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.     

Importance of Gradients in Membrane Properties and Electrical Coupling in Sinoatrial Node Pacing

The sinoatrial node (SAN) is heterogeneous in terms of cell size, ion channels, current densities, connexins and electrical coupling. For example, Na_v1.5 (responsible for I _Na) and Cx43 (responsible for electrical coupling) are absent from the centre of the SAN (normally the leading pacemaker site), but present in the periphery (at SAN-atrial muscle junction). To test whether the heterogeneity is important for the functioning of the SAN, one- and two-dimensional models of the SAN and surrounding atrial muscle were created. Normal functioning of the SAN (in terms of cycle length, position of leading pacemaker site, conduction times, activation and repolarization sequences and space constants) was observed when, from the centre to the periphery, (i) cell characteristics (cell size and ionic current densities) were changed in a gradient fashion from a central-type (lacking I _Na) to a peripheral-type (possessing I _Na) and (ii) coupling conductance was increased in a gradient fashion. We conclude that the heterogeneous nature of the node is important for its normal functioning. The presence of Na_v1.5 and Cx43 in the periphery may be essential for the node to be able to drive the atrial muscle: Na_v1.5 provides the necessary depolarizing current and Cx43 delivers it to the atrial muscle.     

Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 μM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.     

One-Dimensional Mathematical Model of the Atrioventricular Node Including Atrio-Nodal, Nodal, and Nodal-His Cells

Mathematical models are a repository of knowledge as well as research and teaching tools. Although action potential models have been developed for most regions of the heart, there is no model for the atrioventricular node (AVN). We have developed action potential models for single atrio-nodal, nodal, and nodal-His cells. The models have the same action potential shapes and refractoriness as observed in experiments. Using these models, together with models for the sinoatrial node (SAN) and atrial muscle, we have developed a one-dimensional (1D) multicellular model including the SAN and AVN. The multicellular model has slow and fast pathways into the AVN and using it we have analyzed the rich behavior of the AVN. Under normal conditions, action potentials were initiated in the SAN center and then propagated through the atrium and AVN. The relationship between the AVN conduction time and the timing of a premature stimulus (conduction curve) is consistent with experimental data. After premature stimulation, atrioventricular nodal reentry could occur. After slow pathway ablation or block of the L-type Ca²⁺ current, atrioventricular nodal reentry was abolished. During atrial fibrillation, the AVN limited the number of action potentials transmitted to the ventricle. In the absence of SAN pacemaking, the inferior nodal extension acted as the pacemaker. In conclusion, we have developed what we believe is the first detailed mathematical model of the AVN and it shows the typical physiological and pathophysiological characteristics of the tissue. The model can be used as a tool to analyze the complex structure and behavior of the AVN.     

Inhibition of Cardiomyocytes Differentiation of Mouse Embryonic Stem Cells by CD38/cADPR/Ca2+ Signaling Pathway

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca²⁺ mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca²⁺ in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca²⁺ signaling pathway antagonizes the CM differentiation of mouse ES cells.     

Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f)

The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.     

The G-protein–gated K+ channel,IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K⁺ current (I_KACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of I_KACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4^−/− mice), which codes for an integral subunit of the cardiac I_KACh channel. SAN pacemaker cells from Girk4^−/− mice completely lacked I_KACh. Loss of I_KACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4^−/− animals. Although the relative extent of heart rate regulation of Girk4^−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4^−/− animals. We conclude that I_KACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for I_KACh in SAN physiology and heart rate regulation.     

Mouse models for studying pacemaker channel function and sinus node arrhythmia

Pacemaker activity of the heart is generated by a small group of cells forming the sinoatrial node (SAN). Cells of the SAN are spontaneously active and generate action potentials with remarkable regularity and stability under all physiological conditions. The exact molecular mechanisms underlying pacemaker potentials in the SAN have not yet been fully elucidated. Several voltage-dependent ion channels as well as intracellular calcium cycling processes are thought to contribute to the pacemaker activity. Hyperpolarization-activated cation channels, which generate the I_f current, have biophysical properties which seem ideally suited for the initiation of spontaneous electrical activity. This review describes recent work on several transgenic mice lacking different cardiac HCN channel subtypes. The role of I_f for normal pacemaking and sinus node arrhythmia as revealed by these genetic models will be discussed. In addition, a new mouse line is described which enables gene targeting in a temporally-controlled manner selectively in SAN cells. Elucidating the function of HCN and other ion channels in well-controlled mouse models should ultimately lead to a better understanding of the mechanisms underlying human sinoatrial arrhythmias.     

Tbx18-Cre基因敲入小鼠的繁殖、鉴定及其应用

为了探讨Tbx18-Cre基因敲入小鼠(Tbx18:Cre knock-in Mus musculus)的繁殖、鉴定及Tbx18基因敲除小鼠和遗传示踪小鼠模型的应用,将Tbx18-Cre基因敲入杂合子小鼠进行繁殖,应用PCR法鉴定其子代基因型。将子代雌雄杂合子小鼠互交,应用H.E染色观察Tbx18基因敲除胚鼠心的形态学变化。将杂合子小鼠与RosaEYFP报告小鼠交配,应用心冰冻切片技术观察Tbx18:Cre/Rosa26REYFP双转基因遗传示踪胚鼠心内Tbx18阳性心外膜祖细胞发育命运。结果表明,用于繁殖、基因敲除研究及基因遗传示踪的子代基因型均符合孟德尔遗传规律。同时心H.E染色和心冰冻切片发现,Tbx18敲除小鼠心窦房结发育存在缺陷,而Tbx18阳性心外膜祖细胞是心发育重要的祖细胞来源。研究结果揭示,Tbx18-Cre基因敲除小鼠是研究先天性心脏病发病机制的理想模式动物,Tbx18阳性心外膜祖细胞可能是心脏病患者心脏修复和再生潜在的种子细胞。     

Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker

Background

Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (I _f) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K⁺ current (I _K1) flowing through inward rectifier potassium (K_ir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor I _K1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro.

Methods and Results

The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding K_ir2.1, which is essential for generation of I _K1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger I _f currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an I _f inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs.

Conclusions

We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an I _f inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.     

Characterization of the Connexin45 Carboxyl-Terminal Domain Structure and Interactions with Molecular Partners

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.     

Characterization of the Connexin45 Carboxyl-Terminal Domain Structure and Interactions with Molecular Partners

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.