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.

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.

Activation of purified cardiac ryanodine receptors by dihydropyridine agonists.

Prior observations have raised the possibility that dihydropyridine (DHP) agonists directly affect the sarcoplasmic reticulum (SR) cardiac Ca(2+) release channel [i.e., ryanodine receptor (RyR)]. In single-channel recordings of purified canine cardiac RyR, both DHP agonists (-)-BAY K 8644 and (+)-SDZ202-791 increased the open probability of the RyR when added to the cytoplasmic face of the channel. Importantly, the DHP antagonists nifedipine and (-)-SDZ202-791 had no competitive blocking effects either alone or after channel activation with agonist. Thus there is a stereospecific effect of SDZ202-791, such that the agonist activates the channel, whereas the antagonist has little effect on channel activity. Further experiments showed that DHP agonists changed RyR activation by suppressing Ca(2+)-induced inactivation of the channel. We concluded that DHP agonists can also influence RyR single-channel activity directly at a unique allosteric site located on the cytoplasmic face of the channel. Similar results were obtained in human purified cardiac RyR. An implication of these data is that RyR activation by DHP agonists is likely to cause a loss of Ca(2+) from the SR and to contribute to the negative inotropic effects of these agents reported by other investigators. Our results support this notion that the negative inotropic effects of DHP agonists result in part from direct alteration in the activity of RyRs.

Altered E-C coupling in rat ventricular myocytes from failing hearts 6 wk after MI.

Excitation-contraction (E-C) coupling was investigated in rat hearts 6 wk after induction of myocardial infarction (MI) by ligation of the left coronary artery. Heart weight was increased by 74% and left ventricular end-diastolic pressure was 23 +/- 2 mmHg in MI compared with 8 +/- 2 mmHg in sham-operated controls (Sham, P < 0.001). Cell shortening was measured in voltage-clamped myocytes at 36 degrees C. In solutions where Cs(+) had been replaced by K(+), the voltage dependence of contraction was sigmoidal between -20 and +100 mV in Sham and MI cells. Verapamil (20 microM) blocked L-type Ca(2+) current and reduced contraction in Sham cells by approximately 50% (P < 0.01) but did not decrease contraction significantly in MI cells at test potentials above +10 mV. Verapamil-insensitive contractions were blocked by Ni(2+) (5 mM). Na(+)/Ca(2+) exchange current was doubled in MI compared with Sham cells at test potentials between -20 and +80 mV (P < 0.05), whereas mRNA and protein expression increased by 30-40%. Finally, voltage dependence of contraction was bell shaped in Na(+)-free solutions, but contraction was significantly increased in MI cells over a wider voltage range (P < 0.05). The insensitivity to Ca(2+) channel block in MI cells may result from an increased contribution of the Na(+)/Ca(+) exchanger to triggering of E-C coupling. These results suggest significant changes in E-C coupling in the hypertrophy and failure that develop in response to extensive MI.

Modification of cardiac Na(+) current by RWJ 24517 and its enantiomers in guinea pig ventricular myocytes.

We examined the effects of the cardiotonic agent RWJ 24517 (Carsatrin, racemate) and its (S)- and (R)-enantiomers on action potential duration, Na(+) current (I(Na)), and delayed rectifier K(+) current (I(K)) of guinea pig ventricular myocytes. RWJ 24517 (0. 1 and 1 microM) prolongation of action potential duration could not be accounted for by suppression of either the rapid (I(Kr)) or slow (I(Ks),) component of I(K), although RWJ 24517 did reduce I(Kr) at concentrations of 1 microM. A more dramatic effect of RWJ 24517 (0.1-1 microM) and the (S)-enantiomer of RWJ 24517 (0.1-3 microM) was an increase in peak I(Na) and slowing of the rate of I(Na) decay, eliciting a large steady-state current. Neither RWJ 24517 nor the (S)-enantiomer affected the fast time constant for I(Na) decay, but both significantly increased the slow time constant, in addition to increasing the proportion of I(Na) decaying at the slow rate. Both agents elicited a use-dependent decrease of peak I(Na) (3-10 microM), which probably resulted from a slowing of both fast and slow rates of recovery from inactivation. In contrast, the (R)-enantiomer of RWJ 24517 did not induce a steady-state component I(Na) or increase peak I(Na) up to 10 microM, but it decreased peak I(Na) at 30 microM. The (R)-enantiomer displayed little use-dependent reduction of I(Na) during trains of repetitive pulses and had no effect on rates of inactivation or recovery from inactivation. These actions of the racemate and the (S)-stereoisomer to slow inactivation and to prolong both Na(+) influx and action potential duration may contribute to the positive inotropic actions of these agents because the resulting accumulation of intracellular Na(+) would increase intracellular Ca(2+) via Na(+)/Ca(2+) exchange.

