Underlying mechanism of atrial fibrillation-associated Nppa-I137T mutation and cardiac effect of potential drug therapy.

BACKGROUND: More than a hundred genetic loci have been associated with atrial fibrillation (AF). But the exact mechanism remains unclear and the treatment needs to be improved. OBJECTIVE: This study aimed to investigate the mechanism and potential treatment of NPPA mutation-associated AF. METHODS: Nppa knock-in (KI, p.I137T) rats were generated, and cardiac function was evaluated. Blood pressure was recorded using a tail-cuff system. The expression levels were measured using real-time polymerase chain reaction, enzyme-linked immunosorbent assay or Western blot analysis, and RNA-sequence analysis. Programmed electrical stimulation, patch clamp, and multielectrode array were used to record the electrophysical characteristics. RESULTS: Mutant rats displayed downregulated expression of atrial natriuretic peptide but elevated blood pressure and enlarged left atrial end-diastolic diameter. Further, gene topology analysis suggested that the majority of differently expressed genes in Nppa KI rats were related to inflammation, electrical remodeling, and structural remodeling. The expression levels of C-C chemokine ligand 5 and galectin-3 involved in remodeling were higher, while there were declined levels of Nav1.5, Cav1.2, and connexin 40. AF was more easily induced in KI rats. Electrical remodeling included abbreviated action potentials, effective refractory period, increased late sodium current, and reduced calcium current, giving rise to conduction abnormalities. These electrophysiological changes could be reversed by the late sodium current blocker ranolazine and the Nav1.8 blocker A-803467. CONCLUSION: Our findings suggest that structural remodeling related to inflammation and fibrosis and electrical remodeling involved in late sodium current underly the major effects of the Nppa (p.I137T) variant to induce AF, which can be attenuated by the late sodium current blocker and Nav1.8 blocker.

Comparison of restrictive and liberal red blood cell suspension transfusion and analysis of influencing factors on prognosis of premature infants.

OBJECTIVE: To investigate the influence of restrictive and liberal red blood cell suspension (RBCs) transfusions on the prognosis of premature infants and to analyze the influencing factors to provide a reference for the transfusion strategy of preterm infants. METHODS: Retrospective analysis was conducted on 85 cases of anemic premature infants treated in our center, including 63 cases in the restrictive transfusion group and 22 in the liberal transfusion group. RESULTS: RBCs transfusions were effective in both groups, and there were no statistically significant differences in post-transfusion hemoglobin and hematocrit between the two groups (P > 0.05). The outcome events: the duration of ventilatory support was statistically prolonger in the restrictive group compared with the liberal group (P < 0.001); however, the differences in mortality, the increased weight before discharge, and length of stay in the hospital within the two groups were not statistically significant (P = 0.237, 0.36 and 0.771, respectively). Univariate survival analysis showed that age, birth weight, Apgar 1 min and Apgar 10 min scores were the influencing factors for death, with P values of 0.035, 0.004, <0.001, and 0.013, respectively; COX regression analysis showed that Apgar 1 min score was an independent factor of the survival time of preterm infants (P = 0.002). CONCLUSION: Compared with the restrictive transfusion group, liberal transfusion patients presented a shorter duration of ventilatory support, which is more beneficial to the prognosis of premature infants.

[Serological Diagnosis and Clinical Data Analysis of Neonatal Alloimmune Thrombocytopenia].

OBJECTIVE: To investigate the pathogenesis and clinical diagnosis of fetal/neonatal alloimmune thrombocytopenia (FNAIT) and analyze the laboratory test results and clinical data related to the disease, so as to provide reference for clinical treatment and improvement of prognosis. METHODS: The clinical data of six neonatal patients with FNAIT in the Neonatology Department of our hospital from March 2017 to September 2020 were retrospectively analyzed, which included laboratory diagnosis, clinical symptoms, treatment, and prognosis. RESULTS: Among six patients, two cases occurred in the first pregnancy and four cases in the second pregnancy. The platelet count of six cases were decreased at admission or during hospitalization and maternal and neonatal serum autoimmune platelet antibody tests were positive. Five cases were accompanied by different degrees of skin and facial bleeding spots or petechiae and ecchymosis, intracranial hemorrhage. Four cases were treated with immunoglobulin and/or steroid hormone therapy (one of them received cross-matched platelets transfusion), while the symptoms of the other two cases improved spontaneously. Five cases recovered and were discharged from the hospital, while one case had not recovered but the family members requested to be discharged forwardly. Four cases were hospitalized within two weeks, but two cases were hospitalized for more than two weeks due to other diseases or factors (e.g., neonatal sepsis, neonatal enteritis, congenital heart disease, neonatal asphyxia, etc.). CONCLUSION: FNAIT is characterized by decreased platelet count, with or without bleeding symptoms, and may occur in the first and following pregnancy. FNAIT can recover spontaneously or have a good prognosis after treatment. However, the complication with other diseases or factors may affect the prognosis.

