The role of remnant cholesterol beyond low-density lipoprotein cholesterol in diabetes mellitus.

BACKGROUND: Previous research has linked elevated low-density lipoprotein cholesterol (LDL-C) and remnant cholesterol (RC) with diabetes mellitus (DM). The present study aims to estimate the RC-related DM risk beyond LDL-C, and to investigate the extent to which the association of RC and DM is mediated via insulin resistance and inflammation. METHODS: We enrolled 7308 individuals without previous history of DM into the present study from the China Health and Nutrition Survey. Fasting RC was calculated as total cholesterol minus LDL-C and high-density lipoprotein cholesterol. Subjects were divided into four groups according to their LDL-C (100 mg/dL) and RC (24 mg/dL) levels to evaluate the role of LDL-C vs. RC on DM. A logistic regression analysis was then employed to evaluate the relationships between the discordant/concordant LDL-C and RC and DM. A mediation analysis was undertaken to identify potential mediators. RESULTS: Of all the participants, a total of 625 (8.55%) patients were newly diagnosed with DM. Compared to the high LDL-C/low RC group, the low LDL-C/high RC group was more common in DM patients. After a multivariate adjustment, elevated LDL-C and RC were associated with DM. Moreover, the low LDL-C/high RC group and the high LDL-C/low RC group manifested a 4.04-fold (95% CI 2.93-5.56) and 1.61-fold (95% CI 1.21-2.15) higher risk of DM, relative to those with low LDL-C/low RC. The subgroup analysis indicated that low LDL-C/high RC was more likely to be related to DM in females. Similar results were also shown when the sensitivity analyses were performed with different clinical cut-points of LDL-C. Insulin resistance and inflammation partially mediated the association between RC and DM. CONCLUSIONS: Our findings provided evidence for RC beyond the LDL-C associations with DM that may be mediated via insulin resistance and the pro-inflammatory state. In addition, women are more susceptible to RC exposure-related DM.

Myocardial Mechanics Parameters That Predict Left Ventricular Outflow Tract Obstruction in Patients With Hypertrophic Cardiomyopathy: A Cardiovascular Magnetic Resonance Feature Tracking Analysis.

OBJECTIVE: To identify left ventricular (LV) myocardial mechanics predictors of LV outflow tract obstruction (LVOTO) in patients with hypertrophic cardiomyopathy (HCM). METHODS: Thirty-nine adults with HCM and 21 controls underwent cardiovascular magnetic resonance. The feature tracking (FT) analysis results of HCM patients with and without LVOTO and controls were compared. RESULTS: Global radial strain measured on the short-axis slice (GRS-SAX) (odds ratio [OR], 1.09; 95% confidence interval [CI], 1.02-1.15; P < 0.01), global longitudinal strain measured on the long-axis slice (GLS-LAX) (OR, 1.81; 95% CI, 1.21-2.73; P < 0.01) and GRS measured on the long-axis slice (GRS-LAX) (OR, 1.07; 95% CI, 1.01-1.13; P = 0.02) were independent predictors of LVOTO. The combination of GRS-SAX plus GLS-LAX and GRS-LAX showed great discriminatory power for identifying LVOTO with an area under the receiver operating characteristic curve value of 0.91 (95% CI: 0.81-1.00). CONCLUSIONS: In adult HCM patients, GRS-SAX, GLS-LAX, and GRS-LAX were independent predictors of LVOTO. The combination of GRS-SAX plus GLS-LAX and GRS-LAX showed great discriminatory power for identifying LVOTO.

Prediction of pulmonary pressure after Glenn shunts by computed tomography-based machine learning models.

