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Objective. The Japanese guideline for the treatment of chronic constipation recommends that nonpharmacological treatment be applied. However, only a small proportion of patients with constipation seek medical care, and even when they do visit a hospital for constipation in Japan, most are only prescribed medication. This is because the effectiveness of exercise therapy for constipation is still unclear. The purpose of this study was to evaluate the prevalence of constipation in Japanese subjects and the relationship between constipation and exercise. Methods. We conducted a questionnaire survey over a period of four months to determine the prevalence of chronic constipation in nonexercise and exercise groups, as well as the effectiveness of exercise on chronic constipation. Subjective constipation was defined by the constipation scoring system. Multiple regression analysis was performed on risk factors for high constipation scoring system scores, and factors related to constipation symptoms were extracted. Result. We analysed responses regarding 556 participants ranging in mean age 35.6 ± 17.2 years. The constipation scoring system score was significantly higher in the nonexercise group than in the exercise group. Independent predictors for the constipation scoring system score were sex, implementation of exercise, and presence of disease under treatment. Conclusion. The result showed that independent predictors for the constipation scoring system score were sex, implementation of exercise, and presence of disease under treatment. Thus, the present study demonstrated that exercise affects constipation symptom.

Background: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however. Methods: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. Results: We found that cardiac expression of Gα o , an inhibitory G protein encoded in humans by GNAO1 , is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gα o expression increased surface sarcolemmal L-type Ca 2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca 2+ handling in ventricular myocytes, which led to cardiac dysfunction. Conclusions: These findings shed light on a novel function of Gα o in the regulation of cardiac Ca 2+ homeostasis and systolic function and suggest Gα o may be an effective therapeutic target for the treatment of heart failure.

[Purpose] To determine whether short-duration, limited rehabilitation is effective in patients with COVID-19. [Participants and Methods] Single-center, retrospective, observational study. Thirty-six inpatients were classified into the three groups: a close contact (CC) group with a negative polymerase chain reaction (PCR) test (n=14); a PCR-positive (PP) group (n=15); and a PCR-positive and transfer (PT) group with severe COVID-19 patients who were transferred to an acute care hospital for treatment and then returned to our hospital (n=7). Short-duration, limited rehabilitation was provided to the CC and PP groups in isolated rooms by a therapist wearing full personal protective equipment, and we assessed the changes in their activities of daily living. [Results] The patients' clinical characteristics at baseline were similar among the three groups. Functional Independence Measure scores in the CC, PP, and PT groups were not different at baseline (69 ± 29, 53 ± 26, and 63 ± 32), but differed after control of COVID-19 (63 ± 25, 47 ± 24, and 32 ± 19). Multivariate regression analysis showed that the implementation of a customized self-exercise program and the Mini Nutritional Assessment Short-Form at baseline were independently associated with Functional Independence Measure score after control of COVID-19. [Conclusion] These results suggest that even short-duration, limited rehabilitation may be effective for preventing decreases in activities of daily living in patients with COVID-19.

Aims The methodology to distinguish between the heart failure (HF) with recovered ejection fraction (HFrecEF) and those with continuously reduced ejection fraction (EF) (HFcrEF) on admission has not been established. We recently demonstrated that the ratio of plasma levels of pro‐B‐type natriuretic peptide (proBNP) to total BNP (proBNP plus mature BNP) is decreased on admission in patients with mild acute HF, but not in severe acute HF as a compensatory mechanism for activating cyclic GMP via increases of bioactive mature BNP. We aimed to test the hypothesis that the ratio of bioactive mature BNP to total BNP is associated with reverse remodelling capacity in patients with HF with reduced EF. Methods and results Plasma proBNP and total BNP were measured in patients with acute decompensated HF by using specific and sensitive enzyme immunochemiluminescent assay. Estimated percent mature BNP (%emBNP) was calculated as ([total BNP − proBNP]/total BNP) × 100. We retrospectively identified the patients with reduced EF (≤40%, on admission) who had echocardiographic data after discharge (n = 93). We defined patients with increased EF by >10% during the follow‐up term (median, 545 days) after the admission as HFrecEF group. We compared patient characteristics, %emBNP, and other biomarkers between HFrecEF and HFcrEF. Of the enrolled patients with HFrecEF (n = 32) and HFcrEF (n = 61), on admission, %emBNP was significantly higher in HFrecEF than in HFcrEF (44.1% vs. 36.9%; P

B-type (or brain) natriuretic peptide (BNP) is synthesized in cardiac myocytes and released constitutively into the circulation. Pressure/volume overload, neurohumoral factors, cytokines, and ischemia enhance BNP gene expression, and then precursor proBNP is produced. It has been thought that proBNP is cleaved into active BNP molecule and inactive marker molecule NT-proBNP intracellularly by processing enzyme furin, and they are released into the circulation. However, recent studies have shown that considerable amount of uncleaved proBNP circulates in the blood. The commercially available BNP assay kits consist of two antibodies that sandwich the BNP molecule. Therefore, if proBNP is present, BNP assay kit cross-reacts to proBNP and measures it as BNP. Therefore, it should be noted that the current BNP value is proBNP plus BNP. BNP and NT-proBNP have been established as a biomarker for heart failure patients presenting dyspnea. But many pitfalls are present for interpreting the BNP value. For example, the presence of renal dysfunction, age, female sex, atrial fibrillation, inflammation, hyperthyroidism, use of sacubitril/valsartan, and macro-proBNPemia overestimate BNP value, whereas the presence of obesity, immediately after acute coronary syndrome onset, and pericardial effusion underestimate BNP value. In the management for heart failure patients, BNP plays an important role. Therefore, clinicians should note the pitfall of interpretation of BNP and we describe the mechanism involved.

