[Study on mechanism of inhibiting proliferation of head and neck cancer cells by citral, active ingredient of lemon essential oil].

To explore the active substances exerting anti-tumour effect in lemon essential oil and the molecular mechanism inhibiting the proliferation of head and neck cancer cells SCC15 and CAL33, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT) was utilized to identify the active component inhibiting the proliferation of head and neck cancer cells, namely citral. The IC_(50) of citral inhibiting the proliferation of head and neck cancer cells and normal cells were also determined. In addition, a 5-ethynyl-2'-deoxyuridine(EdU) staining assay was used to detect the effect of citral on the proliferation rate of head and neck cancer cells, and a colony formation assay was used to detect the effect of citral on tumor sphere formation of head and neck cancer cells in vitro. The cell cycle arrest and apoptosis induction of head and neck cancer cells by citral were evaluated by flow cytometry, and Western blot was used to detect the effect of citral on the expression levels of cell cycle-and apoptosis-related proteins in head and neck cancer cells. The findings indicated that citral could effectively inhibit the proliferation and growth of head and neck cancer cells, with anti-tumor activity, and its half inhibitory concentrations for CAL33 and SCC15 were 54.78 and 25.23 µg·mL~(-1), respectively. Furthermore, citral arrested cell cycle at G_2/M phase by down-regulating cell cycle-related proteins such as S-phase kinase associated protein 2(SKP2), C-MYC, cyclin dependent kinase 1(CDK1), and cyclin B. Moreover, citral increased the cysteinyl aspartate-specific proteinase-3(caspase-3), cysteinyl aspartate-specific proteinase-9(caspase-9), and cleaved poly ADP-ribose polymerase(PARP). It up-regulated the level of autophagy-related proteins including microtubule associated protein 1 light chain 3B(LC3B), sequestosome 1(P62/SQSTM1), autophagy effector protein Beclin1(Beclin1), and lysosome-associate membrane protein 1(LAMP1), suggesting that citral could effectively trigger cell apoptosis and cell autophagy in head and neck cancer cells. Furthermore, the dual-tagged plasmid system mCherry-GFP-LC3 was used, and it was found that citral impeded the fusion of autophagosomes and lysosomes, leading to autophagic flux blockage. Collectively, our findings reveal that the main active anti-proliferation component of lemon essential oil is citral, and this component has a significant inhibitory effect on head and neck cancer cells. Its underlying molecular mechanism is that citral induces apoptosis and autophagy by cell cycle arrest and ultimately inhibits cell proliferation.

NMDARs activation regulates endothelial ferroptosis via the PP2A-AMPK-HMGB1 axis.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated, voltage-dependent channels of the ionotropic glutamate receptor family. The present study explored whether NMDAR activation induced ferroptosis in vascular endothelial cells and its complicated mechanisms in vivo and in vitro. Various detection approaches were used to determine the ferroptosis-related cellular iron content, lipid reactive oxygen species (LOS), siRNA molecules, RNA-sequence, MDA, GSH, and western blotting. The AMPK activator Acadesine (AICAR), HMGB1 inhibitor glycyrrhizin (GLY), PP2A inhibitor LB-100, and NMDAR inhibitor MK801 were used to investigate the involved in vivo and in vitro pathways. The activation of NMDAR with L-glutamic acid (GLU) or NMDA significantly promoted cellular ferroptosis, iron content, MDA, and the PTGS2 expression, while decreasing GPX4 expression and GSH concentration in human umbilical vein endothelial cells (HUVECs), which was reversed by ferroptosis inhibitors Ferrostatin-1(Fer-1), Liproxstatin-1 (Lip-1), or Deferoxamine (DFO). RNA-seq revealed that ferroptosis and SLC7A11 participate in NMDA or GLU-mediated NMDAR activation. The PP2A-AMPK-HMGB1 pathway was majorly associated with NMDAR activation-induced ferroptosis, validated using the PP2A inhibitor LB-100, AMPK activator AICAR, or HMGB1 siRNA. The role of NMDAR in ferroptosis was validated in HUVECs induced with the ferroptosis activator errasin or RSL3 and counteracted by the NMDAR inhibitor MK-801. The in vivo results showed that NMDA- or GLU-induced ferroptosis and LOS production was reversed by MK-801, LB-100, AICAR, MK-801, and GLY, confirming that the PP2A-AMPK-HMGB1 pathway is involved in NMDAR activation-induced vascular endothelium ferroptosis. In conclusion, the present study demonstrated a novel role of NMDAR in endothelial cell injury by regulating ferroptosis via the PP2A-AMPK-HMGB1 pathway.

