Cross-sectional study of retroperitoneal hematoma after invasive intervention in a Chinese population: Prevalence, characteristics, management and outcomes.

The present study aimed to explore the clinical characteristics and management of retroperitoneal hematoma (RPH) after invasive intervention during a 12-year period in China. A retrospective review of patients with RPH after various invasive interventions was conducted at the China National Center for Cardiovascular Diseases. A total of 42 patients with a mean age of 63.1±2.5 years were continuously recruited in the study between January 2007 and September 2018. The incidence, manifestations and management of RPH were analyzed. A total of 20 patients had punctures in the femoral arterial access under the inguinal ligament and 5 patients had punctures above the inguinal ligament. The majority of RPH occurred within 24 h after intervention, while some occurred after postoperative 24 h. Pain was the most common symptom in patients with RPH. All patients who underwent intervention presented a reduction in hemoglobin (HB) concentration. The overall incidence of nosocomial infection was 38.1% and mortality was 7.1%. The findings demonstrated that RPH is a rare complication after invasive intervention of cardiovascular diseases with non-specific clinical manifestations. The reduction of HB concentration was a vital manifestation for RPH. Most RPH cases could be treated by conservative treatment and blood transfusion. A puncture in the femoral arterial access under the inguinal ligament may result in RPH.

Invasive versus conservative strategy in consecutive patients aged 80 years or older with non-ST-segment elevation myocardial infarction: a retrospective study in China.

OBJECTIVE: To investigate whether the very elderly patients with non-ST-segment elevation myocardial infarction (NSTEMI) will benefit from an invasive strategy versus a conservative strategy. METHODS: 190 consecutive patients aged 80 years or older with NSTEMI were included in the retrospective study from September 2014 to August 2017, of which 69 patients received conservative strategy and 121 patients received invasive strategy. The primary outcome was death. Multivariate Cox regression models were used to assess the statistical association between strategies and mortality. The survival probability was further analyzed. RESULTS: The primary outcome occurred in 17.4% patients in the invasive group and in 42.0% patients in the conservative group (P = 0.0002). The readmission rate in the invasive group (14.9%) was higher than that in the conservative group (7.2%). Creatinine level (OR = 1.01, 95% CI: 0.10-1.03, P = 0.05) and use of diuretic (OR = 3.65, 95% CI: 1.56-8.53, P = 0.003) were independent influential factors for invasive strategy. HRs for multivariate Cox regression models were 3.45 (95% CI: 1.77-6.75, P = 0.0003), 3.02 (95% CI: 1.52-6.01, P = 0.0017), 2.93 (95% CI: 1. 46-5.86, P = 0.0024) and 2.47 (95% CI: 1.20-5.07, P = 0.0137). Compared with the patients received invasive strategy, the conservative group had remarkably reduced survival probability with time since treatment (P < 0.001). CONCLUSIONS: An invasive strategy is superior to a conservative strategy in reducing mortality of patients aged 80 years or older with NSTEMI. Our results suggest that an invasive strategy is more suitable for the very elderly patients with NSTEMI in China.

Treatment outcomes and therapeutic evaluations of patients with left ventricular aneurysm.

OBJECTIVE: This study was performed to analyze and compare the efficacy of three treatment methods for left ventricular aneurysm (LVA): coronary artery bypass grafting (CABG) combined with left ventricular resection, drug treatment, and percutaneous coronary intervention (PCI). METHODS: In total, 183 patients with LVA from Fuwai Hospital were divided into three groups according to the treatment method: 51 patients underwent left ventricular resection combined with CABG (CABG-resection group), 65 underwent drug treatment (drug group), and 67 underwent PCI (PCI group). The clinical characteristics and survival rates of the patients were compared among the three groups. RESULTS: The patients' basic data and medical history were analyzed. The postoperative left ventricular end-diastolic dimension (LVEDD) and left ventricular ejection fraction (LVEF) were significantly higher than those before surgery, indicating that the left ventricular function markedly improved after the operation. CONCLUSION: Surgery is recommended as the first treatment option for LVA, and conservative therapy can be considered for selected patients. Although the difference was not statistically significant, CABG with left ventricular resection was associated with a better LVEF and LVEDD and higher survival and non-recurrence rates than PCI or drug treatment.

