Aligned human cardiac syncytium for in vitro analysis of electrical, structural, and mechanical readouts.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC-CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC-CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two-dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC-CMs.

Drug-induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine.

BACKGROUND AND PURPOSE: Fluoxetine (Prozac) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro. EXPERIMENTAL APPROACH: We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels heterologously expressed in HEK293 cells. KEY RESULTS: Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (IhERG). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of IhERG but did not alter fluoxetine's effect on hERG channel protein trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce IhERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

Transient left ventricular apical ballooning: a review of the literature.

Transient left ventricular apical ballooning is a newly defined syndrome characterized by sudden onset of chest symptoms, electrocardiographic changes characteristic of myocardial ischemia, transient left ventricular dysfunction-particularly in the apical region, low-grade troponin elevation, and no significant coronary stenosis by angiogram. This syndrome is also referred to as Takotsubo cardiomyopathy, "Ampulla" cardiomyopathy, Human Stress cardiomyopathy, and Broken Heart Syndrome. Emergency physicians, family physicians, general internists, and cardiologists may all encounter this syndrome at the point of contact. The similarity to acute coronary syndrome requires all clinicians who may potentially care for these patients to familiarize themselves with this newly recognized disease. We provide a recent case and review the current literature surrounding this syndrome.

Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Anionic phospholipids modulate the activity of inwardly rectifying potassium channels (Fan, Z., and J.C. Makielski. 1997. J. Biol. Chem. 272:5388-5395). The effect of phosphoinositides on adenosine triphosphate (ATP) inhibition of ATP-sensitive potassium channel (K(ATP)) currents was investigated using the inside-out patch clamp technique in cardiac myocytes and in COS-1 cells in which the cardiac isoform of the sulfonylurea receptor, SUR2, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K(ATP) in low [ATP] (1 microM) within 30 s. Phosphoinositides desensitized ATP inhibition with a longer onset period (>3 min), activating channels inhibited by ATP (1 mM). Phosphoinositides treatment for 10 min shifted the half-inhibitory [ATP] (K(i)) from 35 microM to 16 mM. At the single-channel level, increased [ATP] caused a shorter mean open time and a longer mean closed time. Phosphoinositides prolonged the mean open time, shortened the mean closed time, and weakened the [ATP] dependence of these parameters resulting in a higher open probability at any given [ATP]. The apparent rate constants for ATP binding were estimated to be 0.8 and 0.02 mM(-1) ms(-1) before and after 5-min treatment with phosphoinositides, which corresponds to a K(i) of 35 microM and 5.8 mM, respectively. Phosphoinositides failed to desensitize adenosine inhibition of K(ATP). In the presence of SUR2, phosphoinositides attenuated MgATP antagonism of ATP inhibition. Kir6.2DeltaC35, a truncated Kir6.2 that functions without SUR2, also exhibited phosphoinositide desensitization of ATP inhibition. These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition. We propose a model of the ATP binding site involving positively charged residues on the COOH-terminus of Kir6.2, with which phosphoinositides interact to desensitize ATP inhibition.

Kinetic effects of quaternary lidocaine block of cardiac sodium channels: a gating current study.

The interaction of antiarrhythmic drugs with ion channels is often described within the context of the modulated receptor hypothesis, which explains the action of drugs by proposing that the binding site has a variable affinity for drugs, depending upon whether the channel is closed, open, or inactivated. Lack of direct evidence for altered gating of cardiac Na channels allowed for the suggestion of an alternative model for drug interaction with cardiac channels, which postulated a fixed affinity receptor with access limited by the conformation of the channel (guarded receptor hypothesis). We report measurement of the gating currents of Na channels in canine cardiac Purkinje cells in the absence and presence of QX-222, a quaternary derivative of lidocaine, applied intracellularly, and benzocaine, a neutral local anesthetic. These data demonstrate that the cardiac Na channel behaves as a modulated rather than a guarded receptor in that drug-bound channels gate with altered kinetics. In addition, the results suggest a new interpretation of the modulated receptor hypothesis whereby drug occupancy reduces the overall voltage-dependence of gating, preventing full movement of the voltage sensor.

Sodium currents in single cardiac Purkinje cells.

