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Objective The severity of mitral regurgitation (MR) dynamically changes during a stress test. Isometric handgrip is a readily-available stress test in daily practice; however, little is known regarding the response to isometric handgrip in MR patients during right heart catheterization. We aimed to evaluate this issue from our case-series study. Methods We retrospectively investigated consecutive MR patients using the isometric handgrip stress test during right heart catheterization at our institution between October 2019 and April 2021. After resting measurements were obtained, sustained maximum-effort hand dynamometer grasping was maintained for about 2-3 minutes. We investigated the differences in right heart catheterization data between at rest and during handgrip, and evaluated the individual response to the isometric handgrip stress test. Results We investigated a total of 15 patients (mean age: 75±6 years, moderate/severe MR: 7/8, primary/secondary MR: 8/7, mean left ventricular ejection fraction: 56±16%, exertional dyspnea: 10). During the handgrip test, the pulmonary capillary wedge pressure (PCWP) significantly increased [9 (8, 13) mmHg at rest to 20 (15, 27) mmHg during handgrip; p0.001]. PCWP changes varied among individuals (range 2-22 mmHg) and were not correlated with patients' backgrounds including age, the natriuretic peptide levels, left ventricular ejection fraction, left atrial diameter or E/e' (all p0.05). Patients with PCWP ≥25 mmHg during handgrip had a higher prevalence of exertional dyspnea than those without [6 (100%) vs. 4 (44%); p=0.04]. Conclusion We observed dynamic and varied hemodynamic changes during isometric handgrip in MR patients, suggesting that further research is needed to evaluate the clinical value of this maneuver.

Aims Previous studies have shown that proteinuria is independently associated with the incidence of atrial fibrillation (AF), and is also associated with the incidence of cardiovascular events such as stroke and thromboembolism in patients with AF. However, the association of proteinuria with heart failure (HF) events in patients with AF remains unclear. Methods and results The Fushimi AF Registry is a community-based prospective study of patients with AF. Of the entire cohort of 4489 patients, 2164 patients had available data of proteinuria. We compared the clinical background and outcomes between patients with proteinuria (n = 606, 28.0%) and those without (n = 1558, 72.0%). Patients with proteinuria were older and had a higher prevalence of major co-morbidities. During the median follow-up of 5.0 years, the incidence rates of HF events (composite of cardiac death or HF hospitalization) were higher in patients with proteinuria than those without (4.1% vs. 2.1% person-year, P Conclusion Our community-based large prospective cohort suggests that proteinuria is independently associated with the incidence of HF events in Japanese patients with AF.

Background Functional mitral regurgitation (MR) changes dynamically depending on the loading conditions and can cause acute heart failure (HF). Isometric handgrip is a simple stress test and can be performed during early phase of acute HF for the evaluation of MR. Case summary A 70-year-old woman with a prior myocardial infarction four months before, and with history of recurrent HF admission with functional MR, who received optimal HF medications, was hospitalized for acute HF. On the following day of the admission, isometric handgrip stress echocardiography was performed to evaluate functional MR. During the handgrip, MR deteriorated from moderate to severe and the tricuspid regurgitation pressure gradient increased from 45 to 60 mmHg. After HF stabilization 2 weeks after admission, repeat handgrip stress echocardiography showed that the degree of MR did not significantly change being moderate and the tricuspid regurgitation pressure gradient was only mildly elevated from 25 to 30 mmHg. She underwent transcatheter edge-to-edge mitral repair, and thereafter she has not experienced the rehospitalization for acute HF. Discussion Exercise stress test is recommended for the evaluation of functional MR in HF patients; however, exercise tests are difficult to perform during the early phase of acute HF. In this regard, handgrip test is an option to investigate the exacerbating impact of functional MR during early-phase acute HF. This case indicated that response to isometric handgrip can vary depending on HF condition, highlighting the importance of taking into account the timing of the handgrip procedure in patients with functional MR and HF.