Gender differences in treatment strategies among patients ≥80 years old with non-ST-segment elevation myocardial infarction.

BACKGROUND: This study aims to investigate the gender differences in treatment strategies among non-ST-segment elevation myocardial infarction (NSTEMI) patients ≥80 years old in China. METHODS: A total of 190 consecutive NSTEMI patients ≥80 years old in Fuwai Hospital were included from 2014 to 2017. These patients were grouped by gender, and sub-grouped by conservative treatment or invasive treatment. The clinical characteristics, medical history, discharge drug used, and prognosis were collected and compared between these two treatment strategies. RESULTS: There were significant differences between these two treatment strategies in terms of GRACE grade, history of myocardial infarction (MI), after coronary artery bypass grafting (CABG), III grade, renal dysfunction, anemia, and use of diuretic (P<0.05). In addition, the age, creatinine and Killip class of female patients, and the death and good prognosis of male patients were found to be significantly different between these two treatment strategies (P<0.05). The multivariate logistic regression analysis revealed that the death of males was significantly associated with treatment strategies in the multivariable logistic regression analysis (P<0.05). In addition, the Kaplan-Meier survival analyses revealed that the survival rates of invasive strategy were significantly higher, when compared to that of conservative strategy in males (P=0.001) and females (P=0.015). CONCLUSIONS: There were gender differences in treatment strategies among NSTEMI patients ≥80 years old. The difference in treatment strategies in males was more pronounced than in females, in terms of long-term survival rate.

MicroRNA-466 inhibits osteosarcoma cell proliferation and induces apoptosis by targeting CCND1.

Emerging pieces of evidence indicate that microRNA-466 (miR-466) serves as a tumor suppressor in several human tumors, including colorectal cancer and prostate cancer. However, whether miR-466 is involved in osteosarcoma (OS) progression remains largely unknown. The present study demonstrated that miR-466 was significantly downregulated in OS tissues and cell lines. Furthermore, it was revealed that the expression of miR-466 was negatively correlated with OS severity. Moreover, low miR-466 expression in patients with OS predicted poor prognosis. Through functional experiments, miR-466 overexpression significantly inhibited the proliferation and cell cycle of OS cells while inducing cellular apoptosis. In terms of mechanism, it was revealed that CCND1 was a target of miR-466 in OS cells. miR-466 overexpression suppressed CCND1 expression in OS cells. A reverse association was observed between the expression levels of miR-466 and CCND1 in OS tissues. Furthermore, CCND1 restoration in OS cells significantly rescued the effects of miR-466 on cellular proliferation and apoptosis. Overall, the results of the present study demonstrated that miR-466 suppressed OS progression by targeting CCND1, suggesting that miR-466 may be a promising biomarker and therapeutic target for OS prognosis and treatment.

Computer-aided discovery and biological characterization of human lactate dehydrogenase 5 inhibitors with anti-osteosarcoma activity.

Human lactate dehydrogenase 5 (hLDH5) is overexpressed in various tissues of human tumors, which could be a potential therapeutic target for cancer treatment. Herein, we describe the computer-aided discovery and biological characterizations of hLDH5 inhibitors with anti-osteosarcoma activity. Biochemical assay indicated that the identified compounds 3 and 9 strongly inhibited hLDH5 function with EC50 values of 0.67 and 0.39 µM, respectively. The MTT assay revealed that most of the identified inhibitors had little effect on MG-63 cell proliferation at 4 µM, only 9 reduced the cancer cell proliferation at the same concentration, with an IC50 value of 3.18 µM. Our data suggested that 9 could be a starting lead of developing potent hLDH5 inhibitor for the anti-osteosarcoma agents in cancer treatment.

Readthrough of SCN5A Nonsense Mutations p.R1623X and p.S1812X Questions Gene-therapy in Brugada Syndrome.