OBJECTIVES: This study aimed to develop non-invasive machine learning classifiers for predicting post-Glenn shunt patients with low and high risks of a mean pulmonary arterial pressure (mPAP) > 15 mmHg based on preoperative cardiac computed tomography (CT). METHODS: This retrospective study included 96 patients with functional single ventricle who underwent a bidirectional Glenn procedure between November 1, 2009, and July, 31, 2017. All patients underwent post-procedure CT, followed by cardiac catheterization. Overall, 23 morphologic parameters were manually extracted from cardiac CT images for each patient. The Mann-Whitney U or chi-square test was applied to select the most significant predictors. Six machine learning algorithms including logistic regression, Naive Bayes, random forest (RF), linear discriminant analysis, support vector machine, and K-nearest neighbor were used for modeling. These algorithms were independently trained on 100 train-validation random splits with a 3:1 ratio. Their average performance was evaluated by area under the curve (AUC), accuracy, sensitivity, and specificity. RESULTS: Seven CT morphologic parameters were selected for modeling. RF obtained the best performance, with mean AUC of 0.840 (confidence interval [CI] 0.832-0.850) and 0.787 (95% CI 0.780-0.794); sensitivity of 0.815 (95% CI 0.797-0.833) and 0.778 (95% CI 0.767-0.788), specificity of 0.766 (95% CI 0.748-0.785) and 0.746 (95% CI 0.735-0.757); and accuracy of 0.782 (95% CI 0.771-0.793) and 0.756 (95% CI 0.748-0.764) in the training and validation cohorts, respectively. CONCLUSIONS: The CT-based RF model demonstrates a good performance in the prediction of mPAP, which may reduce the need for right heart catheterization in post-Glenn shunt patients with suspected mPAP > 15 mmHg. KEY POINTS: • Twenty-three candidate descriptors were manually extracted from cardiac computed tomography images, and seven of them were selected for subsequent modeling. • The random forest model presents the best predictive performance for pulmonary pressure among all methods. • The computed tomography-based machine learning model could predict post-Glenn shunt pulmonary pressure non-invasively.

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.

A simple risk score model for predicting contrast-induced nephropathy after coronary angiography in patients with diabetes.

BACKGROUND: Contrast-induced nephropathy (CIN) is a common complication in patients undergoing coronary angiography (CAG) or percutaneous coronary intervention (PCI) and associated with poor outcome. Some previous studies have already set up models to predict CIN, but there is no model for patients with diabetes mellitus (DM) especially. Therefore, we aim to develop and validate a simple risk score for predicting the risk of CIN in patients with DM undergoing CAG/PCI. METHODS: A total of 1157 consecutive patients with DM undergoing CAG/PCI were randomly assigned to a development cohort (n = 771) and a validation cohort (n = 386). The primary endpoint was CIN, which was defined as an absolute increase in serum creatinine (SCr) by 0.5 mg/dL from the baseline within 48-72 h after contrast exposure. The independent predictors for CIN were identified by multivariate logistic regression, and the discrimination and calibration of the risk score were assessed by ROC curve and Hosmer-Lemeshow test, respectively. RESULTS: The overall incidence of CIN was 45 (3.9%). The new simple risk score (Chen score), which included four independent variables (age > 75 years, acute myocardial infarction, SCr > 1.5 mg/dL, the use of intra-aortic balloon pump), exhibited a similar discrimination and predictive ability on CIN (AUC 0.813, 0.843, 0.796, P > 0.05, respectively), mortality (AUC 0.735, 0.771, 0.826, respectively) and MACEs when being compared with the classical Mehran or ACEF risk score. CONCLUSION: Our data suggest that the new simple risk score might be a good tool for predicting CIN in patients with DM undergoing CAG/PCI.

Screening for cardiac HERG potassium channel interacting proteins using the yeast two-hybrid technique.

The human ERG protein (HERG or Kv 11.1) encoded by the human ether-a-go-go-related gene (herg) is the pore-forming subunit of the cardiac delayed rectifier potassium current (IKr) responsible for action potential (AP) repolarization. Mutations in HERG lead to long-QT syndrome, a major cause of arrhythmias. Protein-protein interactions are fundamental for ion channel trafficking, membrane localization, and functional modulation. To identify proteins involved in the regulation of the HERG channel, we conducted a yeast two-hybrid screen of a human heart cDNA library using the C-terminus or N-terminus of HERG as bait. Fifteen proteins were identified as HERG amino terminal (HERG-NT)-interacting proteins, including Caveolin-1 (a membrane scaffold protein with multiple interacting partners, including G-proteins, kinases and NOS), the zinc finger protein, FHL2 and PTPN12 (a non-receptor tyrosine phosphatase). Eight HERG carboxylic terminal (HERG-CT)-interacting proteins were also identified, including the NF-κB-interacting protein myotrophin, We have identified multiple potential interacting proteins that may regulate cardiac IKr through cytoskeletal interactions, G-protein modulation, phosphorylation and downstream second messenger and transcription cascades. These findings provide further insight into dynamic modulation of HERG under physiological conditions and arrhythmogenesis.

