Remodeling Promotes Proarrhythmic Disruption of Calcium Homeostasis in Failing Atrial Myocytes.

It is well known that heart failure (HF) typically coexists with atrial fibrillation (AF). However, until now, no clear mechanism has been established that relates HF to AF. In this study, we apply a multiscale computational framework to establish a mechanistic link between atrial myocyte structural remodeling in HF and AF. Using a spatially distributed model of calcium (Ca) signaling, we show that disruption of the spatial relationship between L-type Ca channels (LCCs) and ryanodine receptors results in markedly increased Ca content of the sarcoplasmic reticulum (SR). This increase in SR load is due to changes in the balance between Ca entry via LCCs and Ca extrusion due to the sodium-calcium exchanger after an altered spatial relationship between these signaling proteins. Next, we show that the increased SR load in atrial myocytes predisposes these cells to subcellular Ca waves that occur during the action potential (AP) and are triggered by LCC openings. These waves are common in atrial cells because of the absence of a well-developed t-tubule system in most of these cells. This distinct spatial architecture allows for the presence of a large pool of orphaned ryanodine receptors, which can fire and sustain Ca waves during the AP. Finally, we incorporate our atrial cell model in two-dimensional tissue simulations and demonstrate that triggered wave generation in cells leads to electrical waves in tissue that tend to fractionate to form wavelets of excitation. This fractionation is driven by the underlying stochasticity of subcellular Ca waves, which perturbs AP repolarization and consequently induces localized conduction block in tissue. We outline the mechanism for this effect and argue that it may explain the propensity for atrial arrhythmias in HF.

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers.

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.

Defective excitation-contraction coupling in hearts of rats with congestive heart failure.

AIM: We examined the cellular basis for depressed cardiac contractility in rats with congestive heart failure (CHF) secondary to myocardial infarction. METHODS: Six weeks after ligation of the left coronary artery, CHF was confirmed by haemodynamic measures and echocardiographic demonstration of reduced myocardial contractility in vivo. Papillary muscles from CHF animals developed less force than those from sham operated (SHAM) animals. Cell shortening was measured in isolated ventricular myocytes voltage-clamped with high resistance electrodes. Ca2+ transients were measured in fluo-4 loaded myocytes. RESULTS: Contractions triggered by depolarizing test steps from a post conditioning potential of -70 mV were significantly smaller and had significantly reduced velocity of shortening in CHF compared with SHAM myocytes. However, contractions initiated from -40 mV, were similar in amplitude and velocity of shortening in CHF and SHAM cells. L-type Ca2+ current was not significantly different between CHF and SHAM cells, whether activated from -70 or -40 mV. Therefore, in SHAM cells, excitation-contraction coupling exhibited higher gain when contractions were initiated from negative (-70 mV), as compared with depolarized potentials (-40 mV). However, in CHF myocytes, excitation-contraction coupling gain was selectively depressed with steps from -70 mV. This depression of gain in CHF was not accompanied by a significant reduction in sarcoplasmic reticulum Ca2+ content. Isoproterenol increased Ca2+ transients less in CHF than SHAM myocytes. CONCLUSION: In this post-infarction model of CHF, the contractile deficit was voltage dependent and the gain of excitation-contraction coupling was selectively depressed for contractions initiated negative to -40 mV.

New evidence for similarities in excitation-contraction coupling in skeletal and cardiac muscle.