Atrial fibrillation (AF) patients often have concomitant coronary artery disease (CAD); however, there are little data on clinical characteristics and outcomes of such patients in daily clinical practice in Japan.Methods and Results: The Fushimi AF Registry is a community-based prospective survey of AF patients in Fushimi-ku, Kyoto, Japan. Follow-up data were available for 4,464 patients, and the median follow up was 5.1 (interquartile range: 2.3-8.0) years. History of CAD was present in 647 patients (14%); of those patients, 267 (41%) had history of myocardial infarction (MI). Patients with CAD were older and had more comorbidities than those without CAD. The crude incidences (% per patient-year) of cardiovascular events were significantly higher in patients with CAD than those without CAD (cardiac death: 1.8 vs. 0.7, stroke or systemic embolism [SE]: 2.9 vs. 2.1, MI: 0.6 vs. 0.1, composite of those events: 5.1 vs. 2.8, respectively, all log-rank P0.01). After multivariate adjustment, concomitant CAD was associated with incidence of cardiac events, and history of MI was associated with incidence of MI; however, neither history of CAD nor MI was associated with the incidence of stroke/SE.In Japanese AF patients, concomitant CAD was associated with higher prevalences of major co-morbidities and higher incidences of cardiovascular events; however, history of CAD was not associated with the incidence of stroke/SE.

Data regarding the associations of anemia (hemoglobin level

Atrial fibrillation (AF) increases the risk of stroke and death. Oral anticoagulants (OAC) are highly effective in reducing the risk of stroke, and direct oral anticoagulants (DOAC) became available worldwide in 2011.Methods and Results:The Fushimi AF Registry is an on-going prospective survey of AF patients in Fushimi-ku, Kyoto, Japan. The study cohort consisted of 4,489 patients (mean age 73.6 years, 59.6% male, mean CHADSOver the past 10 years, there has been a major transition in antithrombotic therapy and a decline in the incidence of adverse events in AF patients.

Background: We previously reported that left atrial enlargement (LAE) was an independent predictor of stroke/systemic embolism (SE) in Japanese atrial fibrillation (AF) patients. Mitral regurgitation (MR) likely accompanies LAE; it sometimes causes LAE and conversely LAE can cause secondary MR. However, the relationship between MR and LAE on the risk of stroke/SE is unknown. Purpose: The aim of this study is to investigate the impact of LAE on stroke/SE in AF patients with or without MR. Method: The Fushimi AF Registry is a community-based prospective survey of AF patients in Fushimi-ku, Kyoto, which is a typical urban district of Japan. We started to enroll patients from March 2011, and follow-up data were available for 4,472 patients by the end of October 2020. In the entire cohort, echocardiography data were available for 3,622 patients. 131 AF patients with rheumatic mitral stenosis or prosthetic valve and 63 patients without data of left atrial diameter (LAD) were excluded. We divided eligible 3,428 patients in MR group (n=393) and non-MR group (n=3,035). We compared clinical characteristics and rates of stroke/SE between patients with LAE (LAD > 45mm) and without LAE (Non-LAE) in each group. Result: In both MR and non-MR groups, patients with LAE were, as compared with non-LAE, more in body weight, more often persistent/permanent type, more likely to have chronic kidney disease. LAE group was older, more likely have heart failure, hypertension, diabetes mellitus and more often received oral anticoagulant in non-MR group, but not in MR group. During the median follow-up of 1,804 days, the incidence rate of stroke/SE was higher in LAE than non-LAE in non-MR group (2.13 vs. 1.08 per 100 person-years, respectively, log rank p Conclusion: The incidence rate of stroke/SE was higher in LAE than non-LAE in non-MR group, but not in MR group.

Background: Atrial fibrillation (AF) is associated with an increased risk of cardioembolic stroke and systemic embolism (SE). A secondary analysis of the Systolic Blood Pressure Intervention Trial (SPRINT) demonstrated that low diastolic blood pressure (DBP) was not related to risk of the first episode of stroke in hypertensive patients who received prescription of any antihypertensive agents. However, data regarding the impact of low on-treatment DBP on the first episode of stroke or SE in AF patients are limited. Methods: The Fushimi AF Registry is a community-based prospective survey of AF patients in Fushimi-ku, Kyoto, Japan. Follow-up data were available in 4,472 patients, and hypertensive patients who received prescription of any antihypertensive agents, and without prior stroke or SE were examined (n=1,934).We divided these patients into four groups based on the quartile of DBP; Q1 (DBP< 63 mmHg, n=463), Q2 (63 mmHg≤DBP Results: Patients in Q1 group were older (Q1 vs. Q2 vs. Q3 vs. Q4; 77.2±0.45 vs. 74.2±0.45 vs. 73.6±0.44 vs. 71.0±0.42; p Conclusion: In Japanese AF patients without prior stroke or SE, low on-treatment DBP was associated with the incidence of stroke or SE.

