Rescue of expression and function of long QT syndrome-causing mutant hERG channels by enhancing channel stability in the plasma membrane.

The human ether-a-go-go-related gene (hERG) encodes the Kv11.1 (or hERG) channel that conducts the rapidly activating delayed rectifier potassium current (IKr). Naturally occurring mutations in hERG impair the channel function and cause long QT syndrome type 2. Many missense hERG mutations lead to a lack of channel expression on the cell surface, representing a major mechanism for the loss-of-function of mutant channels. While it is generally thought that a trafficking defect underlies the lack of channel expression on the cell surface, in the present study, we demonstrate that the trafficking defective mutant hERG G601S can reach the plasma membrane but is unstable and quickly degrades, which is akin to WT hERG channels under low K+ conditions. We previously showed that serine (S) residue at 624 in the innermost position of the selectivity filter of hERG is involved in hERG membrane stability such that substitution of serine 624 with threonine (S624T) enhances hERG stability and renders hERG insensitive to low K+ culture. Here, we report that the intragenic addition of S624T substitution to trafficking defective hERG mutants G601S, N470D, and P596R led to a complete rescue of the function of these otherwise loss-of-function mutant channels to a level similar to the WT channel, representing the most effective rescue means for the function of mutant hERG channels. These findings not only provide novel insights into hERG mutation-mediated channel dysfunction but also point to the critical role of S624 in hERG stability on the plasma membrane.

Ubiquitination is involved in PKC-mediated degradation of cell surface Kv1.5 channels.

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K+ current (IKur) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis, and immunocytochemical staining, we demonstrate that ubiquitination is involved in the PMA-mediated degradation of mature Kv1.5 channels. Since the expression of the Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that the N-terminus alone did not but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation.

Hydroxychloroquine Attenuates hERG Channel by Promoting the Membrane Channel Degradation: Computational Simulation and Experimental Evidence for QT-Interval Prolongation with Hydroxychloroquine Treatment.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic has led to millions of confirmed cases and deaths worldwide and has no approved therapy. Currently, more than 700 drugs are tested in the COVID-19 clinical trials, and full evaluation of their cardiotoxicity risks is in high demand. METHODS: We mainly focused on hydroxychloroquine (HCQ), one of the most concerned drugs for COVID-19 therapy, and investigated the effects and underlying mechanisms of HCQ on hERG channel via molecular docking simulations. We further applied the HEK293 cell line stably expressing hERG-wild-type channel (hERG-HEK) and HEK293 cells transiently expressing hERG-p.Y652A or hERG-p.F656A mutants to validate our predictions. Western blot analysis was used to determine the hERG channel, and the whole-cell patch clamp was utilized to record hERG current (IhERG). RESULTS: HCQ reduced the mature hERG protein in a time- and concentration-dependent manner. Correspondingly, chronic and acute treatment of HCQ decreased the hERG current. Treatment with brefeldin A (BFA) and HCQ combination reduced hERG protein to a greater extent than BFA alone. Moreover, disruption of the typical hERG binding site (hERG-p.Y652A or hERG-p.F656A) rescued HCQ-mediated hERG protein and IhERG reduction. CONCLUSION: HCQ can reduce the mature hERG channel expression and IhERG via enhancing channel degradation. The QT prolongation effect of HCQ is mediated by typical hERG binding sites involving residues Tyr652 and Phe656.

Paracondylar process combined with persistent first intersegmental vertebral artery: an anatomic case report and literature review.

The paracondylar process (PCP) and the persistent first intersegmental vertebral artery (PFIA) are both rare variations at the craniovertebral junction. We report the above two variations coexisting in one cadaveric head during the training of far lateral approach in our skull base lab. The specimen simultaneously had a left occipitalized atlas associated with a PFIA and a right PCP. The previous reports, the embryogenesis, and the clinical implications of the two variations were also reviewed. Preoperative recognition of the rare variations is essential to a safe far lateral approach.

