Overexpression of human beta2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes

This study investigated cardiac excitation-contraction coupling at 37 degrees C in transgenic mice with cardiac-specific overexpression of human beta2-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca2+ current (I(Ca-L)) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and I(Ca-L), Ca2+ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca2+ and amplitudes of Ca2+ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca2+ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca2+ was accompanied by increased frequency and amplitudes of spontaneous Ca2+ sparks measured at 37 degrees C with fluo 3. These observations suggest that the gain of Ca(2+)-induced Ca2+ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of I(Ca-L) in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.     

Suppression of electrical alternans by overexpression of HERG in canine ventricular myocytes

Suppression of electrical alternans may be antiarrhythmic. Our previous computer simulations have suggested that increasing the rapid component of the delayed rectifier K(+) current (I(Kr)) suppresses alternans. To test this hypothesis, I(Kr) in isolated canine ventricular myocytes was increased by infection with an adenovirus containing the gene for the pore-forming domain of I(Kr) [human ether-a-go-go gene (HERG)]. With the use of the perforated or whole cell patch-clamp technique, action potentials recorded at different pacing cycle lengths (CLs) were applied to the myocytes as the command waveforms. HERG infection markedly increased peak I(Kr) during the action potential (from 0.54 +/- 0.03 pA/pF in control to 3.60 +/- 0.81 pA/pF). Rate-dependent alterations of peak I(Kr) were similar for freshly isolated myocytes and HERG-infected myocytes. In both cell types, I(Kr) increased when CL decreased from 1,000 to 500 ms and then decreased progressively as CL decreased further. During alternans at CL = 170 ms, peak I(Kr) was larger for the short than for the long action potential for both groups, but the difference in peak I(Kr) was larger for HERG-infected myocytes. The voltage at which peak I(Kr) occurred was significantly less negative in HERG-infected myocytes, in association with shifts of the steady-state voltage-dependent activation and inactivation curves to less negative potentials. Pacing at short CL induced stable alternans in freshly isolated myocytes and in cultured myocytes without HERG infection, but not in HERG-infected myocytes. These data support the idea that increasing I(Kr) may be a viable approach to suppressing electrical alternans.     

兔心室肌细胞电生理异质性研究--缺血对动作电位和钾流的影响

实验用全细胞膜片箝技术,观察正常及缺血条件下,兔心内膜下心室肌细胞与心外膜下心室肌细胞的动作电位和稳态外向钾流及其变化。结果显示：（１）正常条件下,心外膜下心室肌细胞与心内膜下心室肌细胞动作电位形态有差异,心外膜下心室肌细胞动作电位时程（ＡＰＤ）较短,复极１期后有明显的初迹,动作电位形态是“锋和圆顶”,而心内膜下心室肌细胞ＡＰＤ较长,并且没有上述动作电位形态特征。这两类细胞静息电位无差异。（２）在     

Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (I(m)) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of -87 +/- 2 mV and -83.9 +/- 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of I(m) around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of I(m) was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased I(m) at -120 mV from 4.3 pA/pF to 27 pA/pF with an EC(50) of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of I(m) fourfold, shifted its reversal potential from -78 +/- 3 to -84 +/- 3 mV, and stabilized the resting membrane potential at -92 +/- 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or I(m) in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K(+) current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.     

Electrophysiological properties of the membrane of smooth muscle cells from bovine lymphatic vessels

Using single sucrose gap technique, studies have been made on electrophysiological properties of the membrane in myocytes of the lymphatic vessels in the ox Bos taurus. It was shown that electrical stimulation does not induce tetanic contraction in the myocytes. The results obtained indicate strong similarity between electrophysiological properties of the myocytes in the lymphatic vessels and those of the myocardial cells in homoiotherms. Refractory state which follows the action potential, accounts for a possibility of rhythmic activity in the myocytes of the lymphatic vessels. Both single and rhythmic stimulation produce in the myocytes the "all-or-none" response. The main factor determining the level of excitability in the myocytes is the intravascular pressure. The latter exerts its influence on contractile activity via changes in the electrical activity (the membrane potential, duration of the plateau phase and the number of fast peak potentials on this plateau).     

Quantification of calcium entry at the T-tubules and surface membrane in rat ventricular myocytes

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by approximately 60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 micromol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 micromol/L cytosol, respectively). However, more Ca entry occurs per microm(2) of junctional membrane at the cell surface than in the T-tubules (in nM/microm(2): 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions ( approximately 35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.     

