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Aim: To explore the effect of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza) on early renal injury. Materials & methods: Cell damage and inflammation are features of early renal injury. The apoptosis and inflammation in hypoxia/reoxygenation (H/R)-induced human proximal tubular epithelial cells (HK-2) and ischemia–reperfusion kidney were studied, and expression of the protein klotho was investigated. Results: Aza induced HK-2 apoptosis in a dose-dependent manner, but low-dose Aza attenuated the apoptosis and inflammation in H/R-induced HK-2 cells and ischemia–reperfusion kidney. Low-dose Aza ameliorated renal function in mice with renal ischemia–reperfusion injury. Meanwhile, low-dose Aza upregulated klotho expression in H/R-induced HK-2 cells and ischemia–reperfusion kidney. Klotho knockdown abrogated the effects of low-dose Aza on apoptosis and inflammation. Conclusion: Low-dose Aza protects against renal early injury by increasing klotho expression.

Fagaceae species are increasingly used as models to elucidate the process and mechanism of adaptation and speciation by integrating ecology, evolution and genomics. The genus Castanopsis belongs to the family Fagaceae and is mainly distributed across subtropical and tropical Asia. In the present study, we reported the first chromosome-scale genome assembly of Castanopsis tibetana, a common species of evergreen broadleaved forests in subtropical China. The combination of Nanopore sequencing and Hi-C technologies enabled a high-quality genome assembly. The final assembled genome size of C. tibetana was 878.6 Mb (97.6% of the estimated genome size), consisting of 477 contigs with an N50 length of 3.3 Mb. The benchmarking universal single-copy orthologue (BUSCO) assessment indicated a completeness of 93.0%. Hi-C scaffolding generated 12 pseudochromosomes, representing 98.7% of the assembled genome. Subsequently, 40,937 protein-coding genes were predicted and 90.04% of them were functionally annotated. More than 476.9 Mb of repetitive sequences (54.3% of the genome) were identified, and the percentage of the genome covered by TE elements was 39.98%. Comparative genomics analysis revealed that C. tibetana was most closely related to Castanea mollissima and diverged at 18.48 Ma, and that C. tibetana has undergone considerable gene family expansion and contraction. Evidence of positive selection was detected in 53 genes, which showed different arrangement pattern compared to Quercus robur. The chromosome-scale genome assembly of C. tibetana will expand Fagaceae genome resources across the family and provide a powerful comparative framework to study the adaptation and evolution of Fagaceae trees.

Background and objectives Manipulating different signaling pathways via small molecules could efficiently induce cardiomyocytes from human induced pluripotent stem cells (hiPSC). However, the effect of transcription factors on the hiPSC-directed cardiomyocytes differentiation remains unclear. Transcription factor, p53 has been demonstrated indispensable for the early embryonic development and mesendodermal differentiation of embryonic stem cells (ESC). We tested the hypothesis that p53 promotes cardiomyocytes differentiation from human hiPSC. Methods and results Using the well-characterized GiWi protocol that cardiomyocytes are generated from hiPSC via temporal modulation of Wnt signaling pathway by small molecules, we demonstrated that forced expression of p53 in hiPSC remarkably improved the differentiation efficiency of cardiomyocytes from hiPSC, whereas knockdown endogenous p53 decreased the yield of cardiomyocytes. This p53-mediated increased cardiomyocyte differentiation was mediated through WNT3, as evidenced by that overexpression of p53 upregulated the expression of WNT3, and knockdown of p53 decreased the WNT3 expression. Mechanistic analysis showed that the increased cardiomyocyte differentiation partially depended on the amplified mesendodermal specification resulted from p53-mediated activation of WNT3-mediated Wnt signaling. Consistently, endogenous WNT3 knockdown significantly ameliorated mesendodermal specification and subsequent cardiomyocyte differentiation. Conclusions These results provide a novel insight into the potential effect of p53 on the development and differentiation of cardiomyocyte during embryogenesis.