Eleutheroside B, a selective late sodium current inhibitor, suppresses atrial fibrillation induced by sea anemone toxin II in rabbit hearts.

Eleutheroside B (EB) is the main active constituent derived from the Chinese herb Acanthopanax senticosus (AS) that has been reported to possess cardioprotective effects. In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation (AF). Whole-cell recording was conducted in isolated rabbit atrial myocytes. The intracellular calcium ([Ca2+]i) concentration was measured using calcium indicator Fura-2/AM fluorescence. Monophasic action potential (MAP) and electrocardiogram (ECG) synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system. We showed that EB dose-dependently inhibited late sodium current (INaL), transient sodium current (INaT), and sea anemone toxin II (ATX II)-increased INaL with IC50 values of 167, 1582, and 181 µM, respectively. On the other hand, EB (800 µM) did not affect L-type calcium current (ICaL), inward rectifier potassium channel current (IK), and action potential duration (APD). Furthermore, EB (300 µM) markedly decreased ATX II-prolonged the APD at 90% repolarization (APD90) and eliminated ATX II-induced early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activities (TAs). Moreover, EB (200 µM) significantly suppressed ATX II-induced Na+-dependent [Ca2+]i overload in atrial myocytes. In the Langendorff-perfused rabbit hearts, application of EB (200 µM) or TTX (2 µM) substantially decreased ATX II-induced incidences of atrial fibrillation (AF), ventricular fibrillation (VF), and heart death. These results suggest that augmented INaL alone is sufficient to induce AF, and EB exerts anti-AF actions mainly via blocking INaL, which put forward the basis of pharmacology for new clinical application of EB.

Curcumin, a Multi-Ion Channel Blocker That Preferentially Blocks Late Na+ Current and Prevents I/R-Induced Arrhythmias.

Increasing evidence shows that Curcumin (Cur) has a protective effect against cardiovascular diseases. However, the role of Cur in the electrophysiology of cardiomyocytes is currently not entirely understood. Therefore, the present study was conducted to investigate the effects of Cur on the action potential and transmembrane ion currents in rabbit ventricular myocytes to explore its antiarrhythmic property. The whole-cell patch clamp was used to record the action potential and ion currents, while the multichannel acquisition and analysis system was used to synchronously record the electrocardiogram and monophasic action potential. The results showed that 30 µmol/L Cur shortened the 50 and 90% repolarization of action potential by 17 and 7%, respectively. In addition, Cur concentration dependently inhibited the Late-sodium current (I Na.L), Transient-sodium current (I Na.T), L-type calcium current (I Ca.L), and Rapidly delayed rectifying potassium current (I Kr), with IC50 values of 7.53, 398.88, 16.66, and 9.96 µmol/L, respectively. Importantly, the inhibitory effect of Cur on I Na.L was 52.97-fold higher than that of I Na.T. Moreover, Cur decreased ATX II-prolonged APD, suppressed the ATX II-induced early afterdepolarization (EAD) and Ca2+-induced delayed afterdepolarization (DAD) in ventricular myocytes, and reduced the occurrence and average duration of ventricular tachycardias and ventricular fibrillations induced by ischemia-reperfusion injury. In conclusion, Cur inhibited I Na.L, I Na.T, I Ca.L, and I Kr; shortened APD; significantly suppressed EAD and DAD-like arrhythmogenic activities at the cellular level; and exhibited antiarrhythmic effect at the organ level. It is first revealed that Cur is a multi-ion channel blocker that preferentially blocks I Na.L and may have potential antiarrhythmic property.