The regulation of L-type Ca2+ currents in cardiac myocytes of hibernating animals

The action of isoproterenol and BAY K 8644 on voltage-dependent Ca2+ currents in isolated ground squirrel cardiac myocytes was studied in two (active and hibernating) states of the animal. In cardiac myocytes of active animals the effect of both drugs was shown to depend on the holding potential. At Vh of about -50 mV both isoproterenol and BAY K 8644 increased the Ca2+ current and their action was additive. At Vh of about -20 mV, both drugs inhibited the Ca2+ current. In cardiac myocytes from hibernating animals, isoproterenol increased the Ca2+ current at any holding potentials, while the effect of BAY K 8644 did not differ significantly from its effect on active animals. The combined action of the two drugs caused the inhibition of the Ca2+ current at high holding potentials. In terms of the two-site Ca2+ channel model, this means that one of the two pathways of channel phosphorylation is blocked in hibernating animal cardiac cells, and BAY K 8644 restores this pathway.     

DITPA prevents the blunted contraction-frequency relationship in myocytes from infarcted hearts

Loss of the positive force-frequency relationship is a characteristic finding in failing hearts. The mechanisms of this change are not well understood. Myocardial infarction (MI) was induced in rabbits to produce left ventricular (LV) dysfunction. Beginning 1 day after MI, a subgroup of rabbits received diiodothyropropionic acid (DITPA) (3.75 mg x kg(-1) x day(-1) sc) for 3 wk. We measured contractions, Ca(2+) transients, action potentials, and sarcoplasmic reticulum (SR) Ca(2+) content at different stimulation rates in single LV myocytes. The shortening-frequency relationship was markedly flattened in MI myocytes compared with control myocytes. In addition, Ca(2+) transients, action potentials, and contractions were prolonged. Myocytes from DITPA-treated MI rabbits had preserved inotropic responses to increased stimulation rate and normal duration of action potentials and Ca(2+) transients. SR Ca(2+) content increased significantly when stimulation rate was increased from 0.5 to 2.0 Hz in control myocytes but did not change significantly in MI myocytes. Myocytes from DITPA-treated MI rabbits had a greater frequency-dependent increase in SR Ca(2+) content compared with the untreated MI rabbits. Thus single myocytes from infarcted rabbit hearts have frequency-dependent abnormalities of contractility, Ca(2+) cycling, and action potential repolarization. The flattened contraction-frequency relationship can be partially explained by an attenuation of the normal enhancement of SR Ca(2+) content that occurs when stimulation rate is increased. Chronic DITPA administration after MI largely prevents the development of these abnormalities.     

Connexins in mammalian heart function

In heart, the propagation of electrical activity is mediated by intercellular channels, referred to as junctional channels, aggregated into gap junctions and localised between myocytes. These channels consist of structurally related transmembrane proteins, the connexins, three of which (CX43, CX40 and CX45) have been shown to be associated with the myocytes of mammalian heart; a fourth, CX37, was detected exclusively in endothelial cells. In this paper, we review the recent data dealing with the topographical heterogeneity of expression of these connexins in the different cardiac tissues and the unique conductance properties of the channels they form, and attempt to assess the role played by each connexin and the consequences of their multiplicity in the propagation of action potentials.     

Electrical, contractile, and ultrastructural properties of adult rat and guinea-pig ventricular myocytes in long-term primary cultures

The electrical, contractile, and morphological characteristics of ventricular myocytes isolated from adult rat and guinea-pig hearts and maintained in cultures for 7-24 days are described. These cultured cells form different networks, depending on the species, when plated at certain density and maintained under specific conditions; the cells within the networks appear to be electrically coupled. Their resting and action potentials, their contractile activity (shortenings), and their pharmacological responses qualitatively resemble those of freshly isolated myocytes. Cultured cells from both species exhibit near-normal ultrastructural organization of sarcomeres, myofilaments, and mitochondria, as well as formation of intercellular contacts, or gap junctions. These data indicate that cultured adult rat and guinea-pig myocardial cells that make intercellular contacts possess electrical, contractile, and ultrastructural properties and responses to pharmacological agents similar to those of the respective adult myocardial tissues and the functionally intact freshly isolated cells from which these cultures are prepared. Thus, this study indicates that long-term cultures (7-24 days) of networked cardiac myocytes could be used as a valuable experimental model in various investigations of excitation-contraction coupling in cardiac muscle.     

