Isolated infant human atrial cells have a slower early repolarization than adult human atrial cells. In addition, from room temperature voltage-clamp studies, infant cells have lower basal L-type calcium currents than adult cells. We hypothesized that the slower repolarization increases the calcium transient of infant human atrial cells. Atrial myocytes were enzymatically dissociated from biopsies of human right atrial appendages of infant (3–8 mo) patients who were undergoing open-heart surgery. Intracellular calcium transients were measured with fluorescence microscopy with application of either square waves or action potential waveforms at physiologic temperature. After repetitive application (1 Hz) of 100-ms duration conditioning depolarizations to 10 mV (from −80 mV), a test pulse of varying duration (ΔT; 2–100 ms) produced smaller transients (expressed as percentage of the last conditioning pulse) at shorter durations (33 ± 7% for ΔT = 2 ms, 80 ± 4% for ΔT = 25 ms). With repetitive application of either adult or infant prerecorded action potentials to infant cells, the cells had a decreased calcium transient with the adult action potential (F/F 0 2.2 ± 0.4 for infant action potential versus 1.6 ± 0.2 for adult action potential; n = 7; p < 0.05). The delayed early repolarization of infant cells alters the Ca 2+ transient, which may compensate for the lower availability of basal calcium current in infant cells. The steep relationship that we have demonstrated between test-pulse duration and the calcium transient suggests that modulation of the early repolarization phase of the action potential may be of great significance in modulating excitation-contraction coupling.

The activity of the voltage-sensitive K+ (Kv) channels varies as a function of the intracellular redox state and metabolism, and several Kv channels act as oxygen sensors. However, the mechanisms underlying the metabolic and redox regulation of these channels remain unclear. In this study we investigated the regulation of Kv channels by pyridine nucleotides. Heterologous expression of Kvalpha1.5 in COS-7 cells led to the appearance of noninactivating currents. Inclusion of 0.1-1 mM NAD+ or 0.03-0.5 mM NADP+ in the internal solution of the patch pipette did not affect Kv currents. However, 0.5 and 1 mM NAD+ and 0.1 and 0.5 mM NADP+ prevented inactivation of Kv currents in cells transfected with Kvalpha1.5 and Kvbeta1.3 and shifted the voltage dependence of activation to depolarized potentials. The Kvbeta-dependent inactivation of Kvalpha currents was also decreased by internal pipette perfusion of the cell with 1 mM NAD+. The Kvalpha1.5-Kvbeta1.3 currents were unaffected by the internal application of 0.1 mM NADPH or 0.1 or 1 mM NADH. Excised inside-out patches from cells expressing Kvalpha1.5-Kvbeta1.3 showed transient single-channel activity. The mean open time and the open probability of these currents were increased by the inclusion of 1 mM NAD+ in the perfusate. These results suggest that NAD(P)+ prevents Kvbeta-mediated inactivation of Kv currents and provide a novel mechanism by which pyridine nucleotides could regulate specific K+ currents as a function of the cellular redox state [NAD(P)H-to-NAD(P)+ ratio].

Objective: To investigate the hypothesis that acute oxidative stress and abnormal stretch of the myocardium may contribute to the production of early-afterdepolarizations (EADs).

Stretch of the atrium and sympathetic activity have been implicated as substrates for atrial fibrillation. We investigate how a model of stretch in combination with sympathetic stimulation can induce automaticity in atrial cells. We adapted our coupling clamp circuit so that a model ionic current that represents stretch-activated channels (SACs) was injected into an isolated rat atrial cell in real time. This current was calculated as I-SAC=G(SAC) (V-m-E-SAC), where G(SAC) and E-SAC are the conductance and reversal potential of SACs and V-m is the cell's membrane potential. Repetitive automaticity was induced by a sufficiently large G(SAC) and this critical value of G(SAC) was decreased by exposure to isoproterenol. The critical value of G(SAC) decreased from 0.63+/-0.05 nS (mean+/-SE) in control to 0.40+/-0.07 nS in isoproterenol (P<0.05). Additionally, after exposure to isoproterenol, automaticity continued after G(SAC) was no longer applied and was accompanied by delayed after-depolarizations. In three cells, repetitive automaticity could not be induced at any value of G(SAC). Exposure to 10 nM isoproterenol converted these cells to cells with repetitive automaticity in response to G(SAC). We conclude that automaticity can be induced in isolated rat atrial cells by application of a model of SACs. Exposure to isoproterenol enhances this effect.