Kv1.5 channels are regulated by PKC-mediated endocytic degradation.

The voltage-gated potassium channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (IKv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 channel function under PKC activation conditions.

Cardiovascular disease burden attributable to dietary risk factors from 1990 to 2019: A systematic analysis of the Global Burden of Disease study.

BACKGROUND AND AIMS: Dietary risks have always been a major risk factor for cardiovascular diseases (CVDs), especially in young people. This article aimed to provide an updated and comprehensive view of the spatial, temporal and sexual heterogeneity in diet-attributable CVD burdens from 1990 to 2019. METHODS AND RESULTS: Data on diet-attributable CVD burdens were extracted from the Global Burden of Disease (GBD) Study 2019. Numbers and age-standardized rates (ASRs) of deaths, disability-adjusted life years (DALYs) and corresponding estimated annual percentage change (EAPC) were determined. Globally, the number of diet-attributable CVD deaths and DALYs in 2019 were 6.9 million and 153.2 million, marking 43.8% and 34.3% increases since 1990, respectively. However, ASRs of death and DALYs have declined over time. The regions with the highest ASRs of diet-related CVD deaths and DALYs were in Central Asia, whereas the lowest ASRs of CVD deaths and DALYs were observed in the high-income Asia Pacific region. Globally, men suffered higher death and DALY burdens than women. Ischemic heart disease and stroke were the leading causes of CVD deaths and DALYs, globally. Regarding the specific diet group, diets low in whole grains, high in sodium, low in fruits, low in nuts and seeds, low in vegetables and low in seafood omega-3 fatty acids contributed to CVD deaths and DALYs the most. Dietary risks accounted for a higher proportion in people aged less than 65 years old. CONCLUSIONS: Diet-attributable CVDs threaten public health, particularly in low SDI countries and younger generations. As diet-related CVDs are nation-specific, the prioritization of public health interventions should be evidence-based.

Mechanical stretch increases Kv1.5 current through an interaction between the S1-S2 linker and N-terminus of the channel.

The voltage-gated potassium channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic channel inhibition and results in an increase in IKv1.5.

1/2SH: A Simple, Accurate, and Reliable Method of Calculating the Hematoma Volume of Spontaneous Intracerebral Hemorrhage.

Background and Purpose- 1/2ABC has been used widely for assessing the volume of intracerebral hematoma. However, it is only suitable for calculating regular and small volume hematomas. Therefore, we re-explored the formula of hematoma volume to find a method that can calculate hematoma volumes accurately, reliably, and quickly. Methods- Computed tomography imaging data of 257 patients with intracerebral hemorrhage were collected. Hematoma volumes were estimated using 3-dimensional Slicer and 7 formulas (π/6ABC, 1/2ABC, 1/3ABC, 2/3SH, 1/2SH, π/6SH, and 2.5/6ABC). Taking the hematoma volumes measured by 3-dimensional Slicer as the reference standard, the accuracy and reliability of the 7 formulas were evaluated. Furthermore, the time needed to calculate hematoma volumes by the 1/2SH method was noted for further analysis. Results- (1) The accuracy of the 7 formulas based on the error analysis from the highest to the lowest was: π/6SH, 1/2SH, 2.5/6ABC, 1/3ABC, 1/2ABC, and π/6ABC or 2/3SH. According to concordance analysis and receiver operating characteristic curve analysis, the results from the highest to lowest were as follows: 1/2SH, π/6SH, 2.5/6ABC, 1/3ABC, 1/2ABC, 2/3SH, and π/6ABC. After categorizing cases according to size, shape, and location of hematoma, the results were almost the same as the results for overall accuracy evaluation in any subgroup. (2) Intraclass correlation coefficient (ICC) of 1/2SH in intra and inter-researcher were 0.998 and 0.989, respectively. For the formula π/6SH, intraclass correlation coefficient was the same as that of 1/2ABC. Kappa values of 1/2SH for intra- and inter-observer were 0.992 and 0.913, respectively. For π/6SH, kappa values of within- and between-reader were 0.984 and 0.904, respectively. (3) The average time taken to calculate hematoma volumes by 1/2SH was 74 seconds. Conclusions- The 1/2SH and π/6SH are accurate, reliable, and rapid methods for calculating hematoma volumes. The accuracy and reliability of 1/2SH were slightly higher than those of π/6SH.

