Dysregulation of miR-25-3p in Diabetic Nephropathy and Its Role in Inflammatory Response.

To investigate the expression level of miR-25-3p in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN), and its effect on proliferation, apoptosis and inflammatory response of mesangial cells cultured with high glucose. Blood samples of all clinical subjects were collected for RT-qPCR analysis to detect serum miR-25-3p levels. Human mesangial cells (HMCs) cultured with high glucose were used to construct DN model in vitro. MTT assay, flow cytometry and ELISA were used to evaluate the effects of miR-25-3p on the proliferation, apoptosis, and inflammatory response of DN cell models. Serum miR-25-3p was decreased in both T2DM group and DN group, but more in DN group. Serum miR-25-3p was positively correlated with eGFR and negatively correlated with UAER. The expression of miR-25-3p was reduced in HMCs induced by high glucose. Transfection of miR-25-3p mimic could significantly up-regulate the miR-25-3p level in HMCs. Besides, high glucose culture resulted in abnormal proliferation of HMCs, reduced apoptotic cells, and increased inflammation. The addition of miR-25-3p mimic significantly inhibited cell proliferation and promoted cell apoptosis and reduced the production of inflammatory factors. The abnormal reduction of serum miR-25-3p in DN indicates that it may be a potential biomarker for clinical diagnosis of DN. In in vitro experiments, miR-25-3p was involved in the progression of DN by regulating cell proliferation, apoptosis, and inflammatory response.

The clinical utility of circulating cell division control 42 in small-vessel coronary artery disease patients undergoing drug-coated balloon treatment.

BACKGROUND: Cell division control 42 (CDC42) regulates atherosclerosis, blood lipids, and inflammation and thus affects coronary artery disease (CAD), but its utility in drug-coated balloon (DCB)-treated small-vessel CAD (SV-CAD) patients is unclear. This study intended to evaluate the change and prognostic role of CDC42 in SV-CAD patients underwent DCB. METHODS: Serum CDC42 was measured by enzyme-linked immunosorbent assay in 211 SV-CAD patients underwent DCB at baseline, day (D) 1, D3, and D7, as well as in 50 healthy controls (HCs). RESULTS: CDC42 was decreased in SV-CAD patients compared to HCs (P < 0.001), and it was negatively associated with total cholesterol (P = 0.015), low-density lipoprotein cholesterol (P = 0.003), C-reactive protein (P = 0.001), multivessel disease (P = 0.020), and American college of cardiology/American heart association type B2/C lesions (P = 0.039) in SV-CAD patients. Longitudinally, CDC42 decreased from baseline to D1 and then gradually increased to D7 (P < 0.001) in SV-CAD patients after DCB. Interestingly, high CDC42 (cut-off value = 500 pg/mL) at baseline (P = 0.047), D3 (P = 0.046), and D7 (P = 0.008) was associated with a lower accumulating target lesion failure (TLF) rate; high CDC42 at D3 (P = 0.037) and D7 (P = 0.041) was related to a lower accumulating major adverse cardiovascular event (MACE) rate in SV-CAD patients underwent DCB. Importantly, CDC42 at D7 (high vs. low) independently predicted lower accumulating TLF (hazard ratio (HR) = 0.145, P = 0.021) and MACE (HR = 0.295, P = 0.023) risks in SV-CAD patients underwent DCB. CONCLUSIONS: Circulating CDC42 level relates to milder disease conditions and independently estimates lower risks of TLF and MACE in SV-CAD patients underwent DCB, but further validation is still needed.

Apoe-knockout induces strong vascular oxidative stress and significant changes in the gene expression profile related to the pathways implicated in redox, inflammation, and endothelial function.