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.

Mechanisms of cardiomyocyte dysfunction in heart failure following myocardial infarction in rats.

Available information regarding the cellular and molecular mechanisms for reduced myocardial function after myocardial infarction (MI) is scarce. In rats with congestive heart failure (CHF), we examined cardiomyocytes isolated from the non-infarcted region of the left ventricle 6 weeks after ligation of the left coronary artery. Systolic left-ventricular pressure was reduced and diastolic pressure was markedly increased in the CHF-rats. The cardiomyocytes isolated from the CHF-hearts had increased resting length, reduced fractional shortening by 31% and a 34% increase in time to 90% relaxation compared to sham cells (P<0.01 for all). Peak L-type calcium currents were not significantly changed, but peak calcium transients measured with fura-2 were reduced by 19% (P<0.01). Moreover, the decline of the calcium transients as measured by the time constant of a monoexponential function was significantly increased by 26% (P<0.01). We also examined the contribution of the Ca2+-ATPase of the sarcoplasmic reticulum (SR) in the removal of cytosolic Ca2+ during relaxation by superfusing cells with 1 microM thapsigargin that effectively inhibits the Ca2+-ATPase. Relaxation time in CHF-cells was significantly less prolonged when this drug was used (P<0.01). This suggests that other mechanisms, probably the Na+-Ca2+ exchanger, contribute significantly to the relaxation rate in CHF. Simultaneous measurements of fura-2 transients and mechanical shortening did not reveal any alteration in the calcium-myofilament sensitivity in CHF. Our study clearly shows reduced shortening and prolonged relaxation in cardiomyocytes isolated from non-infarcted region of the left ventricle in heart failure. Moreover, we were able to relate the observed cardiomyocyte dysfunction to changes in specific steps in the excitation-contraction coupling.

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.

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.

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.

Characteristics of cocaine block of purified cardiac sarcoplasmic reticulum calcium release channels.

We have examined the effects of cocaine on the SR Ca2+ release channel purified from canine cardiac muscle. Cocaine induced a flicker block of the channel from the cytoplasmic side, which resulted in an apparent reduction in the single-channel current amplitude without a marked reduction in the single-channel open probability. This block was evident only at positive holding potentials. Analysis of the block revealed that cocaine binds to a single site with an effective valence of 0.93 and an apparent dissociation constant at 0 mV (Kd(0)) of 38 mM. The kinetics of cocaine block were analyzed by amplitude distribution analysis and showed that the voltage and concentration dependence lay exclusively in the blocking reaction, whereas the unblocking reaction was independent of both voltage and concentration. Modification of the channel by ryanodine dramatically attenuated the voltage and concentration dependence of the on rates of cocaine block while diminishing the off rates to a lesser extent. In addition, ryanodine modification changed the effective valence of cocaine block to 0.52 and the Kd(0) to 110 mM, suggesting that modification of the channel results in an alteration in the binding site and its affinity for cocaine. These results suggest that cocaine block of the SR Ca2+ release channel is due to the binding at a single site within the channel pore and that modification of the channel by ryanodine leads to profound changes in the kinetics of cocaine block.

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.

