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.

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)

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.

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).

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.

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.

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.

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.

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.

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.

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.

Dantrolene sodium: effects on isolated cardiac tissues.

The effects of dantrolene sodium on dog Purkinje fibers, cat atrial and ventricular muscles were studied. Action potential duration was significantly increased and contractility was significantly decreased by dantrolene in all three types of tissue. The plateau phase of Purkinje fiber and occasionally atrial action potential was slightly depressed. Dantrolene sodium had no significant effect on resting membrane potential, action potential amplitude or upstroke velocity of phase 6. The negative inotropic effects were most pronounced in Purkinje fibers, followed by atrial muscle while papillary muscles were least sensitive. Contractile force of Purkinje fibers was decreased by relatively the same amount at all frequencies of stimulation. At faster rates, atrial and ventricular muscle contractility was depressed relatively more than at slower rates. Slow response action potentials in cat papillary muscle were diminished slightly, but this effect was not significant. All drug effects took 10 to 15 min to develop, reached a steady state after 30 to 40 min, and were irreversible upon washout. Increasing the extracellular calcium concentration reversed the dantrolene-induced changes. These findings suggest that effects of dantrolene are mediated in part by a decrease in the intracellular free calcium concentration in cardiac tissue.

Depolarizing effects of catecholamines in quiescent sheep cardiac Purkinje fibers.

Isoproterenol reversibly depolarizes quiescent sheep cardiac Purkinje fibers, in contrast to its reported hyperpolarizing effect in many excitable tissues. The depolarization is inhibited by drugs that block beta 1-adrenergic receptors. Tetrodotoxin and verapamil have no effect on the isoproterenol-induced depolarization. Cesium reduced the isoproterenol-induced depolarization by 74%. The voltage dependency of activation of a current component called If, measured under voltage clamp, was shifted in the depolarizing direction by isoproterenol. No evidence was found to support the suggestion made for other tissues that the Na+-K+ pump is stimulated by isoproterenol. These data suggest that in quiescent sheep Purkinje fibers the isoproterenol-induced depolarization reflects activation of a pacemaker current.

Effect of catecholamines on intracellular pH in sheep cardiac Purkinje fibres.

1. It has been reported that catecholamines affect intracellular pH (pHi) in a number of tissues, generally by altering the kinetics of the Na(+)-H+ exchanger. We postulated that catecholamines might affect pHi in cardiac tissue. We tested this in resting sheep cardiac Purkinje fibres by measuring transmembrane potential and pHi with standard and H(+)-sensitive microelectrodes. 2. Adrenaline and the beta-adrenergic agonist isoprenaline, both 5.0 x 10(-6) M, resulted in depolarization and intracellular acidification (adrenaline, 0.03 +/- 0.01 pH units, n = 8, P = 0.005; isoprenaline, 0.08 +/- 0.01 pH units, n = 17, P = 0.0001). The alpha-adrenergic agonist phenylephrine, at concentrations up to 200 microM, had no significant effect on membrane potential or pHi. 3. Isoprenaline significantly attenuated the half-time (t0.5) for pHi recovery from intracellular acidification induced via the NH4Cl pulse technique. Isoprenaline also attenuated the hyperpolarization that is normally seen at the onset of pHi recovery. Phenylephrine slightly reduced the t0.5 for recovery, although the reduction did not reach statistical significance. 4. Forskolin, 7.5-10 x 10(-5) M, an agent that raises intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP), also induced depolarization and acidification, similar to that induced by adrenaline and isoprenaline. 5. In the presence of the Na(+)-H+ exchange blocker 5-dimethyl amiloride, 2-6 x 10(-5) M, isoprenaline-induced acidification was blunted but not abolished. When administered in Na(+)-free Tyrode solution, isoprenaline-induced acidification was also not abolished. Buffering power, tested using the NH4Cl method, was not decreased by isoprenaline, but rather, was slightly increased. Reversal of H+ driving force across the cell membrane from the normally inward direction to outward (achieved by increasing pHo to 8.3-8.5 and depolarizing the membrane with 10 mM K+ solutions) did not prevent intracellular acidification from occurring in the presence of isoprenaline. When glycolysis was inhibited by a 60 min exposure to glucose-free solution containing 5.5 mM 2-deoxyglucose, acidification by isoprenaline was nearly abolished. 6. We conclude that, in resting sheep Purkinje fibres, beta- but not alpha-adrenergic stimulation results in intracellular acidification and depolarization, probably mediated via an increase in cyclic AMP. beta- but not alpha-adrenergic stimulation slows the rate of recovery from intracellular acidification and blunts the hyperpolarization associated with this recovery. 7. The intracellular acidification appears to be due both to partial inhibition of Na(+)-H+ exchange and to stimulation of glycolysis by beta-adrenergic agents.