Man-Wen Jin

Acacetin, a Natural Flavone, Selectively Inhibits Human Atrial Repolarization Potassium Currents and Prevents Atrial Fibrillation in Dogs

Background— The development of atrium-selective antiarrhythmic agents is a current strategy for inhibiting atrial fibrillation (AF). The present study investigated whether the natural flavone acacetin from the traditional Chinese medicine Xuelianhua would be an atrium-selective anti-AF agent. Methods and Results— The effects of acacetin on human atrial ultrarapid delayed rectifier K+ current (IKur) and other cardiac ionic currents were studied with a whole-cell patch technique. Acacetin suppressed IKur and the transient outward K+ current (IC50 3.2 and 9.2 &mgr;mol/L, respectively) and prolonged action potential duration in human atrial myocytes. The compound blocked the acetylcholine-activated K+ current; however, it had no effect on the Na+ current, L-type Ca2+ current, or inward-rectifier K+ current in guinea pig cardiac myocytes. Although acacetin caused a weak reduction in the hERG and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells, it did not prolong the corrected QT interval in rabbit hearts. In anesthetized dogs, acacetin (5 mg/kg) prolonged the atrial effective refractory period in both the right and left atria 1 to 4 hours after intraduodenal administration without prolongation of the corrected QT interval, whereas sotalol at 5 mg/kg prolonged both the atrial effective refractory period and the corrected QT interval. Acacetin prevented AF induction at doses of 2.5 mg/kg (50%), 5 mg/kg (85.7%), and 10 mg/kg (85.7%). Sotalol 5 mg/kg also prevented AF induction (60%). Conclusions— The present study demonstrates that the natural compound acacetin is an atrium-selective agent that prolongs the atrial effective refractory period without prolonging the corrected QT interval and effectively prevents AF in anesthetized dogs after intraduodenal administration. These results indicate that oral acacetin is a promising atrium-selective agent for the treatment of AF.

Evidence for functional expression of TRPM7 channels in human atrial myocytes

Transient receptor potential melastatin-7 (TRPM7) channels have been recently reported in human atrial fibroblasts and are believed to mediate fibrogenesis in human atrial fibrillation. The present study investigates whether TRPM7 channels are expressed in human atrial myocytes using whole-cell patch voltage-clamp, RT-PCR and Western blotting analysis. It was found that a gradually activated TRPM7-like current was recorded with a K+- and Mg2+-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca2+ and Mg2+, and the current increase could be inhibited by Ni2+ or Ba2+. The TRPM7-like current was potentiated by acidic pH and inhibited by La3+ and 2-aminoethoxydiphenyl borate. In addition, Ca2+-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca2+ ionophore A23187 in bath solution. RT-PCR and Western immunoblot analysis revealed that in addition to TRPM4, TRPM7 channel current, mRNA and protein expression were evident in human atrial myocytes. Interestingly, TRPM7 channel protein, but not TRPM4 channel protein, was significantly increased in human atrial specimens from the patients with atrial fibrillation. Our results demonstrate for the first time that functional TRPM7 channels are present in human atrial myocytes, and the channel expression is upregulated in the atria with atrial fibrillation.

TRPM7 channels regulate proliferation and adipogenesis in 3T3-L1 preadipocytes.

Transient receptor potential melastatin‐7 (TRPM7) channels are involved in many cellular physiological and pathological processes. The present study was designed to investigate the expression of TRPM7 channels and the potential role in regulating cell proliferation and adipogenesis in 3T3‐L1 preadipocytes with approaches of whole‐cell patch voltage‐clamp, molecular biology, cell proliferation, adipogenesis, etc. We found that a TRPM7‐like current was recorded with Mg2+‐free pipette solution in 3T3‐L1 preadipocytes, and the current was inhibited by intercellular free Mg2+. The TRPM7‐like current was potentiated by acidic pH and inhibited by 2‐aminoethoxydiphenyl borate (2‐APB). RT‐PCR, Western blot and immunocytochemistry revealed that gene and protein of TRPM7 channels were abundant in 3T3‐L1 preadipocytes. Blockade of TRPM7 channels with 2‐APB inhibited cell proliferation in 3T3‐L1 cells. In addition, knockdown of TRPM7 with specific siRNA inhibited both proliferation and adipogenesis. The present study demonstrates for the first time that TRPM7 channels regulate cell cycle and adipogenesis of 3T3‐L1 preadipocytes. J. Cell. Physiol. 229: 60–67, 2014.

The selective estrogen receptor modulator raloxifene inhibits cardiac delayed rectifier potassium currents and voltage-gated sodium current without QTc interval prolongation.

