Kir2.1 channel regulates macrophage polarization via the Ca2+/CaMK II/ERK/NF-κB signaling pathway.

Macrophage polarization plays a key role in the inflammatory response. Various ion channels expressed in macrophages have been documented, but very little is known about their roles in macrophage polarization. We found that knockdown or blockade of the Kir2.1 (also known as KCNJ2) channel significantly inhibited M1 macrophage polarization, but promoted M2 macrophage polarization. Lipopolysaccharide (LPS)-induced M1 polarization was also remarkably suppressed in high extracellular K+ solutions (70 mM K+), and this inhibition was partially abolished by adding Ca2+ to the culture medium. Ca2+ imaging showed that Ca2+ influx was dependent on the hyperpolarized membrane potential generated by the Kir2.1 channel. The upregulation of phospho (p)-CaMK II, p-ERK, and p-NF-κB proteins in macrophages from the RAW264.7 cell line that were stimulated with LPS was significantly reversed by blocking the Kir2.1 channel or culturing the cells with 70 mM K+ medium. Furthermore, in vivo studies showed that mice treated with a Kir2.1 channel blocker were protected from LPS-induced peritonitis. In summary, our data reveal the essential role of the Kir2.1 channel in regulating macrophage polarization via the Ca2+/CaMK II/ERK/NF-κB signaling pathway.

Acacetin, a Natural Flavone with Potential in Improving Liver Disease Based on Its Anti-Inflammation, Anti-Cancer, Anti-Infection and Other Effects.

Liver disease is a global public problem, and the cost of its therapy is a large financial burden to governments. It is well known that drug therapy plays a critical role in the treatment of liver disease. However, present drugs are far from meeting clinical needs. Lots of efforts have been made to find novel agents to treat liver disease in the past several decades. Acacetin is a dihydroxy and monomethoxy flavone, named 5,7-dihydroxy-4'-methoxyflavone, which can be found in diverse plants. It has been reported that acacetin exhibits multiple pharmacological activities, including anti-cancer, anti-inflammation, anti-virus, anti-obesity, and anti-oxidation. These studies indicate the therapeutic potential of acacetin in liver disease. This review discussed the comprehensive information on the pathogenesis of liver disease (cirrhosis, viral hepatitis, drug-induced liver injury, and hepatocellular carcinoma), then introduced the biological source, structural features, and pharmacological properties of acacetin, and the possible application in preventing liver disease along with the pharmacokinetic and toxicity of acacetin, and future research directions. We systemically summarized the latest research progress on the potential therapeutic effect of acacetin on liver disease and existing problems. Based on the present published information, the natural flavone acacetin is an anticipated candidate agent for the treatment of liver disease.

The natural flavone acacetin protects against high-fat diet-induced lipid accumulation in the liver via the endoplasmic reticulum stress/ferroptosis pathway.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. To date, no medication has been approved to treat NAFLD. In this study, we evaluated the therapeutic effect of the natural flavone acacetin on high-fat diet (HFD)-induced NAFLD in mice and the underlying mechanisms. We found that acacetin (10, 20, 50 mg/kg/day) suppressed the increase in body weight, serum total cholesterol, triglycerides, low-density lipoprotein, aspartate aminotransferase, and alanine aminotransferase levels in mice fed with HFD with a dose-dependent manner. Hepatic lipid accumulation, iron overload, and lipid peroxidation were significantly alleviated by acacetin. Quantitative PCR and western blotting revealed that acacetin inhibited endoplasmic reticulum (ER) stress, ferroptosis, and expressions of lipid acid synthesis-related genes in the livers of HFD mice. Similar results were observed in HepG2 cells treated with oleic acid and lipopolysaccharide. The suppressive effects of acacetin on triglycerides and expression of lipid acid synthesis genes were abolished by ER stress and the ferroptosis activators, erastin or TU. Interestingly, the action of TU was more potent than that of erastin. Treatment with the ER stress inhibitor GSK and the ferroptosis inhibitor Fer-1 revealed that ER stress was the upstream signal of ferroptosis for hepatic lipid accumulation. These findings suggest the protective effect of acacetin against lipid accumulation via suppressing ER stress and ferroptosis and provide evidence that ER stress is an upstream signal of ferroptosis in lipid accumulation. Acacetin may be a promising candidate agent for NAFLD treatment.

