IP3R2-mediated Ca2+ release promotes LPS-induced cardiomyocyte pyroptosis via the activation of NLRP3/Caspase-1/GSDMD pathway.

Pyroptosis plays a crucial role in sepsis, and the abnormal handling of myocyte calcium (Ca2+) has been associated with cardiomyocyte pyroptosis. Specifically, the inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is a Ca2+ release channel in the endoplasmic reticulum (ER). However, the specific role of IP3R2 in sepsis-induced cardiomyopathy (SIC) has not yet been determined. Thus, this study aimed to investigate the underlying mechanism by which IP3R2 channel-mediated Ca2+ signaling contributes to lipopolysaccharide (LPS)-induced cardiac pyroptosis. The SIC model was established in rats by intraperitoneal injection of LPS (10 mg/kg). Cardiac dysfunction was assessed using echocardiography, and the protein expression of relevant signaling pathways was analyzed using ELISA, RT-qPCR, and western blot. Small interfering RNAs (siRNA) and an inhibitor were used to explore the role of IP3R2 in neonatal rat cardiomyocytes (NRCMs) stimulated by LPS in vitro. LPS-induced NLRP3 overexpression and GSDMD-mediated pyroptosis in the rats' heart. Treatment with the NLRP3 inhibitor MCC950 alleviated LPS-induced cardiomyocyte pyroptosis. Furthermore, LPS increased ATP-induced intracellular Ca2+ release and IP3R2 expression in NRCMs. Inhibiting IP3R activity with xestospongin C (XeC) or knocking down IP3R2 reversed LPS-induced intracellular Ca2+ release. Additionally, inhibiting IP3R2 reversed LPS-induced pyroptosis by suppressing the NLRP3/Caspase-1/GSDMD pathway. We also found that ER stress and IP3R2-mediated Ca2+ release mutually regulated each other, contributing to cardiomyocyte pyroptosis. IP3R2 promotes NLRP3-mediated pyroptosis by regulating ER Ca2+ release, and the mutual regulation of IP3R2 and ER stress further promotes LPS-induced pyroptosis in cardiomyocytes.

High hydrostatic pressure participates in atrial fibrosis through the p300/p53/Smad3 pathway.

As an independent risk factor of atrial fibrillation (AF), hypertension (HTN) can induce atrial fibrosis through cyclic stretch and hydrostatic pressure. The mechanism by which high hydrostatic pressure promotes atrial fibrosis is unclear yet. p300 and p53/Smad3 play important roles in the process of atrial fibrosis. This study investigated whether high hydrostatic pressure promotes atrial fibrosis by activating the p300/p53/Smad3 pathway. Biochemical experiments were used to study the expression of p300/p53/Smad3 pathway in left atrial appendage (LAA) tissues of patients with sinus rhythm (SR), AF, AF + HTN, and C57/BL6 mice, hypertensive C57/BL6 mice and atrial fibroblasts of mice. To investigate the roles of p300 and p53 in the process of atrial fibrosis, p300 and p53 in mice atrial fibroblasts were knocked in or knocked down, respectively. The expression of p300/p53/Smad3 and fibrotic factors was higher in patients with AF and AF + HTN than those with SR only. The expressions of p300/p53/Smad3 and fibrotic factors increased in hypertensive mice. Curcumin (Cur) and knocking down of p300 reversed the expressions of these factors. 40 mmHg hydrostatic pressure/overexpression of p300 upregulated the expressions of p300/p53/Smad3 and fibrotic factors in mice LAA fibroblasts. While Cur or knocking down p300 reversed these changes. Knocking down/overexpression of p53, the expressions of p53/Smad3 and fibrotic factors also decreased/increased, correspondingly. High hydrostatic pressure promotes atrial fibrosis by activating the p300/p53/Smad3 pathway, which further increases the susceptibility to AF.

Involvement of plasminogen activator inhibitor-1 in p300/p53-mediated age-related atrial fibrosis.

