Chih-Chieh Yu

Risk factors and incidence of ischemic stroke in Taiwanese with nonvalvular atrial fibrillation-- a nation wide database analysis.

BACKGROUND Atrial fibrillation (AF) is a risk factor for ischemic stroke. Stroke-prevention strategies based on risk schemes have been developed but most of the data are from western people. Our goal is to investigate the risk factors of ischemic stroke in Taiwanese with AF in a nation-wide database. METHODS A universal national health insurance (NHI) program has been implemented in Taiwan since 1995. We used system sampling database from 1997 to 2008 with a total of 1,000,000 subjects. By using ambulatory and inpatient claim data, we included subjects with AF and were above 20 years old. We excluded those who had ever taken warfarin or aspirin or had valvular heart diseases. RESULT A total of 7920 patients (3633 women, 4287 men) were included in the final analyses. Cox regression analysis showed that the risk factors for ischemic stroke were age (OR=1.338 for age 65-74 years vs. age 20-64 years, P=0.014; OR=1.652 for age over 75 years vs. age 20-64 years, P<0.001), hypertension (HTN) (OR=2.656, P<0.001), diabetes mellitus (DM) (OR=1.341, P=0.005), heart failure (OR=1.611, P<0.001), previous ischemic stroke or transient ischemic accident (TIA) (OR=2.752, P<0.001) and peripheral arterial disease (PAD) (OR=1.814, P=0.006). The gender, coronary artery disease, history of myocardial infarction and chronic renal insufficiency were not associated with ischemic stroke. The rate of ischemic stroke was much lower in current cohort as compared with that in whites. Frequent used risk schemes including CHADS₂ and CHA₂DS₂-VASC had comparable but only limited ability to predict ischemic stroke in subjects with AF. CONCLUSION Compare with western people, hypertension plays a more important role in ischemic stroke in Taiwanese with AF and the incidence is lower. A substantial number of ischemic strokes cannot be accurately predicted by current risk schemes.

Increased Expression of Mineralocorticoid Receptor in Human Atrial Fibrillation and a Cellular Model of Atrial Fibrillation

OBJECTIVES This study was designed to evaluate the status of steroidogenesis proteins and de novo synthesis of aldosterone in the atrium, and relationships of these factors to atrial fibrillation (AF). BACKGROUND The role of mineralocorticoid in the pathogenesis of AF is unknown. METHODS We studied atrial expression of steroidogenesis proteins and aldosterone level in patients with and without AF, and in HL-1 atrial myocytes. We also investigated the electrophysiologic effects and signal transduction of aldosterone on atrial myocytes. RESULTS We found basal expressions of mineralocorticoid receptors (MRs), glucocorticoid receptors, and 11-beta-hydroxysteroid dehydrogenase type 2 (11bHSD2) but not 11-beta-hydroxylase (CYP11B1) or aldosterone synthase (CYP11B2) in human atria and HL-1 myocytes. There was no significant difference of mean atrial aldosterone level between patients with AF and those with normal sinus rhythm. However, patients with AF had a significantly higher atrial MR expression compared with those with normal sinus rhythm (1.73 +/- 0.24-fold, p < 0.001). Using mouse HL-1 atrial myocytes as a cellular AF model, we found that rapid depolarization increased MR expression (1.97 +/- 0.72-fold, p = 0.008) through a calcium-dependent mechanism, thus augmenting the genomic effect of aldosterone signaling as evaluated by MR reporter. Aldosterone increased intracellular oxidative stress through a nongenomic pathway, which was attenuated by nicotinamide adenine dinucleotide phosphate oxidase inhibitor diphenyleneiodonium, but not by MR-blockade spironolactone. Aldosterone increased expression of the alpha-1G and -1H subunits of the T-type calcium channel and thus increased the T-type calcium current (-13.6 +/- 2.9 pA/pF vs. -4.5 +/- 1.6 pA/pF, p < 0.01) and the intracellular calcium load through a genomic pathway, which were attenuated by spironolactone, but not by diphenyleneiodonium. CONCLUSIONS Expression of MR increased in AF, thus augmenting the genomic effects of aldosterone. Aldosterone induced atrial ionic remodeling and calcium overload through a genomic pathway, which was attenuated by spironolactone. These results suggest that aldosterone may play a role in AF electrical remodeling and provide insight into the treatment of AF with MR blockade.

