Usefulness of the Fibrosis-4 index and alanine aminotransferase at 1 year of nucleos(t)ide analog treatment for prediction of hepatocellular carcinoma in chronic hepatitis B patients.

AIM: Nucleos(t)ide analogs do not completely prevent hepatocellular carcinoma (HCC) in chronic hepatitis B virus infection. This study aimed to evaluate the dynamics of a non-invasive liver fibrosis marker, the Fibrosis-4 (FIB-4) index, for predicting HCC development. METHODS: Among a total of 882 chronically hepatitis B virus infection-infected patients who were treated with nucleos(t)ide analogs, 472 patients without HCC history whose FIB-4 at baseline and 1 year of treatment was obtained were evaluated for the incidence of HCC. RESULTS: The median FIB-4 was 2.00 at baseline and was significantly reduced to 1.58 at 1 year (P < 0.001), but the reduction was small at 2 years or later. When a receiver operating characteristic analysis of FIB-4 was performed to predict HCC within 5 years, the area under the curve of FIB-4 at 1 year was higher than that at baseline (0.676 vs. 0.599). The HCC incidence was significantly higher in patients with FIB-4 ≥1.58 than in those with FIB-4 <1.58 (14.8% vs. 3.6% at 10 years, P < 0.001). Additionally, an abnormal alanine aminotransferase (≥31 U/L) at 1 year was an independent risk for HCC. When a fibrosis and alanine aminotransferase-1 (FAL-1) score was evaluated as an applicable number of FIB-4 ≥1.58, and alanine aminotransferase ≥31 as 0, 1, and 2, the HCC risk in patients with score 2 was significantly higher than in those with score 1 or score 0 (24.1% vs. 9.8% vs. 0.7% at 10 years, P < 0.001). CONCLUSIONS: FIB-4 ≥1.58 and alanine aminotransferase ≥31 at 1 year of nucleos(t)ide analog was an independent risk factor for HCC development, and a score using these factors stratified the risk of HCC.

A Final Report on the Real Impact of the COVID-19 Pandemic on the Diagnosis of Gastrointestinal Cancer in Akita Prefecture, Japan in 2022.

The long-term impact of the coronavirus disease 2019 (COVID-19) pandemic on the disruption of gastrointestinal cancer diagnoses remains unclear. This study investigated the actual impact on esophagogastric cancer (EGC) and colorectal cancer (CRC) diagnoses up to the third year of the pandemic in Akita Prefecture, Japan, using population-based registry data. We collected data on the annual number of EGC and CRC diagnoses using a database from the collaborative Akita Prefecture hospital-based registration. The net number of cancers diagnosed in the first three years of the pandemic (2020-2022) were compared with those diagnosed in the three years before the pandemic (2017-2019). Changes in the proportion of cancer stage and initial treatment for diagnosed EGC and CRC after the pandemic were then compared. The total number of EGCs was 9.3% lower in the first three years of the pandemic than in the three years before, probably due to its long-term declining trend. The total number of CRCs in the first three years of the pandemic exceeded that in the three years before, suggesting successful recovery of the diagnostic procedure. The proportion of cancer stages and initial treatment for EGCs and CRCs remained largely unchanged after the onset of the pandemic. Based on the population-based registry data from the first three years of the pandemic, the disruption of gastrointestinal cancer diagnoses caused by the pandemic is settling down without any substantial disease progression, even in Akita Prefecture, the area with the highest incidence of cancer in all of Japan.

Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle.

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43-/-) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/- mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/- mice (cCx43-/- mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2',7'-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43-/- mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43-/- mice. In cCx43-/- mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43-/- mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43-/- mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.

A Follow-up Report on the Diagnosis of Gastrointestinal Cancer during the COVID-19 Pandemic in Akita Prefecture, Japan in 2021.