Increase in CO2 levels by upregulating late sodium current is proarrhythmic in the heart.

BACKGROUND: Increased CO2 levels in the general circulation and/or in the myocardium are common under pathologic conditions. OBJECTIVE: The purpose of this study was to test the hypothesis that an increase in CO2 levels, and not just the subsequent extra- or intracellular acidosis, would augment late sodium current (INa,L) and contribute to arrhythmogenesis in hearts with reduced repolarization reserve. METHODS: Monophasic action potential durations at 90% completion of repolarization (MAPD90) from isolated rabbit hearts, INa,L, and extra- (pHo) and intracellular pH (pHi) values from cardiomyocytes using the whole-cell patch-clamp techniques and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM), respectively, were measured. RESULTS: Increasing CO2 levels from 5% to 10% and 20% and administration of 1 nM sea anemone toxin (ATX)-II increased INa,L and prolonged both epicardial and endocardial MAPD90 (n = 7 and 10, respectively) without causing arrhythmic activities. Compared to 5% CO2, 10% and 20% CO2 decreased pHo and pHi in hearts treated with 1 nM ATX-II, caused greater prolongation of MAPD90, and elicited ventricular tachycardias. Increasing CO2 levels from 5% to 10% and 20% with pHo maintained at 7.4 produced smaller changes in pHi (P <.05) but similar increases in INa,L, prolongation of MAPD90, and incidence of ventricular tachycardias (n = 8). Inhibition of INa,L reversed the increase in INa,L, suppressed MAPD90 prolongations, and ventricular tachycardias induced by 20% CO2. Increased phospho-calmodulin-dependent protein kinase II-δ (CaMKIIδ) and phospho-NaV1.5 protein levels in hearts treated with 20% CO2 was attenuated by eleclazine. CONCLUSION: Increased CO2 levels enhance INa,L and are proarrhythmic factors in hearts with reduced repolarization reserve, possibly via mechanisms related to phosphorylation of CaMKIIδ and NaV1.5.

Sodium Houttuyfonate Inhibits Voltage-Gated Peak Sodium Current and Anemonia Sulcata Toxin II-Increased Late Sodium Current in Rabbit Ventricular Myocytes.

AIM: Sodium houttuyfonate (SH), a chemical compound originating from Houttuynia cordata, has been reported to have anti-inflammatory, antibacterial, and antifungal effects, as well as cardioprotective effects. In this study, we investigated the effects of SH on cardiac electrophysiology, because to the best of our knowledge, this issue has not been previously investigated. METHODS: We used the whole-cell patch-clamp technique to explore the effects of SH on peak sodium current (INa.P) and late sodium current (INa.L) in isolated rabbit ventricular myocytes. To test the drug safety of SH, we also investigated the effect of SH on rapidly activated delayed rectifier potassium current (IKr). RESULTS: SH (1, 10, 50, and 100 µmol/L) inhibited INa.P in a concentration-dependent manner with an IC50 of 78.89 µmol/L. In addition, SH (100 µmol/L) accelerated the steady state inactivation of INa.P. Moreover, 50 and 100 µmol/L SH inhibited Anemonia sulcata toxin II (ATX II)-increased INa.L by 30.1 and 57.1%, respectively. However, SH (50 and 100 µmol/L) only slightly affected IKr. CONCLUSIONS: The inhibitory effects of SH on ATX II-increased INa.L may underlie the electrophysiological mechanisms of the cardioprotective effects of SH; SH has the potential to be an effective and safe antiarrhythmic drug.

Barbaloin inhibits ventricular arrhythmias in rabbits by modulating voltage-gated ion channels.

