Feline Ventricular Myocytes Research Articles

The effects of ZD6169 on the ATP-dependent K + current ( IK ATP) in isolated cat ventricular myocytes

The effect of the K ATP channel opener ZD6169 [( S)- N-(4-benzoyl-phenyl)-3,3,3-trifluoro-2-hydroxy-2-methyl-propionamide] currently under development for the treatment of urinary incontinence was explored in acutely isolated adult feline ventricular myocytes. ZD6169 activated a current over a wide range of concentrations (0.1–100 μM) that is completely blocked by 10 μM glyburide thereby identifyinga it as I K ATP . The maximum activation of K ATP current was observed at 10 μM; higher concentrations decreased current activation. In contrast, the standard K ATP channel opener cromakalim showed a more usual concentration–response relationship, with increasing current for increased concentrations and no signs of saturation or reversal. The bell-shaped dose–response relationship for ZD6169 activation of I K ATP has also been seen in bladder myocytes, albeit at a lower concentration, and it has been proposed to contribute to the reported lack of in vivo cardiovascular side effects. We compared the effects of ZD6169 to cromakalim and showed that both compounds dramatically shorten cardiac myocyte action potential duration and that ZD6169 does so in spite of the bell-shaped concentration–response relationship for activation of K ATP current.

Slowly inactivating sodium currents are reduced by exposure to oxidative stress.

Exposure of cardiac myocytes to oxidant stress has been implicated in the development of reperfusion arrhythmias. Studies on the effects of free radical generating systems on the fast sodium current have suggested an increase in a "window" current. The resulting increase in sodium influx has been hypothesized to cause an intracellular sodium load that stimulates Na+, Ca2+ exchange and promotes a Ca2+ overload. To test this proposal, the time course for effects of oxidative stress on a sodium current elicited with voltage ramps was investigated in feline ventricular myocytes. No window current was observed; instead, a slowly inactivating sodium current was generated at negative voltages near the sodium threshold potential. At room temperature there were no effects of a 30-min exposure to 1 mm H2O2 on this slowly inactivating sodium current. Likewise, there were no effects of either 1 mm H2O2 or 1.5 mm t-butyl hydroperoxide on fast sodium currents recorded at cool temperatures (12-15 degrees C). Experiments were repeated with t-butyl hydroperoxide at warm temperatures (30-33 degrees C), and the fast sodium current was reduced in magnitude and the reversal potential shifted to more negative voltages. These results demonstrate a temperature dependence for the loss of the fast sodium current during exposure to t-butyl hydroperoxide. Two exponentials were fit to the decaying phase of the fast sodium current and the slow time constant of inactivation was prolonged, suggesting delayed inactivation of the sodium current. Currents elicited with a steady-state inactivation protocol suggested development of a non-inactivating component during exposure to t-butyl hydroperoxide at warm temperatures. Direct evaluation of the slowly inactivating sodium current elicited by voltage ramps at warm temperatures (33-35 degrees C), and analysed as subtraction currents to remove background leak currents, showed a gradual reduction. It is concluded that the non-inactivating component identified during analysis of the fast sodium current was not the result of enhancement of either a slowly inactivating sodium current or a window current. Thus, an increase in sodium influx through voltage-dependent sodium channels does not occur during exposure to oxidative stress, and therefore, cannot induce an intracellular sodium load.

Cellular basis of contractile derangements of hypertrophied feline ventricular myocytes.

