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Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy an optimized cryo-ET platform that enables cellular-structural biology in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using this platform, we reconstructed sub-nanometer resolution structures of the human cardiac muscle thin filament, a central component of the contractile machinery. Reconstructing the troponin complex, a regulatory component of the thin filament, from within cells, we identified previously unobserved conformations that highlight the structural flexibility of this regulatory complex. We next measured the impact of chemical and genetic perturbations associated with cardiovascular disease on the structure of troponin. In both cases, we found changes in troponin structure that are consistent with known disease phenotypes-highlighting the value of our approach for dissecting complex disease mechanisms in the cellular context., Competing Interests: Competing interest statement J.C.W. is a co-founder and is on the SAB of Greenstone Biosciences, but this work was done independently. J.A.S. is a co-founder and consultant for Cytokinetics Inc. and owns stock in the company, which has a focus on therapeutic treatments for cardiomyopathies and other muscle diseases, but this work was done independently. The other authors declare no competing interests.

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity., Competing Interests: Declaration of interests L.S.Q. is a founder and scientific advisory board member of Epic Bio, and a scientific advisory board member of Laboratory of Genomics Research and Kytopen Corp. J.C.W. is a co-founder and scientific advisory board member of Greenstone Biosciences., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cannabis, the most commonly used recreational drug, is illicit in many areas of the world. With increasing decriminalization and legalization, cannabis use is increasing in the United States and other countries. The adverse effects of cannabis are unclear because its status as a Schedule 1 drug in the United States restricts research. Despite a paucity of data, cannabis is commonly perceived as a benign or even beneficial drug. However, recent studies show that cannabis has adverse cardiovascular and pulmonary effects and is linked with malignancy. Moreover, case reports have shown an association between cannabis use and neuropsychiatric disorders. With growing availability, cannabis misuse by minors has led to increasing incidences of overdose and toxicity. Though difficult to detect, cannabis intoxication may be linked to impaired driving and motor vehicle accidents. Overall, cannabis use is on the rise, and adverse effects are becoming apparent in clinical data sets.

Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases., (© 2022. Springer Nature Limited.)

Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs., Competing Interests: Declaration of interests J.C.W. is the cofounder of Khloris Biosciences but has no competing interests as the work presented here is completely independent., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated. Here, we clarified the distributions of TG2 protein/activity and conducted quantitative proteomics analyses of possible substrates crosslinked by TG2 on unfixed lung sections in a mouse pulmonary fibrosis model. We identified 126 possible substrates as markedly TG2-dependently increased in fibrotic lung. Gene ontology analysis revealed that these identified proteins were mostly enriched in the lipid metabolic process, immune system process, and protein transport. In addition, these proteins were enriched in 21 pathways, including phagosome, lipid metabolism, several immune responses, and protein processing in endoplasmic reticulum. Furthermore, the network analyses screened out the six clusters and top 20 hub proteins with higher scores, which are related to endoplasmic reticulum stress and peroxisome proliferator-activated receptor signals. Several enriched pathways and categories were identified, some of which were the same terms based on transcription analysis in IPF. Our results provide novel pathological molecular networks driven by protein crosslinking via TG2, which can lead to the development of new therapeutic targets for IPF.

Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33 -/- mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33 f/f dopamine-β-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA) A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33 f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress.

During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field., Competing Interests: Declaration of Competing Interest JCW is a cofounder of Khloris Biosciences but has no competing interests, as the work presented here is completely independent. LSQ is a cofounder of Refuge Biotechnologies but has no competing interests, as the work presented here is independent., (Copyright © 2020. Published by Elsevier B.V.)

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Coronavirus disease 2019 (COVID-19), caused by a strain of coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic that has affected the lives of billions of individuals. Extensive studies have revealed that SARS-CoV-2 shares many biological features with SARS-CoV, the zoonotic virus that caused the 2002 outbreak of severe acute respiratory syndrome, including the system of cell entry, which is triggered by binding of the viral spike protein to angiotensin-converting enzyme 2. Clinical studies have also reported an association between COVID-19 and cardiovascular disease. Pre-existing cardiovascular disease seems to be linked with worse outcomes and increased risk of death in patients with COVID-19, whereas COVID-19 itself can also induce myocardial injury, arrhythmia, acute coronary syndrome and venous thromboembolism. Potential drug-disease interactions affecting patients with COVID-19 and comorbid cardiovascular diseases are also becoming a serious concern. In this Review, we summarize the current understanding of COVID-19 from basic mechanisms to clinical perspectives, focusing on the interaction between COVID-19 and the cardiovascular system. By combining our knowledge of the biological features of the virus with clinical findings, we can improve our understanding of the potential mechanisms underlying COVID-19, paving the way towards the development of preventative and therapeutic solutions.

