Inhibitory activity of blockers of the slow inward current in rat myocardium, a study in steady state and of rate of action

The inhibitory effects of verapamil (84.4% inhibition) and D-600 (70.4% inhibition) on the conductance of the slow inward current were compared with that of Mn²⁺ (99.7% inhibition). Hill coefficients of 2 for Mn²⁺ and D-600 and one for verapamil were found. For racemic mixtures of verapamil and D-600 the apparent dissociation constants (K_0.5) were 1.4 and 3.6 μm respectively, whereas the apparent dissociation constant of Mn²⁺ was 625 μm. With such high affinities, as compared to Mn²⁺, time constants of inhibition for D-600 and verapamil were larger and their offset rates were slower. It is concluded that Mn²⁺ and the organic blockers have different mechanisms of action, that varapamil and D-600 have to pass through or enter into the membrane, and that the molecules of D-600 and verapamil possess one and two active groups respectively.     

Slow inward current and contraction in frog atrial muscle at various extracellular concentrations of Na and Ca ions.

NaCa antagonism in frog cardiac muscle was investigated under voltage-clamp conditions to obtain further information about the underlying mechanism(s). Specifically the relationship between the slow inward current (I_slow) and that part of contractility activated by this current (phasic tension) was examined in various [Na]₀ and [Ca]₀. When [Na]₀ was reduced by substituting with LiCl or sucrose I_slow was changed only slightly (decreased in LiCl and increased in sucrose), whereas, phasic tension was increased several fold regardless of the type of substitute. When [Ca]₀ was reduced I_slow was again less affected than phasic tension. This decrease in maximal peak tension resulting from reduced [Ca]₀ was partially but not fully compensated for by simultaneously decreasing [Ca]₀ and [Na]₀, while keeping the ratio $\text{[Ca]}{}_0\text{[Na]}{}_0{}^2$ constant. The current decreased almost immediately upon application of these low Na, low Ca solutions but the change in phasic tension was biphasic—an increase followed by a decrease. The results indicate that contractile force in frog myocardium does not simply depend on $\text{[Ca]}{}_0\text{[Na]}{}_0{}^2$. The NaCa antagonism is explained by an interaction between the slow inward current and the intracellular stores of Ca which are regulated by NaCa exchange. A qualitative model of regulation of cardiac contractility is presented.     

8-bromo-guanosine-3',5' -monophosphate mimics the effect of acetylcholine on slow response action potential and contractile force in mammalian atrial myocardium.

In isolated myocardium from the left atrium of guinea pigs, the effect of acetylcholine (ACh) and of 8-Br-cGMP on the slow response action potential (elicited after inactivation of the fast Na current) and on the isometric contractile force was studied. ACh reduced upstroke velocity and overshoot and abbreviated action potential duration. Though the sensitivity towards ACh (1.65 × 10^?8m to 5.5 × 10^?6m) widely differed, the decrease of upstroke velocity was accompanied in the most experiments by similar quantitative changes of contractile force. 8-Br-cGMP caused the same depression of the slow response action potential. The development of the negative inotropic effect was the more pronounced the stronger the upstroke velocity was depressed. 6 × 10^?4m 8-Br-cGMP diminished upstroke velocity by 12 to 58% and isometric contractile force by 8 to 60%. The β-adrenergic compound isoproterenol abolished the inhibitory effects. But the absolute values of upstroke velocity, overshoot, and contractile force reached in the presence of isoproterenol were lower when the preparation had been pretreated with ACh or 8-Br-cGMP. It is concluded that cGMP might mediate the ACh effect on the surface membrane. The decline of upstroke velocity of the atrial slow response action potential suggests that 8-Br-cGMP could exert an inhibitory action on the Ca system of the membrane.     

Papaverine blockade of an inward slow Ca2+ current in guinea pig heart.

