Drug-induced Fatal Arrhythmias: Acquired long QT and Brugada Syndromes

Since the early 1990s, the concept of primary “inherited” arrhythmia syndromes or ion channelopathies has evolved rapidly as a result of revolutionary progresses made in molecular genetics. Alterations in genes coding for membrane proteins such as ion channels or their associated proteins responsible for the generation of cardiac action potentials (AP) have been shown to cause specific malfunctions which eventually lead to cardiac arrhythmias. These arrhythmic disorders include congenital long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, short QT syndrome, progressive cardiac conduction disease, etc. Among these, long QT and Brugada syndromes are the most extensively studied, and drugs cause a phenocopy of these two diseases. To date, more than 10 different genes have been reported to be responsible for each syndrome. More recently, it was recognized that long QT syndrome can be latent, even in the presence of an unequivocally pathogenic mutation (silent mutation carrier). Co-existence of other pathological conditions in these silent mutation carriers may trigger a malignant form of ventricular arrhythmia, the so called torsade de pointes (TdP) that is most commonly brought about by drugs. In analogy to the drug-induced long QT syndrome, Brugada type 1 ECG can also be induced or unmasked by a wide variety of drugs and pathological conditions; so physicians may encounter patients with a latent form of Brugada syndrome. Of particular note, Brugada syndrome is frequently associated with atrial fibrillation whose therapeutic agents such as Vaughan Williams class IC drugs can unmask the dormant and asymptomatic Brugada syndrome. This review describes two types of drug-induced arrhythmias: the long QT and Brugada syndromes

Exploring Hidden Atrial Fibrillation in Patients with Type 2 Diabetes Mellitus Admitted to Shinshu University Hospital

Using the event-triggered recorder, SpiderFlash-t AFIB, which allows us to detect asymptomatic or symptomatic arrhythmia, we examined the prevalence of hidden atrial fibrillation (AF) in patients with type 2 diabetes admitted to our hospital. In total, we enrolled 69 patients with type 2 diabetes mellitus admitted to the hospital due to hyperglycemia. Averages of HbA1c, age, duration of the disease, and BMI were 8.9 ％, 64.8 years old, 14.8 years, and 26.0 kg/m2, respectively. Neuropathy, retinopathy, and nephropathy were found in 37 (53.6 ％), 27 (39.1 ％), and 27 (39.1 ％), respectively. Macroangiopathy was found in 13 (18.9 ％). In the first series of surveys where we attached the recorder for 7 days no sustained AF events were found, and only 2 transient events. We attached the recorder for 14 days with 39 subjects in the second series of the survey, and fail to find any AF rhythm. This study is the first attempt to reveal the frequency of hidden AF in diabetes. This finding suggests that screening of atrial fibrillation with SpiderFlash-t AFIB for patients with type 2 diabetic patients hospitalized for glycemic control may not be useful in assessing the prevalence of atrial fibrillation.Article信州医学雑誌 69(2): 75-81(2021)departmental bulletin pape

CD44 standard isoform is involved in maintenance of cancer stem cells of a hepatocellular carcinoma cell line

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Cancer stem cells (CSCs) have attracted attention as a novel therapeutic target for cancer because they play important roles in the development and aggravation of cancer. CD44 is expressed as a standard isoform (CD44s) and several variant isoforms. CD44v is a major isoform expressed on CSCs of a variety of tumors and has been extensively studied. However, HCC tissues dominantly express CD44s, whose function in CSCs remains unclear. In the present study, we investigated the roles of CD44s in CSCs of HCC. Knock-out of the CD44 gene in HuH7 HCC cells on which only CD44s is expressed resulted in decreased spheroid formation and increased drug sensitivity. The expression of CSC marker genes, including CD133 and EpCAM, was significantly downregulated in the spheroids of CD44-deficient cells compared with those in the spheroids of HuH7 cells. In addition, CD44 deficiency impaired antioxidant capacity, concomitant with downregulation of glutathione peroxidase 1 (GPX1) and thioredoxin. Because GPX1 uses the reduced form of glutathione (GSH) to regenerate oxidized cellular components, GSH levels were significantly increased in the CD44-deficient cells. We also found that NOTCH3 and its target genes were downregulated in the spheroids of CD44-deficient cells. NOTCH3 expression in HCC tissues was significantly increased compared with that in adjacent nontumor liver tissues and was correlated with CD44 expression. These results suggest that CD44s is involved in maintenance of CSCs in a HCC cell line, possibly through the NOTCH3 signaling pathway

Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation

Hypokalemia Promotes Late Phase 3 Early Afterdepolarization and Recurrent Ventricular Fibrillation During Isoproterenol Infusion in Langendorff Perfused Rabbit Ventricles

BACKGROUND Hypokalemia and sympathetic activation are commonly associated with electrical storm (ES) in normal and diseased hearts. The mechanisms remain unclear. OBJECTIVE To test the hypothesis that late phase 3 early afterdepolarization (EAD) induced by IKATP activation underlies the mechanisms of ES during isoproterenol infusion and hypokalemia. METHODS Intracellular calcium (Cai) and membrane voltage were optically mapped in 32 Langendorff-perfused normal rabbit hearts. RESULTS Repeated episodes of electrically-induced VF at baseline did not result in spontaneous VF (SVF). During isoproterenol infusion, SVF occurred in 1 of 15 hearts (7%) studied in normal extracellular potassium ([K+]o) (4.5 mmol/L), 3 of 8 hearts (38%) in 2.0 mmol/L [K+]o, 9 of 10 hearts (90%) in 1.5 mmol/L [K+]o, and 7 of 7 hearts (100%) in 1.0 mmol/L [K+]o (P<0.001). Optical mapping showed isoproterenol and hypokalemia enhanced Cai transient duration (CaiTD) and heterogeneously shortened action potential duration (APD) after defibrillation, leading to late phase 3 EAD and SVF. IKATP blocker (glibenclamide, 5 μmol/L) reversed the post-defibrillation APD shortening and suppressed recurrent SVF in all hearts studied despite no evidence of ischemia. Nifedipine reliably prevented recurrent VF when given before, but not after, the development of VF. IKr blocker (E-4031) and small conductance calcium activated potassium channel blocker (apamin) failed to prevent recurrent SVF. CONCLUSION Beta-adrenergic stimulation and concomitant hypokalemia could cause non-ischemic activation of IKATP, heterogeneous APD shortening and prolongation of CaiTD to provoke late phase 3 EAD, triggered activity and recurrent SVF. IKATP inhibition may be useful in managing ES during resistant hypokalemia

Arrhythmogenic Calmodulin Mutations Impede Activation of Small-conductance Calcium-Activated Potassium Current

Background Apamin sensitive small-conductance Ca2+-activated K+ (SK) channels are gated by intracellular Ca2+ through a constitutive interaction with calmodulin. Objective We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels. Methods We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G and F90L). Plasmids encoding either wild type (WT) or mutant calmodulin were transiently transfected into human embryonic kidney (HEK) 293 cells that stably express SK2 channels (SK2 Cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current (IKAS) densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (N=7) or without (N=3) pretreatment with sea anemone toxin (ATX II). Results SK2 cells transfected with WT calmodulin exhibited IKAS density (in pA/pF) of 33.6 [31.4;36.5] (median and confidence interval 25%-75%), significantly higher than that observed for cells transfected with N54I (17.0 [14.0;27.7], p=0.016), F90L (22.6 [20.3;24.3], p=0.011), D96V (13.0 [10.9;15.8], p=0.003), N98S (13.7 [8.8;20.4], p=0.005) and D130G (17.6 [13.8;24.6], p=0.003). The reduction of SK2 current was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular APD80 (from 79.6 ms [63.4-93.3] to 121.8 ms [97.9-127.2], p=0.010) in hearts pre-treated with ATX-II but not in control hearts. Conclusion Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in HEK 293 cells