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.

Objective: This study aims to investigate the time-dependent prognostic utility of two fibrosis markers representing organ fibrogenesis (N-terminal propeptide of procollagen III (PIIINP) and type IV collagen 7S (P4NP 7S)) in patients with acute heart failure (HF)., Methods: 390 patients with acute HF were dichotomised based on the median value of fibrosis markers at discharge. The primary outcome measure was a composite of cardiac death and HF hospitalisation., Results: P4NP 7S significantly declined during hospitalisation, whereas PIIINP did not. The cumulative 90-day and 365-day incidence of the primary outcome measure was 16.6% vs 16.0% (p=0.42) and 33.3% vs 28.4% (p=0.34) in the patients with high versus low PIIINP; 19.9% vs 13.0% (p=0.04) and 32.3% vs 29.0% (p=0.34) in the patients with high and low P4NP 7S, respectively. After adjusting for confounders, high P4NP 7S correlated with significant excess risk relative to low P4NP 7S for both 90-day and 365-day primary outcome measure (adjusted HR, 1.50; 95% CI, 1.02 to 2.21; p=0.04 and adjusted HR, 1.89; 95% CI, 1.11 to 3.26; p=0.02, respectively), which was driven by significant association of high P4NP 7S with higher incidence of HF hospitalisation. Furthermore, P4NP 7S exhibited an additive value to conventional prognostic factors for predicting 90-day outcome (p=0.038 for net reclassification improvement; p=0.0068 for integrated discrimination improvement). High PIIINP did not correlate with significant excess risk for both 90-day and 365-day outcome., Conclusions: This study suggests a possible role of P4NP 7S in the risk stratification of patients with acute HF., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

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., (© 2019 The Authors.)

A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an in vivo delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs) and promoted angiogenesis in hypoxic conditions, showing significantly lower cytotoxicity than Lipofectamine. Furthermore, one injected dose of hydrogel/miNP in MI rats demonstrated significantly improved cardiac functions: increased ejection fraction from 45% to 64%, reduced scar size from 20% to 10%, and doubled capillary density in the border zone compared to the control group at 4 weeks. As such, our results indicate that this injectable hydrogel/miNP composite can deliver miRNA to restore injured myocardium efficiently and safely.

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.

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., (© 2019 The Author(s).)

Aims: Collagen-derived peptides such as collagen I C-terminal telopeptide (CITP) and procollagen III N-terminal propeptide (PIIINP) have been conventionally used as markers of cardiac fibrosis. Collagen IV 7S domain (P4NP 7S) has been recently reported to be correlated with haemodynamics in patients with acute heart failure. We investigated whether these markers reflect cardiac remodelling and myocardial collagen expression., Methods and Results: In 80 patients with dilated cardiomyopathy, relationships of CITP, PIIINP, and P4NP 7S to clinical and echocardiographic variables were analysed. CITP and PIIINP were inversely correlated with estimated glomerular filtration rate (r = -0.41, P < 0.001 and r = -0.32, P = 0.004, respectively); P4NP 7S was positively correlated with B-type natriuretic peptide (r = 0.32, P = 0.003) and γ-glutamyltransferase (r = 0.38, P < 0.001). These correlations were significant even after adjustment by potential confounders, whereas all three collagen markers were not independently correlated with ejection fraction nor with left ventricular (LV) diastolic diameter. In 33 patients undergoing endomyocardial biopsy, myocardial collagen I and III mRNA expressions were correlated with LV end-diastolic volume index (r = 0.42, P = 0.02 and r = 0.54, P = 0.002, respectively), whereas myocardial collagen IV mRNA expression was not correlated with LV end-diastolic volume index nor with ejection fraction. Each collagen-derived peptide was not significantly correlated with the myocardial expression of their corresponding collagen mRNA., Conclusions: Our study shows that CITP, PIIINP, and P4NP 7S do not reflect myocardial collagen mRNA expression but presumably reflect extra-cardiac organ injury in heart failure., (© 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

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.