The objective of this study was to further explore the cellular basis of the reduced rate and magnitude of contraction of feline left ventricular myocytes with severe hypertrophy induced by slow progressive pressure overload. A 3.0 mm internal diameter band was placed around the ascending aorta of 12 young (8-10 weeks old) cats, and sham operations were performed in 13 others. This caused no major pressure overload initially, but 15 weeks later there was a significant pressure gradient across the band (56+/-14 mmHg) and the heart weight to body weight ratio had increased from 4.2-7 gm/kg. Contraction rates and magnitudes of myocytes isolated from the hearts with hypertrophy (LVH) were significantly slower and smaller, respectively, than those from control (C) animals. Indo-1 fluorescence transients in LVH myocytes were significantly smaller in magnitude and longer in duration than in C, suggesting that contractile defects result from Ca2+ derangements. Elevation of bath Ca2+ increased the peak Indo-1 fluorescence and the rate and magnitude of contraction in all myocytes. At the bath Ca2+ which had maximal inotropic effects there were no differences in the peak Indo-1 fluorescence in LVH and C myocytes, but contraction magnitude remained significantly smaller in LVH. This suggests that there are Ca2+-independent contractile derangements in LVH. In support of this hypothesis, the relationship between contraction magnitude and the peak Indo-1 fluorescence (index of myofibrillar Ca2+ sensitivity) was significantly shifted in LVH myocytes, suggesting that myofibrillar Ca2+ sensitivity was reduced. There was also a significant shift of the terminal portions of hysteresis loops of cell length v indo-1 fluorescence ratio, providing additional support for this idea. Experiments with isoproterenol suggest that it can reduce myofibrillar Ca2+ sensitivity in C, but not LVH myocytes. The idea that increased internal resistance to shortening (internal load) is responsible for the contractile defects of LVH myocytes was examined by defining the relationship between the rate of relengthening and the magnitude of shortening. There was no significant difference in this relation between C and LVH myocytes. In addition, colchicine (which depolymerizes microtubular tubulin) had no significant effect on contraction magnitude in either C or LVH myocytes. These results suggest that the contractile properties of feline LVH myocytes result from changes in cellular Ca2+ regulation and myofibrillar Ca2+ sensitivity, but not from changes in the internal loading.

DNA Synthesis in Adult Feline Ventricular Myocytes

Adult mammalian ventricular myocytes are terminally differentiated cells, and the prevailing perception has been that DNA synthesis and repair are not active. We tested the hypothesis that there is potential for DNA synthesis and repair by studying the ability of whole-cell extracts from adult myocytes to incorporate [alpha-32P]dCTP into damaged plasmids. Left ventricular myocytes were isolated from adult cat hearts by collagenase dissociation. Cells were maintained in room air (control extract, CE) or made ischemic (IE) with N2 displacement of O2 and extracted for total protein. The nicked form of the plasmid was produced by exposure to an Fe3+/ascorbic acid free radical generating system. Both IE and CE degraded the supercoiled form of the plasmid and incorporated [alpha-32P]dCTP into the nicked (32P/DNA mass; CE = 2.2, IE = 3.0) and linear forms (32P/DNA mass; CE = 28.7, IE = 25.2). Exposure of plasmids to UV light did not inhibit incorporation of label. Inhibition studies with the cell extracts suggested a participation of polymerase delta in myocyte DNA repair/synthesis. Myocyte extract was as active as extract from rapidly growing COS cells at incorporating labeled nucleotides into plasmid DNA. The ability of intact myocytes to incorporate [alpha-32P]dCTP into endogenous DNA was measured in isolated cells made permeable with saponin. Studies were done in room air or N2. Permeable cells incorporated [alpha-32P]dCTP into nuclear DNA, but maximal specific activity of DNA was observed at 15 minutes with ischemia and at 60 minutes with room air control cells (ischemia, 1.34 +/- 0.5, 0.86 +/- 0.33, 0.60 +/- 0.04; air, 1.0, 1.28 +/- 0.20, 1.87 +/- 0.38, at 15, 30, and 60 minutes, respectively). These data indicate that mammalian adult ventricular myocytes can actively repair and/or synthesize both exogenous and endogenous DNA. A DNA synthetic response to cellular damage may have important pathological and clinical implications.

Characterization of the acetylcholine-sensitive muscarinic K+ channel in isolated feline atrial and ventricular myocytes

