Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/ΔKPQ hearts suggest an overlap syndrome

Mutations in SCN5A, the gene encoding the pore-forming subunit of cardiac Na(+) channels, cause a spectrum of arrhythmic syndromes. Of these, sinoatrial node (SAN) dysfunction occurs in patients with both loss- and gain-of-function SCN5A mutations. We explored for corresponding alterations in SAN function and intracardiac conduction and clarified possible mechanisms underlying these in an established mouse long QT syndrome type 3 model carrying a mutation equivalent to human SCN5A-ΔKPQ. Electrophysiological characterizations of SAN function in living animals and in vitro sinoatrial preparations were compared with cellular SAN and two-dimensional tissue models exploring the consequences of Scn5a+/ΔKPQ mutations. Scn5a+/ΔKPQ mice showed prolonged electrocardiographic QT and corrected QT intervals confirming long QT phenotypes. They showed frequent episodes of sinus bradycardia, sinus pause/arrest, and significantly longer sinus node recovery times, suggesting compromised pacemaker activity compared with wild-type mice. Electrocardiographic waveforms suggested depressed intra-atrial, atrioventricular node, and intraventricular conduction in Scn5a+/ΔKPQ mice. Isolated Scn5a+/ΔKPQ sinoatrial preparations similarly showed lower mean intrinsic heart rates and overall slower conduction through the SAN to the surrounding atrium than did wild-type preparations. Computer simulations of both single SAN cells as well as two-dimensional SAN-atrial models could reproduce the experimental observations of impaired pacemaker and sinoatrial conduction in terms of changes produced by both augmented tail and reduced total Na(+) currents, respectively. In conclusion, the gain-of-function long QT syndrome type 3 murine Scn5a+/ΔKPQ cardiac system, in overlap with corresponding features reported in loss-of-function Na(+) channel mutations, shows compromised SAN pacemaker and conduction function explicable in modeling studies through a combination of augmented tail and reduced peak Na(+) currents.     

Conduction barriers and pathways of the sinoatrial pacemaker complex: their role in normal rhythm and atrial arrhythmias

Since Keith and Flack's anatomical discovery of the sinoatrial node (SAN), the primary pacemaker of the heart, the question of how such a small SAN structure can pace the entire heart has remained for a large part unanswered. Recent advances in optical mapping technology have made it possible to unambiguously resolve the origin of excitation and conduction within the animal and human SAN. The combination of high-resolution optical mapping and histological structural analysis reveals that the canine and human SANs are functionally insulated from the surrounding atrial myocardium, except for several critical conduction pathways. Indeed, the SAN as a leading pacemaker requires anatomical (fibrosis, fat, and blood vessels) and/or functional barriers (paucity of connexins) to protect it from the hyperpolarizing influence of the surrounding atrium. The presence of conduction barriers and pathways may help explain how a small cluster of pacemaker cells in the SAN pacemaker complex manages to depolarize different, widely distributed areas of the right atria as evidenced functionally by exit points and breakthroughs. The autonomic nervous system and humoral factors can further regulate conduction through these pathways, affecting pacemaker automaticity and ultimately heart rate. Moreover, the conduction barriers and multiple pathways can form substrates for reentrant activity and thus lead to atrial flutter and fibrillation. This review aims to provide new insight into the function of the SAN pacemaker complex and the interaction between the atrial pacemakers and the surrounding atrial myocardium not only in animal models but also human hearts.     

Effect of potential fluctuations on the activity of sinoatrial node cells

The effect of fluctuations of the transmembrane potential on the generation of the action potential is studied by simulating the rabbit sinoatrial node (SAN) pacemaker. It is shown that the effect of fluctuations is enhanced with an increase in the concentration of acetylcholine and becomes most pronounced at the border of spontaneous activity loss and after it. When applying and washing off acetylcholine, the hysteretic effect is observed.     

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.     