We recently reported the decrease in the number of gastrointestinal (GI) cancer diagnoses in 2020 due to disturbance of the healthcare system by the coronavirus disease 2019 (COVID-19) pandemic, using a hospital-based cancer registration system in Akita prefecture, Japan. In this study, we extended the research by showing the latest data (2021) on the number of cancers and examinations. Information on the occurrence and stage of esophageal, gastric, and colorectal cancers was collected from the same database. The number of GI examinations (cancer screening procedures and endoscopic examinations) was also investigated. Following the immediate decrease in the numbers of both GI examinations and GI cancer diagnoses in 2020, a rebound increase in the numbers of GI cancer diagnoses-especially colorectal cancers-was observed in 2021, resulting from an increased number of GI examinations i.e., the total number of colorectal cancers in 2021 increased by 9.0% and 6.8% in comparison to 2020 and pre-pandemic era, respectively. However, the rebound increase in 2021 was largely due to an increase in early-stage cancers, and there was no apparent trend toward the increased predominance of more advanced cancers. It therefore seems that we managed to escape from the worst-case scenario of disturbance of the healthcare system due to pandemic (i.e., an increase in the number of more advanced cancers due to delayed diagnoses). We need to continue to watch the trends in Akita prefecture, which has the highest rate of mortality from the 3 major GI cancers in Japan.

CD98 expression can be a predictive factor of resistance to radiotherapy in head and neck squamous cell carcinoma.

CD98 is a marker of cancer stem cells, and it regulates radiosensitivity in head and neck squamous cell carcinoma (HNSCC). The current study aimed to investigate whether CD98 can be used as a prognostic factor and marker of radioresistance. CD98 immunostaining was performed using biopsy specimens collected from patients diagnosed with HNSCC. The average period of postoperative monitoring was 31.6 months. The treatment options were radiation therapy with either cisplatin or cetuximab, and surgery. The participants were divided into groups of low and high fluorescence intensity. CD98 was an independent prognostic factor of radioresistance. In total, 103 patients were treated with chemoradiotherapy or bioradiotherapy. The overall survival rates of patients receiving chemoradiotherapy or bioradiotherapy were 69.2% in the low group and 36.2% in the high group. The progression-free survival rates were 60.0% and 24.6%, respectively. CD98 expression was considered an independent prognostic factor of overall survival and progression-free survival. In total, 99 patients underwent surgical treatment. The surgery group did not differ according to CD98 expression. Via CD98 immunostaining, sensitivity to radiotherapy can be determined in advance. In HNSCC, knowledge about sensitivity to radiotherapy can significantly improve prognosis.

Roles of stretch-activated channels and NADPH oxidase 2 in the induction of twitch contraction by muscle stretching in rat ventricular muscle.

Mechano-electric feedback means that muscle stretching causes depolarization of membrane potential. We investigated whether muscle stretching induces action potential and twitch contraction with a threshold of sarcomere length (SL) and what roles stretch-activated channels (SACs) and stretch-activated NADPH oxidase (X-ROS signaling) play in the induction. Trabeculae were obtained from the right ventricles of rat hearts. Force, SL, and [Ca2+]i were measured. Various degrees of stretching from the SL of 2.0 µm were applied 0.5 s after the last stimulus of the electrical train with 0.4-s intervals for 7.5 s. The SLtwitch was defined as the minimal SL at which twitch contraction was induced by the stretching. Muscle stretching induced twitch contraction with a threshold of SL at 0.4-s stimulus intervals ([Ca2+]o = 0.7 mmol/L). The SLtwitch was not changed by increasing the stimulus intervals and [Ca2+]o and by adding 1 µmol/L isoproterenol. The SLtwitch was not changed by adding 10 µmol/L Gd3+, 100 µmol/L or 200 µmol/L streptomycin, and 5 µmol/L GsMTx4. The SLtwitch was not changed by adding 1 µmol/L ryanodine and 3 µmol/L diphenyleneiodonium chloride. In contrast, the SLtwitch was increased by elevating extracellular K+ from 5 to 10 mmol/L and by adding the stretching during the refractory period of membrane potential. The addition of the stretching-induced twitch contraction more frequently induced arrhythmias. These results suggest that muscle stretching can induce twitch contraction with a threshold of SL and concern the occurrence of arrhythmias and that SACs and X-ROS signaling play no roles in the induction.

Disturbance of Gastrointestinal Cancers Diagnoses by the COVID-19 Pandemic in a Depopulated Area of Japan: A Population-Based Study in Akita Prefecture.