Plasminogen activator inhibitor-1 (PAI-1), a key regulator of the fibrinolytic system, is also intimately involved in the fibrosis. Although PAI-1 may be involved in the occurrence of atrial fibrillation (AF) and thrombosis in the elderly, but whether it participated in aging-related atrial fibrosis and the detailed mechanism is still unclear. We compared the transcriptomics data of young (passage 4) versus senescent (passage 14) human atrial fibroblasts and found that PAI-1 was closely related to aging-related fibrosis. Aged mice and senescent human and mouse atrial fibroblasts underwent electrophysiological and biochemical studies. We found that p300, p53, and PAI-1 protein expressions were increased in the atrial tissue of aged mice and senescent human and mouse atrial fibroblasts. Curcumin or C646 (p300 inhibitor), or p300 knockdown inhibited the expression of PAI-1 contributing to reduced atrial fibroblasts senescence, atrial fibrosis, and the AF inducibility. Furthermore, p53 knockdown decreased the protein expression of PAI-1 and p21 in senescent human and mouse atrial fibroblasts. Our results suggest that p300/p53/PAI-1 signaling pathway participates in the mechanism of atrial fibrosis induced by aging, which provides new sights into the treatment of elderly AF.

Sacubitril/valsartan reduces susceptibility to atrial fibrillation by improving atrial remodeling in spontaneously hypertensive rats.

AIM: Sacubitril/valsartan (Sac/Val, LCZ696), the world's first angiotensin receptor-neprilysin inhibitor (ARNi), has been widely used in the treatment of heart failure. However, the use of Sac/Val in the treatment of atrial fibrillation (AF), especially AF with hypertension, has been less reported. We investigated the effect of Sac/Val on atrial remodeling and hypertension-related AF. METHODS: The AF induction rate and electrophysiological characteristics of spontaneously hypertensive rats (SHRs) treated with Sac/Val or Val were detected by rapid atrial pacing and electrical mapping/optical mapping. The whole-cell patch-clamp and Western blot were used to observe electrical/structural remodeling of atrial myocytes/tissue of rats and atrium-derived HL-1 cells cultured under 40 mmHg in vitro. RESULTS: Sac/Val was superior to Val in reducing blood pressure, myocardial hypertrophy and susceptibility of AF in SHRs. The shorten action potentials duration (APD), decreased L type calcium channel current (ICa,L) and Cav1.2, increased ultrarapid delayed rectified potassium current (Ikur) and Kv1.5 in atrial myocytes/tissue of SHRs could be better improved by Sac/Val, as well as the levels of atrial fibrosis. While the protein expression of angiotensin-converting enzyme-1 (ACE-1), angiotensin, angiotensin II type I AT1 receptor (AT1R) and neprilysin (NEP) were increased, which could be more effective ameliorated by Sac/Val than Val. Furthermore, Val + Sacubitrilat (LBQ657) (an active NEP inhibitor) was also superior to LBQ657 or Val in improving the electrical and structural remodeling of HL-1 cells through inhibiting NEP. CONCLUSION: Sac/Val can improve atrial structural and electrical remodeling induced by hypertension and reduce the AF susceptibility by inhibiting RAS and NEP. The above effects of Sac/Val were superior to Val alone.

Acetyltransferase p300 regulates atrial fibroblast senescence and age-related atrial fibrosis through p53/Smad3 axis.