Circular RNA circPITX1 knockdown inhibits glycolysis to enhance radiosensitivity of glioma cells by miR-329-3p/NEK2 axis.

BACKGROUND: Numerous circular RNAs (circRNAs) have been recognized as vital modulators of human malignancies, including glioma. Whereas, the functional role of circRNA Pituitary Homeo Box 1 (circPITX1) in the radioresistance of glioma cells remains largely uncertain. METHODS: Quantitative real-time PCR (qRT-PCR) or western blot analysis was employed to examine the expression of circPITX1, microRNA (miR)-329-3p and NIMA-related kinase 2 (NEK2). 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to determine cell viability. Glycolysis was assessed by commercial kits and western blot analysis. Colony formation assay was conducted to analyze cell survival and clonogenicity capacity. The relationship among circPITX1, miR-329-3p and NEK2 was confirmed via dual-luciferase reporter assay. The in vivo function of circPITX1 was evaluated by tumor xenograft assay. RESULTS: Expression of circPITX1 and NEK2 was up-regulated in glioma tissues and cells, while miR-329-3p exhibited reverse trend. CircPITX1 knockdown repressed viability, glycolysis and colony formation, but promoted radiosensitivity of glioma cells, as well as inhibited tumor growth in vivo. MiR-329-3p was a target miRNA of circPITX1 and miR-329-3p deficiency reversed knockdown of circPITX1-mediated glycolysis inhibition and radioresistance reduction. MiR-329-3p exerted inhibitory effects on glycolysis and radioresistance of glioma cells by targeting NEK2. CircPITX1 facilitated NEK2 expression by sponging miR-329-3p. Glycolytic inhibitor 2-deoxy-d-glucose (2-DG) disposition weakened the promoted impact on glycolysis caused by circPITX1. CONCLUSION: CircPITX1 knockdown reduced glycolysis to contribute to radiosensitivity in glioma through miR-329-3p/NEK2 axis, providing a possible mechanism of circPITX1 in the development of glioma.

Exendin-4 regulates endoplasmic reticulum stress to protect endothelial progenitor cells from high-glucose damage.

BACKGROUND: High glucose affects the function of endothelial cells by increasing oxidative stress. Studies have found that exendin-4 can improve wound healing in diabetic mice and mice with normal blood glucose. However, the mechanism of exendin-4 in endothelial progenitor cells under high-glucose condition has not been fully elucidated. METHODS: Diabetic mouse models were established to investigate the effects of exendin-4 on endothelial progenitor cells in diabetic mice. Serum superoxide dismutase (SOD) and malondialdehyde (MDA) were determined by WST-8 and thiobarbituric acid (TBA) colorimetry, respectively. Cell viability, apoptosis and reactive oxygen species (ROS) were detected by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry. Gene and protein expressions were determined by Quantitative reverse transcription PCR (qRT-PCR) assay and Western blot (WB). RESULTS: The results showed that in diabetic mice, exendin-4 did not affect blood glucose or body weight, moreover, it improved aortic diastolic function, increased SOD activity and down-regulated malondialdehyde (MDA) level in the mice. In addition, exendin-4 also increased endothelial progenitor cell (EPCs) viability and reduced cell apoptosis through inhibiting p38 MAPK pathway and reducing endoplasmic reticulum stress and ROS. CONCLUSION: Exndin-4 can alleviate diabetes-caused damage to mice, moreover, it reduced endoplasmic reticulum stress and ROS through inhibiting p38 MAPK pathway in MPCs cells under high-glucose condition, thus increasing cell viability and reducing cell apoptosis.