In the early nineties, Joyner and coworkers introduced the “coupling clamp” technique in which an isolated cardiac cell can be electrically coupled to either another isolated cardiac cell or to an analog model cell (RC circuit). In brief, an amplifier system does a continuous analog computation of the current that would be flowing between the two cells if there had been an intercellular coupling conductance C,, and then provides current inputs to the cells accordingly. Building on this concept, we developed the computer-controlled “model clamp” technique, in which an isolated cardiac cell is dynamically coupled in real time to a comprehensive mathematical cell model (e.g., the phase-2 Luo-Rudy model). With this system we have the ability to vary coupling conductance, effective size of both model cell and real cell, and intrinsic cellular properties of the model cell. In courses on cardiac electrophysiology, the model clamp system provides a useful computer tool to probe action potential transfer between cardiac cells. It can be used to assess alterations in the critical value of coupling conductance required for action potential transfer from a real ventricular cell to the Luo-Rudy model ventricular cell upon exposure of the real cell to, e.g., noradrenaline.

Protein phosphatases play a major role in the regulation of L-type calcium current (ICa) in heart cells. We previously showed developmental differences in the effects of inhibitors of protein phosphatases (PP's) on the modulation of ICa, with greater stimulatory effects on ICa observed in newborn than in adult ventricular cells. We hypothesized that this developmental difference might be due to greater expression and levels of PP 1 and PP 2A in newborn than in adult ventricular cells. We thus determined the mRNA expression of α and β subunits of PP 1 and the α subunit of PP 2A in adult and newborn rabbit ventricles and levels of PP 1 and PP 2A in total homogenates, particulate membranes, and in soluble fraction prepared from isolated ventricular myocytes from adult and newborn rabbits. RT-PCR analysis demonstrated the presence of mRNA of these subunits of PP's in both newborn and adult ventricles. Northern blot analysis using 32P labeled cDNA probes specific for PP 1α, PP 1β and PP 2Aα showed that the expression of steady state mRNA levels for PP 1α, PP 1β and PP 2Aα were much higher in newborn compared to adult rabbit ventricles. mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for sarcoplasmic reticulum Ca2+-ATPase (SERCA) in rabbit ventricles were measured as controls. GAPDH did not show significant developmental changes while mRNA for SERCA was higher in adult compared to newborns. Western blot analysis showed that PP 1 and PP 2A protein levels were also much higher in newborn compared to adult rabbit ventricular cells. Immunoblot analysis in particulate membranes and soluble fraction showed that PP1 was mainly membrane bound while PP 2 was present only in soluble fraction. These findings suggest that the two major protein phosphatases (PP 1 and PP 2A) in heart are expressed at much higher levels in newborn and decline to lower levels in adult ventricular myocytes. The presence of high levels of PP's and particularly PP 1 in newborn cells may be responsible for the greater dependence of newborn cells on the inhibition of PP as a mechanism of action of β-agonist isoproterenol on ICa.

The present study investigated the effects of protein tyrosine kinase inhibitors on the fast sodium current (INa) in rabbit ventricular myocytes. Single rabbit ventricular myocytes were isolated enzymatically using Langendorff perfusion. INa was recorded using the whole-cell patch-clamp technique at room temperature. The protein tyrosine kinase inhibitors genistein, AG957, ST638, and PP2 reversibly inhibited INa in a concentration-dependent manner. At a test pulse potential of −30 mV, genistein (n=7) inhibited INa by 37.7±3.2%, 53.4±2.5%, and 71.8±2.7% at concentrations of 15, 50, and 100 µM, respectively, without changing the voltage dependence of activation, while 100 µM AG957, 100 µM ST638, and 30 µM PP2 inhibited INa by 38.7±2.4, 35.8±3.4, and 21.1±3.9%, respectively. Genistein (100 µM) and AG957 (100 µM) shifted the voltage for half-maximal inactivation of INa from −76.7±2.0 mV (n=10) in control to −88.37±2.6 mV (n=6, P<0.05), and −82.9±1.7 (n=4, P<0.05), respectively, without changing the slope factor. Genistein and AG957 also significantly prolonged the time course of INa recovery from inactivation. Daidzein and PP3, inactive analogs of genistein and PP2, respectively, did not inhibit INa significantly. We conclude that protein tyrosine kinase signaling pathways may play an important role in regulation of INa in cardiac myocytes.

The pore-forming subunits of the voltage-sensitive K+ channel (Kv) associate with ancillary β-subunits that regulate inactivation and voltage-dependence of the channel. The β-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant Kvβ2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine nucleotides regulate the Kvβ-mediated inactivation of K+ channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvα1.5 cDNA led to the appearance of the Kv1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with Kvβ1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, Kvβ1.3 co-transfected with Kvα1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both Kvα1.5 and Kvβ1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD+ in the internal solution of the patch pipette abolished Kvβ-induced inactivation of Kv1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with Kv1.5 alone, indicating that in the absence of Kvβ, NAD+ does not affect the activity of Kvα. The inactivating currents recorded from cells expressing both Kvα1.5 and Kvβ1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD+ impart different conformational states to the Kvβ protein and that only the NADPH bound Kvβ imparts inactivation to non-inactivating K+ currents. Thus, differential binding of pyridine nucleotide coenzymes to Kvβ could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD+/NADPH ratio is sensitive to oxygen concentration, the differential changes in Kvβ-mediated inactivation of K currents by NAD+ and NADPH could represent an oxygen-sensing mechanism.