Atrial fibrosis induced by aging is one of the main causes of atrial fibrillation (AF), but the potential molecular mechanism is not clear. Acetyltransferase p300 participates in the cellular senescence and fibrosis, which might be involved in the age-related atrial fibrosis. Four microarray datasets generated from atrial tissue of AF patients and sinus rhythm (SR) controls were analyzed to find the possible relationship of p300 (EP300) with senescence and fibrosis. And then, biochemical assays and in vivo electrophysiological examination were performed on older AF patients, aging mice, and senescent atrial fibroblasts. The results showed that (1) the left atrial tissues of older AF patients, aging mouse, and senescence human atrial fibroblasts had more severe atrial fibrosis and higher protein expression levels of p300, p53/acetylated p53 (ac-p53)/p21, Smad3/p-Smads, and fibrosis-related factors. (2) p300 inhibitor curcumin and p300 knockdown treated aging mouse and senescence human atrial fibroblasts reduced the senescence ratio of atrial fibroblasts, ameliorated the atrial fibrosis, and decreased the AF inducibility. In contrast, over-expression of p300 can lead to the senescence of atrial fibroblasts and atrial fibrosis. (3) p53 knockdown decreased the expression of aging and fibrosis-related proteins. (4) Co-immunoprecipitation and immunofluorescence showed that p53 forms a complex with smad3 and directly regulates the expression of smad3 in atrial fibroblasts. Our findings suggest that the mechanism of atrial fibrosis induced by aging is, at least, partially dependent on the regulation of p300, which provides new sights into the AF treatment, especially for the elderly.

Contribution of calcium dysregulation to impaired coronary artery contraction in Zucker diabetic fatty rats.

Diabetic coronary artery injury is closely associated with Ca2+ dysregulation, although the underlying mechanism remains unclear. This study explored the role and mechanism of Ca2+ handling in coronary artery dysfunction in type 2 diabetic rats. Zucker diabetic fatty (ZDF) rats were used as the type 2 diabetes mellitus model. The contractility of coronary artery rings induced by KCl, CaCl2 , 5-HT and U46619 was significantly lower in ZDF rats than in Zucker lean rats. Vasoconstriction induced by 5-HT and U46619 was greatly inhibited by nifedipine. However, in the presence of 1 µM nifedipine or in the Ca2+ -free KH solution containing 1 µM nifedipine, there was no difference in the vasoconstriction between Zucker lean and ZDF rats. Store-operated calcium channels (SOCs) were not involved in coronary vasoconstriction. The downregulation of contractile proteins and the upregulation of synthesized proteins were in coronary artery smooth muscle cells (CASMCs) from ZDF rats. Metformin reversed the reduction of vasoconstriction in ZDF rats. Taken together, L-type calcium channel is important for regulating the excitation-contraction coupling of VSMCs in coronary arteries, and dysregulation of this channel contributes to the decreased contractility of coronary arteries in T2DM.

Differential role of STIM1 in calcium handling in coronary and intrarenal arterial smooth muscles.

Calcium (Ca2+) dysregulation contributes to various vascular diseases, but the role and underlying mechanism of stromal interaction molecule-1 (STIM1) in Ca2+ signaling and vasocontraction remain elusive. By using smooth muscle-specific STIM1 knockout (sm-STIM1 KO) mice and a multi myograph system, we investigated the differential role of STIM1 in Ca2+ handling between coronary and intrarenal arterial smooth muscles. After STIM1 deletion, contractile responses to 5-HT were obviously reduced in coronary and intrarenal arteries in the sm-STIM1 KO mice, but not altered in U46619. Phenylephrine barely induced the contraction of coronary arteries, we only detected an effect on the contraction of intrarenal arteries, which was also reduced in the sm-STIM1 KO mice. Then, L-type Ca2+ channel (Cav1.2)- mediated vasocontractions were significantly enhanced in coronary and intrarenal arteries in sm-STIM1 KO mice, similar to treatment with the Cav1.2 agonist Bay K8644 in coronary arteries. However, non-Cav1.2-mediated vasocontractions were remarkably reduced. IP3 receptor- and ryanodine receptor-mediated vasocontractions were both obviously decreased in coronary and intrarenal arteries in sm-STIM1 KO mice. Moreover, STIM1-mediated store operated Ca2+ entry (SOCE) only participated in the contraction of intrarenal arteries. In conclusion, we demonstrate that STIM1 participates in Cav1.2, sarcoplasmic reticulum (SR) Ca2+ release and store-operated Ca2+ (SOC) channels-mediated vasocontraction, which exhibits obvious organ-specificity between coronary and intrarenal arteries.

