A Novel Hemostatic Peptide Solution for Common Acute Gastrointestinal Bleeding Diseases: First Case Series Study on the Treatment Results of Endoscopic Hemostasis by Nonexpert Endoscopists.

INTRODUCTION: We investigated the hemostatic effect and safety of a hemostatic peptide solution for the treatment of gastrointestinal bleeding requiring emergency endoscopy. METHODS: We retrospectively examined the patient backgrounds, hemostatic results, and procedural safety in patients who were treated with a hemostatic peptide solution for hemostasis during emergency endoscopies for gastrointestinal bleeding. All hemostatic procedures were performed by nonexpert physicians with less than 10 years of endoscopic experience. All of the cases were treated at a single institution over the months from January 2022 to January 2023. RESULTS: Twenty-six consecutive patients (17 males and 9 females) with a median age of 74 (45-95) years were included. Their conditions requiring emergency endoscopy were melena in 8 patients, hematochezia in 2, hematemesis in 8, anemia in 6, and bleeding during esophagogastroduodenoscopy in 2. The sites of bleeding were the esophagus in 3 patients, the stomach in 17, the duodenum in 3, the small intestine in 2, and the colon in 1. Hemostasis was obtained with another hemostasis device used in conjunction with the hemostatic peptide solution in 13 cases and with the hemostatic peptide solution alone in 13 cases. The hemostasis success rate was 100%, with no complications. Rebleeding occurred within 1 week in 4 cases. CONCLUSION: Hemostasis with the hemostatic peptide solution was safe and provided a temporary high hemostatic effect in emergency gastrointestinal endoscopy.

Bioinformatic Identification of Potential RNA Alterations on the Atrial Fibrillation Remodeling from Human Pulmonary Veins.

Atrial fibrillation (AF) is the most frequent persistent arrhythmia. Many genes have been reported as a genetic background for AF. However, most transcriptome analyses of AF are limited to the atrial samples and have not been evaluated by multiple cardiac regions. In this study, we analyzed the expression levels of protein-coding and long noncoding RNAs (lncRNAs) in six cardiac regions by RNA-seq. Samples were donated from six subjects with or without persistent AF for left atria, left atrial appendages, right atria, sinoatrial nodes, left ventricles, right ventricles, and pulmonary veins (PVs), and additional four right atrial appendages samples were collected from patients undergoing mitral valve replacement. In total, 23 AF samples were compared to 23 non-AF samples. Surprisingly, the most influenced heart region in gene expression by AF was the PV, not the atria. The ion channel-related gene set was significantly enriched upon analysis of these significant genes. In addition, some significant genes are cancer-related lncRNAs in PV in AF. A co-expression network analysis could detect the functional gene clusters. In particular, the cancer-related lncRNA, such as SAMMSON and FOXCUT, belong to the gene network with the cancer-related transcription factor FOXC1. Thus, they may also play an aggravating role in the pathogenesis of AF, similar to carcinogenesis. In the least, this study suggests that (1) RNA alteration is most intense in PVs and (2) post-transcriptional gene regulation by lncRNA may contribute to the progression of AF. Through the screening analysis across the six cardiac regions, the possibility that the PV region can play a role other than paroxysmal triggering in the pathogenesis of AF was demonstrated for the first time. Future research with an increase in the number of PV samples will lead to a novel understanding of the pathophysiology of AF.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes†.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Molecular identification of HSPA8 as an accessory protein of a hyperpolarization-activated chloride channel from rat pulmonary vein cardiomyocytes.

Pulmonary veins (PVs) are the major origin of atrial fibrillation. Recently, we recorded hyperpolarization-activated Cl- current (ICl, h) in rat PV cardiomyocytes. Unlike the well-known chloride channel protein 2 (CLCN2) current, the activation curve of ICl, h was hyperpolarized as the Cl- ion concentration ([Cl-] i ) increased. This current could account for spontaneous activity in PV cardiomyocytes linked to atrial fibrillation. In this study, we aimed to identify the channel underlying ICl, h Using RT-PCR amplification specific for Clcn2 or its homologs, a chloride channel was cloned from rat PV and detected in rat PV cardiomyocytes using immunocytochemistry. The gene sequence and electrophysiological functions of the protein were identical to those previously reported for Clcn2, with protein activity observed as a hyperpolarization-activated current by the patch-clamp method. However, the [Cl-] i dependence of activation was entirely different from the observed ICl, h of PV cardiomyocytes; the activation curve of the Clcn2-transfected cells shifted toward positive potential with increased [Cl-] i , whereas the ICl, h of PV and left ventricular cardiomyocytes showed a leftward shift. Therefore, we used MS to explore the possibility of additional proteins interacting with CLCN2 and identified an individual 71-kDa protein, HSPA8, that was strongly expressed in rat PV cardiomyocytes. With co-expression of HSPA8 in HEK293 and PC12 cells, the CLCN2 current showed voltage-dependent activation and shifted to negative potential with increasing [Cl-] i Molecular docking simulations further support an interaction between CLCN2 and HSPA8. These findings suggest that CLCN2 in rat heart contains HSPA8 as a unique accessory protein.