Background: The ELDERCARE-AF trial demonstrated that low-dose edoxaban (15 mg once-daily) prevented stroke or systemic embolism (SE) in very elderly Japanese patients with nonvalvular atrial fibrillation (NVAF) who were inappropriate for standard oral anticoagulant therapy due to high bleeding risk. The association between clinical outcomes and bleeding risks in such patients in routine clinical practice are unclear. Methods: From the Fushimi AF registry, we obtained ELDERCARE-eligible patients with NVAF who aged ≥80 years, had CHADS 2 score ≥2, and had one or more bleeding risks as shown in the ELDERCARE-AF trial: low creatinine clearance (15 to 30 ml/min), a history of bleeding from a critical organ, low body weight (≤45 kg), or regular use of antiplatelet drugs. The association of the four bleeding risk factors with the outcomes was evaluated in eligible patients. Results: We obtained 549 ELDERCARE-eligible patients (12.8% of entire cohort, 52.8% of ≥80 years and CHADS 2 score ≥2). The median follow-up period was 1,823 (interquartile range: 809-2,900) days. The incidences (% per patient-year) of stroke/SE, major bleeding, all-cause mortality, and cardiovascular death were significantly higher in eligible patients than in non-eligible patients (stroke/SE: 4.6% vs. 2.0%, major bleeding: 3.5% vs. 1.8%, all-cause mortality: 15.5% vs. 3.9%, cardiovascular death: 2.9% vs. 0.6%, all log-rank P Conclusion: Patients with NVAF who met the inclusion criteria of the ELDERCARE-AF trial had poor clinical outcomes, and low creatinine clearance was associated with the incidences of both all-cause and cardiovascular mortality.

Visual Abstract, Highlights • KUS121 was developed to selectively inhibit the adenosine triphosphatase activity of valosin-containing protein without affecting other cellular functions of valosin-containing protein. • KUS121 preserved adenosine triphosphate levels, reduced endoplasmic reticulum stress, and suppressed cell death in H9C2 rat cardiomyoblast cells, treated with tunicamycin or hydrogen peroxide, or cultured in glucose-free medium. • In murine ischemia and reperfusion injury models, KUS121 treatment after reperfusion attenuated the infarcted size and preserves cardiac function by maintaining adenosine triphosphate levels and reducing ER stress. • In porcine ischemia and reperfusion injury models, intracoronary administration of KUS121 also attenuated the infarcted area in a dose-dependent manner. • These results indicated that KUS121 is a promising novel therapeutic agent for myocardial infarction., Summary No effective treatment is yet available to reduce infarct size and improve clinical outcomes after acute myocardial infarction by enhancing early reperfusion therapy using primary percutaneous coronary intervention. The study showed that Kyoto University Substance 121 (KUS121) reduced endoplasmic reticulum stress, maintained adenosine triphosphate levels, and ameliorated the infarct size in a murine cardiac ischemia and reperfusion injury model. The study confirmed the cardioprotective effect of KUS121 in a porcine ischemia and reperfusion injury model. These findings confirmed that KUS121 is a promising novel therapeutic agent for myocardial infarction in conjunction with primary percutaneous coronary intervention.

Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3′-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.

Background: Obesity, which leads to left arterial remodeling, increases the risk for the development of atrial fibrillation (AF). We previously demonstrated the progression from paroxysmal to sustained AF was associated with an increased risk of ischemic stroke or hospitalization for heart failure (HF). However, the risk of progression from paroxysmal to sustained AF in obese patients has not been fully evaluated. Methods: Based on the initial type of AF and whether paroxysmal AF progressed to sustained AF during follow-up (FU), patients enrolled in the Fushimi AF registry were categorized into 3 groups; i) paroxysmal AF without progression, ii) paroxysmal AF with progression, iii) sustained AF. Obesity was defined as BMI at baseline >30. Results: Obese patients (172/3834) had a higher prevalence of sustained AF at baseline as compared with those without obesity (58.7% vs 51.0%, p=0.047), and they had a higher rate of progression from paroxysmal to sustained AF during a median FU of 4.3 years (2.4% vs 1.5% [per person-year], p Conclusions: Obesity may be the risk of the progression of paroxysmal to sustained AF. The progression may be associated with increased risk of ischemic stroke in both obese and non-obese patients, but the risk of hospitalization for HF might be lower in patients with obesity as compared with those without obesity.

