Kentaro Takahashi

Nitric oxide-dependent modulation of the delayed rectifier K+ current and the L-type Ca2+ current by ginsenoside Re, an ingredient of Panax ginseng, in guinea-pig cardiomyocytes.

Ginsenoside Re, a major ingredient of Panax ginseng, protects the heart against ischemia–reperfusion injury by shortening action potential duration (APD) and thereby prohibiting influx of excessive Ca2+. Ginsenoside Re enhances the slowly activating component of the delayed rectifier K+ current (IKs) and suppresses the L‐type Ca2+ current (ICa,L), which may account for APD shortening. We used perforated configuration of patch‐clamp technique to define the mechanism of enhancement of IKs and suppression of ICa,L by ginsenoside Re in guinea‐pig ventricular myocytes. S‐Methylisothiourea (SMT, 1 μM), an inhibitor of nitric oxide (NO) synthase (NOS), and N‐acetyl‐L‐cystein (LNAC, 1 mM), an NO scavenger, inhibited IKs enhancement. Application of an NO donor, sodium nitroprusside (SNP, 1 mM), enhanced IKs with a magnitude similar to that by a maximum dose (20 μM) of ginseonside Re, and subsequent application of ginsenoside Re failed to enhance IKs. Conversely, after IKs had been enhanced by ginsenoside Re (20 μM), subsequently applied SNP failed to further enhance IKs. An inhibitor of guanylate cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μM), barely suppressed IKs enhancement, while a thiol‐alkylating reagent, N‐ethylmaleimide (NEM, 0.5 mM), clearly suppressed it. A reducing reagent, di‐thiothreitol (DTT, 5 mM), reversed both ginsenoside Re‐ and SNP‐induced IKs enhancement. ICa,L suppression by ginsenoside Re (3 μM) was abolished by SMT (1 μM) or LNAC (1 mM). NEM (0.5 mM) did not suppress ICa,L inhibition and DTT (5 mM) did not reverse ICa,L inhibition, whereas in the presence of ODQ (10 μM), ginsenoside Re (3 μM) failed to suppress ICa,L. These results indicate that ginsenoside Re‐induced IKs enhancement and ICa,L suppression involve NO actions. Direct S‐nitrosylation of channel protein appears to be the main mechanism for IKs enhancement, while a cGMP‐dependent pathway is responsible for ICa,L inhibition.

High-fat diet increases vulnerability to atrial arrhythmia by conduction disturbance via miR-27b.

BACKGROUNDnLifestyle-related diseases, such as obesity and dyslipidemia are important risk factors for atrial fibrillation (AF). However, the underlying mechanism linking these diseases and AF has not been fully investigated.nnnMETHODSnAdult male mice were fed a high-fat diet (HFD) or vehicle (NC) for 2 months. Electrocardiography and in vivo electrophysiological study were performed. Mice were then sacrificed for quantification of mRNA, microRNA, and protein in atria, in addition to histological analysis. Conduction velocity (CV) in right atrium was measured by optical mapping in Langendorff perfused hearts. Cultured atrial cardiomyocytes were treated with palmitate with or without a specific microRNA inhibitor. Twelve hours after stimulation, cells were lysed, and subjected to analysis with qPCR and Western blotting.nnnRESULTSnHFD mice showed prolonged P wave duration, increased inducibility of sustained atrial tachycardia, and reduced atrial CV than NC mice. HFD mice also showed increased expression in inflammatory cytokines, whereas fibrotic area and signals relating fibrosis were not changed. HFD mice demonstrated reduced expression of Cx40 in mRNA and protein levels, and its lateralized expression in atria. MicroRNA array analysis revealed that miR-27b expression was up-regulated in HFD mice, and luciferase assay confirmed the direct interaction between miR-27b and Cx40 3UTR. In palmitate-stimulated atrial cardiomyocytes, miR-27b up-regulation and Cx40 down-regulation were observed, while expression of inflammatory cytokines was not altered. Inhibition of miR-27b with antisense oligonucleotides reversed the alteration caused by palmitate stimulation.nnnCONCLUSIONnHFD may increase the vulnerability to atrial arrhythmia by down-regulation of Cx40 via miR-27b, rather than fibrosis, which is independent of inflammation.

