Protein kinase C and Ca(2+) -calmodulin-dependent protein kinase II mediate the enlarged reverse INCX induced by ouabain-increased late sodium current in rabbit ventricular myocytes.

NEW FINDINGS: What is the central question of this study? What are the effects of protein kinase C (PKC) and Ca(2+) -calmodulin-dependent protein kinase II (CaMKII) on late sodium current (INaL ), reverse Na(+) -Ca(2+) exchange current (reverse INCX ) or intracellular Ca(2+) levels changed by ouabain? What is the main finding and its importance? Ouabain, even at low concentrations (0.5-8.0 µm), can increase INaL and reverse INCX , and these effects may contribute to the effect of the glycoside to increase Ca(2+) transients and contractility. Both PKC and CaMKII activities may mediate or modulate these processes. It has been reported that the cardiac glycoside ouabain can increase the late sodium current (INaL ), as well as the diastolic intracellular calcium concentration and contractile shortening. Whether an increase of INaL participates in a pathway that can mediate the positive inotropic response to ouabain is unknown. We therefore determined the effects of ouabain on INaL , reverse Na(+) -Ca(2+) exchange current (reverse INCX ), intracellular Ca(2+) ([Ca(2+) ]i ) levels and contractile shortening in rabbit isolated ventricular myocytes. Ouabain (0.1-8 µm) markedly increased INaL and reverse INCX in a concentration-dependent manner, with significant effects at concentrations as low as 0.5 and 1 µm. These effects of ouabain were suppressed by the INaL inhibitors TTX and ranolazine, the protein kinase C inhibitor bisindolylmaleimide and the Ca(2+) -calmodulin-dependent protein kinase II inhibitor KN-93. The enhancement by 0.5 µm ouabain of ventricular myocyte contractility and intracellular Ca(2+) transients was suppressed by 2.0 µm TTX. We conclude that ouabain, even at low concentrations (0.5-8.0 µm), can increase INaL and reverse INCX , and these effects may contribute to the effect of the glycoside to increase Ca(2+) transients and contractility. Both protein kinase C and Ca(2+) -calmodulin-dependent protein kinase II activities may mediate or modulate these processes.

Cardiac late Na⁺ current: proarrhythmic effects, roles in long QT syndromes, and pathological relationship to CaMKII and oxidative stress.

Myocyte sodium channel current that persists throughout the plateau of the cardiac action potential is referred to as late sodium current (I(Na-L)). The magnitude of I(Na-L) is normally small, but can increase significantly in common acute and chronic pathological settings as a result of inherited and/or acquired Na(+) channelopathies that alter channel opening and closing (ie, gating), location (trafficking), or anchoring and interactions with cytoskeletal proteins. An increase in I(Na-L) reduces repolarization reserve in atrial and ventricular myocytes and prolongs the action potential duration and the QT interval. An enhanced I(Na-L) is a cause of long QT syndrome 3. I(Na-L) may be a cause of afterdepolarizations, triggered arrhythmias, and spontaneous diastolic depolarization-induced automaticity. In addition, enhancement of I(Na-L) increases both the temporal and the spatial dispersion of repolarization in the myocardium and may lead to spatially discordant action potential duration alternans, wavebreak, and reentrant arrhythmias. Positive feedback loops between increases in I(Na-L) and the activity of Ca(2+)/calmodulin-dependent protein kinase II appear to contribute to the genesis of arrhythmias and to certain abnormalities of the ischemic heart. In this review, we discuss some of the more relevant experimental results, clinical findings, and insights from cellular and animal models that highlight the role of I(Na-L) in the genesis of arrhythmias, long QT syndromes, and intracellular Ca(2+) homeostasis.

Intracellular Na+ overload causes oxidation of CaMKII and leads to Ca2+ mishandling in isolated ventricular myocytes.

An increase of late Na(+) current (INaL) in cardiac myocytes can raise the cytosolic Na(+) concentration and is associated with activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alterations of mitochondrial metabolism and Ca(2+) handling by sarcoplasmic reticulum (SR). We tested the hypothesis that augmentation of INaL can increase mitochondrial reactive oxygen species (ROS) production and oxidation of CaMKII, resulting in spontaneous SR Ca(2+) release and increased diastolic Ca(2+) in myocytes. Increases of INaL and/or of the cytosolic Na(+) concentration led to mitochondrial ROS production and oxidation of CaMKII to cause dysregulation of Ca(2+) handling in rabbit cardiac myocytes.