Concanavalin A promotes angiogenesis and proliferation in endothelial cells through the Akt/ERK/Cyclin D1 axis.

CONTEXT: Concanavalin A (Con A) exhibited multiple roles in cancer cells. However, the role of Con A in endothelial cells was not reported. OBJECTIVE: Our present study investigated the potential angiogenic role of Con A in endothelial cells and ischaemic hind-limb mice. MATERIALS AND METHODS: Human umbilical vein endothelial cells and Ea.hy926 cells were employed to determine the effect of Con A (0.3, 1, and 3 µg/mL) or vehicle on angiogenesis and cell proliferation with tube formation, ELISA, flow cytometry, EdU, and western blot. Hind-limb ischaemic mice were conducted to determine the pro-angiogenic effect of Con A (10 mg/kg) for 7 days. RESULTS: Con A promoted tube formation to about three-fold higher than the control group and increased the secretion of VEGFa, PDGFaa, and bFGF in the medium. The cell viability was promoted to 1.3-fold by Con A 3 µg/mL, and cell cycle progression of G0G1 phase was decreased from 77% in the vehicle group to 70% in Con A 3 µg/mL, G2M was promoted from 15 to 19%, and S-phase was from 7 to 10%. Con A significantly stimulated phosphorylation of Akt and ERK1/2 and expression of cyclin D1 and decreased the expression of p27. These effects of Con A were antagonised by the PI3K inhibitor LY294002 (10 µM) and MEK pathway antagonist PD98059 (10 µM). Moreover, Con A (10 mg/kg) exhibited a repair effect in ischaemic hind-limb mice. DISCUSSION AND CONCLUSIONS: This study will provide a new option for treating ischaemic disease by local injection with Con A.

Regulation of the TRPC1 channel by endothelin-1 in human atrial myocytes.

BACKGROUND: Our recent study demonstrated that the nonselective cation current mediated by the transient receptor potential canonical 1 (TRPC1) channel is activated by endothelin-1 (ET-1) in human atrial myocytes; however, the related signal molecules involved are unknown. OBJECTIVE: The purpose of this study was to investigate how the TRPC1 channel is regulated by ET-1 and whether it is upregulated in human atria from patients with atrial fibrillation (AF). METHODS: Whole-cell patch technique and molecular biology techniques were used in the study. RESULTS: The ET-1-evoked TRPC1 current was inhibited by the ET-1 type A (ETA) receptor antagonist BQ123 and the ET-1 type B (ETB) receptor antagonist BQ788 as well as the protein kinase C inhibitor chelerythrine. ETA receptor-mediated TRPC1 channel activity was selectively inhibited by the phosphoinositide-3-kinase inhibitor wortmannin, while ETB receptor-mediated TRPC1 activity was inhibited by the phospholipase C inhibitor U73122. The messenger RNAs and proteins of the TRPC1 channel and ETA receptor, but not the ETB receptor, were significantly upregulated in atria from patients with AF. The basal TRPC1 current increased in AF myocytes, and the response to ET-1 was greater in AF myocytes than in sinus rhythm myocytes. ET-1 induced a delayed repolarization in 20% of AF myocytes. CONCLUSION: These results demonstrate for the first time that TRPC1 activation by ET-1 is mediated by protein kinase C through the distinct phospholipids pathways phosphoinositide-3-kinase and phospholipase C and that the TRPC1 channel and ETA receptor are upregulated in AF atria, which are likely involved in atrial electrical remodeling in patients with AF.

Comparative study of carvedilol and quinidine for inhibiting hKv4.3 channel stably expressed in HEK 293 cells.