PURPOSE: Nonsense mutation readthrough is used as a gene-specific treatment in some genetic diseases. The response to readthrough treatment is determined by the readthrough efficiency of various nonsense mutations. In this manuscript, we aimed to explore the harmful effects of nonsense mutation suppression. METHODS: HEK293 cells were transfected with two SCN5A (encode cardiac Na+ channel) nonsense mutations, p.R1623X and p.S1812X. We applied two readthrough-enhancing methods (either aminoglycosides or a siRNA-targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1)) to suppress these SCN5A nonsense mutations. When either of readthrough methods was used, the sodium channel proteins were examined by western blot and immunoblotting and recorded by whole cell patch-clamp to observe the functional characterization of the restored channels. RESULTS: Upon readthrough treatment, the sodium currents were restored to the mutant cDNAs. These mutations reduced full-length sodium channel protein levels, and the sodium currents were reduced to 3% of wild-type. The mutant cDNA sodium currents were increased to 30% of wild-type, and the fulllength proteins also increased. However, the functional characterization of these channels from cDNAs carrying p.R1623X and p.S1812X exhibited abnormal biophysical properties, including a negative shift in steady-state sodium channel inactivation, a positive shift in sodium channel activation and robust late sodium currents. The ramp test showed prolonged QT intervals. CONCLUSION: These results demonstrated that readthrough-enhancing methods effectively suppressed nonsense mutations in SCN5A and restored the expression of full-length channels. However, the restored channels may increase the risk of arrhythmia.

Metformin and Resveratrol Inhibited High Glucose-Induced Metabolic Memory of Endothelial Senescence through SIRT1/p300/p53/p21 Pathway.

Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic vascular complications, a phenomenon known as "metabolic memory." Although SIRT1 has been demonstrated to mediate high glucose-induced endothelial senescence, whether and how "metabolic memory" would affect endothelial senescence through SIRT1 signaling remains largely unknown. In this study, we investigated the involvement of SIRT1 axis as well as the protective effects of resveratrol (RSV) and metformin (MET), two potent SIRT1 activators, during the occurrence of "metabolic memory" of cellular senescence (senescent "memory"). Human umbilical vascular endothelial cells (HUVECs) were cultured in either normal glucose (NG)/high glucose (HG) media for 6 days, or 3 days of HG followed by 3 days of NG (HN), with or without RSV or MET treatment. It was shown that HN incubation triggered persistent downregulation of deacetylase SIRT1 and upregulation of acetyltransferase p300, leading to sustained hyperacetylation (at K382) and activation of p53, and subsequent p53/p21-mediated senescent "memory." In contrast, senescent "memory" was abrogated by overexpression of SIRT1 or knockdown of p300. Interestingly, we found that SIRT1 and p300 could regulate each other in response to HN stimulation, suggesting that a delicate balance between acetyltransferases and deacetylases may be particularly important for sustained acetylation and activation of non-histone proteins (such as p53), and eventually the occurrence of "metabolic memory." Furthermore, we found that RSV or MET treatment prevented senescent "memory" by modulating SIRT1/p300/p53/p21 pathway. Notably, early and continuous treatment of MET, but not RSV, was particularly important for preventing senescent "memory." In conclusion, short-term high glucose stimulation could induce sustained endothelial senescence via SIRT1/p300/p53/p21 pathway. RVS or MET treatment could enhance SIRT1-mediated signaling and thus protect against senescent "memory" independent of their glucose lowering mechanisms. Therefore, they may serve as promising therapeutic drugs against the development of "metabolic memory."

[Early clinical outcomes of percutaneous balloon aortic valvuloplasty in treating high-risk patients with severe aortic stenosis].