The Predictive Value of N-Terminal Probrain Natriuretic Peptide for Infection in Patients With Acute Myocardial Infarction.

Background: Infections increase the risk of poor outcomes in patients with ST-elevation myocardial infarction (STEMI) undergoing percutaneous coronary intervention (PCI). However, predicting patients at a high risk of developing infection remains unclear. Moreover, the value of N-terminal probrain natriuretic peptide (NT-proBNP) for predicting infection is still unknown. Thus, we aimed to assess the relationship between NT-proBNP and the following development of infection, and clinical adverse outcomes in patients with STEMI undergoing PCI. Methods: STEMI patients undergoing PCI were consecutively enrolled from January 2010 to July 2016 and divided into groups according to baseline NT-proBNP levels: tertiles T1 (<988 pg/mL), T2 (988-3520 pg/mL), and T3 (≥3520 pg/mL). The primary endpoint was infection during hospitalization. Results: A total of 182 (27%) patients developed in-hospital infection. The incidence of infection increased from T1 to T3 (10.5, 17.7, and 54.5%, P < 0.001). NT-proBNP was an independent risk factor (adjusted odds ratio = 1.39, 95% confidence interval (CI) = 1.12-1.73, P = 0.003) and presented accurately predicting infection (area under curve = 0.774). Multivariate cox analysis showed that NT-proBNP was a significant risk factor for major adverse clinical events (MACE) at follow-up (adjusted HR = 1.92, 95% CI = 1.61-2.29, P < 0.001). Conclusion: The baseline NT-proBNP level has a good predictive value for infection and MACE in STEMI patients undergoing PCI.

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.

Connexin43 interacts with Caveolin-3 in the heart.

Gap junctions (GJs), collections of multiple intercellular channels between neighboring cells, are specialized channels facilitating intercellular electrical and chemical communication. GJs are important for synchronizing coupling and coordinated contraction in the heart, and are crucial regulators of heart gene transcription, cardiac development, and protection of ischemic cardiomyocytes through second messenger communication. Identification of proteins that interact with Connexin43 (Cx43), the predominant protein in cardiac GJs, may contribute to the understanding of GJ functional regulation. Using a yeast two-hybrid system, we identified Caveolin-3 (Cav3) as a new Cx43-interacting protein. This interaction was confirmed by co-immunoprecipitation and co-localization experiments. CX43 interacts with Cav3, suggesting that Cav3 may participate in the functional regulation of GJs.

Role of the ADCY9 gene in cardiac abnormalities of the Rubinstein-Taybi syndrome.

BACKGROUND: Rubinstein-Taybi syndrome (RTS) is a rare, congenital, plurimalformative, and neurodevelopmental disorder. Previous studies have reported that large deletions contribute to more severe RTS phenotypes than those caused by CREBBP point mutations, suggesting a concurrent pathogenetic role of flanking genes, typical of contiguous gene syndromes, but the detailed genetics are unclear. RESULTS: This study presented a rare case of Rubinstein-Taybi (RT) syndrome with serious cardiac abnormalities. Based on the clinical and genetic analysis of the patient, the ADCY9 gene deletion was highlighted as a plausible explanation of cardiac abnormalities. In adcy9 morphant zebrafish, cardiac malformation was observed. Immunofluorescence study disclosed increased macrophage migration and cardiac apoptosis. RNA sequencing in zebrafish model highlighted the changes of a number of genes, including increased expression of the mmp9 gene which encodes a matrix metalloproteinase with the main function to degrade and remodel extracellular matrix. CONCLUSIONS: In this study, we identified a plausible new candidate gene ADCY9 of CHD through the clinical and genetic analysis of a rare case of Rubinstein-Taybi (RT) syndrome with serious cardiac abnormalities. By functional study of zebrafish, we demonstrated that deletion of adcy9 is the causation for the cardiac abnormalities. Cardiac apoptosis and increased expression of the MMP9 gene are involved in the pathogenesis.

