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.

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.

Receptor activator of NF-κB mediates podocyte injury in diabetic nephropathy.

Receptor activator of NF-κB (RANK) expression is increased in podocytes of patients with diabetic nephropathy. However, the relevance of RANK to diabetic nephropathy pathobiology remains unclear. Here, to evaluate the role of podocyte RANK in the development of diabetic nephropathy, we generated a mouse model of podocyte-specific RANK depletion (RANK-/-Cre T), and a model of podocyte-specific RANK overexpression (RANK TG), and induced diabetes in these mice with streptozotocin. We found that podocyte RANK depletion alleviated albuminuria, mesangial matrix expansion, and basement membrane thickening, while RANK overexpression aggravated these indices in streptozotocin-treated mice. Moreover, streptozotocin-triggered oxidative stress was increased in RANK overexpression but decreased in the RANK depleted mice. Particularly, the expression of NADPH oxidase 4, and its obligate partner, P22phox, were enhanced in RANK overexpression, but reduced in RANK depleted mice. In parallel, the transcription factor p65 was increased in the podocyte nuclei of RANK overexpressing mice but decreased in the RANK depleted mice. The relevant findings were largely replicated with high glucose-treated podocytes in vitro. Mechanistically, p65 could bind to the promoter regions of NADPH oxidase 4 and P22phox, and increased their respective gene promoter activity in podocytes, dependent on the levels of RANK. Taken together, these findings suggested that high glucose induced RANK in podocytes and caused the increase of NADPH oxidase 4 and P22phox via p65, possibly together with the cytokines TNF- α, MAC-2 and IL-1 ß, resulting in podocyte injury. Thus, we found that podocyte RANK was induced in the diabetic milieu and RANK mediated the development of diabetic nephropathy, likely by promoting glomerular oxidative stress and proinflammatory cytokine production.

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.

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.

Comparison of Ca2+ Handling for the Regulation of Vasoconstriction between Rat Coronary and Renal Arteries.

BACKGROUND: Ca2+ plays an important role in the regulation of vasoconstriction. Ca2+ signaling is regulated by a number of Ca2+-handling proteins. However, whether differences in Ca2+ handling affect the regulation of vasoconstriction in different arteries remains elusive. OBJECTIVE: To determine whether differences in Ca2+ handling affect the response to vasoconstrictors in different arteries. METHODS: Arterial ring contraction was measured using a Multi Myograph System. Vascular smooth muscle cells (VSMCs) were digested with type 2 collagenase in DMEM, then intracellular calcium concentration was measured with the Ca2+ probe fluo-4/AM in the isolated cells. Calcium-related proteins were assayed by Western blotting. RESULTS: Phenylephrine did not induce -coronary arterial contraction. There were differences in -5-hydroxytryptamine, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2a, and endothelin 1-induced vasoconstriction in different solutions between coronary and renal arteries. Vasoconstrictions in the presence of Bay K8644 were stronger in coronary than in renal arteries. Store-operated calcium (SOC) channels could mediate Ca2+ influx in VSMCs of both groups. SOC channels did not participate in the contraction of coronary arteries. In addition, there were significant differences in the expressions of receptors and ion channels between the two groups. CONCLUSIONS: Ca2+ handling contributed to the different responses to vasoconstrictors between coronary and renal arteries.

Mechanisms of U46619-induced contraction in mouse intrarenal artery.

Thromboxane A2 (TXA2 ) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca2+ ]i ) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo-4/AM dye and a confocal laser scanning microscopy. The results show that the U46619-induced vasoconstriction of renal artery was completely blocked by a TXA2 receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI-PLC) inhibitor U73122 at 10 µmol/L and partially inhibited by a Phosphatidylcholine - specific phospholipase C (PC-PLC) inhibitor D609 at 50 µmol/L. Moreover, the U46619-induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10 µmol/L, and a selective PKCδ inhibitor rottlerin at 10 µmol/L. In addition, the PKC-induced vasoconstriction was partially inhibited by a Rho-kinase inhibitor Y-27632 at 10 µmol/L and was further completely inhibited together with a putative IP3 receptor antagonist and store-operated Ca2+ (SOC) entry inhibitor 2-APB at 100 µmol/L. On the other hand, U46619-induced vasoconstriction was partially inhibited by L-type calcium channel (Cav1.2) inhibitor nifedipine at 1 µmol/L and 2-APB at 50 and 100 µmol/L. Last, U46619-induced vasoconstriction was partially inhibited by a cell membrane Ca2+ activated C1- channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) at 50 and 100 µmol/L. Our results suggest that the U46619-induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane-prostanoid receptors and its downstream signaling pathway.