Disruption of cancer screening programs and diagnoses of gastrointestinal cancers by the COVID-19 pandemic has been reported; however, little attention has been paid to the situation in depopulated areas with low infection rates. Akita Prefecture is one of the most depopulated areas of Japan and has the lowest COVID-19 infection rate per capita; at the same time, the prefecture has been top-ranked for mortality due to gastrointestinal cancer for years. In this population-based study in Akita Prefecture, we investigated the occurrence of gastrointestinal cancers and the number of cancer screening procedures over the five-year period of 2016-2020, employing a database from the collaborative Akita Prefecture hospital-based registration system of cancers. The occurrence of gastrointestinal cancers, especially esophago-gastric cancers, declined by 11.0% in 2020, when the COVID-19 pandemic affected the overall healthcare system, compared with the average of 2016-2019. Nonetheless, the occurrence of advanced-stage (stage IV) esophago-gastric cancers increased by 7.2% in 2020. The decrease in the gastrointestinal cancer diagnosis rate in 2020 coincided with a 30% decline in the total number of regular population-based screening programs. Under the ongoing COVID-19 pandemic, cancer screening was uniformly suspended throughout Japan. Accordingly, the COVID-19 pandemic has substantially disrupted the cancer screening system, leading to delays in diagnoses of gastrointestinal cancer, even in depopulated areas (Akita Prefecture) of Japan with a low prevalence of infection. Suspension of cancer screening procedures during an infectious disease pandemic should be thoroughly considered for each region based on the cancer incidence and infection status in that area.

Effect of transient elevation of glucose on contractile properties in non-diabetic rat cardiac muscle.

In non-diabetic patients with severe disease, such as acute myocardial infarction or acute heart failure, admission blood glucose level is associated with their short-term and long-term mortality. We examined whether transient elevation of glucose affects contractile properties in non-diabetic hearts. Force, intracellular Ca2+ ([Ca2+]i), and sarcomere length were measured in trabeculae from rat hearts. To assess contractile properties, maximum velocity of contraction (Max dF/dt) and minimum velocity of relaxation (Min dF/dt) were calculated. The ratio of phosphorylated troponin I (P-TnI) to troponin I (TnI) was measured. One hour after elevation of glucose from 150 to 400 mg/dL, developed force, Max dF/dt, and Min dF/dt were reduced without changes in [Ca2+]i transients at 2.5 Hz stimulation and 2.0 mM [Ca2+]o, while developed force and [Ca2+]i transients showed no changes at 0.5 Hz stimulation and 0.7 mM [Ca2+]o. In the presence of 1 µM KN-93, a Ca2+/calmodulin-dependent protein kinaseII (CaMKII) inhibitor, or 50 µM diazo-5-oxonorleucine, a L-glutamine-D-fructose-6-phosphate amidotransferase inhibitor, the reduction of contractile properties after elevation of glucose was suppressed. Furthermore, 1 h after elevation of glucose to 400 mg/dL at 2.0 mM [Ca2+]o, the ratio of P-TnI to TnI was increased. These results suggest that in non-diabetic hearts under higher Ca2+-load, transient elevation of glucose for 1 h reduces contractile properties probably by activating CaMKII through O-GlcNAcylation. Thus, in the patients with severe disease, transient elevation of blood glucose, such as due to stress, may worsen cardiac function and thereby affect their mortality without known diabetes.

Different roles of myocardial ROCK1 and ROCK2 in cardiac dysfunction and postcapillary pulmonary hypertension in mice.

Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1-/-) and ROCK2-deficient (cROCK2-/-) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1-/- mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2-/- mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1-/- hearts and down-regulated in cROCK2-/- hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1-/- mice, whereas their expressions were significantly lower in cROCK2-/- mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.

Mechano-electric and mechano-chemo-transduction in cardiomyocytes.