Apolipoprotein E (APOE) was recognized as a key regulator of lipid metabolism, which prompted the Apoe-knockout (Apoe-/-) mouse to be the most widely used atherosclerotic model. However, with more and more important physiological roles of APOE being revealed, it is necessary to reacquaint its comprehensive function in the aorta. In this study, we aimed to reveal how Apoe-knockout impacts the gene pathways and phenotypes in the aorta of mice. We performed transcriptome sequencing to acquire the gene expression profile (GEP) for C57BL/6J and Apoe-/- mouse aorta, and used enrichment analysis to reveal the signal pathways enriched for differentially expressed genes (DEGs). In addition, we used immunofluorescence and ELISA to detect the phenotypic differences of vascular tissues and plasma in the two-group mice. Apoe-knockout resulted in significant changes in the expression of 538 genes, among which about 75% were up-regulated and 134 genes were altered more than twice. In addition to the lipid metabolism pathways, DEGs were also mainly enriched in the pathways implicated in endothelial cell proliferation, migration of epithelial cells, immune regulatory, and redox. GSEA shows that the up-regulated genes are mainly enriched in 'immune regulation pathways' and 'signal regulation' pathways, while the down-regulated genes are enriched in lipid metabolism pathways, 'regulation_of_nitric_oxide_synthase_activity' and the pathways involved in redox homeostasis, including 'monooxygenase regulation', 'peroxisomes' and 'oxygen binding'. A significant increase of reactive oxygen species and a remarkable reduction of GSH/GSSG ratio were respectively observed in the vascular tissues and plasma of Apoe-/- mice. In addition, endothelin-1 significantly increased in the vascular tissue and the plasma of Apoe-/- mice. Taken together, our results suggest that besides functioning in lipid metabolism, APOE may be an important signal regulator that mediates the expression of the genes related to the pathways involved in redox, inflammation, and endothelial function. Apoe-knockout-induced strong vascular oxidative stress is also the key factor contributing to atherosclerosis.

Clinical Efficacy of Blood Ultrafiltration Therapy in Patients with Acute Decompensated Chronic Heart Failure Running Title: Blood Ultrafiltration Therapy for Heart Failure.

In this study, we investigated the clinical effects of blood ultrafiltration therapy in patients with acute decompensated chronic heart failure. We enrolled 78 patients with acute decompensated chronic heart failure who were admitted to a hospital from September 2017 to December 2021, and divided them into two groups based on the digital randomization method. The FQ-16 heart failure ultrafiltration dehydrating device blood ultrafiltration therapy was administered to the observation group (39 patients) for 8-16 hours, while the control group (39 patients) received the stepped drug therapy. Echocardiography was used to assess the changes in cardiac function of the patients in both groups before and after treatment. The changes in urine volume, N-terminal pro-B-type natriuretic peptide (NT-proBNP), plasma renin, and serum creatinine levels were measured before and after the treatment to compare the overall response rate of the patients in both groups. The differences in left ventricular end-systolic dimension and left ventricular end-diastolic dimension and the ejection fraction between the groups before treatment were not statistically significant (P > 0.05), however, the left ventricular end-diastolic dimension in the observation group was significantly lower and the ejection fraction was significantly higher (P < 0.05) compared with that before treatment; the urine volume, N-terminal pro-B-type natriuretic peptide (NT-proBNP), plasma renin, and serum creatinine were significantly improved in both groups after treatment compared with that before treatment. All indexes in the observation group were better than those in the control group (P < 0.05), 74.36%. The overall response rate of the observation group was 94.87%, x2 = 4.843 and the difference between groups was statistically significant (P < 0.05). Blood ultrafiltration therapy for patients with acute decompensated chronic heart failure can improve their cardiac and renal functions, reduce NT-proBNP, reduce volume load, and enhance efficacy while ensuring high safety.

The Acid Sphingomyelinase Inhibitor Amitriptyline Ameliorates TNF-α-Induced Endothelial Dysfunction.