This review describes several new experimental observations indicating that some of the differences thought to distinguish activation of contraction in skeletal and cardiac muscle may be in fact much less profound than are currently considered. Three such areas are considered in particular. First, it now appears that activation of the elementary units of Ca2+ release from the sarcoplasmic reticulum ('Ca2+ sparks') in skeletal muscle may occur not only as the result of voltage activation but also of Ca2+ activation in a process very much like Ca2+-induced Ca2+ release (CICR) in cardiac muscle. Second, there is new evidence that activation of contraction in cardiac muscle may be partly reliant on a voltage-sensitive release mechanism (VSRM) similar to that in skeletal muscle. Third, digitalis binds to a high affinity site on the cardiac sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor RyR) causing an increase in single channel open probability which could contribute to its positive inotropic action; although mammalian skeletal muscle does not appear to share this sensitivity to cardiac glycosides, amphibian skeletal muscle has both cardiac and skeletal isoforms of the channel and does indeed demonstrate a positive inotropic action in response to digitalis. These results raise the possibility that several differences thought to represent 'fundamental' distinctions between the two muscle types and how they generate and regulate contraction, as well as pharmacological sensitivities, may be more similar than are currently considered.

Effects of MDL 19205 (piroximone), a new cardiotonic agent, on electrophysiological, mechanical, and intracellular ionic characteristics of sheep cardiac tissues.

This study was undertaken to characterize the cardiac electrophysiological and mechanical effects of MDL 19205, a new and potent nonglycoside, positive inotropic agent. In addition, intracellular Na+ activity (aiNa) was measured to determine if this agent might produce its inotropic effects by increasing aiNa and secondarily by increasing intracellular calcium via Na+ -Ca2+ exchange. Experiments were conducted in free-running trabecular muscles and Purkinje fibers obtained from sheep hearts. The following were the most significant effects of MDL 19205: a decrease in action potential duration in both ventricular and Purkinje tissues; a cumulative dose-dependent increase in contractile force in ventricular muscle but not in Purkinje strands; no change in aiNa in Purkinje fibers to accompany the positive inotropic effect of this agent; a shift in the dose-response relation by approximately fourfold in the presence of beta-adrenergic blockade with sotalol (10(-7) M); an enhancement of diastolic depolarization in Purkinje fibers resulting in automaticity that is accelerated by overdrive; and a potentiation of the positive inotropic effects of MDL 19205 by 8-bromo-cAMP (1 mM), indicating a potent phosphodiesterase inhibitory action of MDL 19205. These results suggest that MDL 19205 exerts at least part of its positive inotropic and automatic actions through stimulation of beta-adrenergic receptors. This action occurs in conjunction with its ability to inhibit phosphodiesterase, thus promoting an accumulation of cAMP in cardiac cells. Other intracellular actions may also contribute to the effect of this drug, but they do not rely on an increase in aiNa or dramatic changes in the action potential plateau or duration.

Changes in intracellular Na+ during Na,K pump inhibition in sheep cardiac tissues.

The differences in inotropic and toxic sensitivities of cardiac ventricular tissue to cardiac glycosides were investigated in order to determine whether or not the reportedly greater sensitivity of Purkinje fibers compared to myocardium is the result of a fundamentally different response to Na,K pump inhibition. I measured the changes in intracellular Na+ activity (aiNa) in the two tissue types simultaneously during exposure to actodigin (4.0 microM) and ouabain (0.5 microM) under quiescent conditions. A sheep papillary muscle and a Purkinje fiber from the same heart were placed in an experimental chamber and measurements of aiNa from both were obtained with Na+ -sensitive microelectrodes. In five experiments in which all electrode impalements were successfully maintained, actodigin caused similar changes in aiNa in the two tissues (from 7.2 +/- 1.0 mM in control to 12.9 +/- 1.9 mM in the presence of drug in papillary muscles compared to 7.3 +/- 0.3 mM in control and 13.2 +/- 1.0 mM in Purkinje fibers; means +/- S.E.M.). After washout, exposure to ouabain increased aiNa in both papillary muscles and in Purkinje fibers (from 7.2 +/- 0.7 mM in control and 16.2 +/- 1.4 mM during exposure to drug in papillary muscles compared to 7.4 +/- 0.3 mM and 14.9 +/- 0.8 mM in Purkinje fibers). In fact, ouabain caused a greater increase of aiNa in papillary muscles than in Purkinje fibers (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular sodium and the positive inotropic effect of veratridine and cardiac glycoside in sheep Purkinje fibers.