Novel Variants in the CLCN1, RYR2, and DCTN1 Found in Elderly Japanese Dementia Patients: A Case Series

Dementia has an enormous impact on medical and financial resources in aging societies like Japan. Diagnosis of dementia can be made by physical and mental examinations, imaging tests, and findings of high abnormal proteins in cerebrospinal fluids. In addition, genetic tests can be performed in neurodegenerative diseases such as Alzheimer's disease (AD), frontotemporal dementia (FTD), and Parkinson's disease (PD). In this case series, we presented three cases of dementia with unknown causes who carry novel variants in the genes associated with neurodegenerative diseases. Three patients (Patients 1, 2, and 6) were found by screening 18 dementia patients using a gene panel including 63 genes. The age of onset for Patient 1 was 74 years old, and his father had PD and mother had AD. The age of onset for Patient 2 was 75 years old, and her mother had AD. The age of onset for Patient 6 was 83 years old, and her father, two sisters, and daughter had dementia. The Mini-Mental State Examination produced results of 20, 15, and 22, respectively. The suspected diagnosis by neurological examinations and imaging studies for Patients 1 and 2 was AD, and for Patient 6 was FTD. Patient 1 was treated with donepezil; Patient 2 was treated with donepezil and memantine; and Patient 6 was treated with donepezil, galantamine, and rivastigmine. The three rare variants identified were: CLCN1, encoding a chloride channel, c.2848G>A:p.Glu950Lys (Patient 1); RYR2, encoding a calcium releasing ryanodine receptor, c.13175A>G:p.Lys4392Arg (Patient 2); and DCTN1, encoding a subunit of dynactin, c. 3209G>A:p.Arg1070Gln (Patient 6). The detected variants were interpreted according to the American College of Medical Genetics (ACMG) guidelines. The minor allele frequency for each variant was 0.025%, 0.023%, and 0.0004% in East Asians, respectively. The DCTN1 variant found in Patient 6 might be associated with FTD. Although none of them were previously reported in dementia patients, all variants were classified as variants of unknown significance (VUS). Our report suggests that results of genetic tests in elderly patients with dementia need to be carefully interpreted. Further data accumulation of genotype-phenotype relationships and development of appropriate functional models are warranted

Abnormal Cardiac Repolarization After Seizure Episodes in Structural Brain Diseases: Cardiac Manifestation of Electrical Remodeling in the Brain?

Background: Abnormal cardiac repolarization is observed in patients with epilepsy and can be associated with sudden death. We investigated whether structural brain abnormalities are correlated with abnormal cardiac repolarizations in patients with seizure or epilepsy. Methods and Results: We retrospectively analyzed and compared 12-lead ECG parameters following seizures between patients with and without structural brain abnormalities. A total of 96 patients were included: 33 women (17 with and 16 without brain abnormality) and 63 men (44 with and 19 without brain abnormality). Brain abnormalities included past stroke, chronic hematoma, remote bleeding, tumor, trauma, and postsurgical state. ECG parameters were comparable for heart rate, PR interval, and QRS duration between groups. In contrast, corrected QT intervals evaluated by Fridericia, Framingham, and Bazett formulas were prolonged in patients with brain abnormality compared with those without (women: Fridericia [normal versus abnormal], 397.4±32.7 versus 470.9±48.9; P=0.002; Framingham, 351.0±40.1 versus 406.2±46.1; P=0.002; Bazett, 423.8±38.3 versus 507.7±56.6; P<0.0001; men: Fridericia, 403.8±30.4 versus 471.0±47.1; P<0.0001; Framingham, 342.7±36.4 versus 409.4±45.8; P<0.0001; Bazett, 439.3±38.6 versus 506.2±56.8; P<0.0001). QT dispersion and Tpeak-Tend intervals were comparable between groups. We also observed abnormal ST-segment elevation in 5 patients. Importantly, no patients showed fatal arrhythmias during or after seizures. Conclusions: Our study demonstrated that brain abnormalities can be associated with abnormal cardiac repolarization after seizures, which might be a manifestation of electrophysiological remodeling in the brain

