Mechanisms of fever-induced QT prolongation and torsades de pointes in patients with KCNH2 mutation.

AIMS: Patients with particular mutations of type-2 long QT syndrome (LQT2) are at an increased risk for malignant arrhythmia during fever. This study aimed to determine the mechanism by which KCNH2 mutations cause fever-induced QT prolongation and torsades de pointes (TdP). METHODS AND RESULTS: We evaluated three KCNH2 mutations, G584S, D609G, and T613M, in the Kv11.1 S5-pore region, identified in patients with marked QT prolongation and TdP during fever. We also evaluated KCNH2 M124T and R269W, which are not associated with fever-induced QT prolongation. We characterized the temperature-dependent changes in the electrophysiological properties of the mutant Kv11.1 channels by patch-clamp recording and computer simulation. The average tail current densities (TCDs) at 35°C for G584S, WT+D609G, and WT+T613M were significantly smaller and less increased with rising temperature from 35°C to 40°C than those for WT, M124T, and R269W. The ratios of the TCDs at 40°C to 35°C for G584S, WT+D609G, and WT+T613M were significantly smaller than for WT, M124T, and R269W. The voltage dependence of the steady-state inactivation curve for WT, M124T, and R269W showed a significant positive shift with increasing temperature; however, that for G584S, WT+D609G, and WT+T613M showed no significant change. Computer simulation demonstrated that G584S, WT+D609G, and WT+T613M caused prolonged action potential durations and early afterdepolarization formation at 40°C. CONCLUSION: These findings indicate that KCNH2 G584S, D609G, and T613M in the S5-pore region reduce the temperature-dependent increase in TCDs through an enhanced inactivation, resulting in QT prolongation and TdP at a febrile state in patients with LQT2.

The utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of KCNQ1 variants.

Genetic testing for inherited arrhythmias and discriminating pathogenic or benign variants from variants of unknown significance (VUS) is essential for gene-based medicine. KCNQ1 is a causative gene of type 1 long QT syndrome (LQTS), and approximately 30% of the variants found in type 1 LQTS are classified as VUS. We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. We generated homozygous kcnq1 deletion zebrafish (kcnq1del/del) using the CRISPR/Cas9 and expressed human Kv7.1/MinK channels in kcnq1del/del embryos. We dissected the hearts from the thorax at 48 h post-fertilization and measured the transmembrane potential of the ventricle in the zebrafish heart. Action potential duration was calculated as the time interval between peak maximum upstroke velocity and 90% repolarization (APD90). The APD90 of kcnq1del/del embryos was 280 ± 47 ms, which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (168 ± 26 ms, P < 0.01 vs. kcnq1del/del). A study of two pathogenic variants (S277L and T587M) and one VUS (R451Q) associated with clinically definite LQTS showed that the APD90 of kcnq1del/del embryos with these mutant Kv7.1/MinK channels was significantly longer than that of Kv7.1 WT/MinK channels. Given the functional results of the zebrafish model, R451Q could be reevaluated physiologically from VUS to likely pathogenic. In conclusion, functional analysis using in vivo zebrafish cardiac arrhythmia model can be useful for determining the pathogenicity of loss-of-function variants in patients with LQTS.

[Pacemaker Dislocation into the Peritoneal Cavity].

A 16-day-old neonate with congenital complete atrioventricular block underwent epicardial pacemaker implantation under the rectus. Four months later, abodominal X-ray imaging revealed dislocation of the generator from the abdomen to the pelvis. The infant was diagnosed with intraperitoneal pacemaker dislocation. However, there were no abdominal manifestations or complications associated with the bowel, urinary tract, and vascular system. Surgical refixation was performed in a hybrid room. Fluoroscopy helped avoid bowel injury when removing the generator from the peritoneal cavity. The pacing lead, which was adherent and entangled with the omentum, was released under direct vision. The generator was placed in a new pocket created in the subcutaneous layer of the anterior fascia of the rectus.

Impact of Updated Diagnostic Criteria for Long QT Syndrome on Clinical Detection of Diseased Patients: Results From a Study of Patients Carrying Gene Mutations.