Introduction: Hyperlipidemia is a well-established risk factor for cardiovascular disease. However, low cholesterol is also reported to be associated with poor outcome in chronic disease, which is called “cholesterol paradox”, and the impact of cholesterol level on the incidence of stroke or systemic embolism (SE) in atrial fibrillation (AF) remains unclear. Methods: The Fushimi AF Registry is a community-based prospective survey of the AF patients in Fushimi-ku, Kyoto, Japan. We obtained baseline total cholesterol level (TC) in 3,051 patients, and examined the relationship between TC and incidence of stroke/SE. Results: TC was inversely correlated with age and positively correlated with body weight. Patients with lower TC were more likely to be male, and had more diabetes, coronary artery disease, heart failure, prior stroke/SE, chronic kidney disease, and anemia. CHA 2 DS 2 -VASc score was inversely correlated with TC. Prescription of statin was higher in patients with lower TC, but that of oral anti-coagulants was not associated with TC. During the median follow-up of 1,476 days, stroke/SE occurred in 277 (9.1%) patients. After adjustment for the components of CHA 2 DS 2 -VASc score, body weight, and prescription of oral anti-coagulants and statins, TC had significant inverse association with the incidence of stroke/SE (hazard ratio (HR), 0.94; 95% confidence interval (CI) 0.91-0.98 for 10-mg/dl increase) (Table). TC was significantly associated with ischemic stroke/SE (HR, 0.95; 95% CI 0.91-0.99 for 10-mg/dl increase), but was not associated with intracranial bleeding (HR, 0.95; 95% CI 0.88-1.02 for 10-mg/dl increase). When we divided the patients into the elderly (≥75 years) and the young, low TC was a predictor for stroke/SE in the elderly (HR, 0.94; 95% CI 0.90-0.99 for 10-mg/dl increase), but was not in the young (HR, 0.95; 95% CI 0.90-1.01 for 10-mg/dl increase). Conclusions: Low TC might be an independent predictor for stroke/SE in elderly patients with AF.

Recent high-throughput approaches have revealed a vast number of transcripts with unknown functions. Many of these transcripts are long noncoding RNAs (lncRNAs), and intergenic region-derived lncRNAs are classified as long intergenic noncoding RNAs (lincRNAs). Although Myosin heavy chain 6 (Myh6) encoding primary contractile protein is down-regulated in stressed hearts, the underlying mechanisms are not fully clarified especially in terms of lincRNAs. Here, we screen upregulated lincRNAs in pressure overloaded hearts and identify a muscle-abundant lincRNA termed Lionheart. Compared with controls, deletion of the Lionheart in mice leads to decreased systolic function and a reduction in MYH6 protein levels following pressure overload. We reveal decreased MYH6 results from an interaction between Lionheart and Purine-rich element-binding protein A after pressure overload. Furthermore, human LIONHEART levels in left ventricular biopsy specimens positively correlate with cardiac systolic function. Our results demonstrate Lionheart plays a pivotal role in cardiac remodeling via regulation of MYH6., Kuwabara et al. identify a novel long intergenic noncoding RNA (lincRNA), termed Lionheart, upregulated in pressure overloaded hearts in mice. Deleting this gene results in decreased systolic function and reduction in MYH6 protein levels following pressure overload. They demonstrate that Lionheart interacts with PURA, preventing its binding to the promoter region of Myh6 locus, leading to reduced MYH6 protein expression.

The Hippo signaling pathway is involved in the pathophysiology of various cardiovascular diseases. Yes‐associated protein (YAP) and transcriptional enhancer activator domain (TEAD) transcriptional factors, the main transcriptional complex of the Hippo pathway, were recently identified as modulators of phenotypic switching of vascular smooth muscle cells (VSMCs). However, the intrinsic regulator of YAP/TEAD‐mediated gene expressions involved in vascular pathophysiology remains to be elucidated. Here, we identified Homeobox A4 (HOXA4) as a potent repressor of YAP/TEAD transcriptional activity using lentiviral shRNA screen. Mechanistically, HOXA4 interacts with TEADs and attenuates YAP/TEAD‐mediated transcription by competing with YAP for TEAD binding. We also clarified that the expression of HOXA4 is relatively abundant in the vasculature, especially in VSMCs. In vitro experiments in human VSMCs showed HOXA4 maintains the differentiation state of VSMCs via inhibition of YAP/TEAD‐induced phenotypic switching. We generated Hoxa4‐deficient mice and confirmed the downregulation of smooth muscle‐specific contractile genes and the exacerbation of vascular remodeling after carotid artery ligation in vivo. Our results demonstrate that HOXA4 is a repressor of VSMC phenotypic switching by inhibiting YAP/TEAD‐mediated transcription., HOXA4 suppresses vascular smooth muscle cell phenotypic switching and vascular remodeling by inhibiting YAP/TEAD‐mediated transcription.