Comparison of 3D endoscopy and conventional 2D endoscopy in gastric endoscopic submucosal dissection: an ex vivo animal study.

BACKGROUND AND OBJECTIVES: Conventional endoscopy provides two-dimensional (2D) information without depth information. This study compared three-dimensional (3D) endoscopy and 2D endoscopy using an endoscopic submucosal dissection (ESD) training model to evaluate the utility of 3D endoscopy. METHODS: Porcine stomach specimens (7 × 7 cm) were prepared from commercially available resected porcine stomachs and a 10-mm hypothetical lesion was marked at the center of each specimen. Specimens were individually placed in an ESD training model, and subjected to either 2D or 3D ESD. En bloc resection rate, perforation rate, incision time, dissection time, and levels of five eyestrain symptoms (fatigue, pain, blurred vision, head-heaviness, and headache; 100-mm visual analog scale) were compared between the 2D and 3D procedures. In a crossover design, 8 endoscopists each performed two 2D and two 3D procedures. RESULTS: All 32 lesions were resected en block, but perforation occurred in one 2D procedure. Incision time was significantly shorter in 3D ESD than in 2D ESD (102.8 ± 42.1 s vs. 135.8 ± 65.7 s, p < 0.05). Dissection time was also significantly shorter in 3D ESD than in 2D ESD (366.3 ± 187.6 s vs. 517.8 ± 282.3 s, p < 0.05). Differences in levels of all symptoms except blurred vision between before and after ESD were larger in 3D ESD than in 2D ESD. CONCLUSIONS: Incision time and dissection time were significantly shorter in 3D ESD compared with 2D ESD, but eyestrain was increased. Depth information from 3D images appears to facilitate rapid and stable ESD maneuvers.

Operational effectiveness of three-dimensional flexible endoscopy: an ex vivo study using a new model.

BACKGROUND AND OBJECTIVES: Two-dimensional (2D) images lack depth information and thus provide probabilistic recognition that do not completely match the actual three-dimensional (3D) information. Here, we investigated the operability of 3D endoscopes. METHODS: A 3D operation model was developed by passing 20 silk threads through upper and lower plates at 2-mm intervals in front and back rows separated by 1 mm. We evaluated accuracy and time of operating an electrosurgical knife. A successful operation was defined as pulling only a front-row thread; an unsuccessful operation was defined as pulling no thread (miss) or simultaneously pulling front- and back-row threads. Endoscopists (four experts, six trainees) repeated the operation under 2D and 3D conditions until individually accumulating 10 successful attempts under each condition. RESULTS: Operation accuracy was significantly higher for 3D compared with 2D in all endoscopists (88.5% vs. 61.3%; p < 0.01) and in both experience groups (trainees: 84.5% vs. 61.2%; experts: 95.2% vs. 61.5%; both p < 0.01). Operation time was significantly shorter for 3D compared with 2D in all endoscopists (12.5 ± 4.1 s vs. 14.8 ± 4.7 s; p < 0.01) and in both experience groups (trainees: 12.8 ± 4.2 s vs. 15.2 ± 4.9 s; experts: 12.1 ± 4.0 s vs. 14.3 ± 4.3 s; both p < 0.01). DISCUSSION: Compared with 2D endoscopy, 3D endoscopy significantly improved operation accuracy and shortened operation time, suggesting that 3D endoscopy enables accurate operation by depth information, aiding spatial recognition.

Mechanisms Underlying Spontaneous Action Potential Generation Induced by Catecholamine in Pulmonary Vein Cardiomyocytes: A Simulation Study.

