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The underlying mechanisms of ventricular remodeling after myocardial infarction (MI) remain largely unknown. In this study, we performed an integrative analysis of spatial transcriptomics and single-nucleus RNA sequencing (snRNA-seq) in a murine MI model and found that mechanical stress-response genes are expressed at the border zone and play a critical role in left ventricular remodeling after MI. An integrative analysis of snRNA-seq and spatial transcriptome of the heart tissue after MI identified the unique cluster that appeared at the border zone in an early stage, highly expressing mechano-sensing genes, such as Csrp3. AAV9-mediated gene silencing and overexpression of Csrp3 demonstrated that upregulation of Csrp3 plays critical roles in preventing cardiac remodeling after MI by regulation of genes associated with mechano-sensing. Overall, our study not only provides an insight into spatiotemporal molecular changes after MI but also highlights that the mechano-sensing genes at the border zone act as adaptive regulators of left ventricular remodeling.

Using genetically engineered mice lacking estrogen receptor-α non-nuclear signaling, this study demonstrated that estrogen receptor-α non-nuclear signaling activated myocardial cyclic guanosine monophosphate-dependent protein kinase G and conferred protection against cardiac remodeling induced by pressure overload. This pathway was indispensable to the therapeutic efficacy of cyclic guanosine monophosphate-phosphodiesterase 5 inhibition but not to that of soluble guanylate cyclase stimulation. These results might partially explain the equivocal results of phosphodiesterase 5 inhibitor efficacy and also provide the molecular basis for the advantage of using a soluble guanylate cyclase simulator as a new therapeutic option in post-menopausal women. This study also highlighted the need for female-specific therapeutic strategies for heart failure.

The 83rdAnnual Scientific Meeting of the Japanese Circulation Society was held in Yokohama, Japan, on March 29-31, 2019, just as the cherry blossoms came into full bloom. Because the environment around cardiovascular healthcare is rapidly changing, it becomes highly important to make a breakthrough at the dawn of a new era. The main theme of this meeting was "Renaissance of Cardiology for the Creation of Future Medicine". The meeting benefited from the participation of 18,825 people, and there were in-depth and extensive discussions at every session, focusing on topics covering clinical and basic research, medical care provision system, human resource development, and public awareness in cardiovascular medicine. The meeting was completed with great success, and we greatly appreciate the tremendous cooperation and support from all affiliates.

The left ventricular remodeling after myocardial infarction (MI) causes heart failure, but its underlying mechanisms remain largely unknown. Here, we performed an integrative analysis of spatial transcriptomics and single cell RNA-seq in murine MI model and found that mechanical stress-response genes are expressed at the border zone and play a critical role in left ventricular remodeling after MI. Single-cardiomyocyte RNA-seq of the heart after MI divided cardiomyocytes into 8 clusters. We identified the cell cluster which was specifically derived from the ischemic area in an early stage after MI and expressed mechano-sensing genes such as Csrp3, Ankrd1 and Ankrd2. Spatial transcriptomic analysis demonstrated that cardiomyocytes with this gene expression profile were specifically localized at the border zone in post-MI day 1. AAV9-mediated gene silencing of Csrp3 exacerbated cardiac dilatation and dysfunction after MI. Collectively, our datasets and findings not only provide an insight into spatiotemporal molecular pathogenesis of myocardial infarction but also highlight mechano-sensing genes at the border zone as an adaptive regulator of left ventricular remodeling.

Tissue fibrosis and organ dysfunction are hallmarks of age-related diseases including heart failure, but it remains elusive whether there is a common pathway to induce both events. Through single-cell RNA-seq, spatial transcriptomics, and genetic perturbation, we elucidate that high-temperature requirement A serine peptidase 3 (Htra3) is a critical regulator of cardiac fibrosis and heart failure by maintaining the identity of quiescent cardiac fibroblasts through degrading transforming growth factor-β (TGF-β). Pressure overload downregulates expression of Htra3 in cardiac fibroblasts and activated TGF-β signaling, which induces not only cardiac fibrosis but also heart failure through DNA damage accumulation and secretory phenotype induction in failing cardiomyocytes. Overexpression of Htra3 in the heart inhibits TGF-β signaling and ameliorates cardiac dysfunction after pressure overload. Htra3-regulated induction of spatio-temporal cardiac fibrosis and cardiomyocyte secretory phenotype are observed specifically in infarct regions after myocardial infarction. Integrative analyses of single-cardiomyocyte transcriptome and plasma proteome in human reveal that IGFBP7, which is a cytokine downstream of TGF-β and secreted from failing cardiomyocytes, is the most predictable marker of advanced heart failure. These findings highlight the roles of cardiac fibroblasts in regulating cardiomyocyte homeostasis and cardiac fibrosis through the Htra3-TGF-β-IGFBP7 pathway, which would be a therapeutic target for heart failure.

Most seven transmembrane receptors (7TMRs) are G protein-coupled receptors; however, some 7TMRs evoke intracellular signals through β-arrestin as a biased receptor. As several β-arrestin-biased agonists have been reported to be cardioprotective, we examined the role of the chemokine receptor CXCR7 as a β-arrestin-biased receptor in the heart. Among 510 7TMR genes examined, Cxcr7 was the most abundantly expressed in the murine heart. Single-cell RNA-sequencing analysis revealed that Cxcr7 was abundantly expressed in cardiomyocytes and fibroblasts. Cardiomyocyte-specific Cxcr7 null mice showed more prominent cardiac dilatation and dysfunction than control mice 4 weeks after myocardial infarction. In contrast, there was no difference in cardiac phenotypes between fibroblast-specific Cxcr7-knockout mice and control mice even after myocardial infarction. TC14012, a specific agonist of CXCR7, significantly recruited β-arrestin to CXCR7 in CXCR7-expressing cells and activated extracellular signal-regulated kinase (ERK) in neonatal rat cardiomyocytes. Cxcr7 expression was significantly increased and ERK was activated in the border zone of the heart in control, but not Cxcr7 null mice. These results indicate that the abundantly expressed CXCR7 in cardiomyocytes may play a protective role in the heart as a β-arrestin-biased receptor and that CXCR7 may be a novel therapeutic target for myocardial infarction.