The sodium (Na) channel is the fundamental unit of excitability in heart muscle. This channel has been very difficult to study in detail, because the major experimental tool, the voltage clamp, has been difficult to use in multicellular tissue. In the absence of more direct studies in the heart, it has been assumed that the sodium channel in the heart was the same as that in nerve tissue, where it could be studied quantitatively. However, the sodium channel is not likely to be the same as in nerve, because it responds differently to local anesthetics and to other drugs such as tetrodotoxin. It is essential to learn the details of the cardiac sodium channel, because it is the membrane process that underlies many lethal cardiac arrhythmias, and it is the molecular site of action of the most effective antiarrhythmic drugs. Single cardiac Purkinje cells were dialyzed at room temperature through a large bore pipette, and their Na+ currents were studied under voltage clamp control. The peak currents were 0.5 to 1.0 mA/cm2, assuming a 1 mu farad/cm2 membrane. Peak currents near 0 mV were achieved in less than 1 ms. The decay of the Na+ current did not correspond to a single exponential process. This result and the observation that recovery from inactivation occurred with a latency are inconsistent with the original Hodgkin-Huxley model, but they qualitatively fit a model with two sequential inactivated states or a model with two kinetically different types of Na+ channels. The steady state inactivation curve shifted in the negative direction after initiation of intracellular dialysis, stabilizing with a half-availability voltage of -115 mV.

Intrinsic lidocaine affinity for Na channels expressed in Xenopus oocytes depends on alpha (hH1 vs. rSkM1) and beta 1 subunits.

OBJECTIVE: The affinity of lidocaine for the alpha-subunit of the Na channel has been reported to be greater for heart than for non-heart alpha-subunits, and also to be no different. Lidocaine block has a complex voltage dependence caused by a higher affinity for the inactivated state over the resting state. Inactivation kinetics, however, depend upon the alpha-subunit isoform and the presence of the auxiliary beta 1-subunit and will affect measures of block. METHODS: We studied the voltage dependence of lidocaine block of Na currents by a two microelectrode voltage clamp in oocytes injected with RNA for the Na channel alpha-subunits of human heart (hH1a) or a rat skeletal muscle (rSkM1) alone, or coexpressed with the beta 1-subunit. RESULTS: The midpoints of availability for a 25-s conditioning potential in control solutions were -65 mV for rSkM1, -50 for rSkM1 + beta 1, -78 mV for hH1a and -76 for hH1a + beta 1. The Kd of tonic lidocaine block was measured at -90, -100, -110, -120 and -130 mV in the same oocytes. The apparent Kd for both isoforms +/- beta 1 became greater with more negative holding potentials, but tended to reach different plateaus at -130 mV (Kd = 2128 microM for rSkM1, 1760 microM for rSkM1 + beta 1, 433 for hH1a, and 887 microM for hH1a + beta 1). Inactivated state affinities, assessed by fitting the shift in the Boltzmann midpoint of the availability relationship to the modulated receptor model, were 4 microM for rSkM1, 1 microM for rSkM1 + beta 1, 7 microM for hH1a and 9 microM for hH1a + beta 1. CONCLUSION: The heart Na channel alpha-subunits expressed in oocytes have an intrinsically higher rest state affinity for lidocaine compared to rSkM1 after the voltage- and state dependence of block are considered. Coexpression with beta 1 modestly increased the rest affinity of lidocaine for rSkM1, but had the opposite effect for hH1a.

The heart sodium channel phenotype for inactivation and lidocaine block.

The heart Na channel, although resembling other voltage-gated Na channels, has important functional and structural differences. For heart channels expressed in oocytes, the midpoint of the inactivation relationship was 13 mV negative to that of rat skeletal muscle Na channels, and sensitivity to tonic lidocaine block was approximately 5 times more sensitive for heart. Co-expression with the beta subunit increased the difference in inactivation midpoint to 24 mV, largely by changing the midpoint of the rat skeletal muscle channel by 10 mV in the positive direction. Co-expression with beta 1 decreased lidocaine sensitivity for heart but not for skeletal muscle Na channels, and decreased but did not eliminate the greater heart sensitivity to lidocaine block. The differences in inactivation are likely to account for some, but not all, of the differences in lidocaine sensitivity. This cardiac phenotype is important for the role the channel plays in cardiac physiology and pathophysiology, and also may lead to elucidation of structure-function relationships.

Intracellular H+ and Ca2+ modulation of trypsin-modified ATP-sensitive K+ channels in rabbit ventricular myocytes.

