Masaharu Akao

A Novel SCN5A Gain-of-Function Mutation M1875T Associated With Familial Atrial Fibrillation

OBJECTIVES This study describes a novel heterozygous gain-of-function mutation in the cardiac sodium (Na+) channel gene, SCN5A, identified in a Japanese family with lone atrial fibrillation (AF). BACKGROUND SCN5A mutations have been associated with a variety of inherited arrhythmias, but the gain-of-function type modulation in SCN5A is associated with only 1 phenotype, long-QT syndrome type 3 (LQTS3). METHODS We studied a Japanese family with autosomal dominant hereditary AF, multiple members of which showed an onset of AF or frequent premature atrial contractions at a young age. RESULTS The 31-year-old proband received radiofrequency catheter ablation, during which time numerous ectopic firings and increased excitability throughout the right atrium were documented. Mutational analysis identified a novel missense mutation, M1875T, in SCN5A. Further investigations revealed the familial aggregation of this mutation in all of the affected individuals. Functional assays of the M1875T Na(+) channels using a whole-cell patch-clamp demonstrated a distinct gain-of-function type modulation; a pronounced depolarized shift (+16.4 mV) in V(1/2) of the voltage dependence of steady-state inactivation; and no persistent Na+ current, which is a defining mechanism of LQTS3. These biophysical features of the mutant channels are potentially associated with increased atrial excitability and normal QT interval in all of the affected individuals. CONCLUSIONS We identified a novel SCN5A mutation associated with familial AF. The mutant channels displayed a gain-of-function type modulation of cardiac Na+ channels, which is a novel mechanism predisposing to increased atrial excitability and familial AF. This is a new phenotype resulting from the SCN5A gain-of-function mutations and is distinct from LQTS3.

D85N, a KCNE1 Polymorphism, Is a Disease-Causing Gene Variant in Long QT Syndrome

OBJECTIVES This study aims to address whether D85N, a KCNE1 polymorphism, is a gene variant that causes long QT syndrome (LQTS) phenotype. BACKGROUND KCNE1 encodes the beta-subunit of cardiac voltage-gated K(+) channels and causes LQTS, which is characterized by the prolongation of the QT interval and torsades de pointes, a lethal arrhythmia. D85N, a KCNE1 polymorphism, is known to be a functional variant associated with drug-induced LQTS. METHODS In order to elucidate the prevalence and clinical significance of this polymorphism, we performed genetic screening in 317 LQTS probands. For comparison, we examined its presence in 496 healthy control subjects. We also conducted biophysical assays for the D85N variant in mammalian cells. RESULTS The allele frequency for D85N carriers was 0.81% in healthy people. In contrast, among LQTS probands, there were 1 homozygous and 23 heterozygous carriers (allele frequency 3.9%). Seven of 23 heterozygous carriers had additional mutations in LQTS-related genes, and 3 female subjects had documented factors predisposing to the symptom. After excluding these probands, the D85N prevalence was significantly higher compared with control subjects (allele frequency 2.1%, p < 0.05). In a heterologous expression study with Chinese hamster ovarian cells, KCNE1-D85N was found to exert significant loss-of-function effects on both KCNQ1- and KCNH2-encoded channel currents. CONCLUSIONS The KCNE1-D85N polymorphism was significantly more frequent in our LQTS probands. The functional variant is a disease-causing gene variant of LQTS phenotype that functions by interacting with KCNH2 and KCNQ1. Since its allele frequency was approximately 1% among control individuals, KCNE1-D85N may be a clinically important genetic variant.

Current status of clinical background of patients with atrial fibrillation in a community-based survey:The Fushimi AF Registry