Ranolazine attenuates hypoxia- and hydrogen peroxide-induced increases in sodium channel late openings in ventricular myocytes.

Ranolazine attenuates cardiac arrhythmic activity associated with hypoxia and hydrogen peroxide (H2O2) by inhibition of late sodium current (late INa). The mechanism of ranolazine's action on Na channels was investigated using whole-cell and single-channel recording from guinea pig isolated ventricular myocytes. Hypoxia increased whole-cell late INa from -0.48 ± 0.02 to -3.99 ± 0.07 pA/pF. Ranolazine at 3 and 9 µmol/L reduced the hypoxia-induced late INa by 16% ± 3% and 55% ± 3%, respectively. Hypoxia increased the mean open probability and open time of Na-channel late openings from 0.016 ± 0.001 to 0.064 ± 0.007 milliseconds and from 0.693 ± 0.043 to 1.081 ± 0.098 milliseconds, respectively. Ranolazine at 3 and 9 µmol/L attenuated the hypoxia-induced increase of open probability by 19% ± 7% and 61% ± 1%, and increase of open time by 26% ± 19% and 74 ± 21%, respectively. H2O2 increased the mean open probability and open time of Na-channel late openings from 0.013 ± 0.002 to 0.107 ± 0.015 milliseconds and from 0.689 ± 0.075 to 1.487 ± 0.072 milliseconds, respectively. Ranolazine at 3 and 6 µmol/L reduced the H2O2-induced increase of mean open probability by 60% ± 7% and 95% ± 2%, and the increase of mean open time by 31% ± 21% and 82% ± 8%. In conclusion, the inhibition by ranolazine of hypoxia- and H2O2-stimulated late INa is due to reduction of both the open probability and open time of Na-channel late openings.

The role of late I Na in development of cardiac arrhythmias.

Late I Na is an integral part of the sodium current, which persists long after the fast-inactivating component. The magnitude of the late I Na is relatively small in all species and in all types of cardiomyocytes as compared with the amplitude of the fast sodium current, but it contributes significantly to the shape and duration of the action potential. This late component had been shown to increase in several acquired or congenital conditions, including hypoxia, oxidative stress, and heart failure, or due to mutations in SCN5A, which encodes the α-subunit of the sodium channel, as well as in channel-interacting proteins, including multiple ß subunits and anchoring proteins. Patients with enhanced late I Na exhibit the type-3 long QT syndrome (LQT3) characterized by high propensity for the life-threatening ventricular arrhythmias, such as Torsade de Pointes (TdP), as well as for atrial fibrillation. There are several distinct mechanisms of arrhythmogenesis due to abnormal late I Na, including abnormal automaticity, early and delayed after depolarization-induced triggered activity, and dramatic increase of ventricular dispersion of repolarization. Many local anesthetic and antiarrhythmic agents have a higher potency to block late I Na as compared with fast I Na. Several novel compounds, including ranolazine, GS-458967, and F15845, appear to be the most selective inhibitors of cardiac late I Na reported to date. Selective inhibition of late I Na is expected to be an effective strategy for correcting these acquired and congenital channelopathies.

Larger late sodium current density as well as greater sensitivities to ATX II and ranolazine in rabbit left atrial than left ventricular myocytes.

An increase of cardiac late sodium current (INa.L) is arrhythmogenic in atrial and ventricular tissues, but the densities of INa.L and thus the potential relative contributions of this current to sodium ion (Na(+)) influx and arrhythmogenesis in atria and ventricles are unclear. In this study, whole-cell and cell-attached patch-clamp techniques were used to measure INa.L in rabbit left atrial and ventricular myocytes under identical conditions. The density of INa.L was 67% greater in left atrial (0.50 ± 0.09 pA/pF, n = 20) than in left ventricular cells (0.30 ± 0.07 pA/pF, n = 27, P < 0.01) when elicited by step pulses from -120 to -20 mV at a rate of 0.2 Hz. Similar results were obtained using step pulses from -90 to -20 mV. Anemone toxin II (ATX II) increased INa.L with an EC50 value of 14 ± 2 nM and a Hill slope of 1.4 ± 0.1 (n = 9) in atrial myocytes and with an EC50 of 21 ± 5 nM and a Hill slope of 1.2 ± 0.1 (n = 12) in ventricular myocytes. Na(+) channel open probability (but not mean open time) was greater in atrial than in ventricular cells in the absence and presence of ATX II. The INa.L inhibitor ranolazine (3, 6, and 9 µM) reduced INa.L more in atrial than ventricular myocytes in the presence of 40 nM ATX II. In summary, rabbit left atrial myocytes have a greater density of INa.L and higher sensitivities to ATX II and ranolazine than rabbit left ventricular myocytes.