Veratridine is a sodium channel toxin that exerts a powerful positive inotropic effect and prolongs the action potential duration in the heart. To determine the basis of the inotropic action of veratridine and to examine the effects of dissimilar methods of raising intracellular sodium activity on contractility, we measured twitch tension and intracellular sodium activity using sodium-sensitive microelectrodes in stimulated sheep Purkinje fibers exposed to veratridine and in voltage-clamped fibers exposed to veratridine and cardiac glycoside. In stimulated fibers, veratridine (0.1-1 microM) produced coincident increases in intracellular sodium activity, action potential duration, and tension. In voltage-clamped fibers, veratridine (1-2 microM) and acetylstrophanthidin (0.1 microM) raised intracellular sodium activity and tension to a comparable degree. Tetrodotoxin (10 microM) abolished the mechanical, electrophysiological, and ionic changes produced by veratridine. The relationship between intracellular sodium activity and tension in voltage-clamped fibers (n = 6) was indistinguishable for veratridine and acetylstrophanthidin and could be fitted either with a linear function with slopes of 122.8% and 124.2%, respectively, or with a power function with slopes of 4.60 and 4.54, respectively, where the slope represents the exponential power of intracellular sodium activity to which tension is proportional. These results indicate that the positive inotropic action of veratridine is entirely accounted for by accumulation of intracellular sodium, which increases intracellular calcium available for contraction by sodium-calcium exchange. This study is the first direct demonstration that veratridine or any other sodium channel toxin affects intracellular sodium activity and suggests that the inotropic potency of veratridine and cardiac glycoside rely on the same mechanism, namely, elevation of intracellular sodium.

Effects of quinidine on action potentials and ionic currents in isolated canine ventricular myocytes.

We examined the effects of quinidine (5-20 microM) on transmembrane action potentials and ionic currents of isolated canine ventricular myocytes. Collagenase treatment of canine ventricular tissue produced a yield of 40-60% healthy cells. Myocytes had normal resting and action potentials as measured using conventional microelectrodes. Quinidine decreased Vmax, amplitude, overshoot, and the duration of action potentials stimulated by passage of brief current pulses through the recording pipette. Recovery was complete after washout except that action potential duration was prolonged compared with control. A discontinuous single microelectrode voltage ("switch") clamp was used to measure ionic currents. Quinidine irreversibly reduced steady-state outward current as measured with three different voltage clamp protocols. Quinidine reversibly decreased peak calcium current as well as the slowly inactivating and/or steady-state inward currents in the plateau voltage range, presumably both "late" sodium (tetrodotoxin-sensitive) and calcium (tetrodotoxin-insensitive) currents. The effect on calcium current showed both tonic and use-dependent block. Thus, quinidine has a multitude of actions on both inward and outward currents, which combine to produce the net effect of quinidine on action potential configuration.

Basis for tetrodotoxin and lidocaine effects on action potentials in dog ventricular myocytes.

We studied the effects of tetrodotoxin (TTX) and lidocaine on transmembrane action potentials and ionic currents in dog isolated ventricular myocytes. TTX (0.1-1 x 10(-5) M) and lidocaine (0.5-2 x 10(-5) M) decreased action potential duration, but only TTX decreased the maximum rate of depolarization (Vmax). Both TTX (1-2 x 10(-5) M) and lidocaine (2-5 x 10(-5) M) blocked a slowly inactivating toward current in the plateau voltage range. The voltage- and time-dependent characteristics of this current are virtually identical to those described in Purkinje fibers for the slowly inactivating inward Na+ current. In addition, TTX abolished the outward shift in net current at plateau potentials caused by lidocaine alone. Lidocaine had no detectable effect on the slow inward Ca2+ current and the inward K+ current rectifier, Ia. Our results indicate that 1) there is a slowly inactivating inward Na+ current in ventricular cells similar in time, voltage, and TTX sensitivity to that described in Purkinje fibers; 2) both TTX and lidocaine shorten ventricular action potentials by reducing this slowly inactivating Na+ current; 3) lidocaine has no additional actions on other ionic currents that contribute to its ability to abbreviate ventricular action potentials; and 4) although both agents shorten the action potential by the same mechanism, only TTX reduces Vmax. This last point suggests that TTX produces tonic block of Na+ current, whereas lidocaine may produce state-dependent Na+ channel block, namely, blockade of Na+ current only after Na+ channels have already been opened (inactivated-state block).