BACKGROUND Atrial fibrillation (AF) increases the risks of stroke and death, and the prevalence of AF is increasing significantly. Until recently, warfarin was the only oral anticoagulant for stroke prevention, but novel anticoagulants are now under development. METHODS AND RESULTS The Fushimi AF Registry is a community-based survey of AF patients. We aimed to enroll all of the AF patients in Fushimi-ku, which is located at the southern end of the city of Kyoto. Fushimi-ku is densely populated with a total population of 283,000, and is assumed to represent a typical urban community in Japan. On the basis of the general prevalence of AF in the Japanese (0.6%), we estimated the total number of AF patients as 1700. A total of 76 institutions, a large proportion of which were private clinics, participated in the study. At present, we have enrolled 3183 patients from March 2011 to June 2012 (approximately 1.12% of total population). The mean age was 74.2±11.0 years, and 59.3% of subjects were male. The mean body weight was 58.5±13.2 kg, and the proportions with a body weight of less than 50 kg and 60 kg were 25.7% and 55.0%, respectively. The type of AF was paroxysmal in 46.0%, persistent in 7.3%, and permanent in 46.7%. Major co-existing diseases were hypertension (60.6%), heart failure (27.9%), diabetes (23.2%), stroke (19.4%), coronary artery disease (15.0%), myocardial infarction (6.4%), dyslipidemia (42.4%), and chronic kidney disease (26.4%). The mean CHADS2 score was 2.09±1.35: 0 in 11.8% of patients, 1 in 27.1%, and 2 in 29.1%. Warfarin was prescribed in only 48.5% of patients, whereas anti-platelet drugs, mainly aspirin, were prescribed for more than 30% of the patients. CONCLUSIONS The Fushimi AF Registry provides a unique snapshot of current AF management in an urban community in Japan.

Long QT syndrome with compound mutations is associated with a more severe phenotype: A Japanese multicenter study

BACKGROUND Long QT syndrome (LQTS) can be caused by mutations in the cardiac ion channels. Compound mutations occur at a frequency of 4% to 11% among genotyped LQTS cases. OBJECTIVE The purpose of this study was to determine the clinical characteristics and manner of onset of cardiac events in Japanese patients with LQTS and compound mutations. METHODS Six hundred three genotyped LQTS patients (310 probands and 293 family members) were divided into two groups: those with a single mutation (n = 568) and those with two mutations (n = 35). Clinical phenotypes were compared between the two groups. RESULTS Of 310 genotyped probands, 26 (8.4%) had two mutations in the same or different LQTS-related genes (compound mutations). Among the 603 LQTS patients, compound mutation carriers had significantly longer QTc interval (510 ± 56 ms vs 478± 53 ms, P = .001) and younger age at onset of cardiac events (10 ± 8 years vs 18 ± 16 years, P = .043) than did single mutation carriers. The incidence rate of cardiac events before age 40 years and use of beta-blocker therapy among compound mutation carriers also were different than in single mutation carriers. Subgroup analysis showed more cardiac events in LQTS type 1 (LQT1) and type 2 (LQT2) compound mutations compared to single LQT1 and LQT2 mutations. CONCLUSION Compound mutation carriers are associated with a more severe phenotype than single mutation carriers.

Latent Genetic Backgrounds and Molecular Pathogenesis in Drug-induced Long QT Syndrome

Background—Drugs with IKr-blocking action cause secondary long-QT syndrome. Several cases have been associated with mutations of genes coding cardiac ion channels, but their frequency among patients affected by drug-induced long-QT syndrome (dLQTS) and the resultant molecular effects remain unknown. Methods and Results—Genetic testing was carried out for long-QT syndrome–related genes in 20 subjects with dLQTS and 176 subjects with congenital long-QT syndrome (cLQTS); electrophysiological characteristics of dLQTS-associated mutations were analyzed using a heterologous expression system with Chinese hamster ovary cells together with a computer simulation model. The positive mutation rate in dLQTS was similar to cLQTS (dLQTS versus cLQTS, 8 of 20 [40%] versus 91 of 176 [52%] subjects, P=0.32). The incidence of mutations was higher in patients with torsades de pointes induced by nonantiarrhythmic drugs than by antiarrhythmic drugs (antiarrhythmic versus others, 3 of 14 [21%] versus 5 of 6 [83%] subjects, P<0.05). When reconstituted in Chinese hamster ovary cells, KCNQ1 and KCNH2 mutant channels showed complex gating defects without dominant negative effects or a relatively mild decreased current density. Drug sensitivity for mutant channels was similar to that of the wild-type channel. With the Luo-Rudy simulation model of action potentials, action potential durations of most mutant channels were between those of wild-type and cLQTS. Conclusions—dLQTS had a similar positive mutation rate compared with cLQTS, whereas the functional changes of these mutations identified in dLQTS were mild. When IKr-blocking agents produce excessive QT prolongation (dLQTS), the underlying genetic background of the dLQTS subject should also be taken into consideration, as would be the case with cLQTS; dLQTS can be regarded as a latent form of long-QT syndrome.