Immunosenescence can negatively affect cytokine production in elderly and may impair poor antibody responses to influenza vaccination and infection. Herein, the effects of Banafine® administration on influenza vaccine antibody titer in elderly patients (average age ∼80 years) receiving gastrostomy tube feeding were examined. In the double-blind, single-center, randomized clinical studies, 30 elderly bedridden patients were administered Banafine® or placebo for 8 weeks. At week 4, all patients received influenza vaccination against H1N1, H3N2, B/Yamagata, or B/Victoria. Blood biochemical indices and serum antibody titers were assessed. Banafine® administration significantly increased hemagglutination inhibition titers in response to vaccination against H1N1, H3N2, and B/Yamagata in the elderly patients (P < 0.05). Moreover, the seroconversion rate against H1N1 (47.1%) and H3N2 (29.4%) and seroprotection rate against H1N1 (71.4%) and both B strains (31.3% and 12.5%, respectively) were increased for the Banafine® group. These results suggest that Banafine® administration can increase antibody responses to influenza vaccination in bedridden hospitalized patients, and potentially modulate immune function in the elderly. PRACTICAL APPLICATION: Literature review suggested that most of the synbiotics are based on innate immunity, strain specific (probiotics), and are not consistently observed. Herein, in clinical studies we demonstrate that administration of Banafine® , a plant-based glycoconjugate, can increase antibody levels in bedridden hospitalized elderly patients following influenza vaccination.

During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however.We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant.We found that cardiac expression of GαThese findings shed light on a novel function of Gα

The heart has long been considered a pumping organ, consisting of muscles [...]

BACKGROUND & AIMS Heart failure and malnutrition are known to each negatively affect a patient's ability to improve their activities of daily living (ADL) through rehabilitation. Here, we investigated whether the negative effects of malnutrition and heart failure on ADL are additive in patients after hip fracture. METHODS This retrospective observational cohort study included 155 patients with hip fracture admitted to convalescent rehabilitation wards. Nutritional status was measured with the Geriatric Nutritional Risk Index (GNRI). Heart failure was assessed using plasma B-type natriuretic peptide (BNP) levels. Based on their GNRIs and BNP levels, patients were classified into four groups: a high GNRI (≥92)-low BNP (

Immunosenescence can negatively affect cytokine production in elderly and may impair poor antibody responses to influenza vaccination and infection. Herein, the effects of Banafine

Several studies have shown that nutrition and muscle strength were associated with functional recovery in patients with hip fracture. However, the impact of heart failure on the improvement of activity of daily living (ADL) in patients with hip fracture have not been fully investigated. The purpose was investigating the effect of heart failure on the ADL improvement by rehabilitation in patients with hip fracture. A total of 116 patients with hip fracture discharged from our convalescent rehabilitation ward were studied. Heart failure was assessed based on plasma B-type natriuretic peptide (BNP) levels on admission. ADL was assessed based on rehabilitation effectiveness (REs), which was calculated using the FIM instrument. Clinical, demographic, and nutritional variables were measured. Multiple regression analysis was performed with REs as the dependent variable; variables showing significant correlation with REs in univariate analyses were selected as independent variables. Based on plasma BNP levels, we assigned 39 patients to a Low group: 22 (17-25) median (interquartile) pg/mL, 39 to a Middle group: 52 (42-65) pg/mL, and 38 to a High group: 138 (93-209) pg/mL. REs, handgrip strength, Hb, albumin, and GNRI were higher and age was younger in the Low group than High group (each p < 0.01, respectively). Multiple linear regression analysis revealed that age (p < 0.05), sex (p < 0.05), handgrip strength (p < 0.01), FOIS at admission (p < 0.01), rehabilitation time per day (p < 0.01), and BNP (p < 0.05) were significantly associated with REs. The effect of rehabilitation on ADL improvement was significantly blunted in the High group compared to the Low group. In conclusion, these results suggest that heart failure assessed based on plasma BNP levels negatively impacts improvements in ADL achieved through rehabilitation in patients with hip fracture.

Aim: Measurement of B-type natriuretic peptide (BNP) is widely used as a diagnostic and risk assessment tool for cardiovascular disease. Recent studies have demonstrated that BNP-32 and its precursor proBNP circulate in the blood stream, and that most commercial BNP immunoassays measure both forms. However, recombinant or synthetic BNP-32 is used as the standard for those BNP immunoassays. This gap between clinical samples and the standard might be a potential source of variation in BNP measurements among assays. The purpose of this study is to validate a more suitable calibrator for BNP immunoassays. Methods: External BNP calibrators containing both BNP-32 and glycosylated proBNP were prepared at two concentration levels. Target BNP concentrations of the low and high levels were 40 and 160 pg/mL, respectively. And to reflect clinical samples, the molar ratios of BNP-32 to glycosylated proBNP in these concentration levels were adjusted to 50:50 and 25:75, respectively. BNP concentrations of plasma samples along with the external BNP calibrators were measured at two commercial labs and using an automated analyzer MI02 Shionogi® BNP (MI02). These samples and the calibrators were also measured using an immunoradiometric assay (IRMA) as a standard assay procedure. Concentrations of the plasma samples measured at the labs or using the MI02 were adjusted according to a comparison of the measured concentrations of the external BNP calibrators with the IRMA. Results: After measured concentrations of the plasma samples were adjusted using the external BNP calibrators, the correlation between each measurement and the IRMA was improved. The range of the slopes according to Passing-Bablok regression analysis narrowed from 0.628 - 0.955 to 0.911 - 1.005. Conclusions: Our data suggests that a mixture of BNP-32 and glycosylated proBNP at different ratios by concentration level is suitable for calibration to minimize variations in BNP measurements among immunoassays.