Inhibition of the late sodium current slows t-tubule disruption during the progression of hypertensive heart disease in the rat.

The treatment of heart failure (HF) is challenging and morbidity and mortality are high. The goal of this study was to determine if inhibition of the late Na(+) current with ranolazine during early hypertensive heart disease might slow or stop disease progression. Spontaneously hypertensive rats (aged 7 mo) were subjected to echocardiographic study and then fed either control chow (CON) or chow containing 0.5% ranolazine (RAN) for 3 mo. Animals were then restudied, and each heart was removed for measurements of t-tubule organization and Ca(2+) transients using confocal microscopy of the intact heart. RAN halted left ventricular hypertrophy as determined from both echocardiographic and cell dimension (length but not width) measurements. RAN reduced the number of myocytes with t-tubule disruption and the proportion of myocytes with defects in intracellular Ca(2+) cycling. RAN also prevented the slowing of the rate of restitution of Ca(2+) release and the increased vulnerability to rate-induced Ca(2+) alternans. Differences between CON- and RAN-treated animals were not a result of different expression levels of voltage-dependent Ca(2+) channel 1.2, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, ryanodine receptor type 2, Na(+)/Ca(2+) exchanger-1, or voltage-gated Na(+) channel 1.5. Furthermore, myocytes with defective Ca(2+) transients in CON rats showed improved Ca(2+) cycling immediately upon acute exposure to RAN. Increased late Na(+) current likely plays a role in the progression of cardiac hypertrophy, a key pathological step in the development of HF. Early, chronic inhibition of this current slows both hypertrophy and development of ultrastructural and physiological defects associated with the progression to HF.

The arrhythmogenic consequences of increasing late INa in the cardiomyocyte.

This review presents the roles of cardiac sodium channel NaV1.5 late current (late INa) in generation of arrhythmic activity. The assumption of the authors is that proper Na(+) channel function is necessary to the maintenance of the transmembrane electrochemical gradient of Na(+) and regulation of cardiac electrical activity. Myocyte Na(+) channels' openings during the brief action potential upstroke contribute to peak INa and initiate excitation-contraction coupling. Openings of Na(+) channels outside the upstroke contribute to late INa, a depolarizing current that persists throughout the action potential plateau. The small, physiological late INa does not appear to be critical for normal electrical or contractile function in the heart. Late INa does, however, reduce the net repolarizing current, prolongs action potential duration, and increases cellular Na(+) loading. An increase of late INa, due to acquired conditions (e.g. heart failure) or inherited Na(+) channelopathies, facilitates the formation of early and delayed afterpolarizations and triggered arrhythmias, spontaneous diastolic depolarization, and cellular Ca(2+) loading. These in turn increase the spatial and temporal dispersion of repolarization time and may lead to reentrant arrhythmias.

A novel, potent, and selective inhibitor of cardiac late sodium current suppresses experimental arrhythmias.