Combined Analysis of Human and Experimental Murine Samples Identified Novel Circulating MicroRNAs as Biomarkers for Atrial Fibrillation

BACKGROUNDnRecent experimental studies have demonstrated that several microRNAs (miRNAs) expressed in atrial tissue promote a substrate of atrial fibrillation (AF). However, because it has not been fully elucidated whether these experimental data contribute to identifying circulating miRNAs as biomarkers for AF, we used a combined analysis of human serum and murine atrial samples with the aim of identifying these biomarkers for predicting AF.Methodsu2004andu2004Results:Comprehensive analyses were performed to screen 733 miRNAs in serum from 10 AF patients and 5 controls, and 672 miRNAs in atrial tissue from 6 inducible atrial tachycardia model mice and 3 controls. We selected miRNAs for which expression was detected in both analyses, and their expression levels were changed in the human analyses, the murine analyses, or both. This screening identified 11 candidate miRNAs. Next, we quantified the selected miRNAs using a quantitative RT-PCR in 50 AF and 50 non-AF subjects. The individual assessment revealed that 4 miRNAs (miR-99a-5p, miR-192-5p, miR-214-3p, and miR-342-5p) were significantly upregulated in AF patients. A receiver-operating characteristics curve indicated that miR-214-3p and miR-342-5p had the highest accuracy. The combination of the 4 miRNAs modestly improved the predictive accuracy for AF (76% sensitivity, 80% specificity).nnnCONCLUSIONSnNovel circulating miRNAs were upregulated in the serum of AF patients and might be potential biomarkers of AF.

Oxidative Stress Induced Ventricular Arrhythmia and Impairment of Cardiac Function in Nos1ap Deleted Mice

Genome-wide association study has identified that the genetic variations at NOS1AP (neuronal nitric oxide synthase-1 adaptor protein) were associated with QT interval and sudden cardiac death (SCD). However, the mechanism linking a genetic variant of NOS1AP and SCD is poorly understood. We used Nos1ap knockout mice (Nos1ap(-/-)) to determine the involvement of Nos1ap in SCD, paying special attention to oxidative stress.At baseline, a surface electrocardiogram (ECG) and ultrasound echocardiography (UCG) showed no difference between Nos1ap(-/-) and wild-type (WT) mice. Oxidative stress was induced by a single injection of doxorubicin (Dox, 25 mg/kg). After Dox injection, Nos1ap(-/-) showed significantly higher mortality than WT (93.3 versus 16.0% at day 14, P < 0.01). ECG showed significantly longer QTc in Nos1ap(-/-) than WT, and UCG revealed significant reduction of fractional shortening (%FS) only in Nos1ap(-/-) after Dox injection. Spontaneous ventricular tachyarrhythmias were documented by telemetry recording after Dox injection only in Nos1ap(-/-). Ex vivo optical mapping revealed that the action potential duration (APD)90 was prolonged at baseline in Nos1ap(-/-), and administration of Dox lengthened APD90 more in Nos1ap(-/-) than in WT. The expression of Bnp and the H2O2 level were higher in Nos1ap(-/-) after Dox injection. Nos1ap(-/-) showed a reduced amplitude of calcium transient in isolated cardiomyocytes after Dox injection. Administration of the antioxidant N-acetyl-L-cysteine significantly reduced mortality of Nos1ap(-/-) by Dox injection, accompanied by prevention of QT prolongation and a reduction in %FS.Although Nos1ap(-/-) mice have apparently normal hearts, oxidative stress evokes ventricular tachyarrhythmia and heart failure, which may cause sudden cardiac death.