昆虫击倒抗性基因突变对钠通道功能的影响

该文综述了昆虫钠通道基因的表达与功能特性、击倒抗性突变的功能和这些突变对钠通道门控的影响,以及钠通道基因突变与抗性表现型之间的因果关系;还讨论了这些突变增强击倒抗性的分子机理。     

Examination of paralysis in Drosophila temperature-sensitive paralytic mutations affecting sodium channels; a proposed mechanism of paralysis

We have used the identified cells of the Drosophila Giant Fiber System (GFS) to study the defects induced by the temperature-sensitive paralytic mutations no action potential (nap) and paralytic (para). These mutations paralyze at elevated temperatures, reported as due to a block of action potential propagation. We found, however, that the cells of the GFS still were able to respond to stimuli at 7-10 degrees C above the temperature causing mutant paralysis. Stimulus threshold and conduction time both decrease with increasing temperature in the mutants in a manner indistinguishable from wild-type. Since action potentials can propagate efficiently in the mutants at elevated temperatures, we looked for other neural defects that might be involved in producing paralysis. We did find reduced neuronal function at sites such as electrical synapses and axonal branch points where current may be limiting. These sites had weakened following frequency, occasional failures, and increased conduction times. We believe the non-temperature-dependent defects in nap and para uncover the normally temperature-sensitive traits latent within all neurons. Increasing temperature increases the rates of channel activation and inactivation. At higher temperatures, Na+ inactivation and K+ activation encroach upon the Na(+)-activation time, reducing inward sodium current. In addition to this normal temperature-dependent effect, the mutations decrease the number of sodium channels in neurons in a non-temperature-dependent manner. These two reductions in sodium current combine to prevent spiking threshold from being reached at current limited sites. The temperature at which a sufficient number of these sites block should be the temperature of paralysis.     

Extracellular finger domain modulates the response of the epithelial sodium channel to shear stress

The epithelial sodium channel (ENaC) is regulated by multiple extracellular stimuli, including shear stress. Previous studies suggest that the extracellular finger domains of ENaC α and γ subunits contain allosteric regulatory modules. However, the role of the finger domain in the shear stress response is unknown. We examined whether mutations of specific residues in the finger domain of the α subunit altered the response of channels to shear stress. We observed that Trp substitutions at multiple sites within the tract αLys-250-αLeu-290 altered the magnitude or kinetics of channel activation by shear stress. Consistent with these findings, deletion of two predicted peripheral β strands (αIle-251-αTyr-268) led to slower channel activation by shear stress, suggesting that these structures participate in the shear stress response. The effects of mutations on the shear stress response did not correlate with their effects on allosteric Na(+) inhibition (i.e. Na(+) self-inhibition), indicating a divergence within the finger domain regarding mechanisms by which the channel responds to these two external stimuli. This result contrasts with well correlated effects we previously observed at sites near the extracellular mouth of the pore, suggesting mechanistic convergence in proximity to the pore. Our results suggest that the finger domain has an important role in the modulation of channel activity in response to shear stress.     

Regulatory Evolution and Voltage-Gated Ion Channel Expression in Squid Axon: Selection-Mutation Balance and Fitness Cliffs

It has been suggested that optimization of either axonal conduction velocity or the energy efficiency of action potential conduction predominates in the selection of voltage-gated sodium conductance levels in the squid axon. A population genetics model of channel gene regulatory function was used to examine the role of these and other evolutionary forces on the selection of both sodium and potassium channel expression levels. In this model, the accumulating effects of mutations result in degradation of gene regulatory function, causing channel gene expression to fall to near-zero in the absence of positive selection. In the presence of positive selection, channel expression levels fall to the lowest values consistent with the selection criteria, thereby establishing a selection-mutation balance. Within the parameter space of sodium and potassium conductance values, the physiological performance of the squid axon model showed marked discontinuities associated with conduction failure and excitability. These discontinuities in physiological function may produce fitness cliffs. A fitness cliff associated with conduction failure, combined with the effects of phenotypic noise, can account for the selection of sodium conductance levels, without considering either conduction velocity or metabolic cost. A fitness cliff associated with a transition in axonal excitability, combined with phenotypic noise, can explain the selection of potassium channel expression levels. The results suggest that voltage-gated ion channel expression will fall to low levels, consistent with key functional constraints, even in the absence of positive selection for energy efficiency. Channel expression levels and individual variation in channel expression within the population can be explained by regulatory evolution in combination with genetic variation in regulatory function and phenotypic noise, without resorting to more complex mechanisms, such as activity-dependent homeostasis. Only a relatively small region of the large, nominally isofunctional parameter space for channel expression will normally be occupied, because of the effects of mutation.     

