Mindin Activates Autophagy for Lipid Utilization and Facilitates White Spot Syndrome Virus Infection in Shrimp.

Mindin is a secreted extracellular matrix protein that is involved in regulating cellular events through interacting with integrin. Studies have demonstrated its role in host immunity, including phagocytosis, cell migration, and cytokine production. However, the function of Mindin in the host-virus interaction is largely unknown. In the present study, we report that Mindin facilitates virus infection by activating lipid utilization in an arthropod, kuruma shrimp (Marsupenaeus japonicus). Shrimp Mindin facilitates white spot syndrome virus infection by facilitating viral entry and replication. By activating autophagy, Mindin induces lipid droplet consumption, the hydrolysis of triglycerides into free fatty acids, and ATP production, ultimately providing energy for virus infection. Moreover, integrin is essential for Mindin-mediated autophagy and lipid utilization. Therefore, by revealing the mechanism by which Mindin facilitates virus infection through regulating lipid metabolism, the present study reveals the significance of Mindin in the host-virus interaction. IMPORTANCE White spot syndrome virus (WSSV) is an enveloped double-stranded DNA virus that has had a serious influence on worldwide shrimp farming in the last 30 years. We have demonstrated that WSSV hijacks host autophagy and lipid metabolism for reproduction in kuruma shrimp (Marsupenaeus japonicus). These findings revealed the mechanism by which WSSV exploits host machinery for its infection and provided serial targets for WSSV prevention and control in shrimp farming.

Enhanced basal late sodium current appears to underlie the age-related prolongation of action potential duration in guinea pig ventricular myocytes.

Aging hearts have prolonged QT interval and are vulnerable to oxidative stress. Because the QT interval indirectly reflects the action potential duration (APD), we examined the hypotheses that 1) the APD of ventricular myocytes increases with age; 2) the age-related prolongation of APD is due to an enhancement of basal late Na+ current (INaL); and 3) inhibition of INaL may protect aging hearts from arrhythmogenic effects of hydrogen peroxide (H2O2). Experiments were performed on ventricular myocytes isolated from (young) 1-mo- and (old) 1-yr-old guinea pigs (GPs). The APD of myocytes from old GPs was significantly longer than that from young GPs and was shortened by the INaL inhibitors GS967 and tetrodotoxin. The magnitude of INaL was significantly larger in myocytes from old than from young GPs. The CaMKII inhibitors KN-93 and AIP and the NaV1.5-channel blocker methanethiosulfonate ethylammonium blocked the INaL. There were no significant differences between myocytes from young and old GPs in L-type Ca2+ current and the rapidly and slowly activating delayed rectifier K+ currents, although the inward rectifier K+ current was slightly decreased in myocytes from old GPs. H2O2 induced more early afterdepolarizations in myocytes from old than from young GPs. The effect of H2O2 was attenuated by GS967. The results suggest that 1) the APD of myocytes from old GPs is prolonged, 2) a CaMKII-mediated increase in NaV1.5-channel INaL is responsible for the prolongation of APD, and 3) inhibition of INaL may be beneficial for maintaining electrical stability under oxidative stress in myocytes of old GPs.NEW & NOTEWORTHY The action potential duration is significantly longer in ventricular myocytes from old than from young guinea pigs, which may explain, at the cellular level, the increase in QT interval with age. A CaMKII-mediated enhancement of NaV1.5-channel late current is responsible for the age-related prolongation of action potential duration. The enhanced basal late sodium current may predispose cardiac myocytes of old animals to oxidative stress and arrhythmogenesis.

Basal late sodium current is a significant contributor to the duration of action potential of guinea pig ventricular myocytes.