The membrane potentials of the ventricular fibers at the epicardial surface of isolated perfused guinea pig hearts were recorded with intracellular microelectrodes. Papaverine ($\text{15 mg}\text{100 ml}$) markedly depressed the rate of rise (to about 10V/s) within 10–15 min, prolonged the duration of the action potential, and abolished the contractions, i.e., the hearts became electromechanically uncoupled. The rate of rise or overshoot of this slowly-rising action potential was not affected by tetrodotoxin (TTX). Papaverine also abolished within 3 min the slowly-rising overshooting, plateau-like responses (with accompanying contractions) induced by isoproterenol (10^?7m) in hearts perfused with 27 mm K⁺ (cells depolarized to ?40 mV) to inactivate the fast Na⁺ channels. The effects of papaverine were readily reversible. The cyclic AMP levels of the ventricles were elevated nearly 3 fold by papaverine, and the ATP levels were not significantly depressed. Verapamil and metabolic inhibitors did not increase the cyclic AMP levels. These results suggest that papaverine has three major actions: (1) it blocks fast Na⁺ channels; (2) it directly blocks the inward slow Ca²⁺ current (like verapamil does), but leaves a TTX-insensitive slow cation current (presumably Na⁺) operational in 2.7 mm K⁺; and (3) it increases cyclic AMP levels, presumably by acting as a phosphodiesterase inhibitor.     

Frequency dependence of the ionic currents determining the action potential repolarization in rat ventricular muscle

In rat ventricular muscle, the action potential configuration and the ionic currents were studied as a function of frequency under current and voltage clamp conditions. Increase of stimulation frequency produced (a) a progressive loss of the plateau accompanied by a shortening of the action potential; (b) no significant change in the amplitude of the instantaneous outward current in [Ca²⁺]_o between 2.2 and 10.0 mm, and (c) an increase in the amplitude of the slow inward current, I_si and a marked reduction of its inactivation time constant τ_f up to frequency of about 75 stimuli/min. Above this frequency, the amplitude of I_si decreased and τ_f increased again.     

Quantitative differences between the inhibitory action of verapamil and Ni ions on the slow response action potential in mammalian ventricular myocardium.

Some organic compounds and a number of bivalent cations have been found capable of blocking the slow inward current (I_si) that is mainly carried by Ca in mammalian ventricular myocardium. With respect to verapamil and Ni ions, it has been shown that the mode of action of both inhibitory agents is, despite their different chemical structure, quite similar and predominantly arises from a decrease of maximum conductance of the slow channel system, $\text{g}{}_{\mathrm{<mtext>si</mtext>}}$ [2, 3]. Moreover, at a given verapamil or Ni concentration, the degree of inhibition of I_si depends crucially on the external Ca concentration. This supports the hypothesis that the decline of $\text{g}{}_{\mathrm{<mtext>si</mtext>}}$ results from a competitive interaction of these inhibitors with Ca at certain groups of the slow channel, thereby making these (anionic) groups unable to allow Ca inward movements through the slow channel during excitation. In an attempt to further characterize the reaction of verapamil and Ni ions with their receptor molecules of the cardiac membrane, a quantitative analysis of the verapamil and Ni-induced inhibition of the I_si-mediated slow response action potential was performed in isolated papillary muscles of guinea pigs.     

Diastolic transient inward current in long QT syndrome type 3 is caused by Ca overload and inhibited by ranolazine

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na⁺ channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca²⁺)-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized ΔKPQ LQT-3 mutation but the link to altered Ca²⁺ cycling related to mutant Na⁺ channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca²⁺ load and induction of spontaneous diastolic inward current (I_TI) in myocytes expressing ΔKPQ Na⁺ channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca²⁺, and measurement of SR Ca²⁺ content using a caffeine dump methodology. We compared the Ca²⁺ content of ΔKPQ^+/− myocytes displaying I_TI to those without spontaneous diastolic activity and found that I_TI induction correlates with higher sarcoplasmic reticulum (SR) Ca²⁺. Both spontaneous diastolic I_TI and underlying Ca²⁺ waves are inhibited by ranolazine at concentrations that preferentially target I_NaL during prolonged depolarization. Furthermore, ranolazine I_TI inhibition is accompanied by a small but significant decrease in SR Ca²⁺ content. Our results provide the first direct evidence that induction of diastolic transient inward current (I_TI) in ΔKPQ^+/− myocytes occurs under conditions of elevated SR Ca²⁺ load.     