Raloxifene is widely used in the treatment of postmenopausal osteoporosis and also has been shown to be cardioprotective. The effect of raloxifene on cardiac ion channels is not fully understood. The present study investigated whether raloxifene could affect the cloned hERG channel (I(hERG)) and recombinant human cardiac KCNQ1/KCNE1 channel (I(Ks)) stably expressed in HEK 293 cells using a patch-clamp technique. Raloxifene blocked I(hERG) with an IC(50) of 1.1 μM and decreased I(Ks) (IC(50): 4.8 μM) without affecting activation kinetics. In addition, raloxifene significantly decreased I(Na) (IC(50): 2.8 μM) in guinea pig ventricular myocytes. However, this drug (1 μM) did not increase QRS and QTc interval in isolated guinea pig hearts. These results demonstrate that raloxifene, despite its inhibitory action on delayed rectifier potassium currents, does not prolong ECG QTc interval, suggesting that raloxifene is likely a safe selective estrogen receptor modulator with less cardiac toxicity.

Anti-diabetic effects of pentamethylquercetin in neonatally streptozotocin-induced diabetic rats

Pentamethylquercetin (PMQ), a methylated quercetin derivative, was screened for anti-diabetic effects in neonatally streptozotocin (STZ)-induced diabetic rats (n-STZ diabetic rats). Streptozotocin (90 mg/kg) was administered intraperitoneally to 2-day-old male pups to induce diabetes. PMQ was administered at doses of 2.5, 5, 10 and 20mg/kg/day orally when the rats were 6 weeks old and continued for 10 consecutive weeks. Body weights were followed. Fasting and fed glucose, triglyceride and insulin levels were monitored periodically at the 6th and 10th week after PMQ treatment. At the end of the study, oral glucose tolerance test was performed, and kidney and liver function parameters were assayed. It was found that PMQ intervention dose-dependently reduced postprandial glucose and triglyceride levels, prevented the onset of overt diabetes, ameliorated polydipsia symptom induced by diabetes, attenuated glucose intolerance, enhanced insulin sensitivity indices and decreased endogenous creatinine clearance rate, urinary albumin excretion rate and blood alanine aminotransferase levels of n-STZ diabetic rats in comparison to their diabetic counterparts. The results from the present study thus suggest that PMQ exhibits great potential as an antidiabetic agent by improving hyperglycemia and reducing the incidence of peripheral complications.

Functional ion channels in mouse cardiac c-kit cells

Cardiac c-kit(+) cells are generally believed to be the major population of stem/progenitor cells in the heart and can be used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not understood in this type of cells. The present study was designed to investigate functional ion channels in undifferentiated mouse cardiac c-kit(+) cells using approaches of whole cell patch voltage clamp, RT-PCR, and cell proliferation assay. It was found that three types of ionic currents were present in mouse cardiac c-kit(+) cells, including a delayed rectifier K(+) current (IK(DR)) inhibited by 4-aminopyridine (4-AP), an inward rectifier K(+) current (I(Kir)) decreased by Ba(2+), and a volume-sensitive chloride current (I(Cl.vol)) inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB). RT-PCR revealed that the corresponding ion channel genes, Kv1.1, Kv1.2, and Kv1.6 (for IK(DR)), Kir.1.1, Kir2.1, and Kir2.2 (likely responsible for I(Kir)), and Clcn3 (for I(Cl.vol)), were significant in mouse cardiac c-kit(+) cells. The inhibition of I(Cl.vol) with NPPB and niflumic acid, but not IK(DR) with 4-AP and tetraethylammonium, reduced cell proliferation and accumulated the cell progression at G(0)/G(1) phase in mouse cardiac c-kit(+) cells. Our results demonstrate that three types of functional ion channel currents (i.e., IK(DR), I(Kir), and I(Cl.vol)) are present in mouse cardiac c-kit(+) cells, and I(Cl.vol) participates in regulating cell proliferation.

SKF-96365 blocks human ether-à-go-go-related gene potassium channels stably expressed in HEK 293 cells.