The four and a half LIM domain protein 2 interacts with and regulates the HERG channel.

Protein-protein interactions are critical for protein trafficking, localization and the regulation of ion channels. The human ether-a-go-go-related gene (herg) encodes the alpha-subunit of the potassium channel underlying the rapid component of the cardiac delayed rectifier current. To identify the cellular proteins involved in the regulation of the HERG channel, a human heart cDNA library was screened using a yeast two-hybrid system, with the N-terminus of HERG as bait. The four and a half LIM domain protein 2 (FHL2) was identified as a potential HERG partner. The interaction between these two proteins was confirmed by co-immunoprecipitation and glutathione transferase pull-down assays and immunocytochemical analysis. The physiological implication of HERG-FHL2 interaction, assessed by whole-cell, patch-clamp electrophysiology experiments, showed a significant increase in the HERG current amplitude and a faster deactivation of the tail current in human embryonic kidney 293 cells co-expressing HERG and FHL2. These data indicate that FHL2 interacts with and regulates the HERG channel. Our findings may aid in the further understanding of the molecular basis of HERG channel diversity and arrhythmogenesis in the long-QT syndrome.

[Na+-H+ exchanger protein changes in vascular smooth muscle and effect of Na+-H+ exchange inhibitor on vessel stenosis after balloon injuries in rabbits].

OBJECTIVE: To explore the mechanism and effect of sodium/hydrogen exchanger (Na(+)-H(+) exchanger, NHE), amiloride, on vessel stenosis. METHODS: Thirty-two adult male New Zealand white rabbits were randomly divided into groups of amiloride intervention (IG, n = 12), balloon injury (BG, n = 10) and sham-operation (SG, n = 10). A 2.5 mm x 20 mm Foley's tube was used to injury left side iliac artery in the IG and BG groups, whereas a same Foley's tube was inserted into the vessel without any injuries in the SG group. Amiloride (5 mg.kg(-1).d(-1)) was intraperitoneally injected 3 days before balloon injuries and the same dosage normal saline was used in the same way in the BG group for 28 days after operation. The rabbits were killed and the iliac arteries were stained with Hematoxylin-Eosin, alpha-actin and Masson's trichrome to observe the morphologic changes in the vessel cava, neointima, media layer, and vascular smooth muscle cells (VSMCs) migration into the neointima and extracellular matrixes (ECMs). RESULTS: Four weeks after balloon injuries in rabbits, a cave narrow of the iliac artery and neointima were found and the media layer (VSM layer) was proliferated. The quantities of NHE-1 protein from artery smooth muscle in all the groups were 0.21 +/- 0.02, 0.25 +/- 0.04 and 0.11 +/- 0.03 (P < 0.01), respectively. The difference between the BG and SG groups was significant, which indicated that the NHE-1 proteins increased after balloon injury. The quantities of NHE-1 protein from the IG group were lower than those from the BG group. The cave areas were 0.91 mm(2) +/- 0.23 mm(2), 0.68 mm(2) +/- 0.19 mm(2) and 1.08 mm(2) +/- 0.17 mm(2) (P < 0.01), respectively. The intima areas were 0.27 mm(2) +/- 0.15 mm(2), 0.67 mm(2) +/- 0.24 mm(2) and 0.05 mm(2) +/- 0.03 mm(2), respectively (P < 0.01). The ratios of intima to media area were 1.21 +/- 0.24, 1.39 +/- 0.26 and 0.15 +/- 0.08 (P < 0.01), respectively. Amiloride increased vessel cave areas, but decreased intima areas and intima to media ratios in the IG group. In the IG group, alpha-actin positive areas in neointima was higher (16,328.31 microm(2) +/- 6220.27 microm(2)) than those in the SG group (4164.15 microm(2) +/- 1788.37 microm(2)) (P < 0.01). ECMs areas in neointima in the IG group were lower (8910.62 microm(2) +/- 7041.62 microm(2)) than those in the SG group (33,358.76 microm(2) +/- 7290.17 microm(2)) (P < 0.01). CONCLUSIONS: Balloon injuries of iliac artery in rabbits induce VSMCs proliferation, migration, narrowed cave and vessel stenosis. Amiloride, a NHE-1 inhibitor, may relieve this vessel stenosis.

Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography.

Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 degrees C for 30min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.

[Clinical study on changes of heme oxygenase-1 expression in patients with acute myocardial infarction].

OBJECTIVE: To investigate changes in heme oxygenase-1(HO-1) in patients with acute myocardial infarction. METHODS: Forty-five patients with acute myocardial infarction and 50 with coronary heart disease (diagnosed by coronary angiography) but without acute myocardial infarction were included in this study, and another 40 patients with normal coronary artery as controls. Levels of HO-1 protein expression in monocyte and lymphocyte isolated from the patients were determined by immunohistochemistry and Western blot. Computer picture analyzing system was also used to measure levels of HO-1 protein expression. RESULTS: HO-1 protein expression was located in the cytoplasm. The levels of HO-1 protein expression in patients with acute myocardial infarction were significantly higher than those without acute myocardial infarction (P<0.01). In addition, low levels of HO-1 protein expression were observed in patients with normal coronary artery. CONCLUSION: There is a higher expression of HO-1 in patients with acute myocardial infarction, and a lower expression in patients with normal coronary artery.

[Protein tyrosine phosphatase non-receptor type 12 negatively regulates cardiac HERG channel currents].

OBJECTIVE: To study the effect of protein tyrosine phosphatase non-receptor type 12 (PTPN12) in regulating cardiac HERG channel currents. METHODS: The plasmids pcDNA3.1-PTPN12-RFP and herg mutant constructed by PCR technique were transfected into HEK293 cells via Lipofectamine 2000, and the cells stably expressing PTPN12 selected with G418 were identified by Western blotting with anti-PTPN12 antibody. HERG channel current in cells expressing HERG alone (HEK293/HERG cells), cells overexpressing PTPN12 (HEK293/HERG cells transfected with pCDNA3.1-PTPN12-RFP), PAO-treated cells (PTPN12/HERG cells treated with PAO), and herg mutant cells (HEK293/HERGY327A-Y700A-Y845A cells transfected with pcDNA3.1-PTPN12-RFP) were recorded by patch-clamp technique. RESULTS: The plasmids pcDNA3.1-PTPN12-RFP and herg mutant were successfully constructed, and the stable expressing cell lines were established. Red fluorescence was obversed in HEK293/HERG cells transfected with pcDNA3.1-PTPN12-RFP, and the protein expression of PTPN12 was detected. Overexpression of PTPN12 significantly decreased HERG current density in HEK293/HERG cells, and this change was significantly weakened in the inhibitor group and herg mutant group. CONCLUSION: PTPN12 negatively regulates cardiac HERG channel cerrent possibly by decreasing the phosphorylation level of HERG tyrosine residues. This finding provides further insight into the regulatory mechanism of HERG channel and the pathogenesis of long QT syndrome.

Down-regulation of the human ether-a-go-go-related gene in rat cardiac hypertrophy.

