN was studied by histology and immunolabelling of Cx43 and hyperpolarization-activated, cyclic nucleotide-binding channels (HCN). The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activities recorded from mouse intact SAN and AVN preparations have been investigated. RESULTS: Immuno-histological characterization delineated the structure of the AVN showing the similar molecular phenotype of the SAN. The effects of these inhibitors on the cycle length (CL) of the spontaneous pacemaker activity of the SAN and the AVN were characterized. Inhibition of RyR by 0.2 and 2 microM Ryanodine prolonged CL by 42+/-12.3% and 64+/-18.1% in SAN preparations by 163+/-72.3% and 241+/-91.2% in AVN preparations. Inhibition of TTX-sensitive iNa by 100 nM TTX prolonged CL by 22+/-6.0% in SAN preparations and 53+/-13.6% in the AVN preparations. Block of iKr by E-4031 prolonged CL by 68+/-12.5% in SAN preparations and 28+/-3.4% in AVN preparations. Inhibition of iKur by 50 microM 4-AP prolonged CL by 20+/-3.4% in SAN preparations and 18+/-3.0% in AVN preparations. CONCLUSION: Mouse SAN and AVN showed distinct different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the cardiac nodes in the mouse. Our data provide valuable information for developing virtual tissue models of mouse SAN and AVN.     

Cryoablation for atrioventricular nodal re-entrant tachycardia associated with persistent left superior vena cava

Catheter ablation for atrioventricular nodal re-entrant tachycardia (AVNRT) in patients with persistent left superior vena cava (PLSVC) is challenging because of anatomical abnormalities of Koch's triangle associated with the enlarged coronary sinus ostium. We present the Case of successful ablation in a patient with PLSVC using the cryoablation technique. The ablation was successfully performed without damaging the conduction system by virtue of “cryomapping” and “cryoadhesion.” Cryoablation is a safe and efficacious alternative to radiofrequency catheter ablation for the treatment of AVNRT associated with PLSVC.     

VEGF signaling has distinct spatiotemporal roles during heart valve development

Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.     

Importance of the relationship between sinus cycle length and junctional rhythm cycle length (occured during radiofrequency ablation) in predicting the successful modification of the slow pathway in Atrioventricular Nodal Re-entrant Tachycardias

Background

In atrioventricular nodal re-entrant tachycardias (AVNRT), the achievement of Junctional Rhythms (JR) during Radiofrequency Ablation (RF) is a sensitive but non-specific marker of success. Our aim is to analyze prospectively the predictors of non-inducibility of AVNRT, focusing on the characteristics of the JR.

Methods

We included 75 patients with reproducibly inducible AVNRT. Ablation was performed following an electro-anatomical approach. After each application, the induction protocol was repeated.

Results

A total of 341 applications were performed. Although the achievement of ≥1 JR was necessary to obtain the non-inducibility, and the cumulative number of junctional beats (CJB) was higher in effective applications, no CJB cut-off was associated with a success rate higher than 75%. After the observation of a significant correlation between the sinus cycle length (CL) pre-RF and the CL of the JR (JR-CL) (c=0.52; p<0.001), the sinus CL pre-RF/JR-CL ratio (CL-ratio) adequately differentiated the successful vs. unsuccessful applications: 1.41±0.23 vs. 1.17±0.2 (p<0.001). In a multivariate analysis, a CBJ 11 (p<0.001) and a CL-ratio 1.25 (p<0.001) were found to be the only independent predictors of success. The combination of ≥ 11 of CJB with a CL ratio ≥ 1.25 achieved non-inducibility in 97% of our patients.

Conclusion

1) The specificity of the occurrence of JR as a marker of the successful ablation of AVNRT is increased by the CL-ratio. 2) The achievement of ≥ 11 of CJB with a CL ratio ≥ 1.25 predicts non-inducibility in almost all patients.     

Endocardial cell epithelial-mesenchymal transformation requires Type III TGFβ receptor interaction with GIPC

An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFβR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFβR3-mediated endothelial cell EMT.     