Cardiac excitation-contraction (E-C) coupling is influenced by (at least) three dynamic systems that couple and feedback to one another (see Abstract Figure). Here we review the mechanical effects on cardiomyocytes that include mechano-electro-transduction (commonly referred to as mechano-electric coupling, MEC) and mechano-chemo-transduction (MCT) mechanisms at cell and molecular levels which couple to Ca2+ -electro and E-C coupling reviewed elsewhere. These feedback loops from muscle contraction and mechano-transduction to the Ca2+ homeodynamics and to the electrical excitation are essential for understanding the E-C coupling dynamic system and arrhythmogenesis in mechanically loaded hearts. This white paper comprises two parts, each reflecting key aspects from the 2018 UC Davis symposium: MEC (how mechanical load influences electrical dynamics) and MCT (how mechanical load alters cell signalling and Ca2+ dynamics). Of course, such separation is artificial since Ca2+ dynamics profoundly affect ion channels and electrogenic transporters and vice versa. In time, these dynamic systems and their interactions must become fully integrated, and that should be a goal for a comprehensive understanding of how mechanical load influences cell signalling, Ca2+ homeodynamics and electrical dynamics. In this white paper we emphasize current understanding, consensus, controversies and the pressing issues for future investigations. Space constraints make it impossible to cover all relevant articles in the field, so we will focus on the topics discussed at the symposium.

Transient Elevation of Glucose Increases Arrhythmia Susceptibility in Non-Diabetic Rat Trabeculae With Non-Uniform Contraction.

BACKGROUND: In non-diabetic patients with acute coronary syndrome, stress hyperglycemia occasionally occurs and is related to their mortality. Whether transient elevation of glucose affects arrhythmia susceptibility in non-diabetic hearts with non-uniform contraction was examined.Methods and Results:Force, intracellular Ca2+([Ca2+]i), and membrane potential were measured in trabeculae from rat hearts. Non-uniform contraction was produced by a jet of paralyzing solution. Ca2+waves and arrhythmias were induced by electrical stimulation (2.0 mmol/L [Ca2+]o). The activity of Ca2+/calmodulin-dependent protein kinaseII (CaMKII) was measured. An elevation of glucose from 150 to 400 mg/dL increased the velocity of Ca2+waves and the number of spontaneous action potentials triggered by electrical stimulation. Besides, the elevation of glucose increased the CaMKII activity. In the presence of 1 µmol/L KN-93, the elevation of glucose did not increase the velocity of Ca2+waves and the number of triggered action potentials. In addition, in the presence of 1 µmol/L autocamtide-2 related inhibitory peptide or 50 µmol/L diazo-5-oxonorleucine, the elevation of glucose did not increase the number of triggered action potentials. Furthermore, the elevation of glucose by adding L-glucose did not increase their number. CONCLUSIONS: In non-diabetic hearts with non-uniform contraction, transient elevation of glucose increases the velocity of Ca2+waves by activating CaMKII,probably through glycosylation with O-linked ß-N-acetylglucosamine, thereby increasing arrhythmia susceptibility.

Regional increase in ROS within stretched region exacerbates arrhythmias in rat trabeculae with nonuniform contraction.

In diseased hearts, impaired muscle within the hearts is passively stretched by contractions of the more viable neighboring muscle during the contraction phase. We investigated whether in the myocardium with nonuniform contraction such passive stretch regionally generates ROS within the stretched region and exacerbates arrhythmias. In trabeculae from rat hearts, force, intracellular Ca2+, and membrane potential were measured. To assess regional ROS generation, the slope of the change in the 2',7'-dichlorofluorescein fluorescence (DCFslope) was calculated at the each pixel position along the long axis of trabeculae using DCF fluorescence images. Ca2+ waves and arrhythmias were induced by electrical stimulation. A H2O2 (1 mmol/L) jet regionally increased the DCFslope within the jet-exposed region. A blebbistatin (10 µmol/L) jet caused passive stretch of the muscle within the jet-exposed region during the contraction phase and increased the DCFslope within the stretched region, the velocity of Ca2+ waves, and the number of beats after electrical stimulation (0.2 µmol/L isoproterenol), while 3 µmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, decreased them. A jet of a solution containing 0.2 mmol/L H2O2 in addition to 10 µmol/L blebbistatin also increased them. A H2O2 jet within the region where Ca2+ waves propagated increased their velocity. In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca2+ waves.

Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2+ release system of Purkinje fibers in large mammalian heart.