Switching and Emergence of CTL Epitopes in HIV-1 Infection

Background Human Leukocyte Antigen (HLA) class I restricted Cytotoxic T Lymphocytes (CTLs) exert substantial evolutionary pressure on HIV-1, as evidenced by the reproducible selection of HLA-restricted immune escape mutations in the viral genome. An escape mutation from tyrosine to phenylalanine at the 135th amino acid (Y135F) of the HIV-1 nef gene is frequently observed in patients with HLA-A*24:02, an HLA Class I allele expressed in ~70% of Japanese persons. The selection of CTL escape mutations could theoretically result in the de novo creation of novel epitopes, however, the extent to which such dynamic “CTL epitope switching” occurs in HIV-1 remains incompletely known. Results Two overlapping epitopes in HIV-1 nef, Nef126-10 and Nef134-10, elicit the most frequent CTL responses restricted by HLA-A*24:02. Thirty-five of 46 (76%) HLA-A*24:02-positive patients harbored the Y135F mutation in their plasma HIV-1 RNA. Nef codon 135 plays a crucial role in both epitopes, as it represents the C-terminal anchor for Nef126-10 and the N-terminal anchor for Nef134-10. While the majority of patients with 135F exhibited CTL responses to Nef126-10, none harboring the “wild-type” (global HIV-1 subtype B consensus) Y135 did so, suggesting that Nef126-10 is not efficiently presented in persons harboring Y135. Consistent with this, peptide binding and limiting dilution experiments confirmed F, but not Y, as a suitable C-terminal anchor for HLA-A*24:02. Moreover, experiments utilizing antigen specific CTL clones to recognize endogenously-expressed peptides with or without Y135F indicated that this mutation disrupted the antigen expression of Nef134-10. Critically, the selection of Y135F also launched the expression of Nef126-10, indicating that the latter epitope is created as a result of escape within the former. Conclusions Our data represent the first example of the de novo creation of a novel overlapping CTL epitope as a direct result of HLA-driven immune escape in a neighboring epitope. The robust targeting of Nef126-10 following transmission (or in vivo selection) of HIV-1 containing Y135F may explain in part the previously reported stable plasma viral loads over time in the Japanese population, despite the high prevalence of both HLA-A*24:02 and Nef-Y135F in circulating HIV-1 sequences

Compound heterozygous SCN5A gene mutations in asymptomatic Brugada syndrome child

BACKGROUND. Loss-of-function mutations in the SCN5A gene, encoding the cardiac Nav1.5 sodium channel, have been previously associated with Brugada syndrome (BrS). Despite the low prevalence of the disease, we identified a patient carrying two SCN5A mutations. We aimed at establishing a correlation between genotype, clinical phenotype and in vitro sodium current.MATERIALS AND METHODS. A 3 years old boy presented with right bundle branch block and ST-segment elevation. Genetic analysis and electrophysiology studies in transfected HEK293 cells were performed to identify possibly disease-causing variants and assess their effect on sodium channel function.RESULTS. Two SCN5A variants were identified: a new frameshift deletion causing premature truncation of the putative protein (c.3258_3261del4) and a missense substitution (p.F1293S). In vitro studies revealed that the truncated mutant did not produce functional channels and decreased total sodium current when co-expressed with p.F1293S channels compared to p.F1293S alone. In addition, p.F1293S channels presented with a steep slope of steady-state activation voltage-dependency, which was shifted towards more positive potentials by the co-expression with the truncated channel. p.F1293S channels also showed shift towards more positive potentials of the steady-state inactivation both alone and co-expressed with the deletion mutant.CONCLUSIONS. Our data identified a severe reduction of sodium channel current associated with two distinct SCN5A changes. However, all mutation carriers were asymptomatic and BrS ECG was observed only transiently in the compound heterozygous subject. These observations underline the difficulty of genotype/phenotype correlations in BrS patients and support the idea of a polygenic disorder, where different mutations and variants can contribute to the clinical phenotype