Cardiomyocytes and myocardial sleeves dissociated from pulmonary veins (PVs) potentially generate ectopic automaticity in response to noradrenaline (NA), and thereby trigger atrial fibrillation. We developed a mathematical model of rat PV cardiomyocytes (PVC) based on experimental data that incorporates the microscopic framework of the local control theory of Ca2+ release from the sarcoplasmic reticulum (SR), which can generate rhythmic Ca2+ release (limit cycle revealed by the bifurcation analysis) when total Ca2+ within the cell increased. Ca2+ overload in SR increased resting Ca2+ efflux through the type II inositol 1,4,5-trisphosphate (IP3) receptors (InsP3R) as well as ryanodine receptors (RyRs), which finally triggered massive Ca2+ release through activation of RyRs via local Ca2+ accumulation in the vicinity of RyRs. The new PVC model exhibited a resting potential of -68 mV. Under NA effects, repetitive Ca2+ release from SR triggered spontaneous action potentials (APs) by evoking transient depolarizations (TDs) through Na+/Ca2+ exchanger (APTDs). Marked and variable latencies initiating APTDs could be explained by the time courses of the α1- and ß1-adrenergic influence on the regulation of intracellular Ca2+ content and random occurrences of spontaneous TD activating the first APTD. Positive and negative feedback relations were clarified under APTD generation.

ERK5 Phosphorylates Kv4.2 and Inhibits Inactivation of the A-Type Current in PC12 Cells.

Extracellular signal-regulated kinase 5 (ERK5) regulates diverse physiological responses such as proliferation, differentiation, and gene expression. Previously, we demonstrated that ERK5 is essential for neurite outgrowth and catecholamine biosynthesis in PC12 cells and sympathetic neurons. However, it remains unclear how ERK5 regulates the activity of ion channels, which are important for membrane excitability. Thus, we examined the effect of ERK5 on the ion channel activity in the PC12 cells that overexpress both ERK5 and the constitutively active MEK5 mutant. The gene and protein expression levels of voltage-dependent Ca2+ and K⁺ channels were determined by RT-qPCR or Western blotting. The A-type K⁺ current was recorded using the whole-cell patch clamp method. In these ERK5-activated cells, the gene expression levels of voltage-dependent L- and P/Q-type Ca2+ channels did not alter, but the N-type Ca2+ channel was slightly reduced. In contrast, those of Kv4.2 and Kv4.3, which are components of the A-type current, were significantly enhanced. Unexpectedly, the protein levels of Kv4.2 were not elevated by ERK5 activation, but the phosphorylation levels were increased by ERK5 activation. By electrophysiological analysis, the inactivation time constant of the A-type current was prolonged by ERK5 activation, without changes in the peak current. Taken together, ERK5 inhibits an inactivation of the A-type current by phosphorylation of Kv4.2, which may contribute to the neuronal differentiation process.

Effect of skip lymphovascular invasion on hepatic metastasis in colorectal carcinomas.

BACKGROUND: "Skip" lymphovascular invasion presenting as discontinuous foci of tumor cells within the colon wall is now excluded from consideration when determining T stage in the TNM classification. The purpose of this study was to assess the clinicopathological characteristics of colorectal cancer (CRC) patients with such skip lymphovascular invasion. METHODS: First, a retrospective questionnaire survey of the incidence of skip lymphovascular invasion was performed for a total of 1,868 patients with CRCs at ten institutions. Next, we comparatively assessed clinicopathological data for 896 CRC patients with or without skip lymphovascular invasion. RESULTS: The incidence of skip lymphovascular invasion was 1.1 % (20 out of 1,868). Most of the affected cases were rectal, pT2, and node negative, with moderately differentiated histology. Skip lymphovascular invasion was present in the muscularis propria and subserosa, with the tumors directly invading submucosa (pT1) or muscularis propria (pT2). Hepatic metastasis was greater in CRC with skip lymphovascular invasion (25 %) than in pT1/2 CRC (0 %; P < 0.001) or pT3 CRC without such invasion (13.8 %; P = 0.185). CONCLUSIONS: Our study suggests that skip lymphovascular invasion is associated with hepatic metastasis in CRC cases. Thus, definition of a T category including such invasion would be useful for clinical practice.

Pathological impact of hyperpolarization-activated chloride current peculiar to rat pulmonary vein cardiomyocytes.

Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs(+) resistant, and was not affected by removal of external Na(+) or K(+). The current was inhibited with Cd(2+), and the reversal potential was sensitive to changes in [Cl(-)] on either side of the membrane in a manner consistent with a Cl(-) selective channel. Cl(-) channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl(-) current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl(-) current in rat PVs was shifted to negative potentials as [Cl(-)]i increased, (ii) the Cl(-) current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl(-) channel, and that the channel may play a functional role in norepinephrine-induced automaticity.

Stochasticity intrinsic to coupled-clock mechanisms underlies beat-to-beat variability of spontaneous action potential firing in sinoatrial node pacemaker cells.

Recent evidence indicates that the spontaneous action potential (AP) of isolated sinoatrial node cells (SANCs) is regulated by a system of stochastic mechanisms embodied within two clocks: ryanodine receptors of the "Ca(2+) clock" within the sarcoplasmic reticulum, spontaneously activate during diastole and discharge local Ca(2+) releases (LCRs) beneath the cell surface membrane; clock crosstalk occurs as LCRs activate an inward Na(+)/Ca(2+) exchanger current (INCX), which together with If and decay of K(+) channels prompts the "M clock," the ensemble of sarcolemmal-electrogenic molecules, to generate APs. Prolongation of the average LCR period accompanies prolongation of the average AP beating interval (BI). Moreover, the prolongation of the average AP BI accompanies increased AP BI variability. We hypothesized that both the average AP BI and AP BI variability are dependent upon stochasticity of clock mechanisms reported by the variability of LCR period. We perturbed the coupled-clock system by directly inhibiting the M clock by ivabradine (IVA) or the Ca(2+) clock by cyclopiazonic acid (CPA). When either clock is perturbed by IVA (3, 10 and 30 µM), which has no direct effect on Ca(2+) cycling, or CPA (0.5 and 5 µM), which has no direct effect on the M clock ion channels, the clock system failed to achieve the basal AP BI and both AP BI and AP BI variability increased. The changes in average LCR period and its variability in response to perturbations of the coupled-clock system were correlated with changes in AP beating interval and AP beating interval variability. We conclude that the stochasticity within the coupled-clock system affects and is affected by the AP BI firing rate and rhythm via modulation of the effectiveness of clock coupling.

Histological assessment of intra- and inter-institutional reliabilities in detection of desmoplastic reaction in biopsy specimens of early colorectal carcinomas.

We previously reported a relationship between depth of submucosal invasion of early colorectal carcinomas and desmoplastic reaction (DR). However, poor inter-observer agreement on the histopathological diagnosis of DR in biopsy specimens with hematoxylin and eosin (H&E) staining has been the major critique of this tool. In this study, reproducibility of the histopathological diagnosis of DR was evaluated. Furthermore, we investigated the possible improvement of the reproducibility after education about histological characteristics and tried to identify histological characteristics that are most important in the recognition of DR. A total of 34 H&E stained slides were included in this study and analyzed by three pathologists. Slides were reviewed before and after education about histological characteristics of DR. Kappa statistics were used to compare the inter-observer variability. We investigated the relationship between DR and histopathological factor. The inter-observer agreement during the first session varied between 0.30 and 0.63, which improved during the second session toward an agreement between 0.58 and 0.71. Myofibroblast proliferation associated with cancer invasion was found to be the most useful in the diagnosis of DR. In conclusion, the correct detection of myofibroblasts may facilitate the standardization of diagnosis of DR.

Diagnosis of desmoplastic reaction by immunohistochemical analysis, in biopsy specimens of early colorectal carcinomas, is efficacious in estimating the depth of invasion.

The aim of our study was to evaluate the diagnosis of desmoplastic reaction (DR) by immunostaining for α-smooth muscle actin (αSMA) and desmin, for predicting the depth of submucosal invasion in biopsy specimens of early colorectal carcinomas (CRCs). Thirty-eight cases of non-pedunculated early CRCs were included in this study. Positive for DR was defined as αSMA-positive and desmin-negative stroma in the CRC. The depth of submucosal invasion was measured in endoscopically or surgically resected specimens and the lesions were subsequently divided into two groups: Group A (carcinoma in situ/intramucosal carcinoma and submucosal invasive carcinoma with a depth <1000 µm) and Group B (submucosal invasion with a depth ≥1000 µm). Twenty-one cases were DR-positive and 17 were DR-negative. No statistical significance was found between the DR with regard to tumor size, location and histological type. All DR-positive cases belonged to Group B whereas 14 (82.4%) DR-negative lesions belonged to Group A (p < 0.001). The sensitivity, specificity, positive and negative predictive values and accuracy of DR positivity for diagnosis of Group B were 87.5%, 100%, 100%, 82.4% and 92.1%, respectively. Conclusively, detection of DR in biopsy specimens with ancillary immunohistochemistry (αSMA/desmin) would help in preoperative diagnosis for the depth of submucosal invasion of early CRC.