Barbaloin (10-ß-D-glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone) is extracted from the aloe plant and has been reported to have anti-inflammatory, antitumor, antibacterial, and other biological activities. Here, we investigated the effects of barbaloin on cardiac electrophysiology, which has not been reported thus far. Cardiac action potentials (APs) and ionic currents were recorded in isolated rabbit ventricular myocytes using whole-cell patch-clamp technique. Additionally, the antiarrhythmic effect of barbaloin was examined in Langendorff-perfused rabbit hearts. In current-clamp recording, application of barbaloin (100 and 200 µmol/L) dose-dependently reduced the action potential duration (APD) and the maximum depolarization velocity (Vmax), and attenuated APD reverse-rate dependence (RRD) in ventricular myocytes. Furthermore, barbaloin (100 and 200 µmol/L) effectively eliminated ATX II-induced early afterdepolarizations (EADs) and Ca2+-induced delayed afterdepolarizations (DADs) in ventricular myocytes. In voltage-clamp recording, barbaloin (10-200 µmol/L) dose-dependently inhibited L-type calcium current (ICa.L) and peak sodium current (INa.P) with IC50 values of 137.06 and 559.80 µmol/L, respectively. Application of barbaloin (100, 200 µmol/L) decreased ATX II-enhanced late sodium current (INa.L) by 36.6%±3.3% and 71.8%±6.5%, respectively. However, barbaloin up to 800 µmol/L did not affect the inward rectifier potassium current (IK1) or the rapidly activated delayed rectifier potassium current (IKr) in ventricular myocytes. In Langendorff-perfused rabbit hearts, barbaloin (200 µmol/L) significantly inhibited aconitine-induced ventricular arrhythmias. These results demonstrate that barbaloin has potential as an antiarrhythmic drug.

Occurrence of Plasmid- and Chromosome-Carried mcr-1 in Waterborne Enterobacteriaceae in China.

The aim of this study was to investigate the prevalence of the polymyxin resistance gene mcr-1 in Enterobacteriaceae from environmental water sources in Hangzhou, China. Colistin-resistant bacteria were isolated from environmental water samples using an enrichment broth culture method, were screened for mcr-1, and then were analyzed for the location and transferability of mcr-1 Isolates positive for mcr-1 were further examined to determine their susceptibility profiles and were screened for the presence of additional resistance genes. Twenty-three mcr-1-positive isolates (16 Escherichia coli, two Citrobacter freundii, two Klebsiella oxytoca, two Citrobacter braakii, and one Enterobacter cloacae) were isolated from 7/9 sampling locations; of those, eight mcr-1-positive isolates also contained ß-lactamase-resistance genes, eight contained qnrS, and 10 contained oqx No mcr-2-positive isolates were identified. The majority of isolates demonstrated a low to moderate level of colistin resistance. Transconjugation was successfully conducted from 14 of the 23 mcr-1-positive isolates, and mcr-1 was identified on plasmids ranging from 60 to 220 kb in these isolates. Conjugation and hybridization experiments revealed that mcr-1 was chromosome-borne in only three isolates. Pulsed-field gel electrophoresis showed that the majority of E. coli isolates belonged to different clonal lineages. Multilocus sequence typing analysis revealed that sequence type 10 (ST10) was the most prevalent, followed by ST181 and ST206. This study demonstrates the utility of enrichment broth culture for identifying environmental mcr-1-positive isolates. Furthermore, it highlights the importance of responsible agriculture and clinical use of polymyxins to prevent further widespread dissemination of polymyxin-resistant pathogens.

Tolterodine reduces veratridine-augmented late INa, reverse-INCX and early afterdepolarizations in isolated rabbit ventricular myocytes.