Identifying the Specific Subtype of Intracerebral Hemorrhage that is Indicated for Minimally Invasive Craniopuncture.

BACKGROUND: Surgeries for intracerebral hemorrhage (ICH) remain controversial. Our previous study found that postoperative cerebrospinal fluid (CSF) outflow was associated with high hematoma evacuation efficiency in ICH cases with intraventricular involvement (ICHV) treated with minimally invasive craniopuncture (MIC). This study was designed to identify factors that predict postoperative CSF outflow and the specific subtype of ICHV that may benefit from MIC. METHODS: A total of 189 MIC needles applied to 125 ICHV patients were retrospectively analyzed. Univariate and multivariate analyses were used to identify independent predictive factors of postoperative CSF outflow. RESULTS: A density of the whole hematoma of ≤ 59 HU [odds ratio (OR) = 8.572, 95% confidence interval (CI) 3.235-22.714, P < 0.001, standardization regression coefficients B' = 0.576] and a distance between the needle tip and the ventricular tear (tip-tear distance) of 21.79-34.15 mm (OR = 25.566, 95% CI 8.707-75.074, P < 0.001, B' = 0.883) were identified as independent predictive factors of postoperative CSF outflow. The density of the hematoma within 34.15 mm of the tear (clot 3.4) showed no statistical difference from that of the whole hematoma (P = 0.571). A density of clot 3.4 ≤ 60 HU was also a predictive factor of postoperative CSF outflow (area under curve: 0.771). CONCLUSIONS: ICHV patients who meet the following conditions may benefit from MIC: (1) The MIC needle tip can be placed in the hematoma 21.79-34.15 mm from the ventricular tear; (2) the density of the whole hematoma is low (≤ 59 HU); and (3) the density of clot 3.4 is also low (≤ 60 HU). Future perspective studies should be conducted on this specific patient subtype.

Differential Regulation of Human Ether-à-Go-Go-Related Gene (hERG) Current and Expression by Activation of Protein Kinase C.

The human ether-à-go-go-related gene (hERG) encodes the channel that conducts the rapidly activating delayed rectifier potassium current (IKr) in the heart. Reduction in IKr causes long QT syndrome, which can lead to fatal arrhythmias triggered by stress. One potential link between stress and hERG function is protein kinase C (PKC) activation; however, seemingly conflicting results regarding PKC regulation of hERG have been reported. We investigated the effects of PKC activation using phorbol 12-myristate 13-acetate (PMA) on hERG channels expressed in human embryonic kidney cell line 293 (HEK293) cells and IKr in isolated neonatal rat ventricular myocytes. Acute activation of PKC by PMA (30 nM, 30 minutes) reduced both hERG current (IhERG) and IKr Chronic activation of PKC by PMA (30 nM, 16 hours) increased IKr in cardiomyocytes and the expression level of hERG proteins; however, chronic (30 nM, 16 hours) PMA treatment decreased IhERG, which became larger than untreated control IhERG after PMA removal for 4 hours. Deletion of amino acid residues 2-354 (Δ2-354 hERG) or 1-136 of the N terminus (ΔN 136 hERG) abolished acute PMA (30 nM, 30 minutes)-mediated IhERG reduction. In contrast to wild-type hERG channels, chronic activation of PKC by PMA (30 nM, 16 hours) increased both Δ2-354 hERG and ΔN136 hERG expression levels and currents. The increase in hERG protein was associated with PKC-induced phosphorylation (inhibition) of Nedd4-2, an E3 ubiquitin ligase that mediates hERG degradation. We conclude that PKC regulates hERG in a balanced manner, increasing expression through inhibiting Nedd4-2 while decreasing current through targeting a site(s) within the N terminus.

Subclinical thyroid dysfunction is associated with adverse prognosis in heart failure patients with reduced ejection fraction.