Mechanical Stretch of Atrial Myocyte Monolayer Decreases Sarcoplasmic Reticulum Calcium Adenosine Triphosphatase Expression and Increases Susceptibility to Repolarization Alternans

OBJECTIVES The purpose of this study was to investigate the effect of stretch (the major risk factor for atrial fibrillation [AF]) on spatial and temporal alternations of action potential duration (APD-ALT) and calcium transient in cultured atrial myocyte monolayer. BACKGROUND How rapid firings or premature beats trigger AF is not completely understood. Discordant repolarization alternans, characterized by simultaneous prolongation and shortening of APD in different myocardial regions, is central to the genesis of ventricular fibrillation. We hypothesized that repolarization alternans also is central to the initiation of multiple re-entry circuits and AF. METHODS Confluent HL-1 atrial myocyte monolayer with spontaneous depolarization was cultured in silicone membrane and subjected to mechanical stretch. Rapid field pacing was used to induce alternans. A high-resolution dual optical mapping system was used to record action potentials and calcium transients. RESULTS High-rate pacing induced APD-ALT and calcium transient in atrial myocyte monolayer. Mechanical stretch significantly decreased the thresholds for APD-ALT and calcium transient. Mechanical stretch decreased the expression of sarcoplasmic reticulum adenosine triphosphatase 2, and thus slower calcium reuptake kinetics, which was responsible for the susceptibility to alternans. Mechanical stretch did not alter the APD restitution kinetics. Mechanical stretch also enhanced spatially discordant alternans. Overexpression of sarcoplasmic reticulum adenosine triphosphatase 2 reversed all the effects of stretch on susceptibility to alternans. In intact atrium, mechanical stretch also enhanced discordant alternans. CONCLUSIONS Mechanical stretch increased the susceptibility to alternans in atrial myocytes, which may explain the susceptibility to AF in conditions of atrial stretch, such as mitral valvular heart disease, heart failure, and hypertension.

In-vitro recording of adult zebrafish heart electrocardiogram — A platform for pharmacological testing

BACKGROUND Recently zebrafish has become a powerful vertebrate model system for cardiac arrhythmia and another advantage is that the heart could be rapidly excised for in-vitro electrophysiological recording. METHODS We made direct in-vitro recordings of adult zebrafish heart ECG using the microelectrode and tested the effects of various drugs on zebrafish heart ECG. RESULTS The QT interval change in the ECG was verified by optically mapped action potential duration (APD). The mean in-vitro heart rate (HR) of the adult zebrafish was 118 ± 22 beats/min. The mean QT interval was 312 ± 36 ms and mean HR corrected QT interval (QTc) 439 ± 39 ms. The mean optically mapped APD was 308 ± 30 ms, which was close to the QT interval in ECG (p=0.815). We found that sympathomimetic drug isoproterenol increased HR, whereas L-type calcium channel blocker verapamil decreased HR. Sodium channel blocker quinidine and L-type calcium channel activator BayK8644 prolonged QTc interval in a dose-dependent manner (515 ± 24 ms and 519 ± 27 ms, respectively, both p<0.01). The APD was also prolonged accordingly. Both rapidly and slowly activating delayed rectifier potassium channel (IKr and IKs) blockers E4031 and chromanol 293B, respectively, also prolonged QTc interval in a dose-dependent manner (523 ± 25 ms and 529 ± 27 ms, respectively, both p<0.01). Quinidine, E4031 and chromanol 293B also decreased HR. CONCLUSIONS Direct in-vitro recording of adult zebrafish heart is an efficient platform for test of drugs which exert electrophysiological effects on cardiomyocytes, and perturbation of ionic channels that are responsible for human long QT syndrome also modulates QT interval in adult zebrafish heart.