Introduction: Vasoconstriction is triggered by an increase in intracellular-free calcium concentration. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC), and so on. In this study, we studied the changes of vascular reactivity as well as the underlying signaling pathways in aging spontaneously hypertensive rats (SHRs). Methods: The artery tension induced by α1-adrenergic receptor activator (α1-AR) phenylephrine (PE) was measured in the absence or presence of myosin light chain kinase (MLCK), PKC, and ROCK inhibitors. The α1-AR, PKC, ROCK, phosphorylation of myosin light chain (MLC), and PKC-potentiated phosphatase inhibitors of 17 kDa (CPI-17) of rat mesenteric arteries were analyzed at the mRNA level or protein level. Results: The vascular tension measurements showed that there was a significant increase in the mesenteric artery contraction induced by PE in old SHR. MLCK inhibitor ML-7 can similarly inhibit PE-induced vasoconstriction. PKC inhibitor GF109203X has the weakest inhibitory effect on PE-induced contraction in old SHR. At the presence of ROCK inhibitor H1152, PE-induced contraction was significantly reduced in young Wistar-Kyoto (WKY) rats, but this phenomenon disappeared in other rats. Furthermore, in old SHR the protein expression of α1-AR decreased and phosphorylation of MLC and CPI-17 were upregulated and MLC phosphatase (MLCP) activity was significantly lower. The expressions of PKC were upregulated in SHR and old rats. In addition, the expression of ROCK-1 was decreased and ROCK-2 was significantly upregulated with age in SHR. Conclusion: In aging hypertension, the expression/activity of PKC or ROCK-2/CPI-17 excessively increased, MLCP activity decreased and MLC phosphorylation enhanced, leading to increased α1-AR-induced vasoconstriction.

In this paper, selenized Artemisia sphaerocephala polysaccharides (SePAS) were obtained through employing N-methyl-2-pyrrolidone hydrosulfate as catalyst, which showed a maximum Se content enhanced to 8744 μg/g. FT-IR, 1D/2D NMR, X-ray photoelectron spectroscopy (XPS) and size-exclusion chromatograph analysis exhibited that Se had been successfully introduced into PAS and existed in the form of selenate group (Se4+) with the substitution position at C-6. Furthermore, immunostimulating assays indicated that SePAS with high Se content exhibited stronger immunomodulatory activities by upregulated the phosphorylation level of ERK, JNK and p38, thus enhancing RAW264.7 cells proliferation, phagocytosis, levels of interleukin-6, nitric oxide, tumor necrosis factor and interleukin-1β. The current outcome suggested that Se content might be a critical factor affecting the immunomodulatory effects of selenized PAS on macrophage RAW264.7.

Intracellular calcium is related to cardiac hypertrophy. The CaV1.2 channel and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaM regulate the intracellular calcium content. However, the differences in CaMKII and CaM in cardiac hypertrophy are still conflicting and are worthy of studying as drug targets. Therefore, in this study, we aim to investigate the roles and mechanism of CaM and CaMKII on CaV1.2 in pathological myocardial hypertrophy. The results showed that ISO stimulation caused SD rat heart and cardiomyocyte hypertrophy. In vivo, the HW/BW, LVW/BW, cross-sectional area, fibrosis ratio and ANP expression were all increased. There were no differences in CaV1.2 channel expression in the in vivo model or the in vitro model, but the ISO stimulation induced channel activity, and the [Ca2+]i increased. The protein expression levels of CaMKII and p-CaMKII were all increased in the ISO group, but the CaM expression level decreased. AIP inhibited ANP, CaMKII and p-CaMKII expression, and ISO-induced [Ca2+]i increased. AIP also reduced HDAC4, p-HDAC and MEF2C expression. However, CMZ did not play a cardiac hypertrophy reversal role in vitro. In conclusion, we considered that compared with CaM, CaMKII may be a much more important drug target in cardiac hypertrophy reversal.