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm × 2 mm or 10 mm x 10 mm, with a 100 × 100 resolution (20 µm/pixel or 100 µm/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

Differential Wnt-mediated programming and arrhythmogenesis in right versus left ventricles

Several inherited arrhythmias, including Brugada syndrome and arrhythmogenic cardiomyopathy, primarily affect the right ventricle and can lead to sudden cardiac death. Among many differences, right and left ventricular cardiomyocytes derive from distinct progenitors, prompting us to investigate how embryonic programming may contribute to chamber-specific conduction and arrhythmia susceptibility. Here, we show that developmental perturbation of Wnt signaling leads to chamber-specific transcriptional regulation of genes important in cardiac conduction that persists into adulthood. Transcriptional profiling of right versus left ventricles in mice deficient in Wnt transcriptional activity reveals global chamber differences, including genes regulating cardiac electrophysiology such as Gja1 and Scn5a. In addition, the transcriptional repressor Hey2, a gene associated with Brugada syndrome, is a direct target of Wnt signaling in the right ventricle only. These transcriptional changes lead to perturbed right ventricular cardiac conduction and cellular excitability. Ex vivo and in vivo stimulation of the right ventricle is sufficient to induce ventricular tachycardia in Wnt transcriptionally inactive hearts, while left ventricular stimulation has no effect. These data show that embryonic perturbation of Wnt signaling in cardiomyocytes leads to right ventricular arrhythmia susceptibility in the adult heart through chamber-specific regulation of genes regulating cellular electrophysiology.

Calcification of joints and arteries with novel NT5E mutations with involvement of upper extremity arteries.

A 66-year-old man with a 30-year history of bilateral intermittent claudication was referred to our hospital. His medical history included hypertension, dyslipidemia, stroke, and impaired glucose tolerance, treated with standard medications. He had a 46 pack-year smoking history, but never used illicit drugs. He had no family history of vascular diseases. Both lower legs were cold and the ankle–brachial index was markedly decreased (0.49 on the right and 0.59 on the left). Laboratory examination was unremarkable, including serum calcium, phosphate, and parathyroid hormone. Computed tomography showed severely calcified lower extremity arteries, in contrast to little calcification in the aorta (Panel A, left). Conventional X-ray displayed slight pericapsular calcification of the finger joints (Panel B, upper panels: white arrows; R, right; L, left), and bilateral calcified aneurysmal popliteal and brachial arteries (Panel A, right and Panel B, lower panels). Considering these findings, genetic screening of the ecto-5’-nucleotidase (NT5E) gene was conducted with the patient’s informed consent. Genomic DNA was extracted and amplified by polymerase chain reaction (PCR) for each exon of the NT5E gene (NM_002526.3) and analyzed by direct sequencing. Four novel homozygous mutations were identified (3G>C, 488C>T (silent mutation; not shown), 1086A>G, and 1096T>C) in the proband (Panel C), and he was diagnosed with calcification of joints and arteries (CALJA). Familial screening identified that his mother had one homozygous (1086A>G) and three heterozygous (3G>C, 488C>T, and 1096T>C) mutations, while his brother had one homozygous mutation (1086A>G). The families had no symptoms or findings of CALJA. CALJA was originally described by Magnus-Levy in 1914,1 and St Hilaire et al.2 identified the causal mutations in the NT5E gene in 2011. Since then, 13 cases of CALJA from six families with NT5E mutations have been reported. The NT5E gene encodes CD73, which is thought to be a key inhibitor of tissue calcification. The classical characteristics of CALJA are late-onset profound calcification of lower extremity arteries and hand and foot joint capsules that spares the arteries in the trunk. Zhang et al.3 reported a case of CALJA involving the upper extremity arteries, and our report is the second case to document a similar distribution. In this case, we identified novel mutations in the NT5E gene. As 488C>T was a silent mutation (Y176Y), and his family members with 1086A>G seemed unaffected, 3G>C and/or 1096T>C are candidates for the causal mutation in this case. We speculated 3G>C was the causal mutation because it resulted in substitution of the start codon of the NT5E gene. Calcification of joints and arteries with novel NT5E mutations with involvement of upper extremity arteries