Regional and frequency-dependent changes in action potentials and transient outward K<Superscript>+</Superscript> currents in ventricular myocytes from J-2-K cardiomyopathic hamsters

Abstract. Objectives Although lengthening of action potential duration (APD) and decreased transient outward K⁺ currents (I_to) have been observed in ventricular myocytes from cardiomyopathic hamsters, epi- and endo-cardial differences in I_to and their roles in frequency-dependent changes in APD have not been claried. Methods The patch-clamp technique of whole-cell conguration was used to record membrane potentials and currents in epicardial and endocardial myocytes of the J-2 hamster germline without (control) and with cardiomyopathy (CM). Results In control, APD in endocardial myocytes was longer than that in epicardial myocytes at 0.1 Hz. APD significantly lengthened with increased frequencies of stimulation from 0.1 to 6.0 Hz in both groups with the longer APD in endocardial myocytes. In CM, APD lengthened in epicardial myocytes exceeding the endocardial APD without a frequency-dependent prolongation. Pretreatment with 4 mM 4-aminopyridine completely abolished the frequency-dependent changes and abolished APD differences between epicardial and endocardial myocytes, and between control and CM hamsters. The transient outward K⁺ current (I_to) significantly decreased in epicardial myocytes from CM hamsters compared with that of control (17.5 ± 1.5 pA/pF in control vs. 9.5 ± 2.5 pA/pF in CM at +60 mV) with altered recovery from inactivation, without changes in the endocardial I_to. Moreover, the inward rectifier K⁺ current decreased in epicardial myocytes from CM hamsters and the L-type Ca2⁺ current reduced in both regions from CM compared to control. Conclusion Results indicate that differences in APD between epi- and endocardial myocytes in CM hamsters are mainly caused by a decreased current density and altered recovery from inactivation of I_to in epicardial myocytes.     

Unloaded rat hearts in vivo express a hypertrophic phenotype of cardiac repolarization

Cardiac unloading with left ventricular assist devices is increasingly used to treat patients with severe heart failure. Unloading has been shown to improve systolic and diastolic function, but its impact on the repolarization of left ventricular myocytes is not known. Unloaded hearts exhibit similar patterns of gene expression as hearts subjected to an increased hemodynamic load. We therefore hypothesized that cardiac unloading also replicates the alterations in action potential and underlying repolarizing ionic currents found in pressure-overload induced cardiac hypertrophy. Left ventricular unloading was induced by heterotopic heart transplantation in syngenic male Lewis rats. Action potentials and underlying K⁺ and Ca²⁺ currents were investigated using whole-cell patch-clamp technique. Real-time RT-PCR was used to quantify mRNA expression of Kv4.2, Kv4.3, and KChIP2. Unloading markedly prolonged cardiac action potentials and suppressed the amplitude of several repolarizing K⁺ currents, in particular of the transient outward K⁺ current I_to, in both, epicardial and endocardial myocytes. The reduction of I_to was associated with significantly lower levels of Kv4.2 and Kv4.3 mRNAs in epicardial myocytes, and of KChIP2 mRNA in endocardial myocytes. Concomitantly, the L-type Ca²⁺ current was increased in myocytes of unloaded hearts. Collectively, these results show that left ventricular unloading induces a profound remodelling of cardiac repolarization with action potential prolongation, downregulation of repolarizing K⁺ currents and upregulation of the L-type Ca²⁺ current. This indicates that unloaded rat hearts in vivo express a hypertrophic phenotype of cardiac repolarization at the cellular and the molecular level.     

Ventricular K currents are reduced in mice with elevated levels of serum TNFα

In the present study mice were treated with tumor necrosis factor alpha (TNFα) for 6 weeks to determine if chronic TNFα treatment could produce serum levels of TNFα similar to what has been observed in disease states (heart failure, HIV) and to determine if these levels of TNFα alter ventricular K⁺ currents. Mice chronically treated with TNFα and sham treated mice were utilized for experiments. Serum levels were measured with a Searchlight® protein array. Patch-clamp techniques, real-time PCR and Western blot analysis were used to study K⁺ current densities and K⁺ channel expression. Results showed that serum concentrations of TNFα were significantly higher in TNFα treated mice compared to controls (control: 9.5 ± 1.5 pg/ml, TNFα: 27.4 ± 5.0 pg/ml; p < 0.05) and comparable to serum TNFα levels observed in heart failure and HIV models. In ventricular myocytes from TNFα treated mice the outward K⁺ currents I_to and I_Kur were significantly reduced (at + 30 mV: I_to: control: 45.0 ± 2.9 pA/pF, TNFα: 34.5 ± 2.9 pA/pF; p < 0.05; I_Kur: control 34.1 ± 2.7 pA/pF, TNFα: 25.0 ± 2.2 pA/pF; p < 0.05). Expression studies revealed that ventricular mRNA and protein expression for the channels underlying I_to and I_Kur did not differ between the two groups. However, the recovery from inactivation for I_Kur was significantly longer in TNFα treated mice. Overall, this study shows that pathologically relevant levels of serum TNFα modulate K⁺ currents in mouse ventricle. These findings could help to explain the role of TNFα in the pathogenesis of cardiac arrhythmia.     