OBJECTIVE: To explore the procedural feasibility and early clinical outcomes of percutaneous balloon aortic valvuloplasty (PBAV) in patients with severe aortic stenosis, who were considered transiently unsuitable for surgical aortic valve replacement (sAVR) and transcatheter aortic valve replacement (TAVR). METHODS: Between March 2011 and January 2014, datas of 20 patients underwent PBAV in Fuwai Hospital were retrospectively analyzed. Mean patients age was (72 ± 8) years.Initial procedural and clinical outcomes were evaluated. RESULTS: PBAV was successfully performed in all cases. Post-procedure, aortic valve area increased from (0.55 ± 0.09) m(2) to (0.77 ± 0.15) m(2)(P < 0.001), left ventricle ejection fraction from (31.7 ± 9.0) % to (39.0 ± 11.0) % (P = 0.018), mean transaortic valve gradient decreased from (49.5 ± 15.0) mmHg (1 mmHg = 0.133 kPa) to (31.7 ± 12.0) mmHg (P < 0.001), and pulmonary artery systolic pressure decreased from (55.1 ± 18.0) mmHg to (38.7 ± 11.0) mmHg (P = 0.025) . There was no significant change in the aortic regurgitation grade (P = 0.854). The most common complications were hypotension (n = 4) and transient left bundle branch block (n = 5). Overall 24-hour and 30-day mortality was 5% (n = 1) and 15% (n = 3), respectively. Within 30 days after PBAV procedure, five patients underwent successful sAVR, one patient underwent TAVR, and five patients awaited TAVR. CONCLUSION: In high-risk patients with severe aortic stenosis and temporary contraindication to sAVR or TAVR, PBAV can be safely used as a bridging intervention procedure and the short-term procedural and clinic outcomes are satisfactory.

The expression of Ubc9 and the intensity of SERCA2a-SUMOylation were reduced in diet-induced obese rats and partially restored by trimetazidine.

BACKGROUND: Reduced expression of sarcoplasmic reticulum calcium-transporting ATPase isoform 2a (SERCA2a) has been shown to play a significant role in the cardiac dysfunction of obese animal models. It was reported recently that SUMOylation enhances the stability and activity of SERCA2a. We hypothesized that SERCA2a-SUMOylation might be involved in obesity-mediated reduction of SERCA2a. METHOD AND RESULTS: Trimetazidine (TMZ), the drug that inhibits fatty acid oxidation, was used in diet-induced obese (DIO) rats and palmitic acid (PA)-treated cardiomyocytes. The intensity of SERCA2a-SUMOylation and proteins involved in SERCA2a-SUMOylation were investigated in vivo and in vitro. DIO rats presented cardiac dysfunction, which was alleviated by TMZ treatment. Reductions of SERCA2a protein and the intensity of SERCA2a-SUMOylation were observed in DIO rats and PA-treated cardiomyocytes. These reductions were partially restored by TMZ. However, TMZ itself did not alter the intensity of SERCA2a-SUMOylation in control cardiomyocytes. The variations of protein and messenger RNA levels of Ubiquitin carrier protein 9 are in accordance with the intensity of SERCA2a-SUMOylation. Whereas the other proteins involved in SERCA2a-SUMOylation were not changed by DIO and PA. CONCLUSIONS: TMZ alleviates the DIO- and PA-induced reductions of SERCA2a-SUMOylation. Ubiquitin carrier protein 9 is involved in the reductions.

Remote ischemic preconditioning reduces perioperative cardiac and renal events in patients undergoing elective coronary intervention: a meta-analysis of 11 randomized trials.