The natriuretic peptide family consists of three biologically active peptides: atrial natriuretic peptide (ANP), brain (or B-type) natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Among these, ANP and BNP are secreted by the heart and act as cardiac hormones. Both ANP and BNP preferentially bind to natriuretic peptide receptor-A (NPR-A or guanylyl cyslase-A) and exert similar effects through increases in intracellular cyclic guanosine monophosphate (cGMP) within target tissues. Expression and secretion of ANP and BNP are stimulated by various factors and are regulated via multiple signaling pathways. Human ANP has three molecular forms, α-ANP, β-ANP, and proANP (or γ-ANP), with proANP predominating in healthy atrial tissue. During secretion proANP is proteolytically processed by corin, resulting in secretion of bioactive α-ANP into the peripheral circulation. ProANP and β-ANP are minor forms in the circulation but are increased in patients with heart failure. The human BNP precursor proBNP is proteolytically processed to BNP1-32 and N-terminal proBNP (NT-proBNP) within ventricular myocytes. Uncleaved proBNP as well as mature BNP1-32 and NT-proBNP is secreted from the heart, and its secretion is increased in patients with heart failure. Mature BNP, its metabolites including BNP3-32, BNP4-32, and BNP5-32, and proBNP are all detected as immunoreactive-BNP by the current BNP assay system. We recently developed an assay system that specifically detects human proBNP. Using this assay system, we observed that miR30-GALNTs-dependent O-glycosylation in the N-terminal region of proBNP contributes to regulation of the processing and secretion of proBNP from the heart.

Brain (or B-type) natriuretic peptide (BNP) is a cardiac hormone produced in the heart and an established biochemical marker for heart failure (HF) because the level in plasma increases in proportion to disease severity. Recently, the diversity of BNP molecular forms in the peripheral circulation, which includes mature BNP (BNP1-32) and its metabolites (BNP3-32, BNP4-32, and BNP5-32), was demonstrated. Moreover, studies showed that unprocessed BNP prohormone (proBNP) is also secreted from the heart, and its secretion is increased in patients with HF. Interestingly, BNP1-32, its metabolites, and proBNP are all detected as immunoreactive BNP by the currently available BNP assay system. Current N-terminal proBNP (NT-proBNP) assay systems also can react to both NT-proBNP and proBNP. In addition, the N-terminal region of proBNP and NT-proBNP are often O-glycosylated, which may result in underestimation of total NT-proBNP level, which includes both glycosylated and non-glycosylated NT-proBNP, by the NT-proBNP assay system. More recently, we have shown that miR30-GALNT-dependent O-glycosylation in the N-terminal region of proBNP affects the processing of proBNP and contributes to its secretion from the heart. The level of proBNP relative to BNP (proBNP/BNP ratio) in the coronary sinus is higher in patients with more severe HF. The proBNP/BNP ratio and the deglycosylated NT-proBNP level may be new and clinically useful biomarkers of HF.

A recent study showed that both glycosylation of pro-B-type natriuretic peptide (BNP) and the proBNP/total BNP ratio are decreased in acute decompensated heart failure (ADHF). However, the following points regarding the proBNP/total BNP ratio have not been determined in patients with ADHF: 1) the relationship with the severity of ADHF, 2) the changes in the ratio during treatment, and 3) the relationship with cyclic guanosine monophosphate (cGMP)-generating activity.Plasma proBNP and total BNP (proBNP+mature BNP) were measured in patients with ADHF (n=154). Measurement was performed on admission, 3 and 7days after admission, and before discharge using recently developed sandwich chemiluminescence enzyme immunoassays. The percent proBNP was calculated as: (proBNP/total BNP)×100.On admission, %proBNP was higher in patients with severe ADHF than in patients with mild ADHF (median: 61.7% vs. 56.2%, respectively; p0.01), while the plasma cGMP/total BNP ratio, an index of the biological activity of BNP, was lower (p0.001). In patients with severe ADHF, the higher %proBNP and lower cGMP/total BNP ratio were unchanged during hospitalization, whereas %proBNP increased gradually in patients with mild ADHF and the cGMP/total BNP ratio also increased at 3days after admission.These findings suggest that in patients with mild ADHF, compensation for heart failure occurs via increased proBNP processing, leading to increase of mature BNP and activation of the BNP/cGMP cascade. In contrast, this compensatory mechanism may be impaired in patients with severe ADHF and a vicious cycle can potentially occur.

B-type natriuretic peptide (BNP) is a cardiac hormone widely used as a biomarker for heart failure. Here, we present the first report of extremely high levels of immunoreactive BNP caused by formation of macro-proBNP. A 70-year-old woman with left ventricular hypertrophy and normal systolic function presented with extremely high plasma levels of BNP (35,374pg/ml) and N-terminal proBNP (NT-proBNP; 30,600pg/ml). Our recently developed proBNP immunoassay showed that nearly 100% of her immunoreactive BNP was proBNP. Polyethylene glycol precipitation tests reported extremely low BNP recovery (1.3%), while protein G addition tests also reported a remarkably low BNP fraction (3.3%). Gel filtration chromatography with normal elution buffer combined with BNP immunoassays showed a BNP peak with a retention time slightly shorter than that of IgG. With acidic elution buffer (pH3.0), however this peak disappeared and a new BNP peak consistent with glycosylated human proBNP appeared. These results suggest that in this case most BNP immunoreactivity consisted of macro-proBNP, which is an immune complex composed of proBNP and an anti-proBNP autoantibody. Gel filtration chromatography combined with NT-proBNP immunoassays revealed that the NT-proBNP assay cross-reacts with both the proBNP-IgG complex and proBNP. In addition, with acidic buffer, a new large peak appeared with a retention time the same as that of glycosylated NT-proBNP. These results suggest spuriously high levels of BNP and NT-proBNP are caused by macro-proBNP. Macro-NT-proBNP is not detected by the currently available NT-proBNP assay system.