Interest in galactomannans and its derivatives as a functional health supplement is growing based on physicochemical properties. In this work, the optimized conditions of sulfated tara gum (STG) with a maximum DS of 0.66 by box-behnken design (BBD) were obtained as following: ratio of chlorosulfonic acid/pyridine 3:1, reaction time 4 h and reaction temperature 40 °C. The structure features of STG such as the degree of substitution (DS), substitution position, weight average molar mass (MW), monosaccharide components and chain conformation were investigated. Decreasing of MW, the increasing of Z-average radius of gyration (〈S2〉Z1/2) and specific volume for gyration (SVg) were obtained by SEC-MALLS. In addition, the structural properties of four sulfated galactomannans were comparatively investigated and analyzed based on our earlier reports of sulfated fenugreek gum, guar gum and locust bean gum. A conclusion was drown that higher galactose branch could enhance steric hindrance, which was inferred as one of the significant factors for the derivatization efficiency, thus affecting the DS, MW and conformational transition of sulfated galactomannans. This study will provide valuable information for further research on the comparison of bioactivities and medical application of galactomannans family.

Effects of hypertrophic challenge on small-conductance, Ca2+-activated K+(SK2) channel expression were explored in intact murine hearts, isolated ventricular myocytes and neonatal rat cardiomyocytes (NRCMs). An established experimental platform applied angiotensin II (Ang II) challenge in the presence and absence of reduced p21-activated kinase (PAK1) (PAK1cko vs. PAK1f/f, or shRNA-PAK1 interference) expression. SK2 current contributions were detected through their sensitivity to apamin block. Ang II treatment increased such SK2 contributions to optically mapped action potential durations (APD80) and their heterogeneity, and to patch-clamp currents. Such changes were accentuated in PAK1cko compared to PAK1f/f, intact hearts and isolated cardiomyocytes. They paralleled increased histological and echocardiographic hypertrophic indices, reduced cardiac contractility, and increased SK2 protein expression, changes similarly greater with PAK1cko than PAK1f/f. In NRCMs, Ang II challenge replicated such increases in apamin-sensitive SK patch clamp currents as well as in real-time PCR and western blot measures of SK2 mRNA and protein expression and cell hypertrophy. Furthermore, the latter were enhanced by shRNA-PAK1 interference and mitigated by the PAK1 agonist FTY720. Increased CaMKII and CREB phosphorylation accompanied these effects. These were rescued by both FTY720 as well as the CaMKII inhibitor KN93, but not its inactive analogue KN92. Such CREB then specifically bound to the KCNN2 promoter sequence in luciferase assays. These findings associate Ang II induced hypertrophy with increased SK2 expression brought about by a CaMKII/CREB signaling convergent with the PAK1 pathway thence upregulating the KCNN2 promoter activity. SK2 may then influence cardiac electrophysiology under conditions of cardiac hypertrophy and failure.

The embedding of small peptide ligands within large inactive pre-pro-precursor proteins encoded by orphan open reading frames (ORFs) makes them difficult to identify and study. To address this problem, we generated oligonucleotide ( 1200 amino acid EGF precursor protein or the orphan ECRG4 ORF, that encodes a 148 amino acid Esophageal Cancer Related Gene 4 (ECRG4), a putative cytokine precursor protein of up to eight ligands. After phage display and 3–4 rounds of biopanning for phage internalization into prostate cancer epithelial cells, sequencing identified the 53-amino acid EGF ligand encoded by the 5′ region of the EGF ORF and three distinct domains within the primary sequence of ECRG4: its membrane targeting hydrophobic signal peptide, an unanticipated amino terminus domain at ECRG437–63 and a C-terminus ECRG4133–148 domain. Using HEK-blue cells transfected with the innate immunity receptor complex, we show that both ECRG437–63 and ECRG4133–148 enter cells by interaction with the TLR4 immune complex but neither stimulate NFkB. Taken together, the results help establish that phage display can be used to identify cryptic domains within ORFs of the human secretome and identify a novel TLR4-targeted internalization domain in the amino terminus of ECRG4 that may contribute to its effects on cell migration, immune cell activation and tumor suppression.