BACKGROUND: Results from randomized controlled trials (RCT) concerning cardiac and renal effect of remote ischemic preconditioning(RIPC) in patients with stable coronary artery disease(CAD) are inconsistent. The aim of this study was to explore whether RIPC reduce cardiac and renal events after elective percutaneous coronary intervention (PCI). METHODS AND RESULTS: RCTs with data on cardiac or renal effect of RIPC in PCI were searched from Pubmed, EMBase, and Cochrane library (up to July 2014). Meta-regression and subgroup analysis were performed to identify the potential sources of significant heterogeneity(I(2) ≥ 40%). Eleven RCTs enrolling a total of 1713 study subjects with stable CAD were selected. Compared with controls, RIPC significantly reduced perioperative incidence of myocardial infarction (MI) [odds ratio(OR) = 0.68; 95% CI, 0.51 to 0.91; P = 0.01; I(2) = 41.0%] and contrast-induced acute kidney injury(AKI) (OR = 0.61; 95% CI, 0.38 to 0.98; P = 0.04; I(2) = 39.0%). Meta-regression and subgroup analyses confirmed that the major source of heterogeneity for the incidence of MI was male proportion (coefficient  = -0.049; P = 0.047; adjusted R(2) = 0.988; P = 0.02 for subgroup difference). CONCLUSIONS: The present meta-analysis of RCTs suggests that RIPC may offer cardiorenal protection by reducing the incidence of MI and AKI in patients undergoing elective PCI. Moreover, this effect on MI is more pronounced in male subjects. Future high-quality, large-scale clinical trials should focus on the long-term clinical effect of RIPC.

[Safety and feasibility of carotid artery stenting in patients with coexisting carotid and coronary artery disease].

OBJECTIVE: To evaluate the safety and feasibility of carotid artery stenting (CAS) for treating patients with coexisting carotid and coronary artery disease. METHODS: The clinical data of 237 consecutive patients [(66.1 ± 7.7) years old, 79.7% male] with coexisting carotid and coronary artery disease undergoing CAS in Fuwai hospital from January 2005 to June 2010. The patients were analyzed retrospectively.Indication for CAS was defined as carotid artery diameter reduction of > 60% (symptomatic) or > 80% (asymptomatic) with suitable carotid artery anatomy for stenting. Thirty-day rates of stroke, death and myocardial infarction after CAS were assessed. RESULTS: All patients suffered from coronary artery disease, of whom 87(36.7%) had unstable angina pectoris and 82(34.6%) had recent myocardial infarction (< 30 days). The procedural success rate of CAS was 99.2 % (235/237). Cerebral protection devices were used in 234 patients (99.6%). Among them, 36(15.2%) patients received simultaneous bilateral CAS and 79(33.3%) patients underwent simultaneous percutaneous intervention of other non-coronary arteries.Within 30 days after CAS, 127(53.6%) patients underwent coronary revascularization, including 118(49.6%) coronary artery bypass grafting and 9 (3.8%) percutaneous coronary intervention. The rate of major stroke, minor stroke, death and myocardial infarction from time of CAS to 30 days was 2.1% (5/237), 3.0% (7/237),0.4% (1/237) and 0.4% (1/237) respectively. CONCLUSION: Data from this study indicate that CAS is safe and feasible for treating patients with coexisting carotid and coronary artery disease with a low incidence of periprocedural complication rate.

A heterozygous missense SCN5A mutation associated with early repolarization syndrome.

The genetic background of early repolarization syndrome (ERS) has not been fully understood. In this study, we identified a missense SCN5A mutation and a polymorphism in a patient with ERS and characterized the functional consequences of the two variants. The functional consequences of mutant channels were investigated with the patch-clamp technique, immunocytochemical studies and real-time PCR. A 19-year-old female proband with recurrent syncope had a documented electrocardiogram with ventricular fibrillation (VF) proceeded by large J waves in leads I, II, III, aVF and V2-V6. Genetic analysis revealed that the patient carried a missense mutation of c.4297 G>C and a synonymous polymorphism of T5457C on the same allele of the SCN5A gene. Patch-clamp experiments demonstrated that the c.4297 G>C mutation significantly reduced the sodium current (INa) density and altered the channel kinetics. Immunocytochemical studies demonstrated that the mutation dramatically inhibited the expression of sodium channels in the cell membrane and in the cytoplasm, although the mRNA levels remained in the normal range. Noteworthy, the reduction in INa density may be partially restored from the co-existence of the T5457C polymorphism on the same allele by the upregulation of mRNA levels. In conclusion, our study indicated that the c.4297 G>C mutation caused the 'loss-of-function' of sodium channels that may account for the clinical phenotype of ERS. The reduction in INa density was due to a decreased number of sodium channels caused by abnormal translation processes. The T5457C polymorphism partially rescued the INa density of the mutant channels by the upregulation of mRNA levels.