In the early nineties, Joyner and coworkers introduced the "coupling clamp" technique in which an isolated cardiac cell can be electrically coupled to either another isolated cardiac cell or to an analog model cell (RC circuit). In brief, an amplifier system does a continuous analog computation of the current that would be flowing between the two cells if there had been an intercellular coupling conductance G,, and then provides current inputs to the cells accordingly. Building on this concept, we developed the computer-controlled "model clamp" technique, in which an isolated cardiac cell is dynamically coupled in real time to a comprehensive mathematical cell model (e.g., the phase-2 Luo-Rudy model). With this system we have the ability to vary coupling conductance, effective size of both model cell and real cell, and intrinsic cellular properties of the model cell. In courses on cardiac electrophysiology, the model clamp system provides a useful computer tool to probe action potential transfer between cardiac cells. It can be used to assess alterations in the critical value of coupling conductance required for action potential transfer from a real ventricular cell to the Luo-Rudy model ventricular cell upon exposure of the real cell to, e.g., noradrenaline.

The anisotropy that normally exists in the myocardium may be either enhanced in peri-infarction zones by loss of lateral cell connections or reduced by redistribution of gap junctions. To test how the degree of anisotropy affects the development of ectopic focal activity, we carried out computer simulations in which a model of an ectopic focus is incorporated as the central element of a two-dimensional sheet of ventricular cells. At low values of intercellular coupling conductance (G c), the focus region is spontaneously active, but the limited intercellular current flow inhibits propagation. At high G c, automaticity is suppressed by the loading effects of the surrounding cells. At intermediate G c, the ectopic activity may propagate into the sheet. In the case of isotropic coupling, the minimum size of the focus region for propagation to occur (in terms of number of collaborating cells within the focus) is as small as approximately ten cells, and this number decreases with increasing anisotropy. Thus, the presence of anisotropy facilitates the development of ectopic focal activity. We conclude that the remodeling that occurs in peri-infarction zones may create a substrate that either facilitates (enhanced anisotropy) or inhibits (reduced anisotropy) the development of cardiac arrhythmias associated with ectopic focal activity.

Atrial activation involves interactions between cells with automaticity and slow-response action potentials with cells that are intrinsically quiescent with fast-response action potentials. Understanding normal and abnormal atrial activity requires an understanding of this process. We studied interactions of a cell with spontaneous activity, represented by a "real-time" simulation of a model of the rabbit sinoatrial (SA) node cell, simultaneously being electrically coupled via our "coupling clamp" circuit to a real, isolated atrial myocyte with variations in coupling conductance (G(c)) or stimulus frequency. The atrial cells were able to be driven at a regular rate by a single SA node model (SAN model) cell. Critical G(c) for entrainment of the SAN model cell to a nonstimulated atrial cell was 0.55 +/- 0.05 nS (n = 7), and the critical G(c) that allowed entrainment when the atrial cell was directly paced at a basic cycle length of 300 ms was 0.32 +/- 0.01 nS (n = 7). For each atrial cell we found periodic phenomena of synchronization other than 1:1 entrainment when G(c) was between 0.1 and 0.3 nS, below the value required for frequency entrainment, when the atrial cell was directly driven at a basic cycle length of either 300 or 600 ms. In conclusion, the high input resistance of the atrial cells allows successful entrainment of nodal and atrial cells at low values of G(c), but further uncoupling produces arrhythmic interactions.

Previous work with model systems for action potential conduction have been restricted to conduction between two real cells or conduction between a model cell and a real cell. The inclusion of additional elements to make a linear strand has allowed us to investigate the interactions between cells at a higher level of complexity. When, in the simplest case of a linear strand of three elements, the conductance between elements 2 and 3 (GC2) is varied, this affects the success or failure of propagation between elements 1 and 2 (coupled by GC1) as well as the success or failure of propagation between elements 2 and 3. Several major features were illustrated. 1) When GC1 was only slightly greater than the coupling conductance required for successful propagation between a model cell and a real cell, addition of a third element of the strand either prevented conduction from element 1 to element 2 (when GC2 was high) or allowed conduction from element 1 to element 2 but not conduction from element 2 to element 3 (when GC2 was low). 2) For higher levels of GC1, there was an allowable "window" of values of GC2 for successful conduction from element 1 through to element 3. The size of this allowable window of GC2 values increased with increasing values of GC1, and this increase was produced by increases in the upper bound of GC2 values. 3) When the size of the central element of the strand was reduced, this facilitated conduction through the strand, increasing the range of the allowable window of GC2 values. The overall success or failure of conduction through a structure of cells that has a spatially inhomogeneous distribution of coupling conductances cannot be predicted simply by the average or the minimum value of coupling conductance but may depend on the actual spatial distribution of these conductances.