PURPOSE: Inflammation associated endothelial cell (EC) dysfunction is key to atherosclerotic disease. Recent studies have demonstrated a protective role of amitriptyline in cardiomyocytes induced by hypoxia/reoxygenation. However, the mechanism by which amitriptyline regulates the inflammatory reaction in ECs remains unknown. Thus, the aim of this study was to investigate whether amitriptyline protects against inflammation in TNF-α-treated ECs. METHODS: HUVECs were incubated with amitriptyline (2.5 µM) or TNF-α (20 ng/ml) for 24 h. EdU, tube formation, transwell, DHE fluorescence staining, and monocyte adhesion assays were performed to investigate endothelial function. Thoracic aortas were isolated from mice, and vascular tone was measured with a wire myograph system. The levels of ICAM-1, VCAM-1, MCP-1, phosphorylated MAPK and NF-κB were detected using western blotting. RESULTS: Amitriptyline increased the phosphorylation of nitric oxide synthase (eNOS) and the release of NO. Amitriptyline significantly inhibited TNF-α-induced increases in ASMase activity and the release of ceramide and downregulated TNF-α-induced expression of proinflammatory proteins, including ICAM-1, VCAM-1, and MCP-1 in ECs, as well as the secretion of sICAM-1 and sVCAM-1. TNF-α treatment obviously increased monocyte adhesion and ROS production and impaired HUVEC proliferation, migration and tube formation, while amitriptyline rescued proliferation, migration, and tube formation and decreased monocyte adhesion and ROS production. Additionally, we demonstrated that amitriptyline suppressed TNF-α-induced MAPK phosphorylation as well as the activity of NF-κB in HUVECs. The results showed that the relaxation response of aortic rings to acetylcholine in the WT-TNF-α group was much lower than that in the WT group, and the sensitivity of aortic rings to acetylcholine in the WT-TNF-α group and WT-AMI-TNF-α group was significantly higher than that in the WT-TNF-α group. CONCLUSION: These results suggest that amitriptyline reduces endothelial inflammation, consequently improving vascular endothelial function. Thus, the identification of amitriptyline as a potential strategy to improve endothelial function is important for preventing vascular diseases.

Impaired coronary flow reserve in patients with supra-normal left ventricular ejection fraction at rest.

PURPOSE: Recently, a "U" hazard ratio curve between resting left ventricular ejection fraction (LVEF) and prognosis has been observed in patients referred for routine clinical echocardiograms. The present study sought to explore whether a similar "U" curve existed between resting LVEF and coronary flow reserve (CFR) in patients without severe cardiovascular disease (CVD) and whether impaired CFR played a role in the adverse outcome of patients with supra-normal LVEF (snLVEF, LVEF ≥ 65%). METHODS: Two hundred ten consecutive patients (mean age 52.3 ± 9.3 years, 104 women) without severe CVD underwent clinically indicated rest/dipyridamole stress electrocardiography (ECG)-gated 13 N-ammonia positron emission tomography/computed tomography (PET/CT). Major adverse cardiac events (MACE) were followed up for 27.3 ± 9.5 months, including heart failure, late revascularization, re-hospitalization, and re-coronary angiography for any cardiac reason. Clinical characteristics, corrected CFR (cCFR), and MACE were compared among the three groups categorized by resting LVEF detected by PET/CT. Dose-response analyses using restricted cubic spline (RCS) functions, multivariate logistic regression, and Kaplan-Meier survival analysis were conducted to evaluate the relationship between resting LVEF and CFR/outcome. RESULTS: An inverted "U" curve existed between resting LVEF and cCFR (p = 0.06). Both patients with snLVEF (n = 38) and with reduced LVEF (rLVEF, LVEF < 55%) (n = 66) displayed a higher incidence of reduced cCFR than those with normal LVEF (nLVEF, 55% ≤ LVEF < 65%) (n = 106) (57.9% vs 54.5% vs 34.3%, p < 0.01, respectively). Both snLVEF (p < 0.01) and rLVEF (p < 0.05) remained independent predictors for reduced cCFR after multivariable adjustment. Patients with snLVEF encountered more MACE than those with nLVEF (10.5% vs 0.9%, log-rank p = 0.01). CONCLUSIONS: Patients with snLVEF are prone to impaired cCFR, which may be related to the adverse prognosis. Further investigations are warranted to explore its underlying pathological mechanism and clinical significance.

miRNA-483-5p Targets HDCA4 to Regulate Renal Tubular Damage in Diabetic Nephropathy.