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.     

Oxidoreductase regulation of Kv currents in rat ventricle

Oxidative stress contributes to the arrhythmogenic substrate created by myocardial ischemia–reperfusion partly through a shift in cell redox state, a key modulator of protein function. The activity of many oxidation-sensitive proteins is controlled by oxidoreductase systems that regulate the redox state of cysteine thiol groups, but the impact of these systems on ion channel function is not well defined. Thus, we examined the roles of the thioredoxin and glutaredoxin systems in controlling K⁺ channels in the ventricle. An oxidative shift in redox state was elicited in isolated rat ventricular myocytes by brief exposure to diamide, a thiol-specific, membrane-permeable oxidant. Voltage-clamp studies showed that diamide decreased peak outward K⁺ current (I_peak) evoked by depolarizing test pulses by 41% (+ 60 mV; p < 0.05) while steady-state outward current (I_ss) measured at the end of the test pulse was decreased by 45% (p < 0.05). These electrophysiological effects were not prevented by protein kinase C blockers, but the tyrosine kinase inhibitors genistein or lavendustin A blocked the suppression of both K⁺ currents by diamide. Moreover, inhibition of I_peak and I_ss by diamide was reversed by dichloroacetate and an insulin-mimetic. The effect of dichloroacetate to normalize I_peak after diamide was blocked by the thioredoxin system inhibitors auranofin or 13-cis-retinoic acid, but I_ss was not affected by either compound. A pan-specific inhibitor of glutaredoxin and thioredoxin systems, 1,3-bis-(2-chloroethyl)-1-nitrosourea, also blocked the dichloroacetate effect on I_peak but only partially inhibited the recovery of I_ss. These data suggest that acute regulation of cardiac K⁺ channels by oxidoreductase systems is mediated by redox-sensitive tyrosine kinase/phosphatase pathways. The pathways controlling I_peak channels are targets of the thioredoxin system whereas those regulating I_ss channels are likely controlled by the glutaredoxin system. Thus, cardiac oxidoreductase systems may be important regulators of ion channels affected by pathogenic oxidative stress.     

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.     

Role of K+ATP Channels in Ischemic Preconditioning and Cardioprotection

Summary. Since the phenomenon of ischemic preconditioning was first described some 15 years ago, interest in strategies aimed at reducing infarct size has increased. During the past 10 years, investigations into the mechanism of ischemic preconditioning have clearly demonstrated the cardioprotective effect of K⁺ _ATP channel opening. Thus, K⁺ _ATP channel activation has been shown to be involved in this protection by a variety of stimuli, including a brief period of complete ischemia (classic ischemic preconditioning) or a partial coronary artery occlusion. In addition, ischemia in remote organs and nonischemic stimuli in the heart such as ventricular pacing, stretch, and heat stress also confer protection via K⁺ _ATP channel activation. Pharmacological agents that open K⁺ _ATP channels reduce infarct size, but K⁺ _ATP channel opening must occur prior to or early during the sustained infarct-producing coronary artery occlusion, while the degree and memory of cardioprotection are less than those produced by classic ischemic preconditioning. Although the exact mechanism by which K⁺ _ATP channel activation protects is still incompletely understood, recent studies indicate a role for the mitochondrial K⁺ _ATP channels. Before K⁺ _ATP channel opening can be employed in patients at increased risk of developing myocardial infarction (e.g., unstable angina), it is mandatory to determine whether tolerance (tachyphylaxia) occurs with repeated administration of K⁺ _ATP channel openers in a fashion similar to what occurs with ischemic preconditioning.     