Peripheral nerve at extreme low temperatures 2: Pharmacologic modulation of temperature effects

Changes in temperature have profound and clinically important effects on the peripheral nerve. In a previous paper, the effects of temperature on many properties of the peripheral nerve action potential (NAP) were explored including the NAP amplitude, conduction velocity and response to paired pulse stimulation. In this paper, the effects of pharmacologic manipulations on these parameters were explored in order to further understand the mechanisms of these effects.The reduction in conduction velocity with temperature was shown to be independent of the ionic composition of the perfusate and was unaffected by potassium or sodium channel blockade. This implies that the phenomenon of reduced conduction velocities at low temperature may be related to changes in the passive properties of the axon with temperature. Blockade of sodium channels and chronic membrane depolarization produced by high perfusate potassium concentrations or high dose 4-aminopyridine impair the resistance of the nerve to hypothermia and enhance the injury to the nerve produced by cycles of cooling and rewarming. This suggests the possibility that changes in the sodium inactivation channel may be responsible for the changes in the NAP amplitude with temperature and that prolonged sodium inactivation may lead more permanent changes in excitability.     

Cardiac responses to exercise in the dog before and after destruction of the sinoatrial node

Chronotropic and dromotropic responses to treadmill exercise were compared in conscious dogs prior to and following excision of the sinoatrial node (SAN). The initial junctional rhythm accompanying removal of the SAN region was replaced within hours to days by subsidiary atrial pacemaker (SAP) foci located in the inferior right atrium along the sulcus terminalis. With SAN intact, cardiac acceleration was immediate at onset of exercise and the tachycardia was directly proportional to work intensity. Atrioventricular (AV) conduction concurrently accelerated during exercise as manifest by shortening in P-R and atrioventricular (A-V) intervals. Following SAN excision, subsidiary atrial pacemaker foci likewise demonstrated prompt tachycardias during exercise, although heart rate was significantly reduced at rest and during steady state exercise. In the SAP state, tachycardia during exercise was related to work intensity and was mediated by changes in cardiac autonomic nerve activity. Combined propranolol-atropine blockade increased heart rate at rest in the SAP state, and significantly attenuated the tachycardia accompanying treadmill exercise. Following SAN excision the P-R (A-V) interval was significantly reduced in the resting animal. In response to exercise, AV conduction time decreased in the SAP state, though the absolute levels during steady state exercise were not significantly different from prior control runs with SAN intact. Blood pressure response to exercise was similar during both SAN and SAP states. We conclude that following an initial unstable period, SAP foci maintain adequate heart rate increases in response to dynamic exercise, primarily mediated via autonomic nerve regulation.     

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 effect of acetylcholine on inositol lipid metabolism and intracellular calcium concentrations in bovine anterior pituitary cells

Acetylcholine (ACh) increased the intracellular calcium concentration in bovine anterior pituitary cells. In the presence of the calcium channel antagonists verapamil (20 microM) or nitrendepine (1 microM) the increase in calcium was partially inhibited but showed both transient and sustained components. In the presence of EGTA (2.5 mM) only the transient component was observed. ACh also decreased inositol radioactivity in phosphatidylinositides and increased it in inositol phosphates. It is concluded that the increase in calcium caused by acetylcholine requires both the entry of external calcium and mobilisation of internal calcium. Replacement of external sodium by N-methyl-D-glucamine inhibited the rises in calcium and inositol phosphate labelling in response to ACh. Tetrodotoxin (3 microM) or ouabain (50 microM) did not affect either response to ACh. Verapamil did not affect the calcium rise induced by ACh in the absence of external sodium. The phorbol ester PMA (10 nM) caused a transient rise in calcium and inhibited the calcium rise caused by acetylcholine: it did not modify the effect of acetylcholine on inositol phosphates. The dependence of the stimulation of external calcium entry and inositol phosphate production on external sodium ions and protein kinase C is discussed.     