This study was designed to evaluate the diagnostic value of miR-483-5p in diabetic nephropathy (DN), and its effect and mechanism on apoptosis and inflammation of human proximal renal tubular cells (HK2) induced by high glucose (HG). Thirty healthy controls, 30 types 2 diabetes mellitus (T2DM) patients, and 28 DN patients were enrolled. miR-483-5p mRNA levels in serum were analyzed by RT-qPCR assays. The receiver operating characteristic curve (ROC) was used to analyze the diagnostic value of miR-483-5p in DN. HK2 cells were induced by HG to establish an in vitro study model. CCK-8 and flow cytometry was used to detect cell viability, apoptosis, and reactive oxygen species (ROS) generation. Inflammation levels were measured by ELISA. Luciferase reporter assay was used to detect target genes of miR-483-5p. miR-483-5p was decreased in DN patients. The decreased level of miR-483-5p was positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with proteinuria. miR-483-5p can significantly distinguish DN patients from healthy controls and T2DM and has a high diagnostic value. miR-483-5p decreased in HK2 cells induced by HG, and overexpression of miR-483-5p reversed HG-induced decreased cell activity, increased apoptosis, ROS production, and inflammation. Histone deacetylase 4 (HDCA4) was markedly increased in DN patients and HG-induced HK2 cells. miR-483-5p directly targeted HDCA4, and increasing miR-483-5p inhibited HDCA4 increased in HG-induced HK2. In conclusion, the results indicate that reduction of miR-483-5p has a high diagnostic value in DN, and overexpression of miR-483-5p has a certain protective effect on HK2 cells induced by HG by targeting HDCA4.

Plasma-derived exosomal miR-183 associates with protein kinase activity and may serve as a novel predictive biomarker of myocardial ischemic injury.

Myocardial infarction (MI) is primarily caused by ischemic heart or coronary artery disease and is a major cause of mortality worldwide. Thus, it is necessary to establish reliable biochemical markers for the early diagnosis of MI. MicroRNAs (miRNAs or miR) have been demonstrated to circulate in biological fluids and are enclosed in extracellular vesicles, including exosomes. The current study assessed the differential expression of exosomal miRNAs in the plasma of patients with MI and healthy individuals, and the possible mechanism involved. Plasma-derived exosomes were isolated from patients with MI and healthy control individuals, and vesicles with a membrane were observed using transmission electron microscopy. Furthermore, an exosomal miRNA expression profile was compared between patients with MI and healthy individuals using a miRNA microarray. Significantly differentially expressed miRNAs were validated using reverse transcription-quantitative polymerase chain reaction. To the best of our knowledge, the present study was the first to demonstrate that miR-183 was markedly upregulated in patients with MI compared with healthy individuals. In addition, the relative exosomal miR-183 level increased with the degree of myocardial ischemic injury. Additionally, GO and KEGG analyses demonstrated that miR-183 is primarily involved in cell communication, protein kinase activity regulation and adrenergic signaling in cardiomyocytes. Furthermore, a protein-protein interaction network of all the miR-183 target genes was constructed. The results demonstrated that five core genes (PPP2CB, PPP2CA, PRKCA, PPP2CA, PPP2R5C and PPP2R2A) in the PPI network were also associated with protein kinase activity regulation and adrenergic signaling in cardiomyocytes. Taken together, these data demonstrate that exosomal miR-183 derived from the serum of patients with MI may be a novel diagnostic biomarker involved in the regulation of protein kinase activity. miR-183 may therefore be further developed for clinical use to benefit patients with coronary artery diseases.

Protective Effect and Potential Mechanism of Simvastatin on Myocardial Injury Induced by Diabetes with Hypoglycemia.

BACKGROUND: Simvastatin has been reported to reduce cardiovascular related morbidity and mortality in clinical trials which was independent of its cholesterol-lowering effect. This study aims to investigate the protective effect of simvastatin on myocardial injury caused by diabetic hypoglycemia and the possible underlying mechanism. METHODS: We used streptozotocin (STZ) at a dose of 55 mg/kg to induce diabetes mellitus (DM) and over-dose insulin to induce hypoglycemia to establish diabetic hypoglycemia rat models. The cardiac protective effect of simvastatin treatment (at a dose of 40 mg/kg·d) upon diabetic hypoglycemia models was assessed. Ex vivo cardiac function, cardiomyocyte inotropic property and calcium transient were evaluated. The expression levels of microRNA (miRNA) and NF-κB in heart tissues were investigated. RESULTS: Both systolic and diastolic functions in diabetic rats were weakened, and further worsened in diabetic hypoglycemia rats, partially restored after administration of simvastatin. The protective effect of simvastatin on cardiac function under diabetic hypoglycermia was associated with decreased intracellular calcium and increased calcium sensitivity. miRNA array showed a differential profile of miRNA expression existed in diabetic hypoglycemia rats compared with diabetic non-hypoglycemia group, and that simvastatin could reverse the altered miRNA expression in diabetic hypoglycemia group. CONCLUSION: Simvastatin has protective effect on myocardial injury caused by diabetes with hypoglycemia, which is associated with increased calcium sensitivity, decreased NF-κB expression and altered miRNA expression profile.