Inhibition of cardiac late sodium current (late I(Na)) is a strategy to suppress arrhythmias and sodium-dependent calcium overload associated with myocardial ischemia and heart failure. Current inhibitors of late I(Na) are unselective and can be proarrhythmic. This study introduces GS967 (6-[4-(trifluoromethoxy)phenyl]-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine), a potent and selective inhibitor of late I(Na), and demonstrates its effectiveness to suppress ventricular arrhythmias. The effects of GS967 on rabbit ventricular myocyte ion channel currents and action potentials were determined. Anti-arrhythmic actions of GS967 were characterized in ex vivo and in vivo rabbit models of reduced repolarization reserve and ischemia. GS967 inhibited Anemonia sulcata toxin II (ATX-II)-induced late I(Na) in ventricular myocytes and isolated hearts with IC(50) values of 0.13 and 0.21 µM, respectively. Reduction of peak I(Na) by GS967 was minimal at a holding potential of -120 mV but increased at -80 mV. GS967 did not prolong action potential duration or the QRS interval. GS967 prevented and reversed proarrhythmic effects (afterdepolarizations and torsades de pointes) of the late I(Na) enhancer ATX-II and the I(Kr) inhibitor E-4031 in isolated ventricular myocytes and hearts. GS967 significantly attenuated the proarrhythmic effects of methoxamine+clofilium and suppressed ischemia-induced arrhythmias. GS967 was more potent and effective to reduce late I(Na) and arrhythmias than either flecainide or ranolazine. Results of all studies and assays of binding and activity of GS967 at numerous receptors, transporters, and enzymes indicated that GS967 selectively inhibited late I(Na). In summary, GS967 selectively suppressed late I(Na) and prevented and/or reduced the incidence of experimentally induced arrhythmias in rabbit myocytes and hearts.

An apoptosis signal-regulating kinase 1 inhibitor reduces cardiomyocyte apoptosis and infarct size in a rat ischemia-reperfusion model.

PURPOSES: We determined whether a small molecule inhibitor of apoptosis signal-regulating kinase 1 (ASK1-i) could reduce myocardial infarct size in a rat ischemia/reperfusion model. METHODS AND RESULTS: Sprague-Dawley rats were randomized to 3 groups: ASK1-i infusion (n = 16), vehicle infusion (n = 16), or ischemic preconditioning (IPC; n = 15). Infusion of ASK1-i (10 mg/kg, iv) or vehicle commenced 45 minutes before myocardial ischemia. IPC consisted of 3 cycles of 3 minutes of coronary occlusion followed by 5 minutes of reperfusion immediately before index myocardial ischemia, which consisted of 30-minute left coronary occlusion followed by 180 minutes of reperfusion. Pathologic analysis revealed no significant difference in the ischemic risk size among the 3 groups. ASK1-I and IPC significantly reduced myocardial infarct size (27.7% ± 3.3%, 16.5% ± 3.4%, and 41.5% ± 4.8% in the ASK1-i group, the IPC group, and the vehicle group, respectively; P = 0.0002) and apoptosis (the percentage of apoptotic nuclei averaged 11.6% ± 1.0%, 10.2% ± 1.7%, and 17.7% ± 2.0% in the ASK1-i group, IPC group, and vehicle group, respectively, P = 0.0055). CONCLUSIONS: A small molecule inhibitor of ASK1 was shown for the first time to reduce apoptosis and myocardial infarct size in a rat model of ischemia/reperfusion.

Block of Na+ currents and suppression of action potentials in embryonic rat dorsal root ganglion neurons by ranolazine.

Ranolazine, an anti-anginal drug, reduces neuropathic and inflammatory-induced allodynia in rats. However, the mechanism of ranolazin's anti-allodynic effect is not known. We hypothesized that ranolazine would reduce dorsal root ganglion (DRG) Na(+) current (I(Na)) and neuronal firing by stabilizing Na(+) channels in inactivated states to cause voltage- and frequency-dependent block. Therefore, we investigated the effects of ranolazine on tetrodotoxin-sensitive (TTXs) and tetrodotoxin-resistant (TTXr) I(Na) and action potential parameters of small diameter DRG neurons from embryonic rats. Ranolazine (10 and 30 µM) significantly reduced the firing frequency of evoked action potentials in DRG neurons from 19.2 ± 1.4 to 9.8 ± 2.7 (10 µM) and 5.7 ± 1.3 (30 µM) Hz at a resting membrane potential of -40 mV. Ranolazine blocked TTXs and TTXr in a voltage- and frequency-dependent manner. Furthermore, ranolazine (10 µM) blocked hNa(v)1.3 (expressed in HEK293 cells) and caused a hyperpolarizing shift in the voltage dependence of steady-state intermediate and slow inactivation Na(v)1.3 current. Taken together, the results suggest that ranolazine suppresses the hyperexcitability of DRG neurons by interacting with the inactivated states of Na(+) channels and these actions may contribute to its anti-allodynic effect in animal models of neuropathic pain.