Fast sodium influx provides an initial step to trigger contractions in cat ventricle.

We examined the possibility that Na+ current (INa) may play a role in excitation-contraction coupling in cat ventricular myocytes. A voltage step from -70 to -40 mV produced a fast INa, followed by a small transient inward current, a brief loss in voltage control to more positive potentials, and a transient contraction (reduction in cell length, delta L). We established that 10 microM nifedipine completely blocked Ca2+ current but did not prevent delta L; nifedipine reduced it by approximately 15%. This nifedipine-insensitive delta L was abolished by 1-10 microM ryanodine, 1-10 microM saxitoxin (STX), and 0.1-1.0 mM Cd2+. The size of delta L increased with more negative holding potential (VH; delta L-VH relation). Maximal delta L was achieved at a VH of approximately -70 mV. Anthopleura toxin A (APA, 3-10 nM), which selectively slows inactivation of INa, increased the size of the nifedipine-insensitive delta L at all VH, thus producing a +7-mV shift in the delta L-VH relation that was not affected by the state of the sarcoplasmic reticulum (SR). APA also produced an increase in maximal delta L, which was no longer observed once the SR was significantly loaded. These effects of APA were prevented by preexposure to STX. The state of the SR Ca2+ stores did not affect the presence of a nifedipine-insensitive delta L but determined its magnitude, suggesting that delta L was not associated with Ca2+ overload. In summary, cat and guinea pig ventricular myocytes are alike in that they both exhibit distinct INa-dependent contractions. Whether these contractions are due to a sudden increase in subsarcolemmal Na+ as a result of fast INa or the depolarization and thus reversal of the Na+/Ca2+ exchange remains undetermined.

Voltage dependence of digitalis afterpotentials, aftercontractions, and inotropy.

The effects of membrane potential on digitalis-induced oscillatory afterpotentials (OAP) and aftercontractions (AC) were investigated in isolated canine false tendons and canine and feline papillary muscles. Transmembrane potential and contractions were measured simultaneously. Membrane potential was varied by current applied through an extracellular pipet. The amplitudes of OAP and AC induced by acetylstrophanthidin (0.5-1.0 X 10(-7) g/ml) or actodigin (0.5-1.0 X 10(-6) g/ml) were increased by depolarization and decreased or abolished by hyperpolarization in all tissues. Prior to the appearance of OAP and AC in preparations exposed to the drugs, depolarization caused both phenomena to appear. As in muscle, the strength of beats superimposed on the ascending limb of AC was potentiated, and the strength of beats superimposed on the relaxation phase was depotentiated. When the amplitude of AC was decreased by hyperpolarization, both potentiation and depotentiation were diminished. This effect was accompanied by a partial reversal of the changes induced by acetylstrophanthidin in the course of restitution of contractility and the configuration of the force-frequency relation.

Acetylcholine-sensitive muscarinic K+ channels in mammalian ventricular myocytes.