OBJECTIVES: In this study, we scored patients with long QT syndrome (LQTS) according to the different Schwartz diagnostic criteria from 1993, 2006, and 2011, and to examine the validation of the criteria in relevance to the frequency of LQTS-related gene mutation. BACKGROUND: Although updated diagnostic criteria have been used in clinical settings, few data exist regarding their impact on the diagnosis of LQTS. METHODS: We used a cohort of 132 patients who presented with prolonged QTc intervals and/or abnormal clinical history in cardiac screening and who underwent exercise stress testing. LQTS scores of ≥3.5 points according to the 2006 and the 2011 criteria were considered to indicate a high probability of LQTS, as opposed to the 4 points used by the 1993 criteria. The 2011 criteria were updated by adding the evaluation of the recovery phase of exercise. RESULTS: The 2011 criteria significantly increased the number of high probability patients (n = 62) compared with the 1993 criteria (n = 32; p = 0.0002) or the 2006 criteria (n = 36; p = 0.0014). The percentage of mutation carriers in those with an intermediate score, which was rather high using the 1993 (53%) and 2006 criteria (53%), was greatly reduced with the 2011 criteria (15%, p = 0.0014 vs. the 1993 criteria, and p = 0.0013 vs. the 2006 criteria). Among 54 mutation carriers, the 1993, the 2006, and the 2011 criteria identified a high probability of carriers in 25 patients (46% sensitivity and 91% specificity), 27 patients (50% sensitivity and 88% specificity), and 48 patients (89% sensitivity and 82% specificity), respectively. CONCLUSIONS: The use of the 2011 criteria will facilitate the diagnosis of LQTS and will decrease the number of false negative results.

The critical role of lipopolysaccharide in the upregulation of aquaporin 4 in glial cells treated with Shiga toxin.

BACKGROUND: In 2011, there was an outbreak of Shiga toxin-producing Escherichia coli (STEC) infections in Japan. Approximately 62 % of patients with hemolytic-uremic syndrome also showed symptoms of encephalopathy. To determine the mechanisms of onset for encephalopathy during STEC infections, we conducted an in vitro study with glial cell lines and primary glial cells. RESULTS: Shiga toxin 2 (Stx-2) in combination with lipopolysaccharide (LPS), or LPS alone activates nuclear factor-κB (NF-κB) signaling in glial cells. Similarly, Stx-2 in combination with LPS, or LPS alone increases expression levels of aquaporin 4 (AQP4) in glial cells. It is possible that overexpression of AQP4 results in a rapid and increased influx of osmotic water across the plasma membrane into cells, thereby inducing cell swelling and cerebral edema. CONCLUSIONS: We have showed that a combination of Stx-2 and LPS induced apoptosis of glial cells recently. Glial cells are indispensable for cerebral homeostasis; therefore, their dysfunction and death impairs cerebral homeostasis and results in encephalopathy. We postulate that the onset of encephalopathy in STEC infections occurs when Stx-2 attacks vascular endothelial cells of the blood-brain barrier, inducing their death. Stx-2 and LPS then attack the exposed glial cells that are no longer in contact with the endothelial cells. AQP4 is overexpressed in glial cells, resulting in their swelling and adversely affecting cerebral homeostasis. Once cerebral homeostasis is affected in such a way, encephalopathy is the likely result in STEC patients.

Early diagnosis of Danon disease: Flow cytometric detection of lysosome-associated membrane protein-2-negative leukocytes.

INTRODUCTION: Danon disease is an extremely rare X-linked dominant disorder characterized by progressive cardiomyopathy, muscle weakness, and mild mental retardation. Most cases harbor nonsense, frameshift, or splice-site mutations in LAMP2 that result in lysosome-associated membrane protein-2 (LAMP-2) deficiency and lysosomal defects. The identification of LAMP2 mutations makes it possible to detect female carriers with significant cardiomyopathy. Therefore, it is of paramount importance to develop useful carrier detection methods. METHODS: To screen for diminished LAMP-2 expression among female patients with progressive cardiomyopathy, we developed a flow cytometric method to detect LAMP-2-deficient leukocytes. RESULTS: In healthy controls, all circulating leukocyte populations, including granulocytes, monocytes, and lymphocytes, expressed significant levels of LAMP-2. In contrast, cells from a male patient with Danon disease lacked detectable LAMP-2. His younger twin sisters showed reduced levels of LAMP-2 expression with characteristic bimodal fluorescence intensity patterns. The percentage of LAMP-2-negative cells in the asymptomatic sibling was nearly the same as that in the symptomatic sibling. CONCLUSION: We developed a flow cytometric assay for LAMP-2 expression that can serve as a rapid primary screening method to detect carriers of LAMP-2 deficiencies. This assay will narrow the target population before subjecting patients to more laborious and expensive gene mutation analysis.

Lactobacillus casei cell wall extract directly stimulates the expression of COX2 independent of Toll-like receptor 2 in rat glial cells.

Kawasaki disease is an acute illness of early childhood that is characterized by prolonged fever and vasculitis of unknown pathogenesis. Lactobacillus casei cell wall extract (LCWE)-induced vasculitis in mice is a well-validated model of Kawasaki disease. In the nervous system, glial cells play an important role in fever development. This study investigated whether LCWE directly stimulates glial cells, resulting in the induction of cyclooxygenase-2 (COX2), which is required for prostaglandin synthesis and fever development. We found that LCWE induced COX2 expression and activated the nuclear factor-κB signaling pathway in rat B92 glial cells, but Toll-like receptor-2, which is one of the receptors for LCWE, could not be detected in the cells. These results suggest that LCWE activates the nuclear factor-κB signaling pathway and induces COX2 in rat B92 glial cells through another LCWE receptor other than Toll-like receptor-2.