M2-cholinergic receptor activation by acetylcholine (ACh) is known to cause a negative inotropic and chronotropic action in atrial tissues. This effect is still controversial in ventricular tissues. The ACh-sensitive muscarinic K+ channel (IK(ACh)) activity was characterized in isolated feline atrial and ventricular myocytes using the patch-clamp technique. Bath application of ACh (1 microM) caused shortening of action potential duration without prior stimulation with catecholamines in atrial and ventricular myocytes. Resting membrane potential was slightly hyperpolarized in both tissues. These effects of ACh were greater in atrium than in ventricle. ACh increased whole-cell membrane current in atrial and ventricular myocytes. The current-voltage (I-V) 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 induced in the pipette solution in both tissues. The channel exhibited a slope conductance of 47 +/- 1 pS (mean +/- SD, n = 14) in atrium and 47 +/- 2 pS (n = 10) in ventricle (not different statistically; NS). The open times were distributed according to a single exponential function with mean open lifetime of 2.0 +/- 0.3 msec (n = 14) in atrium and 1.9 +/- 0.3 msec (n = 10) in ventricle (NS); these conductance and kinetic properties were similar between the two tissues. However, the relationship between the concentration of ACh and single channel activity showed a higher sensitivity to ACh in atrium (IC50 = 0.03 microM) than in ventricle (IC50 = 0.15 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Block of the delayed rectifier current (IK) by the 5‐HT3 antagonists ondansetron and granisetron in feline ventricular myocytes

1. We investigated the effects of two 5-HT3 antagonists, ondansetron and granisetron, on the action potential duration (APD) and the delayed rectifier current (IK) of feline isolated ventricular myocytes. Whole-cell current and action potential recordings were performed at 37 degrees C with the patch clamp technique. 2. Ondansetron and granisetron blocked IK with a KD of 1.7 +/- 1.0 and 4.3 +/- 1.7 microM, respectively. At a higher concentration (30 microM), both drugs blocked the inward rectifier (IKl). 3. The block of IK was dependent on channel activation. Both drugs slowed the decay of IK tail currents and produced a crossover with the pre-drug current trace. These results are consistent with block and unblock from the open state of the channel. 4. Granisetron showed an intrinsic voltage-dependence as the block increased with depolarization. The equivalent voltage-dependency of block (delta) was 0.10 +/- 0.04, suggesting that granisetron blocks from the intracellular side at a binding site located 10% across the transmembrane electrical field. 5. Ondansetron (1 microM) and granisetron (3 microM) prolonged APD by about 30% at 0.5 Hz. The prolongation of APD by ondansetron was abolished at faster frequencies (3 Hz) showing reverse rate dependence. 6. In conclusion, the 5-HT3 antagonists, ondansetron and granisetron, are open state blockers of the ventricular delayed rectifier and show a clear class III action.

Effects of membrane lipid peroxidation by tert butyl hydroperoxide on the sodium current in isolated feline ventricular myocytes.

Membrane lipid peroxidation is known to play a pivotal role in the genesis of coronary reperfusion arrhythmias in both experimental and clinical settings. To elucidate the electrophysiological mechanisms underlying these arrhythmias, the effects of tert butyl hydroperoxide (TBH) on the Na+ current (INa) in isolated feline ventricular myocytes were studied using whole-cell patch clamp techniques under 100% O2 bubbling. This agent at 20 mM inhibited INa from 2.2 +/- 1.3 to 1.7 +/- 1.0 nA (P < 0.01, n = 7) without changing time courses of INa inactivation. Twenty millimoles TBH shifted the steady-state inactivation curve for INa from -77.4 +/- 1.7 to -81.3 +/- 1.8 mV when measured at INa half inhibition voltage (P < 0.01, n = 7), but did not affect the slope factor. The kinetics of INa recovery from inactivation remained unchanged. These findings suggest that lipid peroxidation in the membrane by TBH reduces INa conductance and voltage-dependent INa availability, most likely as a result of structural damage to the Na+ channels.

Block of Na + channel by moricizine hydrochloride in isolated feline ventricular myocytes

The effects of moricizine hydrochloride, a potent class I antiarrhythmic agent, on Na + current (I Na) of single feline ventricular myocytes were studied using whole cell patch clamp techniques. Moricizine inhibited I Na in a concentration-dependent manner without altering the current-voltage relationship for I Na. I Na inhibition was expressed by the Hill equation with a Hill coefficient of 1.3 and dissociation constant of 105 μM in the resting state (holding potential = −140 mV). Moricizine 30 μM shifted the steady state inactivation curve for I Na toward more negative potentials by 7.3 ± 2.4 mV without causing significant changes in the slope factor. Recovery of I Na from inactivation was retarded (time constant = 8 s) at a holding potential of −140 mV in the presence of 30 μM moricizine. When the start of I Na block was studied in experiments using a double pulse protocol, moricizine reduced I Na by only 4% after a 4-ms prepulse, but strongly inhibited it after prepulses longer than 200 ms. Intracellular application of 100 μM moricizine did not produce significant resting or use-dependent I Na block. These results suggest that (1) moricizine blocks I Na by binding to the Na + channel with a 1:1 stoichiometry, (2) the drug has a higher affinity to the inactivated state than to the activated and resting states of the Na + channel, (3) recovery kinetics of moricizine from Na + channel inactivation, or drug dissociation observed during the transition from inactivated to resting state was relatively slow, (4) the drug binding site appeared to be located on the external side of the membrane.