Acetylcholine (ACh) is known to increase K+ conductance in the atrium and in pacemaker tissues in the heart. This effect has not been well defined in mammalian ventricular tissues. We have identified and characterized the ACh-sensitive muscarinic K+ channel [IK(ACh)] activity in isolated human, cat, and guinea pig ventricular myocytes using the patch-clamp technique. Application of ACh increased whole cell membrane current in human ventricular myocytes. Current-voltage relationship of the ACh-induced current in ventricle exhibited inward-rectification whose slope conductance was smaller than that in atrium. In single-channel recording from cell-attached patches, IK(ACh) activity was observed when ACh was included in the solution. The channel exhibited a slope conductance of 43 +/- 2 pS. Open times were distributed according to a single exponential function with mean open lifetime of 1.8 +/- 0.3 ms. The channel had conductance and kinetic characteristics similar to human atrial IK(ACh), which had a slope conductance of 43 +/- 3 pS and mean open lifetime of 1.6 +/- 0.3 ms. However, concentration of ACh at half-maximal stimulation (KD) of the channel in ventricle was greater (KD = 0.13 microM) than that in atrium (KD = 0.03 microM). Adenosine caused activation of the same K+ channel. After formation of an excised inside-out patch, channel activity disappeared. Application of GTP (100 microM) or GTP gamma S (100 microM) to the solution caused reactivation of the channel. When myocytes were preincubated with pertussis toxin (PTX), ACh failed to activate these channels, indicating that the PTX-sensitive G protein, Gi, is essential for activation of IK(ACh). IK(ACh) channel activity was also found in cat and guinea pig ventricular myocytes. We conclude that ACh directly activates the IK(ACh) in mammalian ventricular myocytes via Gi in a fashion almost identical to atrial myocytes.

A matrical approach to the basic and clinical pharmacology of antiarrhythmic drugs.

In summary, the lethal cardiac arrhythmias remain a major public health problem and their treatment is a major challenge to the clinician. We possess rapidly increasing knowledge of the electrophysiologic events which underly arrhythmogenesis and the antiarrhythmic as well as the proarrhythmic actions of drugs. Much of this electrophysiologic knowledge is irrelevant to the practicing physician. While complex, we believe that the matrical approach provides the clinician with a useful intellectual framework within which to consider the actions of arrhythmogenic influences and antiarrhythmic drugs. The matrical approach is scientifically sound, reflects clinical realities, and serves as a rational guide to the treatment of cardiac arrhythmias. The traditional classifications of antiarrhythmic drugs have served a useful purpose, but they are clearly outmoded.

Activation of contraction in cat ventricular myocytes: effects of low Cd(2+) concentration and temperature.

The effects of Cd(2+) (20 microM) and different bath temperatures were used to study the contributions of two separate triggering mechanisms, L-type Ca(2+) current (I(Ca)) and reverse mode Na(+)/Ca(2+) exchange, to excitation-contraction (E-C) coupling in cat ventricular myocytes. Ionic currents and cell shortening were studied with patch pipettes filled with K(+)-containing internal solution and discontinuous ("switch") voltage clamp. Superfusion with Cd(2+) blocked cell shortening that closely mirrored the block of I(Ca); the voltage dependence of Cd(2+)-induced reduction in contraction was bell-shaped, displaying minima at test potentials below -10 mV and above +50 mV and a maximum at about +20 mV. Cd(2+)-insensitive cell shortening was blocked by ryanodine (10 microM) and Ni(2+) (4-5 mM). When an action potential was used as the command waveform for the voltage clamp (action potential clamp), Cd(2+) reduced contraction to approximately 60 +/- 7% of control cell shortening (n = 7). The remaining contraction was blocked by ryanodine and Ni(2+). Superfusion with nifedipine (10 microM) caused nearly identical effects to Cd(2+). The voltage dependence of contraction was sigmoidal at temperatures above 34 degrees C but bell-shaped below 30 degrees C. When Cd(2+) was added to superfusate, contraction was abolished at 25 degrees C (to 6 +/- 3% of control) but reduced only modestly at 34 degrees C (to 65 +/- 13% of control, test potential +10 mV, n = 4, P < 0.01). These results indicate that 1) there is a component of contraction that is sensitive to I(Ca) antagonists, and the block is equivalent with either organic or inorganic antagonists; 2) the contribution of Na(+)/Ca(2+) exchange to triggering of contraction under our experimental conditions is fairly linear throughout the entire voltage range tested; 3) the contribution of I(Ca) is superimposed on this background component contributed by the Na(+)/Ca(2+) exchanger; and 4) triggering via the exchanger is temperature-dependent, providing a major contribution at physiological temperatures but failing at temperatures below 30 degrees C in a nearly all-or-none fashion.