AIM: The augmentation of late sodium current (INa.L) not only causes intracellular Na+ accumulation, which results in intracellular Ca2+ overload via the reverse mode of the Na+/Ca2+ exchange current (reverse-INCX), but also prolongs APD and induces early afterdepolarizations (EAD), which can lead to arrhythmia and cardiac dysfunction. Thus, the inhibition of INa.L is considered to be a potential way for therapeutic intervention in ischemia and heart failure. In this study we investigated the effects of tolterodine (Tol), a competitive muscarinic receptor antagonist, on normal and veratridine (Ver)-augmented INa.L, reverse-INCX and APD in isolated rabbit ventricular myocytes, which might contribute to its cardioprotective activity. METHODS: Rabbit ventricular myocytes were prepared. The INa.L and reverse-INCX were recorded in voltage clamp mode, whereas action potentials and Ver-induced early afterdepolarizations (EADs) were recorded in current clamp mode. Drugs were applied via superfusion. RESULTS: Tol (3-120 nmol/L) concentration-dependently inhibited the normal and Ver-augmented INa.L with IC50 values of 32.08 nmol/L and 42.47 nmol/L, respectively. Atropine (100 µmol/L) did not affect the inhibitory effects of Tol (30 nmol/L) on Ver-augmented INa.L. In contrast, much high concentrations of Tol was needed to inhibit the transient sodium current (INa.T) with an IC50 value of 183.03 µmol/L. In addition, Tol (30 nmol/L) significantly shifted the inactivation curve of INa.T toward a more depolarizing membrane potential without affecting its activation characteristics. Moreover, Tol (30 nmol/L) significantly decreased Ver-augmented reverse-INCX. Tol (30 nmol/L) increased the action potential duration (APD) by 16% under the basal conditions. Ver (20 µmol/L) considerably extended the APD and evoked EADs in 18/24 cells (75%). In the presence of Ver, Tol (30 nmol/L) markedly decreased the APD and eliminated EADs (0/24 cells). CONCLUSION: Tol inhibits normal and Ver-augmented INaL and decreases Ver-augmented reverse-INCX. In addition, Tol reverses the prolongation of the APD and eliminates the EADs induced by Ver, thus prevents Ver-induced arrhythmia.

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents.

AIM: Intracellular Ca(2+) ([Ca(2+)]i) overload occurs in myocardial ischemia. An increase in the late sodium current (INaL) causes intracellular Na(+) overload and subsequently [Ca(2+)]i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca(2+)]i overload. The aim of this study was to investigate the effects of ketamine on Na(+)-dependent Ca(2+) overload in ventricular myocytes in vitro. METHODS: Ventricular myocytes were enzymatically isolated from hearts of rabbits. INaL, NCX current (INCX) and L-type Ca(2+) current (ICaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca(2+)]i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system. RESULTS: Ketamine (20, 40, 80 µmol/L) inhibited INaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), INaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented INaL. Ketamine (40 µmol/L) also significantly suppressed hypoxia or H2O2-induced enhancement of INaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode INCX. In addition, ketamine (40 µmol/L) inhibited ICaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca(2+)]i, and the rate and amplitude of [Ca(2+)]i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 µmol/L) or by ketamine (40 µmol/L). CONCLUSION: Ketamine protects isolated rabbit ventricular myocytes against [Ca(2+)]i overload by inhibiting INaL and ICaL.

Ranolazine attenuates the enhanced reverse Na⁺-Ca²âº exchange current via inhibiting hypoxia-increased late sodium current in ventricular myocytes.

Ranolazine (RAN), a novel antianginal agent, inhibits the increased late sodium current (INa.L) under many pathological conditions. In this study, the whole-cell patch-clamp technique was used to explore the effects of RAN on INa.L and reverse Na(+)/Ca(2+) exchange current (INCX) in rabbit ventricular myocytes during hypoxia.Tetrodotoxin (TTX) at 2 µM or RAN at 9 µM decreased significantly INa.L and reverse INCX under normoxia and RAN had no further effects on both currents in the presence of TTX. RAN (3, 6, and 9 µM) attenuated hypoxia-increased INa.L and reverse INCX in a concentration-dependent manner. Hypoxia-increased INa.L and reverse INCX were inhibited by 2 µM TTX, whereas 9 µM RAN applied sequentially did not further decrease both currents. In another group, after both currents were decreased by 9 µM RAN, 2 µM TTX had no further effects in the presence of Ran. In monophasic action potential (MAP) recording, early after-depolarizations (EADs) were suppressed by RAN (9 µM) during hypoxia. In conclusion, RAN decreased reverse INCX by inhibiting INa.L in normoxia, concentration-dependently attenuated the increase of INa.L, which thereby decreased the reverse INCX, and obviously relieved EADs during hypoxia.

Procaine, a state-dependent blocker, inhibits HERG channels by helix residue Y652 and F656 in the S6 transmembrane domain.

