Non-missense variants of KCNH2 show better outcomes in type 2 long QT syndrome

AIMS: More than one-third of type 2 long QT syndrome (LQT2) patients carry KCNH2 non-missense variants that can result in haploinsufficiency (HI), leading to mechanistic loss-of-function. However, their clinical phenotypes have not been fully investigated. The remaining two-thirds of patients harbour missense variants, and past studies uncovered that most of these variants cause trafficking deficiency, resulting in different functional changes: either HI or dominant-negative (DN) effects. In this study, we examined the impact of altered molecular mechanisms on clinical outcomes in LQT2 patients. METHODS AND RESULTS: We included 429 LQT2 patients (234 probands) carrying a rare KCNH2 variant from our patient cohort undergoing genetic testing. Non-missense variants showed shorter corrected QT (QTc) and less arrhythmic events (AEs) than missense variants. We found that 40% of missense variants in this study were previously reported as HI or DN. Non-missense and HI-groups had similar phenotypes, while both exhibited shorter QTc and less AEs than the DN-group. Based on previous work, we predicted the functional change of the unreported variants-whether they cause HI or DN via altered functional domains-and stratified them as predicted HI (pHI)- or pDN-group. The pHI-group including non-missense variants exhibited milder phenotypes compared to the pDN-group. Multivariable Cox model showed that the functional change was an independent risk of AEs (P = 0.005). CONCLUSION: Stratification based on molecular biological studies enables us to better predict clinical outcomes in the patients with LQT2

High-Density Lipoprotein Suppresses Neutrophil Extracellular Traps Enhanced by Oxidized Low-Density Lipoprotein or Oxidized Phospholipids

Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized low-density lipoprotein (oxLDL) promotes NET formation of neutrophils, and that the resulting NETs increase the inflammatory responses of endothelial cells. In this study, we investigated the effects of high-density lipoproteins (HDL) on NET formation. HL-60-derived neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and further incubated with oxLDL and various concentrations of HDL for 2 h. NET formation was evaluated by quantifying extracellular DNA and myeloperoxidase. We found that the addition of native HDL partially decreased NET formation of neutrophils induced by oxLDL. This effect of HDL was lost when HDL was oxidized. We showed that oxidized phosphatidylcholines and lysophosphatidylcholine, which are generated in oxLDL, promoted NET formation of PMA-primed neutrophils, and NET formation by these products was completely blocked by native HDL. Furthermore, we found that an electronegative subfraction of LDL, LDL(–), which is separated from human plasma and is thought to be an in vivo oxLDL, was capable of promoting NET formation. These results suggest that plasma lipoproteins and their oxidative modifications play multiple roles in promoting NET formation, and that HDL acts as a suppressor of this response

A novel SCN5A mutation associated with drug induced Brugada type ECG

Background: Class IC antiarrhythmic agents may induce acquired forms of Brugada Syndrome. We have identified a novel mutation in SCN5A, the gene that encodes the α-subunit of the human cardiac sodium channel (hNav1.5), in a patient who exhibited Brugada- type ECG changes during pharmacotherapy of atrial arrhythmias. Objective: To assess whether the novel mutation p.V1328M can cause drug induced Brugada Syndrome. Methods: Administration of pilsicainide, a class IC antiarrhythmic agent, caused Brugada- type ST elevation in a 66-year-old Japanese male who presented with paroxysmal atrial fibrillation (PAF), type I atrial flutter and inducible ventricular fibrillation (VF) during electrophysiological study. Genetic screening using direct sequencing identified a novel SCN5A variant, p. V1328M. Electrophysiological parameters of WT and p.V1328M and their effects on drug pharmacokinetics were studied using the patch-clamp method. Results: Whole-cell sodium current densities were similar for WT and p.V1328M channels. While p. V1328M mutation did not affect the voltage-dependence of the activation kinetics, it caused a positive shift of voltage-dependent steady-state inactivation by 7 mV. The tonic block in the presence of pilsicainide was similar in WT and p.V1328M, when sodium currents were induced by a low frequency pulse protocol (q15s). On the contrary, p.V1328M mutation enhanced pilsicainide induced use-dependent block at 2 Hz. (Ki: WT, 35.8 μM; V1328M, 19.3 μM). Conclusion: Our study suggests that a subclinical SCN5A mutation, p.V1328M, might predispose individuals harboring it to drug-induced Brugada Syndrome