Objective— Atherosclerosis is a common disease caused by a variety of metabolic and inflammatory disturbances. MicroRNA (miR)-33a within SREBF2 (sterol regulatory element-binding factor 2) is a potent target for treatment of atherosclerosis through regulating both aspects; however, the involvement of miR-33b within SREBF1 remains largely unknown. Although their host genes difference could lead to functional divergence of miR-33a/b, we cannot dissect the roles of miR-33a/b in vivo because of lack of miR-33b sequences in mice, unlike human. Approach and Results— Here, we analyzed the development of atherosclerosis using miR-33b knock-in humanized mice under apolipoprotein E–deficient background. MiR-33b is prominent both in human and mice on atheroprone condition. MiR-33b reduced serum high-density lipoprotein cholesterol levels and systemic reverse cholesterol transport. MiR-33b knock-in macrophages showed less cholesterol efflux capacity and higher inflammatory state via regulating lipid rafts. Thus, miR-33b promotes vulnerable atherosclerotic plaque formation. Furthermore, bone marrow transplantation experiments strengthen proatherogenic roles of macrophage miR-33b. Conclusions— Our data demonstrated critical roles of SREBF1 -miR-33b axis on both lipid profiles and macrophage phenotype remodeling and indicate that miR-33b is a promising target for treating atherosclerosis.

Recent evidence suggests that the accumulation of macrophages as a result of obesity-induced adipose tissue hypoxia is crucial for the regulation of tissue fibrosis, but the molecular mechanisms underlying adipose tissue fibrosis are still unknown. In this study, we revealed that periostin (Postn) is produced at extraordinary levels by adipose tissue after feeding with a high-fat diet (HFD). Postn was secreted at least from macrophages in visceral adipose tissue during the development of obesity, possibly due to hypoxia. Postn−/− mice had lower levels of crown-like structure formation and fibrosis in adipose tissue and were protected from liver steatosis. These mice also showed amelioration in systemic insulin resistance compared with HFD-fed WT littermates. Mice deficient in Postn in their hematopoietic compartment also had lower levels of inflammation in adipose tissue, in parallel with a reduction in ectopic lipid accumulation compared with the controls. Our data indicated that the regulation of Postn in visceral fat could be beneficial for the maintenance of healthy adipose tissue in obesity.

Objective— Abdominal aortic aneurysm (AAA) is an increasingly prevalent and ultimately fatal disease with no effective pharmacological treatment. Because matrix degradation induced by vascular inflammation is the major pathophysiology of AAA, attenuation of this inflammation may improve its outcome. Previous studies suggested that miR-33 (microRNA-33) inhibition and genetic ablation of miR-33 increased serum high-density lipoprotein cholesterol and attenuated atherosclerosis. Approach and Results— MiR-33a-5p expression in central zone of human AAA was higher than marginal zone. MiR-33 deletion attenuated AAA formation in both mouse models of angiotensin II– and calcium chloride–induced AAA. Reduced macrophage accumulation and monocyte chemotactic protein-1 expression were observed in calcium chloride–induced AAA walls in miR-33 −/− mice. In vitro experiments revealed that peritoneal macrophages from miR-33 −/− mice showed reduced matrix metalloproteinase 9 expression levels via c-Jun N-terminal kinase inactivation. Primary aortic vascular smooth muscle cells from miR-33 −/− mice showed reduced monocyte chemotactic protein-1 expression by p38 mitogen-activated protein kinase attenuation. Both of the inactivation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were possibly because of the increase of ATP-binding cassette transporter A1 that is a well-known target of miR-33. Moreover, high-density lipoprotein cholesterol derived from miR-33 −/− mice reduced expression of matrix metalloproteinase 9 in macrophages and monocyte chemotactic protein-1 in vascular smooth muscle cells. Bone marrow transplantation experiments indicated that miR-33–deficient bone marrow cells ameliorated AAA formation in wild-type recipients. MiR-33 deficiency in recipient mice was also shown to contribute the inhibition of AAA formation. Conclusions— These data strongly suggest that inhibition of miR-33 will be effective as a novel strategy for treating AAA.