Properties of Xenopus Kv1.10 channels expressed in HEK293 cells

Voltage-gated K+ channels play important roles in shaping the characteristics of action potentials and electrical activity. In a previous study, we isolated cDNAs encoding several distinct K+ channel isoforms, including a novel isoform (XKv1.10) expressed in Xenopus laevis spinal cord neurons and myocytes. Here, we report the biophysical characterization of XKv1.10 expressed in transiently transfected HEK293 cells. Whole cell patch clamp recordings revealed a voltage-gated, rapidly activating and inactivating K+ current. Interestingly, the rate of inactivation of XKv1.10 channels showed apparent voltage dependence, with time constants between 77.7-213.3 ms. The predicted protein sequence of XKv1.10 does not appear to encode an N-terminal inactivating "ball and chain" domain, and instead these channels may inactivate via a C/P-type mechanism. Consistent with this, either increasing the external concentration of K+ or external application of tetraethylammonium caused a decrease in the rate of inactivation. Pharmacologically, XKv1.10 K+ channels were sensitive to 4-aminopyridine and tetraethylammonium with apparent IC50 values of 68.5 microM and 17.1 mM, respectively. When simulated action potentials were used as a voltage command, XKv1.10 was similar to XKv1.4 in that it carried more repolarizing current during the action potential than XKv1.2. However, while XKv1.4 was active during the interspike interval, XKv1.10 and XKv1.2 were not. Overall, the data suggest that XKv1.10 channels make a unique contribution to the developmental maturation of electrical signaling in Xenopus laevis.     

Effects of bimoclomol, the novel heat shock protein coinducer, in dog ventricular myocardium

The effects of the novel HSP-coinducer bimoclomol was studied on action potentials, ionic currents and [Ca2+]i transients in isolated canine ventricular myocytes using conventional microelectrode techniques and whole cell voltage clamp combined with fluorescent [Ca2+]i measurements. Contractility was studied in right ventricular trabeculae. All preparations were paced with a frequency of 0.2 Hz. Bimoclomol (100 microM) shortened action potential duration measured at 50% repolarization, but lengthened action potentials at the 90% repolarization level, decreased action potential amplitude and maximum depolarization velocity in a reversible manner. In voltage clamped myocytes, the drug activated a steady-state outward current at positive membrane potentials leaving the peak inward current unaffected. [Ca2+]i transients, measured under voltage clamp control, were increased in amplitude and had accelerated decay kinetics in the presence of the compound, in addition to reduction of diastolic [Ca2+]i. Bimoclomol significantly decreased the force of contraction in right ventricular trabeculae. Comparison of present data to previous results indicate that the cardiac effects of bimoclomol strongly depend on actual experimental conditions. The reduced contractility in spite of the increased amplitude of [Ca2+]i transients suggests that 100 microM bimoclomol may decrease calcium sensitivity of the contractile apparatus.     

Characterization of the tissue‐level Ca2+ signals in spontaneously contracting human myometrium

In the labouring uterus, millions of myocytes forming the complex geometrical structure of myometrium contract in synchrony to increase intrauterine pressure, dilate the cervix and eventually expel the foetus through the birth canal. The mechanisms underlying the precise coordination of contractions in human myometrium are not completely understood. In the present study, we have characterized the spatio‐temporal properties of tissue‐level [Ca²⁺]_i transients in thin slices of intact human myometrium. We found that the waveform of [Ca²⁺]_i transients and isotonic contractions recorded from thin slices was similar to the waveform of isometric contractions recorded from the larger strips in traditional organ bath experiments, suggesting that the spatio‐temporal information obtained from thin slices is representative of the whole tissue. By comparing the time course of [Ca²⁺]_i transients in individual cells to that recorded from the bundles of myocytes we found that the majority of myocytes produce rapidly propagating long‐lasting [Ca²⁺]_i transients accompanied by contractions. We also found a small number of cells showing desynchronized [Ca²⁺]_i oscillations that did not trigger contractions. The [Ca²⁺]_i oscillations in these cells were insensitive to nifedipine, but readily inhibited by the T‐type Ca²⁺ channel inhibitor NNC55‐0396. In conclusion, our data suggest that the spread of [Ca²⁺]_i signals in human myometrium is achieved via propagation of long‐lasting action potentials. The propagation was fast when action potentials propagated along bundles of myocytes and slower when propagating between the bundles of uterine myocytes.     