Determinants of heterogeneity, excitation and conduction in the sinoatrial node: a model study

The sinoatrial node (SAN) is a complex structure that exhibits anatomical and functional heterogeneity which may depend on: 1) The existence of distinct cell populations, 2) electrotonic influences of the surrounding atrium, 3) the presence of a high density of fibroblasts, and 4) atrial cells intermingled within the SAN. Our goal was to utilize a computer model to predict critical determinants and modulators of excitation and conduction in the SAN. We built a theoretical "non-uniform" model composed of distinct central and peripheral SAN cells and a "uniform" model containing only central cells connected to the atrium. We tested the effects of coupling strength between SAN cells in the models, as well as the effects of fibroblasts and interspersed atrial cells. Although we could simulate single cell experimental data supporting the "multiple cell type" hypothesis, 2D "non-uniform" models did not simulate expected tissue behavior, such as central pacemaking. When we considered the atrial effects alone in a simple homogeneous "uniform" model, central pacemaking initiation and impulse propagation in simulations were consistent with experiments. Introduction of fibroblasts in our simulated tissue resulted in various effects depending on the density, distribution, and fibroblast-myocyte coupling strength. Incorporation of atrial cells in our simulated SAN tissue had little effect on SAN electrophysiology. Our tissue model simulations suggest atrial electrotonic effects as plausible to account for SAN heterogeneity, sequence, and rate of propagation. Fibroblasts can act as obstacles, current sinks or shunts to conduction in the SAN depending on their orientation, density, and coupling.     

降钙素基因相关肽对家兔离体窦房结电生理活动的影响

用常规微电极方法研究了降钙素基因相关肽（ＣＧＲＰ）对家兔窦房结起搏细胞的电生理作用,并进一步探讨这种作用与钙电流的关系。结果：⑴低浓度ＣＧＲＰ（１ｎｍｏｌ／Ｌ）对窦房结动作电位各参数无显著影响;中等浓度ＣＧＲＰ（１０ｎｍｏｌ／Ｌ）可增加最大舒张期电位、动作电位幅度、０期最大除极化速率和４期自动除极速率,缩短窦性周期、动作电位复极化５０％和９０％时间,这些作用经２０ｍｉｎ达到高峰;高浓度ＣＧＲＰ（２     

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.     

Identification and detection of the isolated sinus venosus from the Asian toad

The pacemaker activity of mammalian sinoatrial node (SAN) of the heart plays a fundamental role in the integration of vital functions. Studying factors such as drugs that influence pacemaker activity of SAN has its significance. In this study, we isolated sinus venosus, SAN from toads (Bufo gargarizans), and analysed its electronic signal, histological characteristics and the influence of acetylcholine (ACh) and ivabradine on its pacemaker activity using PowerLab® and Chart® 5.0 software. We found that when isolated sinus venosus was treated with ACh, its histological distribution was disorganized and inter‐beat (RR) interval was also broadened. The high frequency normalized unit (HFnu) and Poincaré plot of heart rate variability (HRV) of the isolated sinus venosus was also altered upon ACh treatment in a time‐dependent and dose‐dependent manner. When treated with ivabradine, these parameters of HRV such as square root of the mean of the squared differences between adjacent NN intervals (RMSSD) and HFnu were in the upward tendency, but low frequency normalized unit and low frequency/high frequency were in the opposite tendency. Taken together, we have developed a new model for studying the influences of drugs on autorhythmicity using isolated sinus venosus of the toad. With this model, we showed that ACh and ivabradine may affect the pacemaker activity by stimulating muscarinic receptor or inhibiting I_f current, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Complete Atrial-Specific Knockout of Sodium-Calcium Exchange Eliminates Sinoatrial Node Pacemaker Activity

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by “funny” current (I_f) through HCN4 channels (the “Membrane Clock“ hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two (“Coupled Clock”). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of I_f in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.     

Vascular smooth muscle contractile properties in the turtle Pseudemys scripta elegans

1. Turtle aortic rings were characterized by high frequency spontaneous contractile activity and variable responsiveness to constrictor agents.2. The tissue response was remarkably insensitive to temperature at a range of 37°–15°C.3. The contractile response was effectively blocked by the calcium channel antagonist nifedipine and was substantially dependent on extracellular calcium concentrations.4. Lowering the sodium concentration of the bath medium resulted in a strong, transient contraction followed by reduced responsiveness to norepinephrine and the absence of spontaneous activity.5. Disruption of the vessel endothelium resulted in enhanced and reduced responsiveness to norepinephrine (NE) and acetylcholine (ACh), respectively.6. The results indicate that the regulation of contractile function in turtle vascular smooth muscle differs in several respects from that of mammalian tissue, perhaps, reflecting the adaptation of the vasculature to low pressure and ectothermic conditions.