Adrenomedullin (AM) is a vasodilatory peptide originally discovered in human pheochromocytoma tissue. Although AM is highly expressed in the adrenal glands, heart, lungs, and kidneys, vascular endothelium and smooth muscle are thought to be the main source of plasma AM. The AM precursor is processed to AM-glycine, which is then converted to AM-mature through C-terminal amidation. In this process, mid-regional pro-adrenomedullin (MR-proAM) is also produced. Plasma AM, AM-mature, AM-glycine, and MR-proAM levels are all higher in patients with heart failure than healthy subjects in proportional to the disease severity. All molecular forms of AM are prognostic markers for heart failure.

We previously reported the secretion of C-type natriuretic peptide (CNP) from vascular endothelial cells and proposed the existence of a vascular natriuretic peptide system composed of endothelial CNP and smooth muscle guanylyl cyclase-B (GC-B), the CNP receptor, and involved in the regulation of vascular tone, remodeling, and regeneration. In this study, we assessed the functional significance of this system in the regulation of blood pressure in vivo using vascular endothelial cell–specific CNP knockout and vascular smooth muscle cell–specific GC-B knockout mice. These mice showed neither the skeletal abnormality nor the early mortality observed in systemic CNP or GC-B knockout mice. Endothelial cell–specific CNP knockout mice exhibited significantly increased blood pressures and an enhanced acute hypertensive response to nitric oxide synthetase inhibition. Acetylcholine-induced, endothelium-dependent vasorelaxation was impaired in rings of mesenteric artery isolated from endothelial cell–specific CNP knockout mice. In addition, endothelin-1 gene expression was enhanced in pulmonary vascular endothelial cells from endothelial cell–specific CNP knockout mice, which also showed significantly higher plasma endothelin-1 concentrations and a greater reduction in blood pressure in response to an endothelin receptor antagonist than their control littermates. By contrast, vascular smooth muscle cell–specific GC-B knockout mice exhibited blood pressures similar to control mice, and acetylcholine-induced vasorelaxation was preserved in their isolated mesenteric arteries. Nonetheless, CNP-induced acute vasorelaxation was nearly completely abolished in mesenteric arteries from vascular smooth muscle cell–specific GC-B knockout mice. These results demonstrate that endothelium-derived CNP contributes to the chronic regulation of vascular tone and systemic blood pressure by maintaining endothelial function independently of vascular smooth muscle GC-B.

Background We previously demonstrated that a transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability. Transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg) exhibit systolic dysfunction with cardiac dilation and premature death due to lethal arrhythmias like human dilated cardiomyopathy (DCM). Underlining mechanisms remain to be elucidated, however. Purpose We studied underling mechanisms by which NRSF maintains normal cardiac function to identify novel therapeutic targets for heart failure. Methods and results We generated cardiac-specific NRSF knockout mice (NRSFcKO) and confirmed that cardiac phenotypes of NRSFcKO are similar to those of dnNRSF-Tg. cDNA microarray analysis revealed that cardiac gene expression of GNAO1 that encodes Gαo, a member of inhibitory G protein Gαi family, is increased in both dnNRSF-Tg and NRSFcKO ventricles. We confirmed that GNAO1 is a direct target of NRSF through ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay. In dnNRSF-Tg, pharmacological inhibition of Gαo with pertussis toxin improved systolic dysfunction and knockdown of Gαo by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates. Electrophysiological and biochemical analysis in ventricular myocytes obtained from dnNRSF-Tg demonstrated that genetic reduction of Gαo ameliorated abnormalities in Ca2+ handling, which include increased current density in surface sarcolemmal L-type Ca2+ channel, reduced content of sarcoplasmic reticulum Ca2+ and lowered peak of Ca2+ transient. Furthermore, genetic reduction of Gαo attenuated increased phosphorylation levels of CAMKII in dnNRSF-Tg ventricles, which presumably underlies the improvement in Ca2+ handling. In addition, we identified increased Gαo expression in ventricles of heart failure model mice induced by transverse aortic constriction and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gαo ameliorated cardiac dysfunction. Figure 1 Conclusions We found that increased expression of Gαo, induced by attenuation of NRSF-mediated repression, plays a crucial role in the progression of cardiac dysfunction and lethal arrhythmias by evoking Ca2+ handling abnormality. These data demonstrate that Gαo is a potential therapeutic target for heart failure.

Background Early detection for worsening renal function ( WRF ) is indispensable in patients with acute decompensated heart failure (HF). We tested the hypothesis that the difference in the circulating levels of each B‐type or brain natriuretic peptide ( BNP ) molecular form is associated with the occurrence of WRF . Methods and Results Circulating levels of pro BNP , the NT‐proBNP (N‐terminal pro BNP ), and total BNP (pro BNP +mature BNP ) were prospectively measured in patients with acute decompensated HF using specific and sensitive enzyme immunochemiluminescent assays. An estimated mature BNP (em BNP ) concentration was calculated by subtracting pro BNP levels from total BNP levels. WRF was defined as a >20% decrease in the estimated glomerular filtration rate during the hospitalization. One‐way repeated‐measures ANOVA was used to compare the changes of variables between the patients with and without WRF . In patients with acute decompensated HF (New York Heart Association class III – IV ; 96%) hospitalized for HF , NT ‐pro BNP levels did not differ during the hospitalization between patients with and without WRF (n=42 and 140, respectively). By contrast, em BNP levels were lower in patients with WRF than in those without WRF on day 3 after admission. NT ‐pro BNP /em BNP molar ratios were elevated on day 3 after admission in the patients with WRF , before estimated glomerular filtration rate declined, but were unchanged in patients without WRF . On day 3 after hospital admission, NT ‐pro BNP /em BNP ratios were strongly associated with percentage decreases in estimated glomerular filtration rate. Conclusions These findings suggest that elevation of NT ‐pro BNP /em BNP ratio precedes WRF in patients with acute HF and can be a potentially useful biomarker for risk stratification of cardiorenal syndrome.

