Metabolic remodeling and calcium handling abnormality in induced pluripotent stem cell-derived cardiomyocytes in dilated phase of hypertrophic cardiomyopathy with MYBPC3 frameshift mutation.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder characterized by left ventricular hypertrophy and diastolic dysfunction, and increases the risk of arrhythmias and heart failure. Some patients with HCM develop a dilated phase of hypertrophic cardiomyopathy (D-HCM) and have poor prognosis; however, its pathogenesis is unclear and few pathological models exist. This study established disease-specific human induced pluripotent stem cells (iPSCs) from a patient with D-HCM harboring a mutation in MYBPC3 (c.1377delC), a common causative gene of HCM, and investigated the associated pathophysiological mechanisms using disease-specific iPSC-derived cardiomyocytes (iPSC-CMs). We confirmed the expression of pluripotent markers and the ability to differentiate into three germ layers in D-HCM patient-derived iPSCs (D-HCM iPSCs). D-HCM iPSC-CMs exhibited disrupted myocardial sarcomere structures and an increased number of damaged mitochondria. Ca2+ imaging showed increased abnormal Ca2+ signaling and prolonged decay time in D-HCM iPSC-CMs. Cell metabolic analysis revealed increased basal respiration, maximal respiration, and spare-respiratory capacity in D-HCM iPSC-CMs. RNA sequencing also showed an increased expression of mitochondrial electron transport system-related genes. D-HCM iPSC-CMs showed abnormal Ca2+ handling and hypermetabolic state, similar to that previously reported for HCM patient-derived iPSC-CMs. Although further studies are required, this is expected to be a useful pathological model for D-HCM.

Selective O-Acylation of Enol Silyl Ethers with Acyl Fluorides Catalyzed by Fluoride Ions Derived from Potassium Fluoride and 18-Crown-6.

The fluoride ion-catalyzed selective O-acylation of enol silyl ethers with acyl fluorides using KF and 18-Crown-6 is described herein. This catalytic system facilitated the practical and facile reaction of a variety of enol silyl ethers derived from aromatic/aliphatic ketones and aldehydes with acyl fluorides to afford useful and valuable enol esters.

Identification and evolution of a diterpenoid phytoalexin oryzalactone biosynthetic gene in the genus Oryza.

The natural variation of plant-specialized metabolites represents the evolutionary adaptation of plants to their environments. However, the molecular mechanisms that account for the diversification of the metabolic pathways have not been fully clarified. Rice plants resist attacks from pathogens by accumulating diterpenoid phytoalexins. It has been confirmed that the composition of rice phytoalexins exhibits numerous natural variations. Major rice phytoalexins (momilactones and phytocassanes) are accumulated in most cultivars, although oryzalactone is a cultivar-specific compound. Here, we attempted to reveal the evolutionary trajectory of the diversification of phytoalexins by analyzing the oryzalactone biosynthetic gene in Oryza species. The candidate gene, KSLX-OL, which accounts for oryzalactone biosynthesis, was found around the single-nucleotide polymorphisms specific to the oryzalactone-accumulating cultivars in the long arm of chromosome 11. The metabolite analyses in Nicotiana benthamiana and rice plants overexpressing KSLX-OL indicated that KSLX-OL is responsible for the oryzalactone biosynthesis. KSLX-OL is an allele of KSL8 that is involved in the biosynthesis of another diterpenoid phytoalexin, oryzalexin S and is specifically distributed in the AA genome species. KSLX-NOL and KSLX-bar, which encode similar enzymes but are not involved in oryzalactone biosynthesis, were also found in AA genome species. The phylogenetic analyses of KSLXs, KSL8s, and related pseudogenes (KSL9s) indicated that KSLX-OL was generated from a common ancestor with KSL8 and KSL9 via gene duplication, functional differentiation, and gene fusion. The wide distributions of KSLX-OL and KSL8 in AA genome species demonstrate their long-term coexistence beyond species differentiation, suggesting a balancing selection between the genes.

Inhibitory Actions of Antidepressants, Hypnotics, and Anxiolytics on Recombinant Human Acetylcholinesterase Activity.