In cardiac myocytes, an enhancement of late sodium current (INaL) under pathological conditions is known to cause prolongation of action potential duration (APD). This study investigated the contribution of INaL under basal, physiological conditions to the APD Whole-cell INaL and the APD of ventricular myocytes isolated from healthy adult guinea pigs were measured at 36°C. The INaL inhibitor GS967 or TTX was applied to block INaL The amplitude of basal INaL and the APD at 50% repolarization in myocytes stimulated at a frequency of 0.17 Hz were -0.24 ± 0.02 pA/pF and 229 ± 6 msec, respectively. GS967 (0.01-1 µmol/L) concentration dependently reduced the basal INaL by 18 ± 3-82 ± 4%. At the same concentrations, GS967 shortened the APD by 9 ± 2 to 25 ± 1%. Similarly, TTX at 0.1-10 µmol/L decreased the basal INaL by 13 ± 1-94 ± 1% and APD by 8 ± 1-31 ± 2%. There was a close correlation (R2 = 0.958) between the percentage inhibition of INaL and the percentage shortening of APD caused by either GS967 or TTX MTSEA (methanethiosulfonate ethylammonium, 2 mmol/L), a NaV1.5 channel blocker, reduced the INaL by 90 ± 5%, suggesting that the NaV1.5 channel isoform is the major contributor to the basal INaL KN-93 (10 µmol/L) and AIP (2 µmol/L), blockers of CaMKII, moderately reduced the basal INaL Thus, this study provides strong evidence that basal endogenous INaL is a significant contributor to the APD of cardiac myocytes. In addition, the basal INaL of guinea pig ventricular myocytes is mainly generated from NaV1.5 channel isoform and is regulated by CaMKII.

Modulation of late sodium current by Ca2+ -calmodulin-dependent protein kinase II, protein kinase C and Ca2+ during hypoxia in rabbit ventricular myocytes.

NEW FINDINGS: What is the central question of this study? Hypoxia-induced increase in late sodium current (INa,L ) is associated with conditions causing cellular Ca2+ overload and contributes to arrhythmogenesis in the ventricular myocardium. The INa,L is an important drug target. We investigated intracellular signal transduction pathways involved in modulation of INa,L during hypoxia. What is the main finding and its importance? Hypoxia caused increases in INa,L , reverse Na+ -Ca2+ exchange current and diastolic [Ca2+ ], which were attenuated by inhibitors of Ca2+ -calmodulin-dependent protein kinase II (CaMKII) and protein kinase C and by a Ca2+ chelator. The findings suggest that CaMKII, protein kinase C and Ca2+ all participate in mediation of the effect of hypoxia to increase INa,L . Hypoxia leads to augmentation of the late sodium current (INa,L ) and cellular Na+ loading, increased reverse Na+ -Ca2+ exchange current (reverse INCX ) and intracellular Ca2+ loading in rabbit ventricular myocytes. The purpose of this study was to determine the intracellular signal transduction pathways involved in the modulation of INa,L during hypoxia in ventricular myocytes. Whole-cell and cell-attached patch-clamp techniques were used to record INa,L , and the whole-cell mode was also used to record reverse INCX and to study intercellular signal transduction mechanisms that mediate the increased INa,L . Dual excitation fluorescence photomultiplier systems were used to record the calcium transient in ventricular myocytes. Hypoxia caused increases of INa,L and reverse INCX . These increases were attenuated by KN-93 (an inhibitor of Ca2+ -calmodulin-dependent protein kinase II), bisindolylmaleimide VI (BIM; an inhibitor of protein kinase C) and BAPTA AM (a Ca2+ chelator). KN-93, BIM and BAPTA AM had no effect on INa,L in normoxia. In studies of KN-93, hypoxia alone increased the density of INa,L from -0.31 ± 0.02 to -0.66 ± 0.03 pA pF-1 (n = 6, P < 0.01 versus control) and the density of reverse INCX from 1.02 ± 0.06 to 1.91 ± 0.20 pA pF-1 (n = 7, P < 0.01 versus control) in rabbit ventricular myocytes. In the presence of 1 µm KN-93, the densities of INa,L and reverse INCX during hypoxia were significantly attenuated to -0.44 ± 0.03 (n = 6, P < 0.01 versus hypoxia) and 1.36 ± 0.15 pA pF-1 (n = 7, P < 0.01 versus hypoxia), respectively. In studies of BIM, hypoxia increased INa,L from -0.30 ± 0.03 to -0.60 ± 0.03 pA pF-1 (n = 6, P < 0.01 versus control) and reverse INCX from 0.91 ± 0.10 to 1.71 ± 0.27 pA pF-1 (n = 6, P < 0.01 versus control). In the presence of 1 µm BIM, the densities of INa,L and reverse INCX during hypoxia were significantly attenuated to -0.48 ± 0.02 (n = 6, P < 0.01 versus hypoxia) and 1.33 ± 0.21 pA pF-1 (n = 6, P < 0.01 versus hypoxia), respectively. In studies of BAPTA AM, hypoxia increased INa,L from -0.26 ± 0.04 to -0.63 ± 0.05 pA pF-1 (n = 6, P < 0.01 versus control) and reverse INCX from 0.86 ± 0.09 to 1.68 ± 0.35 pA pF-1 (n = 6, P < 0.01 versus control). The effects of hypoxia on INa,L and reverse INCX were significantly attenuated in the presence of 1 mm BAPTA AM to -0.39 ± 0.02 (n = 6, P < 0.01 versus hypoxia) and 1.12 ± 0.27 pA pF-1 (n = 6, P < 0.01 versus hypoxia), respectively. Results of single-channel studies showed that hypoxia apparently increased the mean open probability and mean open time of sodium channels. These effects were inhibited by either 1 µm KN-93 or 1 mm BAPTA AM. The suppressant effects of drug interventions were reversed upon washout. In addition, KN-93, BIM and BAPTA AM also reversed the hypoxia-enhanced diastolic Ca2+ concentration and the attenuated amplitude of the [Ca2+ ]i transient, maximal velocities of Ca2+ increase and Ca2+ decay. In summary, the findings suggest that Ca2+ -calmodulin-dependent protein kinase II, protein kinase C and Ca2+ all participate in mediation of the effect of hypoxia to increase INa,L .

