Adipocytes promote tumor progression and induce PD-L1 expression via TNF-α/IL-6 signaling.

BACKGROUND: Obesity confers increased risk for various types of cancer. PD-L1 is a key molecule in tumor immune evasion by inducing T cell exhaustion. The relationship between obesity and PD-L1 is still ambiguous. This study was designed to reveal the development of hepatocellular carcinoma and melanoma in obese mice and to investigate if adipocytes regulate PD-L1 expression and the underlying mechanism. METHODS: Monosodium glutamate-induced obese mice were inoculated with H22 tumor cells and High fat diet (HFD)-induced obese mice were inoculated with B16-F1 mouse melanoma cells. Human hepatoma HepG2 cells and B16-F1 cells were treated with conditional media from 3T3-L1 adipocytes (adi-CM). Neutralized anti-TNF-α and anti-IL-6 antibodies and inhibitor of NF-κB or STAT3 were used to reveal the mechanism of effect of adi-CM. RESULTS: In obese mice, H22 and B16-F1 tumor tissues grew faster and PD-L1 expression in tumor tissue was increased. Adi-CM up-regulated PD-L1 level in HepG2 and B16-F1 cells in vitro. Differentiated 3T3-L1 adipocytes secreted TNF-α and IL-6, and neutralizing TNF-α and/or IL-6 reduced PD-L1 expression in adi-CM-treated cells. p-NF-κB/NF-κB level was downregulated in HepG2 and B16-F1 cells, and p-STAT3/STAT3 level was also decreased in HepG2 cells. In addition, inhibitor of NF-κB or STAT3 reversed the effect of adi-CM on PD-L1 expression. CONCLUSIONS: TNF-α and IL-6 secreted by adipocytes up-regulates PD-L1 in hepatoma and B16-F1 cells, which may be at least partially involved in the role of obesity in promoting tumor progression.

Pentamethylquercetin protects against cardiac remodeling via activation of Sestrin2.

Oxidative stress is widely involved in pathophysiological processes of cardiac remodeling. Molecules associated with antioxidant functions may be ideal targets for reversing cardiac remodeling. Sestrin2 is the important component of endogenous antioxidant defense, while there is little information on the pathophysiological roles of it in cardiac remodeling. The aim of this study was to investigate whether Sestrin2 is closely involved in cardiac remodeling, and whether the protective effect of pentamethylquercetin (PMQ) on cardiac remodeling is related to upregulation of the Sestrin2 endogenous antioxidant system. We generated a transverse aorta constriction (TAC)-induced pressure-overload cardiac-remodeling model in mice, and also established an isoproterenol (ISO)-induced neonatal rat cardiomyocyte (NRCM) hypertrophy model. The data showed Sestrin2 expression was downregulated significantly, and Nrf2 and HO-1 expression was also reduced in myocardial tissue or NRCM of model group, whereas keap1 expression was upregulated. PMQ significantly ameliorated cardiac remodeling and rectified the abnormal expression of Sestrin2/Nrf2/keap1. Sestrin2 small interfering RNA (SiRNA) reduced the protective effect of PMQ on NRCMs, as well as abolished its regulating effect on the Nrf2/keap1 pathway. In conclusion, Sestrin2 may be an important target in the anti-myocardial remodeling of PMQ.

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.

Functional ion channels and cell proliferation in 3T3-L1 preadipocytes.

Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K(+) current (I(Kir), recorded in 15% of cells) inhibited by Ba(2+), a Ca(2+)-activated intermediate K(+) current (IK(Ca), recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I(Cl)) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive I(Cl) (I(Cl.vol)). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK(Ca)), Kir2.1 (for I(Kir)), and Clcn3 (for I(Cl.vol)). Blockade of IK(Ca) with TRAM-34 or I(Cl.vol) with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I(KCa), I(Cl.vol), and I(Kir), are heterogeneously present in 3T3-L1 preadipocytes. I(KCa) and I(Cl.vol) participate in the regulation of cell proliferation.

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.

In vivo and in vitro protective effects of pentamethylquercetin on cardiac hypertrophy.