The article evaluated the inhibitory action of procaine on wild-type and mutated HERG potassium channel current (I(HERG)) to determine whether mutations in the S6 region are important for the inhibition of I(HERG) by procaine. HERG channels (WT, Y652A, and F656A) were expressed in Xenopus laevis oocytes and studied using the standard two-microelectrode voltage-clamp technique. The results revealed that WT HERG is blocked in a concentration-, voltage-, and state-dependent manner by procaine ([IC50] = 34.79 µM). The steady state activation curves slightly move to the negative, while inactivation parameters move to the positive in the presence of procaine. Time-dependent test reveals that voltage-dependent I(HERG) blockade occurs extremely rapidly. Furthermore, the mutation to Ala of Y652 and F656 produce about 11-fold and 18-fold increases in IC50 for I(HERG) blockade, respectively. Simultaneously, for Y652A, the steady state activation and inactivation parameters are shifted to more positive values after perfusion of procaine. Conclusively, procaine state-dependently inhibits HERG channels (WT, Y652A, and F656A). The helix residues Y652 and F656 in the S6 transmembrane domain might play a role in interaction of the drug with the channel.

Sophocarpine attenuates the Na(+)-dependent Ca2(+) overload induced by Anemonia sulcata toxin-increased late sodium current in rabbit ventricular myocytes.

Many studies indicate that an increase in late sodium current (I(Na.L)) of cardiomyocytes causes intracellular Na overload and subsequently raises the reverse Na/Ca exchanger current (INCX), ultimately resulting in intracellular Ca overload. Therefore, using drugs to inhibit the increased INa.L under various pathological conditions can lower intracellular Ca overload. This study was intended to explore the effect of sophocarpine (SOP) on the increase in INa.L, INCX, calcium transient and contraction in rabbit ventricular myocytes induced by Anemonia sulcata toxin II (ATX II), an opener of sodium channel, with the application of whole-cell patch-clamp techniques, the video-based motion edge detection system, and the intracellular calcium concentration determination system. The results indicate that tetrodotoxin (TTX, 4 µM ) obviously decreased INa.L and INCX enlarged by ATX II (30 nM), and SOP (20, 40, and 80 µM) also inhibited both the parameters concentration dependently in rabbit ventricular myocytes. However, transient sodium current remained unaffected by the above-mentioned concentrations of ATX II, TTX, and SOP. In addition, SOP also reversed diastolic calcium concentration, calcium transient amplitude, and ventricular muscle contractility augmented by ATX II. Its effects were similar to those of TTX, a specific inhibitor of the sodium channel. In conclusion, SOP inhibits INa.L, INCX, diastolic Ca concentration, and contractility in rabbit ventricular myocytes, which suggests that relief of intracellular Ca overload through inhibiting INa.L is likely to become a new therapeutic mechanism of SOP against arrhythmia and myocyte damage associated with intracellular Ca overload.

[Involvement of veratridine-induced increase of reverse Na(+)/Ca(2+) exchange current in intracellular Ca(2+) overload and extension of action potential duration in rabbit ventricular myocytes].