Rationale: Heart failure and atherosclerosis share the underlying mechanisms of chronic inflammation followed by fibrosis. A highly conserved microRNA (miR), miR-33, is considered as a potential therapeutic target for atherosclerosis because it regulates lipid metabolism and inflammation. However, the role of miR-33 in heart failure remains to be elucidated. Objective: To clarify the role of miR-33 involved in heart failure. Methods and Results: We first investigated the expression levels of miR-33a/b in human cardiac tissue samples with dilated cardiomyopathy. Increased expression of miR-33a was associated with improving hemodynamic parameters. To clarify the role of miR-33 in remodeling hearts, we investigated the responses to pressure overload by transverse aortic constriction in miR-33–deficient (knockout [KO]) mice. When mice were subjected to transverse aortic constriction, miR-33 expression levels were significantly upregulated in wild-type left ventricles. There was no difference in hypertrophic responses between wild-type and miR-33KO hearts, whereas cardiac fibrosis was ameliorated in miR-33KO hearts compared with wild-type hearts. Despite the ameliorated cardiac fibrosis, miR-33KO mice showed impaired systolic function after transverse aortic constriction. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart. Deficiency of miR-33 impaired cardiac fibroblast proliferation, which was considered to be caused by altered lipid raft cholesterol content. Moreover, cardiac fibroblast–specific miR-33–deficient mice also showed decreased cardiac fibrosis induced by transverse aortic constriction as systemic miR-33KO mice. Conclusion: Our results demonstrate that miR-33 is involved in cardiac remodeling, and it preserves lipid raft cholesterol content in fibroblasts and maintains adaptive fibrotic responses in the remodeling heart.

Background Micro RNA (miR)‐33 targets cholesterol transporter ATP ‐binding cassette protein A1 and other antiatherogenic targets and contributes to atherogenic progression. Its inhibition or deletion is known to result in the amelioration of atherosclerosis in mice. However, mice lack the other member of the miR‐33 family, miR‐33b, which exists in humans and other large mammals. Thus, precise evaluation and comparison of the responsibilities of these 2 miRs during the progression of atherosclerosis has not been reported, although they are essential. Methods and Results In this study, we performed a comprehensive analysis of the difference between the function of miR‐33a and miR‐33b using genetically modified mice. We generated 4 strains with or without miR‐33a and miR‐33b. Comparison between mice with only miR‐33a (wild‐type mice) and mice with only miR‐33b (miR‐33a −/− /miR‐33b +/+ ) revealed the dominant expression of miR‐33b in the liver. To evaluate the whole body atherogenic potency of miR‐33a and miR‐33b, we developed apolipoprotein E–deficient/miR‐33a +/+ /miR‐33b −/− mice and apolipoprotein E–deficient/miR‐33a −/− /miR‐33b +/+ mice. With a high‐fat and high‐cholesterol diet, the apolipoprotein E–deficient/miR‐33a −/− /miR‐33b +/+ mice developed increased atherosclerotic plaque versus apolipoprotein E–deficient/miR‐33a +/+ /miR‐33b −/− mice, in line with the predominant expression of miR‐33b in the liver and worsened serum cholesterol profile. By contrast, a bone marrow transplantation study showed no significant difference, which was consistent with the relevant expression levels of miR‐33a and miR‐33b in bone marrow cells. Conclusions The miR‐33 family exhibits differences in distribution and regulation and particularly in the progression of atherosclerosis; miR‐33b would be more potent than miR‐33a.

Acute cardiac rupture and adverse left ventricular (LV) remodeling causing heart failure are serious complications of acute myocardial infarction (MI). While cardio-hepatic interactions have been recognized, their role in MI remains unknown. We treated cultured cardiomyocytes with conditioned media from various cell types and analyzed the media by mass spectrometry to identify α1-microglobulin (AM) as an Akt-activating hepatokine. In mouse MI model, AM protein transiently distributed in the infarct and border zones during the acute phase, reflecting infiltration of AM-bound macrophages. AM stimulation activated Akt, NFκB, and ERK signaling and enhanced inflammation as well as macrophage migration and polarization, while inhibited fibrogenesis-related mRNA expression in cultured macrophages and cardiac fibroblasts. Intramyocardial AM administration exacerbated macrophage infiltration, inflammation, and matrix metalloproteinase 9 mRNA expression in the infarct and border zones, whereas disturbed fibrotic repair, then provoked acute cardiac rupture in MI. Shotgun proteomics and lipid pull-down analysis found that AM partly binds to phosphatidic acid (PA) for its signaling and function. Furthermore, systemic delivery of a selective inhibitor of diacylglycerol kinase α-mediated PA synthesis notably reduced macrophage infiltration, inflammation, matrix metalloproteinase activity, and adverse LV remodeling in MI. Therefore, targeting AM signaling could be a novel pharmacological option to mitigate adverse LV remodeling in MI.