Functional Potassium Channels in Macrophages.

Macrophages are the predominant component of innate immunity, which is an important protective barrier of our body. Macrophages are present in all organs and tissues of the body, their main functions include immune surveillance, bacterial killing, tissue remodeling and repair, and clearance of cell debris. In addition, macrophages can present antigens to T cells and facilitate inflammatory response by releasing cytokines. Macrophages are of high concern due to their crucial roles in multiple physiological processes. In recent years, new advances are emerging after great efforts have been made to explore the mechanisms of macrophage activation. Ion channel is a class of multimeric transmembrane protein that allows specific ions to go through cell membrane. The flow of ions through ion channel between inside and outside of cell membrane is required for maintaining cell morphology and intracellular signal transduction. Expressions of various ion channels in macrophages have been detected. The roles of ion channels in macrophage activation are gradually caught attention. K+ channels are the most studied channels in immune system. However, very few of published papers reviewed the studies of K+ channels on macrophages. Here, we will review the four types of K+ channels that are expressed in macrophages: voltage-gated K+ channel, calcium-activated K+ channel, inwardly rectifying K+ channel and two-pore domain K+ channel.

K2P1 leak cation channels contribute to ventricular ectopic beats and sudden death under hypokalemia.

Hypokalemia causes ectopic heartbeats, but the mechanisms underlying such cardiac arrhythmias are not understood. In reduced serum K+ concentrations that occur under hypokalemia, K2P1 two-pore domain K+ channels change ion selectivity and switch to conduct inward leak cation currents, which cause aberrant depolarization of resting potential and induce spontaneous action potential of human cardiomyocytes. K2P1 is expressed in the human heart but not in mouse hearts. We test the hypothesis that K2P1 leak cation channels contribute to ectopic heartbeats under hypokalemia, by analysis of transgenic mice, which conditionally express induced K2P1 specifically in hearts, mimicking K2P1 channels in the human heart. Conditional expression of induced K2P1 specifically in the heart of hypokalemic mice results in multiple types of ventricular ectopic beats including single and multiple ventricular premature beats as well as ventricular tachycardia and causes sudden death. In isolated mouse hearts that express induced K2P1, sustained ventricular fibrillation occurs rapidly after perfusion with low K+ concentration solutions that mimic hypokalemic conditions. These observed phenotypes occur rarely in control mice or in the hearts that lack K2P1 expression. K2P1-expressing mouse cardiomyocytes of transgenic mice much more frequently fire abnormal single and/or rhythmic spontaneous action potential in hypokalemic conditions, compared to wild type mouse cardiomyocytes without K2P1 expression. These findings confirm that K2P1 leak cation channels induce ventricular ectopic beats and sudden death of transgenic mice with hypokalemia and imply that K2P1 leak cation channels may play a critical role in human ectopic heartbeats under hypokalemia.

Protective Effect of Water-Soluble Acacetin Prodrug on APAP-Induced Acute Liver Injury Is Associated with Upregulation of PPARγ and Alleviation of ER Stress.