BACKGROUND: Three distinct Ca2+ release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca2+ release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca2+ cycling of P-cells is unclear. Recent modeling of Ca2+ mobilization in P-cells suggested that Ca2+ sensitivity of Ca2+induced Ca2+release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca2+ release exists in live P-cells. We compared the regional Ca2+ dynamics with the arrangement of intracellular Ca2+ release (CR) channels. METHODS: Gene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca2+ activity and electrically-evoked Ca2+ mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca2+ events. We used the Ca2+ propagation velocities as indicators of channel Ca2+ sensitivity. RESULTS: RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP3R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca2+ release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca2+-transients: (1) large asymmetrical Ca2+ sparks under the membrane, and (2) typical Ca2+-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca2+ waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3µM) and xestospongin C (XeC; 50µM) dramatically reduced their frequency. The Ca2+ wavelets propagated in a 5-10µm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10µM). The velocity of wavelets was two times larger (86±16µm/s; n=14) compared to CWWs' (46±10µm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca2+ concentration under the membrane which preceded the propagation of Ca2+ into the interior of the cell. Elevated Cai propagated at 150µm/s (147±34µm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24µm/s; n=5) in the central region wherein predominant RyR2 expression was detected. CONCLUSION: We identified two layers of distinct Ca2+ release channels in the periphery of Pcell: an outer layer of InsP3Rs under the membrane and an inner layer of RyR3s. The propagation of Ca2+ events in these layers revealed that Ca2+ sensitivity of Ca2+ release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.

Full-length genomic sequence analysis of new subtype 3k hepatitis E virus isolates with 99.97% nucleotide identity obtained from two consecutive acute hepatitis patients in a city in northeast Japan.

Full-length genomic sequences of hepatitis E virus (HEV) obtained from two consecutive cases of acute self-limiting hepatitis E in a city in northeast Japan were determined. Interestingly, two HEV isolates from each patient shared nucleotide identity of 99.97% in 7 225 nucleotides, and a phylogenetic analysis showed that they formed a cluster of Japanese isolates that is considered as a new HEV subtype 3k. The high similarity of HEV sequences of two isolates from these patients in this study suggested that a subtype 3k HEV strain had spread via a commonly distributed food in the city, possibly pig liver.

Identification of Genotype 2 HCV in Serotype-1 Hepatitis C Patients Unresponsive to Daclatasvir plus Asunaprevir Treatment.

It is important to determine the genotypes or serotypes of hepatitis C virus (HCV) in patients before treatment with direct-acting antiviral agents (DAAs), because the effects of DAAs differ among genotypes. In Japan, two tests for HCV typing are available clinically, but only serotyping, not genotyping, is approved by the public health insurance. Although most serotype-1 Japanese patients are infected with genotype 1b HCV, it is known that a small proportion of patients show different results from two typing methods. This study focused on such patients and the effectiveness of treatment with daclatasvir plus asunaprevir (DCV/ASV) was evaluated. We analyzed 644 DCV/ASV-treated patients with serotype 1 or genotype 1b, and among them, 166 serotype-1 patients received a commercial-based direct sequencing (DS) test for resistant-associated variants of genotype 1b HCV. We found four patients (2.4%) with DS test failure, suggesting that the PCR primers targeting genotype 1b may not match. Importantly, none of the four patients achieved a sustained virological response. Our in-house DS test analyzing the 5'-untranslated region and coding regions for NS4 and NS5B of HCV showed that three of the four patients were infected with genotype 2 HCV, and one patient was infected with genotype 1a HCV. No recombinant virus of different genotypes was found. This study indicates that a subset of serotype-1 hepatitis C patients is infected with HCV of genotype 2 or 1a in Japan and that DCV/ASV is not effective for such patients. Thus, attention should be paid to DAA treatment without HCV genotyping.

Shifting hepatitis B virus genotypes of acute hepatitis B patients in northeast Japan.