Ionic basis of ischemia-induced bradycardia in the rabbit sinoatrial node

To investigate the basis of ischemia-induced bradycardia (<60 beats/min), we isolated pacemaker cells from the rabbit sinoatrial node and exposed them to ischemic-like conditions, including omission of glucose, pH 6.6, and either 5.4 or 10 mM KCl to evaluate the role of increased serum [K]. A perforated-patch technique was employed to test the hypothesis that the arrhythmia is caused by attenuation of inward currents that contribute to the diastolic depolarization. After exposure to "ischemic" Tyrode containing 5.4 mM KCl, the pacemaker cells exhibited 13% slower beat rates and action potentials with 6-mV greater overshoots and 44% longer durations. In contrast, after exposure to "ischemic" Tyrode containing 10 mM KCl, the pacemaker cells exhibited a 7-mV depolarization of the maximum diastolic potential but no significant change in the overshoot. Beat rates were slowed by 43%, and the action potentials were prolonged by 46%. "Ischemic" Tyrode containing 5.4 mM KCl increased L-type Ca current, decreased T-type Ca current and reduced Ni-sensitive inward current tails (presumably Na-Ca exchange current), even after treatment with 40 muM ryanodine to block Ca release from the sarcoplasmic reticulum. "Ischemic" Tyrode containing 10 mM KCl increased hyperpolarization-activated inward current at diastolic potentials and reduced the slowly activating component, but not the rapidly activating component, of delayed rectifier K current. Our results suggest that reductions of inward Na-Ca exchange current and T-type Ca current contribute to "ischemia"-induced "bradycardia" in sinoatrial node pacemaker cells.     

Dual Effect of Nitric Oxide on the Hyperpolarization-activated Inward Current (If) in Sino-atrial Node Cells of the Rabbit

Using the whole-cell voltage-clamp technique, we have investigated the effect of nitric oxide (NO) donor (sodium nitroprusside, SNP) on hyperpolarization-activated inward current,I_f, in isolated rabbit sinoatrial node (SAN) cells.I_fin the basal state increased when NO was applied but decreased whenI_fwas pre-stimulated by isoproterenol (ISO) or by adding cAMP to the pipette solution. Both the stimulatory and the inhibitory effects of NO were abolished by guanylyl cyclase inhibitor, methylene blue (MB), suggesting that the effect of NO is mediated by cGMP. The inhibitory effect of NO was abolished whenI_fwas pre-stimulated by 3-isobutyl-1-methylxanthine (IBMX), which is a phosphodiesterase (PDE) inhibitor, or by adding 8Br-cAMP (which is resistant to PDE) to the pipette solution. An analogue of cGMP, 8Br-cGMP, which is a potent stimulator of cGMP-dependent protein kinase (PKG) but has little effect on PDE, did not inhibitI_fwhenI_fwas pre-stimulated by ISO. In its basal state,I_fwas still increased by 8Br-cGMP, and this effect was not prevented by the pretreatment with H-7, PKG inhibitor. The effect of acetylcholine (ACh) was not identical to that of NOI_fdecreased when pre-stimulated not only by ISO, but also by IBMX. The above results suggest that via cGMP, NO exerts a dual effect onI_f: the inhibitory effect is mediated by cGMP-stimulated PDE, and the stimulatory effect may be attributable to direct binding of cGMP toI_fchannels.     

Atrial-selective approaches for the treatment of atrial fibrillation

Atrial-selective pharmacologic approaches represent promising novel therapeutic options for the treatment of atrial fibrillation (AF). Medical treatment for AF is still more widely applied than interventional therapies but is hampered by several important weaknesses. Besides limited clinical efficacy (cardioversion success and sinus-rhythm maintenance), side effects like ventricular proarrhythmia and negative inotropy are important limitations to present class I and III drug therapy. Although no statistically significant detrimental survival consequences have been documented in trials, constitutional adverse effects might also limit applicability. Cardiac targets for novel atrial-selective antiarrhythmic compounds have been identified, and a large-scale search for safe and effective medications has begun. Several ionic currents (I(KACh), I(Kur)) and connexins (Cx-40) are potential targets, because atrial-selective expression makes them attractive in terms of reduced ventricular side-effect liability. Data on most agents are still experimental, but some clinical findings are available. Atrial fibrillation generates a specifically remodeled atrial milieu for which other therapeutic interventions might be effective. Some drugs show frequency-dependent action, whereas others target structurally remodeled atria. This review focuses on potential atrial-selective compounds, summarizing mechanisms of action in vitro and in vivo. It also mentions favorable interventions on the milieu in terms of conventional (such as antifibrotic effects of angiotensin-system antagonism) and innovative gene-therapy approaches that might add to future AF therapeutic options.     