A water-soluble acacetin prodrug has been synthesized and reported by our group previously. Acetaminophen (APAP) overdose is a leading cause of acute liver injury. We found that subcutaneous injection of acacetin prodrug (5, 10, 20 mg/kg) decreased serum ALT, AST, and ALP, corrected the abnormal MDA and GSH in liver, and improved intrahepatic hemorrhage and destruction of liver structures in APAP (300 mg/kg)-treated mice. Molecular mechanism analysis revealed that the expressions of endoplasmic reticulum (ER) stress markers ATF6, CHOP, and p-PERK, apoptosis-related protein BAX, and cleaved caspase 3 were decreased by acacetin in a dose-dependent manner in vivo and in vitro. Moreover, via the acacetin-upregulated peroxisome-proliferator-activated receptor gamma (PPARγ) of HepG2 cells and liver, the suppressive effect of acacetin on ER stress and apoptosis was abolished by PPARγ inhibitor (GW9662) or PPARγ-siRNA. Molecular docking revealed that acacetin can bind to three active pockets of PPARγ, mainly by hydrogen bond. Our results provide novel evidence that acacetin prodrug exhibits significant protective effect against APAP-induced liver injury by targeting PPARγ, thereby suppressing ER stress and hepatocyte apoptosis. Acacetin prodrug is likely a promising new drug candidate for treating patients with acute liver injury induced by APAP.

Kir2 inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate pacemaking activity.

Spontaneous rhythmic action potential or pacemaking activity of pacemaker cells controls rhythmic signaling such as heartbeat. The mechanism underlying the origin of pacemaking activity is not well understood. In this study, we created human embryonic kidney (HEK) 293 cells that show pacemaking activity through heterologous expression of strong inward rectifier K+ subfamily 2 isoform 1 (Kir2.1) channels, hyperpolarization-activated cyclic nucleotide-gated isoform 2 (HCN2) nonselective cation channels, and voltage-gated Na+ subfamily 1 isoform 5 or Ca2+ subfamily 3 isoform 1 (Nav1.5 or Cav3.1) channels. A range of relative levels of Kir2.1 and HCN2 currents dynamically counterbalance, generating spontaneous rhythmic oscillation of resting membrane potential between -64 and -34 mV and determining oscillation rates. Each oscillation cycle begins with an autodepolarization phase, which slowly proceeds to the threshold potential that activates Nav1.5 or Cav3.1 channels and triggers action potential, causing engineered HEK293 cells to exhibit pacemaking activity at a rate of ≤67 beats/min. Engineered HEK293 cells with Kir2.1 and either HCN3 or HCN4 also show the oscillation. Engineered HEK293 cells expressing HCN2 and other Kir2 channels, which lack Kir2.1-like complete inward rectification, do not show the oscillation. Therefore, Kir2.1-like inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate spontaneous rhythmic action potential and form an origin of pacemaking activity; Kir2 and HCN channels play essential roles in pacemaking activity.-Chen, K., Zuo, D., Wang, S.-Y. Chen, H. Kir2 inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate pacemaking activity.

Kir2.1 channels set two levels of resting membrane potential with inward rectification.

Strong inward rectifier K+ channels (Kir2.1) mediate background K+ currents primarily responsible for maintenance of resting membrane potential. Multiple types of cells exhibit two levels of resting membrane potential. Kir2.1 and K2P1 currents counterbalance, partially accounting for the phenomenon of human cardiomyocytes in subphysiological extracellular K+ concentrations or pathological hypokalemic conditions. The mechanism of how Kir2.1 channels contribute to the two levels of resting membrane potential in different types of cells is not well understood. Here we test the hypothesis that Kir2.1 channels set two levels of resting membrane potential with inward rectification. Under hypokalemic conditions, Kir2.1 currents counterbalance HCN2 or HCN4 cation currents in CHO cells that heterologously express both channels, generating N-shaped current-voltage relationships that cross the voltage axis three times and reconstituting two levels of resting membrane potential. Blockade of HCN channels eliminated the phenomenon in K2P1-deficient Kir2.1-expressing human cardiomyocytes derived from induced pluripotent stem cells or CHO cells expressing both Kir2.1 and HCN2 channels. Weakly inward rectifier Kir4.1 or inward rectification-deficient Kir2.1•E224G mutant channels do not set such two levels of resting membrane potential when co-expressed with HCN2 channels in CHO cells or when overexpressed in human cardiomyocytes derived from induced pluripotent stem cells. These findings demonstrate a common mechanism that Kir2.1 channels set two levels of resting membrane potential with inward rectification by balancing inward currents through different cation channels such as hyperpolarization-activated HCN channels or hypokalemia-induced K2P1 leak channels.