Alzheimer's disease (AD) is accompanied by behavioral and psychological symptoms of dementia (BPSD), which is often alleviated by treatment with psychotropic drugs, such as antidepressants, hypnotics, and anxiolytics. If these drugs also inhibit acetylcholinesterase (AChE) activity, they may contribute to the suppression of AD progression by increasing brain acetylcholine concentrations. We tested the potential inhibitory effects of 31 antidepressants, 21 hypnotics, and 12 anxiolytics on recombinant human AChE (rhAChE) activity. At a concentration of 10-4 M, 22 antidepressants, 19 hypnotics, and 11 anxiolytics inhibited rhAChE activity by <20%, whereas nine antidepressants (clomipramine, amoxapine, setiptiline, nefazodone, paroxetine, sertraline, citalopram, escitalopram, and mirtazapine), two hypnotics (triazolam and brotizolam), and one anxiolytic (buspirone) inhibited rhAChE activity by ≥20%. Brotizolam (≥10-6 M) exhibited stronger inhibition of rhAChE activity than the other drugs, with its pIC50 value being 4.57 ± 0.02. The pIC50 values of the other drugs were <4, and they showed inhibitory activities toward rhAChE at the following concentrations: ≥3 × 10-6 M (sertraline and buspirone), ≥10-5 M (amoxapine, nefazodone, paroxetine, citalopram, escitalopram, mirtazapine, and triazolam), and ≥3 × 10-5 M (clomipramine and setiptiline). Among these drugs, only nefazodone inhibited rhAChE activity within the blood concentration range achievable at clinical doses. Therefore, nefazodone may not only improve the depressive symptoms of BPSD through its antidepressant actions but also slow the progression of cognitive symptoms of AD through its AChE inhibitory actions.

AIM/CD5L ameliorates autoimmune arthritis by promoting removal of inflammatory DAMPs at the lesions.

The hallmark of autoimmune arthritis is the preceding autoantibody production and the following synovial inflammation with hyperplasia and tissue destruction of the joints. The joint inflammation is mediated not only by effector lymphocytes and auto-antibodies but also chronic activation of innate immunity, particularly promoted by the danger-associated molecular patterns (DAMPs). Here we show that apoptosis inhibitor of macrophage (AIM, also called CD5L) protein regulates arthritis by promoting removal of lesional DAMPs both physiologically and therapeutically. When the autoimmune arthritis was promoted by injecting a cocktail of anti-collagen antibodies without type-II collagen immunization, AIM-deficient (AIM-/-) mice exhibited more exacerbated and sustained swelling at multiple joints with greater synovial hyperplasia and bone erosion than wild-type mice. Administration of recombinant AIM (rAIM) reduced S100A8/9, a major DAMP known to be involved in arthritis progression, and decreased various inflammatory cytokines at the lesions in antibody-injected AIM-/- mice, leading to marked prevention of arthritis symptoms. In human rheumatoid arthritis (RA) patients, AIM was more activated via dissociating from IgM-pentamer in response to DAMPs-mediated inflammation both in serum and synovial fluid than in healthy individuals or non-autoimmune osteoarthritis patients, suggesting a disease-regulatory potency of AIM also in human RA patients. Thus, our study implied a therapeutic availability of rAIM to prevent arthritis symptoms targeting DAMPs.

Increased angiotensin II coupled with decreased Adra1a expression enhances cardiac hypertrophy in pregnancy-associated hypertensive mice.

Cardiac hypertrophy is a crucial risk factor for hypertensive disorders during pregnancy, but its progression during pregnancy remains unclear. We previously showed cardiac hypertrophy in a pregnancy-associated hypertensive (PAH) mouse model, in which an increase in angiotensin II (Ang II) levels was induced by human renin and human angiotensinogen, depending on pregnancy conditions. Here, to elucidate the factors involved in the progression of cardiac hypertrophy, we performed a comprehensive analysis of changes in gene expression in the hearts of PAH mice and compared them with those in control mice. We found that alpha-1A adrenergic receptor (Adra1a) mRNA levels in the heart were significantly reduced under PAH conditions, whereas the renin-angiotensin system was upregulated. Furthermore, we found that Adra1a-deficient PAH mice exhibited more severe cardiac hypertrophy than PAH mice. Our study suggests that Adra1a levels are regulated by renin-angiotensin system and that changes in Adra1a expression are involved in progressive cardiac hypertrophy in PAH mice.

Identification of potential dilated cardiomyopathy-related targets by meta-analysis and co-expression analysis of human RNA-sequencing datasets.