[Simvastatin prevented myocardium of diabetes rats from apoptosis through inhibition of oxidative stress].

OBJECTIVE: To investigate the protective effects and the possible mechanisms of simvastatin on myocardial injury induced by diabetes. METHODS: Twenty-four SD rats (180~220)g were randomly divided into control group (control, n=8) and modeled groups(n=16), the modeled groups were injected with streptozotocin intraperitoneally to induce diabetes. Then the modeled rats were randomly divided into diabetes mellitus group (DM group, n=8) and diabetes mellitus + simvastatin group (DM+S group, n=8). Rats in DM+S group were treated with simvastatin at the dose of 40 mg/(kg·d)by gavage for 4 weeks, and the other two groups were treated with the same amount of saline. At the end of experiments, the heart tissues were collected for further observation. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in heart tissues were measured by spectrophotometry; HE staining of rat heart slides was used to observe the pathological changes; TUNEL assay was used to determine the apoptosis index of myocardial cells in each groups; The distribution of p53 in the heart tissues was evaluated by immunohistochemistry; Western blot was used to detect the expressions of p53, p53-phospho-serine 15, Bax and Bcl-2 in the heart tissues. RESULTS: ①Compared with control group, the content of malondialdehyde (MDA) was increased while the activity of superoxide dismutase (SOD) was decreased significantly in DM group (P<0.01). After simvastatin administration, the activity of SOD was increased and the content of MDA was decreased significantly (P<0.01). ② HE staining results showed that the myocardial cells in the DM group were disorganized, with unclear morphological structure and a large number of inflammatory cells infiltration. Compared with DM group, the myocardial morphology in DM+S group was improved significantly. ③TUNEL staining results showed that the apoptosis index of myocardial cells in DM group was increased significantly compared with that of control group, and the apoptosis index was decreased significantly after the treatment of simvastatin (P<0.01).④ Immunohistochemistry showed that compared with control group,the expression of p53 in DM group was increased significantly, and was expressed in both cytoplasm and nucleus, while the expression of p53 in DM+S group was decreased and the expression of p53 in nucleus was decreased significantly (P<0.01). ⑤ The results of Western blot showed that the expression levels of p53, p53-phospho-serine15 and Bax were higher than those in control group, and the expression of Bcl-2 was lower than that in control group (P<0.01). After simvastatin administration, the expression levels of p53,p53-phospho-serine 15 (P<0.01) and Bax were decreased significantly (P<0.05) and the expression of Bcl-2 was increased (P<0.05). CONCLUSIONS: Simvastatin exerted protective effects on myocardial injury caused by diabetes through improving the abnormal morphological changes of diabetic myocardium, alleviating oxidative stress and inhibiting apoptosis of myocardial cells. The mechanism is related to the regulation of apoptosis pathway mediated by p53.

[Protective effect of simvastatin on kidney of rats with diabetes mellitus and the possible mechanism].