Differential effects of the transient outward K current activator NS5806 in the canine left ventricle

To examine the electrophysiological and molecular properties of the transient outward current (I_to) in canine left ventricle using a novel I_to activator, NS5806, I_to was measured in isolated epicardial (Epi), midmyocardial (Mid) and endocardial (Endo) cells using whole-cell patch-clamp techniques. NS5806 activation of K_v4.3 current was also studied in CHO-K1 cells and Xenopus laevis oocytes. In CHO-K1 cells co-transfected with K_v4.3 and KChIP2, NS5806 (10 μM) caused a 35% increase in current amplitude and a marked slowing of current decay with τ increasing from 7.0 ± 0.4 to 10.2 ± 0.3 ms. In the absence of KChIP2, current decay was unaffected by NS5806. In ventricular myocytes, NS5806 increased I_to density by 80%, 82%, and 16% in Epi, Mid, and Endo myocytes, respectively (at + 40 mV) and shifted steady-state inactivation to negative potentials. NS5806 also significantly slowed decay of I_to, increasing total charge to 227%, 192% and 83% of control in Epi, Mid and Endo cells, respectively (+ 40 mV, p < 0.05). Quantification of K_v4.3 and KChIP2 mRNA in the 3 ventricular cell types revealed that levels of K_v4.3 message was uniform but those of KChIP2 were significantly greater in Epi and Mid cells. The KChIP2 gradient was confirmed at the protein level by Western blot. Our results suggest that NS5806 augments I_to by increasing current density and slowing decay and that both depend on the presence of KChIP2. I_to and its augmentation by NS5806 are greatest in Epi and Mid cells because KChIP2 levels are highest in these cell types.     

KB130015, A New Amiodarone Derivative with Multiple Effects on Cardiac Ion Channels

KB130015 (KB015), a new drug structurally related to amiodarone, has been proposed to have antiarrhythmic properties. In contrast to amiodarone, KB015 markedly slows the kinetics of inactivation of Na⁺ channels by enhancing concentration‐dependently (K_0.5# 2 μM) a slow‐inactivating I_Na component (#_slow# 50 ms) at the expense of the normal, fast‐inactivating component (#_fast# 2 to 3 ms). However, like amiodarone, KB015 slows the recovery from inactivation and causes a shift (K_0.5. # 6.9 μM) of the steady‐state voltage‐dependent inactivation to more negative potentials. Despite prolonging the opening of Na⁺ channels KB015 does not lengthen but often shortens the action potential duration (APD) in pig myocytes or in multicellular preparations. Only short APDs in mouse are markedly prolonged by KB015, which frequently induces early afterdepolarizations. KB015 has also an effect on other ion channels. It decreases the amplitude of the L‐type Ca²⁺ current (I^Ca‐L) without changing its time course, and it inhibits G‐protein gated and ATP‐gated K⁺ channels. Both the receptor‐activated I^k(ACH) (induced in atrial myocytes by either ACh, adenosine or sphingosylphosphorylcholine) and the receptor‐independent (GTPγS‐induced or background) I^k(ACH) are concentration‐dependently (K_0.5# 0.6 – 0.9 μM) inhibited by KB015. I^k(ATP), induced in atrial myocytes during metabolic inhibition with 2,4‐dinitrophenol (DNP), is equally suppressed. However, KB015 has no effect on I_K1 or on I_to. Consistent with the effects in K⁺ currents, KB015 does not depolarize the resting potential but antagonizes the APD shortening by muscarinic receptor activation or by DNP. Intracellular cell dialysis with KB015 has marginal or no effect on Na⁺ or K⁺ channels and does not prevent the effect of extracellularly applied drug, suggesting that KB015 interacts directly with channels at sites more easily accessible from the extracellular than the intracellular side of the membrane. At high concentrations KB015 exerts a positive inotropic action. It also interacts with thyroid hormone nuclear receptors. Its toxic effects remain largely unexplored, but it is well tolerated during chronic administration.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

Molecular and functional remodeling of Ito by angiotensin II in the mouse left ventricle