Background: A transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability. Transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg) exhibit systolic dysfunction and premature death due to lethal arrhythmias like human dilated cardiomyopathy(DCM). Underlining mechanisms remain to be elucidated, however. Purpose: To clarify how NRSF maintains normal cardiac homeostasis. Methods and Results: cDNA microarray analysis of dnNRSF-Tg and newly generated cardiac-specific NRSF knockout mice (NRSFcKO), which show similar cardiac phenotypes to those of dnNRSF-Tg, revealed that cardiac gene expression of GNAO1 that encodes Gα o , a member of inhibitory G protein Gα i family, is increased in both dnNRSF-Tg and NRSFcKO ventricles. ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that GNAO1 is a direct target of NRSF. In dnNRSF-Tg, pharmacological inhibition of Gα o by pertussis toxin improved systolic dysfunction and knockdown of Gα o by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates. Electrophysiological analysis in ventricular myocytes obtained from dnNRSF-Tg demonstrated that genetic reduction of Gα o ameliorated abnormalities in Ca 2 + handling, which include increased current density in surface sarcolemmal L-type Ca 2 + channel, reduced content of sarcoplasmic reticulum Ca 2+ and lowered peak of Ca 2+ transient. In addition, genetic reduction of Gα o attenuated increased phosphorylation levels of CAMKII and RyR2 in dnNRSF-Tg ventricles, which presumably underlies the improvement in Ca 2 + handling. Furthermore, we identified increased Gα o expression in ventricles of heart failure model mice induced by transverse aortic constriction and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gα o ameliorated cardiac dysfunction. Conclusions: We found that increased expression of Gα o , induced by attenuation of NRSF-mediated repression, plays a pivotal role in the progression of heart failure by evoking Ca 2+ handling abnormality. Gα o is a potential therapeutic target for heart failure.

AimsThere are significant differences in how atrial (A-type) and B-type natriuretic peptide (ANP and BNP) are secreted and metabolised, but there is little information available about the relative clinical significance of the two peptides. The aim of the present study was to investigate: (1) the association between the circulating level of each ANP molecular form and patient clinical background and (2) their prognostic power for patients with acute decompensated heart failure (ADHF).MethodsWe used specific chemiluminescence enzyme immunoassays to prospectively evaluate the levels of six bioactive molecular forms of ANP (pro-ANP, β-ANP and total ANP) and BNP (pro-BNP, N-terminal pro-BNP (NT-pro-BNP) and total BNP) in plasma samples collected from 173 patients with ADHF on their hospital admission.ResultsWe found that pro-ANP levels were strongly associated with left ventricular (LV) size and ejection fraction (pConclusionCirculating levels of pro-ANP are strongly associated with LV function and clinical outcomes of patients with ADHF. These findings suggest that during the acute phases of HF, pro-ANP has a prognostic power comparable with NT-pro-BNP independently of renal function.

[Aims]The progression of pathological left ventricular remodelling leads to cardiac dysfunction and contributes to the occurrence of malignant arrhythmias and sudden cardiac death. The underlying molecular mechanisms remain unclear, however. Our aim was to examine the role of the renin–angiotensin system (RAS) in the mechanism underlying arrhythmogenic cardiac remodelling using a transgenic mouse expressing a cardiac-specific dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). This mouse model exhibits progressive cardiac dysfunction leading to lethal arrhythmias. [Methods and results]Subcutaneous administration of aliskiren, a direct renin inhibitor, significantly suppressed the progression of pathological cardiac remodelling and improved survival among dnNRSF-Tg mice while reducing arrhythmogenicity. Genetic deletion of the angiotensin type 1a receptor (AT1aR) similarly suppressed cardiac remodelling and sudden death. In optical mapping analyses, spontaneous ventricular tachycardia (VT) and fibrillation (VF) initiated by breakthrough-type excitations originating from focal activation sites and maintained by functional re-entry were observed in dnNRSF-Tg hearts. Under constant pacing, dnNRSF-Tg hearts exhibited markedly slowed conduction velocity, which likely contributes to the arrhythmogenic substrate. Aliskiren treatment increased conduction velocity and reduced the incidence of sustained VT. These effects were associated with suppression of cardiac fibrosis and restoration of connexin 43 expression in dnNRSF-Tg ventricles. [Conclusion]Renin inhibition or genetic deletion of AT1aR suppresses pathological cardiac remodelling that leads to the generation of substrates maintaining VT/VF and reduces the occurrence of sudden death in dnNRSF-Tg mice. These findings demonstrate the significant contribution of RAS activation to the progression of arrhythmogenic substrates.