The objectives of this study were to investigate the effects of veratridine (VER) on persistent sodium current (I(Na.P)), Na(+)/Ca(2+) exchange current (I(NCX)), calcium transients and the action potential (AP) in rabbit ventricular myocytes, and to explore the mechanism in intracellular calcium overload and myocardial contraction enhancement by using whole-cell patch clamp recording technique, visual motion edge detection system, intracellular calcium measurement system and multi-channel physiological signal acquisition and processing system. The results showed that I(Na.P) and reverse I(NCX) in ventricular myocytes were obviously increased after giving 10, 20 µmol/L VER, with the current density of I(Na.P) increasing from (-0.22 ± 0.12) to (-0.61 ± 0.13) and (-2.15 ± 0.14) pA/pF (P < 0.01, n = 10) at -20 mV, and that of reverse I(NCX) increasing from (1.62 ± 0.12) to (2.19 ± 0.09) and (2.58 ± 0.11) pA/pF (P < 0.05, n = 10) at +50 mV. After adding 4 µmol/L tetrodotoxin (TTX), current density of I(Na.P) and reverse I(NCX) returned to (-0.07 ± 0.14) and (1.69 ± 0.15) pA/pF (P < 0.05, n = 10). Another specific blocker of I(Na.P), ranolazine (RAN), could obviously inhibit VER-increased I(Na.P) and reverse I(NCX). After giving 2.5 µmol/L VER, the maximal contraction rate of ventricular myocytes increased from (-0.91 ± 0.29) to (-1.53 ± 0.29) µm/s (P < 0.01, n = 7), the amplitude of contraction increased from (0.10 ± 0.04) to (0.16 ± 0.04) µm (P < 0.05, n = 7), and the baseline of calcium transients (diastolic calcium concentration) increased from (1.21 ± 0.08) to (1.37 ± 0.12) (P < 0.05, n = 7). After adding 2 µmol/L TTX, the maximal contraction rate and amplitude of ventricular myocytes decreased to (-0.86 ± 0.24) µm/s and (0.09 ± 0.03) µm (P < 0.01, n = 7) respectively. And the baseline of calcium transients reduced to (1.17 ± 0.09) (P < 0.05, n = 7). VER (20 µmol/L) could extend action potential duration at 50% repolarization (APD(50)) and at 90% repolarization (APD(90)) in ventricular myocytes from (123.18 ± 23.70) to (271.90 ± 32.81) and from (146.94 ± 24.15) to (429.79 ± 32.04) ms (P < 0.01, n = 6) respectively. Early afterdepolarizations (EADs) appeared in 3 out of the 6 cases. After adding 4 µmol/L TTX, APD(50) and APD(90) were reduced to (99.07 ± 22.81) and (163.84 ± 26.06) ms (P < 0.01, n = 6) respectively, and EADs disappeared accordingly in 3 cases. It could be suggested that: (1) As a specific agonist of the I(Na.P), VER could result in I(Na.P) increase and intracellular Na(+) overload, and subsequently intracellular Ca(2+) overload with the increase of reverse I(NCX). (2) The VER-increased I(Na.P) could further extend the action potential duration (APD) and induce EADs. (3) TTX could restrain the abnormal VER-induced changes of the above-mentioned indexes, indicating that these abnormal changes were caused by the increase of I(Na.P). Based on this study, it is concluded that as the I(Na.P) agonist, VER can enhance reverse I(NCX) by increasing I(Na.P), leading to intracellular Ca(2+) overload and APD abnormal extension.

Blocking effect of methylflavonolamine on human Na(V)1.5 channels expressed in Xenopus laevis oocytes and on sodium currents in rabbit ventricular myocytes.

AIM: To investigate the blocking effects of methylflavonolamine (MFA) on human Na(V)1.5 channels expressed in Xenopus laevis oocytes and on sodium currents (I(Na)) in rabbit ventricular myocytes. METHODS: Human Na(V)1.5 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. I(Na) and action potentials in rabbit ventricular myocytes were studied using the whole-cell recording. RESULTS: MFA and lidocaine inhibited human Na(V)1.5 channels expressed in Xenopus oocytes in a positive rate-dependent and concentration-dependent manner, with IC(50) values of 72.61 micromol/L and 145.62 micromol/L, respectively. Both of them markedly shifted the steady-state activation curve of I(Na) toward more positive potentials, shifted the steady-state inactivation curve of I(Na) toward more negative potentials and postponed the recovery of the I(Na) inactivation state. In rabbit ventricular myocytes, MFA inhibited I(Na) with a shift in the steady-state inactivation curve toward more negative potentials, thereby postponing the recovery of the I(Na) inactivation state. This shift was in a positive rate-dependent manner. Under current-clamp mode, MAF significantly decreased action potential amplitude (APA) and maximal depolarization velocity (V(max)) and shortened action potential duration (APD), but did not alter the resting membrane potential (RMP). The demonstrated that the kinetics of sodium channel blockage by MFA resemble those of class I antiarrhythmic agents such as lidocaine. CONCLUSION: MFA protects the heart against arrhythmias by its blocking effect on sodium channels.

Modulation of K(ATP) currents in rat ventricular myocytes by hypoxia and a redox reaction.