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.

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 Ly6C high 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 Ly6C high 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 Ly6C high monocytes and that nonhematopoietic miR-33 deficiency caused reduced peripheral Ly6C high 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 Ly6C high 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 Ly6C high monocytes, and the other involves the elevation of HDL-C to decrease peripheral Ly6C high monocytes., (Copyright © 2018 Baba et al.)

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., (© 2017 American Heart Association, Inc.)

Aims: Recent studies have shown that serum microRNA (miR) abundance is informative for the diagnosis or prognosis of heart failure. However, the dynamics and kinetics of miRs in acute heart failure are largely unknown. Serial measurement and analysis of serum miRs changes in individuals along their therapeutic course could reduce inter-individual variation and should detect potentially important serum miRs related to disease mechanisms. Based on this concept, we profiled serum miR signatures of blood samples that were obtained sequentially on the day of admission and on hospital Day 7., Methods and Results: This prospective, observational study included 42 consecutive acute heart failure patients (74 ± 1 years old, 24 male). From admission to Day 7, most of the patients showed clinical improvement. In such a cohort, we detected several fluctuations of serum miRs by two distinct screening methods (quantitative PCR and high-throughput sequencing). One of these fluctuating serum miRs, miR-122-5p, decreased significantly from Day 1 to Day 7 [median arbitrary unit (1st:3rd quantile value); 4.62 [2.39:12.3] to 3.07 [1.67:5.39], P  = 0.007]. This fluctuation was significantly correlated with changes in serum liver function markers (estimated coefficient and 95% confidence interval; vs change in aspartate aminotransferase 1.69, 0.890-2.484, P  < 0.001 and r  = 0.560, vs change in alanine aminotransferase 1.09, 0.406-1.771, P  = 0.007 and r  = 0.428)., Conclusions: The serum miR signature of patients with acute heart failure might indicate the severity of the disease or patients' response to therapeutic intervention. Notably, serum miR-122-5p levels reflect liver damage in this condition.

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., (© 2016 American Heart Association, Inc.)

Background: Despite recent progress with drug-eluting stents, restenosis and thrombosis after endovascular intervention are still major limitations in the treatment of cardiovascular diseases. These problems are possibly caused by inappropriate inhibition of neointimal formation and retardation of re-endothelialization on the surface of the stents. miR-126 has been shown to have the potential to enhance vascular endothelial cell proliferation., Methods and Results: We designed and constructed a 27-nt double strand RNA (dsRNA) conjugated to cholesterol, which has high membrane permeability, and formed mature miR-126 after transfection. For site-specific induction of miR-126, we utilized poly (DL-lactide-co-glycolide) nanoparticles (NPs). miR-126-dsRNA-containing NPs (miR-126 NPs) significantly reduced the protein expression of a previously identified miR-126 target, SPRED1, in human umbilical vascular endothelial cells (HUVECs), and miR-126 NPs enhanced the proliferation and migration of HUVECs. On the other hand, miR-126 NPs reduced the proliferation and migration of vascular smooth muscle cells, via the suppression of IRS-1. Finally, we developed a stent system that eluted miR-126. This delivery system exhibited significant inhibition of neointimal formation in a rabbit model of restenosis., Conclusions: miR-126 NP-conjugated stents significantly inhibited the development of neointimal hyperplasia in rabbits. The present study may indicate the possibility of a novel therapeutic option to prevent restenosis after angioplasty.

Background: The optimal timing of aortic valve replacement (AVR) is controversial in patients with asymptomatic severe aortic stenosis (AS) except when very severe. Prediction of progression of severe AS is helpful in deciding on the timing of AVR. The purpose of this study was to clarify the predictors of progression rate and clinical outcomes of severe AS., Methods and results: We retrospectively investigated 140 consecutive patients with asymptomatic severe AS (aortic valve area [AVA], 0.75-1.0 cm(2)). First-year progression rate and annual progression rate of AVA and of aortic jet velocity (AV-Vel) were calculated. Cardiac events were examined and the predictors of rapid progression and cardiac events were analyzed. The median follow-up period was 36 months. The median annual progression rate was -0.05 cm(2)/year for AVA and 0.22 m/s/year for AV-Vel. Dyslipidemia, moderate-severe calcification, and first-year AV-Vel progression ≥0.22 m/s/year were independent predictors of cardiac events. Cardiac event-free rate was lower in patients with AV-Vel first-year progression rate ≥0.22 m/s/year than in those with a lower rate. Diabetes and moderate-severe calcification were related to first-year rapid progression., Conclusions: The annual progression rate of severe AS was -0.05 cm(2)/year for AVA and 0.22 m/s/year for AV-Vel. Patients with first-year rapid progression or severely calcified aortic valve should be carefully observed while considering an early operation. (Circ J 2016; 80: 1863-1869).