Angiotensin II (Ang II) modulates blood pressure via Ang II type 1 receptor (AT1R) and type 2 receptor (AT2R). The activation of AT2R relaxes vascular tone through opening large-conductance Ca2+-activated potassium (BKCa) channels in vascular smooth muscle cells (SMCs). In the present study, we studied the role of the AT2R-BKCa pathway in patients with hypertension. The mesenteric arterial SMCs (MSMCs) were obtained from normotensive patients (NP) and hypertensive patients (HP). BKCa currents were recorded with patch clamp and the expressions of mRNAs and proteins of AT1R/AT2R were analyzed by RT-PCR and Western blotting, respectively. Ang II significantly increased the macroscopic BKCa currents at the whole cell level, while increased the open probability and decreased the mean close time of BKCa channels at the single channel level with AT1R blockade by valsartan in NP. However, Ang II had no effect on the BKCa currents at the same condition in HP. Furthermore, the expressions of mRNA and protein of AT2R but not AT1R were markedly decreased in the MSMCs of HP compared to that of NP. The data suggest that AT2R is well functioned in the MSMCs in NP but not in HP and deficiency in the AT2R-BKCa pathway may contribute to the development of hypertension.

Castanopsis &times, kuchugouzhui Huang et Y. T. Chang was recorded in Flora Reipublicae Popularis Sinicae (FRPS) as a hybrid species on Yuelushan mountain, but it is treated as a hybrid between Castanopsis sclerophylla (Lindl.) Schott. and Castanopsis tibetana Hance in Flora of China. After a thorough investigation on Yuelushan mountain, we found a population of C. sclerophylla and one individual of C. &times, kuchugouzhui, but no living individual of C. tibetana. We collected C. &times, kuchugouzhui, and we sampled 42 individuals of C. sclerophylla from Yuelushan and Xiushui and 43 individuals of C. tibetana from Liangyeshan and Xiushui. We used chloroplast DNA sequences and 29 nuclear microsatellite markers to investigate if C. &times, kuchugouzhui is a natural hybrid between C. sclerophylla and C. tibetana. The chloroplast haplotype analysis showed that C. &times, kuchugouzhui shared haplotype H2 with C. sclerophylla on Yuelushan. The STRUCTURE analysis identified two distinct genetic pools that corresponded well to C. sclerophylla and C. tibetana, revealing the genetic admixture of C. &times, kuchugouzhui. Furthermore, the NewHybrids analysis suggested that C. &times, kuchugouzhui is an F2 hybrid between C. sclerophylla and C. tibetana. Our results confirm that C. &times, kuchugouzhui recorded in FRPS is a rare hybrid between C. sclerophylla and C. tibetana.

Intracellular calcium is related to cardiac hypertrophy. The Ca

OBJECTIVE: To analyze the correlation of annexin A2 with coronary atherosclerotic heart disease (CAD) and the severity of CAD. METHODS: We collected data from a total of 200 inpatients admitted in our department between August, 2017 and August, 2019. According to the Results of coronary angiography, the patients were divided into CAD group (n=150) and non-CAD (n=50), and the CAD patients was further divided, according to their clinical stability, into stable angina (SAP) group and acute coronary syndrome (ACS) group. Serum levels of annexin A2, MPO and PON1 were detected in all these patients, and their correlations with CAD, disease severity, and degree of coronary artery stenosis were analyzed. RESULTS: The levels of annexin A2 and MPO were significantly higher in CAD patients than in non-CAD patients (P < 0.05). Among the CAD patients, those with ACS had significantly higher levels of annexin A2 (P < 0.05) and lower levels of PON-1 (P < 0.05) than those with SAP, but annexin A2 level was not significantly correlated with coronary lesion count, Gensini score, or the co-morbidity of diabetes. CONCLUSION: Annexin A2 is significantly elevated in patients with CAD, especially in those with ACS, and can be used as a predictor of clinical instability.

BACKGROUND Increased small-conductance Ca2+-activated K+ current (SK), abnormal intracellular Ca2+ handling, and enhanced expression and activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been found in clinical and/or experimental models of atrial fibrillation (AF), but the cumulative effect of these phenomena and their mechanisms in AF are still unclear. This study aimed to test the hypothesis that CaMKII increases SK current in human chronic AF. MATERIAL AND METHODS Right atrial appendage tissues from patients with either sinus rhythm (SR) or AF and neonatal rat atrial myocytes were used. Patch clamp, qRT-PCR, and Western blotting techniques were used to perform the study. RESULTS Compared to SR, the apamin-sensitive SK current (IKAS) was significantly increased, but the mRNA and protein levels of SK1, SK2, and SK3 were significantly decreased. In AF, the steady-state Ca2+ response curve of [i]IKAS[/i] was shifted leftward and the [Ca2+]i level was significantly increased. CaMKII inhibitors (KN-93 or autocamtide-2-related inhibitory peptide (AIP)) reduced the IKAS in both AF and SR. The inhibitory effect of KN-93 or AIP on [i]IKAS[/i] was greater in AF than in SR. The expression levels of calmodulin, CaMKII, and autophosphorylated CaMKII at Thr287 (but not at Thr286) were significantly increased in AF. Furthermore, KN-93 inhibited the expression of (Thr287)p-CaMKII and SK2 in neonatal rat atrial myocytes. CONCLUSIONS SK current is increased via the enhanced activation of CaMKII in patients with AF. This finding may explain the difference between SK current and channels expression in AF, and thus may provide a therapeutic target for AF.