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.

A computational modelling approach combined with cellular electrophysiology data provides insights into the therapeutic benefit of targeting the late Na+ current.

KEY POINTS: The ventricular action potential plateau is a phase of high resistance, which makes ventricular myocytes vulnerable to small electrical perturbations. We developed a computationally based model of GS-458967 interaction with the cardiac Na+ channel, informed by experimental data recorded from guinea pig isolated single ventricular myocytes. The model predicts that the therapeutic potential of GS-458967 derives largely from the designed property of significant potent selectivity for INaL. ABSTRACT: Selective inhibition of the slowly inactivating or late Na(+) current (INaL) in patients with inherited or acquired arrhythmia syndrome may confer therapeutic benefit by reducing the incidence of triggers for arrhythmia and suppressing one component of arrhythmia-promoting cardiac substrates (e.g. prolonged refractoriness and spatiotemporal dispersion of action potential duration). Recently, a novel compound that preferentially and potently reduces INaL, GS-458967 (IC50 for block of INaL = 130 nM) has been studied. Experimental measurements of the effects of GS-458967 on endogenous INaL in guinea pig ventricular myocytes demonstrate a robust concentration-dependent reduction in action potential duration (APD). Using experimental data to calibrate INaL and the rapidly activating delayed rectifier K(+) current, IKr, in the Faber-Rudy computationally based model of the guinea pig ventricular action potential, we simulated effects of GS-458967 on guinea pig ventricular APD. GS-458967 (0.1 µM) caused a 28.67% block of INaL and 12.57% APD shortening in experiments, while the model predicted 10.06% APD shortening with 29.33% block of INaL. An additional effect of INaL block is to reduce the time during which the membrane potential is in a high resistance state (i.e. the action potential plateau). To test the hypothesis that targeted block of INaL would make ventricular myocytes less susceptible to small electrical perturbations, we used the computational model to test the degree of APD prolongation induced by small electrical perturbations in normal cells and in cells with simulated long QT syndrome. The model predicted a substantial dose-dependent reduction in sensitivity to small electrical perturbations as evidenced by action potential duration at 90% repolarization variability in the presence of GS-458967-induced INaL block. This effect was especially potent in the 'disease setting' of inherited long QT syndrome. Using a combined experimental and theoretical approach, our results suggest that INaL block is a potent therapeutic strategy. This is because reduction of INaL stabilizes the action potential waveform by reducing depolarizing current during the plateau phase of the action potential. This reduces the most vulnerable phase of the action potential with high membrane resistance. In summary, by reducing the sensitivity of the myocardial substrate to small electrical perturbations that promote arrhythmia triggers, agents such as GS-458967 may constitute an effective antiarrhythmic pharmacological strategy.

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.

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.

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).

A slowly inactivating sodium current contributes to spontaneous diastolic depolarization of atrial myocytes.