BACKGROUND: Subclinical thyroid dysfunction whose typical patterns include subclinical hypothyroidism and subclinical hyperthyroidism, has been indicated to be associated with an increased risk of heart failure (HF). However, the relationship between subclinical thyroid dysfunction and the clinical outcomes of HF patients is uncertain. This meta-analysis was conducted to assess the association between subclinical thyroid dysfunction and the clinical outcomes of HF patients. METHODS: Pubmed, Embase, Web of Science and Cochrane Central Register of Clinical Trials were searched for eligible studies published up to August 1, 2018 which reported the association between subclinical thyroid dysfunction and the clinical outcomes of HF patients. The pooled hazard ratio (HR) with the corresponding 95% confidence interval (CI) was used to assess the association. RESULTS: Fourteen studies met the eligibility criteria and a total of 21,221 patients with heart failure were included in the meta-analysis. Compared with HF patients with euthyroidism, the pooled HR of subclinical hypothyroidism for all-cause mortality was 1.45 (95% CI 1.26-1.67) in a randomized effects model with mild heterogeneity (I2 = 40.1, P = 0.073). The pooled HR of subclinical hypothyroidism for cardiac death and/or hospitalization was 1.33 (1.17-1.50) in a randomized effects model with moderate heterogeneity (I2 = 69.4, P < 0.001). Subclinical hyperthyroid can increase the risk of all-cause mortality without heterogeneity (HR 1.31, 95% CI 1.10-1.55, I2 = 25.5%, P = 0.225) but have no influence on the risk of cardiac death and/or hospitalization (HR 1.03, 95% CI 0.87-1.23, I2 = 0.0%, P = 0.958). These significant adverse associations were also retained in subgroup analysis. Sensitivity analysis demonstrated the stability of the results of our meta-analysis. CONCLUSIONS: Both subclinical hypothyroidism and subclinical hyperthyroidism are associated with adverse prognosis in patients with HF. Subclinical thyroid dysfunction may be a useful and promising predictor for the long-term prognosis in HF patients.

Lack of influence of sex hormones on Brugada syndrome-associated mutant Nav1.5 sodium channel.

Brugada syndrome (BS) is an autosomal dominant disease. The most common causes of BS are loss-of-function mutations occur in the SCN5A gene which encodes the sodium channel protein Nav1.5. BS has a higher incidence rate in males and the underlying mechanisms of the gender inequality are not yet fully understood. Considering sex hormones are among the most important factors behind gender differences and have previously been shown to regulate the activity of multiple cardiac ion channels, we hypothesized that sex hormones also affect Nav1.5 function which lead to BS predominantly affecting males. In this study, we investigate the protein expression level and current of Nav1.5 in the HEK293 cells cotransfected with SCN5A and sex hormone receptor plasmids using both wild-type SCN5A and BS-associated SCN5A channel mutants R878C and R104W. Our findings showed that sex hormones have no effects on the protein expression level and current of the wild-type Nav1.5, neither does it affect the protein expression level and current of BS-associated Nav1.5 mutants R878C and R104W, regardless of homozygous or heterozygous state. Our results suggest that the male preponderance of BS does not arise from the effects of the sex hormones on Nav1.5. Further studies are needed to explain the male preponderance of this disease.

Gremlin1 promotes carcinogenesis of glioma in vitro.

As the most prevalent and lethal type of brain tumours, gliomas, especially malignant ones, are relatively resistant to conventional therapies. Gremlin 1 (GREM1) is a secreted glycoprotein that is implicated in the maintenance of cancer stem cells in tumour hierarchy. In the current study, the role of GREM1 in the carcinogenesis of glioma was studied using a knockdown approach. We first examined the expression level of GREM1 in the clinical samples, and then evaluated the effect of GREM1 knockdown on the viability and colony formation capacity of U87-MG cells. Moreover, the migration ability, invasiveness, cell cycle, and apoptosis of GREM1-silenced cells were assessed. Furthermore, the involvement of functional GREM1 in the epithelial-mesenchymal transition (EMT) process of glioma was investigated by detecting the expression levels of glioma-associated oncogene homologue 3 (GLI3) and EMT-related molecules. Our results demonstrated that knockdown of GREM1 reduced cell viability, suppressed migration and invasion, and inhibited GLI3 expression and the EMT process in U87-MG cells. Meanwhile, GREM1 silencing promoted apoptosis in U87-MG cells through the accumulation of Bax, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) as well as the downregulation of Bcl-2. In addition, GREM1 knockdown abolished transforming growth factor (TGF)-ß1-mediated activation of the Smad pathway, which may underlie the mechanism of GREM1-regulated EMT. In conclusion, GREM1 plays an important role in the development of glioma, and it may serve as a potential target in glioma therapy.