Kir2.1 and K2P1 channels reconstitute two levels of resting membrane potential in cardiomyocytes.

KEY POINTS: Outward and inward background currents across the cell membrane balance, determining resting membrane potential. Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the resting membrane potential of cardiomyocytes. Human cardiomyocytes exhibit two levels of resting membrane potential at subphysiological extracellular K+ concentrations or pathological hypokalaemia, however, the underlying mechanism is unclear. In the present study, we show that human cardiomyocytes derived from induced pluripotent stem cells with enhanced expression of isoform 1 of Kir2 (Kir2.1) channels and mouse HL-1 cardiomyocytes with ectopic expression of two pore-domain K+ channel isoform 1 (K2P1) recapitulate two levels of resting membrane potential, indicating the contributions of Kir2.1 and K2P1 channels to the phenomenon. In Chinese hamster ovary cells that express the channels, Kir2.1 currents non-linearly counterbalance hypokalaemia-induced K2P1 leak cation currents, reconstituting two levels of resting membrane potential. These findings support the hypothesis that Kir2 currents non-linearly counterbalance inward background cation currents, such as K2P1 currents, accounting for two levels of resting membrane potential in human cardiomyocytes and demonstrating a novel mechanism that regulates excitability. ABSTRACT: Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the normal resting membrane potential of cardiomyocytes. At subphysiological extracellular K+ concentrations or pathological hypokalaemia, human cardiomyocytes show both hyperpolarized and depolarized resting membrane potentials; these depolarized potentials cause cardiac arrhythmia; however, the underlying mechanism is unknown. In the present study, we show that inward rectifier K+ channel subfamily 2 isoform 1 (Kir2.1) currents non-linearly counterbalance hypokalaemia-induced two pore-domain K+ channel isoform 1 (K2P1) leak cation currents, reconstituting two levels of resting membrane potential in cardiomyocytes. Under hypokalaemic conditions, both human cardiomyocytes derived from induced pluripotent stem cells with enhanced Kir2.1 expression and mouse HL-1 cardiomyocytes with ectopic expression of K2P1 channels recapitulate two levels of resting membrane potential. These cardiomyocytes display N-shaped current-voltage relationships that cross the voltage axis three times and the first and third zero-current potentials match the two levels of resting membrane potential. Inhibition of K2P1 expression eliminates the phenomenon, indicating contributions of Kir2.1 and K2P1 channels to two levels of resting membrane potential. Second, in Chinese hamster ovary cells that heterologously express the channels, Kir2.1 currents non-linearly counterbalance hypokalaemia-induced K2P1 leak cation currents, yielding the N-shaped current-voltage relationships, causing the resting membrane potential to spontaneously jump from hyperpolarization at the first zero-current potential to depolarization at the third zero-current potential, again recapitulating two levels of resting membrane potential. These findings reveal ionic mechanisms of the two levels of resting membrane potential, demonstrating a previously unknown mechanism for the regulation of excitability, and support the hypothesis that Kir2 currents non-linearly balance inward background cation currents, accounting for two levels of resting membrane potential of human cardiomyocytes.

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.

SKF-96365 strongly inhibits voltage-gated sodium current in rat ventricular myocytes.