BackgroundRecent studies have shown that plasma levels of the biologically inactive prohormone for brain natriuretic peptide (proBNP) are increased in patients with heart failure. This can contribute to a reduction in the effectiveness of circulating BNP and exacerbate heart failure progression. The precise mechanisms governing the increase in proBNP remain unclear, however.Methods and ResultsWe used our recently developed, highly sensitive human proBNP assay system to investigate the mechanisms underlying the increase in plasma proBNP levels. We divided 53 consecutive patients hospitalized with heart failure into 2 groups based on their aortic plasma levels of immunoreactive BNP. Patients with higher levels exhibited more severe heart failure, a higher proportion of proBNP among the immunoreactive BNP forms secreted from failing hearts, and a weaker effect of BNP as estimated from the ratio of plasma cyclic guanosine monophosphate levels to log‐transformed plasma BNP levels. Glycosylation at threonines 48 and 71 of human proBNP contributed to the increased secretion of proBNP by attenuating its processing, and GalNAc‐transferase (GALNT) 1 and 2 mediated the glycosylation‐regulated increase in cardiac human proBNP secretion. Cardiac GALNT1 and 2 expression was suppressed by microRNA (miR)‐30, which is abundantly expressed in the myocardium of healthy hearts, but is suppressed in failing hearts.ConclusionsWe have elucidated a novel miR‐30‐GALNT1/2 axis whose dysregulation increases the proportion of inactive proBNP secreted by the heart and impairs the compensatory actions of BNP during the progression of heart failure., Article, JOURNAL OF THE AMERICAN HEART ASSOCIATION. 6(2): e003601(2017)

Aims Dysregulation of autonomic nervous system activity can trigger ventricular arrhythmias and sudden death in patients with heart failure. N-type Ca2+ channels (NCCs) play an important role in sympathetic nervous system activation by regulating the calcium entry that triggers release of neurotransmitters from peripheral sympathetic nerve terminals. We have investigated the ability of NCC blockade to prevent lethal arrhythmias associated with heart failure. Methods and results We compared the effects of cilnidipine, a dual N- and L-type Ca2+ channel blocker, with those of nitrendipine, a selective L-type Ca2+ channel blocker, in transgenic mice expressing a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). In this mouse model of dilated cardiomyopathy leading to sudden arrhythmic death, cardiac structure and function did not significantly differ among the control, cilnidipine, and nitrendipine groups. However, cilnidipine dramatically reduced arrhythmias in dnNRSF-Tg mice, significantly improving their survival rate and correcting the imbalance between cardiac sympathetic and parasympathetic nervous system activity. A β-blocker, bisoprolol, showed similar effects in these mice. Genetic titration of NCCs, achieved by crossing dnNRSF-Tg mice with mice lacking CACNA1B , which encodes the α1 subunit of NCCs, improved the survival rate. With restoration of cardiac autonomic balance, dnNRSF-Tg; CACNA1B +/− mice showed fewer malignant arrhythmias than dnNRSF-Tg; CACNA1B +/+ mice. Conclusions Both pharmacological blockade of NCCs and their genetic titration improved cardiac autonomic balance and prevented lethal arrhythmias in a mouse model of dilated cardiomyopathy and sudden arrhythmic death. Our findings suggest that NCC blockade is a potentially useful approach to preventing sudden death in patients with heart failure.

Background and Purpose Angiotensin II has been implicated in the development of various cardiovascular ailments, including cardiac hypertrophy and heart failure. The fact that inhibiting its signalling reduced the incidences of both sudden cardiac death and heart failure in several large-scale clinical trials suggests that angiotensin II is involved in increased cardiac arrhythmogenicity during the development of heart failure. However, because angiotensin II also promotes structural remodelling, including cardiomyocyte hypertrophy and cardiac fibrosis, it has been difficult to assess its direct contribution to cardiac arrhythmogenicity independently of the structural effects. Experimental Approach We induced cardiac hypertrophy in wild-type (WT) and angiotensin II type 1a receptor knockout (AT1aR-KO) mice by transverse aortic constriction (TAC). The susceptibility to ventricular tachycardia (VT) assessed in an in vivo electrophysiological study was compared in the two genotypes. The effect of acute pharmacological blockade of AT1R on the incidences of arrhythmias was also assessed. Key Results As described previously, WT and AT1aR-KO mice with TAC developed cardiac hypertrophy to the same degree, but the incidence of VT was much lower in the latter. Moreover, although TAC induced an increase in tyrosine phosphorylation of connexin 43, a critical component of gap junctional channels, and a reduction in ventricular levels of connexin 43 protein in both genotypes, the effect was significantly ameliorated in AT1aR-KO mice. Acute pharmacological blockade of AT1R also reduced the incidence of arrhythmias. Conclusions and Implications Our findings demonstrate that AT1aR-mediated signalling makes a direct contribution to the increase in arrhythmogenicity in hypertrophied hearts independently of structural remodelling.

Background: Growth promoting signals from the epicardium are essential for driving myocardial proliferation during embryogenesis. In adults, these signals become reactivated following injury and promote angiogenesis and myocardial repair. Therefore, identification of such paracrine factors could lead to novel therapeutic strategies. The multi-functional peptide adrenomedullin (Adm = gene, AM = protein) is required for normal heart development. Moreover, elevated plasma AM following myocardial infarction offers beneficial cardioprotection and serves as a powerful diagnostic and prognostic indication of disease severity. Results: Here, we developed a new model of Adm overexpression by stabilizing the Adm mRNA through gene-targeted replacement of the endogenous 3′ untranslated region. As expected, Admhi/hi mice express three-times more AM than controls in multiple tissues, including the heart. Despite normal blood pressures, Admhi/hi mice unexpectedly showed significantly enlarged hearts due to increased cardiac hyperplasia during development. The targeting vector was designed to allow for reversion to wild-type levels by means of Cre-mediated modification. Using this approach, we demonstrate that AM derived from the epicardium, but not the myocardium or cardiac fibroblast, is responsible for driving cardiomyocyte hyperplasia. Conclusions: AM is produced by the epicardium and drives myocyte proliferation during development, thus representing a novel and clinically relevant factor potentially related to mechanisms of cardiac repair after injury. Developmental Dynamics 243:243–256, 2014. © 2013 Wiley Periodicals, Inc.