Fluid-structure interaction simulation of artificial textile reinforced aortic heart valve: Validation with an in-vitro test

Prosthetic heart valves deployed in the left heart (aortic and mitral) are subjected to harsh hemodynamical conditions. Most of the tissue engineered heart valves have been developed for the low pressure pulmonary position because of the difficulties in fabricating a mechanically strong valve, able to withstand the systemic circulation. This necessitates the use of reinforcing scaffolds, resulting in a tissue-engineered textile reinforced tubular aortic heart valve. Therefore, to better design these implants, material behaviour of the composite, valve kinematics and its hemodynamical response need to be evaluated. Experimental assessment can be immensely time consuming and expensive, paving way for numerical studies. In this work, the material properties obtained using the previously proposed multi-scale numerical method for textile composites was evaluated for its accuracy. An in silico immersed boundary (IB) fluid structure interaction (FSI) simulation emulating the in vitro experiment was set-up to evaluate and compare the geometric orifice area and flow rate for one beat cycle. Results from the in silico FSI simulation were found to be in good coherence with the in vitro test during the systolic phase, while mean deviation of approximately 9% was observed during the diastolic phase of a beat cycle. Merits and demerits of the in silico IB-FSI method for the presented case study has been discussed with the advantages outweighing the drawbacks, indicating the potential towards an effective use of this framework in the development and analysis of heart valves.     

Fine structural examination of the single heart tube in the fifteen day ferret embryo

Summary The different segments of the embryonic heart tube of the ferret were examined with light and transmission electron microscopy. The cells of bulbus cordis, bulboventricular junction, primitive ventricle, atrioventricular junction, and primitive atria were in the process of differentiating into myocardial cells. The ventricular muscle cells were the most developed cells; the least mature muscle cells were those located at the arterial and venous ends of the heart tube. The cells between the ventricle and the two ends of the heart tube showed a spectrum of developmental stages, especially with respect to the morphological development of the myofibrils. Other organelles as well as surface specializations did not permit a distinction between cells of the different regions of the heart tube. There was no striking difference in the size and shape of the developing muscle cells of the atrioventricular and bulboventricular junctions compared to the developing ventricular muscle cells. Morphologically there was no evidence to suggest that the tissue of the atrioventricular and bulboventricular rings was specialized or different from any of the other segments of the single heart tube of the embryonic ferret.This work was supported by NIH Grant#HL26893-01 and by Biomedical Research Support Grant S07RR05417 from the Division of Research Resources, NIH     

The development of innervation in the rat atrioventricular node

Summary The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day and vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed.Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day.These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.     

Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice

Transformation of endocardial endothelial cells into invasive mesenchyme is a critical antecedent of cardiac cushion tissue formation. The message for bone morphogenetic protein (BMP)-2 is known to be expressed in myocardial cells in a manner consistent with the segmental pattern of cushion formation [Development 109(1990) 833]. In the present work, we localized BMP-2 protein in atrioventricular (AV) myocardium in mice at embryonic day (ED) 8.5 (12 somite stage) before the onset of AV mesenchymal cell formation at ED 9.5. BMP-2 protein expression was absent from ventricular myocardium throughout the stages examined. After cellularization of the AV cushion at ED 10.5, myocardial BMP-2 protein expression was diminished in AV myocardium, whereas cushion mesenchymal cells started expressing BMP protein. Expression of BMP-2 in cushion mesenchyme persisted during later stages of development, ED 13.5-16, during valuvulogenesis. Intense expression of BMP-2 persisted in the valve tissue in adult mice. Based on the expression pattern, we performed a series of experiments to test the hypothesis that BMP-2 mediates myocardial regulation of cardiac cushion tissue formation in mice. When BMP-2 protein was added to the 16-18 somite stage (ED 9.25) AV endocardial endothelium in culture, cushion mesenchymal cells were formed in the absence of AV myocardium, which invaded into collagen gels and expressed the mesenchymal marker, smooth muscle (SM) alpha-actin; whereas the endothelial marker, PECAM-1, was lost from the invaded cells. In contrast, when noggin, a specific antagonist to BMPs, was applied together with BMP-2 to the culture medium, AV endothelial cells remained as an epithelial monolayer with little expression of SM alpha-actin, and expression of PECAM-1 was retained in the endocardial cells. When noggin was added to AV endothelial cells cocultured with associated myocardium, it blocked endothelial transformation to mesenchyme. AV endothelium treated with BMP-2 expressed elevated levels of TGFbeta-2 in the absence of myocardium, as observed in the endothelium cocultured with myocardium. BMP-2-supported elevation of TGFbeta-2 expression in endocardial cells was abolished by noggin treatment. These data indicated that BMP signaling is required in and BMP-2 is sufficient for myocardial segmental regulation of AV endocardial cushion mesenchymal cell formation in mice.