Arrhythmogenic coupling between the Na+ -Ca2+ exchanger and inositol 1,4,5-triphosphate receptor in rat pulmonary vein cardiomyocytes.

Atrial fibrillation, the most common sustained arrhythmia, is believed to be triggered by ectopic electrical activity originating in the myocardial sleeves surrounding the pulmonary veins (PVs). It has been reported that myocardial sleeves have the potential to generate automaticity in response to norepinephrine. This study investigated the cellular mechanisms underlying norepinephrine-induced automaticity in PV cardiomyocytes isolated from rats. Application of 10 µM norepinephrine to PV cardiomyocytes induced repetitive and transient increases in intracellular Ca(2+) concentrations. The Ca(2+) transient was accompanied by depolarization, and induced automatic rhythmic action potentials at approximately 4Hz in perforated patch clamp preparations in 27% of myocytes were observed. When the recording mode was switched from current-clamp to voltage-clamp mode during the continuous presence of automaticity, an oscillatory current was observed. The oscillatory current was always inward, irrespective of the membrane potential, indicating that the current was derived mainly from the Na(+)-Ca(2+) exchanger (NCX). The norepinephrine-induced automaticity was suppressed by blocking either the ß(1)- or α(1)-adrenoceptor. Additionally, this automaticity was blocked by inhibitors of phospholipase C and the inositol 1,4,5-triphosphate receptor (IP(3)R) but not by a protein kinase C inhibitor. We observed that the transverse-tubule system was enriched in cardiomyocytes in the PV, in contrast to those of the atrium, and that the NCX and IP(3)R were co-localized along transverse tubules. These findings suggest that a functional coupling between the NCX and IP(3)R causes arrhythmic excitability of the PV during the presence of combined ß(1)- and α(1)-adrenoceptor stimulation.

Direct effects of esmolol and landiolol on cardiac function, coronary vasoactivity, and ventricular electrophysiology in guinea-pig hearts.

The ultra-short acting, selective ß(1)-adrenergic antagonists landiolol and esmolol are widely used perioperatively; however, little is known about their acute direct actions on the heart. The current study utilized the Langendorff perfused heart system to measure changes in cardiac function and hemodynamics in response to each drug. Furthermore, electrophysiological analysis was performed on isolated ventricular myocytes. Direct application of esmolol significantly decreased systolic left ventricular pressure and heart rate at concentrations > 10 µM, while it dose-dependently increased coronary perfusion pressure. Esmolol also shortened the action potential duration (APD) in a concentration-dependent manner, an action maintained even when the delayed rectifier K(+) current or ATP sensitive K(+) current was blocked. Moreover, esmolol inhibited both the inward rectifier K(+) current (I(K1)) and the L-type Ca(2+) current (I(CaL)) and increased the outward current dose-dependently. In contrast, landiolol had minimal cardiac effects. In the Kyoto Model computer simulation, inhibition of either I(K1) or I(CaL) alone failed to shorten the APD; however, an additional increase in the time-independent outward current caused shortening of the APD, equal to that induced by esmolol. In conclusion, esmolol directly inhibits cardiac performance significantly more so than landiolol, an effect revealed to be at least in part mediated by esmolol-induced APD shortening.

Molecular Pathogenesis of Cardiac Arrhythmia.

The heart is a necessary organ for sustaining life in mammals, and it is the first organ to function during early development [...].

Comparative Study of Transcriptome in the Hearts Isolated from Mice, Rats, and Humans.

The heart is a significant organ in mammalian life, and the heartbeat mechanism has been an essential focus of science. However, few studies have focused on species differences. Accordingly, challenges remain in studying genes that have universal functions across species and genes that determine species differences. Here, we analyzed transcriptome data in mouse, rat, and human atria, ventricles, and sinoatrial nodes (SA) obtained from different platforms and compared them by calculating specificity measure (SPM) values in consideration of species differences. Among the three heart regions, the species differences in SA were the greatest, and we searched for genes that determined the essential characteristics of SA, which was SHOX2 in our criteria. The SPM value of SHOX2 was prominently high across species. Similarly, by calculating SPM values, we identified 3 atrial-specific, 11 ventricular-specific, and 17 SA-specific markers. Ontology analysis identified 70 cardiac region- and species-specific ontologies. These results suggest that reanalyzing existing data by calculating SPM values may identify novel tissue-specific genes and species-dependent gene expression. This study identified the importance of SHOX2 as an SA-specific transcription factor, a novel cardiac regional marker, and species-dependent ontologies.