Transfer and Enzyme-Mediated Metabolism of Oxidized Phosphatidylcholine and Lysophosphatidylcholine between Low- and High-Density Lipoproteins

Oxidized low-density lipoprotein (oxLDL) and oxidized high-density lipoprotein (oxHDL), known as risk factors for cardiovascular disease, have been observed in plasma and atheromatous plaques. In a previous study, the content of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) species stayed constant in isolated in vivo oxLDL but increased in copper-induced oxLDL in vitro. In this study, we prepared synthetic deuterium-labeled 1-palmitoyl lysoPC and palmitoyl-glutaroyl PC (PGPC), a short chain-oxPC to elucidate the metabolic fate of oxPC and lysoPC in oxLDL in the presence of HDL. When LDL preloaded with d13-lysoPC was mixed with HDL, d13-lysoPC was recovered in both the LDL and HDL fractions equally. d13-LysoPC decreased by 50% after 4 h of incubation, while d13-PC increased in both fractions. Diacyl-PC production was abolished by an inhibitor of lecithin-cholesterol acyltransferase (LCAT). When d13-PGPC-preloaded LDL was incubated with HDL, d13-PGPC was transferred to HDL in a dose-dependent manner when both LCAT and lipoprotein-associated phospholipase A2 (Lp-PLA2) were inhibited. Lp-PLA2 in both HDL and LDL was responsible for the hydrolysis of d13-PGPC. These results suggest that short chain-oxPC and lysoPC can transfer between lipoproteins quickly and can be enzymatically converted from oxPC to lysoPC and from lysoPC to diacyl-PC in the presence of HDL

Additional file 1: Figure S1. of TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression

TSG101 depletion does not affect MMP-9 degradation in HT1080 cells. Cells transfected with control (con) or TSG101 (TSG#1) siRNA were incubated with 10 μM MG132 or 100 nM bafilomycin A1 (BafA1) in fresh serum-free medium containing or not 200 nM PMA for 7 h. MMPs in conditioned media (A) and cell lysates (B) were measured using gelatin zymography. The gels shown are representative of three independent experiments. (PDF 1033 kb

Additional file 2: Figure S2. of TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression

TSG101 depletion does not affect MMP-9 mRNA stability in either HT1080 or HeLaS3 cells. Stability of MMP-9 mRNA in TSG101-depleted HT1080 cells (A and B) and TSG101-depleted HeLaS3 cells (C and D). Cells transfected with control or TSG101 (TSG#1) siRNA were incubated in fresh serum-free medium containing 1 μg/ml actinomycin D for various time points and RT-PCR was performed to monitor turnover of MMP-9 mRNA. Expression levels of MMP-9 (A and C) and 18S rRNA (B and D) mRNAs were analyzed by RT-PCR. Results are plotted as the percentage of mRNA remaining relative to the starting amounts at 0 h individually in control and TSG101 (TSG#1) siRNA transfected cells. The results shown are the means ± S.D. of three independent experiments. Ns, not significant, by Student’s t-test. (PDF 351 kb

Additional file 3: Figure S3. of TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression

TSG101 depletion leads to increased levels of MMP-9 mRNA in U2OS cells. (A) Depletion of TSG101 by siRNA. Total cell lysates of cells transfected with control (con) or TSG101 (TSG#1) siRNA were analyzed by western blot using the indicated antibodies. (B) and (C) Secretion and expression of MMP-9 in TSG101-depleted cells. Cells transfected with control (con) or TSG101 (TSG#1) siRNA were incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. MMPs in conditioned media (B) and cell lysates (C) were measured using gelatin zymography. (D) MMP-9 mRNA expression in PMA-treated cells. Subconfluent cells were serum starved for 16 h and subsequently incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. Expression levels of MMP-9 and GAPDH mRNAs were analyzed by RT-PCR. The ratio of MMP-9 mRNA level relative to the GAPDH mRNA level is expressed as arbitrary units. MMP-9 mRNA level in non-treated cells is set to 1.0. (E) MMP-9 mRNA expression in TSG101-depleted cells. Cells transfected with control (con) or TSG101 (TSG#1) siRNA were incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. The ratio of MMP-9 mRNA level relative to the GAPDH mRNA level is expressed as arbitrary units. MMP-9 mRNA level in the cells transfected with control (con) siRNA is set to 1.0 individually in non- and PMA-treated cells. The blots and gels shown are representative of three independent experiments. The results shown are means ± S.D. of three independent experiments. **, p < 0.005, by a Student’s t-test. (PDF 592 kb