AIMS: Dilated cardiomyopathy (DCM) remains among the most refractory heart diseases because of its complicated pathogenesis, and the key molecules that cause it remain unclear. MAIN METHODS: To elucidate the molecules and upstream pathways critical for DCM pathogenesis, we performed meta-analysis and co-expression analysis of RNA-sequencing (RNA-seq) datasets from publicly available databases. We analyzed three RNA-seq datasets containing comparisons of RNA expression in left ventricles between healthy controls and DCM patients. We extracted differentially expressed genes (DEGs) and clarified upstream regulators of cardiovascular disease-related DEGs by Ingenuity Pathway Analysis (IPA). Weighted Gene Co-expression Network Analysis (WGCNA) and Protein-Protein Interaction (PPI) analysis were also used to identify the hub gene candidates strongly associated with DCM. KEY FINDINGS: In total, 406 samples (184 healthy, 222 DCM) were used in this study. Overall, 391 DEGs [absolute fold change (FC) ≥ 1.5; P < 0.01], including 221 upregulated and 170 downregulated ones in DCM, were extracted. Seven common hub genes (LUM, COL1A2, CXCL10, FMOD, COL3A1, ADAMTS4, MRC1) were finally screened. IPA showed several upstream transcriptional regulators, including activating (NFKBIA, TP73, CALR, NFKB1, KLF4) and inhibiting (CEBPA, PPARGC1A) ones. We further validated increased expression of several common hub genes in the transverse aortic constriction-induced heart failure model. SIGNIFICANCE: In conclusion, meta-analysis and WGCNA using RNA-seq databases of DCM patients identified seven hub genes and seven upstream transcriptional regulators.

Generation of a human induced pluripotent stem cell line derived from a patient with dilated cardiomyopathy carrying LMNA nonsense mutation.

Dilated cardiomyopathy (DCM) is a refractory heart disease characterized by dilation of the left ventricle and systolic dysfunction. LMNA, the gene encoding lamin A/C (a nuclear envelope protein), is the second leading causative gene associated with familial DCM. LMNA-related DCM is likely to develop severe heart failure, various types of arrhythmias, and poor prognosis. We established a human induced pluripotent stem cell line, derived from a patient with DCM carrying a nonsense mutation in LMNA. This line should be a useful resource for elucidating disease mechanisms and developing fundamental treatments for LMNA-related DCM.

Electrical, structural, and autonomic atrial remodeling underlies atrial fibrillation in inflammatory atrial cardiomyopathy.

Background: There is growing evidence indicating a close relationship between inflammation and atrial fibrillation (AF). Although underlying inflammatory atrial cardiomyopathy may contribute to the development of AF, the arrhythmogenic remodeling caused by atrial inflammation has not been elucidated in detail. Herein, we examined electrical, structural, and autonomic changes in the atria in a mouse model of autoimmune myocarditis. Methods: BALB/c mice were immunized with cardiac myosin peptide (MyHC-α614-629) conjugated with complete Freund's adjuvant on days 0 and 7. Susceptibility to AF was assessed using right-atrial burst pacing. Results: The mice immunized with MyHC-α614-629 showed an inflammatory atrial cardiomyopathy phenotype, with enlarged atria; a high degree of inflammatory cell infiltration primarily consisting of CD4+ T cells, CD8+ T cells, Ly6GlowCD11b+ macrophages, and CD11c+ dendritic cells; and severe interstitial fibrosis with collagen deposition. These mice demonstrated significantly enhanced susceptibility to AF, as indicated by their increased AF induction rate and duration. In addition, the expression of potassium channels (Kcnh2, Kcnd3, and Kcnj2) and calcium handling-associated genes (Cacna1c, Camk2, Ryr2, and Atp2a2) was downregulated. Connexin 40 expression was significantly downregulated, leading to frequent lateralization to the inflamed atrium. Sympathetic and parasympathetic innervation and neurotrophin expression (nerve growth factor and brain-derived neurotrophic factor) were upregulated in the inflamed atria. Conclusion: Inflammatory atrial cardiomyopathy promotes susceptibility to AF via arrhythmogenic electrical, structural, and autonomic remodeling of the atria.

Xanthine oxidase inhibitor febuxostat reduces atrial fibrillation susceptibility by inhibition of oxidized CaMKII in Dahl salt-sensitive rats.

Oxidative stress could be a possible mechanism and a therapeutic target of atrial fibrillation (AF). However, the effects of the xanthine oxidase (XO) inhibition for AF remain to be fully elucidated. We investigated the effects of a novel XO inhibitor febuxostat on AF compared with allopurinol in hypertension rat model. Five-week-old Dahl salt-sensitive rats were fed either low-salt (LS) (0.3% NaCl) or high-salt (HS) (8% NaCl) diet. After 4 weeks of diet, HS diet rats were divided into three groups: orally administered to vehicle (HS-C), febuxostat (5 mg/kg/day) (HS-F), or allopurinol (50 mg/kg/day) (HS-A). After 4 weeks of treatment, systolic blood pressure (SBP) was significantly higher in HS-C than LS, and it was slightly but significantly decreased by treatment with each XO inhibitor. AF duration was significantly prolonged in HS-C compared with LS, and significantly suppressed in both HS-F and HS-A (LS; 5.8 ± 3.5 s, HS-C; 33.9 ± 23.7 s, HS-F; 15.0 ± 14.1 s, HS-A; 20.1 ± 11.9 s: P<0.05). Ca2+ spark frequency was obviously increased in HS-C rats and reduced in the XO inhibitor-treated rats, especially in HS-F group. Western blotting revealed that the atrial expression levels of Met281/282-oxidized Ca2+/Calmodulin-dependent kinase II (CaMKII) and Ser2814-phosphorylated ryanodine receptor 2 were significantly increased in HS-C, and those were suppressed in HS-F and HS-A. Decreased expression of gap junction protein connexin 40 in HS-C was partially restored by treatment with each XO inhibitor. In conclusion, XO inhibitor febuxostat, as well as allopurinol, could reduce hypertension-related increase in AF perpetuation by restoring Ca2+ handling and gap junction.