The inhibition of transient outward potassium current (Ito) is the major ionic mechanism for quinidine to treat Brugada syndrome; however, quinidine is inaccessible in many countries. The present study compared the inhibitory effect of the nonselective ß-adrenergic blocker carvedilol with quinidine on human Kv4.3 (hKv4.3, encoding for Ito) channel and action potential notch using a whole-cell patch technique in HEK 293 cell line expressing KCND3 as well as in ventricular epicardial myocytes of rabbit hearts. It was found that carvedilol and quinidine inhibited hKv4.3 current in a concentration-dependent manner. The IC50 of carvedilol was 1.2 µM for inhibiting hKv4.3 charge area, while the IC50 of quinidine was 2.9 µM (0.2 Hz). Both carvedilol and quinidine showed typical open channel blocking properties (i.e. decreasing the time to peak of activation and increasing the inactivation of hKv4.3), negatively shifted the V1/2 of activation and inactivation, and slowed the recovery from inactivation of the channel. Although carvedilol had weaker in use- and rate-dependent inhibition of hKv4.3 peak current than quinidine, its reduction of the charge area was more than quinidine at all frequencies (0.2-3.3 Hz). Moreover, the inhibitory effect of carvedilol on action potential notch was greater than quinidine. These results provide the novel information that carvedilol, like quinidine, significantly inhibits hKv4.3 and action potential notch, suggesting that carvedilol is likely an alternative drug for preventing malignant ventricular arrhythmias in patients with Brugada syndrome in countries where quinidine is unavailable.

Distribution patterns and variation analysis of simple sequence repeats in different genomic regions of bovid genomes.

As the first examination of distribution, guanine-cytosine (GC) pattern, and variation analysis of microsatellites (SSRs) in different genomic regions of six bovid species, SSRs displayed nonrandomly distribution in different regions. SSR abundances are much higher in the introns, transposable elements (TEs), and intergenic regions compared to the 3'-untranslated regions (3'UTRs), 5'UTRs and coding regions. Trinucleotide perfect SSRs (P-SSRs) were the most frequent in the coding regions, whereas, mononucleotide P-SSRs were the most in the introns, 3'UTRs, TEs, and intergenic regions. Trifold P-SSRs had more GC-contents in the 5'UTRs and coding regions than that in the introns, 3'UTRs, TEs, and intergenic regions, whereas mononucleotide P-SSRs had the least GC-contents in all genomic regions. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs showed significantly different distributions in different regions (P < 0.01). Except for the coding regions, mononucleotide P-SSRs had the most RCNs, followed by the pattern: di- > tri- > tetra- > penta- > hexanucleotide P-SSRs in the same regions. The analysis of coefficient of variability (CV) of SSRs showed that the CV variations of RCN of the same mono- to hexanucleotide SSRs were relative higher in the intronic and intergenic regions, followed by the CV variation of RCN in the TEs, and the relative lower was in the 5'UTRs, 3'UTRs, and coding regions. Wide SSR analysis of different genomic regions has helped to reveal biological significances of their distributions.

Bradykinin-mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c-Kit+ progenitor cells.

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ ( Cai2+ ) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store-operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Cai2+ via ER-IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit+ progenitor cells.

Noradrenaline up-regulates volume-regulated chloride current by PKA-independent cAMP/exchange protein activated by cAMP pathway in human atrial myocytes.

BACKGROUND AND PURPOSE: Adrenergic regulation of cell volume-regulated chloride current (ICl.vol ) is species-dependent. The present study investigates the mechanism underlying adrenergic regulation of ICl.vol in human atrial myocytes. EXPERIMENTAL APPROACH: Conventional whole-cell patch voltage-clamp techniques were used to record membrane current in human atrial myocytes. ICl.vol was evoked by hyposmotic bath solution (0.6 times isosmotic, 0.6 T). KEY RESULTS: ICl.vol was augmented by noradrenaline (1 µM) during cell swelling in 0.6 T but not under isosmotic (1 T) conditions. Up-regulation of ICl.vol in 0.6 T was blocked by the ß-adrenoceptor antagonist propranolol (2 µM), but not by the α1 -adrenoceptor antagonist prazosin (2 µM). This ß-adrenergic response involved cAMP but was independent of PKA; the protein kinase inhibitor H-89 (2 µM) or PKI (10 µM in pipette solution) failed to prevent ICl.vol up-regulation by noradrenaline. Moreover, the PI3K/PKB inhibitor LY294002 (50 µM) and the PKG inhibitor KT5823 (10 µM) did not affect noradrenaline-induced increases in ICl.vol . Interestingly, the exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2'-O-Me-cAMP (50 µM) also up-regulated ICl.vol , and the noradrenaline-induced increase of ICl.vol in 0.6 T was reversed or prevented by the Epac inhibitor ESI-09 (10 µM). CONCLUSION AND IMPLICATIONS: These data show that ICl.vol evoked by cell swelling of human atrial myocytes is up-regulated by noradrenaline via a PKA-independent cAMP/Epac pathway in human atrial myocytes. cAMP/Epac-induced ICl.vol is expected to shorten action potential duration during human atrial myocytes swelling and may be involved in abnormal cardiac electrical activity during cardiac pathologies that evoke ß-adrenoceptor signalling.