Background: ST-segment elevation (STE) in leads V1-2 is often observed in patients with severe aortic stenosis (AS), but its significance remains unknown., Methods and results: We retrospectively evaluated baseline ECGs and 5-year clinical outcomes in 211 consecutive patients with severe AS, defined as peak aortic jet velocity (Aortic Vmax) >4.0 m/s, or mean aortic pressure gradient >40 mmHg, or aortic valve area (AVA) <1.0 cm(2). The primary outcome measure was a composite of death or surgical aortic valve replacement (AVR). Patients with STE in leads V1-2(≥0.15 mV) had greater Aortic Vmax and smaller AVA than patients without. With a median follow-up of 4.9 years, the cumulative 5-year incidence of death or AVR was significantly higher in patients with STE in leads V1-2 than in patients without (91.4% vs. 77.1%; P=0.003). After adjusting for confounders, STE in leads V1-2 was independently associated with higher risk for death or AVR (hazard ratio, 1.53; 95% confidence interval, 1.06-2.22; P=0.02). In 64 asymptomatic patients without any indication for AVR at initial diagnosis of severe AS, the cumulative incidence of AVR was significantly higher in patients with STE in leads V1-2 than in patients without (57.6% vs. 30.5%; P<0.001)., Conclusions: STE in leads V1-2 independently predicted poorer prognosis and more frequent need for AVR in patients with severe AS.

Background: Recently, it has been reported that specific microRNA (miRNA) levels are elevated in serum and can be used as biomarkers in patients with cardiovascular diseases. However, miRNAs expression profiles and their sources in pericardial fluid (PF) are unclear., Methods and Results: The purpose of this study was to identify the levels of miRNAs in PF in relation to those in the serum in patients undergoing cardiac surgery. Serum (S) and PF from patients undergoing coronary artery bypass graft (CABG) due to stable angina pectoris (sAP) and unstable AP (uAP) and aortic valve replacement due to aortic stenosis (AS) were analyzed for the detection of miRNAs. We named these samples S-sAP, S-uAP, S-AS, PF-sAP, PF-uAP, and PF-AS, respectively. We first measured the levels of miR-423-5p, which was recognized previously as a biomarker for heart failure. miR-423-5p levels were significantly higher in PF than serum. Although there was no difference in miR-423-5p levels among the PF-AS, PF-sAP, and PF-uAP, its levels were significantly elevated in S-uAP compared with those in S-AS and S-sAP. In order to clarify the source of miR-423-5p in PF, we measured the levels of muscle-enriched miR-133a and vascular-enriched miR-126 and miR-92a in the same samples. miR-133a levels were significantly higher in serum than in PF, and it was elevated in S-uAP compared with S-AS. miR-126 level was significantly increased in serum compared with PF, and the level of miR-92a the similar tendency. miR-423-5p is located in the first intron of NSRP1. There is another miRNA, miR-3184, encoded in the opposite direction in the same region. In vitro experiments indicated that the duplex of miR-423-5p and miR-3184-3p was more resistant to RNase than the duplex of miR-423-5p and miR-133-3p, which may help to stabilize miR-423-5p in the PF., Conclusions: Our results suggested that miR-423-5p is enriched in PF, and serum miR-423-5p may be associate with uAP. Its expression pattern was different to that of muscle- and vascular-enriched miRNAs, miR-133a, miR-126, and miR-92a.

The number of adult patients with coronary artery disease caused by Kawasaki disease (KD) is gradually increasing, but some patients drop out of clinical follow-up. However, careful, long-term follow-up and establishment of an optimal treatment strategy are needed in these patients, as well as cooperation between pediatric and adult cardiologists. We report a case of acute myocardial infarction in a young adult patient with KD whose serial coronary angiography was followed up for over 30 years. Successful reperfusion therapy was performed with thrombus aspiration, rotational atherectomy, and implantation of a drug-eluting stent.