Diastolic depolarization (DD) of atrial myocytes can lead to spontaneous action potentials (APs) and, potentially, atrial tachyarrhythmias. This study examined the hypotheses that 1) a slowly inactivating component of the Na(+) current (referred to as late I(Na)) may contribute to DD and initiate AP firing and that 2) blocking late I(Na) will reduce spontaneous and induced firing of APs by atrial myocytes. Guinea pig atrial myocytes without or with DD and spontaneous AP firing were studied using the whole cell patch-clamp technique. In experiments using cells with a stable resting membrane potential (no spontaneous DD or firing), hydrogen peroxide (H(2)O(2), 50 micromol/l) caused DD and AP firing. The H(2)O(2)-induced activity was suppressed by the late I(Na) inhibitors tetrodotoxin (TTX, 1 micromol/l) and ranolazine (5 micromol/l). In cells with DD but no spontaneous APs, the late I(Na) enhancer anemone toxin II (ATX-II, 10 nmol/l) accelerated DD and induced APs. In cells with DD and spontaneous AP firing, TTX and ranolazine (both, 1 micromol/l) significantly reduced the slope of DD by 81 +/- 12% and 75 +/- 11% and the frequency of spontaneous firing by 70 +/- 15% and 74 +/- 9%, respectively. Ramp voltage-clamp simulating DD elicited a slow inward current. TTX at 1, 3, and 10 micromol/l inhibited this current by 41 +/- 4%, 73 +/- 2%, and 91 +/- 1%, respectively, suggesting that a slowly inactivating I(Na) underlies the DD. ATX-II and H(2)O(2) increased the amplitude of this current, and the effects of ATX-II and H(2)O(2) were attenuated by ranolazine or TTX. In conclusion, late I(Na) can contribute to the DD of atrial myocytes and the inhibition of this current suppresses atrial DD and spontaneous APs.

The late Na+ current (INa) inhibitor ranolazine attenuates effects of palmitoyl-L-carnitine to increase late INa and cause ventricular diastolic dysfunction.

Palmitoyl-L-carnitine (PC), an ischemic metabolite, causes cellular Na(+) and Ca(2+) overload and cardiac dysfunction. This study determined whether ranolazine [(+/-)-1-piperazineacetamide, N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-] attenuates PC-induced Na(+) current and ventricular contractile dysfunction of the isolated heart. PC (4 microM, 30 min) increased late Na(+) current by 1034 +/- 349% in guinea pig isolated ventricular myocytes; ranolazine (10 microM) and tetrodotoxin (TTX, 3 microM) significantly attenuated this effect of PC. PC increased left ventricular end-diastolic pressure (LVEDP), coronary perfusion pressure (CPP), wall stiffness, and cardiac lactate and adenosine release from the isolated heart. Ranolazine (10 microM) significantly reduced the PC-induced increase in LVEDP by 72 +/- 6% (n = 6, p < 0.001), reduced left ventricular wall stiffness, and attenuated the PC-induced increase of CPP by 53 +/- 10% (n = 6-7, p < 0.05). Ranolazine (10 microM) reduced the PC-induced increases of lactate and adenosine release by 70 +/- 8 and 81 +/- 5%, respectively (n = 6, p

Contribution of frequency-augmented inward Ca2+ current to myocardial contractility.

The sarcoplasmic reticular Ca2+ pump (SERCA) is thought to be the primary determinant of heart rate-dependent increases in myocardial contractile [Ca2+]i and force (force-frequency relationship (FFR)), an important mechanism to increase cardiac output. This report demonstrates a rate-dependent role for inward Ca2+ current (ICa) in the human and rat FFR. Human action potential plateau height increased linearly with contractility when heart rate increased in vivo, as measured by monophasic action potential catheter and echocardiography. Rat rate-dependent developed force and cytosolic [Ca2+]i transients were quantified in isolated left ventricular papillary muscles, and ICa and action potential duration in cardiomyocytes. ICa and SERCA measurements better reflected [Ca2+]i and force transients than SERCA activity alone. These data support a direct and (or) indirect contribution to myocardial contractility by ICa at heart rates from approximately 1 to 3-4 Hz (60 to 180-240 bpm) in tandem with SERCA to sustain the typical 'bell shape' of the FFR across species.

An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes.