Epidemiogical evidence has shown that the incidence of atrial fibrillation in tumor patients is higher than non-tumor patients and general population. The potential risk factors predisposing tumor patients to atrial fibrillation include advanced age, comorbidities, direct anatomic local occupying effect of tumors in the heart or adjacent organs, paraneoplastic manifestations of some tumors, tumor-induced dys-regulation of metabolism, radio-, bio- and chemo-therapeutics, disturbance of autonomous nerve system because of physical pain and psychological sufferings, chronic inflammation typical of most tumors, and surgical interventions among others. However, whether tumor suppressor genes commonly mutated or dys-regulated in tumor play any roles in the pathogenesis of atrial fibrillation remain largely unexplored. Tumor suppressor genes or genes possessing tumor suppressing function have been reported to be constitutively expressed in quiescent heart, and mutations, small nucleotide polymorphisms, or disturbed expression of tumor suppressor genes has been implicated in the pathogenesis of atrial fibrillation. Here, we provide a state-of-the-art overview of the unrecognized roles of tumor suppressor genes in the pathogenesis of atrial fibrillation, focusing mainly on the two well-characterized tumor suppressor genes, zinc finger homeobox protein-3 and esophageal cancer related gene-4.

Uridine adenosine tetraphosphate (Up4A) exerts potent relaxation in porcine coronary arteries that is reduced following myocardial infarction, suggesting a crucial role for Up4A in the regulation of coronary flow (CF) in cardiovascular disorders. We evaluated the vasoactive effects of Up4A on CF in atherosclerosis using ApoE knockout (KO) mice ex vivo and in vivo. Functional studies were conducted in isolated mouse hearts using the Langendorff technique. Immunofluorescence was performed to assess purinergic P2X1 receptor (P2X1R) expression in isolated mouse coronary arteries. In vivo effects of Up4A on coronary blood flow (CBF) were assessed using ultrasound. Infusion of Up4A (10-9-10-5 M) into isolated mouse hearts resulted in a concentration-dependent reduction in CF in WT and ApoE KO mice to a similar extent; this effect was exacerbated in ApoE KO mice fed a high-fat diet (HFD). The P2X1R antagonist MRS2159 restored Up4A-mediated decreases in CF more so in ApoE KO + HFD than ApoE KO mice. The smooth muscle to endothelial cell ratio of coronary P2X1R expression was greater in ApoE KO + HFD than ApoE KO or WT mice, suggesting a net vasoconstrictor potential of P2X1R in ApoE KO + HFD mice. In contrast, Up4A (1.6 mg/kg) increased CBF to a similar extent among the three groups. In conclusion, Up4A decreases CF more in ApoE KO + HFD mice, likely through a net upregulation of vasoconstrictor P2X1R. In contrast, Up4A increases CBF in vivo regardless of the atherosclerotic model.

Epidemiological studies have shown a strong correlation between tumor and AF. However, the molecular link between tumor and AF remains unknown. ECRG4, a tumor suppressor gene that is expressed in the A-V node and in sporadic ventricular myocytes, inhibits tumorigenesis and monitors tissue homeostasis by functioning as a ‘sentinel’ molecule gauging inflammatory and cell proliferative responses. To explore the potential physiological function of Ecrg4 in heart, we evaluated its distribution in heart, analyzed its expression in patients with persistent AF and in a canine AF model, and dissected the molecular events downstream of Ecrg4. The results showed that the level of Ecrg4 expression is homogenously high in atria and the conduction systems and in sporadic ventricular myocytes. Importantly, the expression of Ecrg4 was significantly decreased in atrial appendages of AF patients than patients with SR. Moreover, in rapid pacing canine AF models, the expression of ECRG4 in atria was significantly decreased compared to that of the controls. Mechanistically, knockdown ECRG4 in atrial myocytes significantly shortened the APDs, inhibited the expression of Gja1, and activated pro-inflammatory cascades and genes involved in cardiac remodeling. These results suggest that Ecrg4 may play a critical role in the pathogenesis of AF.