Arrhythmogenic Right Ventricular Dysplasia: Clinical Characteristics and Identification of Novel Desmosome Gene Mutations

BACKGROUND/PURPOSE Desmosome gene mutations have been reported in patients with arrhythmogenic right ventricular dysplasia (ARVD). However, there are hardly any genetic studies in Asians. We studied the clinical characteristics, cardiac manifestations and desmosome gene mutations in ARVD patients in Taiwan. METHODS Medical records of five ARVD patients were reviewed and genomic DNA was obtained from peripheral blood samples. Mutation screening in desmoplakin (DSP), plakophilin-2, desmoglein-2 (DSG2) and desmocollin-2 genes was performed using polymerase chain reaction and DNA sequencing techniques. RESULTS Among the five patients, three presented with palpitations followed by loss of consciousness, and the other two had palpitations or chest tightness without loss of consciousness. Electrocardiogram (ECG), magnetic resonance imaging and signal averaged ECG results were similar to those reported in Western countries. Mutations in the desmosome genes were identified in four of the five patients (three with a DSG2 mutation and one with a DSP mutation). Five gene mutations were noted in four patients and all mutations were novel (one patient had a DSG2 double mutation). The mutation types were missense in four and splicing mutation in one. CONCLUSION Patients with ARVD in Taiwan had similar clinical and cardiac manifestations as reported in the Western literature. More than half of the patients had desmosome gene mutations.

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background Apamin sensitive potassium current (I KAS), carried by the type 2 small conductance Ca2+-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles. Objective To test the hypothesis that amiodarone inhibits I KAS in human embryonic kidney 293 (HEK-293) cells. Methods We used the patch-clamp technique to study I KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration. Results Amiodarone inhibited IKAS in a dose-dependent manner (IC50, 2.67±0.25 µM with 1 µM intrapipette Ca2+). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of IKAS induced with 1 µM intrapipette Ca2+ (n = 3). IKAS inhibition by amiodarone was not voltage-dependent, but was Ca2+-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I KAS induced with 1 µM Ca2+ (n = 4), and 16.4±4.9% with 250 nM Ca2+ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca2+ dependent inhibition of I KAS. Conclusion Both amiodarone and desethylamiodarone inhibit I KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca2+ concentration. SK2 current inhibition may in part underlie amiodarones effects in preventing electrical storm in failing ventricles.

Mutation spectrum of the fibrillin‐1 (FBN1) gene in Taiwanese patients with Marfan syndrome

The aim of this study was to establish a national database of mutations in the fibrillin‐1 (FBN1) gene that cause Marfan syndrome (MFS) in the Taiwanese population. In this study, we screened 294 patients from 157 families for the presence of FBN1 mutations using polymerase chain reaction/ denaturing high performance liquid chromatography (PCR/DHPLC). We identified 56 mutations in 62 of the 157 (40%) families including 49 single‐base substitutions (36 missense mutations, seven nonsense mutations, and six splicing sites), one small insertion, four small deletions, one small indel (insertion and deletion), and one exonic deletion (Exon 36). When family history was taken into consideration, the mutation detection rate rose to 91% (29 of 32). We further investigated the phenotypic data and found that one third (47 of 157) of the families fit the Ghent criteria for MFS. Based on that data, the mutation rate was 98% (46/47). That finding implies that family history and the Ghent criteria play a more important role than clinical manifestations in establishing a clinical diagnosis of Marfan syndrome. Among the 56 mutations found in this study, 40 (71%) have not been registered in the Human Gene Mutation Database (HGMD) or in the Universal Mutation Database (UMD). This is the first study of the mutation spectrum of MFS in a cohort of patients in Taiwan. The database is expected to considerably improve genetic counseling for and medical care of MFS families.

Disease-Targeted Sequencing of Ion Channel Genes identifies de novo mutations in Patients with Non-Familial Brugada Syndrome

Brugada syndrome (BrS) is one of the ion channelopathies associated with sudden cardiac death (SCD). The most common BrS-associated gene (SCN5A) only accounts for approximately 20–25% of BrS patients. This study aims to identify novel mutations across human ion channels in non-familial BrS patients without SCN5A variants through disease-targeted sequencing. We performed disease-targeted multi-gene sequencing across 133 human ion channel genes and 12 reported BrS-associated genes in 15 unrelated, non-familial BrS patients without SCN5A variants. Candidate variants were validated by mass spectrometry and Sanger sequencing. Five de novo mutations were identified in four genes (SCNN1A, KCNJ16, KCNB2, and KCNT1) in three BrS patients (20%). Two of the three patients presented SCD and one had syncope. Interestingly, the two patients presented with SCD had compound mutations (SCNN1A:Arg350Gln and KCNB2:Glu522Lys; SCNN1A:Arg597* and KCNJ16:Ser261Gly). Importantly, two SCNN1A mutations were identified from different families. The KCNT1:Arg1106Gln mutation was identified in a patient with syncope. Bioinformatics algorithms predicted severe functional interruptions in these four mutation loci, suggesting their pivotal roles in BrS. This study identified four novel BrS-associated genes and indicated the effectiveness of this disease-targeted sequencing across ion channel genes for non-familial BrS patients without SCN5A variants.