Advanced glycation end products induce senescence of atrial myocytes and increase susceptibility of atrial fibrillation in diabetic mice.

Diabetes mellitus (DM) is a common chronic metabolic disease caused by significant accumulation of advanced glycation end products (AGEs). Atrial fibrillation (AF) is a common cardiovascular complication of DM. Here, we aim to clarify the role and mechanism of atrial myocyte senescence in the susceptibility of AF in diabetes. Rapid transesophageal atrial pacing was used to monitor the susceptibility of mice to AF. Whole-cell patch-clamp was employed to record the action potential (AP) and ion channels in single HL-1 cell and mouse atrial myocytes. More importantly, anti-RAGE antibody and RAGE-siRNA AAV9 were used to investigate the relationship among diabetes, aging, and AF. The results showed that elevated levels of p16 and retinoblastoma (Rb) protein in the atrium were associated with increased susceptibility to AF in diabetic mice. Mechanistically, AGEs increased p16/Rb protein expression and the number of SA-ß-gal-positive cells, prolonged the action potential duration (APD), reduced protein levels of Cav1.2, Kv1.5, and current density of ICa,L , IKur in HL-1 cells. Anti-RAGE antibody or RAGE-siRNA AAV9 reversed these effects in vitro and in vivo, respectively. Furthermore, downregulating p16 or Rb by siRNA prevented AGEs-mediated reduction of Cav1.2 and Kv1.5 proteins expression. In conclusion, AGEs accelerated atrial electrical remodeling and cellular senescence, contributing to increased AF susceptibility by activating the p16/Rb pathway. Inhibition of RAGE or the p16/Rb pathway may be a potential therapeutic target for AF in diabetes.

Comparison of the Serum Total 25-Hydroxyvitamin D Concentrations Using Chemiluminescent Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry in Children.

We aimed to assess the difference and agreement between the CL-series Vitamin D Total assay (Mindray), which was a kind of chemiluminescent immunoassay (CLIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the measurement of serum 25-hydroxyvitamin D [25(OH)D] concentrations in children. We compared the 25(OH)D concentrations of 92 children using the CLIA and LC-MS/MS. Paired samples t-test was used to compare the two groups. Linear regression was used to show the correlation between CLIA and LC-MS/MS. The difference and bias between 2 methods were revealed in Bland-Altman plot. Agreement in classification of deficiency between CLIA and LC-MS/MS was assessed using Cohen's Kappa. p value<0.05 was considered statistically significant. Using Shapiro-Wilk Test to assess whether the data follows a normal distribution. Using 95% children's serum 25(OH)D concentrations by LC-MS/MS as the reference interval. The regression equation was CLIA=1.185×LC-MS/MS-3.328. The fitness adjusted r2 was 0.589. The CLIA showed positive bias compared to LC-MS/MS, p<0.05, bias=(1.94±16.56) ng/mL. Cohen's Kappa=0.53, p<0.001. The agreement of 2 methods in diagnosing "deficiency" was good. According to Shapiro-Wilk Test, the data followed a normal distribution (W=0.99). The reference interval of children's serum 25(OH)D concentrations by LC-MS/MS was 11.35-44.57 ng/mL. In measuring 25(OH)D concentration of children, CLIA represented higher levels than LC-MS/MS. The two methods were consistent in diagnosing vitamin D deficiency. The reference interval of children's serum 25(OH)D concentrations by LC-MS/MS was 11.35-44.57 ng/mL in our area in summer.

Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via. CaM/Src/Pitx2 Activation in Atrial Myocytes.