ATP-sensitive K+ current (IK.ATP) channels are thought to play a role in the K+ efflux observed in cardiac ischemia. Intracellular acidosis is a prominent early effect in ischemia; therefore, the effects of acidosis on IK.ATP may have certain pathophysiological implications. Increased intracellular proton concentration (pHi) is known to regulate IK.ATP in frog skeletal muscle by increasing open probability. The pHi effect on IK.ATP is not clearly understood in heart because, unlike frog skeletal muscle, low pHi causes IK.ATP run-down in inside-out patches. This would tend to mask any opening effect of low pHi if it exists. Trypsin modification of IK.ATP has recently been shown to prevent run-down in inside-out patches. We used single channel recordings in inside-out patches to study IK.ATP after exposure to trypsin. After trypsin treatment, the open probability of IK.ATP was not sensitive to pHi in the absence of ATP. In the presence of ATP, however, a decrease in pHi consistently increased the open probability of trypsin-modified IK.ATP by reducing ATP inhibition. In the absence of ATP the mean open probability was 0.43 +/- 0.07 at pHi 7.4, and 0.5 mM ATP decreased the mean open probability to 0.03 +/- 0.04 at pHi 7.4, but mean open probability was significantly increased to 0.20 +/- 0.07 at pHi 6.3 (n = 7, p < 0.01). Ca2+ did not affect the activity of trypsin-modified IK.ATP in either the absence or presence of ATP at pHi 7.4. However, Ca2+ was able to antagonize the low pHi effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Block of sodium current by heptanol in voltage-clamped canine cardiac Purkinje cells.

Heptanol blocks sodium current (INa) in nerve, but its effects on cardiac INa have not been well characterized. Block of INa by heptanol was studied in 16 internally perfused voltage-clamped cardiac Purkinje cells at reduced Na+ (45 mM outside, 0 mM inside). Heptanol block of peak sodium conductance was well described by a single-site binding curve with half block at 1.3 mM (20 degrees C) and showed no "use dependence." With 1.5 mM heptanol, block increased slightly by 0.7%/degrees C from 10 degrees C to 27 degrees C. With 3.0 mM heptanol, steady-state availability shifted by 9.4 +/- 1.3 mV (n = 6) in the hyperpolarizing direction, and steady-state activation shifted by 8.3 +/- 2.2 mV (n = 5) in the depolarizing direction, thus closing off the INa "window current." Heptanol also decreased the time to peak and accelerated the decay of INa. Similar results were found with octanol at lower concentrations. These alcohols have important effects on cardiac INa at concentrations used in studies for cellular uncoupling in heart.

Temperature dependence of sodium current block by lidocaine in cardiac Purkinje cells.

Cardiac Na current block by antiarrhythmic drugs has usually been studied at lower than physiological temperatures to achieve adequate voltage control, but the effect of temperature on block is not well characterized. Using a large suction pipette, we studied the effect of 50 microM lidocaine on blocking peak Na current between 10 and 25 degrees C in cells isolated from canine cardiac Purkinje fibers using three voltage clamp protocols: 1) development of block during a prolonged depolarization, 2) recovery from block after a prolonged depolarization, and 3) development of block during trains of depolarizations. At least two exponential components [a fast component with time constant (tau f) and relative amplitude (as) and a slow component with time constant (tau f) and relative amplitude (as)] were required to describe both development and recovery in lidocaine. For development of block between 10 and 25 degrees C, tau s decreased from 1,100 to 200 ms but tau f remained about the same (less than 30 ms), and as approximately equal to 0.8 and af approximately equal to 0.2 did not change with temperature. For recovery between 10 and 25 degrees C, tau a decreased from approximately 2,500 to 35 ms, tau f was relatively constant at approximately 25 ms, af decreased from 0.46 to 0.16, and as increased from 0.54 to 0.84. The fractional block developed during pulse trains decreased at higher temperatures (from 0.55 at 10 degrees C to 0.22 at 25 degrees C) and developed more quickly with time constant 6 pulses at 10 degrees C to 1.3 pulses at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of lysophosphatidylcholine-modified cardiac Na channels by a lidocaine derivative QX-222.

Single Na channels from rat and rabbit ventricular cells were studied with use of the excised inside-out patch-clamp technique. To investigate local anesthetic interactions with Na channels modified by the ischemic metabolite lysophosphatidylcholine (LPC), the quaternary ammonium lidocaine derivative QX-222 [2-(trimethylamino)-N-(2,6-dimethylphenyl)acetamide] was applied to the cytoplasmic side of patches from untreated cells and from those treated with LPC for approximately 1 h. Single-channel amplitudes and kinetics for unmodified channels were similar to those reported previously for cardiac cells with a single-component, mean-channel open time. LPC-modified channels showed prolonged open channel bursting with a two-component, mean open time, suggesting two open states. Conductance sublevels to the 60-70% level of the main conductance were found in both unmodified and LPC-modified channels and also with and without QX-222 present. QX-222 reversibly shortened the open time of the unmodified channel and for both open times of the LPC-modified channel without decreasing single-channel amplitude. Calculated association rates for QX-222 with the channel were found to be greater for the open states of the modified channel than those for the unmodified channel. Thus the lidocaine analogue QX-222 interacts with and blocks the open state of both unmodified and LPC-modified, cardiac Na channels. The blocking effect on LPC-modified channels would be predicted to be greater both because of the longer dwell time in the high-affinity open states for modified channels and also because of an intrinsically greater association rate in the modified channels.