Hypothyroid-like effect of amiodarone in the ventricular myocardium of the rat

Summary Due to the similar electrophysiological effects of amiodarone and hypothyroidism in the myocardium, the induction of a local hypothyroid state has been proposed as the mechanism of action of amiodarone. To examine this hypothesis we have studied the influence of amiodarone on the distribution of ventricular isomyosins — a sensitive parameter of the thyroid state in the rat heart — and the effects of amiodarone on 3,5,3-triiodothyronine (T₃) myocardial nuclear receptor binding in vivo.Amiodarone induced a dosage-dependent redistribution of isomyosins similar to hypothyroidism, while simultaneously inducing a low T₃ syndrome at the higher dose level. In hypothyroid rats, which were pretreated with amiodarone, substitution of T₃ (2 g/100 g) led to a complete reversal of the myosin pattern not differing from control hypothyroid rats which were only given T₃; the effect of T₃ (0.5 g/100 g) was however partially inhibited by amiodarone. Nuclear receptor binding of T₃ determined in hypothyroid rats in vivo was unaffected by amiodarone.We conclude that amiodarone induces a hypothyroid-like state in the ventricular myocardium of rats by inhibiting the action of T₃ — an effect which cannot be attributed to an antagonism at the T₃ nuclear receptor level.     

Importance of sodium ions in the protective effects of high-potassium, high-glucose solution on electromechanical activities in the guinea-pig myocardium

The protective effects of high-K, high-glucose solution (GK solution) on electromechanical activity in the isolated guinea-pig ventricular muscle were studied. The basal test solution (basal GK solution) contained 30 mM KCl, 230 mM glucose and 4 mM NaHCO3, that is nominally Ca-free. Various types of GK solutions were also prepared by modifying the composition of this solution. When the muscle was exposed to the basal GK solution, the resting potential fell to about -40 mV, the muscle lost the excitability, and an irreversible contracture gradually developed. This contracture was prevented by elevating [Na]o above 60 mM. Reduction of [K]o to 20 mM, addition of EGTA (0.4 mM), or lowering the temperature (23 degrees C) also suppressed the above contracture. When the GK solution with high Na (106-115 mM NaCl added to basal GK solution in exchange for glucose) was applied, contractures frequently developed upon reintroduction of Tyrode solution (Ca-paradox-like phenomenon). Thus the Ca-free GK solution with 60 mM Na (56 mM NaCl added to basal GK solution in exchange for glucose) induced no contracture, either during or after the test period. Recovery of the action potential after this application was all but complete. On the other hand, addition of 0.9 to 1.8 mM Ca to this solution produced another type of contracture which was sensitive to verapamil. The cardioplegic effects of the Ca-free GK solution with 60 mM Na persisted under hypoxic conditions, and glucose appeared to play a significant role in preventing the hypoxia-related contracture. In contrast, the high-Na (110 mM Na) GK solution containing 0.2 to 0.5 mM Ca and Tyrode solution, both of which produced no contracture under normoxic conditions, did produce contractures under hypoxic conditions. Therefore, Ca-free GK solution containing an appropriate concentration (around 60 mM) of Na may protect the normoxic and hypoxic myocardium against intracellular Ca overload. The related mechanisms involved were discussed with special reference to membrane functions and intracellular Ca-regulating systems.     

Magnifying gastroendoscopy for diagnosis of histologic gastritis in the gastric antrum