Clenbuterol Attenuates hERG Channel by Promoting the Mature Channel Degradation.

Clenbuterol, a ß2-selective adrenergic receptor agonist, is illicitly used in weight loss and performance enhancement and animal production. Increasing evidence demonstrates that clenbuterol induces various kinds of arrhythmias and QTc interval prolongation. However, little is known about the underlying mechanism. Most drugs are associated with QTc prolongation through interfering with human ether-a-go-go-related gene (hERG) K+ channels. The present study aims to investigate the effects and underlying mechanisms of clenbuterol on the hERG channel. HEK 293 cells were transfected with wild type and Y652A or F656A mutants of the hERG channel and treated with clenbuterol. The hERG current was recorded using whole-cell patch-clamp technique, and protein level was evaluated by Western blot. We found that clenbuterol decreases the mature form of the hERG protein at the cell membrane in a concentration- and time-dependent manner, without affecting the immature form. Correspondingly, clenbuterol chronic treatment reduced hERG current to a greater extent compared to acute treatment. In the presence of Brefeldin A (BFA), which was used to block hERG channel trafficking to cell membrane, clenbuterol reduced hERG on plasma membrane to a greater extent than BFA alone. In addition, the hERG channel's drug binding sites mutant Y652A and F656A abolished clenbuterol-mediated hERG reduction and current blockade. In conclusion, clenbuterol reduces hERG channel expression and current by promoting the channel degradation. The effect of clenbuterol on the hERG channel is related to the drug-binding sites, Tyr-652 and Phe-656, located on the S6 domain. This biophysical mechanism may underlie clenbuterol-induced QTc prolongation or arrhythmia.

The RyR2-P2328S mutation downregulates Nav1.5 producing arrhythmic substrate in murine ventricles.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca(2+) homeostasis and can arise from ryanodine receptor (RyR2) mutations including RyR2-P2328S. Previous reports established that homozygotic murine RyR2-P2328S (RyR2 (S/S)) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2 (S/S) hearts to connexin-43 (Cx43) and Nav1.5 expression and Na(+) current (I Na). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2 (S/S) hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2 (S/S) than WT, but comparable changes in AP durations (APD90) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2 (S/S) compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced I Na in RyR2 (S/S) ventricles. We thus attribute arrhythmogenesis in RyR2 (S/S) ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing I Na, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak I Na of the AP but nonlinear relationships between peak I Na and maximum Na(+) permeability.

Effect of MTHFR Gene Polymorphism Impact on Atherosclerosis via Genome-Wide Methylation.

BACKGROUND: Atherosclerosis seriously threats human health. Homocysteine is an independent risk factor closely related to DNA methylation. MTHFR C667T loci polymorphism is closely associated with homocysteine level. This study aimed to investigate the relationship among MTHFR C667T loci polymorphism, genome-wide methylation, and atherosclerosis. MATERIAL/METHODS: Blood sample was collected from 105 patients with coronary atherosclerosis and 105 healthy controls. Pyrosequencing methylation was used to detect LINE-1 methylation level. Polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) was used to test MTHFR. RESULTS: LINE-1 methylation level in the patient group was significantly lower than in the controls (t=5.007, P<0.001). MTHFR C667T genotype distribution presented marked differences in the 2 groups. TT genotype carriers had significantly increased risk of atherosclerosis (OR=3.56, P=0.009). Three different genotypes of MTHFR C667T loci showed different LINE-1 methylation level between the 2 groups (P<0.01). LINE-1 methylation level in TT and CT genotype carriers was obviously lower than in CC genotype carriers (P<0.05). CONCLUSIONS: MTHFR C667T loci polymorphism may affect atherosclerosis by regulating genome methylation level.