SKF-96365 (1-(beta-[3-(4-methoxy-phenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride) is a general TRPC channel antagonist commonly used to characterize the potential functions of TRPC channels in cardiovascular system. Recent reports showed that SKF-96365 induced a reduction in cardiac conduction. The present study investigates whether the reduced cardiac conduction caused by SKF-96365 is related to the blockade of voltage-gated sodium current (I Na) in rat ventricular myocytes using the whole-cell patch voltage-clamp technique. It was found that SKF-96365 inhibited I Na in rat ventricular myocytes in a concentration-dependent manner. The compound (1 µM) negatively shifted the potential of I Na availability by 9.5 mV, increased the closed-state inactivation of I Na, and slowed the recovery of I Na from inactivation. The inhibition of cardiac I Na by SKF-96365 was use-dependent and frequency-dependent, and the IC50 was decreased from 1.36 µM at 0.5 Hz to 1.03, 0.81, 0.61, 0.56 µM at 1, 2, 5, 10 Hz, respectively. However, the selective TRPC3 antagonist Pyr3 decreased cardiac I Na by 8.5% at 10 µM with a weak use and frequency dependence. These results demonstrate that the TRPC channel antagonist SKF-96365 strongly blocks cardiac I Na in use-dependent and frequency-dependent manners. Caution should be taken for interpreting the alteration of cardiac electrical activity when SKF-96365 is used in native cells as a TRPC antagonist.

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 Mg(2+) -free pipette solution in 3T3-L1 preadipocytes, and the current was inhibited by intercellular free Mg(2+) . 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.

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 Mg(2+)-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca(2+) and Mg(2+), and the current increase could be inhibited by Ni(2+) or Ba(2+). The TRPM7-like current was potentiated by acidic pH and inhibited by La(3+) and 2-aminoethoxydiphenyl borate. In addition, Ca(2+)-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca(2+) 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.

Exosomal microRNAs: potential targets for the prevention and treatment of diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM), a condition in which myocardial dysfunction is caused by diabetes mellitus, has become an epidemic disorder in the world. DCM initially presents as diastolic relaxation dysfunction and will progress to heart failure in the absence of coronary artery disease, valvular disease, and other conventional cardiovascular risk factors such as hypertension and dyslipidemia. However, the underlying molecular mechanisms of DCM are poorly understood. Recent studies reveal that exosomal miRNAs are associated with multiple DCM risk factors and may act as potential therapeutic targets. Therefore, this review summarizes the recent advancements to understand the role of exosomal miRNAs in DCM development and explores potential preventative and therapeutic strategies.

PPARα mediates night neon light-induced weight gain: role of lipid homeostasis.

Rationale: Light pollution leads to high risk of obesity but the underlying mechanism is not known except for the influence of altered circadian rhythm. Peroxisome proliferator-activated receptor α (PPARα) regulates lipid metabolism, but its role in circadian-related obesity is not clear. Methods: Wild-type (WT) and Ppara-null (KO) mice on a high-fat diet (HFD) were treated with neon light at night for 6 weeks. Body weights were recorded and diet consumption measured. The hypothalamus, liver, adipose and serum were collected for mechanism experimentation. Results: WT mice on a HFD and exposed to night neon light gained about 19% body weight more than the WT control mice without light exposure and KO control mice on a HFD and exposed to night neon light. The increase in adipose tissue weight and adipocyte size led to the differences in body weights. Biochemical analysis suggested increased hepatic lipid accumulated and increased transport of lipid from the liver to peripheral tissues in the WT mice that gained weight under neon light exposure. Unlike KO mice, the expression of genes involved in lipid metabolism and the circadian factor circadian locomotor output cycles kaput (CLOCK) in both liver and adipose tissues were elevated in WT mice under neon light exposure. Conclusions: PPARα mediated weight gain of HFD-treated mice exposed to night neon light. More lipids were synthesized in the liver and transported to peripheral tissue leading to adaptive metabolism and lipid deposition in the adipose tissue. These data revealed an important mechanism of obesity induced by artificial light pollution where PPARα was implicated.

The Natural Flavone Acacetin Blocks Small Conductance Ca2+-Activated K+ Channels Stably Expressed in HEK 293 Cells.