INTRODUCTION: Cardiac hypertrophy is a risk factor for QT prolongation and cardiac sudden death. In this study, the authors examined the expressional regulation on the rat human ether-a-go-go-related gene (HERG), which encodes a structural subunit of the rapid component of the delayed rectifier potassium current (I(Kr)), during myocardial hypertrophy using rat as a model system. METHODS: Cardiac hypertrophy was established in Sprague-Dawley rats by coarctation of the abdominal aorta [left ventricular hypertrophy (LVH) group]. Sham-operated rats were defined as control group (Ctrl group). Hemodynamic, morphologic and histologic parameters were recorded 6 weeks after operation. In addition, the expression of HERG was also determined using a combination of real-time polymerase chain reaction, Western blot and immunohistochemical analyses. RESULTS: Compared with the sham-operated Ctrl group, abdominal aortic coarctation induced LVH in the LVH group, as evidenced by significantly increased ratios of heart weight/left ventricular weight to body weight and enlarged left ventricular myocytes in the histologic sections. The hemodynamic profile revealed significant increases in heart rate and left ventricular end-diastolic pressure, as well as a decrease in the maximal rate of left ventricular pressure fall in the LVH rats, when compared with the Ctrl rats. Electrocardiograms showed prolonged QT and corrected QT intervals. On the molecular level, a significant reduction of HERG, messengerRNA and protein was observed in LVH group, which was inversely correlated with prolonged corrected QT (r = -0.842, P = 0.000). CONCLUSION: The expressional down-regulation of HERG gene may constitute a novel mechanism for QT prolongation during cardiac hypertrophy.

The regulation of the cardiac potassium channel (HERG) by caveolin-1.

Protein-protein interaction plays a key role in the regulation of biological processes. The human potassium (HERG) channel is encoded by the ether-à-go-go-related gene (herg), and its activity may be regulated by association with other cellular proteins. To identify cellular proteins that might play a role in the regulation of the HERG channel, we screened a human heart cDNA library with the N terminus of HERG using a yeast 2-hybrid system, and identified caveolin-1 as a potential HERG partner. The interaction between these 2 proteins was confirmed by coimmunoprecipitation assay, and their overlapping subcellular localization was demonstrated by fluorescence immunocytochemistry. The physiologic implication of the protein-protein interaction was studied in whole-cell patch-clamp electrophysiology experiments. A significant increase in HERG current amplitude and a faster deactivation of tail current were observed in HEK293/HERG cells in a membrane lipid rafts disruption model and caveolin-1 knocked down cells by RNA interference. Alternatively, when caveolin-1 was overexpressed, the HERG current amplitude was significantly reduced and the tail current was deactivated more slowly. Taken together, these data indicate that HERG channels interact with caveolin-1 and are negatively regulated by this interaction. The finding from this study clearly demonstrates the regulatory role of caveolin-1 on HERG channels, and may help to understand biochemical events leading to arrhythmogenesis in the long QT syndrome in cardiac patients.

The effect of delayed preconditioning on connexin 43 in ischemic myocardium.

The objective of this study is to investigate the effects of preconditioning on the restoration and distribution of connexin 43 (Cx43) in ischemic myocardium in dogs. In this study, 40 dogs were randomly divided into 5 groups of 8 as follows: control, 0hI-R (ischemia followed by 0 h reperfusion), 6hI-R (ischemia followed by 6 h reperfusion), 24hI-R (ischemia followed by 24 h reperfusion), and 48hI-R (ischemia followed by 48 h reperfusion). Four dogs in each group were preconditioned with brief episodes of ischemia prior to the respective treatments and were referred as the PC groups, while the other 4 were not preconditioned and were referred as the nonPC groups. The myocardial ischemia was induced by ligation of the left anterior descending coronary artery. The expression and distribution of Cx43 within the ischemic myocardium were measured by Western blot analysis and studied using laser confocal microscopy using a double-label immunohistochemistry technique. Compared with the control group, there was a significant reduction in Cx43 content within ischemic myocardium of all test groups both with and without PC (P < 0.01, P < 0.05). Within the 0hI-R, 6hI-R, and 24hI-R groups, an insignificant difference was found in the expression and distribution of Cx43 within the ischemic region between the PC and the nonPC groups. However, in the 48hI-R group, the area and intensity of Cx43 staining within the ischemic region of the PC dogs were significantly larger and more intense than those of the nonPC dogs (P < 0.01), and the ratio of Cx43 pixel density in intercalated disk areas to that in side-to-side junction areas in the PC dogs was significantly greater than that in nonPC dogs (P < 0.01). Our results suggest that preconditioning has a significant effect on the restoration and distribution of Cx43 in the ischemic myocardium in dogs after 48 h. Hence, preconditioning may be a plausible cause for the observed reductions in cardiac arrhythmias.