MicroRNA 33 (miR-33) targets ATP-binding cassette transporter A1 (ABCA1), and its deficiency increases serum high-density lipoprotein (HDL)-cholesterol (HDL-C) and ameliorates atherosclerosis. Although we previously reported that miR-33 deficiency increased peripheral Ly6Chigh monocytes on an ApoE-deficient background, the effect of miR-33 on the monocyte population has not been fully elucidated, especially in a wild-type (WT) background. We found that Ly6Chigh monocytes in miR-33−/− mice were decreased in peripheral blood and increased in bone marrow (BM). Expansion of myeloid progenitors and decreased apoptosis in Lin− Sca1+ c-Kit+ (LSK) cells were observed in miR-33−/− mice. A BM transplantation study and competitive repopulation assay revealed that hematopoietic miR-33 deficiency caused myeloid expansion and increased peripheral Ly6Chigh monocytes and that nonhematopoietic miR-33 deficiency caused reduced peripheral Ly6Chigh monocytes. Expression of high-mobility group AT-hook 2 (HMGA2) targeted by miR-33 increased in miR-33-deficient LSK cells, and its knockdown abolished the reduction of apoptosis. Transduction of human apolipoprotein A1 and ABCA1 in WT mouse liver increased HDL-C and reduced peripheral Ly6Chigh monocytes. These data indicate that miR-33 deficiency affects distribution of inflammatory monocytes through dual pathways. One pathway involves the enhancement of Hmga2 expression in hematopoietic stem cells to increase Ly6Chigh monocytes, and the other involves the elevation of HDL-C to decrease peripheral Ly6Chigh monocytes.

Changes in the surface morphology of salt crystals composed of 4-(2,5-diisopropylbenzoyl)benzoic acid with (S)-phenylethylamine and 2,4-dichlorobenzylamine were observed upon photoirradiation. The reaction processes were monitored using Fourier transform infrared (FT-IR) spectroscopy and surface morphologies determined using atomic force microscopy. The crystals underwent enantiospecific and enantioselective photocyclization of the benzophenone derivative via a single-crystal-to-single-crystal transformation. UV irradiation of the salt microcrystals at wavelengths greater than 290 nm induced the formation of hemispheric features on the initially platelike (001) face, which gradually grew to heights of tens of nanometers. The maximum relative surface roughness was several percent of the 1 μm crystal thickness. Prolonged irradiation after the reactions had reached completion decreased the average feature height, and the surface eventually returned to its initial smooth state. On the basis of X-ray crystallo...

Background: MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33 (miR-33a) located within the intron of sterol regulatory element-binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for treating atherosclerosis. Primates, but not rodents, express a second miR-33 gene (miR-33b) from an intron of SREBF1. To address miR-33b function in vivo, we developed humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron. Methods and Results: The human miR-33b sequence was introduced into intron 16 of mouse Srebf1 by conventional gene targeting methods, because miR-33b is located in intron 16 of human SREBF1 and there are high homologies in exons 16 and 17 between human and mouse. We successfully established miR-33b knock-in (KI) mice with C57BL/6 background and this miR-33b KI strategy did not alter Srebf1 intron 16 splicing, which was confirmed by RT-PCR and sequencing. The expression of miR-33b in miR-33b KI hetero mice were almost half of those in miR-33b KI homo mice. An LXR agonist T0901317, which induces Srebf1 expression, enhanced miR-33b expression in primary hepatocytes and the liver of miR-33 KI homo mice. The protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. Peritoneal macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Serum HDL-C levels were reduced by almost 35% even in miR-33b KI hetero mice compared with wild-type mice. HPLC elution analysis showed that the decreased HDL levels were mainly composed of very large-, large-, medium sized HDL, which was compatible with the previous results of miR-33a deficient mice. Conclusions: miR-33b KI mice for an intron of Srebf1 showed reduced HDL-C levels. These results indicate that miR-33b can be a potential target for raising HDL-C in humans and may account for lower HDL-C levels in humans than those in mice. These mice will aid in elucidating the roles of miR-33s in different disease models and in screening of the drugs that can alter miR-33a and miR-33b levels and activities.