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.

Effects of equol on multiple K+ channels stably expressed in HEK 293 cells.

The present study investigated the effects of equol on cardiovascular K+ channel currents. The cardiovascular K+ channel currents were determined in HEK 293 cells stably expressing cloned differential cardiovascular K+ channels with conventional whole-cell patch voltage-clamp technique. We found that equol inhibited hKv1.5 (IC50: 15.3 µM), hKv4.3 (IC50: 29.2 µM and 11.9 µM for hKv4.3 peak current and charge area, respectively), IKs (IC50: 24.7 µM) and IhERG (IC50: 31.6 and 56.5 µM for IhERG.tail and IhERG.step, respectively), but not hKir2.1 current, in a concentration-dependent manner. Interestingly, equol increased BKCa current with an EC50 of 0.1 µM. It had no significant effect on guinea pig ventricular action potentials at concentrations of ≤3 µM. These results demonstrate that equol inhibits several cardiac K+ currents at relatively high concentrations, whereas it increases BKCa current at very low concentrations, suggesting that equol is a safe drug candidate for treating patients with cerebral vascular disorders.

Tyrphostin AG556 increases the activity of large conductance Ca2+ -activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase.

The present study was designed to investigate whether large conductance Ca2+ -activated K+ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK-HEK 293 cells expressing both functional α-subunits and the auxiliary ß1-subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK-HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α- and ß1-subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre-contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

BACKGROUND AND PURPOSE: The ultra-rapidly activating delayed rectifier K+ current IKur (encoded by Kv 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). EXPERIMENTAL APPROACH: Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial IKur and hKv1.5 channels stably expressed in HEK 293 cells. KEY RESULTS: Human atrial IKur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of IKur induced by 30 µM genistein or 10 µM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hKv 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 µM) slightly enhanced IKur and hKv 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hKv 1.5 channels and that the decrease was countered by orthovanadate. CONCLUSION AND IMPLICATIONS: The PTK inhibitors genistein and AG556 decrease human atrial IKur and cloned hKv 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases IKur and hKv 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial IKur .

Clemizole hydrochloride blocks cardiac potassium currents stably expressed in HEK 293 cells.

BACKGROUND AND PURPOSE: Clemizole, a histamine H1 receptor antagonist has a potential therapeutic effect on hepatitis C infection and also potently inhibits TRPC5 ion channels. The aim of the present study was to investigate whether clemizole blocks cardiac K+ currents and thus affects cardiac repolarization. EXPERIMENTAL APPROACH: Whole-cell patch techniques was used to examine the effects of clemizole on hERG channel current, IKs and Kv 1.5 channel current in HEK 293 cell expression systems as well as on ventricular action potentials of guinea pig hearts. Isolated hearts from guinea pigs were used to determine the effect on the ECG. KEY RESULTS: Clemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (IC50 : 0.07 µM). The S631A, S636A, Y652A and F656V hERG mutant channels reduced the inhibitory effect of clemizole (IC50 : 0.82, 0.89, 1.49 and 2.98 µM, respectively), suggesting that clemizole is a pore blocker of hERG channels. Clemizole also moderately decreased IKs and human Kv 1.5 channel current. Moreover, clemizole increased the duration of the ventricular action potential in guinea pig hearts and the QTc interval in isolated perfused hearts from guinea pigs, in a concentration-dependent manner (0.1-1.0 µM). CONCLUSION AND IMPLICATIONS: Our results provide the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac IKs , delays cardiac repolarization and thereby prolongs QT interval. Thus, caution should be taken when clemizole is used as a TRPC5 channel blocker or for treating hepatitis C infection.

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.

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.

Functional TRPV2 and TRPV4 channels in human cardiac c-kit(+) progenitor cells.

The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells.

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.

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.

Roles of store-operated Ca2+ channels in regulating cell cycling and migration of human cardiac c-kit+ progenitor cells.

Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.

Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells.

Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa), a voltage-gated TTX-sensitive sodium current (INa.TTX), and an inward rectifier K+ current (IKir) were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.