Hypertension is a major cardiovascular risk factor for atrial fibrillation (AF) worldwide. However, the role of mechanical stress caused by hypertension on downregulating the L-type calcium current (ICa,L), which is vital for AF occurrence, remains unclear. Therefore, the aim of the present study was to investigate the role of Piezo1, a mechanically activated ion channel, in the decrease of ICa,L in response to high hydrostatic pressure (HHP, one of the principal mechanical stresses) at 40 mmHg, and to elucidate the underlying pathways. Experiments were conducted using left atrial appendages from patients with AF, spontaneously hypertensive rats (SHRs) treated with valsartan (Val) at 30 mg/kg/day and atrium-derived HL-1 cells exposed to HHP. The protein expression levels of Piezo1, Calmodulin (CaM), and Src increased, while that of the L-type calcium channel a1c subunit protein (Cav1.2) decreased in the left atrial tissue of AF patients and SHRs. SHRs were more vulnerable to AF, with decreased ICa,L and shortened action potential duration, which were ameliorated by Val treatment. Validation of these results in HL-1 cells in the context of HHP also demonstrated that Piezo1 is required for the decrease of ICa,L by regulating Ca2+ transient and activating CaM/Src pathway to increase the expression of paired like homeodomain-2 (Pitx2) in atrial myocytes. Together, these data demonstrate that HHP stimulation increases AF susceptibility through Piezo1 activation, which is required for the decrease of ICa,L via. the CaM/Src/Pitx2 pathway in atrial myocytes.

Identification of Hub Genes Associated with Immune Infiltration in Cardioembolic Stroke by Whole Blood Transcriptome Analysis.

Cardioembolic stroke (CS) is the most common type of ischemic stroke in the clinic, leading to high morbidity and mortality worldwide. Although many studies have been conducted, the molecular mechanism underlying CS has not been fully grasped. This study was aimed at exploring the molecular mechanism of CS using comprehensive bioinformatics analysis and providing new insights into the pathophysiology of CS. We downloaded the public datasets GSE58294 and GSE16561. Differentially expressed genes (DEGs) were screened via the limma package using R software. CIBERSORT was used to estimate the proportions of 22 immune cells based on the gene expression profiling of CS patients. Using weighted gene correlation network analysis (WGCNA) to cluster the genes into different modules and detect relationships between modules and immune cell types, hub genes were identified based on the intersection of the protein-protein interaction (PPI) network analysis and WGCNA, and their clinical significance was then verified using another independent dataset GSE16561. Totally, 319 genes were identified as DEGs and 5413 genes were clustered into nine modules using WGCNA. The blue module, with the highest correlation coefficient, was identified as the key module associated with stroke, neutrophils, and B cells naïve. Based on the PPI analysis and WGCNA, five genes (MCEMP1, CLEC4D, GPR97, TSPAN14, and FPR2) were identified as hub genes. Correlation analysis indicated that hub genes had general association with infiltration-related immune cells. ROC analysis also showed they had potential clinical significance. The results were verified using another dataset, which were consistent with our analysis. Five crucial genes determined using integrative bioinformatics analysis might play significant roles in the pathophysiological mechanism in CS and be potential targets for pharmaceutic therapies.

Connexin 43 participates in atrial electrical remodelling through colocalization with calcium channels in atrial myocytes.

Atrial fibrillation (AF) is associated with atrial conduction disturbances caused by electrical and/or structural remodelling. In the present study, we hypothesized that connexin might interact with the calcium channel through forming a protein complex and, then, participates in the pathogenesis of AF. Western blot and whole-cell patch clamp showed that protein levels of Cav1.2 and connexin 43 (Cx43) and basal ICa,L were decreased in AF subjects compared to sinus rhythm (SR) controls. In cultured atrium-derived myocytes (HL-1 cells), knocking-down of Cx43 or incubation with 30 mmol/L glycyrrhetinic acid significantly inhibited protein levels of Cav1.2 and Cav3.1 and the current density of ICa,L and ICa,T . Incubation with nifedipine or mibefradil decreased the protein level of Cx43 in HL-1 cells. Moreover, Cx43 was colocalized with Cav1.2 and Cav3.1 in atrial myocytes. Therefore, Cx43 might regulate the ICa,L and ICa,T through colocalization with calcium channel subunits in atrial myocytes, representing a potential pathogenic mechanism in AF.

Involvement of plasminogen activator inhibitor-1 and its related molecules in atrial fibrosis in patients with atrial fibrillation.