Beta-adrenergic and cholinergic modulation of inward rectifier K+ channel function and phosphorylation in guinea-pig ventricle.

1. To clarify the nature of the inhibition of whole-cell inwardly rectifying K+ current (IK1) by isoprenaline (Iso) and its antagonism by acetylcholine (ACh), we studied the effects of Iso and ACh and their surrogates on single channel currents (iK1) carried by inwardly rectifying K+ channels in cell-attached and excised inside-out patches obtained from guinea-pig ventricular myocytes. 2. Bath application of Iso suppressed iK1 channel activity in cell-attached patches. This was inhibited by propranolol. Bath-applied forskolin or dibutyryl cAMP mimicked the effect of bath-applied Iso. 3. Exposure of the cytosolic face of inside-out patches to purified catalytic subunit of the cAMP-dependent protein kinase (PKA) also suppressed iK1 channel activity, mimicking the effect of bath-applied Iso on iK1 recorded from cell-attached patches. 4. When applied directly to cell-attached patches via the patch pipette solution, ACh antagonized Iso-induced (1 microM applied via the bath) suppression of iK1 channels. In contrast, bath-applied ACh (10 microM) partially antagonized the effect of low concentrations of Iso (e.g. < 50 nM) on iK1 channels in cell-attached patches but had no detectable effect when 1 microM or more Iso was used. 5. In myocytes pretreated with pertussis toxin (PTX), ACh failed to antagonize Iso-induced suppression of iK1 channels. When inside-out patches were used, bath-applied preactivated exogenous inhibitory G protein subunit, G1 alpha, antagonized the suppression of iK1 channels induced by bath-applied catalytic subunit of PKA (PKA-CS), suggesting that a PTX-sensitive G1 alpha mediates ACh-induced antagonism of Iso-induced suppression of iK1. 6. Neither GTP gamma S nor G1 alpha antagonized the suppression of iK1 produced by bath-applied PKA-CS in inside-out patches when okadaic acid was present in the bath. In addition, bath application of alkaline phosphatase also reactivated iK1 channels suppressed by PKA-CS. 7. Findings in guinea-pig ventricular myocytes suggest that iK1 can be suppressed by a PKA-mediated phosphorylation of the iK1 channel occurring in response to Iso-induced beta-adrenergic receptor activation and that ACh can antagonize the suppression by mechanisms that involve both intracellular and membrane-delimited pathways. The membrane-delimited pathway appears to involve M2-cholinergic receptors, their associated G protein, G1, and a protein phosphatase, all located in the sarcolemma in close proximity to the involved iK1 channels.

An analysis of lidocaine block of sodium current in isolated human atrial and ventricular myocytes.

Lidocaine is a Na+ channel blocker that is highly effective for the treatment of ventricular tachyarrhythmias, but is largely ineffective against atrial arrhythmias. If is not known if this differential efficacy is the result of differences in lidocaine inhibition of atrial v ventricular Na+ channels. The purpose of the present study was to characterize lidocaine block of Na+ channels in human atrium and ventricle. We used the whole cell voltage clamp technique with low external and internal Na+ concentrations (5 mM) to study the Na+ current (INa) in single human atrial and ventricular cells isolated enzymatically from specimens obtained during surgery. We found that tonic block of peak INa by lidocaine (200 microM, holding potential = -140 mV, 0.1 Hz, at 17 degrees C) was not voltage dependent in either cell type. Reduction of maximal peak Na+ conductance in 41 atrial cells (19.8 +/- 2.7%) and nine ventricular cells (22.6 +/- 1.7%) was virtually identical. The rate of onset of block development was determined during depolarization to either -80 mV or -20 mV. The time course of onset of block was described by a single exponential at -80 mV and by a double exponential at -20 mV. When the rate of block onset during a single conditioning depolarization was compared to that which developed during conditioning by a train of brief pulses (3 ms, 30 Hz), onset was faster during the pulse train. The results were nearly identical for atrial and ventricular INa. The time constants of recovery from block following either single pulse or multiple-pulse conditioning did not differ. These data suggest that lidocaine binds to both the activated and inactivated states of the human cardiac Na+ channel. Using an analytical method based upon the Guarded Receptor Hypothesis, we calculated apparent rate constants describing lidocaine's interaction with the three primary states of the human Na+ channel (resting, activated and inactivated). Rate constants were similar to those reported for other mammalian species. Our results demonstrate that lidocaine block of INa is virtually identical for human atrial and ventricular cells; thus additional mechanisms must be invoked to explain the differential efficacy of lidocaine against ventricular as compared to atrial dysrhythmias.