It has been reported that acute hepatitis B (AHB) patients with genotype A HBV (HBV/A) have been increasing since the 1990s in metropolitan areas in Japan. However, little is known about the trends of HBV genotypes in AHB patients in northeast Japan where genotype B HBV (HBV/B) prevails more than in other areas. In this study, we aimed to clarify the changes in the HBV genotypes and clinical characteristics of AHB patients in this area. HBV genotypes were determined by direct sequencing (n = 125) or enzyme immunoassay (n = 9) using serum samples from AHB patients including fulminant hepatitis in 1987-2014. Among 134 patients, 26 (19%), 33 (25%), and 75 (56%) patients were infected with HBV of genotypes A, B, and C, respectively. HBV/A emerged from 2001 and the percentage was increased gradually up to 48% in 2010-2014, whereas HBV/B was reduced from 40% in 1987-1994 to 10% in 2010-2014. Phylogenetic analysis showed that three major subgenotype A2 strains had come into this area serially. The levels of HBV DNA and prothrombin time were higher in HBV/A patients than other genotypes. This study could not show significant difference in the HBsAg-positive period among genotypes nor the effect of nucleoside analogues to shorten the HBsAg-positive period. A higher level of initial HBV DNA was associated with late disappearance of HBsAg. In conclusion, the percentage of HBV/A in AHB patients has been increasing in northeast Japan since 2001, which is later than metropolitan areas, whereas that of HBV/B is decreasing.

Rho-Kinase Inhibition During Early Cardiac Development Causes Arrhythmogenic Right Ventricular Cardiomyopathy in Mice.

OBJECTIVE: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty changes of the right ventricle, ventricular arrhythmias, and sudden death. Though ARVC is currently regarded as a disease of the desmosome, desmosomal gene mutations have been identified only in half of ARVC patients, suggesting the involvement of other associated mechanisms. Rho-kinase signaling is involved in the regulation of intracellular transport and organizes cytoskeletal filaments, which supports desmosomal protein complex at the myocardial cell-cell junctions. Here, we explored whether inhibition of Rho-kinase signaling is involved in the pathogenesis of ARVC. APPROACH AND RESULTS: Using 2 novel mouse models with SM22α- or αMHC-restricted overexpression of dominant-negative Rho-kinase, we show that mice with Rho-kinase inhibition in the developing heart (SM22α-restricted) spontaneously develop cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, resulting in premature sudden death, phenotypes fulfilling the criteria of ARVC in humans. Rho-kinase inhibition in the developing heart results in the development of ARVC phenotypes in dominant-negative Rho-kinase mice through 3 mechanisms: (1) reduction of cardiac cell proliferation and ventricular wall thickness, (2) stimulation of the expression of the proadipogenic noncanonical Wnt ligand, Wnt5b, and the major adipogenic transcription factor, PPARγ (peroxisome proliferator activated receptor-γ), and inhibition of Wnt/ß-catenin signaling, and (3) development of desmosomal abnormalities. These mechanisms lead to the development of cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, ultimately resulting in sudden premature death in this ARVC mouse model. CONCLUSIONS: This study demonstrates a novel crucial role of Rho-kinase inhibition during cardiac development in the pathogenesis of ARVC in mice.

Effect of Carbenoxolone on Arrhythmogenesis in Rat Ventricular Muscle.

BACKGROUND: Connexin43 (Cx43) is a major connexin that forms gap junction (GJ) channels in the heart and is also present in the cell membrane as unopposed/non-junctional hemichannels and in the inner mitochondrial membrane. By using carbenoxolone (CBX), a blocker of Cx43, the effect of the blockade of Cx43 on Ca(2+)waves and triggered arrhythmias in the myocardium with non-uniform contraction was examined. METHODS AND RESULTS: Trabeculae were obtained from rat hearts. Force, [Ca(2+)]i, and the diffusion coefficient were measured. Non-uniform contraction was produced with a 2,3-butanedione monoxime jet. Ca(2+)waves were induced by electrical stimulation. Inducibility of arrhythmias was estimated based on the minimal [Ca(2+)]oat which arrhythmias were induced. The Ca(2+)spark rate was measured in isolated single rat ventricular myocytes. CBX reduced the GJ permeability, whereas it did not change force and [Ca(2+)]itransients. CBX increased the Ca(2+)leak from the sarcoplasmic reticulum in trabeculae and increased the Ca(2+)spark rate in isolated single myocytes. CBX increased the velocity of Ca(2+)waves and further increased the inducibility of arrhythmias. Modulation of mitochondrial KATPchannels by diazoxide, cromakalim and 5-hydroxydecanoic acid affected the inducibility of arrhythmias increased by CBX. CONCLUSIONS: These results suggest that in diseased hearts, Cx43 plays an important role in the occurrence of triggered arrhythmias, probably under the modulation of mitochondrial KATPchannels.