OBJECTIVE: To observe the protective effect of simvastatin on renal injury in diabetic rats and to explore the possible molecular mechanism. METHODS: Twenty-four SD rats were randomly divided into normal control (NC) group (n=8) and modeling group (n=16).The rats in modeling group were injected with streptozotocin intraperitoneally at a dose of 55 mg/kg to establishing diabetic rat model. After diabetic ratmodel established successfully, the diabetic rats were randomly subdivided into diabetes mellitus (DM) group and diabetes mellitus + simvastatin (DM+Sim) group (n=8).Rats in DM+Sim group were given simvastatin at a dose of 40 mg/kg by oral gavages, once a day for 4 weeks. Morphological changes and interstitial fibrosis of kidney were observed by histopathological method. The expressions of relative protein in endoplasmic reticulum stress, inflammatory molecules in renal tissues and cells apoptosis were detected by molecular biology method. RESULTS: ① Compared with NC group, the pathological changes of glomerulus and tubulointerstitium were obvious, and the collagen fibers were obviously erythrophilous and unevenly distributed in DM group. Compared with DM group, the morphological changes and fibrosis were significantly improved in DM+Sim group. ② The expressions of GRP78, p-IRE1α, NF-κB p65 and MCP-1 in DM group were significantly higher than those in NC group (P<0.05), while the expressions of GRP78, p-IRE1α, NF-κB p65 and MCP-1in DM + Sim group were decreased (P<0.05). ③ There were a small number of apoptotic nuclei in the glomeruli and adjunctive renal tubules in NC group detected by TUNEL assay, while there were a large number of apoptotic nuclei in DM group (P<0.01). The number of apoptotic nuclei was decreased significantly in DM+Sim group (P<0.01). CONCLUSIONS: Morphologicalchanges and fibrosis of renal tissue are improved obviously, and the number of apoptotic cells is decreased significantly after administration of simvastatin in diabetic rats. Simvastatin exertsthe protective effect on diabetic nephropathy by inhibiting endoplasmic reticulum stress and NF-κB inflammatory signaling pathway, and reducing renal cell apoptosis.

A partially penalty immersed Crouzeix-Raviart finite element method for interface problems.

The elliptic equations with discontinuous coefficients are often used to describe the problems of the multiple materials or fluids with different densities or conductivities or diffusivities. In this paper we develop a partially penalty immersed finite element (PIFE) method on triangular grids for anisotropic flow models, in which the diffusion coefficient is a piecewise definite-positive matrix. The standard linear Crouzeix-Raviart type finite element space is used on non-interface elements and the piecewise linear Crouzeix-Raviart type immersed finite element (IFE) space is constructed on interface elements. The piecewise linear functions satisfying the interface jump conditions are uniquely determined by the integral averages on the edges as degrees of freedom. The PIFE scheme is given based on the symmetric, nonsymmetric or incomplete interior penalty discontinuous Galerkin formulation. The solvability of the method is proved and the optimal error estimates in the energy norm are obtained. Numerical experiments are presented to confirm our theoretical analysis and show that the newly developed PIFE method has optimal-order convergence in the [Formula: see text] norm as well. In addition, numerical examples also indicate that this method is valid for both the isotropic and the anisotropic elliptic interface problems.

A critical role of DDRGK1 in endoplasmic reticulum homoeostasis via regulation of IRE1α stability.

Disturbance of endoplasmic reticulum (ER) homoeostasis induces ER stress and leads to activation of the unfolded protein response (UPR), which is an adaptive reaction that promotes cell survival or triggers apoptosis, when homoeostasis is not restored. DDRGK1 is an ER membrane protein and a critical component of the ubiquitin-fold modifier 1 (Ufm1) system. However, the functions and mechanisms of DDRGK1 in ER homoeostasis are largely unknown. Here, we show that depletion of DDRGK1 induces ER stress and enhances ER stress-induced apoptosis in both cancer cells and hematopoietic stem cells (HSCs). Depletion of DDRGK1 represses IRE1α-XBP1 signalling and activates the PERK-eIF2α-CHOP apoptotic pathway by targeting the ER-stress sensor IRE1α. We further demonstrate that DDRGK1 regulates IRE1α protein stability via its interaction with the kinase domain of IRE1α, which is dependent on its ufmylation modification. Altogether, our results provide evidence that DDRGK1 is essential for ER homoeostasis regulation.

Role of ß3 adrenoceptor in rat thoracic aorta contractility.