Alterations in muscarinic K+ channel response to acetylcholine and to G protein-mediated activation in atrial myocytes isolated from failing human hearts.

BACKGROUND: A variety of previous studies have demonstrated reduced diastolic potential and electrical activity in atrial specimens from patients with heart disease. Although K+ channels play a major role in determining resting membrane potential and repolarization of the action potential, little is known about the effects of preexisting heart disease on human atrial K+ channel activity. METHODS AND RESULTS: We characterized the inwardly rectifying K+ channel (IKI) and the muscarinic K+ channel [IK(ACh)] in atrial myocytes isolated from patients with heart failure (HF) and compared electrophysiological characteristics with those from donors (control) by the patch-clamp technique. Resting membrane potentials of isolated atrial myocytes from HF were more depolarized (-51.1 +/- 9.7 mV, mean +/- SD, n = 30 patients) than those from donors (-73.0 +/- 7.2 mV, n = 4 patients, P < .001). The action potential duration in HF was longer than that in donors. Although acetylcholine (ACh) shortened the action potential, reduced the overshoot, and hyperpolarized the atrial cell membrane in HF, these effects were attenuated compared with those observed in donors. The whole-cell membrane current slope conductance in HF was small, the reversal potential was more positive, and the sensitivity to ACh was less compared with donors. In single-channel recordings from cell-attached patches, IK1 channel conductance and gating characteristics were the same in HF and donor atria. When ACh was included in the pipette solution, IK(ACh) was activated in both groups. Single-channel slope conductance of IK(ACh) averaged 42 +/- 3 pS (n = 28) in HF and 44 +/- 2 pS (n = 4) in donors, and mean open lifetime was 1.3 +/- 0.3 milliseconds (n = 24) in HF and 1.5 +/- 0.4 milliseconds (n = 4) in donors. These values were virtually identical in the two groups (not significantly different, NS), although both single IK1 and IK(ACh) channel densities were less in HF. Channel open probability of IK(ACh) was also less in HF (4.0 +/- 1.2%, n = 24) than in donors (6.8 +/- 1.1%, n = 3, P < .01). The concentration of ACh at half-maximal activation was 0.11 mumol/L in HF and 0.03 mumol/L in donors. In excised inside-out patches, IK(ACh) from HF required higher concentrations of GTP and GTP gamma S to activate the channel compared with donors. These results suggest a reduced IK(ACh) channel sensitivity to M2 cholinergic receptor-linked G protein (Gi) in HF compared with donors. CONCLUSIONS: Atrial myocytes isolated from failing human hearts exhibited a lower resting membrane potential and reduced sensitivity to ACh compared with donor atria. Whole-cell and single-channel measurements suggest that these alterations are caused by reduced IK1 and IK(ACh) channel density and reduced IK(ACh) channel sensitivity to Gi-mediated channel activation in HF.

Lactate enhances sodium channel conductance in isolated guinea pig ventricular myocytes.