Correlations between MTP and ROS Levels of Peripheral Blood Lymphocytes and Readmission in Patients with Chronic Heart Failure.

BACKGROUND: Peripheral blood lymphocytes exhibit changes that parallel those in failing cardiomyocytes. We hypothesised that mitochondrial transmembrane potential (MTP) and reactive oxygen species (ROS) levels of lymphocytes are associated with serum NT-proBNP and short-term prognosis in patients with chronic heart failure (CHF). METHODS: Fifty-four CHF patients and 30 controls were enrolled in this prospective study. Mitochondrial transmembrane potential and ROS levels of lymphocytes were evaluated by flow cytometry and reported as the JC-1 fluorescence ratio and DCF fluorescence intensity, respectively. Serum NT-proBNP levels and biochemical parameters were also examined. All the participants received follow-up to evaluate clinical end-points after eight months. RESULTS: Chronic heart failure patients had higher levels of DCF fluorescence intensity of lymphocytes and serum NT-proBNP, as well as lower levels of JC-1 fluorescence ratios compared with those of controls (all P<0.05). A closer relationship was found between Lg(DCF fluorescence intensity of lymphocytes) or JC-1 fluorescence ratio of lymphocytes and Lg(NT-proBNP) (both P<0.05) in CHF patients. During the eight-month follow-up period, 14 CHF patients (25.9%) were readmitted for severe HF, but none died. A logistic regression analysis showed that both ROS level and MTP of the lymphocytes were independent predictors (B=7.03, P=0.006; B= - 0.32, P=0.029, respectively) of readmission of CHF patients. CONCLUSIONS: In CHF patients at low risk, MTP and ROS levels of the lymphocytes showed a significant change that is associated with serum NT-proBNP and patient readmission.

Apamin-Sensitive K+ Current Upregulation in Volume-Overload Heart Failure is Associated with the Decreased Interaction of CK2 with SK2.

Recent studies have shown that the sensitivity of apamin-sensitive K(+) current (I KAS, mediated by apamin-sensitive small conductance calcium-activated potassium channels subunits) to intracellular Ca(2+) is increased in heart failure (HF), leading to I KAS upregulation, action potential duration shortening, early after depolarization, and recurrent spontaneous ventricular fibrillation. We hypothesized that casein kinase 2 (CK2) interacted with small conductance calcium-activated potassium channels (SK) is decreased in HF, and protein phosphatase 2A (PP2A) is increased on the opposite, upregulating the sensitivity of I KAS to intracellular Ca(2+) in HF. Rat model of volume-overload HF was established by an abdominal arteriovenous fistula procedure. The expression of SK channels, PP2A and CK2 was detected by Western blot analysis. Interaction and colocalization of CK2 with SK channel were detected by co-immunoprecipitation analysis and double immunofluorescence staining. In HF rat left ventricle, SK3 was increased by 100 % (P < 0.05), and SK2 was not significantly changed. PP2A protein was increased by 94.7 % in HF rats (P < 0.05), whereas the level of CK2 was almost unchanged. We found that CK2 colocalized with SK2 and SK3 in rat left ventricle. With anti-CK2α antibody, SK2 and SK3 were immunoprecipitated, the level of precipitated SK2 decreased by half, whereas precipitated SK3 was almost unchanged. In conclusion, the increased expression of total PP2A and decreased interaction of CK2 with SK2 may underlie enhanced sensitivity of I KAS to intracellular Ca(2+) in volume-overload HF rat.