Adult ventricular myocytes isolated from CHF 146 and CHF 147 cardiomyopathic hamsters

Single ventricular myocytes have been isolated from normal and cardiomyopathic hamsters using a collagenase method. The procedure produces calcium-tolerant myocytes that are viable and appear on light and electron microscopic examination to be healthy. These cells respond to electrical stimulus and have normal intracellular resting and action potentials.     

海葵毒素对心肌钠通道开放模式，动作电位及心电图QT周期的影响

应用膜片箝技术记录游离豚鼠心肌细胞的钠通道电流,细胞内微电极技术记录心室乳头肌的动作电位和心电图机记录豚鼠的心电图,使用与心肌;细胞钠通道有高度亲和力的海葵毒素（sea anemone toxin,ATXⅡ）改变钠通道开放的动力过程,从三个水平来研究钠通道,动作电位,心电图变化的关系,并试图探讨长QT综合征（long QT syndrome,LQTs)的发病机制,结果显示,ATXⅡ使钠通道的开放频率增加,钠通道中“长时间开放模式”的开放时间常数增大,动人电位的持续时间APD50和APD50也分别增加了23％和27％,ATXⅡ使动物心电图QT间期延长18．6％,QTc(校正的QT间期）增大18．9％,这些结果提示,钠通道动力过程的变化对动作电位和心电图QT间期有重要影响,钠通道功能或结构的变异可能是临床上部分长QT综合征产生的原因。     

Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio)

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.     

A model for propagation of action potentials in smooth muscle

A modified Hodgkin & Huxley (1952) model for axons was used to simulate smooth muscle action potentials. The modifications were such as to match our own experimental results and published data on the passive and active behavior of smooth muscle.A brief account of the modifications introduced to the HH model is as follows. The resting ionic conductances were obtained from the data of Casteels (1969). Chloride conductance was replaced by an ad hoc leakage conductance ($\text{g}\text{?}{}_{\mathrm{L}}$) in order to obtain a resting membrane resistance of about 11 kΩcm². The ionic equilibrium potentials were according to Kao & Nishiyama (1969). The rate constants m, n and h have similar form to those in axons, but their different numerical values produce action potentials that match the duration of the smooth muscle action potential (about 16 ms) at half its maximum amplitude. The effective membrane capacitance was taken as 2.5 μF/cm².The results obtained by implementing those smooth muscle parameters in the HH formulation include: (a) a membrane potential that matches the main characteristics of experimentally recorded action potentials in uterine smooth muscle and guinea-pig taenia-coli, and (b) a propagated action potential which, on a cable diameter of 5 μm (similar to the diameter of a single smooth muscle cell), has a speed of propagation within the range of the values experimentally recorded in smooth muscle. This observed velocity of propagation is not compatible with a large cable and it is concluded that “functional units” are not required to sustain propagation of action potentials in smooth muscle.     

Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes

A large conductance (～300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.     

Influence of sympathetic innervation on the membrane electrical properties of neonatal rat cardiomyocytes in culture

Co-cultures of rat ventricular myocytes and sympathetic neurons were established. Superior cervical ganglia and ventricles from newborn rats were enzymatically dissociated and plated in a culture dish. Experiments were done between the 3rd (when evidence of neuron-myocyte proximity arises) and the 5th day in culture (before the myocytes become confluent). Simultaneous intracellular recording from a cardiomyocyte and an attached neuron was done using conventional microelectrode techniques (resistance of 60-100 Mohm). The myocytes in co-culture were either quiescent or spontaneously contracting. The contracting cells were either latent pacemaker or ventricular-like myocytes. The action potential (AP) characteristics of cardiomyocytes in co-cultures were comparable to those recorded in cardiomyocytes in pure cultures. Sympathetic innervation of the cardiomyocytes in co-cultures was evidenced by stimulating the neuron and observing an increase in rate of beating in latent pacemaker myocytes (average increase of 19.4 +/- 4.6%). In quiescent cardiomyocytes, neural stimulation evoked a slow depolarization that can reach threshold and initiate APs in the cell. This response is similar to slow excitatory postsynaptic potentials (EPSPs) observed in other synapses. Slow ESPSs could also be recorded in spontaneous beating cells, made quiescent by nifedipine (1x10(-6)-1x10(-7) M). These results indicate that functional synaptic contacts are developed in co-culture of sympathetic neurons and cardiac myocytes, and slow EPSPs can be evoked in cardiomyocytes as well as in other excitable cells. The sympathetic innervation occurring in culture did not significantly modify the spontaneous AP characteristics of the cardiomyocytes.