Atrial fibrillation is the most common form of cardiac arrhythmia. Atrial fibrosis is a significant feature of atrial fibrillation though its mechanism is not well understood. We searched the Gene Expression Omnibus database to compare mRNA expression patterns between atrial fibrillation and sinus rhythm samples; one hundred and forty eight differentially expressed genes were identified. Most of these genes were significantly enriched in the extracellular matrix organization process and collagen-activated tyrosine kinase receptor signaling pathway. To screen hub genes involved in atrial fibrosis, we constructed a protein-protein interaction network and found that three hub genes (SERPINE1/plasminogen activator inhibitor-1/PAI-1, TIMP Metallopeptidase Inhibitor 3/TIMP3 and decorin/DCN) play vital roles in atrial fibrosis, especially plasminogen activator inhibitor-1. Elevated plasminogen activator inhibitor-1 expression was positively correlated with the p53 signaling pathway. Plasminogen activator inhibitor-1 and p53 protein expression levels were verified in patients with sinus rhythm and atrial fibrillation by Western blot analysis. Compared with the sinus rhythm controls, p53 and plasminogen activator inhibitor-1 protein expressions were upregulated in the atrial tissues of patients with atrial fibrillation. p53 was also found to regulate plasminogen activator inhibitor-1 based on the results of cellular and molecular experiments. Thus, the p53/plasminogen activator inhibitor-1 signaling axis may participate in the pathophysiological processes of atrial fibrillation, and plasminogen activator inhibitor-1 may serve as a new therapeutic biomarker in atrial fibrillation.

High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy.

Mitochondrial dysfunction and impaired Ca2+ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca2+ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca2+ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca2+ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca2+ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1-Drp1 axis is a novel target for treating DCM.

Effect of BTP2 on agonist-induced vasoconstriction in the mouse aorta in vitro.

BTP2 is a potent inhibitor of store-operated Ca2+ entry (SOCE), which plays a vital role in vasoconstriction. However, the direct effect of BTP2 on the contractile response remains unclear. Here, we investigated the effects and mechanisms of action of BTP2 in the mouse aorta. Isometric tension was measured using a Multi Myograph System with two stainless steel wires. Ca2+ transient was recorded by confocal laser scanning microscope. The results showed that BTP2 markedly suppressed vasoconstriction mediated by SOCE and Ca2+ influx mediated by SOCE. The cumulative concentration of BTP2 had no effect on the baseline of mouse aortic rings, whereas it increased vasoconstriction stimulated by 3 µmol/L Phenylephrine. BTP2 (1 µmol/L) significantly increased vasoconstriction induced by 3 µmol/L Phe or cumulative concentration. BTP2 also promoted noradrenaline-induced aortic contraction. However, Phe- and noradrenaline-induced contraction was not affected by 0.3 or 3 µmol/L BTP2, and BTP2 at 10 µmol/L significantly suppressed aortic contraction. BTP2 inhibited 5-HT-evoked contraction in a concentration-dependent manner. BTP2 at higher concentrations (>3 µmol/L) inhibited CaCl2 -induced and 60 mmol/L K+ -induced contraction with progressive reduction of maximal contraction in a concentration-dependent manner. These results suggest that 1 µmol/L BTP2 increases contraction evoked by α1 adrenoreceptor activation. BTP2 at higher concentrations may inhibit Cav1.2 channels.

Activation of PKCα participates in the reduction of Ikur in atrial myocytes induced by tumour necrosis factor-α.