Effect of myofilament Ca(2+) sensitivity on Ca(2+) wave propagation in rat ventricular muscle.

BACKGROUND: The propagation velocity of Ca(2+) waves determines delayed afterdepolarization and affects the occurrence of triggered arrhythmias in cardiac muscle. We focused on myofilament Ca(2+) sensitivity, investigating how the velocity of Ca(2+) waves responds to its increased sensitivity resulting from muscle stretch or the addition of a myofilament Ca(2+) sensitizer, SCH00013. We further investigated whether production of reactive oxygen species (ROS) may be involved in the change in velocity. METHODS: Trabeculae were obtained from rat hearts. Force, sarcomere length, and [Ca(2+)]i were measured. ROS production was estimated from 2',7'-dichlorofluorescein (DCF) fluorescence. Trabeculae were exposed to a 10 mM Ca(2+) jet for the induction of Ca(2+) leak from the sarcoplasmic reticulum in its exposed region. Ca(2+) waves were induced by 2.5-Hz stimulus trains for 7.5s (24 °C, 2.0 mM [Ca(2+)]o). Muscle stretch of 5, 10, and 15% was applied 300 ms after the last stimulus of the train. RESULTS: Muscle stretch increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca(2+) waves depending on the degree of stretch. After preincubation with 3 µM diphenyleneiodonium (DPI), muscle stretch increased only the amplitude of aftercontractions but not the DCF fluorescence nor the velocity of Ca(2+) waves. SCH00013 (30 µM) increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca(2+) waves. DPI suppressed these increases. CONCLUSIONS: Muscle stretch increases the velocity of Ca(2+) waves by increasing ROS production, not by increasing myofilament Ca(2+) sensitivity. In the case of SCH00013, ROS production increases myofilament Ca(2+) sensitivity and the velocity of Ca(2+) waves. These results suggest that ROS rather than myofilament Ca(2+) sensitivity plays an important role in the determination of the velocity of Ca(2+) waves, that is, arrhythmogenesis.

Role of reactive oxygen species and Ca(2+) dissociation from the myofilaments in determination of Ca(2+) wave propagation in rat cardiac muscle.

Ca(2+) waves are initiated not only by Ca(2+) leak from the sarcoplasmic reticulum (SR), but also by Ca(2+) dissociation from the myofilaments in the myocardium with nonuniform contraction. We investigated whether contractile properties and the production of reactive oxygen species (ROS) affect Ca(2+) wave propagation. Trabeculae were obtained from 76 rat hearts. Force was measured with a strain gauge, sarcomere length with a laser diffraction technique, and [Ca(2+)](i) with fura-2 and a CCD camera (24°C, 2.0mmol/L [Ca(2+)](o)). ROS production was estimated from 2',7'-dichlorofluorescein (DCF) fluorescence. Trabeculae were regionally exposed to a jet of solution containing 1) 10mmol/L Ca(2+) to initiate Ca(2+) waves by SR Ca(2+) leak due to Ca(2+) overload within the jet-exposed region, and 2) 0.2mmol/L Ca(2+) or 5mmol/L caffeine to initiate such waves by Ca(2+) dissociation from the myofilaments due to nonuniform contraction. Ca(2+) waves were induced by stimulus trains for 7.5s. Ten-percent muscle stretch increased DCF fluorescence and accelerated Ca(2+) waves initiated due to both Ca(2+) overload and nonuniform contraction. Preincubation with 3µmol/L diphenyleneiodonium or 10µmol/L colchicine suppressed the increase in DCF fluorescence but suppressed acceleration of Ca(2+) waves initiated only due to Ca(2+) overload. Irrespective of preincubation with colchicine, reduction of force after the addition of 10µmol/L blebbistatin did not decelerate Ca(2+) waves initiated due to Ca(2+) overload, while it did decelerate waves initiated due to nonuniform contraction. These results suggest that Ca(2+) wave propagation is modulated by ROS production through an intact microtubule network only during stretch and may be additionally modulated by Ca(2+) dissociated from the myofilaments in the case of nonuniform contraction.