BACKGROUND: We investigated the potential of magnifying endoscopy for diagnosis of histologic gastritis in the gastric antrum. In addition, we investigated whether magnifying endoscopy can be applied for evaluation of Helicobacter pylori eradication therapy. METHODS: We examined 176 Japanese patients including 53 with H. pylori eradication. We evaluated the antrum by magnifying observation and ordinary endoscopic findings, and compared these results. Biopsy specimens were taken from the sites observed. RESULTS: The magnified views were classified into four types. Histology of the biopsy specimens allowed us to match the four magnified views with normal mucosa with fundic glands, normal mucosa with pyloric glands, mucosa with gastritis and intestinal metaplasia/epithelial hyperplasia. The types of magnifying appearances were specific enough for the diagnosis of histologic gastritis (148 out of the 176 (82.4%) cases; sensitivity, 96.3%; specificity, 73.7%). We could accurately diagnose the histologic gastritis by magnifying endoscopy in 49 out of the 53 (92%) cases with H. pylori eradication, while only in 38% by ordinary endoscopy. The accuracy of diagnosis was statistically higher with the use of magnifying endoscopy than with ordinary endoscope (P < 0.001). CONCLUSION: Magnifying gastroendoscopy is useful to judge the histologic gastritis, especially, in cases with H. pylori eradication.     

Augmented potassium current is a shared phenotype for two genetic defects associated with familial atrial fibrillation

Mutations in multiple genes have been implicated in familial atrial fibrillation (AF), but the underlying mechanisms, and thus implications for therapy, remain ill-defined. Among 231 participants in the Vanderbilt AF Registry, we found a mutation in KCNQ1 (encoding the α-subunit of slow delayed rectifier potassium current [I_Ks]) and separately a mutation in natriuretic peptide precursor A (NPPA) gene (encoding atrial natriuretic peptide, ANP), both segregating with early onset lone AF in different kindreds. The functional effects of these mutations yielded strikingly similar I_Ks “gain-of-function.” In Chinese Hamster Ovary (CHO) cells, coexpression of mutant KCNQ1 with its ancillary subunit KCNE1 generated ∼ 3-fold larger currents that activated much faster than wild-type (WT)-I_Ks. Application of the WT NPPA peptide fragment produced similar changes in WT-I_Ks, and these were exaggerated with the mutant NPPA S64R peptide fragment. Anantin, a competitive ANP receptor antagonist, completely inhibited the changes in I_Ks gating observed with NPPA S64R. Computational simulations identified accelerated transitions into open states as the mechanism for variant I_Ks gating. Incorporating these I_Ks changes into computed human atrial action potentials (AP) resulted in 37% shortening (120 vs. 192 ms at 300 ms cycle length), reflecting loss of the phase II dome which is dependent on L-type calcium channel current. We found striking functional similarities due to mutations in KCNQ1 and NPPA genes which led to I_Ks “gain-of-function”, atrial AP shortening, and consequently altered calcium current as a common mechanism between diverse familial AF syndromes.     

Gender-related differences in ventricular myocyte repolarization in the guinea pig

Abstract. It is well established that gender-differences exist in cardiac electrophysiology and these are thought to contribute to the increased risk of women, compared to men, for the potentially lethal ventricular arrhythmia, torsades de pointes. Data from animal models with abbreviated estrus cycles suggest that androgens may play a protective role in males. However, the role of female sex hormones in gender-differences in cardiac electrophysiology is less clear. This report describes gender differences in ventricular electrophysiology, investigated using the guinea pig heart. Ionic currents and action potentials were compared between ventricular myocytes isolated from male guinea pig hearts and those from females on the day of estrus (day 0) and 4 days post-estrus (day 4). The density of inward rectifier K⁺ current (I_K1) at –120 mV was significantly greater in male myocytes than in female myocytes either at day 0 or day 4. The peak L-type Ca²⁺ current (I_Ca) at +10 mV was also significantly larger in male myocytes than in day 0 and day 4 female myocytes. Moreover, I_Ca differed significantly between day 0 and day 4 female myocytes, strongly suggesting that I_Ca density varies around the estrus cycle. Delayed rectifier (I_K) tail currents were significantly different between male and female day 4 myocytes. Action potential duration (at 90% repolarization; APD₉₀) was significantly shorter in male myocytes than in female myocytes at day 0, but not at day 4, broadly consistent with the combined differences in I_K and I_Ca between the three groups. Taken together, our data are consistent with the contribution of multiple factors, rather than a single hormone, to gender differences in ventricular repolarization. Since female guinea pigs possess a conventional estrus cycle, our data suggest that this species may be well suited to elucidating the modulatory influence of ovarian steroids on ventricular repolarization and arrhythmic risk. Our findings suggest that further work examining the basis to gender differences in ventricular repolarization in the guinea pig is warranted.