Clinical and angiographic correlates of left ventricular dysfunction in patients with three vessel coronary disease.

BACKGROUND: Among patients with advanced multivessel coronary disease, left ventricular (LV) function is widely variable, and clinical and angiographic correlates of ventricular dysfunction remain to be defined. METHODS: Among 73 339 patients undergoing diagnostic cardiac catheterization at a single center in China, patients with left ventriculographic assessment were identified with three-vessel coronary disease with or without left main involvement. Clinical and angiographic characteristics were examined among patients with normal or varying extent of LV dysfunction, and predictors of LV impairment (ejection fraction (EF): < 25%, 25% - 40% or > 40%) were determined. RESULTS: Among 11 950 patients identified with three-vessel coronary disease, the sample distribution of LVEF was > 40%, n = 10 776; 25% - 40%, n = 948; < 25%, n = 226. Patients with reduced LV function (< 40%) more commonly were male and had a history of myocardial infarction (MI), diabetes or unstable angina. Hypertension was more frequent in those with LVEF ≥ 40%. In a multivariate Logistic regression analysis, prior MI (odds ratio (OR), 3.37; 95% confidence interval (CI), 2.96 - 3.84) was most predictive of LVEF < 40%, followed by male gender, diabetes, and presentation with unstable angina. For LVEF < 25%, only prior MI was identified as a significant correlate of severe LV dysfunction (OR 4.06, 95%CI 3.06 - 5.39). Following exclusion of patients with previous MI (n = 7416), male gender and diabetes were predictive of LVEF < 40%, yet presentation with unstable angina was the only factor significantly associated with LVEF < 25%. CONCLUSION: Among individuals identified with three-vessel coronary disease with or without left main involvement, previous MI was the most significant risk factor of LV dysfunction.

[Clinical analysis of simultaneous bilateral carotid stenting for treating patients with bilateral atherosclerotic carotid stenosis].

OBJECTIVE: To evaluate the safety and feasibility of simultaneous bilateral carotid stenting for treating patients with bilateral atherosclerotic carotid stenosis. METHODS: The clinical data of 39 consecutive patients with bilateral atherosclerotic carotid stenosis undergoing simultaneous bilateral carotid artery stenting in Fuwai hospital from January 2005 to December 2009 were collected and analyzed retrospectively. The reduction of the angiographic diameter stenosis after stenting and clinical outcomes of 30 days after stenting including hyperperfusion syndrome, hemodynamic depression, stroke, myocardial infarction and death were assessed. RESULTS: The patients were 43 - 78 (65.9 ± 8.5) years old, and there were 25 (64.1%) male. Carotid stenting procedure success rate was 100%. Distal embolic protection devices were used in all patients, and 20 (51.3%) out of 39 patients underwent coronary artery bypass surgery after carotid stenting. The angiographic diameter stenosis reduced from (87.0 ± 5.8)% to (10.2 ± 5.6)% after stenting (P < 0.01). Up to 30 days after carotid artery stenting, the incidence of hyperperfusion syndrome, hemodynamic depression, minor stroke, major stroke, myocardial infarction and death was 2.6% (1/39), 28.2% (11/39), 5.1% (2/29), 0, 2.6% (1/39), 2.6% (1/39), respectively. CONCLUSION: The data show that simultaneous bilateral carotid stenting is a technically feasible and safe alternative for patients with severe bilateral atherosclerotic carotid stenosis.

[Readthrough of nonsense mutation W822X in the SCN5A gene can effectively restore expression of cardiac Na+ channels W822X].