SKF-96365 is a TRPC channel antagonist commonly used to characterize the potential functions of TRPC channels in different systems, which was recently reported to induce QTc prolongation on ECG by inhibiting TRPC channels. The present study investigates whether the blockade of cardiac repolarization currents would be involved in the increase of QTc interval. Cardiac repolarization currents were recorded in HEK 293 cells stably expressing human ether-à-go-go-related gene potassium (hERG or hKv11.1) channels, hKCNQ1/hKCNE1 channels (IKs) or hKir2.1 channels and cardiac action potentials were recorded in guinea pig ventricular myocytes using a whole-cell patch technique. The potential effect of SKF-96365 on QT interval was evaluated in ex vivo guinea pig hearts. It was found that SKF-96365 inhibited hERG current in a concentration-dependent manner (IC50, 3.4μM). The hERG mutants S631A in the pore helix and F656V of the S6 region reduced the inhibitory sensitivity with IC50s of 27.4μM and 11.0μM, suggesting a channel pore blocker. In addition, this compound inhibited IKs and hKir2.1currents with IC50s of 10.8 and 8.7μM. SKF-96365 (10μM) significantly prolonged ventricular APD90 in guinea pig ventricular myocytes and QTc interval in ex vivo guinea pig hearts. These results indicate that the TRPC channel antagonist SKF-96365 exerts blocking effects on hERG, IKs, and hKir2.1 channels. Prolongation of ventricular APD and QT interval is related to the inhibition of multiple repolarization potassium currents, especially hERG channels.

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Allitridi (diallyl trisulfide) is an active compound (volatile oil) from garlic. The previous studies reported that allitridi had anti-arrhythmic effect. The potential ionic mechanisms are, however, not understood. The present study was designed to determine the effects of allitridi on cardiac potassium channels expressed in HEK 293 cells using a whole-cell patch voltage-clamp technique and mutagenesis. It was found that allitridi inhibited hKv4.3 channels (IC50 = 11.4 µM) by binding to the open channel, shifting availability potential to hyperpolarization, and accelerating closed-state inactivation of the channel. The hKv4.3 mutants T366A, T367A, V392A, and I395A showed a reduced response to allitridi with IC50s of 35.5 µM, 44.7 µM, 23.7 µM, and 42.4 µM. In addition, allitridi decreased hKv1.5, hERG, hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells with IC50s of 40.2 µM, 19.6 µM and 17.7 µM. However, it slightly inhibited hKir2.1 current (100 µM, inhibited by 9.8% at −120 mV). Our results demonstrate for the first time that allitridi preferably blocks hKv4.3 current by binding to the open channel at T366 and T367 of P-loop helix, and at V392 and I395 of S6 domain. It has a weak inhibition of hKv1.5, hERG, and hKCNQ1/hKCNE1 currents. These effects may account for its anti-arrhythmic effect observed in experimental animal models.

Distinctive property and pharmacology of voltage-gated sodium current in rat atrial vs ventricular myocytes

BACKGROUND Several mammalian species display distinct biophysical properties between atrial and ventricular voltage-gated sodium current (INa); however, the potential mechanism behind this phenomenon is unknown. OBJECTIVE The purpose of this study was to investigate the potential molecular identities of the different INa in atrial and ventricular myocytes of rat hearts. METHODS Whole-cell patch voltage-clamp and molecular biology techniques were used in the study. RESULTS Ventricular INa exhibited a slower inactivation, more positive potential of inactivation, and quicker recovery from inactivation compared to atrial INa. Real-time polymerase chain reaction and western blot analysis revealed that mRNA and protein levels of NaVβ2 and NaVβ4 subunits, but not NaV1.5, were greater in ventricular myocytes than in atrial myocytes. INa in heterologous HEK 293 cell expression system with coexpressing hNaV1.5 and hNaVβ2/hNaVβ4 showed similar biophysical properties to ventricular INa. Greater protein expression of NaVβ2 and NaVβ4 subunits was also observed in human ventricles. Interestingly, pharmacologic study revealed that the antiarrhythmic drug dronedarone (10 μM) inhibited atrial INa more (by 73%) than ventricular INa (by 42%), and shifted its inactivation to more negative voltages (-4.6 mV) compared to ventricular INa. CONCLUSION The results of this study demonstrate the novel information that the distinctive biophysical properties of INa in atrial and ventricular myocytes can be attributed to inhomogeneous expression of NaVβ2 and NaVβ4 subunits, and that atrial INa is more sensitive to inhibition by dronedarone.

The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide directly blocks human ether à-go-go-related gene potassium channels stably expressed in human embryonic kidney 293 cells

BACKGROUND AND PURPOSE N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalene sulphonamide (W‐7) is a well‐known calmodulin inhibitor used to study calmodulin regulation of intracellular Ca2+ signalling‐related process. Here, we have determined whether W‐7 would inhibit human ether gene (hERG or Kv11.1) potassium channels, hKv1.5 channels or hKIR2.1 channels expressed in human embryonic kidney (HEK) 293 cells.