Protein kinase-dependent Cl- currents in feline ventricular myocytes.

A Cl- current (ICl) induced by isoproterenol (ISO) has been identified in isolated guinea pig ventricular myocytes. This ISO-induced ICl can be inhibited by propranolol and mimicked by forskolin (FSK), suggesting that beta-receptors, cAMP, and protein kinase A (PKA) are involved in regulating the involved Cl- channel. Because activation of protein kinase C (PKC) mediated via alpha-adrenergic receptor stimulation is also known to regulate several ion channels, the idea that activation of PKC also can induce ICl was investigated by using isolated feline ventricular myocytes and the whole-cell patch-clamp technique. We found that extracellularly applied phorbol 12-myristate 13-acetate (PMA) could activate ICl in feline ventricular cells. Control experiments indicated that in the absence of PMA or other interventions, the steady-state current-voltage relation of patches maintained for more than 40 minutes was unchanged over a voltage range from -100 to +80 mV. This suggests that the present findings are not complicated by the development over time after patching of a steady-state ICl, similar to the findings reported for canine atrial myocytes. When induced by PMA, ICl was noninactivating and outwardly rectifying; it reversed polarity at approximately the equilibrium potential for Cl- and was sensitive to the Cl- channel blocker 9-anthracene carboxylic acid. In contrast, PMA failed to induce ICl when either staurosporine or calphostin C was added to the patch pipette solution used to internally dialyze the myocytes. The kinetic properties of PMA- and FSK-induced ICl were similar. When supramaximal concentrations of both ISO (1 mumol/L) and PMA (6 mumol/L) were applied simultaneously, the size of the induced ICl was the same as that induced by the same concentrations of either agonist applied alone. In addition, maximal induction of ICl with PMA (6 mumol/L) prevented the effects of FSK (1 mumol/L, the concentration causing approximately 40% of the maximal response [approximately EC40]), yet the effects of simultaneously applied submaximal concentrations (eg, approximately EC25 to approximately EC40) of both 0.5 mumol/L PMA and 1 mumol/L FSK were roughly additive. The results suggest that (1) both PMA and ISO or FSK can induce ICl with approximately equal efficacy, (2) the PMA- and ISO- or FSK-induced ICls are similar, and (3) they all flow through the same set of Cl- channels, implying that channel phosphorylation via either PKA or PKC can activate this feline cardiac ICl.

Interactions of H 2O 2, EGTA and Patch Pipette Recording Methods in Feline Ventricular Myocytes

Free radical generating systems produce a sequence of changes in the action potentials of isolated cardiac myocytes. Efforts to understand the nature of the alterations in ionic currents have yielded mixed results. The use of patch-pipettes for voltage-clamp experiments and manipulation of pipette solution composition may contribute to the confusion. In this study, action potentials were recorded from feline ventricular myocytes during exposure to 100 μM H 2O 2. Action potential changes recorded with conventional, high resistance microelectrodes occurred in three stages as previously reported. The action potential changes recorded with patch-pipettes showed increases in action potential duration, notch and plateau potentials over time. These cells did not display after depolarizations or loss of excitability as seen with conventional microelectrodes. With the patch-pipette method, untreated control cells had time-dependent changes in the action potentials. When the pipette filling solution was modified by the addition of 5 mM EGTA, the H 2O 2-induced changes appeared reduced but not eliminated. The initial control values of action potential parameters before exposure to H 2O 2 varied with the presence or absence of EGTA. These data show both direct effects of EGTA and of time on action potential parameters independent of H 2O 2-exposure. These artifacts of the patch-pipette method and the composition of the pipette solutions affect the sequence of changes in the action potential during exposure to H 2O 2.

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes.

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non-beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.

T-type Ca2+ current is expressed in hypertrophied adult feline left ventricular myocytes.