Rationale: In some patients with type 2 diabetes mellitus (DM) without hypertension, cardiac hypertrophy and attenuated cardiac function are observed, and this insult is termed diabetic cardiomyopathy. To date, microRNA (miRNAs or miR) functions in diabetic cardiomyopathy remain to be elucidated., Objective: To clarify the functions of miRNAs involved in diabetic cardiomyopathy caused by type 2 DM., Methods and Results: C57BL/6 mice were fed a high-fat diet (HFD) for 20 weeks, which induced obesity and type 2 DM. miRNA microarray analyses and real-time polymerase chain reaction revealed that miR-451 levels were significantly increased in the type 2 DM mouse hearts. Because excess supply of saturated fatty acids is a cause of diabetic cardiomyopathy, we stimulated neonatal rat cardiac myocytes with palmitic acid and confirmed that miR-451 expression was increased in a dose- and time-dependent manner. Loss of miR-451 function ameliorated palmitate-induced lipotoxicity in neonatal rat cardiac myocytes. Calcium-binding protein 39 (Cab39) is a scaffold protein of liver kinase B1 (LKB1), an upstream kinase of AMP-activated protein kinase (AMPK). Cab39 was a direct target of miR-451 in neonatal rat cardiac myocytes and Cab39 overexpression rescued the lipotoxicity. To clarify miR-451 functions in vivo, we generated cardiomyocyte-specific miR-451 knockout mice. HFD-induced cardiac hypertrophy and contractile reserves were ameliorated in cardiomyocyte-specific miR-451 knockout mice compared with control mice. Protein levels of Cab39 and phosphorylated AMPK were increased and phosphorylated mammalian target of rapamycin (mTOR) was reduced in cardiomyocyte-specific miR-451 knockout mouse hearts compared with control mouse hearts., Conclusions: Our results demonstrate that miR-451 is involved in diabetic cardiomyopathy through suppression of the LKB1/AMPK pathway., (© 2014 American Heart Association, Inc.)

Background Context: Chemonucleolysis has been proposed as a less invasive technique than surgery for patients with lumbar disc herniation. Once chymopapain had been approved as a chemonucleolysis drug, it was withdrawn because of serious complications. A novel agent with fewer complications would be desirable., Purpose: The purpose of this study was to investigate the effects of recombinant human matrix metalloproteinase 7 (rhMMP-7) in experimental chemonucleolysis in vitro and in vivo and examine its effects on tissue damage., Study Design: The study design is the experimental study using human herniated discs and enzyme substrates in vitro and dogs in vivo., Methods: The effects of rhMMP-7 on the degradation of human herniated discs were examined by measuring the wet weight in vitro. The correlations between the decrease in wet weight by rhMMP-7 and the conditions associated with herniated discs were also analyzed. The effects of rhMMP-7 on the proteoglycan and water contents were respectively examined with alcian blue staining and T2-weighted magnetic resonance imaging at 7 days after intradiscal injection in dogs. The distribution of [125I]-labeled rhMMP-7 was investigated by autoradioluminography at 7 days after intradiscal injection in dogs. An epidural injection study with rhMMP-7 was performed to evaluate the effects on the tissue damage around the discs at 1 and 13 weeks after the treatment in dogs. The Type 1 and 2 collagen cleavage rates were measured and compared with those of aggrecan in vitro., Results: Recombinant human matrix metalloproteinase 7 concentration dependently decreased the wet weight of herniated discs in vitro. The decrease in wet weight of the discs by rhMMP-7 did not significantly correlate with the conditions associated with herniated discs. Intradiscal injection of rhMMP-7 reduced the proteoglycan and water contents, with an increase in the serum keratan sulfate levels. Radioactivity of [125I]-labeled rhMMP-7 was detected in the nucleus pulposus and annulus fibrosus but not in the muscle. Epidural injection of rhMMP-7 had no effect on the injection site or the nerve tissues. The Type 1 and 2 collagen cleavage rates of rhMMP-7 were 1,000-fold weaker than those of aggrecan., Conclusions: This study demonstrated experimental chemonucleolysis with rhMMP-7 in vitro and in vivo. The effects of rhMMP-7 were not affected by the conditions associated with herniated discs. The epidural injection study together with the autoradioluminography and in vitro enzyme assay suggests that intradiscal injection of rhMMP-7 may not induce tissue damage around the discs because of its distribution and substrate selectivity. Recombinant human matrix metalloproteinase 7 may be a novel and promising chemonucleolysis agent., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33(-/-)Srebf1(+/-) mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33(-/-) mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo.