Real-Time 2-Photon Imaging of Mitochondrial Function in Perfused Rat Hearts Subjected to Ischemia/Reperfusion

Background— Mitochondria play pivotal roles in cell death; the loss of mitochondrial membrane potential (&Dgr;&PSgr;m) is the earliest event that commits the cell to death. Here, we report novel real-time imaging of &Dgr;&PSgr;m in individual cardiomyocytes within perfused rat hearts using 2-photon laser-scanning microscopy, which has unique advantages over conventional confocal microscopy: greater tissue penetration and lower tissue toxicity. Methods and Results— The Langendorff-perfused rat heart was loaded with a fluorescent indicator of &Dgr;&PSgr;m, tetramethylrhodamine ethyl ester. Tetramethylrhodamine ethyl ester was excited with an 810-nm line of a Ti:sapphire laser, and its fluorescence in the heart cells was successfully visualized up to ≈50 &mgr;m from the epicardial surface. Taking advantage of this system, we monitored the spatiotemporal changes of &Dgr;&PSgr;m in response to ischemia/reperfusion at the subcellular level. No-flow ischemia caused progressive &Dgr;&PSgr;m loss and a more prominent &Dgr;&PSgr;m loss on reperfusion. During ischemia/reperfusion, cells maintained a constant &Dgr;&PSgr;m for the cell-to-cell specific period of latency, followed by a rapid, complete, and irreversible &Dgr;&PSgr;m loss, and this process did not affect the neighboring cells. Within a cell, &Dgr;&PSgr;m loss was initiated in a particular area of mitochondria and rapidly propagated along the longitudinal axis. These spatiotemporal changes in &Dgr;&PSgr;m resulted in marked cellular and subcellular heterogeneity of mitochondrial function. Ischemic preconditioning reduced the number of cells undergoing &Dgr;&PSgr;m loss, whereas cyclosporin A partially inhibited &Dgr;&PSgr;m loss in each cell. Conclusions— Investigation of cellular responses in the natural environment will increase knowledge of ischemia/reperfusion injury and provide deeper insights into antiischemia/reperfusion therapy that targets mitochondria.

Coordinate interaction between ATP-sensitive K+ channel and Na+, K+-ATPase modulates ischemic preconditioning

BACKGROUND We reported that digoxin abolishes the infarct size (IS)-limiting effect of ischemic preconditioning (IPC). Because ATP-sensitive K+ (KATP) channels are involved in IPC, we studied whether Na+,K+-ATPase and KATP channels functionally interact, thereby modulating IPC. METHODS AND RESULTS Rabbits received 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. IPC was elicited by 5 minutes of occlusion followed by 10 minutes of reperfusion. The IS, expressed as a percentage of the area at risk, was 40.2+/-2.8% in control and 39.8+/-5.0% in digoxin pretreatment rabbits. Both IPC and pretreatment with cromakalim, a KATP channel opener, reduced IS to 11.8+/-1.8% and 13.4+/-2.6% (P<0. 05 versus control). Digoxin abolished the reduction in IS induced by IPC (33.5+/-3.3%), whereas it did not change that induced by cromakalim (18.8+/-3.0%). In patch-clamp experiments, digoxin was found to inhibit the opening of KATP channels in single ventricular myocytes in which ATP depletion had been induced by metabolic stress. In contrast, digoxin had little effect on the channel opening induced by cromakalim. Moreover, the inhibitory action of digoxin on channel activities was dependent on subsarcolemmal ATP concentration. CONCLUSIONS The IS-limiting effect of IPC is modulated by an interaction between KATP channels and Na+,K+-ATPase through subsarcolemmal ATP.

A novel gain-of-function KCNJ2 mutation associated with short-QT syndrome impairs inward rectification of Kir2.1 currents