Adrenomedullin (AM) is a potent long-acting vasodilatory peptide originally discovered in the acid extract of human pheochromocytoma tissue. Adrenomedullin and its mRNA are widely distributed in the cardiovascular system, including the ventricles, atria, blood vessels, lungs, and kidneys. The components of the AM receptor, which include calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP) 2 and RAMP3, colocalize with AM in the cardiovascular system, suggesting AM serves as an important regulator of cardiovascular function. Adrenomedullin gene expression and/or immunoreactivity are increased in the ventricles of hypertrophied and failing hearts and after myocardial infarction, as are the mRNA levels of CLR, RAMP2, and RAMP3. In vitro, cardiomyocytes and cardiofibroblasts produce and secrete two molecular forms of AM, AM-mature and AM-glycine, and express CLR, RAMP2, and RAMP3. Within the heart, AM has inhibitory effects on collagen synthesis and fibroblast proliferation. In addition, AM inhibits cardiac hypertrophy and exhibits antiapoptotic, angiogenic, antiinflammatory, and antioxidant effects. Adrenomedullin also exerts inotropic effects in vitro and in vivo. Cellular signaling by AM is mediated via cyclic AMP/protein kinase A, nitric oxide/cyclic GMP, phosphoinositide 3-kinase/Akt, and/or the extracellular-signaling-regulated kinase pathway. It thus appears that AM may exert antifibrotic, antihypertrophic, and positive inotropic effects in heart failure, myocardial infarction and myocardial ischemia/reperfusion. Several pilot studies have now demonstrated the beneficial effects of AM in patients with heart failure, acute myocardial infarction, and pulmonary hypertension. Clinical trials also showed the diverse biological effects of AM.

Background: It has been reported that biological inactive pro-brain natriuretic peptide(proBNP), precursor peptide of bioactive BNP, is increased in patients with heart failure, as well as mature BNP-32. However, underlying mechanisms of increased plasma proBNP levels in heart failure remain unclear. Methods: We used the conventional BNP assay system, which detects both proBNP and BNP-32, and our recently developed specific human proBNP assay system. We assessed cardiac production of proBNP and BNP(=proBNP+BNP-32) in patients with heart failure. To elucidate the molecular mechanisms underlying the processing and secretion of proBNP, we generated lentivirus vectors carrying several mutations in glycosylation sites of human proBNP. We evaluated the proBNP/BNP ratio, as an efficacy of processing of proBNP to BNP-32. Results: In coronary sinus, plasma proBNP/BNP ratios were significantly elevated in heart failure patients in accordance with the disease severity. In conditioned medium of cultured ventricular myocytes expressing wild type human proBNP, proBNP/BNP ratio was 37%, while in those of myocytes expressing glycosylation-resistant mutant of proBNP, proBNP/BNP ratio was only 3%, demonstrating the essential contribution of glycosylation to the secretion of proBNP. Among 7 glycosylation sites, we found that glycosylation of Thr48 and Thr71 in proBNP is critical for the secretion of proBNP from ventricular myocytes by attenuating proBNP processing. GalNAc-transferase(GALNT) is the largest glycosyltransferase enyme family, which controll O-glycosylasion. We identified GALNT 1 and 2 mediated the glycosylation-regulated increase in cardiac proBNP secretion by screening with siRNAs. Indeed, GALNT1 and 2-expression was increased in FCS-treated ventricular myocytes and failing hearts. We further found that GALNT1 and 2 expression is suppressed by miR-30 family, and that miR-30s expression is decreased in FCS-treated ventricular myocytes and failing hearts. Conclusions: We have elucidated novel molecular pathways that miR-30-GALNTs axis-mediated O -glycosylation in proBNP contributes to increased proportion of proBNP among secreted BNP in the pathological myocardium.

Myocardin-related transcription factor (MRTF)-A is a Rho signalling-responsive co-activator of serum response factor (SRF). Here, we show that induction of MRTF-A expression is key to pathological vascular remodelling. MRTF-A expression was significantly higher in the wire-injured femoral arteries of wild-type mice and in the atherosclerotic aortic tissues of ApoE−/− mice than in healthy control tissues, whereas myocardin expression was significantly lower. Both neointima formation in wire-injured femoral arteries in MRTF-A knockout (Mkl1−/−) mice and atherosclerotic lesions in Mkl1−/−; ApoE−/− mice were significantly attenuated. Expression of vinculin, matrix metallopeptidase 9 (MMP-9) and integrin β1, three SRF targets and key regulators of cell migration, in injured arteries was significantly weaker in Mkl1−/− mice than in wild-type mice. In cultured vascular smooth muscle cells (VSMCs), knocking down MRTF-A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF-A expression in dedifferentiated VSMCs was the downregulation of microRNA-1. Moreover, the MRTF-A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. MRTF-A could thus be a novel therapeutic target for the treatment of vascular diseases.

Reactivation of the fetal cardiac gene program in adults is a reliable marker of cardiac hypertrophy and heart failure. Normally, genes within this group are expressed in the fetal ventricles during development, but are silent after birth. However, their expression is re-induced in the ventricular myocardium in response to various cardiovascular diseases, and potentially plays an important role in the pathological process of cardiac remodeling. Thus, analysis of the molecular mechanisms that govern the expression of fetal cardiac genes could lead to the discovery of transcriptional regulators and signaling pathways involved in both cardiac differentiation and cardiac disease. In this review we will summarize what is currently known about the transcriptional regulation of the fetal cardiac gene program. Keywords:: transcription, cardiac remodeling, fetal cardiac gene, heart failure