Anionic phospholipids activate ATP-sensitive potassium channels.

The ATP-sensitive potassium channel (KATP) controls insulin release in pancreatic beta-cells and also modulates important functions in other cell types. In this study we report that anionic phospholipids activated KATP in pancreatic beta-cells, cardiac myocytes, skeletal muscle cells, and a cloned KATP composed of two subunits (SUR/Kir6. 2) stably expressed in a mammalian cell line. The effectiveness was proportional to the number of negative charges on the head group of the anionic phospholipid. Screening negative charges with polyvalent cations antagonized the effect. Enzymatic treatment with phospholipases that reduced charge on the lipids also reduced or eliminated the effect. These results suggest that intact phospholipids with negative charges are the critical requirement for activation of KATP, in distinction from the usual cell signaling pathway through phospholipids that requires cleavage. Mutations of two positively charged amino acid residues at the C terminus of Kir6. 2 accelerated loss of channel activity and reduced the activating effects of phospholipids, suggesting involvement of this region in the activation. Metabolism of anionic phospholipids in plasmalemmal membrane may be a novel and general mechanism for regulation of KATP and perhaps other ion channels in the family of inward rectifiers.

The electrophysiology of acute myocardial ischemia.

The cellular events leading to clinical manifestations of acute (less than 30 minutes) ischemia are discussed, with emphasis on the role of early extracellular K+ accumulation and its effect on sodium current kinetics, which causes changes in excitability, conduction, and refractory periods. These are the cellular phenomena responsible for ST elevation, reentry, and enhanced automaticity, which characterize the early clinical events in ischemia. During this early period, ATP levels are normal and calcium metabolism appears normal.

Post-repolarization block of cardiac sodium channels by saxitoxin.

Phasic block of rat cardiac Na+ current by saxitoxin was assessed using pulse trains and two-pulse voltage clamp protocols, and the results were fit to several kinetic models. For brief depolarizations (5 to 50 ms) the depolarization duration did not affect the rate of development or the amplitude of phasic block for pulse trains. The pulse train data were well described by a recurrence relation based upon the guarded receptor model, and it provided rate constants that accurately predicted first-pulse block as well as recovery time constants in response to two-pulse protocols. However, the amplitudes and rates of phasic block development at rapid rates (> 5 Hz) were less than the model predicted. For two pulse protocols with a short (10 ms) conditioning step to -30 mV, block developed only after repolarization to -150 mV and then recovered as the interpulse interval was increased. This suggested that phasic block under these conditions was caused by binding with increased affinity to a state that exists transiently after repolarization to -150 mV. This "post-repolarization block" was fit to a three-state model consisting of a transient state with high affinity for the toxin, the toxin bound state, and the ultimate resting state of the channel. This model accounted for the biphasic post-repolarization block development and recovery observed in two-pulse protocols, and it more accurately described phasic block in pulse trains. The transient state after repolarization was predicted to have a dwell time of 570 ms, an on rate for saxitoxin of 16 s-1 micro M-1, and an off rate of 0.2 s-1 (KD = 12 nM). These results and the proposed model suggest a novel variation on phasic block mechanisms and suggest a long-lived transient Na+ channel conformation during recovery.

External site for local anesthetic block of cardiac Na+ channels.

We report patch clamp studies of single Na+ channels from cardiac ventricular and Purkinje cells that support the hypothesis that local anesthetics can act from the outside of the membrane, and that demonstrate some aspects of their mechanism of action. Inclusion of lidocaine (0.1 mM) or QX-314 (0.5 mM), a membrane-impermeant, quaternary ammonium derivative of lidocaine, in the pipette solution for on-cell single channel recording demonstrated four important findings. (1) The open probability of the channel is reduced by drug in a use-dependent way. (2) Late openings are preferentially reduced. (3) Mean open time is shortened. (4) Hyperpolarization enhances recovery of the drug-bound channels. These findings are consistent with a hyperpolarizing shift of the transition rates for drug-bound channel. Further, we postulate that there is a drug-bound channel conformation which conducts current. At least some of the properties of local anesthetic interaction with the cardiac Na+ channels may be the result of kinetic effects mediated by binding to an external site.

Two components of use-dependent block of sodium current by lidocaine in voltage clamped cardiac Purkinje cells.