To clarify the role of ß3-AR in rat thoracic aorta contractility and underlying mechanisms. BRL 37344 (BRL) was used to detect the role of ß3-AR on rat thoracic aorta. 40 rats were randomly divided into Sham control group, Sham+SR group with SR 59230A (SR) injected, chronic heart failure (CHF) control group, and CHF+SR group. The effects of SR on thoracic aorta structure, function and NF-κB expression were estimated. BRL produced relaxant effect in both endothelium-intact and endothelium-free aorta rings, which was antagonized by SR and partially by L-NAME, but not changed by Propranolol. Similar results were obtained on thoracic aorta smooth muscle of CHF rats. ß3-AR was located in both vascular smooth muscle layer and endothelial layer. After SR injection, the aorta rings in Sham+SR group showed reduced endothelium-dependent relaxation response to Ach compared with Sham control group. The aorta rings in CHF control group showed reduced endothelium-dependent relaxation to Ach, with increased endothelium-dependent relaxation in CHF+SR group. Besides, SR injection showed increased contraction to NA. Meanwhile, NF-κB expression in Sham+SR group was higher than Sham control group, with increased expression in CHF control group but decreased in CHF+SR group. Microarray screening showed 48 and 42 differentially expressed miRNAs in Sham+SR rats and CHF+SR rats respectively with 19 of them associated with NF-κB pathways. ß3-AR is expressed in rat aorta and exerts relaxant effects through NOS-dependent pathway. ß3-AR Inhibition delayed damage of vessels in development of heart failure possibly through regulation of NF-κB signaling pathway.

Carbachol exhibited positive inotropic effect on rat ventricular myocytes via M2 muscarinic receptors.

The present study was aimed to investigate the positive inotropic mechanism of carbachol (CCh) on rat ventricular myocytes. The effects of CCh on L-type calcium current (I(Ca,L)) and Na(+)/Ca(2+) exchange current (I(Na/Ca)) were investigated in isolated rat ventricular myocytes. After loading myocytes with Fura-2/AM, electrically triggered Ca(2+) transient and cell shortening in single myocyte were measured simultaneously using ion imaging system with charge-coupled device (CCD) camera. CCh (100 mumol/L) increased I(Na/Ca) in forward mode from (1.18 +/- 0.57) pA/pF in the control group to (1.65 +/- 0.52) pA/pF (P<0.01) and that in reverse mode from (1.11 +/- 0.49) pA/pF in the control group to (1.53 +/- 0.52) pA/pF (P<0.01), respectively. CCh had no effect on I(Ca,L). The stimulatory effect of CCh on I(Na/Ca) was blocked by application of atropine, a non-selective M muscarinic receptor antagonist, and methoctramine, a selective M(2) muscarinic receptor antagonist. CCh (100 mumol/L) increased cell shortening from (3.00 +/- 0.67) mum in the control group to (3.55 +/- 1.21) mum. Ca(2+) transient was also increased from 203.8 +/- 50.0 in the control group to 234.8 +/- 64.3 in 100 mumol/L CCh group. KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchange, did not change the baseline level of cell shortening and Ca(2+) transient, while completely abolished CCh-induced increments of both Ca(2+) transient and cell shortening. CCh increased cell shortening and Ca(2+) transient in the presence of nicardipine, indicating that the positive inotropic effect of CCh was through activation of Na(+)/Ca(2+) exchange. Calcium sensitivity was not changed by CCh. Both atropine and methoctramine abolished the positive inotropic effects of CCh, demonstrating that CCh induced positive inotropism via the M(2) muscarinic receptor. The results suggest that CCh increases cell contraction and Ca(2+) transient in rat ventricular myocytes. This positive inotropic effect of CCh is through activation of reverse mode Na(+)/Ca(2+) exchange, and M(2) receptors are involved in mediating CCh-induced contraction.

Carbachol augments Na/Ca exchange current via M2 muscarinic receptors in guinea pig ventricular myocytes.

Stimulation of cardiac mAChRs by carbachol (CCh) produces a biphasic inotropic response. The mechanisms of the positive inotropic response by higher concentration of CCh appear to be paradoxical. This article was aimed to study the mechanism of the positive inotropic effect of CCh in guinea pig ventricular myocytes. The effects of CCh on L-type calcium current (I(Ca)) and Na/Ca exchange current (I(Na/Ca)) were observed in voltage-clamped guinea pig ventricular myocytes by using Axon 200A amplifier. The results showed that CCh (100 micromol/L) increased both forward mode and reverse mode I(Na/Ca) from (1.2+/-0.1) pA/pF to (2.0+/-0.3) pA/pF for forward mode (P<0.01) and from (1.3+/-0.5) pA/pF to (2.1+/-0.8) pA/pF for reverse mode (P<0.01), respectively. CCh had no effect on I(Ca). The stimulating effect of CCh on I(Na/Ca) could be blocked by application of atropine, a nonselective blocker of muscarinic receptors, which means that the stimulating effect of CCh is through the activation of muscarinic receptors. We made a further study by using methoctramine, a selective antagonist of M2 muscarinic receptors. It completely abolished I(Na/Ca) induced by 100 micromol/L CCh, indicating that the effect of CCh on I(Na/Ca) was mediated by M2 muscarinic receptors. It is generally accepted that contraction in cardiac myocytes results from elevation of intracellular Ca2+ concentration. Ca2+ enters the cells through two pathways: L-type Ca2+ channels and, less importantly, reverse mode Na/Ca exchange. The calcium influx via both pathways promotes the contraction of cardiac myocytes. Because CCh had no effect on L-type Ca2+ current, the increase in Na/Ca exchange current might be the main factor in the positive inotropism of CCh. These results suggest that the positive inotropic effect of CCh in guinea pig heart is through stimulation of Na/Ca exchange and is mediated by M2 muscarinic receptors.