Involvement of Orai1 in tunicamycin-induced endothelial dysfunction.

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

Involvement of ERK1/2 in Cx43 depression induced by macrophage migration inhibitory factor in atrial myocytes.

Connexin 43 (Cx43) plays an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate the effect of macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, on Cx43 expression and activity and determine the intracellular signalling pathways. Cx43 protein and mRNA levels were assayed using immunofluorescence, real-time polymerase chain reaction (PCR), and western blot. We found that increased MIF and extracellular regulated protein kinases (ERK) expression was accompanied by a significant reduction in Cx43 protein expression in atrial tissues from patients with AF compared with those with sinus rhythm. In cultured atrium-derived myocytes (HL-1 cells), mouse recombinant-MIF (rMIF, 20 or 40 nmol/L, 24 hours) down-regulated gene and protein expression of Cx43 in a concentration-dependent manner. U0126, a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK) could reverse the decrease in expression of Cx43 protein induced by rMIF. Further studies revealed that rMIF (40 nmol/L, 15, 30, and 45 minutes) was able to stimulate phospho-Erk1/2 (Thr202/Tyr204) production in a time-dependent manner. These results suggest that MIF is involved in the pathogenesis of AF, probably by down-regulating the protein and gene expression of Cx43 via ERK1/2 kinase activation. Our findings represent a potential pathogenic mechanism in AF.

The bifunctional effect of propofol on thromboxane agonist (U46619)-induced vasoconstriction in isolated human pulmonary artery.

Propofol is known to cause vasorelaxation of several systemic vascular beds. However, its effect on the pulmonary vasculature remains controversial. In the present study, we investigated the effects of propofol on human pulmonary arteries obtained from patients who had undergone surgery. Arterial rings were mounted in a Multi-Myograph system for measurement of isometric forces. U46619 was used to induce sustained contraction of the intrapulmonary arteries, and propofol was then applied (in increments from 10-300 µM). Arteries denuded of endothelium, preincubated or not with indomethacin, were used to investigate the effects of propofol on isolated arteries. Propofol exhibited a bifunctional effect on isolated human pulmonary arteries contracted by U46619, evoking constriction at low concentrations (10-100 µM) followed by secondary relaxation (at 100-300 µM). The extent of constriction induced by propofol was higher in an endothelium-denuded group than in an endothelium-intact group. Preincubation with indomethacin abolished constriction and potentiated relaxation. The maximal relaxation was greater in the endothelium-intact than the endothelium-denuded group. Propofol also suppressed CaCl2-induced constriction in the 60 mM K+-containing Ca2+-free solution in a dose-dependent manner. Fluorescent imaging of Ca2+ using fluo-4 showed that a 10 min incubation with propofol (10-300 µM) inhibited the Ca2+ influx into human pulmonary arterial smooth muscle cells induced by a 60 mM K+-containing Ca2+-free solution. In conclusion, propofol-induced arterial constriction appears to involve prostaglandin production by cyclooxygenase in pulmonary artery smooth muscle cells and the relaxation depends in part on endothelial function, principally on the inhibition of calcium influx through L-type voltage-operated calcium channels.

Left ventricular deformation associated with cardiomyocyte Ca(2+) transients delay in early stage of low-dose of STZ and high-fat diet induced type 2 diabetic rats.

BACKGROUND: In the early stage of diabetes, the cardiac ejection fraction is preserved, despite the existence of the subclinical cardiac dysfunction to some extent. However, the detailed phenotype of this dysfunction and the underlying mechanism remain unclear. To improve our understanding of this issue, we used low-dose STZ and high-fat diet to induce type 2 diabetic models in rats. The effects and the mechanism associated with the early stages of the disease were analyzed. METHODS: The type 2 diabetic mellitus (T2DM) in SD rats were induced through 30 mg/kg STZ and high-fat diet. Two-dimensional spackle-tracking echocardiography (STE) and the dobutamine test were performed to examine the cardiac function. Calcium transients of left ventricular myocytes were detected and the related intracellular signalling factors were analyzed by western blotting. RESULTS: After 6-weeks, T2DM rats in left ventricular (LV) diastole showed decreased global and segment strain(S) levels (P < 0.05), both in the radial and circumferential directions. Strain rate (Sr) abatement occurred in three segments in the radial and circumferential directions (P < 0.05), and the radial global Sr also decreased (P < 0.05). In the systolic LV, radial Sr was reduced, except the segment of the anterior septum, and the Sr of the lateral wall and post septum decreased in the circumferential direction (P < 0.05). Conventional M-mode echocardiography failed to detect significant alterations of cardiac performance between the two groups even after 12 weeks, and the decreased ejection fraction (EF%), fractional shortening (FS%) and end-systolic diameters (ESD) could be detected only under stress conditions induced by dobutamine (P < 0.05). In terms of calcium transients in cardiac myocytes, the Tpeak in model rats at 6 weeks was not affected, while the Tdecay1/2 was higher than that of the controls (P < 0.05), and both showed a dose-dependent delay after isoproterenol treatment (P < 0.05). Western blot analysis showed that in 6-week T2DM rats, myocardial p-PLB expression was elevated, whereas p-CaMKII, p-AMPK and Sirt1 were significantly down-regulated (P < 0.05). CONCLUSION: A rat model of T2DM was established by low dose STZ and a high-fat diet. LV deformation was observed in the early stages of T2DM in association with the delay of Ca(2+) transients in cardiomyocytes due to the decreased phosphorylation of CaMKII. Myocardial metabolism remodeling might contribute to the early LV function and calcium transportation abnormalities.