Macroscopic T-type Ca2+ currents, which are often observed in fetal and neonatal cardiac muscle cells, were not found in normal (0 of 17) adult feline ventricular myocytes. However, they were present in most (15 of 21) myocytes isolated from adult feline left ventricles with long-standing pressure-overload-induced hypertrophy. This is the first study to provide evidence in a large mammal, such as the cat, that T-type Ca2+ channels may be reexpressed in adults in association with hypertrophy resulting from slow progressive pressure overload. Importantly, this expression was stable for the duration of the hypertrophy process and was not associated with abrupt pressure overload. T-type Ca2+ currents were separated from L-type Ca2+ currents by exploiting the differences in their voltage dependence of steady-state inactivation. Depolarizations from -80 mV revealed a rapidly activating inward current that peaked in magnitude at -30 mV (-1.8 +/- 0.9 [mean +/- SD] pA/pF) and fully inactivated within 100 milliseconds in 15 of 21 hypertrophied myocytes studied. Further depolarizations activated progressively less T-type Ca2+ current, so that at +10 mV the L-type Ca2+ current predominated. In the hypertrophied myocytes that demonstrated both T-type and L-type Ca2+ currents, two distinct peaks occurred in their current-voltage relations. T-type Ca2+ currents were not evident in any of the 17 normal adult feline left ventricular myocytes studied. The purpose of T-type Ca2+ currents in hypertrophy is unclear. However, their presence may make hypertrophied myocardium more prone to spontaneous action potentials and increase the likelihood for arrhythmias in partially depolarized hypertrophied myocardium.

Sodium-calcium exchange-mediated contractions in feline ventricular myocytes.

The hypothesis that Ca entry by the sarcolemmal Na-Ca exchange mechanism induces sarcoplasmic reticulum (SR) Ca release, loads the SR with Ca, and/or directly induces contractions by elevating cytosolic free Ca was tested in voltage-clamped feline ventricular myocytes. Intracellular Na concentration was increased by cellular dialysis to enhance Ca influx via "reverse-mode" Na-Ca exchange at positive membrane potentials, at which the "L-type" Ca current (ICa) should be small. Contractions were induced in the presence of Ca channel antagonists by depolarization to these potentials, suggesting that Ca influx via reverse-mode Na-Ca exchange was involved. These contractions had both phasic (SR related) and tonic components of shortening. They were smaller and began with more delay after depolarization than contractions which involved ICa. The magnitude of shortening was graded by the amount and duration of depolarization, suggesting that Ca influx via reverse-mode Na-Ca exchange has the capacity to induce and grade SR Ca release. Small slow contractions could be evoked in the presence of ryanodine (to impair SR function) and verapamil (to block ICa), supporting the idea that Ca influx via Na-Ca exchange is sufficient to directly activate the contractile proteins. Contractions induced by voltage steps to +10 mV, which were usually small when ICa was blocked, were potentiated if preceded by a voltage step to strongly positive potentials. This potentiation was inhibited by ryanodine, suggesting that Ca entry that occurs by Na-Ca exchange may be important for normal SR Ca loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Verapamil diminishes action potential changes during metabolic inhibition by blocking ATP-regulated potassium currents.

Verapamil has beneficial effects on ischemic myocardium, including reduction in electrophysiological derangements, prevention of intracellular K+ loss, and preservation of high-energy phosphates, but the mechanisms underlying these actions are not clear. Recent studies have demonstrated a role of ATP-regulated K+ (KATP) current in action potential shortening and K+ loss during ischemia and metabolic inhibition. Therefore, we studied the effects of verapamil on KATP current in feline ventricular myocytes to test the hypothesis that the drug prevents ischemic electrophysiological disturbances by affecting the KATP channel. Membrane potentials and currents were recorded using standard patch-clamp techniques. During 15-minute superfusion with 1 mM CN-, action potential duration measured at 90% repolarization was reduced from 259 +/- 12 to 98 +/- 15 msec (62% reduction) in the absence of verapamil and from 266 +/- 11 to 183 +/- 16 msec (31% reduction) in the presence of 2 microM verapamil (p less than 0.01). In inside-out membrane patches, the KATP current, activated in the absence of ATP, was significantly suppressed by intracellular application of 2 microM verapamil, but the single-channel conductance was not changed. Verapamil did not change the mean open and closed times of the channel within bursts (e.g., the mean open time was 1.92 +/- 0.18 and 1.82 +/- 0.21 msec in the absence and presence of 2 microM verapamil, respectively), but it shortened the mean lifetime of bursts from 41.1 +/- 3.5 to 24.9 +/- 2.8 msec (p less than 0.01) and prolonged the closed time between bursts from 39.4 +/- 4.6 to 78.5 +/- 5.1 msec (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Early afterdepolarizations and triggered activity induced by cocaine. A possible mechanism of cocaine arrhythmogenesis.