Structure-activity relationships of 2-amino-3-aroyl-4-[(4-arylpiperazin-1-yl)methyl]thiophenes. Part 2: Probing the influence of diverse substituents at the phenyl of the arylpiperazine moiety on allosteric enhancer activity at the A1 adenosine receptor.

In a preliminary article, we reported the potent allosteric enhancer activity at the A(1) adenosine receptor of a small series of 2-amino-3-(4-chlorobenzoyl)-4-[4-(aryl)piperazin-1-yl)methyl]thiophene derivatives bearing electron-withdrawing or electron-releasing groups at the para-position of the phenylpiperazine moiety. In the present study, we report the development of the compounds previously studied by modifying both the number and position of substituents on the phenylpiperazine moiety, aimed at establishing a structure-activity relationship identifying additional compounds with improved activity. The nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to exert a fundamental influence on the allosteric enhancer activity, with the 3,4-difluoro 4i, 3-chloro-4-fluoro 4o, and 4-trifluoromethoxy 4ak derivatives being the most active compounds in binding (saturation and competition experiments) and functional cAMP studies. This study shows that it is also possible to obtain a good separation between allosteric enhancement and antagonistic activity at the A(1) adenosine receptor.

Calmodulin kinase II and protein kinase C mediate the effect of increased intracellular calcium to augment late sodium current in rabbit ventricular myocytes.

An increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) augments late sodium current (I(Na.L)) in cardiomyocytes. This study tests the hypothesis that both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) mediate the effect of increased [Ca(2+)](i) to increase I(Na.L). Whole cell and open cell-attached patch clamp techniques were used to record I(Na.L) in rabbit ventricular myocytes dialyzed with solutions containing various concentrations of [Ca(2+)](i). Dialysis of cells with [Ca(2+)](i) from 0.1 to 0.3, 0.6, and 1.0 µM increased I(Na.L) in a concentration-dependent manner from 0.221 ± 0.038 to 0.554 ± 0.045 pA/pF (n = 10, P < 0.01) and was associated with an increase in mean Na(+) channel open probability and prolongation of channel mean open-time (n = 7, P < 0.01). In the presence of 0.6 µM [Ca(2+)](i), KN-93 (10 µM) and bisindolylmaleimide (BIM, 2 µM) decreased I(Na.L) by 45.2 and 54.8%, respectively. The effects of KN-93 and autocamtide-2-related inhibitory peptide II (2 µM) were not different. A combination of KN-93 and BIM completely reversed the increase in I(Na.L) as well as the Ca(2+)-induced changes in Na(+) channel mean open probability and mean open-time induced by 0.6 µM [Ca(2+)](i). Phorbol myristoyl acetate increased I(Na.L) in myocytes dialyzed with 0.1 µM [Ca(2+)](i); the effect was abolished by Gö-6976. In summary, both CaMKII and PKC are involved in [Ca(2+)](i)-mediated augmentation of I(Na.L) in ventricular myocytes. Inhibition of CaMKII and/or PKC pathways may be a therapeutic target to reduce myocardial dysfunction and cardiac arrhythmias caused by calcium overload.

Selective action of metoprolol to attenuate regadenoson-induced tachycardia in conscious dogs.

BACKGROUND: Regadenoson is a coronary vasodilator that causes tachycardia via activation of the sympathetic nervous system. We determined whether ß(1)-adrenergic blockade can attenuate tachycardia without significantly reducing coronary vasodilation induced by regadenoson. METHODS AND RESULTS: Hemodynamics and coronary blood flow (CBF) were measured in conscious dogs. Baseline CBF and heart rate (HR) were 42 ± 2 mL/min and 87 ± 8 bpm (mean ± SEM), respectively. Regadenoson (1, 2.5, and 5 µg/kg) increased peak CBF by 129 ± 10, 149 ± 7, and 174 ± 10 mL/min and HR by 48 ± 6, 67 ± 5, and 85 ± 11 bpm, respectively, (all P < .05 vs baseline). In the presence of metoprolol (1.5 mg/kg), the peak increases in CBF caused by these three doses of regadenoson were reduced by only 11 ± 7%, 10 ± 4%, and 21 ± 2% (P = NS, <.05, and <.05 vs regadenoson alone), respectively, whereas the regadenoson-induced tachycardia was significantly reduced by 55 ± 8%, 55 ± 4%, and 52 ± 5% (all P < .05). In the presence of metoprolol, the duration of the regadenoson-induced increase in CBF was reduced, but the duration of the 2-fold increase in CBF caused by 5 µg/kg regadenoson was still nearly 6 minutes. CONCLUSION: ß(1)-Adrenergic blockade with metoprolol attenuated the regadenoson-induced increase in HR more than the increase in CBF.