Programmed Death-Ligand 2 Deficiency Exacerbates Experimental Autoimmune Myocarditis in Mice.

Programmed death ligand 2 (PD-L2) is the second ligand of programmed death 1 (PD-1) protein. In autoimmune myocarditis, the protective roles of PD-1 and its first ligand programmed death ligand 1 (PD-L1) have been well documented; however, the role of PD-L2 remains unknown. In this study, we report that PD-L2 deficiency exacerbates myocardial inflammation in mice with experimental autoimmune myocarditis (EAM). EAM was established in wild-type (WT) and PD-L2-deficient mice by immunization with murine cardiac myosin peptide. We found that PD-L2-deficient mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4+ T cell proliferation in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects against autoreactive CD4+ T cell expansion and severe inflammation in mice with EAM.

Neutrophil Elastase Deficiency Ameliorates Myocardial Injury Post Myocardial Infarction in Mice.

Neutrophils are recruited into the heart at an early stage following a myocardial infarction (MI). These secrete several proteases, one of them being neutrophil elastase (NE), which promotes inflammatory responses in several disease models. It has been shown that there is an increase in NE activity in patients with MI; however, the role of NE in MI remains unclear. Therefore, the present study aimed to investigate the role of NE in the pathogenesis of MI in mice. NE expression peaked on day 1 in the infarcted hearts. In addition, NE deficiency improved survival and cardiac function post-MI, limiting fibrosis in the noninfarcted myocardium. Sivelestat, an NE inhibitor, also improved survival and cardiac function post-MI. Flow cytometric analysis showed that the numbers of heart-infiltrating neutrophils and inflammatory macrophages (CD11b+F4/80+CD206low cells) were significantly lower in NE-deficient mice than in wild-type (WT) mice. At the border zone between intact and necrotic areas, the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells was lower in NE-deficient mice than in WT mice. Western blot analyses revealed that the expression levels of insulin receptor substrate 1 and phosphorylation of Akt were significantly upregulated in NE-knockout mouse hearts, indicating that NE deficiency might improve cardiac survival by upregulating insulin/Akt signaling post-MI. Thus, NE may enhance myocardial injury by inducing an excessive inflammatory response and suppressing Akt signaling in cardiomyocytes. Inhibition of NE might serve as a novel therapeutic target in the treatment of MI.

Serotonin modulates behavior-related neural activity of RID interneuron in Caenorhabditis elegans.

Animals change their behaviors in response to external stimuli, and numerous neurotransmitters are involved in these behavioral changes. In Caenorhabditis elegans, serotonin (5-HT) affects various behaviors such as inhibition of locomotion, stimulation of egg laying, and pharyngeal pumping. Previous research has shown that the neural activity of the RID interneuron increases when the worm moves forward, and the RID is necessary for sustaining forward locomotion. However, the relationship between 5-HT and neural activity of RID, and how it modulates the behavior of the worm has not been investigated. In this article, we reveal the relationship among 5-HT, RID activity, and the behavior of worms using a custom-made tracking and imaging system. We simultaneously measured the neural activity of the RID and behavior in worms with three conditions: mock animals, animals pre-exposed to 5-HT, and 5-HT receptor mod-1 mutants. As shown in previous research, the neural activity of the RID increased during the transition from backward to forward, whereas it decreased during the transition from forward to backward in mock animals. These changes in neural activity were not observed in animals pre-exposed to 5-HT and mod-1 mutants. Moreover, RID activity was correlated with the velocity of the worm in mock animals. However, this correlation was not observed in animals pre-exposed to 5-HT and mod-1 mutants. Our results demonstrate that 5-HT modulates the activity of the RID interneuron, and we infer that the RID plays a role in modulating forward locomotion by changing its activity through 5-HT.