The atrial-specific ultra-rapid delayed rectifier K+ current (Ikur) plays an important role in the progression of atrial fibrillation (AF). Because inflammation is known to lead to the onset of AF, we aimed to investigate whether tumour necrosis factor-α (TNF-α) played a role in regulating Ikur and the potential signalling pathways involved. Whole-cell patch-clamp and biochemical assays were used to study the regulation and expression of Ikur in myocytes and in tissues from left atrial appendages (LAAs) obtained from patients with sinus rhythm (SR) or AF, as well as in rat cardiomyocytes (H9c2 cells) and mouse atrial myocytes (HL-1 cells). Ikur current density was markedly reduced in atrial myocytes from AF patients compared with SR controls. Reduction of Kv1.5 protein levels was accompanied by increased expression of TNF-α and protein kinase C (PKC)α activation in AF patients. Treatment with TNF-α dose-dependently reduced Ikur and protein expression of Kv1.5 but not Kv3.1b in H9c2 cells and HL-1 cells. TNF-α also increased activity of PKCα. Specific PKCα inhibitor Gö6976 alleviated the reduction in Ikur induced by TNF-α, but not the reduction in Kv1.5 protein. TNF-α was involved in the electrical remodelling associated with AF, probably by depressing Ikur in atrial myocytes via activation of PKCα.

Abnormal Ca2+ handling contributes to the impairment of aortic smooth muscle contractility in Zucker diabetic fatty rats.

Vascular dysfunction is a common pathological basis for complications in individuals affected by diabetes. Previous studies have established that endothelial dysfunction is the primary contributor to vascular complications in type 2 diabetes (T2DM). However, the role of vascular smooth muscle cells (VSMCs) in vascular complications associated with T2DM is still not completely understood. The aim of this study is to explore the potential mechanisms associated with Ca2+ handling dysfunction and how this dysfunction contributes to diabetic vascular smooth muscle impairment. The results indicated that endothelium-dependent vasodilation was impaired in diabetic aortae, but endothelium-independent vasodilation was not altered. Various vasoconstrictors such as phenylephrine, U46619 and 5-HT could induce vasoconstriction in a concentration-dependent manner, such that the dose-response curve was parallel shifted to the right in diabetic aortae, compared to the control. Vasoconstrictions mediated by L-type calcium (Cav1.2) channels were attenuated in diabetic aortae, but effects mediated by store-operated calcium (SOC) channels were enhanced. Intracellular Ca2+ concentration ([Ca2+]i) in VSMCs was detected by Fluo-4 calcium fluorescent probes, and demonstrated that SOC-mediated Ca2+ entry was increased in diabetic VSMCs. VSMC-specific knockout of STIM1 genes decreased SOC-mediated and phenylephrine-induced vasoconstrictive response in mice aortae. Additionally, Orai1 expression was up-regulated, Cav1.2 expression was downregulated, and the phenotypic transformation of diabetic VSMCs was determined in diabetic aortae. The overexpression of Orai1 markedly promoted the OPN expression of VSMCs, whereas SKF96365 (SOC channel blocker) reversed the phenotypic transformation of diabetic VSMCs. Our results demonstrated that the vasoconstriction response of aortic smooth muscle was weakened in type 2 diabetic rats, which was related to the downregulation of the Cav1.2 channel and the up-regulation of the SOC channel signaling pathway.

High hydrostatic pressure induces atrial electrical remodeling through angiotensin upregulation mediating FAK/Src pathway activation.

Hypertension is an independent risk factor for atrial fibrillation (AF), although its specific mechanisms remain unclear. Previous research has been focused on cyclic stretch, ignoring the role of high hydrostatic pressure. The present study aimed to explore the effect of high hydrostatic pressure stimulation on electrical remodeling in atrial myocytes and its potential signaling pathways. Experiments were performed on left atrial appendages from patients with chronic AF or sinus rhythm, spontaneously hypertensive rats (SHRs) treated with or without valsartan (10 mg/kg/day) and HL-1 cells were exposed to high hydrostatic pressure using a self-developed device. Whole-cell patch-clamp recordings and western blots demonstrated that the amplitudes of ICa,L, Ito, and IKur were reduced in AF patients with corresponding changes in protein expression. Angiotensin protein levels increased and Ang1-7 decreased, while focal adhesion kinase (FAK) and Src kinase were enhanced in atrial tissue from AF patients and SHRs. After rapid atrial pacing, AF inducibility in SHR was significantly higher, accompanied by a decrease in ICa,L, upregulation of Ito and IKur, and a shortened action potential duration. Angiotensin upregulation and FAK/Src activation in SHR were inhibited by angiotensin type 1 receptor inhibitor valsartan, thus, preventing electrical remodeling and reducing AF susceptibility. These results were verified in HL-1 cells treated with high hydrostatic pressure, and demonstrated that electrical remodeling regulated by the FAK-Src pathway could be modulated by valsartan. The present study indicated that high hydrostatic pressure stimulation increases AF susceptibility by activating the renin-angiotensin system and FAK-Src pathway in atrial myocytes.