Antiadrenergic and hemodynamic effects of ranolazine in conscious dogs.

Effects of ranolazine alone and in the presence of phenylephrine (PE) or isoproterenol (ISO) on hemodynamics, coronary blood flow and heart rate (HR) in the absence and presence of hexamethonium (a ganglionic blocker) were studied in conscious dogs. Ranolazine (0.4, 1.2, 3.6, and 6 mg/kg, intravenous) alone caused transient (<1 minute) and reversible hemodynamic changes. PE (0.3-10 µg/kg) caused a dose-dependent increase in blood pressure and decrease in HR. ISO (0.01-0.3 µg/kg) caused a dose-dependent decrease in blood pressure and an increase in HR. Ranolazine at high (11-13 mM), but not at moderate (4-5 mM) concentrations partially attenuated changes in mean arterial blood pressure and HR caused by either PE or ISO in normal conscious dogs. However, in dogs treated with hexamethonium (20 mg/kg) to cause autonomic blockade, ranolazine (both 4-5 and 11-13 µM) significantly attenuated both the PE- and ISO-induced changes in mean arterial blood pressure. The results suggest that a potential antiadrenergic effect of ranolazine was masked by autonomic control mechanisms in conscious dogs but could be observed when these mechanisms were inhibited (eg, in the hexamethonium-treated dog). Ranolazine, at plasma concentrations <10 µM and in conscious dogs with intact autonomic regulation, had minimal antiadrenergic (α and ß) effects.

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice.

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.

Late sodium current contributes to the reverse rate-dependent effect of IKr inhibition on ventricular repolarization.

BACKGROUND: The reverse rate dependence (RRD) of actions of I(Kr)-blocking drugs to increase the action potential duration (APD) and beat-to-beat variability of repolarization (BVR) of APD is proarrhythmic. We determined whether inhibition of endogenous, physiological late Na(+) current (late I(Na)) attenuates the RRD and proarrhythmic effect of I(Kr) inhibition. METHODS AND RESULTS: Duration of the monophasic APD (MAPD) was measured from female rabbit hearts paced at cycle lengths from 400 to 2000 milliseconds, and BVR was calculated. In the absence of a drug, duration of monophasic action potential at 90% completion of repolarization (MAPD(90)) and BVR increased as the cycle length was increased from 400 to 2000 milliseconds (n=36 and 26; P<0.01). Both E-4031 (20 nmol/L) and d-sotalol (10 µmol/L) increased MAPD(90) and BVR at all stimulation rates, and the increase was greater at slower than at faster pacing rates (n=19, 11, 12 and 7, respectively; P<0.01). Tetrodotoxin (1 µmol/L) and ranolazine significantly attenuated the RRD of MAPD(90,) reduced BVR (P<0.01), and abolished torsade de pointes in hearts treated with either 20 nmol/L E-4031 or 10 µmol/L d-sotalol. Endogenous late I(Na) in cardiomyocytes stimulated at cycle lengths from 500 to 4000 milliseconds was greater at slower than at faster stimulation rates, and rapidly decreased during the first several beats at faster but not at slower rates (n=8; P<0.01). In a computational model, simulated RRD of APD caused by E-4031 and d-sotalol was attenuated when late I(Na) was inhibited. CONCLUSION: Endogenous late I(Na) contributes to the RRD of I(Kr) inhibitor-induced increases in APD and BVR and to bradycardia-related ventricular arrhythmias.

Reducing the late sodium current improves cardiac function during sodium pump inhibition by ouabain.