AIM: The present study investigated the possible regulatory mechanisms of redox agents and hypoxia on the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes. METHODS: Single-channel and whole-cell patch-clamp techniques were used to record the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes. RESULTS: Oxidized glutathione (GSSG, 1 mmol/L) increased the I(KATP), while reduced glutathione (GSH, 1 mmol/L) could reverse the increased I(KATP) during normoxia. To further corroborate the effect of the redox agent on the K(ATP) channel, we employed the redox couple DTT (1 mmol/L)/H2O2 (0.3, 0.6, and 1 mmol/L) and repeated the previous processes, which produced results similar to the previous redox couple GSH/GSSG during normoxia. H2O2 increased the I(KATP) in a concentration dependent manner, which was reversed by DTT (1 mmol/L). In addition, our results have shown that 15 min of hypoxia increased the I(KATP), while GSH (1 mmol/L) could reverse the increased I(KATP). Furthermore, in order to study the signaling pathways of the I(KATP) augmented by hypoxia and the redox agent, we applied a protein kinase C(PKC) inhibitor bisindolylmaleimide VI (BIM), a protein kinase G(PKG) inhibitor KT5823, a protein kinase A (PKA) inhibitor H-89, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-62 and KN-93. The results indicated that BIM, KT5823, KN-62, and KN-93, but not H-89, inhibited the I(KATP) augmented by hypoxia and GSSG; in addition, these results suggest that the effects of both GSSG and hypoxia on K(ATP) channels involve the activation of the PKC, PKG, and CaMK II pathways, but not the PKA pathway. CONCLUSION: The present study provides electrophysiological evidence that hypoxia and the oxidizing reaction are closely related to the modulation of I(KATP).

Quercetin activates human Kv1.5 channels by a residue I502 in the S6 segment.

1. The aims of the present study were to investigate the pharmacological effects of quercetin on wild-type (WT) and mutant (I502A) human (h) Kv1.5 channel currents (I(kur)) and to identify whether mutation in the S6 segment is critical to activation of I(kur) by quercetin. 2. Experiments were performed on WT and site-directed mutant hKv1.5 channels, which were stably expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. 3. Quercetin increased WT hKv1.5 channel current in a concentration-, voltage- and time-dependent manner, with an EC(50) of 37.8 micromol/L and a negative shift in the steady state activation and inactivation curves. Quercetin accelerated channel activation and inactivation, significantly decreasing activation and inactivation time constants. However, mutating the I502 residue to Ala abolished the activating effect of quercetin. Quercetin did not modify the activation and inactivation kinetics of I502A channels. As an anti-oxidant, tanshinone IIA (4 micromol/L) inhibited the H(2)O(2)-induced activation of WT hKv1.5 channels. In contrast, quercetin had no significant effect. 4. We conclude that: (i) quercetin preferentially binds to and increases the current amplitude of WT hKv1.5 channels; (ii) Ile502, an aliphatic and neutral amino acid residue residing in the S6 segment, is important in quercetin binding; and (iii) quercetin-induced changes in the properties of WT hKv1.5 channels may be foreign to its own anti-oxidant action.

Verapamil blocks HERG channel by the helix residue Y652 and F656 in the S6 transmembrane domain.

AIM: The objectives of this study were to investigate the inhibitory action of verapamil on wild-type(WT) and mutation HERG K+ channel current (I(HERG)), and to determine whether mutations in the S6 region are important for the inhibition of I(HERG) by verapamil. METHODS: HERG channels (WT, Y652A, and F656A) were expressed in oocytes of Xenopus laevis and studied using the 2-electrode voltage- clamp technique. RESULTS: WT HERG is blocked in a concentration-dependent manner by verapamil (half-maximal inhibition concentration [IC(50)]=5.1 micromol/L), and the steady state activation and inactivation parameters are shifted to more negative values. However, mutation to Ala of Y652 and F656 located on the S6 domain produced 16-fold and 20-fold increases in IC(50) for IHERG blockade, respectively. Simultaneously, the steady state activation and inactivation parameters for Y652A are also shifted to more negative values in the presence of the blockers. CONCLUSION: Verapamil preferentially binds to and blocks open HERG channels. Tyr-652 and Phe-656, 2 aromatic amino-acid residues in the inner (S6) helix, are critical in the verapamil-binding site.