Myocardial hypoxia and ischemia result in the production of lactate. To study the effect of lactate on the rapid Na+ current (INa), we used the whole cell voltage-clamp technique in enzymatically isolated guinea pig ventricular myocytes. Experiments were conducted at 16 degrees C. Extracellular Na+ concentration ([Na+]o) was maintained in control and test solutions and extracellular pH was 7.4. Lactate (4-10 mM, either sodium lactate or lactic acid) augmented INa in each of eight experiments, increasing the peak Na+ conductance from 75.4 to 84.7 nS (13-16% at all test voltages in the linear portion of the conductance curve). The voltage dependence of steady-state availability and the time course of inactivation remained unchanged. The increase in peak Na+ conductance was concentration dependent, with an apparent dissociation constant of 1.8 mM and Hill coefficient of 1.8. Lactate in the range of 1-10 mM did not significantly reduce the Ca2+ activity of test solutions. These effects of lactate were still observed in Mg(2+)-free test solutions and when the buffering capacity of internal solution was reinforced by increasing N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid concentration from 5 to 20 mM. In conclusion, lactate enhances INa via a mechanism that does not involve chelation of Ca2+ or Mg2+ or changes in intracellular pH. These effects of lactate on the Na+ channel might alter electrophysiological properties during myocardial ischemia and could protect the heart from ischemia-induced conduction abnormalities or, alternatively, could lead to arrhythmias.

Amiodarone blocks the inward rectifier potassium channel in isolated guinea pig ventricular cells.

We examined the effects of amiodarone (5-20 microM) on both whole-cell inward rectifier potassium current (IK1) and single IK1 channel activity in isolated guinea pig ventricular myocytes using patch-clamp techniques. In whole-cell voltage-clamp experiments (n = 8), amiodarone (10-20 microM) caused only a small reduction of outward current at -50 mV (12 +/- 6%, no significant difference, N.S.). However, inward current was significantly reduced at -120 mV (21 +/- 7%; P < .05). When CdCl2 (100 microM) and tetrodotoxin (10 microM) were used to block inward Ca++ and Na+ current, respectively, amiodarone significantly reduced IK1 in both the inward (14 +/- 5% at -120 mV; P < .02) and outward (12 +/- 5% at -50 mV; P < .05; n = 11) directions. However, block required high drug concentrations (10-20 microM) and was slow in onset. In contrast, amiodarone did not affect membrane current when IK1 had been previously blocked by Ba++ (5 mM). In inside-out patch-clamp experiments, amiodarone (5 microM) reduced single IK1 channel open probability by increasing interburst interval (from 0.6 +/- 0.03 to 3.1 +/- 0.9 sec; n = 5; P < .05) with no significant difference in the duration of mean open and closed times or the number of shut events within a burst. The net result was that there was only a small change in both burst duration and single-channel kinetics within a burst. Complete channel block occurred after the increase in interburst interval (n = 6 of six cells).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Sodium current in isolated human ventricular myocytes.

Although fast sodium current (INa) plays a major role in the generation and conduction of the cardiac impulse, the electrophysiological characteristics of INa in isolated human ventricular myocytes have not yet been fully described. We characterized the human ventricular INa of enzymatically isolated myocytes using whole cell voltage-clamp techniques. Sixty myocytes were isolated from ventricular specimens obtained from 22 patients undergoing open-heart surgery. A low temperature (17 degrees C) and Na+ concentration in the external solution (5 or 10 mM) allowed good voltage control and facilitated the measurement of INa. Cs+ was substituted for K+ in both internal and external solutions to block K+ currents, and F- was added to the internal solution to block Ca2+ current. INa was activated at a voltage threshold of approximately -70 mV, and maximal inward current was obtained at approximately -30 mV (holding potential = -140 mV). The voltage dependence of steady-state INa availability (h infinity) was sigmoidal with half inactivation occurring at -97.3 +/- 1.1 mV and a slope factor of 5.77 +/- 0.10 mV (n = 60). We did not detect any significant differences in these parameters in cells from patients with a variety of disease states, with or without congestive heart failure. The overlap in voltage dependence of h infinity and Na+ conductance suggested the presence of a Na+ "window" current. An inactivation time course was voltage dependent and was fitted best by the sum of two exponentials. The rate of recovery from inactivation also was voltage dependent and fitted by the sum of two exponentials.(ABSTRACT TRUNCATED AT 250 WORDS)