Natural history of medium-sized atrial septal defect in pediatric cases.

BACKGROUND: The indication for surgical repair of atrial septal defect (ASD) is pulmonary to systemic blood flow ratio (Qp/Qs)>2.0, and therapeutic strategy depends on the facility in cases of Qp/Qs 1.5-2.0. Defect size increases with age, but hemodynamic changes of medium-sized ASD (Qp/Qs 1.5-2.0) are unknown. METHODS AND RESULTS: From April 1, 1985 to March 31, 2008, we experienced 125 cases of cardiac catheterization for ASD. Twelve cases were re-evaluated without surgical repair. The first and second catheterizations were performed at median ages of 7 years (range, 2-13 years) and 16 years (range, 5-19 years), respectively. The mean follow-up period was 7 years. Qp/Qs increased from 1.6 to 2.0 during follow-up (p<0.05). Of four cases with Qp/Qs<1.5 at initial presentation, three had Qp/Qs≥1.5 at second inspection. Right ventricle diastolic volume (RVEDV/LVEDV) also increased. CONCLUSIONS: Qp/Qs and RVEDV/LVEDV of medium-sized ASD increase together in childhood. Re-evaluation before adulthood should be considered in patients with no indications of ASD closure in childhood.

Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

Successful treatment with bosentan for pulmonary hypertension and reduced peripheral circulation in juvenile systemic sclerosis.

Pulmonary arterial hypertension (PAH) when associated with systemic sclerosis (SSc) (SSc-PAH) is one of the leading causes of mortality and is found in 10-15% of adult patients with SSc. The ET receptor antagonist bosentan has been shown to be effective in the treatment of adult patients with SSc-PAH. Furthermore, it has been shown that bosentan ameliorates decreased skin perfusion and digital ulceration secondary to SSc. However, the effectiveness and safety of bosentan for treatment of juvenile SSc still remains unclear. We describe a case of juvenile SSc-PAH successfully treated with bosentan. The present case shows that bosentan ameliorated PAH and peripheral circulation as evaluated by cold stress thermography. No bosentan-related adverse events such as liver dysfunction were observed. Prospective randomized trials are required to validate the effectiveness of bosentan for patients with juvenile SSc; however, bosentan might be useful for the management of patients with juvenile SSc.

Effects of antithrombin III treatment in vascular injury model of mice.

BACKGROUND: Balloon angioplasty has recently been adopted as an acceptable form of treatment for stenotic vessel lesions of congenital heart diseases. However, precise mechanisms of restenosis and thrombosis, which are the most common complications after these procedures, are unknown. METHODS: We examined the effects of antithrombin III (ATIII) on inflammation, thrombus formation, and remodeling of vascular wall after guidewire-induced injury in the femoral artery of mice. ATIII or saline was administered as a bolus intravenous infusion before injury. RESULTS: Seventy-two hours after injury, approximately half of the saline-treated vessels showed macroscopic thrombus formation. In contrast, no thrombi were seen in the arteries pretreated with ATIII. Significantly higher levels of inflammation were induced in the injured vessels than in the sham-operated controls, as determined by CD11b-positive cell density in the adventitial area. ATIII treatment resulted in marked reduction of inflammatory cell infiltration. Twenty-eight days after injury, similar levels of neointimal proliferation were found in the injured arteries in both groups. CONCLUSIONS: Our results suggested that a high dose of ATIII may influence the sequelae of arterial injury by reducing mural thrombus formation and limiting the inflammatory reaction of the vessel wall without altering the process of vascular remodeling.

Corticosteroid enhances heme oxygenase-1 production by circulating monocytes by up-regulating hemoglobin scavenger receptor and amplifying the receptor-mediated uptake of hemoglobin-haptoglobin complex.

This study examined the relationship between steroid treatment and CD163-mediated downstream pathways linked to inflammatory resolution. Twelve patients referred for congenital heart disease surgery were divided into two groups based on the severity of intravascular hemolysis during cardiopulmonary bypass surgery. Patients with severe intravascular hemolysis were administered haptoglobin during the procedure. Flow cytometry indicated a peak in monocyte CD163 expression on post-operative day 1 in both groups. Enhanced and prolonged heme oxygenase-1 (HO-1) mRNA expression levels were observed in patients who received haptoglobin. Binding of hemoglobin-haptoglobin complex (Hb/Hp) to CD163 resulted in significant induction of HO-1 by peripheral blood mononuclear cells after exposure to dexamethasone prior to culture. This effect was significantly inhibited by anti-CD163 antibody. Our results demonstrated up-regulation of CD163 expression on the monocyte surface by steroid treatment. Steroid treatment was suggested to facilitate CD163-mediated endocytosis of hemoglobin to monocytes/macrophages and thereby induce acceleration of HO-1 synthesis.