This study determined the role of a slowly inactivating component of sodium current (I(Na)), late I(Na), to induce delayed afterdepolarizations (DADs) and triggered activity. We hypothesized that an increase of late I(Na) may induce not only early afterdepolarizations (EADs), but also intracellular calcium overload and DADs. Guinea pig atrial myocytes were studied using the whole cell patch-clamp technique. Anemone toxin II (ATX-II) (5-10 nmol/l) was used to enhance late I(Na). Ranolazine (10 micromol/l) and TTX (2 micromol/l) were applied to block ATX-II-induced late I(Na). ATX-II prolonged action potential duration and induced EADs. In the continuous presence of ATX-II, following the appearance of EADs, both DADs and sustained triggered activity occurred. Triggered activity was abolished and DADs were reduced by either ranolazine or TTX. Consistent with induction of DADs, ATX-II induced the transient inward current (I(TI)). The amplitude of I(TI) was significantly reduced by ranolazine. ATX-II induced only EADs, but no DADs, in the presence of the sodium-calcium exchange inhibitor KB-R7943 or the sarcoplasmic reticulum calcium release channel inhibitor ryanodine, or when the calcium chelator EGTA or BAPTA was included in the pipette solution. In conclusion, an increase of late I(Na), in addition to inducing EADs, can cause cellular calcium overload and induce DADs and sustained triggered activity in atrial myocytes. The data reveal that an increase of late I(Na) is a novel mechanism for initiation of atrial arrhythmic activity.

Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction.

Reactive oxygen species (ROS), including H2O2, cause intracellular calcium overload and ischemia-reperfusion damage. The objective of this study was to examine the hypothesis that H2O2-induced arrhythmic activity and contractile dysfunction are the results of an effect of H2O2 to increase the magnitude of the late sodium current (late INa). Guinea pig and rabbit isolated ventricular myocytes were exposed to 200 microM H2O2. Transmembrane voltages and currents and twitch shortening were measured using the whole-cell patch-clamp technique and video edge detection, respectively. [Na+]i and [Ca2+]i were determined by fluorescence measurements. H2O2 caused a persistent late INa that was almost completely inhibited by 10 microM tetrodotoxin (TTX). H2O2 prolonged the action potential duration (APD), slowed the relaxation rate of cell contraction, and induced early afterdepolarizations (EADs) and aftercontractions. H2O2 also caused increases of [Na+]i and [Ca2+]i. Ranolazine (10 microM), a novel inhibitor of late INa, attenuated H2O2-induced late INa by 51+/-9%. TTX (2 microM) or 10 microM ranolazine attenuated H2O2-induced APD prolongation and suppressed EADs. Ranolazine accelerated the twitch relaxation rate in the presence of H2O2 and abolished H2O2-induced aftercontractions. Pretreatment of myocytes with ranolazine delayed and reduced the increases of APD, [Na+]i, and [Ca2+]i caused by H2O2. In conclusion, the results confirm the hypothesis that an increase in late INa during exposure of ventricular myocytes to H2O2 contributes to electrical and contractile dysfunction and suggest that inhibition of late INa may offer protection against ROS-induced Na+ and Ca2+ overload.

An increase in late sodium current potentiates the proarrhythmic activities of low-risk QT-prolonging drugs in female rabbit hearts.

Assessment of the proarrhythmic risk associated with drugs that prolong the QT interval is difficult. We hypothesized that the proarrhythmic activities of drugs with very low to moderate risk of causing torsades de pointes would be well differentiated when the late sodium current (I(NaL)) was greater than normal. The effects of selected QT-prolonging drugs on electrical activity of female rabbit isolated hearts were determined in the absence and presence of sea anemone toxin (ATX-II; an enhancer of I(NaL)). I(NaL) recorded from ventricular myocytes isolated from female rabbit hearts was slightly increased by 1 and 3 nM ATX-II (n = 13, P < 0.01). ATX-II (1 nM) prolonged the duration of the monophasic action potential (MAPD(90)) the isolated heart by of 19 +/- 3% (P < 0.001, n = 31) and shifted the concentration-response relationships for cisapride (1-30 nM), ziprasidone (0.01-3 microM), quinidine (0.1-1 microM), and moxifloxacin (0.01-1 microM) to prolong MAPD to the left by 2- to 12-fold. In contrast, the increases in MAPD(90) caused by 1 nM ATX-II and pentobarbital were only additive, and the increases in MAPD(90) caused by ATX-II and ranolazine [(+/-)-N-(2,6-dimethylphenyl)-(4[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1-piperazine] were less than additive. Episodes of arrhythmic activity were commonly observed, and beat-to-beat variability of action potential duration was increased, during exposure of hearts to cisapride, ziprasidone, quinidine, and moxifloxacin but not during exposure of hearts to ranolazine or pentobarbital, in the presence of ATX-II. Thus, in the female rabbit heart, ATX-II potentiated the effects of QT-prolonging drugs to increase MAPD(90) and unmasked the proarrhythmic activities of these drugs at clinically relevant drug concentrations.