Inotropic effects of MCI-154 on rat cardiac myocytes.

Calcium sensitizers exert positive inotropic effects without increasing intracellular Ca(2+). Thus, they avoid the undesired effects of Ca(2+) overload such as arrhythmias and cell injury, but most of them may impair myocyte relaxation. However, MCI-154, also a calcium sensitizer, has no impairment to cardiomyocyte relaxation. To clarify the underlying mechanisms, we examined the effects of MCI-154 on Ca(2+) transient and cell contraction using ion imaging system, and its influence on L-type Ca(2+) current and Na(+)/ Ca(2+) exchange current with patch clamp technique in rat ventricular myocytes as well. The results showed that: (1) MCI-154 (1-100 micromol/L) had no effect on L-type Ca(2+) current; (2) MCI-154 concentration-dependently increased cell shortening from 5.00+/-1.6 microm of control to 6.2+/-1.6 microm at 1 micromol/L, 8.7+/-1.6 microm at 10 micromol/L and 14.0+/-1.4 microm at 100 micromol/L, respectively, with a slight increase in Ca(2+) transient amplitude and an abbreviation of Ca(2+) transient restore kinetics assessed by time to 50% restore (TR(50)) and time to 90% restore (TR(90)); (3) MCI-154 dose-dependently increased the electrogenic Na(+)/ Ca(2+) exchange current both in the inward and the outward directions in rat ventricular myocytes. These results indicate that MCI-154 exerted a positive inotropic action without impairing myocyte relaxation. The stimulation of inward Na(+)/ Ca(2+) exchange current may accelerate the Ca(2+) efflux, leading to abbreviations of TR(50) and TR(90) in rat myocytes. The findings suggest that the improvement by MCI-154 of myocyte relaxation is attributed to the forward mode of Na(+)/ Ca(2+) exchange.

Enhancement of Ca(2+) transients and contraction of single ventricular myocytes of rats by 5-(N,N-dimethyl) amiloride.

The effects of 5-(N,N-dimethyl)amiloride (DMA) (a blocker of Na(+)/H(+) exchanger or Na(+)/Ca(2+) exchanger) on calcium transient and cell contraction in isolated ventricular myocytes in normal rats and rats with myocardial hypertrophy were examined using ion imaging system with a charge coupled digital camera (CCD camera). Loading myocytes with Fura-2, electrically triggered Ca(2+) transients and cell shortening were measured simultaneously. The results showed that 10 micromol/L DMA increased Ca(2+) transient and cell shortening from 209.60+/-54.96 and 3.07+/-0.97 micrometer to 238.50+/-80.41 and 4.07+/-1.02 micrometer, respectively (P<0.05), which was completely abolished by application of KB-R7943, a specific reverse mode Na(+)/Ca(2+) exchanger blocker. After blocking L-type Ca(2+) channels by nicardipine, DMA also enhanced Ca(2+) transient and cell shortening. In rats with myocardial hypertrophy, DMA showed the common pharmacologic profile as in normal rats but more intense stimulating effects on Ca(2+) transient and cell contraction. The results suggest that DMA increase Ca(2+) transient and cell contraction via stimulating reverse mode Na(+)/ Ca(2+) exchange, and the stimulating effect is more pronounced in rats with myocardial hypertrophy than in normal ones.