The natural flavone acacetin inhibits several voltage-gated potassium currents in atrial myocytes, and has anti-atrial fibrillation (AF) effect in experimental AF models. The present study investigates whether acacetin inhibits the Ca2+-activated potassium (KCa) currents, including small conductance (SKCa1, SKCa2, and SKCa3), intermediate conductance (IKCa), and large-conductance (BKCa) channels stably expressed in HEK 293 cells. The effects of acacetin on these KCa channels were determined with a whole-cell patch voltage-clamp technique. The results showed that acacetin inhibited the three subtype SKCa channel currents in concentration-dependent manner with IC50 of 12.4 µM for SKCa1, 10.8 µM for SKCa2, and 11.6 µM for SKCa3. Site-directed mutagenesis of SKCa3 channels generated the mutants H490N, S512T, H521N, and A537V. Acacetin inhibited the mutants with IC50 of 118.5 µM for H490N, 275.2 µM for S512T, 15.3 µM for H521N, and 10.6 µM for A537V, suggesting that acacetin interacts with the P-loop helix of SKCa3 channel. However, acacetin at 3-10 µM did not decrease, but induced a slight increase of BKCa (+70 mV) by 8% at 30 µM. These results demonstrate the novel information that acacetin remarkably inhibits SKCa channels, but not IKCa or BKCa channels, which suggests that blockade of SKCa by acacetin likely contributes to its anti-AF property previously observed in experimental AF.

Water-soluble acacetin prodrug confers significant cardioprotection against ischemia/reperfusion injury.

The morbidity and mortality of patients with ischemic cardiomyopathy resulted from ischemia/reperfusion injury are very high. The present study investigates whether our previously synthesized water-soluble phosphate prodrug of acacetin was cardioprotective against ischemia/reperfusion injury in an in vivo rat model. We found that intravenous administration of acacetin prodrug (10 mg/kg) decreased the ventricular arrhythmia score and duration, reduced ventricular fibrillation and infarct size, and improved the impaired heart function induced by myocardial ischemia/reperfusion injury in anesthetized rats. The cardioprotective effects were further confirmed with the parent compound acacetin in an ex vivo rat regional ischemia/reperfusion heart model. Molecular mechanism analysis revealed that acacetin prevented the ischemia/reperfusion-induced reduction of the anti-oxidative proteins SOD-2 and thioredoxin, suppressed the release of inflammation cytokines TLR4, IL-6 and TNFα, and decreased myocyte apoptosis induced by ischemia/reperfusion. Our results demonstrate the novel evidence that acacetin prodrug confer significant in vivo cardioprotective effect against ischemia/reperfusion injury by preventing the reduction of endogenous anti-oxidants and the release of inflammatory cytokines, thereby inhibiting cardiomyocytes apoptosis, which suggests that the water-soluble acacetin prodrug is likely useful in the future as a new drug candidate for treating patients with acute coronary syndrome.

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 NaVß2 and NaVß4 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 hNaVß2/hNaVß4 showed similar biophysical properties to ventricular INa. Greater protein expression of NaVß2 and NaVß4 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 NaVß2 and NaVß4 subunits, and that atrial INa is more sensitive to inhibition by dronedarone.

Pentamethylquercetin ameliorates fibrosis in diabetic Goto-Kakizaki rat kidneys and mesangial cells with suppression of TGF-ß/Smads signaling.

Pentamethylquercetin (PMQ) has been shown to possess glucose-lowering properties, but its effect on renal fibrosis in diabetes is still unclear. This study was designed to investigate the effect of PMQ on renal fibrosis and the underlying mechanisms in spontaneous type II diabetic Goto-Kakizaki rats and mesangial cells in high glucose. We found that in Goto-Kakizaki rats, PMQ treatment attenuated glomerular volume, glycogen deposition, renal collagen and fibronectin accumulation, in addition to amelioration of diabetic symptoms, including reduction of urine volume and urine glucose levels. In mesangial cells, PMQ remarkably inhibited the cell proliferation and total collagen accumulation, and suppressed cell hypertrophy. Further experiments showed that PMQ treatment down-regulated the expression of TGF-ß1, up-regulated Smad7 and inhibited Smad2/3 activation in vivo and vitro. Our results demonstrated that PMQ ameliorated renal fibrosis in diabetes, which may be associated with suppressed TGF-ß/Smads signaling.

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 (IC(50) = 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 IC(50)s 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 IC(50)s 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.