Introduction: MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote degradation. Recent reports, including ours, indicated that miR-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for treating atherosclerosis. Unexpectedly, miR-33 deficient (miR-33 -/- ) mice fed high fat diet (HFD) developed severe fatty liver and the mechanisms were investigated. Methods and Results: The liver weight of miR-33 -/- mice were about 1.5 times heavier than that of miR-33 +/+ mice and histological examination revealed that miR-33 -/- mice developed severe fatty liver under HFD feeding. In order to determine the cause of the fatty liver observed in miR-33 -/- mice fed HFD, we analysed the gene expression profiles using the liver of miR-33 +/+ and miR-33 -/- mice fed normal chow at the age of 16 weeks when they didn’t show fatty liver. As a result, genes involved in fatty acid metabolism were upregulated in miR-33 -/- mice. Among them we found SREBP-1 as a new potential target gene of miR-33 in silico and confirmed that miR-33 targets the 3’UTR of SREBP-1 in vitro . The expression of SREBP-1 and de novo fatty acid production were significantly increased in the liver of miR-33 -/- mice. We further intercrossed miR-33 -/- mice with Srebf1 +/- mice and fed them HFD. Hepatic steatosis was reversed in miR-33 -/- Srebf1 +/- mice compared with miR-33 -/- Srebf1 +/+ mice by histological analysis and measurement of triglyceride levels. The expression levels of genes involved in fatty acid synthesis, including Scd1, Fasn, Acc1 , and Pparg were increased in miR-33 -/- Srebf1 +/+ mice compared with miR-33 +/+ Srebf1 +/+ mice, and those increase were reversed in miR-33 -/- Srebf1 +/- mice. Conclusions: miR-33 regulates lipogenic pathway via regulating SREBP-1 as a novel target in vivo . In sterol-depleted conditions, acetyl-CoA might be preferred as a substrate for cholesterol production and not for fatty acid production by the downregulation of SREBP-1 through the upregulation of miR-33. Conversely, in cholesterol-rich condition, acetyl-CoA might be preferred as a substrate for fatty acid production through the downregulation of miR-33.

MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for treating atherosclerosis. Primates, but not rodents, express miR-33b from an intron of SREBF1. Therefore, humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron, are required to address its function in vivo. We successfully established miR-33b knock-in (KI) mice and found that protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. As a consequence, macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Moreover, HDL-C levels were reduced by almost 35% even in miR-33b KI hetero mice compared with the control mice. These results indicate that miR-33b may account for lower HDL-C levels in humans than those in mice and that miR-33b is possibly utilized for a feedback mechanism to regulate its host gene SREBF1. Our mice will also aid in elucidating the roles of miR-33a/b in different genetic disease models., マウスへのマイクロRNA-33bの導入に成功 -ヒトでのHDL-コレステロールの質の改善に期待-. 京都大学プレスリリース. 2014-06-17.

The changes in the surface morphology of a salt crystal of 4-(2,5-diisopropylbenzoyl)benzoic acid with (S)-phenylethylamine upon photoirradiation were determined under atomic force microscopy. The crystal underwent enantiospecific photocyclization of the benzophenone derivative to give (S)-cyclobutenol as the sole product via single-crystal-to-single-crystal transformation. Upon UV irradiation, numbers of hemispheric unevennesses appeared on the (001) and (010) single-crystal surfaces with heights of tens of nanometers. Prolonged irradiation caused the hemispheres to merge, restoring essentially flat surfaces at the completion of the reaction. In contrast, the roughness of the (100) surface resulting from cutting with a razor blade decreased on photoirradiation, and a smooth surface was obtained. The morphological changes likely resulted from the changes in the molecular structure of the benzophenone derivative; the movement of the salt bond chains near the crystal surface then became easier as the meltin...

Evidences of the carina area between the superior and inferior pulmonary veins (PVs) have been shown the importance on catheter ablation for atrial fibrillation (AF). However, electrical structure of the intervenous isthmus of PVs has not been elucidated fully.Methods: Thirty-six AF patients underwent extensive PV isolation (EPVI). Electrical connections of contiguous intervenous area were estimated with two ring catheters and 3D navigation system before and immediately after completion of EPVI.Results: Continuous pacing at the multisite of ipsilateral PVs revealed intervenous connection at the carina zone in 62.5% of left and 84.6% of right PVs. The courses of electrical connections were classified into three types. Cross band conduction from superior-anterior to inferior-posterior was observed in 31.3% and 38.5%, superior-posterior to inferior-anterior in 12.5% and 15.4%, and presumed epicardial conduction in 12.5% and 38.5% of left and right PVs, respectively. Completion of simultaneous isolation of ipsilateral PVs were higher observed in PVs with intervenous conduction than in PVs without connection (88.9% vs 28.6% in left, 100% vs 71.4% in right, respectively p