Small Conductance Calcium-Activated Potassium Current Is Important in Transmural Repolarization of Failing Human Ventricles

Background—The transmural distribution of apamin-sensitive small conductance Ca2+-activated K+ (SK) current (IKAS) in failing human ventricles remains unclear. Methods and Results—We optically mapped left ventricular wedge preparations from 12 failing native hearts and 2 rejected cardiac allografts explanted during transplant surgery. We determined transmural action potential duration (APD) before and after 100 nmol/L apamin administration in all wedges and after sequential administration of apamin, chromanol, and E4031 in 4 wedges. Apamin prolonged APD from 363 ms (95% confidence interval [CI], 341–385) to 409 (95% CI, 385–434; P<0.001) in all hearts, and reduced the transmural conduction velocity from 36 cm/s (95% CI, 30–42) to 32 cm/s (95% CI, 27–37; P=0.001) in 12 native failing hearts at 1000 ms pacing cycle length (PCL). The percent APD prolongation is negatively correlated with baseline APD and positively correlated with PCL. Only 1 wedge had M-cell islands. The percentages of APD prolongation in the last 4 hearts at 2000 ms PCL after apamin, chromanol, and E4031 were 9.1% (95% CI, 3.9–14.2), 17.3% (95% CI, 3.1–31.5), and 35.9% (95% CI, 15.7–56.1), respectively. Immunohistochemical staining of subtype 2 of SK protein showed increased expression in intercalated discs of myocytes. Conclusions—SK current is important in the transmural repolarization in failing human ventricles. The magnitude of IKAS is positively correlated with the PCL, but negatively correlated with APD when PCL is fixed. There is abundant subtype 2 of SK protein in the intercalated discs of myocytes.

Apamin does not inhibit human cardiac Na+current, L-type Ca2+current or other major K+currents

Background Apamin is commonly used as a small-conductance Ca2+-activated K+ (SK) current inhibitor. However, the specificity of apamin in cardiac tissues remains unclear. Objective To test the hypothesis that apamin does not inhibit any major cardiac ion currents. Methods We studied human embryonic kidney (HEK) 293 cells that expressed human voltage-gated Na+, K+ and Ca2+ currents and isolated rabbit ventricular myocytes. Whole-cell patch clamp techniques were used to determine ionic current densities before and after apamin administration. Results Ca2+ currents (CACNA1c+CACNB2b) were not affected by apamin (500 nM) (data are presented as median [25th percentile;75th percentile] (from –16 [–20;–10] to –17 [–19;–13] pA/pF, P = NS), but were reduced by nifedipine to –1.6 [–3.2;–1.3] pA/pF (p = 0.008). Na+ currents (SCN5A) were not affected by apamin (from –261 [–282;–145] to –268 [–379;–132] pA/pF, P = NS), but were reduced by flecainide to –57 [–70;–47] pA/pF (p = 0.018). None of the major K+ currents (I Ks, I Kr, I K1 and I to) were inhibited by 500 nM of apamin (KCNQ1+KCNE1, from 28 [20]; [37] to 23 [18]; [32] pA/pF; KCNH2+KCNE2, from 28 [24]; [30] to 27 [24]; [29] pA/pF; KCNJ2, from –46 [–48;–40] to –46 [–51;–35] pA/pF; KCND3, from 608 [505;748] to 606 [454;684]). Apamin did not inhibit the I Na or I CaL in isolated rabbit ventricular myocytes (I Na, from –67 [–75;–59] to –68 [–71;–59] pA/pF; I CaL, from –16 [–17;–14] to –14 [–15;–13] pA/pF, P = NS for both). Conclusions Apamin does not inhibit human cardiac Na+ currents, L-type Ca2+ currents or other major K+ currents. These findings indicate that apamin is a specific SK current inhibitor in hearts as well as in other organs.