In this paper, selenized Artemisia sphaerocephala polysaccharides (SePAS) were obtained through employing N-methyl-2-pyrrolidone hydrosulfate as catalyst, which showed a maximum Se content enhanced to 8744 μg/g. FT-IR, 1D/2D NMR, X-ray photoelectron spectroscopy (XPS) and size-exclusion chromatograph analysis exhibited that Se had been successfully introduced into PAS and existed in the form of selenate group (Se

N-methyl berbamine (N-MB) is a berberine derivative. Its analogue berbamine has been reported to have remarkable antiarrhythmic and ischemic protective effects. However, the pharmacological effects of N-MB are ill-defined. In this study, molecular docking was used to evaluate the binding of N-MB to CaV1.2 Ca2+ and KV11.1 K+ channels, and the effects of N-MB on action potential and ionic currents were observed in the ventricular myocytes of rabbits, HEK293 cells stably transfected with the hCaV1.2 gene and CHO cells stably transfected with hERG (human ether-a-go-go related gene). The results showed that N-MB was able to bind to both CaV1.2 and KV11.1 channels. Following a perfusion with N-MB, the durations of action potentials (APD20, APD50 and APD90) were extended, and the outward tail current, Itail, as well as the hERG current, IhERG, were inhibited, while the amplitude of action potential (APA) was only slightly reduced. N-MB also decreased the peak amplitude of the L-type Ca2+ channel current, ICaL, as well as the CaV1.2 current, ICaV1.2; this may limit the prolongation of APD. In conclusion, N-MB is a potent and natural antiarrhythmic multitarget drug that may elicit its antiarrhythmic effect through blocking both Ca2+ and K+ channel currents. Keywords: N-methyl berbamine, Action potential, Ca2+ current, K+ current, Multitarget

Some drugs exert curative effects intracellularly, but their hydrophilic property prohibits the membrane-penetrating process and thus limits the curative efficacies, although this property guarantee their solubility in aqueous phase. An example is sodium tanshinone II-A sulfonate (DS-201), a derivative of Chinese medical herb Danshen (Salvia miltiorrhiza) which is a BKCa channel opener and a vasodilator. This study established and optimized a liposome delivery system which could pack and deliver DS-201 into HEK293 cells transfected with BKCa channels, and DS-201 given this way significantly increased the open probability of BKCa channel from baseline 0.013 ± 0.004 to 0.036 ± 0.011 at +40 mV membrane potential (P

Angiotensin II (Ang II) modulates blood pressure via Ang II type 1 receptor (AT

Background: We previously have proved that sodium tanshinone II-A sulfonate (DS-201), a derivative of traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), is an opener and vasodilator of BKCa channel in the vascular smooth muscle cells (VSMCs). Vascular tension is closely associated with Ca2+ dynamics and activation of BKCa channel may not be the sole mechanism for the relaxation of the vascular tension by DS-201. Therefore, we hypothesized that the vasorelaxing effect of DS-20 may be also related to Ca2+ channel and cytoplasmic Ca2+ level in the VSMCs. Methods: Arterial tension was measured by Danish Myo Technology (DMT) myograph system in the mesentery vessels of rats, intracellular Ca2+ level by fluorescence imaging system in the VSMCs of rats, and L-type Ca2+ current by patch clamp technique in Ca2+ channels transfected human embryonic kidney 293 (HEK-293) cells. Results: DS-201 relaxed the endothelium-denuded artery rings pre-constricted with PE or high K+ and the vasorelaxation was reversible. Blockade of K+ channel did not totally block the effect of DS-201 on vasorelaxation. DS-201 suppressed [Ca2+]i transient induced by high K+ in a concentration-dependent manner in the VSMCs, including the amplitude of Ca2+ transient, the time for Ca2+ transient reaching to the [Ca2+]i peak and the time to remove Ca2+ from the cytoplasm. DS-201 inhibited L-type Ca2+ channel with an EC50 of 59.5 μM and at about 40% efficacy of inhibition. However, DS-201did not significantly affect the kinetics of Ca2+ channel. The effect of DS-201 on L-type Ca2+ channel was rate-independent. Conclusion: The effect of DS-201 on vasorelaxation was not only via activating BKCa channel, but also blocking Ca2+ channel and inhibiting Ca2+ influx in the VSMCs of rats. The results favor the use of DS-201 and Danshen in the treatment of cardiovascular diseases clinically.