AIM: To investigate the in vivo and in vitro protective effects of pentamethylquercetin (PMQ), a member of polymethoxy flavonoids (PMFs), on cardiac hypertrophy. METHODS: An in vivo cardiac hypertrophy model established by abdominal aorta banding technique in rats was treated with PMQ in increasing dosages (2.5, 5, and 10 mg x kg(-1) x d(-1)). An in vitro cardiomyocyte hypertrophy model was induced by treating neonatal cardiomyocytes with endothelin-1 (ET-1, 0.1 µM). An in vitro fibrosis model was developed in cardiac fibroblasts by aldosterone (Ald, 20 nM) and treated with PMQ (0.3, 1, 3 and 10 µM). Hemodynamic, morphological, histological, and biochemical changes were evaluated at corresponding time points. RESULTS: The abdominal aorta constriction (AAC) rats demonstrated a significantly elevated blood pressure and profound systolic and diastolic cardiac dysfunction. The resultant cardiac hypertrophy and heart failure were characterized by a significant increase in the heart and lung indices (3.51 ± 0.30 vs 2.35 ± 0.24, 5.58 ± 0.85 vs 3.94 ± 0.54; both P < 0.01), cardiomyocyte cross-sectional areas (153 ± 33% vs 100 ± 5%, P < 0.01) and myocardial fibrosis (9.09 ± 1.30% vs 1.49 ± 0.20%, P < 0.01) with concomitant elevation of B-type natriuretic peptide and cardiac collagen mRNA level. Daily oral administration of PMQ (2.5, 5, and 10 mg/kg for 7 weeks) prevented the foregoing histology, gene and protein changes secondary to AAC procedure. In addition, the up-regulated inflammation factors such as TNF-α and IL-6, and the down-regulated PPAR α and PPAR ß were normalizd by PMQ treatment. CONCLUSION: PMQ has significant protective effects on cardiac hypertrophy through up-regulating the mRNA and protein levels of PPAR α and PPAR ß involved in the process of inflammation response and cardiac fibrosis.

Pentamethylquercetin improves adiponectin expression in differentiated 3T3-L1 cells via a mechanism that implicates PPARγ together with TNF-α and IL-6.

Adiponectin is an adipocyte-derived hormone that plays a pivotal role in the regulation of lipid and glucose metabolism. Up-regulation of adiponectin expression and production has been shown to benefit for metabolic disorders, including type 2 diabetes, hyperlipidemia, etc. The present study investigated whether the novel polymethoxylated flavonoid pentamethylquercetin (PMQ), a member of polymethoxylated flavonoids family which is present in seabuckthorn (Hippophae L.) would affect adiponectin production in differentiated 3T3-L1 adipocytes. It was found that PMQ increased the adiponectin mRNA and protein expressions in adipocytes in time- and concentration-dependent manners. The PPARγ pathway plays a important roles in this effect of PMQ because blockade of PPARγ by GW9662 eliminates the PMQ-induced up-regulation of adiponectin expression. Furthermore, significant decreases of mRNA expression and secretion of TNF-α and IL-6 were also observed in PMQ-treated cells. Taken together, our study demonstrated that PMQ up-regulates adiponectin expression via a mechanism that implicates PPARγ together with TNF-α and IL-6, suggesting that PMQ might be a potential candidate for the treatment of metabolic diseases.

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.

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.

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 (I(Kur)) and other cardiac ionic currents were studied with a whole-cell patch technique. Acacetin suppressed I(Kur) and the transient outward K(+) current (IC(50) 3.2 and 9.2 mumol/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 Ca(2+) 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.

The membrane permeable calcium chelator BAPTA-AM directly blocks human ether a-go-go-related gene potassium channels stably expressed in HEK 293 cells.

BAPTA-AM is a well-known membrane permeable Ca(2+) chelator. The present study found that BAPTA-AM rapidly and reversibly suppressed human ether a-go-go-related gene (hERG or Kv11.1) K(+) current, human Kv1.3 and human Kv1.5 channel currents stably expressed in HEK 293 cells, and the effects were not related to Ca(2+) chelation. The externally applied BAPTA-AM inhibited hERG channels in a concentration-dependent manner (IC(50): 1.3 microM). Blockade of hERG channels was dependent on channel opening, and tonic block was minimal. Steady-state activation V(0.5) of hERG channels was negatively shifted by 8.5 mV (from -3.7+/-2.8 of control to -12.2+/-3.1 mV, P<0.01), while inactivation V(0.5) was negatively shifted by 6.1 mV (from -37.9+/-2.0 mV of control to -44.0+/-1.6 mV, P<0.05) with application of 3 microM BAPTA-AM. The S6 mutant Y652A and the pore helix mutant S631A significantly attenuated blockade by BAPTA-AM at 10 microM causing profound blockade of wild-type hERG channels. In addition, BAPTA-AM inhibited hKv1.3 and hKv1.5 channels in a concentration-dependent manner (IC(50): 1.45 and 1.23 microM, respectively), and the blockade of these two types of channels was also dependent on channel opening. Moreover, EGTA-AM was found to be an open channel blocker of hERG, hKv1.3, hKv1.5 channels, though its efficacy is weaker than that of BAPTA-AM. These results indicate that the membrane permeable Ca(2+) chelator BAPTA-AM (also EGTA-AM) exerts an open channel blocking effect on hERG, hKv1.3 and hKv1.5 channels.

Raloxifene inhibits transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes.