Role of tumour necrosis factor-a in the regulation of T-type calcium channel current in HL-1 cells.

Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation (AF). Although tumour necrosis factor (TNF)-α levels are increased in patients with AF, the role of TNF-α in the pathogenesis of AF remains unclear. Besides L-type Ca(2+) currents (IC a,L ), T-type Ca(2+) currents (IC a,T ) also plays an important role in the pathogenesis of AF. This study was designed to use the whole-cell voltage-clamp technique and biochemical assays to explore if TNF-α is involved in the pathogenesis of AF through regulating IC a,T in atrial myocytes. It was found that compared with sinus rhythm (SR) controls, T-type calcium channel (TCC) subunit mRNA levels were decreased, while TNF-α expression levels were increased, in human atrial tissue from patients with AF. In murine atrial myocyte HL-1 cells, after culturing for 24 h, 12.5, 25 and 50 ng/mL TNF-α significantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner. The peak current was reduced by the application of 12.5 or 25 ng/mL TNF-α in a concentration-dependent manner (from -15.08 ± 1.11 pA/pF in controls to -11.89 ± 0.83 pA/pF and -8.54 ± 1.55 pA/pF in 12.5 or 25 ng/mL TNF-α group respectively). TNF-α application also inhibited voltage-dependent inactivation of IC a,T, shifted the inactivation curve to the left. These results suggest that TNF-α is involved in the pathogenesis of AF, probably via decreasing IC a,T current density in atrium-derived myocytes through impaired channel function and down-regulation of channel protein expression. This pathway thus represents a potential pathogenic mechanism in AF.

Attenuation of microRNA-16 derepresses the cyclins D1, D2 and E1 to provoke cardiomyocyte hypertrophy.

Cyclins/retinoblastoma protein (pRb) pathway participates in cardiomyocyte hypertrophy. MicroRNAs (miRNAs), the endogenous small non-coding RNAs, were recognized to play significant roles in cardiac hypertrophy. But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy. This study investigates the potential role of microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy. An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC), and in a mouse with transverse aortic constriction (TAC) and in a mouse with subcutaneous injection of phenylephrine (PE) respectively. In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte and based on Ang-II-induced neonatal mouse ventricular cardiomyocyte respectively. We demonstrated that miR-16 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats and mice. Overexpression of miR-16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes, and inhibition of miR-16 induced a hypertrophic phenotype in cardiomyocytes. Expressions of cyclins D1, D2 and E1, and the phosphorylated pRb were increased in hypertrophic myocardium and hypertrophic cardiomyocytes, but could be reversed by enforced expression of miR-16. Cyclins D1, D2 and E1, not pRb, were further validated to be modulated post-transcriptionally by miR-16. In addition, the signal transducer and activator of transcription-3 and c-Myc were activated during myocardial hypertrophy, and inhibitions of them prevented miR-16 attenuation. Therefore, attenuation of miR-16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2 and E1, and activating cyclin/Rb pathway, revealing that miR-16 might be a target to manage cardiac hypertrophy.

Inhibition of LDHA suppresses tumor progression in prostate cancer.

A key hallmark of cancer cells is their altered metabolism, known as Warburg effect. Lactate dehydrogenase A (LDHA) executes the final step of aerobic glycolysis and has been reported to be involved in the tumor progression. However, the function of LDHA in prostate cancer has not been studied. In current study, we observed overexpression of LDHA in the clinical prostate cancer samples compared with benign prostate hyperplasia tissues as demonstrated by immunohistochemistry and real-time qPCR. Attenuated expression of LDHA by siRNA or inhibition of LDHA activities by FX11 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of PC-3 and DU145 cells. Mechanistically, decreased Warburg effect as demonstrated by reduced glucose consumption and lactate secretion and reduced expression of MMP-9, PLAU, and cathepsin B were found after LDHA knockdown or FX11 treatment in PC-3 and DU145 cells. Taken together, our study revealed the oncogenic role of LDHA in prostate cancer and suggested that LDHA might be a potential therapeutic target.