Background The incidence of atrial fibrillation (AF) in rheumatic heart diseases (RHD) is very high and increases with age. Occurrence and maintenance of AF are very complicated process accompanied by many different mechanisms. Ion-channel remodeling, including the voltage-gated potassium channel Kv1.5, plays an important role in the pathophysiology of AF. However, the changes of Kv1.5 channel expression in Han Chinese patients with RHD and AF remain poorly understood. The aim of the present study was to investigate whether the Kv1.5 channels of the right atria may be altered with RHD, age, and sex to contribute to AF. Material/Methods Right atrial appendages were obtained from 20 patients with normal cardiac functions who had undergone surgery, and 26 patients with AF. Subjects were picked from 4 groups: adult and aged patients in normal sinus rhythm (SR) and AF. Patients were divided into non-RHD and RHD groups or men and women groups in normal SR and AF, respectively. The expression of Kv1.5 protein and messenger RNA (mRNA) were measured using Western blotting and polymerase chain reaction (PCR) method, respectively. Results Compared with the SR group, the expression of Kv1.5 protein decreased significantly in the AF group. However, neither Kv1.5 protein nor KCNA5 mRNA had significant differences in adult and aged groups, non-RHD and RHD group, and men and women group of AF. Conclusions The expression of Kv1.5 channel protein changes with AF but not with age, RHD, and sex in AF.

The human cytokine precursor ECRG4 has been associated with multiple physiological, developmental and pathophysiological processes involving cell proliferation, cell migration, innate immunity, inflammation, cancer progression and metastases. Although down-regulation of ECRG4 gene expression has been largely attributed to hypermethylation of CpG islands in the 5'untranslated region of the ECRG4 promoter, the mechanisms that underlie the dynamics of its regulation have never been systematically described. Here we show that the ECRG4 gene is widely expressed in human tissues and report that its core promoter lies between the -780 to +420 base pairs relative to the ATG start codon of the ECRG4 open reading frame. This sequence, which contains several CpG islands, also includes multiple overlapping Sp1 consensus binding sequences and a putative binding site for NF-kB activation. 5'RACE of mRNA derived from human leukocytes shows that ECRG4 transcription initiates from the guanidine at -11 from the initiation ATG of the ECRG4 open reading frame. While there is no canonical TATA- or CAAT-boxes proximal to this translational initiation site, there is a distal TATA-sequence in the 5'UTR. This region was identified as the sequence targeted by hypermethylation because in vitro methylation of plasmids encoding the ECRG4 promoter abolish promoter activity and the treatment of Jurkat cells (which naturally express ECRG4) with the methylation inhibitor 5-AzaC, increases endogenous ECRG4 expression. Because ChIP assays show that Sp1 binds the ECRG4 promoter, that forced Sp1 expression trans-activates the ECRG4 promoter and Sp1 inhibition with mithramycin inhibits ECRG4 expression, we conclude that the dynamic positive and negative regulatory elements controlling ECRG4 expression include a counter regulation between promoter methylation and Sp1 activation.

Aim: Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BKCa channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BKCa channels. Methods: Human BKCa channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BKCa currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BKCa subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy. Results: DS-201 (40–160 μmol/L) concentration-dependently increased the total open probability of BKCa channels in HEK293 cells, associated with enhancements of Ca2+ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC50 for BKCa channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity. Conclusion: DS-201 selectively targets the pore-forming α subunit of human BKCa channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.