Cocaine may produce life-threatening cardiac arrhythmias, but it is not clear whether this is an indirect effect of coronary vasoconstriction and ischemia or a direct myocardial effect of the substance. Except for its effects on the Na+ current as a local anesthetic, little is known about the direct electrophysiological actions on cardiac cells. Therefore, we studied the effects of cocaine on action potentials and membrane currents in isolated feline ventricular myocytes to test the hypothesis that cocaine-induced arrhythmogenesis may be based on cellular and ionic mechanisms. Action potentials and membrane currents were recorded using the patch clamp technique. Single cells were isolated from feline left ventricles by enzymatic digestion. Exposure to cocaine (10 or 50 microM) depressed the plateau phase of the action potential and prolonged action potential duration. Action potential duration measured at 90% repolarization (APD90) was increased from 280 +/- 12 msec to 325 +/- 17 msec (p less than 0.01) by 5-minute exposure to 10 mumol cocaine, when the cells were stimulated at 1 Hz. During exposure to 50 mumol cocaine, APD90 was markedly increased from 298 +/- 13 msec to 437 +/- 35 msec (p less than 0.01) in seven of 16 cells, and early afterdepolarizations (EADs) developed in these cells. The take-off potential and the amplitude of EADs were -28.3 +/- 2.3 mV and 16.8 +/- 1.2 mV, respectively. Triggered activity arising from EADs was induced in four of the seven cells. Addition of 1 nmol isoproterenol augmented EADs and induced sustained triggered activity, whereas they were suppressed by exposure to 2 microM verapamil. Whole-cell voltage clamp experiments revealed that cocaine (50 microM) reduced the peak L-type Ca2+ current from 1.03 +/- 0.13 nA to 0.79 +/- 0.11 nA (23% reduction, p less than 0.05). Cocaine also reduced the peak delayed rectifier K+ current from 362 +/- 51 pA to 113 +/- 32 pA (69% reduction, p less than 0.01). However, cocaine did not affect activation and inactivation kinetics of these channels. Cocaine had no effect on the inward rectifier K+ current. We conclude that cocaine can prolong action potential duration and induce EADs and triggered activity by blocking the delayed rectifier K+ current, and that cocaine-induced abnormalities of repolarization, modulated by its inhibitory effects on catecholamine reuptake, may play a role in the potential of cocaine for induction of acute fatal arrhythmias.

Factors regulating transient outward current in feline ventricular myocytes

Factors regulating transient outward current in feline ventricular myocytes

Effect of H+ on ATP-regulated K+ channels in feline ventricular myocytes

Using patch-clamp techniques, we examined the effects of pH on properties of ATP-regulated K+ channels in single myocytes isolated from cat left ventricles. ATP-K+ channels of inside-out patches were bilaterally exposed to 140 mM K+ solutions (22 degrees C). In the absence of ATP and Mg2+, the channels had a linear current-voltage relationship during hyperpolarizing pulses (20-100 mV negative to the reversal potential) at both intracellular pH (pHi) 7.4 and 6.5, but the slope conductance was 66 +/- 2 pS at pHi 7.4 and 46 +/- 2 pS at pHi 6.5. Lowering pHi from 7.4 to 6.5 increased the mean open time (from 15.9 +/- 4.6 to 35.9 +/- 7.9 ms, P less than 0.01) but decreased the open-state probability measured at 50 mV positive to the reversal potential (from 0.35 +/- 0.04 to 0.16 +/- 0.04, P less than 0.01). However, in the presence of both 0.2 mM ATP and 1 mM MgCl2, lowering pHi from 7.4 to 6.5 increased the mean open time (from 5.0 +/- 2.6 to 17.9 +/- 5.9 ms, P less than 0.01) and the open-state probability (from 0.025 +/- 0.010 to 0.098 +/- 0.024, P less than 0.01). These data indicate that increases in intracellular H+ concentration modulate cardiac ATP-K+ channel properties. Ischemia-associated decreases in pHi may enhance the opening of cardiac ATP-regulated K+ channels and resultant action potential shortening.