Murine double minute 2 rs2279744 polymorphism and hepatocellular carcinoma risk in East Asians: a meta-analysis.

Murine double minute 2 (MDM2) is a crucial negative regulator of p53 function through several mechanisms. There are many studies performed to assess the association between MDM2 rs2279744 polymorphism and hepatocellular carcinoma risk, but the impact of MDM2 rs2279744 polymorphism on hepatocellular carcinoma in East Asians is unclear owing to the inconsistent findings from previous studies. We conducted a comprehensive meta-analysis of epidemiological studies to shed some light on these contradicting results. We used pooled odds ratio (OR) with its 95 % confidence intervals (95 % CI) to assess the association. Overall, seven studies with a total of 4,993 subjects were finally included. The meta-analysis suggested that MDM2 rs2279744 polymorphism was significantly associated with increased risk of hepatocellular carcinoma in East Asians (G versus T: OR = 1.27, 95 % CI 1.06-1.52, P = 0.01; GG versus TT: OR = 1.59, 95 % CI 1.11-2.27, P = 0.01; GG/GT versus TT: OR = 1.41, 95 % CI 1.07-1.87, P = 0.02; GG versus TT/GT: OR = 1.32, 95 % CI 1.08-1.62, P = 0.008). Sensitivity analysis by excluding low-quality study still suggested that the association above was still significant. Thus, the findings from the meta-analysis support that MDM2 rs2279744 polymorphism is significantly associated with increased risk of hepatocellular carcinoma in East Asians.

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.

Mechanism of macrophage migration inhibitory factor-induced decrease of T-type Ca(2+) channel current in atrium-derived cells.

The T-type Ca(2+) current (I(Ca,T)) plays an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate the role of macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, in the regulation of T-type Ca(2+) channels (TCCs) in atrial myocytes. We used the whole-cell voltage-clamp technique and biochemical assays to study the regulation and expression of I(Ca,T) in atrial myocytes. Gene levels of the α1G and α1H subunit of TCCs were decreased in human atrial tissue of patients with AF. In cultured atrium-derived myocytes (HL-1 cells), mouse recombinant MIF (20 or 40 nm, 24 h) suppressed peak I(Ca,T) in a concentration-dependent manner, impaired the voltage-dependent activation of I(Ca,T) and downregulated TCC α1G and α1H mRNA. The Src inhibitors genistein and PP1 significantly enhanced I(Ca,T). The reduction of I(Ca,T) and TCC subunit mRNA induced by recombinant MIF could be reversed by genistein and PP1. The TCC α1G associated with Src in HL-1 cells and mouse cardiomycytes. Macrophage migration inhibitory factor is involved in the pathogenesis of AF, probably by decreasing the T-type calcium current in atrium-derived myocytes through impairment of channel function and activation of c-Src kinases, representing a potential pathogenic mechanism in atrial fibrillation.

COMMD5 is involved in the mechanisms of hypotension after parathyroidectomy in patients receiving hemodialysis.

BACKGROUND: Secondary hyperparathyroidism (SHPT) is a common complication of end-stage renal disease. Parathyroidectomy (PTx) is often employed for treatment of severe SHPT. However, PTx may cause hypotension via unknown mechanisms. COMM domain-containing protein 5 (COMMD5) in the parathyroid glands has been linked to blood pressure regulation of spontaneously hypertensive rats. OBJECTIVE: To explore the relationship between COMMD5 levels and reduced BP after PTx in patients receiving hemodialysis (HD). METHODS AND RESULTS: (1) The study cohort included 31 patients receiving HD who underwent PTx. Serum COMMD5 levels were higher post-PTx vs. pre-PTx. (2) Sprague-Dawley rats (n = 22) were assigned to a 5/6 nephrectomy group or sham surgery group, vascular rings of the thoracic aorta from rats with CKD were incubated with COMMD5, and changes in vascular tension were compared. COMMD5 inhibited vasoconstriction of vascular rings with intact endothelium, but had no effect on vascular rings without the endothelium. (3) Human umbilical vein endothelial cells were stimulated with COMMD5 or small interfering RNA (siRNA). The expression levels of atrial natriuretic peptide (ANP) and endothelial nitric oxide synthase (eNOS) were up-regulated and down-regulated, respectively. CONCLUSIONS: Serum COMMD5 levels were increased after PTx in SHPT patients. COMMD5 promoted high expression of ANP and eNOS in endothelial cells, leading to vasodilation and resulting in hypotension.

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.

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.