A novel UTMD system facilitating nucleic acid delivery into MDA-MB-231 cells.

Gene therapy is emerging as a promising method for the treatment of various diseases. The safe and efficient delivery of therapeutic nucleic acids is a gene therapy prerequisite. Ultrasound, particularly in combination with microbubbles composed of biocompatible materials such as lipid, PLGA and chitosan, is a novel non-viral tool for gene transportation. Under ultrasound irradiation, microbubbles explode and generate pores in the cell membrane. Hence, genes can enter cells more easily. In order to transfect nucleic acids into MDA-MB-231 cells in a low-cost and non-viral manner for further breast cancer gene therapy studies, we explored ultrasound targeted microbubble destruction (UTMD) technology and evaluated the efficiency and safety of the delivery of plasmid encoding enhanced green fluorescent protein (pEGFP) and a microRNA-34a (miR-34a) mimic by UTMD. Sonovitro ultrasonic apparatus was employed to generate ultrasonic field, which was developed by our group. Ultrasonic parameters, including acoustic intensity (AI), exposure time (ET) and duty cycle (DC), were optimized at 0.6 W/cm2 AI, 20 s ET and 20% DC, the cell viability was not obviously impaired. Under these conditions, the UTMD-mediated transfection efficiency of pEGFP was greater than 40%. In addition to plasmid DNA, an miR-34a mimic was also successfully introduced into the cytoplasm by UTMD and found to inhibit proliferation, induce apoptosis of MDA-MB-231 cells and regulate downstream molecules. The present study indicates that further in vivo UTMD-mediated gene therapy studies are warranted.

Mechanism of contractile dysfunction induced by serotonin in coronary artery in spontaneously hypertensive rats.

Hypertension is one of the risk factors for coronary heart disease. The present study investigated the mechanism of contractile dysfunction induced by serotonin (5-HT) in coronary artery in spontaneously hypertensive rats (SHRs). Coronary arteries were isolated form SHRs and Wistar rats. Arterial ring contraction was measured using a multi myograph system. Intracellular calcium concentration was measured with a Ca2+ probe fluo-4/AM in vascular smooth muscle cells (VSMCs) isolated from coronary arteries. Signaling pathway-related proteins were assayed by western blotting. A 5-HT2A receptor blocker, sarpogrelate, completely eliminated coronary artery contraction induced by 5-HT. PLCß inhibitor U73122 also significantly inhibited the response to 5-HT. Compared with the Wistar rats, serotonin (5-HT)- and CaCl2-induced coronary vasoconstriction in the SHRs was significantly reduced. Rho-associated protein kinase inhibitor Y27632, PKC inhibitor rottlerin, and L-type calcium channel blocker nifedipine inhibited the 5-HT-induced coronary artery contraction in a dose-dependent manner in SHRs and Wistar rats. However, the inhibitory effects were reduced in SHRs. In addition, store-operated Ca2+ (SOC) induced an obvious Ca2+ influx in coronary arterial smooth muscle cells, whereas SOC-mediated contraction was very slight in coronary arteries. At the same time, it was found that 5-HT2AR, IP3R, and Cav1.2 protein expression and PKCδ activity were decreased, and STIM1 and Orai1 were increased in VSMCs from coronary arteries of SHRs compared with Wistar rats. These results implicate calcium-handling dysfunction mediated by the 5-HT2A receptor and downstream signaling pathway might lead to a reduction in 5-HT-induced contraction in SHR coronary arteries.