Voltage dependence of contraction and calcium current in severely hypertrophied feline ventricular myocytes

In the present study the voltage dependence of contraction and the characteristics of the “L-type” Ca 2+ current (I Ca) were compared in control (C) and severely hypertrophied (H) myocytes (M). Hypertrophy was induced in young cats by slow progressive pressure overload of the feline right ventricle. The amount of hypertrophy induced in this study was more severe than previously examined in this laboratory. The major findings of this study were: (1) The voltage dependence of contraction was not significantly different in C and HM. Peak shortening occurred at 6.2 ± 1.2 mV in HM and at 9.5 ± 1.3 mV in CM. (2) The magnitude of peak I Ca density was significantly smaller in HM (5.56 ± 0.53 pA/pF; n = 17) than in Cm (7.09 ± 0.42 pA/pF; n = 20). In both groups of cells I Ca reached a maximum at + 10 mV. (3) There were no significant changes in the voltage dependence of both I Ca activation and steady-state inactivation. This is the first sutdy to provide evidence that I Ca density is reduced in severe hypertrophy. The 21% decrease in peak I Ca density could reduce the contractile state of the heart if calcium-induced calcium release is normal and the reduction of I Ca alters the Ca 2+ released from the sacroplasmic reticulum. The reduction in “L-type” Ca 2+ current density in severely hypertrophied myocytes may play a role in the transition from the compensated hypertrophic state to congestive heart failure. Data are means ± standard error.

Mechanical properties of adult feline ventricular myocytes in culture

The contractile and electrophysiological properties of cultured adult feline ventricular myocytes were studied. Cells were field stimulated and contraction was measured using a video-based edge detector. The magnitude of contraction decreased by 36% and the rate of contraction decreased by 52% 2 h after the cells were plated on laminin-coated cover slips. The magnitude and rate of contraction then remained stable for 1 wk. The duration of contraction prolonged and a second component to the twitch frequently, but not invariably, developed after 5 days in culture. This was associated with prolongation of the action potential duration. After 7 days in culture, cells could be divided into two groups based on resting membrane potential. Norepinephrine increased the magnitude of contraction for 5 days after plating. Cultured ventricular myocytes became unresponsive to the effects of norepinephrine after 7 days. Adult cardiac myocytes maintained in primary culture continue to respond to field stimulation and retain many contractile properties for up to 7 days; however, the functional characteristics of these cells do not remain uniform during this time period.

Rest decay of calcium transients and contractility in feline ventricular myocytes

To define the respective contributions of sarcoplasmic reticulum Ca2+ release and action potential-dependent Ca2+ influx to the activator Ca2+, rest decay of the cytosolic free Ca2+ (Ca2+i) transient and contractility was measured in feline ventricular myocytes loaded with the Ca2(+)-sensitive fluorescent dye indo-1. Myocytes were stimulated to a steady state and then rested for periods ranging from 2 to 120 s. Rest decay was assayed with both resumption of stimulation and rapid extracellular applications of caffeine. The protocol was then repeated after exposure to ryanodine. Ryanodine changed the Ca2+i transients during steady-state stimulation from large, rapidly rising transients starting near the resting Ca2+i level to small, slowly rising Ca2+i transients superimposed on an elevated diastolic Ca2+i. Under control conditions the stimulation- and caffeine-induced Ca2+i transients exhibited slow monotonic rest decay with similar time constants of decay (180 and 202 s, respectively, from a representative cell). After ryanodine, the rest decay of stimulation-induced Ca2+i transients was seemingly no different. However, no Ca2+i transient could be elicited by caffeine after 15 s of rest. These results suggest that 1) under normal conditions the primary source of activator Ca2+ is the sarcoplasmic reticulum, and 2) the rest decay of contractility seen in feline ventricular myocytes results from time-dependent loss of Ca2+ from this organelle.