Preferential Expression of Ca2+-Stimulable Adenylyl Cyclase III in the Supraventricular Area, including Arrhythmogenic Pulmonary Vein of the Rat Heart.

Ectopic excitability in pulmonary veins (PVs) is the major cause of atrial fibrillation. We previously reported that the inositol trisphosphate receptor in rat PV cardiomyocytes cooperates with the Na+-Ca2+ exchanger to provoke ectopic automaticity in response to norepinephrine. Here, we focused on adenylyl cyclase (AC) as another effector of norepinephrine stimulation. RT-PCR, immunohistochemistry, and Western blotting revealed that the abundant expression of Ca2+-stimulable AC3 was restricted to the supraventricular area, including the PVs. All the other AC isotypes hardly displayed any region-specific expressions. Immunostaining of isolated cardiomyocytes showed an enriched expression of AC3 along the t-tubules in PV myocytes. The cAMP-dependent response of L-type Ca2+ currents in the PV and LA cells is strengthened by the 0.1 mM intracellular Ca2+ condition, unlike in the ventricular cells. The norepinephrine-induced automaticity of PV cardiomyocytes was reversibly suppressed by 100 µM SQ22536, an adenine-like AC inhibitor. These findings suggest that the specific expression of AC3 along t-tubules may contribute to arrhythmogenic automaticity in rat PV cardiomyocytes.

Azelnidipine treatment reduces the expression of Cav1.2 protein.

AIMS: Azelnidipine, a third-generation dihydropyridine calcium channel blocker (DHP CCB), has a characteristic hypotensive effect that persists even after it has disappeared from the plasma, which is thought to be due to its high hydrophobicity. However, because azelnidipine is unique, it might have other unknown effects on L-type Cav1.2 channels that result in the long-lasting decrease of blood pressure. The aim of this study was to investigate the potential quantitative modification of Cav1.2 by azelnidipine. MAIN METHODS: HEK293 cells were used to express Cav1.2 channels. Immunocytochemical analysis was performed to detect changes in the surface expression of the pore-forming subunit of the Cav1.2 channel, Cav1.2α1c. Western blotting analysis was performed to evaluate changes in expression levels of total Cav1.2α1c and Cavß2c. KEY FINDINGS: The surface expression of Cav1.2α1c was markedly reduced by treatment with azelnidipine, but not with other DHP CCBs (amlodipine and nicardipine). Results obtained with a dynamin inhibitor and an early endosome marker suggested that the reduction of surface Cav1.2α1c was not likely caused by internalization. Azelnidipine reduced the total amount of Cav1.2α1c protein in HEK293 cells and rat pulmonary artery smooth muscle cells. The reduction of Cav1.2α1c was rescued by inhibiting proteasome activity. In contrast, azelnidipine did not affect the amount of auxiliary Cavß2c subunits that function as a chaperone of Cav1.2. SIGNIFICANCE: This study is the first to demonstrate that azelnidipine reduces the expression of Cav1.2α1c, which might partly explain its long-lasting hypotensive effect.

E44Q mutation in NaV1.7 in a patient with infantile paroxysmal knee pain: electrophysiological analysis of voltage-dependent sodium current.

Gain-of-function mutations in voltage-gated sodium channels (NaV1.7, NaV1.8, and NaV1.9) are known causes of inherited pain disorders. Identification and functional assessment of new NaV1.7 mutations could help elucidate the phenotypic spectrum of NaV1.7 channelopathies. We identified a novel NaV1.7 mutation (E44Q in exon 2) that substitutes a glutamic acid residue for glutamine in the cytoplasmic N-terminus of NaV1.7 in a patient with paroxysmal pain attacks during childhood and his family who experienced similar pain episodes. To study the sodium channel's function, we performed electrophysiological recordings. Voltage-clamp recordings revealed that the mutation increased the amplitude of the non-inactivating component of the sodium current, which might facilitate channel opening. These data demonstrate that E44Q is a gain-of-function mutation in NaV1.7, which is consistent with our patient's pain phenotype.