AIMS Short-QT syndrome (SQTS) is a recently recognized disorder associated with atrial fibrillation (AF) and sudden death due to ventricular arrhythmias. Mutations in several ion channel genes have been linked to SQTS; however, the mechanism remains unclear. This study describes a novel heterozygous gain-of-function mutation in the inward rectifier potassium channel gene, KCNJ2, identified in SQTS. METHODS AND RESULTS We studied an 8-year-old girl with a markedly short-QT interval (QT = 172 ms, QTc = 194 ms) who suffered from paroxysmal AF. Mutational analysis identified a novel heterozygous KCNJ2 mutation, M301K. Functional assays displayed no Kir2.1 currents when M301K channels were expressed alone. However, co-expression of wild-type (WT) with M301K resulted in larger outward currents than the WT at more than -30 mV. These results suggest a gain-of-function type modulation due to decreased inward rectification. Furthermore, we analysed the functional significance of the amino acid charge at M301 (neutral) by changing the residue. As with M301K, in M301R (positive), the homozygous channels were non-functional, whereas the heterozygous channels demonstrated decreased inward rectification. Meanwhile, the currents recorded in M301A (neutral) showed normal inward rectification under both homo- and heterozygous conditions. Heterozygous overexpression of WT and M301K in neonatal rat ventricular myocytes exhibited markedly shorter action potential durations than the WT alone. CONCLUSION In this study, we identified a novel KCNJ2 gain-of-function mutation, M301K, associated with SQTS. Functional assays revealed no functional currents in the homozygous channels, whereas impaired inward rectification demonstrated under the heterozygous condition resulted in larger outward currents, which is a novel mechanism predisposing SQTS.

Endothelin-1–Dependent Nuclear Factor of Activated T Lymphocyte Signaling Associates With Transcriptional Coactivator p300 in the Activation of the B Cell Leukemia-2 Promoter in Cardiac Myocytes

Endothelin-1 (ET-1) is a potent survival factor that protects cardiac myocytes from apoptosis. ET-1 induces cardiac gene transcription and protein expression of antiapoptotic B cell leukemia-2 (bcl-2) in a calcineurin-dependent manner. A cellular target of adenovirus early region 1A (E1A) oncoprotein, p300 also activates bcl-2 transcription in cardiac myocytes and is required for their survival. p300 acts as a calcineurin-regulated nuclear factors of activated T lymphocytes (NFATc), downstream targets of calcineurin. In addition, the bcl-2 promoter contains multiple NFAT consensus sequences. These findings prompted us to investigate the role of NFATc in ET-1–dependent and p300-dependent bcl-2 transcription in cardiac myocytes. In primary cardiac myocytes prepared from neonatal rats, mutation of 2 NFAT sites within the bcl-2 promoter completely abolished the ET-1– and p300-induced increases in the activity of this promoter. We show here that p300 markedly potentiates the binding of NFATc1 to the bcl-2 NFAT element by interacting with NFATc1 in an E1A-dependent manner. On the other hand, stimulation of cardiac myocytes with ET-1 causes nuclear translocation of NFATc1, which interacts with p300 and increases DNA binding. Expression of E1A did not change the cardiac nuclear localization of NFATc1 but blocked its interaction with p300, DNA binding, and bcl-2 promoter activation. These findings suggest that ET-1–dependent NFATc signaling associates with p300 in the transactivation of bcl-2 gene in cardiac myocytes.

Incidence of Ischemic Stroke in Japanese Patients With Atrial Fibrillation Not Receiving Anticoagulation Therapy

BACKGROUND The incidence rate of ischemic stroke in Japanese non-valvular atrial fibrillation (NVAF) patients without anticoagulation therapy remains unclear. METHODS AND RESULTS We performed a pooled analysis of 3,588 patients from the Shinken Database (n=1,099), J-RHYTHM Registry (n=1,002), and Fushimi AF Registry (n=1,487) to determine the incidence rate of ischemic stroke in Japanese NVAF patients without anticoagulation therapy. Average patient age was 68.1 years. During the follow-up period (total, 5,188 person-years; average, 1.4 years), 69 patients suffered from ischemic stroke (13.3 per 1,000 person-years; 95% confidence intervals [CI]: 10.5-16.8). The incidence rates of ischemic stroke were 5.4, 9.3, and 24.7 per 1,000 person-years and 5.3, 5.5, and 18.4 per 1,000 person-years in patients with low (0), intermediate (1), and high (≥ 2) CHADS2 and CHA2DS2-VASc scores, respectively. History of ischemic stroke or transient ischemic attack (hazard ratio [HR], 3.25; 95% CI: 1.86-5.67), age ≥ 75 years (HR, 2.31; 95% CI: 1.18-4.52), and hypertension (HR, 1.69; 95% CI: 1.01-2.86) were independent risk factors for ischemic stroke. CONCLUSIONS A low incidence rate of ischemic stroke was observed in Japanese NVAF patients except for those with CHADS2 score ≥ 2. In this pooled analysis, history of ischemic stroke or transient ischemic attack, advanced age, and hypertension were identified as independent risk factors for ischemic stroke.