The transient outward potassium current (I_to) in cardiac myocytes is mainly mediated by members of the Kv4 subfamily of voltage-gated potassium channels. Several in vitro studies have shown that angiotensin II (Ang II), which plays an important role in the development of cardiac hypertrophy, rapidly downregulates Kv4.3 mRNA expression. However, it is not clear whether Ang II regulates I_toin vivo and whether this regulation may depend on alterations in Kv4.3 gene expression. To address this question, we determined the effects of acute (24 h) and chronic (14 days) exogenous infusions of Ang II on I_to and the expression of its channel subunits in the mouse left ventricle. Ang II rapidly increased blood pressure and reduced Kv4.2 but not Kv4.3 mRNA levels in the absence of cardiac hypertrophy. In response to chronically elevated Ang II levels cardiac hypertrophy developed, which was associated with a downregulation of Kv4.2 and Kv4.3 mRNA levels, and an upregulation of Kv1.4 mRNA levels. In contrast, neither KChIP2 mRNA levels nor amplitude or macroscopic inactivation kinetics of I_to were affected by the acute or chronic Ang II treatments. Consistent with the unchanged I_to amplitude, Kv4.2, Kv4.3, and KChIP protein expression levels were similar after chronic Ang II and sham treatment. Our findings demonstrate that elevations of Ang II concentrations that induce hypertension and cardiac hypertrophy do not alter the amplitude of I_to in the mouse left ventricle. Furthermore, they suggest that functional expression of cardiac I_to in mice is stabilized by KChIP2.     

Calcitonin gene-related peptide activates the K+ channels of vascular smooth muscle cells via adenylate cyclase

The aim of the present study was to examine the effects of calcitonin gene-related peptide (CGRP) on the K⁺ channels of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 nM CGRP activated two types of K⁺ channels the Ca²⁺-activated K⁺ channel (K_Ca channel) and the ATP-sensitive K⁺ channel (K_ATP channel) in cell-attached patch configurations. In cells pretreated with Rp-cAMPS, a membrane-permeable inhibitor of cAMP-dependent protein kinase (PKA), extracellular application of 100 nM CGRP could not activate the K_Ca or K_ATP channel, indicating that the activation of the K⁺ channels by CGRP occurs in connection with PKA. In the cell-attached patch configurations, extracellular application of 1 mM dibutyryl cAMP, a membrane permeable cAMP, activated K_Ca and K_ATP channels. In inside-out patch configurations, application of PKA to the cytosolic side activated both the K_Ca and K_ATP channels. These results indicate that CGRP modulates the K⁺ channels of vascular smooth muscle cells via adenylate cyclase, i.e., cAMP-PKA pathway, and contributes to control of vascular tone.     

Constitutive CaMKII activity regulates Na channel in rat ventricular myocytes

The cardiac voltage-gated Na⁺ channel controls the upstroke of action potential and membrane excitability. The Na⁺ channel associates with Ca²⁺/CaM-dependent protein kinase (CaMKII), but the role of CaMKII on Na⁺ channel activity in the resting state is not clear. In this report, we investigated whether CaMKII constitutively regulates Na⁺ currents (I_Na), independent of Ca²⁺ influx in rat ventricular myocytes using patch clamp technique. CaMKII inhibition (by KN93 or autocamtide-related inhibitory peptide) caused a negative shift in I_Na steady-state inactivation and delayed recovery from slow inactivation, limiting channel availability. The reduction of I_Na was 29.47 ± 3.01% at a holding potential (V_h) of − 120 mV and it increased to 77.70 ± 7.92% when V_h was − 70 mV, suggesting that near the resting membrane potential, three-quarters of I_Na depends on CaMKII action. CaMKII inhibition also enhanced intermediate inactivation, as well as delayed recovery from fast inactivation, and decreased late I_Na. KN92, an inactive analog of KN93, had no effect on I_Na. Using an antibody against phosphorylated (activated) CaMKII, we found that constitutively active CaMKII co-immunoprecipitated with Na⁺ channels under resting conditions. CaMKII inhibitors reduced the level of phosphorylated CaMKII, which correlated with the degree of reduction in channel availability. These data suggest that CaMKII in an active form contributes to regulating I_Na. Finally, we observed a drastic reduction in the upstroke velocity of action potentials upon CaMKII inhibition. In conclusion, CaMKII constitutively regulates cardiac Na⁺ channel and this regulatory mechanism is important for the maintenance of Na⁺ channel characteristics under physiological conditions.     