Aim: The optimal treatment strategy for patients with aortic atheroma is not well established because data regarding medical treatment for such patients are lacking, especially with respect to the Japanese population. The purpose of this study was to clarify the effects of medical treatment on the risk of embolic events and mortality in patients with severe aortic plaque., Methods: We retrospectively investigated 75 consecutive patients with severe aortic plaque detected on transesophageal echocardiography (TEE) between 1995 and 2005. The occurrence of embolic events and all-cause death in the period after TEE was assessed. The cumulative incidence of subsequent embolic events and death was evaluated in relation to specific medical treatments, including statins, antiplatelet drugs and warfarin., Results: Embolic events occurred in 27 patients (36%) and death occurred in 37 patients (49%) during follow-up (5.6±3.0 years). The patients who experienced embolic events had a significantly higher prevalence of previous embolic events, atrial fibrillation and hemodialysis than the patients who did not experience embolic events. Univariate and multivariate analyses showed that the use of statins and/or antiplatelet drugs was significantly associated with a low incidence of death but not with a low incidence of embolic events. On the other hand, warfarin exhibited neither beneficial nor harmful effects on the incidence of embolic events or death., Conclusions: Statin and antiplatelet drugs have beneficial effects on the prognosis of patients with severe aortic plaque diagnosed on TEE.

We report a rare case in which mitral regurgitation (MR) was exacerbated to a severe level early after atrial septal defect (ASD) closure, even though the female patient had preoperatively mild MR and mild changes in mitral valve (MV) and sinus rhythm. The mechanism of increased MR was considered as poor coaptation and tethering of the MV due to the restricted motion of the posterior leaflet in addition to geometric changes of the left ventricle (LV) after ASD closure.

Background: Severe tricuspid regurgitation (TR) sometimes develops late after left-sided valve surgery without left heart failure, pulmonary hypertension or rheumatic tricuspid valve. The purpose of the present study was to investigate clinical characteristics and mechanisms of severe isolated TR late after left-sided valve surgery., Methods and Results: A total of 372 consecutive patients who underwent left-sided valve surgery between 1990 and 2003 and who were followed up with echocardiography for at least 5 years, were retrospectively investigated. The mean follow-up period was 9.4 years. Clinical background, preoperative and postoperative echocardiographic parameters were evaluated. Among the 372 patients, severe isolated TR was detected in 23 patients, which developed at a mean of 8.6 years after surgery. Twenty-two of 23 patients had undergone mitral valve surgery. Multivariate logistic regression analysis identified the presence of preoperative atrial fibrillation and preoperative ejection fraction as independent determinants for the development of severe isolated TR. In patients with severe isolated TR, the tricuspid annular diameter and the right atrial area were already enlarged early after surgery and both of these increased prior to TR progression., Conclusions: Severe isolated TR developing late after mitral valve surgery is not uncommon, thus it is important to recognize this disease entity. Annular dilatation was the main cause of isolated TR and serial echocardiographic data are important to detect progression of isolated TR and to assess its mechanisms.

A 65-year-old man with rheumatic combined valvular heart disease showed a persistent fever after cardiac catheterization. He was diagnosed with cholesterol embolism due to multiple mobile plaques in the descending thoracic aorta by transesophageal echocardiography (TEE) along with persistent eosinophilia, deteriorating renal function, and blue toe sign. He was treated with intensive cholesterol-lowering therapy for 3 years, resulting in marked regression of the aortic plaque on TEE.

An 81-year-old man with a history of diabetes mellitus and end-stage renal disease was admitted because of infective endocarditis. During transesophageal echocardiography (TEE), pericardial effusion rapidly increased and led to cardiac tamponade. Despite intensive therapy, the patient did not recover. Autopsy showed hemopericardium, ruptured sinus of Valsalva, and vegetation on the aortic valve. Our case suggests that cardiac tamponade due to the rupture of a sinus of Valsalva can occur in patients with aortic valve endocarditis complicated by perivalvular abscess. Therefore, we must be aware of this devastating complication and take preventive measures when performing TEE in such patients.