Lidocaine is an effective antiarrhythmic and local anesthetic agent, and it has also been a useful tool for characterizing drug interactions with the Na channel. We studied the block of sodium current (INa) caused by lidocaine (20 microM to 2 mM) in canine cardiac Purkinje cells at reduced Na (45 mM outside, 0 mM inside) and temperature (14-17 degrees C). Lidocaine block of INa developed in response to strains of depolarizations, was greater with increasing drug concentration, faster pulsing frequency, longer depolarization duration, and less negative holding potential. The time course of the fall in peak INa was best fitted by two exponentials. In contrast, block of peak INa in pulse trains by the permanently charged lidocaine analogue QX-314 was always best fitted by a single, slow exponential. When the pH was lowered from 7.4 to 6.8 to increase the proportion of the charged form of lidocaine, the contribution of the slow component increased both absolutely and relative to the fast component. The time course of the development of lidocaine block was characterized with a two pulse protocol, and it showed that block developed in a biexponential manner with initial rapid binding to a transiently available state (such as the open state) and with slower binding to a long-lived state (such as the inactivated state). We conclude that the complex binding and unbinding of lidocaine to different conformations of the cardiac Na channel is related to different affinities for the charged and uncharged forms of lidocaine.(ABSTRACT TRUNCATED AT 250 WORDS)

Lidocaine alters activation gating of cardiac Na channels.

The class IB antiarrhythmic drug, lidocaine, interacts strongly with depolarized sodium (Na) channels, an action that is thought to underlie its clinical efficacy. Previously, we have reported Na channel gating current (Ig) experiments with a quaternary form of lidocaine, QX-222, which binds preferentially to open Na channels and modifies the gating-charge/voltage (Q/V) relationship of cardiac Na channels by reducing maximal gating charge (Qmax) and lessening its voltage dependence. We report here investigations with lidocaine itself on Ig of native canine and cloned human cardiac Na channels. Although the state dependence of lidocaine binding to Na channels differs from that of quaternary drugs, Ig measurements demonstrated that lidocaine produced changes in the Q/V relationships similar to those elicited by QX-222, with a reduction in Qmax by 33% and a corresponding decrease in the slope factor. Concentration/response curves for the reduction in gating charge by lidocaine matched those for the block of sodium current (I(Na)), as would be expected if modification of Na channel voltage sensors by lidocaine underlied its action. The application of site-3 toxins, which inhibit movement of the voltage sensor associated with inactivation, to lidocaine-bound Na channels elicits an additional reduction in Qmax suggesting that lidocaine does not "stabilize" the Na channel in an inactivated state. We conclude that lidocaine blocks I(Na) by modification of the Na channel's voltage sensors predominately associated with channel activation leading to channel opening.

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart.

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

Slowly recovering cardiac sodium current in rat ventricular myocytes: effects of conditioning duration and recovery potential.

INTRODUCTION: Recovery of the Na channel from inactivation is essential to the normal conduction and refractoriness of the myocardium. In addition to fast recovery, occurring within several milliseconds at hyperpolarized potentials, a component of the current exhibits slow recovery occurring over hundreds of milliseconds. Long conditioning depolarizations potentiate slow recovery. METHODS AND RESULTS: This study was designed to test conditioning durations (tc) between 0.25 and 4 seconds (s) as to whether recovery was slowed by an effect on the fast (tau f) and slow (tau s) time constants of recovery, the relative amplitude of the slow component (As), or both. We studied Na channel recovery at -150 mV from inactivation using whole cell voltage clamp of rat ventricular cells at 23 degrees C using a two-pulse recovery protocol. Longer conditioning durations dramatically increased A2 (from 12% for tc = 500 msec to 37% for tc = 4000 msec, P < 0.01). Neither tau f (6 vs 5 msec) nor tau s (115 vs 140 msec) were significantly affected. In a second set of experiments, the recovery potential was depolarized to a potential at which the sodium current was 70% available (approximately equal to - 105 mV). This recovery potential had no significant effect on A2, but both tau f and tau s were significantly slower (e.g., at tc = 2 s, tau s = 147 msec and As = 28% at Vr = - 150 mV, and tau s = 456 msec and As = 29% at Vr approximately equal to - 105 mV). In addition, a 1- to 2-msec lag in the onset of recovery was prominent at the depolarized recovery potentials. CONCLUSIONS: Our results support a model for slow recovery where conditioning duration determines entry into an inactivated state from which Na channels recover slowly, and recovery potential determines the rate of recovery from this state. A kinetic scheme with at least three inactivated states is proposed. These results also have implications for cardiac excitability under conditions, such as ischemia, where membranes are depolarized.