The role of Na(+)-Ca2+ exchange in activation of excitation-contraction coupling in rat ventricular myocytes.

1. The purpose of this study was to determine whether mechanisms other than Ca2+ influx via L-type Ca2+ current (ICa) might contribute to activation of contraction in rat ventricular myocytes. The whole-cell voltage-clamp technique was used with normal transmembrane K+ and Na+ gradients at 34 degrees C. The sarcoplasmic reticulum (SR) was conditioned with one to three prepulses to +100 mV for 100 ms. 2. Cell shortening (delta L) increased with test voltage up to a plateau level at about +20 mV, beyond which cell shortening remained fairly constant, thus describing a sigmoidal voltage dependence. This relationship was obtained when holding potential (Vh) was either -40 or -70 mV; however, greater shortening was obtained at the more negative Vh. 3. The sigmoidal V-delta L relationship was converted to a bell shape following the magnitude of ICa when internal Cs+ was substituted for K+ and when the temperature was reduced to 22 degrees C. 4. At 34 degrees C, block of ICa with nifedipine (10 microM) decreased shortening by about 50% but did not alter the voltage dependence of delta L when Vh was either -40 or -70 mV. Addition of Ni2+ (4-5 mM) blocked all remaining contractions. 5. When cell shortening was triggered by an action potential voltage clamp, there was again about 50% of the contraction that was insensitive to nifedipine but was blocked by Ni2+. 6. Our results demonstrate that there is a significant contribution of a nifedipine-insensitive mechanism to the activation of contraction. This mechanism is likely to be reverse mode Na(+)-Ca2+ exchange since it appears to be sensitive to both voltage and Ni2+. We conclude that a contribution of reverse Na(+)-Ca2+ exchange to activation of excitation-contraction coupling occurs in rat heart at near-physiological conditions which include warm temperatures, normal transmembrane Na+ and K+ gradients and activation in response to an action potential.

Catecholamine effects on intracellular sodium activity and tension in dog heart.

Because catecholamines have been reported to stimulate the sodium pump and Na+-K+-ATPase in skeletal and cardiac muscle, we examined the effects of isoproterenol (0.2-1.0 X 10(-6) M) and norepinephrine (2-4 X 10(-7) M) on intracellular sodium activity (aiNa) and twitch tension in dog Purkinje strands and ventricular muscles, aiNa was measured with Na+-sensitive microelectrodes. During the initial rapid increase in tension induced by catecholamines in Purkinje strands, no changes in aiNa were found. After 5-10 min aiNa decreased by about 2 mM, coincident with a small decline in twitch tension. When the catecholamine was removed, tension declined rapidly to a level less than control. Recovery of tension to its control level less than control. Recovery of tension to its control level occurred simultaneously with recovery of aiNa. Comparable changes in aiNa occurred in ventricular muscle, but the biphasic effect of catecholamines on tension was not seen. These results are consistent with sodium pump stimulation in cardiac muscle. In Purkinje strands the resulting changes in aiNa may alter the direct positive inotropic effect of catecholamines, probably by influencing Na+-Ca2+ exchange.

Relation between intracellular sodium and twitch tension in sheep cardiac Purkinje strands exposed to cardiac glycosides.