The selective estrogen receptor modulator raloxifene is widely used in the treatment of postmenopausal osteoporosis, and has cardioprotective properties. However, effects of raloxifene on cardiac ion channels are unclear. The present study was designed to investigate the effects of raloxifene and beta-estradiol on transient outward and ultra-rapid delayed rectifier potassium currents (Ito1 and IKur) in human atrial myocytes with a whole cell patch-clamp technique. Ito1 was inhibited by raloxifene in a concentration-dependent manner with an IC50 of 0.9 microM. Raloxifene at 1 microM decreased Ito1 by 40.2+/-1.9% (at +50 mV, n=14, P<0.01 vs control). Time-dependent recovery from inactivation was slowed, and time to peak and time-dependent inactivation of Ito1 were significantly accelerated, while steady-state voltage dependent activation and inactivation of Ito1 were not affected by raloxifene. In addition, raloxifene remarkably suppressed IKur (IC50=0.7 microM). Raloxifene at 1 microM decreased IKur by 57.3+/-3.3% (at +50 mV, n=10, P<0.01 vs control). However, beta-estradiol inhibited Ito1 (IC50=10.3 microM) without affecting IKur. The inhibitory effects of raloxifene and beta-estradiol on Ito1 and/or IKur were unaffected by the estrogen receptor antagonist ICI 182,780. Our results indicate that raloxifene directly inhibits the human atrial repolarization potassium currents Ito1 and IKur. Whether raloxifene is beneficial for supraventricular arrhythmias remains to be studied.

Pentamethylquercetin induces adipose browning and exerts beneficial effects in 3T3-L1 adipocytes and high-fat diet-fed mice.

Browning white adipocytes may be a new target in anti-obesity therapy. Pentamethylquercetin (PMQ) has been shown to have anti-obesity effects in monosodium glutamate-induced obese mice. Here, we aimed to study the anti-obesity effects of PMQ in vitro and in vivo and to determine if adipose browning is involved in the mechanism underlying the anti-obesity effects of PMQ. We evaluated the effects of PMQ on cell proliferation, cell differentiation, glucose consumption, cellular lipid metabolism, and related brown gene expression in 3T3-L1 adipocytes. We also investigated the effects of PMQ in a mouse model of high-fat diet (HFD)-induced obesity. Our results demonstrated that PMQ increased the consumption of glucose, inhibited the accumulation of cellular triglycerides (TGs), and induced the expression of brown adipocyte-specific genes, such as uncoupling protein 1 (UCP-1), during the early stage of differentiation in 3T3-L1 adipocytes. In HFD mice, PMQ treatment reduced waist circumference, LEE index, white adipose tissue (WAT) weight and white adipocyte size and increased brown adipose tissue (BAT) weight. Moreover, PMQ treatment induced mitochondrial biogenesis and upregulated UCP-1 expression in WAT. These findings suggest that PMQ may induce browning of adipose tissue, a phenomenon that is at least partly related to its anti-obesity effects.

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.

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.

Synthesis of a highly water-soluble acacetin prodrug for treating experimental atrial fibrillation in beagle dogs.

We previously reported that duodenal administration of the natural flavone acacetin can effectively prevent the induction of experimental atrial fibrillation (AF) in canines; however, it may not be used intravenously to terminate AF due to its poor water-solubility. The present study was to design a water-soluble prodrug of acacetin and investigate its anti-AF effect in beagle dogs. Acacetin prodrug was synthesized by a three-step procedure. Aqueous solubility, bioconversion and anti-AF efficacy of acacetin prodrug were determined with different methodologies. Our results demonstrated that the synthesized phosphate sodium salt of acacetin prodrug had a remarkable increase of aqueous solubility in H2O and clinically acceptable solution (5% glucose or 0.9% NaCl). The acacetin prodrug was effectively converted into acacetin in ex vivo rat plasma and liver microsome, and in vivo beagle dogs. Intravenous infusion of acacetin prodrug (3, 6 and 12 mg/kg) terminated experimental AF without increasing ECG QTc interval in beagle dogs. The intravenous LD50 of acacetin prodrug was 721 mg/kg in mice. Our preclinical study indicates that the synthesized acacetin prodrug is highly water-soluble and safe; it effectively terminates experimental AF in beagle dogs and therefore may be a promising drug candidate for clinical trial to treat patients with acute AF.

[Effects of hydrocortisone sodium succinate on sodium current in human and guinea pig cardiac myocytes].

AIM: To study the effects of hydrocortisone sodium succinate on sodium current in human atrial myocytes and in guinea pig ventricular myocytes. METHODS: Single cardiac myocytes were isolated by enzyme. The effects of hydrocortisone sodium succinate on sodium current (INa) were assessed by applying whole-cell patch clamp techniques. RESULTS: Hydrocortisone sodium succinate (1, 3, 10 micromol x L(-1)) was shown to inhibit INa of both human atrial myocytes and guinea pig ventricular myocytes in concentration dependent manner and the IC50 were 6.97 and 8.74 micromol x L(-1), respectively. The inhibition effects acted quickly (1-3 min) and the maximal activating voltage of INa was not changed in both human and guinea pig cardiac myocytes. CONCLUSION: Hydrocortisone sodium succinate can exhibit inhibitory effects on INa in both human and guinea pig cardiac myocytes, and its inhibitory effects act rapidly, which are not consistent with genomic effects, so there may be nongenomic effects.

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.