Use of preclinical assays to predict risk of drug-induced torsades de pointes.

Numerous medications, including drugs prescribed for noncardiac indications, can induce electrophysiologic changes that trigger the rare, malignant polymorphic ventricular tachyarrhythmia known as torsades de pointes (TdP). Although the exact relationship between electrophysiologic events and the development of TdP is not defined, prolongation of the QT interval and inhibition of the rapidly activating potassium current I(Kr) by drugs may be associated with an increased risk of TdP. The ability of a drug to reduce I(Kr) and prolong the QT interval often is considered to predict the likelihood that the drug will cause TdP in humans. However, these surrogate measures of the drug-induced risk of causing TdP, and therefore of drug safety, now are recognized to be imperfect predictors of drug safety. New preclinical models should be used to assess drug risk, including preparations, conditions, and measurements used by basic research scientists to produce ventricular polymorphic arrhythmias in the laboratory. In this review, we discuss the task of assessing the arrhythmogenic potential of a drug. Assays of drug effect to induce early afterdepolarizations and ectopic beats and/or to increase the dispersion of ventricular repolarization when "repolarization reserve" is reduced appear to be the best predictors of the drug-induced risk of TdP. Current experimental models and protocols, especially those using conditions wherein the net repolarizing current is reduced, can detect the potential for a drug to induce TdP, even when the potential is extremely low.

A mechanistic approach to assess the proarrhythmic risk of QT-prolonging drugs in preclinical pharmacologic studies.

Drugs with diverse structures and from several therapeutic classes are reported to increase the risk that a patient will experience ventricular tachyarrhythmias (e.g., torsades de pointes [TdP]) during drug therapy. This review discusses the use of preclinical assays to assess the risk that a QT-prolonging drug will cause TdP. The mechanisms underlying the development of TdP and the factors that increase the risk of TdP are described and applied to the design of preclinical experimental models for detection of proarrhythmic drug actions. Recommended assays, conditions, and preparations for preclinical assessment of the drug-induced risk to TdP are given. No single preparation can simulate all conditions that cause TdP in patients. However, the assays described herein are capable of detecting the proarrhythmic effects of currently used drugs, even when these effects are reported to be extremely rare in clinical practice.

Antagonism by ranolazine of the pro-arrhythmic effects of increasing late INa in guinea pig ventricular myocytes.

The new anti-anginal drug ranolazine causes a slight (<10 milliseconds) prolongation of the QT interval, raising the concern that its use may be associated with an increased incidence of torsades de pointes ventricular tachyarrhythmias. The goal of this study was to show that ranolazine inhibits the late component of INa and attenuates prolongation of action potential duration when late INa is increased, both in the absence and presence of IK-blocking drugs. Currents and action potentials of guinea pig isolated ventricular myocytes were measured by whole-cell patch clamp. Sea anemone toxin (ATX)-II was used to increase late INa and mimic the effect of an SCN5A gene mutation. ATX-II (3-5 nmol/L) increased late INa by 5-fold; ranolazine attenuated this increase of late INa by up to 61 +/- 8%. ATX-II (10-20 nmol/L) increased action potential duration (APD) by > 1 seconds, and caused early afterdepolarizations; both actions were attenuated by ranolazine (0.1-30 micromol/L). Ranolazine (10 micromol/L) reduced by 89% the 13.6-fold increase in variability of APD caused by 10 nmol/L ATX-II. The effects of ATX-II (3 nmol/L) in combinations with either the IKr blocker E-4031 or the IKs blocker chromanol 293B to increase APD were attenuated 76 +/- 5% and 71 +/- 4%, respectively, by 10 micromol/L ranolazine. The results demonstrate that ranolazine reduces late INa and has an anti-arrhythmic effect when late INa is increased.