Adenosine is one of the most important inhibitory modulators of heart function, producing negative inotropic, chronotropic and dromotropic effects and is also a major regulator of coronary circulation. The decrease in contractility by adenosine is mediated through inhibition of adenylyl cyclase by Gi-proteins coupled to adenosine receptors. However, little is known about the developmental differences in the effect of adenosine on cardiac cells. We have now shown that there is a striking developmental difference in the inhibitory effect of adenosine on isoproterenol-stimulated ICa between adult and newborn rabbit ventricular cells. Adenosine had no significant inhibitory effect on 0.1 muM isoproterenol-stimulated ICa in adult cells, while it completely blocked the 10 muM isoproterenol-stimulated ICa in newborn cells with an inhibitory potency similar to carbachol in newborn cells. Similarly, adenosine did not decrease the isoproterenol-stimulated cAMP levels in adult cells while it inhibited isoproterenol-stimulated cAMP levels significantly and equipotently to carbachol in newborn. However, for forskolin-stimulated ICa and cAMP levels in newborn cells, adenosine had a much lower inhibitory potency than carbachol. In adult cells, forskolin-stimulated ICa and cAMP levels were not affected by adenosine. We showed previously that the Gia3 isoform of inhibitory G protein was present in newborn cell membranes, but not detectable in adult cell membranes. We have now used a synthetic decapeptide corresponding to the C-terminal sequence of Gia3 in the patch pipette and have shown a selective partial block of the inhibitory action of adenosine for isoproterenol-stimulated ICa, suggesting that the inhibitory action of adenosine on ICa is mediated primarily through the Gia3 pathway.

An ischemic myocardial region contains cells with a depolarized resting membrane potential. This depolarization leads to an intercellular current flow between the ischemic region and the surrounding normal myocardial cells which has been termed an “injury current”. We have devised an experimental model system in which an isolated guinea pig ventricular cell is electrically coupled to a model depolarized cell in order to evaluate the effects of this injury current on the electrical properties of a normal ventricular cell exposed to drugs which increase calcium current or decrease potassium current. Using low doses of isoproterenol, forskolin, or Bay K 8644 (or 8-bromo-cyclic adenosine monophosphate in the pipette) we found that the action potential duration of the isolated cell was lengthened, but that early after depolarizations (EADs) were not produced unless the cell was also coupled to a depolarized cell model representing an adjacent ischemic region. A similar prolongation of the action potential was produced by low doses of quinidine, but EADs were not produced unless coupling to a depolarized cell model was added. EADs could not be produced in any cells in the absence of the drugs even though the coupling to the depolarized cell model was increased up to the level at which the action potential was indefinitely prolonged. At higher isoproterenol concentrations, EADs or spontaneous activity were produced without coupling to the depolarized cell model. Under these conditions, coupling of the cell to a cell model with normal resting membrane potential stopped the spontaneous activity and prevented the occurrence of EADs even with high levels of resistive coupling. These findings suggest that the electrotonic influences of a localized depolarized region can produce EADs if the calcium current magnitude is increased, which would be the case for sympathetic innervation.

We used whole-cell voltage clamp to compare the modulation of calcium current density (ICa, picoampere per picofarad) of freshly isolated, adult and newborn rabbit heart in response to intracellular application of microcystin and okadaic acid, both of which block phosphatase activity of phosphatase types 1 and 2A. Newborn cells showed a much larger response to the intracellular application of either microcystin or okadaic acid than did adult cells. In newborn cells, the application of microcystin produced an increase in ICa which appeared to maximize ICa, as shown by the rise in ICa to levels which could be reached by application of 10 μM forskolin or by the intracellular application of 200 μM 3′,5′-cyclic adenosine monophosphate (cAMP). In adult cells, the maximal response to microcystin was considerably less than that obtainable with forskolin or cAMP. After achieving a maximal response with microcystin, the addition of forskolin increased ICa further in adult cells but elicited no additional response in newborn cells. The treatment of cells with 0.1 μM isoproterenol, a concentration approximately equal to that required for a half-maximal response, strongly potentiated the effect of microcystin in newborn cells, but not in adult cells. We propose that newborn rabbit heart cells compared with adult rabbit heart cells have a greater level of protein phosphatase activity (perhaps combined with a somewhat greater kinase activity), a greater proportion of the protein phosphatase activity in the form of protein phosphatase type 1 (which is inhibited by isoproterenol) and a greater dependence on the inhibition of protein phosphatase as a mechanism of action of isoproterenol, compared with the increase in kinase activity on calcium channels.