Many neurohumoral factors play an important role in regulation of the cardiovascular system and in the pathophysiology of heart failure. Adrenomedullin (AM) is a potent long-lasting vasodilatory peptide that was discovered in acid extracts of human pheochromocytoma tissues. Both AM and its gene expression are widely distributed in the cardiovascular system, including the heart, vessels, and kidneys. AM co-localizes with its receptor components such as calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP)2 and RAMP3 in the heart, vessels and kidneys, suggesting that it plays an important role in the regulation of cardiovascular function through an autocrine and/or paracrine mechanism. AM has inotropic action in vitro and in vivo and inhibits cardiac hypertrophy in myocytes as well as proliferation and collagen production in cardiac fibroblasts. These results suggest that AM functions as an antifibrotic, antihypertrophic and positive inotropic factor in the failing heart. In addition, AM has anti-apoptotic, angiogenic, anti-inflammatory and anti-oxidant effects. Several mechanisms such as cAMP/PKA, NO/cGMP, PI-3K/Akt and/or ERK are thought to mediate cellular AM signaling, suggesting that increased AM levels play a protective role in heart failure. Indeed, acute AM administration exerts beneficial vasodilatory, diuretic, natriuretic and inotropic effects on experimental and human heart failure. A recent pilot study has shown that AM has potential as a therapeutic drug in the clinical setting of acute decompensated heart failure.

Pro-brain natriuretic peptide (proBNP)-108 and N-terminal proBNP-76 (NT-BNP) contain seven sites for O-linked oligosaccharide attachment. Currently, levels of glycosylated NT-BNP are probably underestimated because it is not recognised by one antibody in the sandwich assay system. The pathophysiological significance of cardiac and plasma levels of non-glycosylated (nonglyNT-BNP) and glycosylated NT-BNP (glyNT-BNP) in heart failure (HF) and chronic renal failure (CRF) was investigated.Plasma samples from 186 patients with HF and 76 patients with CRF on haemodialysis were studied, together with 11 atrial tissue samples. To measure nonglyNT-BNP and glyNT-BNP, samples were incubated with or without deglycosylating enzymes and NT-BNP was measured using Roche Elecsys proBNP I. The percentage glyNT-BNP was calculated as glyNT-BNP/(glyNT-BNP + nonglyNT-BNP).In HF, plasma BNP, nonglyNT-BNP and glyNT-BNP levels all increased with increasing disease severity (New York Heart Association class; p0.0001), though the molar ratio remained constant (molar ratio, BNP:nonglyNT-BNP:glyNT-BNP = 1:2.4:9.6). Before haemodialysis for CRF, plasma BNP and nonglyNT-BNP were somewhat elevated, and glyNT-BNP was markedly increased (molar ratio, BNP:nonglyNT-BNP:glyNT-BNP = 1:8.5:82). After haemodialysis, plasma BNP, nonglyNT-BNP, atrial natriuretic protein and cGMP all declined (p0.0001), but glyNT-BNP was unchanged. Notably, the percentage of glyNT-BNP was elevated before haemodialysis, and was further increased after haemodialysis (p0.0001). Atrial tissue levels of BNP, nonglyNT-BNP and glyNT-BNP were similar.THE findings suggest that most endogenous plasma NT-BNP is glycosylated and therefore undetectable with the current assay system, and that the relative glycosylation level is increased by haemodialysis.

Neuron-restrictive silencer factor (NRSF) is a zinc-finger transcription factor that binds to specific DNA sequences (NRSE) to repress transcription. By down-regulating the transcription of its target genes, NRSF contributes to the regulation of various biological processes, including neuronal differentiation, carcinogenesis and cardiovascular homeostasis. We previously reported that NRSF regulates expression of the cardiac fetal gene program, and that attenuation of NRSF-mediated repression contributes to genetic remodeling in hearts under pathological conditions. The precise molecular mechanisms and signaling pathways via which NRSF activity is regulated in pathological conditions of the heart remain unclear, however. In this study, to search for regulators of NRSF, we carried out yeast two-hybrid screening using NRSF as bait and identified zinc-finger protein (Zfp) 90 as a novel NRSF-binding protein. NRSF and Zfp90 colocalized in the nucleus, with the zinc-finger DNA-binding domain of the former specifically interacting with the latter. Zfp90 inhibited the repressor activity of NRSF by inhibiting its binding to DNA, thereby derepressing transcription of NRSF-target genes. Knockdown of Zfp90 by siRNA led to reduced expression of NRSF-target fetal cardiac genes, atrial and brain natriuretic peptide genes, and conversely, overexpression of Zfp90 in ventricular myocardium resulted in significant increases in the expression of these genes. Notably, expression of Zfp90 mRNA was significantly upregulated in mouse and human hearts with chronic heart failure. Collectively, these results suggest that Zfp90 functions as a negative regulator of NRSF and contributes to genetic remodeling during the development of cardiac dysfunction.

Recent studies have shown that not only plasma B-type natriuretic peptide (BNP)-32, but also plasma proBNP-108 is increased in heart failure (HF), and that the current BNP-32 assay kit crossreacts with proBNP-108. It also was shown that both BNP-32 and proBNP-108 were higher in HF than in normal. The proBNP-108/total BNP (BNP-32 + proBNP-108) ratio was widely distributed and patients with HF with ventricular overload had higher proBNP-108/total BNP ratio than HF patients with atrial overload. Consistent with this finding, proBNP-108 was the major molecular form in ventricular tissue, and BNP-32 was the major molecular form in atrial tissue. In addition, proBNP-108 was the major molecular form of BNP in pericardial fluid. The proBNP-108/total BNP ratio increased with deterioration of HF and decreased with improvement of HF. Thus, not only BNP-32, but also proBNP-108 is increased in HF and the proBNP-108/total BNP ratio also rises in association with pathophysiological conditions such as ventricular overload. A new hypothesis that O-glycosylation at Thr71 in a region close to the cleavage site impairs proBNP-108 processing was proposed. In the future, the precise mechanism of increased proBNP-108 in HF should be elucidated.