Physiological testosterone stimulates tissue plasminogen activator and tissue factor pathway inhibitor and inhibits plasminogen activator inhibitor type 1 release in endothelial cells.

There is a striking gender difference in atherosclerotic vascular disease. For decades, testosterone was considered detrimental to the cardiovascular system. Recent studies, however, have presented some alternative results. The aim of this study was to evaluate the effect of testosterone, using physiological and supraphysiological concentrations, on antigen and mRNA levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue factor pathway inhibitor (TFPI) released by human umbilical vein endothelial cells and to investigate the cellular mechanism. Cells within 2-3 passages were cultured in 25 cm(2) flasks or plated onto 96-well plates with a density of about 1 x 10(5) cells/mL as recommended. The cells were incubated in the presence or absence of testosterone (3, 30, 3 x 10(3), 3 x 10(4) nmol/L) for 48 h. Levels of tPA, PAI-1, and TFPI antigen were assayed with ELISA kits. Reverse transcriptase PCR was carried out to detect tPA, PAI-1, and TFPI mRNA levels. Cells were incubated in androgen-receptor antagonist (flutamide 10 micromol/L) or aromatase inhibitor (aminoglutethimide 50 micromol/L) for 3 h, and then the experiments were repeated. Testosterone at a physiologic concentration (30 nmol/L) increased the antigen levels of tPA and TFPI significantly (P < 0.05). However, tPA and TFPI levels were markedly reduced (P < 0.05) at a larger dose (3 x 10(4) nmol/L). On the other hand, PAI-1 antigen levels decreased significantly at the testosterone concentrations ranging from 3 to 3 x 10(4) nmol/L (P < 0.05). The change in the levels of tPA and TFPI were reflected in the corresponding change in mRNA levels. Flutamide attenuated the effect of testosterone at physiological concentration (30 nmol/L). The results demonstrated that testosterone at physiological concentrations may have a beneficial influence on the haemostatic system through enhancement of anticoagulant activity, resulting from stimulation of TFPI and tPA expression and inhibition of PAI-1 secretion by the endothelium.

Modulation of ICa-L by alpha1-adrenergic stimulation in rat ventricular myocytes.

We found when L-type calcium current (ICa-L) was recorded with the perforated patch-clamp method in rat ventricular myocytes that bath application of phenylephrine (with propranolol) evoked a biphasic response characterized by an initial transient suppression followed by a sustained potentiation. The transient suppression occurred 30-60 s after phenylephrine perfusion and reached peak inhibition at approximately 2 min. The biphasic modulation of ICa-L was also elicited by methoxamine, and the effects of phenylephrine were blocked by prazosin, indicating that the responses were mediated through alpha1-adrenoceptors. Pretreatment of cells with H7 (100 micromol/L), a broad-spectrum protein kinase inhibitor that inhibits both protein kinase C and A, eliminated potentiation but did not affect transient suppression. The transient suppression occurred concurrently with the acceleration of the fast component of ICa-L inactivation. Depletion of intracellular Ca2+ stores by ryanodine plus caffeine or thapsigargin eliminated the transient suppression. When ICa-L was recorded with whole-cell patch-clamp and with 0.05 mmol/L EGTA in the pipette solution to allow intracellular Ca2+ to fluctuate, phenylephrine evoked a transient suppression as in the perforated patch recordings. Heparin, a specific blocker of IP3 (inositol 1,4,5-trisphosphate) receptors, eliminated the phenylephrine-induced transient suppression of ICa-L when added to the pipette solution. Intensive chelation of intracellular Ca2+ by 5 mmol/L BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) in the pipette solution also eliminated the phenylephrine-induced transient suppression of ICa-L. We conclude that transient increase in the concentration of intracellular calcium ([Ca2+]i) caused by Ca2+ release from intracellular stores underlies the transient suppression of ICa-L, whereas the potentiation of ICa-L is a result of activation of protein kinases.