OBJECTIVE: In this study we investigated the functional restoration of nonsense mutations in the SCN5A gene. METHODS: The readthrough-enhancing reagents were introduced to HEK293 cells to suppress one nonsense mutation W822X in the SCN5A gene. Patch-clamp was used to record the whole-cell current and dynamics. Western blot and immunofluorescence staining were used to certify the expression and the location of the sodium channel. RESULTS: In transfected HEK293 cells, the nonsense mutation in SCN5A inhibited the expression level of full-length protein, and the sodium currents from the mutant channels were less than 3% of the wild-type level. Readthrough enhancement by decreasing translation termination efficiency with a siRNA targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1), the sodium current from the mutant cDNAs was restored to as much as 30% of the wild-type. After the treatment by the readthrough-enhancing reagents, the channels from cDNA carrying W822X remained the features of wild-type phenotype, and Western blot and immunochemical staining also showed the expression of full-length channel proteins. CONCLUSION: Readthrough-enhancing reagents could effectively suppress nonsense mutations in SCN5A and partially restore the function of sodium channel and the expression of full-length channels.

Novel compound heterozygous mutations T2C and 1149insT in the KCNQ1 gene cause Jervell and Lange-Nielsen syndrome.

Mutations in the KCNQ1 gene account for more than 90% of the individuals with Jervell and Lange-Nielsen syndrome (JLNS). In this study, we identified and characterized two novel KCNQ1 mutations that caused JLNS. A 6-year-old deaf girl suffering from recurrent syncope had a documented electrocardiogram with polymorphic ventricular fibrillation since the age of 4 years. The baseline electrocardiogram showed a significantly prolonged corrected QT interval (524 msec). Genetic analysis revealed that the proband carried two heterozygous mutations of T2C and 1149insT in the KCNQ1 gene on separate alleles. Patch-clamp analysis demonstrated that the T2C mutation resulted in significant reduction in the slowly activated delayed rectifier current (IKs). Furthermore, western blot analysis and confocal imaging revealed that the T2C mutation produced a truncated protein with trafficking defects. In contrast, the 1149insT mutation failed to generate any measurable current, consistent with no protein expression in both the cell membrane and cytoplasm. Moreover, co-expression of the T2C and 1149insT mutations significantly reduced the peak tail current density to 8.27% of the wild-type (WT) current value, while co-transfected WT channels with either T2C or 1149insT mutant channels produced comparable current and channel kinetics to that of WT channels. Our study demonstrates that the compound heterozygous mutations T2C and 1149insT cause the 'loss-of-function' of the IKs that may account for the clinical phenotype of the proband. Multiple mechanisms have been involved in the pathogenesis of 'loss-of-function' of IKs.

Readthrough of nonsense mutation W822X in the SCN5A gene can effectively restore expression of cardiac Na+ channels.

AIMS: Nonsense mutations in the SCN5A gene result in truncated, non-functional derivatives of the cardiac Na+ channel and thus cause arrhythmias. Studies of other genes suggest that pathogenic phenotypes of nonsense mutations may be alleviated by enhancing readthrough, which enables ribosomes to ignore premature termination codons and produce full-length proteins. Thus, we studied the functional restoration of nonsense-mutated SCN5A. METHODS AND RESULTS: HEK293 cells were transfected with SCN5A cDNA or its mutant carrying W822X, a nonsense mutation associated with Brugada syndrome and sudden cardiac death. The effects of readthrough-enhancing reagents on Na+ channel expression and function were examined in the transfected cells. W822X robustly reduced Na+ current, decreasing maximal Na+ current to <3% of the wild-type level, and inhibited the expression of full-length Na+ channels. When readthrough was enhanced by either reducing translational fidelity with aminoglycosides or decreasing translation termination efficiency with small-interfering RNA against eukaryotic release factor eRF3a, Na+ current of the mutant was restored to approximately 30% of the wild-type level; western blot and immunochemical staining analyses showed the increased expression of full-length channels. When the wild-type and mutant cDNAs were co-transfected, readthrough-enhancing reagents increased Na+ current from 56% to 74% of the wild-type level. Analysis of Na+ channel kinetics showed that the channels expressed from the mutant cDNA under readthrough-enhancing conditions retained the functions of wild-type channels. CONCLUSION: Readthrough-enhancing reagents can effectively suppress SCN5A nonsense mutations and may restore the expression of full-length Na+ channels with normal functions, which might prevent sudden cardiac death in mutation carriers.