Changes in intracellular sodium ion activity (aiNa) produced by several cardiac glycosides were correlated with twitch tension in sheep cardiac Purkinje strands. Simultaneous measurements of aiNa and twitch tension were obtained through the use of Na-sensitive intracellular microelectrodes (ETH 227) in Purkinje preparations stimulated at a frequency of 1 Hz. All concentrations of ouabain, acetylstrophanthidin, and actodigin that were tested caused an increase in aiNa immediately before, or coincident with, a positive inotropic effect. No fall in aiNa was observed at any positive inotropic concentration of digitalis in these beating fibers. In all cases, the onset and washout of the positive inotropic effect were paralleled by the rise and fall in aiNa, respectively. No dissociation between changes in aiNa and twitch tension occurred at any concentration of any of the agents used. The relation between changes in aiNa and twitch tension was linear with 1 mM increase in aiNa producing about a 100% increase in the twitch magnitude. Propranolol did not significantly alter this relationship. The increase in aiNa with digitalis was also associated with a reduction in the maximum depolarization rate of the action potential, presumably as a consequence of a reduction in the transmembrane Na electrochemical gradient. These results indicate that the positive inotropic action of digitalis in sheep Purkinje strands is always associated with a rise in aiNa secondary to inhibition of the Na pump. This increase in aiNa could increase calcium available for contraction via the Na-Ca equilibrium exchange process. In addition, the increase in aiNa reduces Vmax, as a consequence of decreasing the electrochemical gradient for Na.

beta-adrenergic and cholinergic modulation of the inwardly rectifying K+ current in guinea-pig ventricular myocytes.

1. Whole-cell patch-clamp technique was used to study the beta-adrenergic and cholinergic regulation of the inwardly rectifying K+ conductance (gK1) in isolated guinea-pig ventricular myocytes. 2. In Cl(-)-free solutions or in the presence of 9-anthracenecarboxylic acid or Co2+, bath-applied isoprenaline (Iso) partially inhibited the steady-state whole-cell conductance (gss) calculated from the steady-state current (Iss)-voltage (Iss-V) curve at membrane voltages (Vm) negative to the equilibrium potential for potassium (EK). Iss was also inhibited at Vm positive to EK when the extracellular [K+] was 20 mM. The Iso-sensitive component of gss exhibited the characteristics of the inwardly rectifying K+ conductance (gK1). 3. The Iso-induced inhibition of gK1 was reversible, concentration dependent, blocked by propranolol, mimicked by both forskolin and dibutyryl cAMP, and prevented by including a cAMP-dependent protein kinase (PKA) inhibitor in the pipette solution. These findings suggest that PKA mediates the Iso-induced inhibition of gK1. 4. The apparent dissociation constant (KD) for the concentration dependence of Iso-induced inhibition was 0.035 microM and the Hill coefficient was approximately 1.0. A maximal Iso concentration (1 microM) inhibited gK1 by 40 +/- 4.1% (mean +/- S.E.M.; n = 13). 5. Bath application of acetylcholine (ACh, 0.1 microM or more) antagonized the Iso-induced (1 microM) inhibition of gK1; [ACh] > 1.0 microM antagonized 88 +/- 2.1% (n = 10) of the inhibition. ACh increased the KD for Iso to inhibit Iso-sensitive gK1 and also reduced the maximal Iso-induced inhibition. 6. ACh-induced antagonism could be abolished by pre-incubating myocytes with pertussis toxin (PTX), suggesting that a muscarinic receptor-coupled, PTX-sensitive G protein, Gi, is involved. 7. ACh (10 microM) also antagonized approximately 70% of the dibutyryl cyclic AMP (1 mM)-induced inhibition of gK1 (n = 3), suggesting that the ACh-induced antagonism involves more than simply inhibiting the Iso-mediated activation of adenylyl cyclase via the activated Gi. 8. Intracellularly applied okadaic acid (OkA, 1 microM) did not alter gK1 (control = 134 +/- 5.1 nS vs. OkA = 136 +/- 6.1 nS), but the Iso-induced decrease in gK1 was less (P < 0.001) with OkA present (42.1 +/- 2.4 nS, n = 5) than when absent (54.0 +/- 2.2 nS, n = 10). However, ACh (10 microM) failed to antagonize Iso-induced inhibition with OkA present, suggesting involvement of a protein phosphatase.