It is well known that histamines play an important role as a neurotransmitter in the central nervous system and participate in several physiological functions, such as the regulation of body temperature, food intake, circadian rhythm and analgesia. Since the first report by de Almeida and Izquierdo that histamines facilitate memory performance in mice, it has been revealed that the histaminergic nervous system in the brain plays a crucial role in learning and memory functions. First, we demonstrate the effects of histamines and histaminergic drugs on learning and memory using active avoidance and 8-arm radial maze tests. Many reports suggest that acetylcholine and N-methyl-D-aspartate (NMDA) receptors play an important role in learning and memory. In this report, we also describe the interaction between histamine-induced memory facilitation and acetylcholine or NMDA receptors.

We examined the effect of propolis granular A. P. C on lung tumorigenesis in female A/J mice. Lung tumors were induced by the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) administered in drinking water for 7 weeks in mice maintained on an AIN-76A semi-synthetic diet. Propolis granular A. P. C (100 mg/kg body wt.) was administered orally daily for 6 days/week from 1 week before NNK administration and throughout the experiment. Sixteen weeks after the NNK treatment, the mice were killed and the number of surface lung tumors was measured. The number of lung tumors in mice treated with NNK alone for 7 weeks (9.4 mg/mouse) was significantly more than in that observed in control mice. Propolis granular A. P. C significantly decreased the number of lung tumors induced by NNK. These results indicate that propolis granular A. P. C is effective in suppressing NNK-induced lung tumorigenesis in mice.

Rationale: Histamine plays an important role in modulating acquisition and retention in learning and memory process in experimental animals., Objectives: We examined the effects of polyamine and histamine on the N-methyl- d-aspartate (NMDA) receptor glycine site antagonist 7-chlorokynurenic acid-induced spatial memory deficits in radial maze performance in rats., Method: Effects of histamine (0.5 or 1 nmol/site intracerebroventricularly), spermidine (1 nmol/site, intracerebroventricularly) and spermine (1 nmol/site, intracerebroventricularly) on spatial memory deficit in 9-week-old-male Wistar rats were observed. Both reference and working memory errors occurred in radial maze performance in rats, following intracerebroventricular injection of 7-chlorokynurenic acid (10 nmol/site)., Results: Spermidine (1 nmol/site, intracerebroventricularly) or spermine (1 nmol/site, intracerebroventricularly) antagonized 7-chlorokynurenic acid-induced deficits on working memory but not on reference memory errors. Intracerebroventricular histamine (0.5 or 1 nmol/site) or thioperamide (100 nmol/site) also ameliorated 7-chlorokynurenic acid-induced working memory deficits. To determine whether the effects of histamine involve histamine receptors, the effects of some methylhistamines were examined. The effects of R-alpha-methylhistamine on radial maze performance were mimicked by histamine. N(alpha)-methylhistamine had no effect on 7-chlorokynurenic acid-induced memory deficits, whereas 1-methylhistamine, but not 3-methylhistamine reversed 7-chlorokynurenic acid-induced working memory deficits., Conclusion: These results suggest that the amelioration of 7-chlorokynurenic acid-induced working memory deficits by histamine may involve a direct action of histamine at the polyamine sites on NMDA receptors.

1. The aim of the present study was to establish a new schedule of active avoidance response in rats to estimate the central effects of second-generation histamine H1 receptor antagonists. 2. With the new schedule, a rat was placed into a dark room. A sliding door was opened after a delay of 5 s and, unless the animal moved into the lit room, an electric shock was delivered for 3 s. With the conventional schedule, the sliding door was opened immediately after the rat was placed into the dark room. 3. Ketotifen, at a dose of 50 mg/kg, showed no significant effect on the retrieval of active avoidance response with the conventional schedule. However, with the new schedule, the drug caused significant inhibition of retrieval of the response, even at a dose of 10 mg/kg. 4. Epinastine showed no significant effect on retrieval of the active avoidance response, even at a dose of 50 mg/kg with the new schedule. 5. Cetirizine, at a dose of 50 mg/kg, caused a significant effect, indicating that cetirizine, at this dose, markedly inhibits memory retrieval. 6. Both olopatadine and loratadine had potent effects; at doses of 20 and 50 mg/kg, respectively, these agents showed significant inhibitory effects on retrieval of the response. 7. In conclusion, we have developed a new schedule of active avoidance response that can be used to estimate the central effects of second-generation histamine H1 receptor antagonists.