Thyroid stimulating hormone directly modulates cardiac electrical activity

BackgroundThe electrocardiogram of hypothyroid patients shows a series of abnormalities of cardiac repolarization due to a reduction of some repolarizing K⁺ currents and an increase of the L-type calcium current. Experimental and clinical works call into question the unique role of T3 and T4 in these mechanisms and correlate increased serum TSH levels with the repolarization abnormalities in patients with both subclinical and overt hypothyroidism. In this context, the aim of the present study was to investigate the direct effects of TSH upon cardiac electrical properties.MethodsThe action potential recording and the ion channel subunits mRNA expression were obtained from left ventricle of adult rats. Additionally, the repolarizing K⁺ currents and the L-type Ca²⁺ current (ICa-L) were recorded in isolated rat adult ventricular myocytes by the patch-clamp technique.Results24 h exposure to TSH lengthened the action potential and slightly depolarized the resting membrane potential. TSH- receptor activation causes a reduction of the amplitude of I_to and I_K1 currents caused by a reduction in channels expression. However, TSH had no effect on I_Ca-L, I_K or I_Kur.ConclusionThese results support the idea that some of the electrical disturbances seen in hypothyroid hearts, such as the I_to and I_K1 current reduction, could be caused not by low T3 but by the elevation of circulating TSH.     

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

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

Lysophosphatidylcholine enhances I(Ks) currents in cardiac myocytes through activation of G protein, PKC and Rho signaling pathways

Lysophosphatidylcholine (LPC) is a bioactive phospholipid that accumulates rapidly in the ischemic myocardium. In recent years, it has been shown that some of the actions of LPC are mediated through the activation of the membrane G proteins. However, the precise mechanism(s) responsible for the LPC-related intracellular signaling in the regulation of cardiac ion channels are still poorly understood. The present study was undertaken to examine whether LPC regulates the slow component of the delayed rectifier K⁺ current (I_Ks) and, if so, what intracellular signals are important for this process. Isolated guinea pig cardiac myocytes were voltage-clamped using the whole-cell configuration of the patch-clamp method. The bath application of 1-palmitoyl-lysophosphatidylcholine (LPC-16) concentration-dependently (EC₅₀ = 0.7 μM) and reversibly increased I_Ks in atrial cells, but failed to potentiate I_Ks in ventricular myocytes. In contrast, 1-oleoyl-lysophosphatidylcholine (LPC-18:1) only produced a slight I_Ks increase, and 1-caproyl-lysophosphatidylcholine (LPC-6) or the LPC-16 precursor (phosphatidylcholine) had no effect on I_Ks. Pretreatment of atrial cells with an antibody against the N-terminus of the G2A receptor significantly reduced the LPC-16-induced potentiation of I_Ks. The inhibition of heterotrimeric G protein, phospholipase C (PLC) and protein kinase C (PKC) significantly reduced LPC-16-induced enhancement of I_Ks. Moreover, the blockade of Rho and Rho-kinase by specific inhibitors also inhibited the activity of LPC-16. Immunohistochemical studies demonstrated that G2A was densely distributed in the plasma membrane of atrial myocytes. Therefore, the present study suggests that the activation of a G protein (probably Gα_q) by LPC-16 potentiates I_Ks currents through the PLC-PKC and Rho-kinase pathways.     

Pancreatic islets from hypothalamic obese rats maintain K+ATP channel-dependent but not -independent pathways on glucose-induced insulin release process

One of the main features of obesity is hyperinsulinemia, which is related to insulin oversecretion. Glucose is by far the major physiological stimulator of insulin secretion. Glucose promotes an increase in the ATP/ADP ratio, which inactivates ATP-sensitive K⁺ channels (K⁺ _ATP) and induces beta cell depolarization with consequent calcium influx. Increased intracellular calcium concentration triggers insulin exocytosis. K⁺ _ATP channel function is important for K⁺ _ATP channel-dependent pathways involved in glucose-stimulated insulin secretion (GSIS). However, K⁺ _ATP channel-independent pathway has been identified and it has been found that this pathway sustains GSIS. Both pathways are critical to better GSIS control. GSIS was studied in pancreatic islets from hyperinsulinemic adult obese rats obtained by monosodium l-glutamate (MSG) neonatal treatment. Islets from MSG-obese rats were more glucose responsive than control ones. Diazoxide, a drug which maintains the K⁺ _ATP channels open without interfering with cell metabolism, blocked GSIS in islets from both groups. High extracellular potassium concentration plus diaz-oxide was used to study an alternative to the K⁺ _ATP channel pathway; in these conditions islets from MSG-obese rats did not respond, while islets from control animals showed enhanced GSIS. Results indicate that MSG-obese rats oversecreted insulin, even though the K⁺ _ATP channel-independent pathway is impaired in their beta cells.