Inhibition by cardiac glycosides of Na(+), K(+)-ATPase reduces sodium efflux from myocytes and may lead to Na(+) and Ca(2+) overload and detrimental effects on mechanical function, energy metabolism, and electrical activity. We hypothesized that inhibition of sodium persistent inward current (late I(Na)) would reduce ouabain's effect to cause cellular Na(+) loading and its detrimental metabolic (decrease of ATP) and functional (arrhythmias, contracture) effects. Therefore, we determined effects of ouabain on concentrations of intracellular sodium (Na(+)(i)) and high-energy phosphates using (23)Na and (31)P NMR, the amplitude of late I(Na) using the whole-cell patch-clamp technique, and contractility and electrical activity of guinea pig isolated hearts, papillary muscles, and ventricular myocytes in the absence and presence of inhibitors of late I(Na). Ouabain (1-1.3 µM) increased Na(+)(i) and late I(Na) of guinea pig isolated hearts and myocytes by 3.7- and 4.2-fold, respectively. The late I(Na) inhibitors ranolazine and tetrodotoxin significantly reduced ouabain-stimulated increases in Na(+)(i) and late I(Na). Reductions of ATP and phosphocreatine contents and increased diastolic tension in ouabain-treated hearts were also markedly attenuated by ranolazine. Furthermore, the ouabain-induced increase of late I(Na) was also attenuated by the Ca(2+)-calmodulin-dependent kinase I inhibitors KN-93 [N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide] and autocamide-2 related inhibitory peptide, but not by KN-92 [2-[N-(4'-methoxybenzenesulfonyl)]amino-N-(4'-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate]. We conclude that ouabain-induced Na(+) and Ca(2+) overload is ameliorated by the inhibition of late I(Na).

LQTS mutation N1325S in cardiac sodium channel gene SCN5A causes cardiomyocyte apoptosis, cardiac fibrosis and contractile dysfunction in mice.

OBJECTIVE: Mutations in the cardiac sodium channel gene SCN5A cause long QT syndrome (LQTS). We previously generated an LQTS mouse model (TG-NS) that overexpresses the LQTS mutation N1325S in SCN5A. The TG-NS mice manifested the clinical features of LQTS including spontaneous VT, syncope and sudden death. However, the long-term prognosis of LQTS on the structure of the heart has not been investigated in this or any other LQTS models and human patients. METHODS AND RESULTS: Impaired systolic function and reduced left ventricular fractional shortening were detected by echocardiography, morphological and histological examination in two lines of adult mutant transgenic mice. Histological and TUNEL analyses of heart sections revealed fibrosis lesions and increased apoptosis in an age-dependent manner. Cardiomyocyte apoptosis was associated with the increased activation of caspases 3 and 9 in TG-NS hearts. Western blot analysis showed a significantly increased expression of the key Ca(2+) handling proteins L-type Ca(2+) channel, RYR2 and NCX in TG-NS hearts. Increased apoptosis and an altered expression of Ca(2+) handling proteins could be detected as early as 3months of age when echocardiography showed little or no alterations in TG-NS mice. CONCLUSIONS: Our findings revealed for the first time that the LQTS mutation N1325S in SCN5A causes cardiac fibrosis and contractile dysfunction in mice, possibly through cellular mechanisms involving aberrant cardiomyocyte apoptosis. Therefore, we provide the experimental evidence supporting the notion that some LQTS patients have an increased risk of structural and functional cardiac damage in a prolonged disease course.

Sulfonylureas uncouple glucose-dependence for GPR40-mediated enhancement of insulin secretion from INS-1E cells.

Activation of GPR40 is reported to enhance insulin secretion in the presence of glucose. We determined whether sulfonylureas could replace glucose for GPR40-mediated enhancement of insulin secretion and investigated underlying mechanisms using INS-1E cells. GW9508, a specific agonist of GPR40, significantly enhanced insulin secretion in the presence of high concentrations of glucose. In contrast, sulfonylureas increased insulin secretion in the absence of glucose. In the presence of sulfonylureas, activation of GPR40 significantly enhanced insulin secretion. The L-type calcium channel (LTCC) activator S-(-)-Bay K8644 also concentration-dependently increased insulin secretion in the absence of glucose. In the presence of 10 micromol/L S-(-)-Bay K8644, GW9508 significantly increased insulin secretion. On the other hand, the LTCC blocker nifedipine significantly inhibited insulin secretion mediated by either glucose, glipizide or glucose plus GW9508. Thus, sulfonylureas could replace glucose to support GPR40-mediated enhancement of insulin secretion, whereas blockage of LTCC reduced both glucose and sulfonylurea-mediated insulin secretion.