Percutaneous coronary intervention (PCI) is sometimes considered as an alternative therapeutic strategy to surgical revascularization in patients with coronary artery disease (CAD) and reduced left ventricular ejection fraction (LVEF). However, the types or conditions of patients that receive the clinical benefit of left ventricular reverse remodelling (LVRR) remain unknown. The purpose of this study was to investigate the determinants of LVRR following PCI in CAD patients with reduced LVEF. From 4394 consecutive patients who underwent PCI, a total of 286 patients with reduced LV systolic function (LVEF

Background Beta blockers and angiotensin II receptor blockers take effect through G protein-coupled receptors (GPCRs) and their protective roles in heart failure are partially attributable to beta-arrestin biased agonism. CXCR7, a chemokine receptor, is beta-arrestin biased receptor and one of the most expressing GPCRs in the heart. CXCL12 is a common ligand of CXCR4 and CXCR7 and is known to ameliorate myocardial infarction (MI), reportedly through CXCR4 dependent mechanisms. However, the role of another receptor, CXCR7 and its downstream target including beta-arrestin is not fully elucidated in MI. Purpose The aim of this study is to uncover the role of CXCR7 in cardiomyocytes after MI. Methods First, we quantified CXCR7 mRNA expressions in neonate rat cardiomyocytes (NRCM) in a dish by qRT-PCR. NRCMs were treated with CXCR7 agonist: TC14012 and phosphorylation of extracellular signal regulated kinase (ERK) was measured as readout of the downstream of CXCR7, with immunoblotting. Second, MI was induced by left anterior descending artery (LAD) ligation in male 12-week-old mice. We explored spatial expressions of CXCR7 by qRT-PCR in infarct, peri-infarct and remote zones of wild-type MI mice. Finally, we developed cardiomyocyte-specific CXCR7 knockout mice (cKO) by the Cre/loxP system and analyzed CXCR7 expression in cKO by qRT-PCR. LAD ligation was also performed in cKO and littermate controls (Ctl). Heart weight (HW) was measured and systolic function was examined by echocardiography 4 weeks after ligation. Phosphorylated ERK was evaluated with immunoblotting one-day after ligation. Results First, we found that CXCR7 expression was significantly higher in NRCM than neonatal rat fibroblasts (NRFB) and ERK was phosphorylated by CXCR7 stimulation in NRCM. Second, CXCR7 expression was higher in infarct and peri-infarct zones than remote zones. Finally, cardiomyocyte-specific knockout of CXCR7 resulted in 78±21% reduction of CXCR7 expression in the whole heart. HW and left ventricular area was significantly greater (HW: Ctl 190.7±18.4, cKO 220.3±26.4 mg) and fractional area change of left ventricle was significantly lower in cKO than those in Ctl 4 weeks after MI (LV FAC: Ctl 20.6±4.9%, cKO 13.9±5.4%), indicating that loss of CXCR7 in cardiomyocytes caused left ventricle enlargement and systolic dysfunction. One day after MI of Ctl heart, ERK was more phosphorylated in peri-infarct zone than remote zone. However, this ERK phosphorylation in peri-infarct zone was reduced in cKO MI heart. Conclusion We revealed that CXCR7 is expressed in cardiomyocytes and deletion of this chemokine receptor in cardiomyocytes resulted in ventricle enlargement and systolic dysfunction possibly through ERK phosphorylation in peri-infarct zone. Therefore, CXCR7 in cardiomyocytes could prevent cardiac dysfunction after myocardial infarction, which may be another pathway of CXCL12 dependent-protective effect. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI

Visual Abstract, Highlights • New CMs in adult mammalians are generated at a low rate, and Hippo–YAP signaling plays crucial roles in the postnatal cardiac regeneration. • After chemical screenings to identify compounds that activate YAP–TEADs activities and CM proliferation in vitro, the authors synthesized a novel multifaceted fluorinated compound TT-10 (C11H10FN3OS2) from a biologically hit compound. • TT-10 induces proliferation of cultured CMs via nuclear translocation of YAP and activation of Wnt/β-catenin signaling, and also activates NRF2-mediated antioxidant and antiapoptotic effects. • The intraperitoneal injection of TT-10 ameliorates cardiac remodeling after MI in mice, which is partially mediated by CM proliferation and by direct antioxidant and antiapoptotic effects in vivo. • Stimulating CM proliferation and/or protection with TT-10 might complement current therapies for MI., Summary Accumulating data suggest that new cardiomyocytes in adults are generated from existing cardiomyocytes throughout life. To enhance the endogenous cardiac regeneration, we performed chemical screenings to identify compounds that activate pro-proliferative YES-associated protein and transcriptional enhancer factor domain activities in cardiomyocytes. We synthesized a novel fluorine-containing TT-10 (C11H10FN3OS2) from the biologically hit compound. TT-10 promoted cardiomyocyte proliferation and simultaneously exerted antioxidant and antiapoptotic effects in vitro. TT-10 treatment in mice ameliorated myocardial infarction–induced cardiac dysfunction at least in part via enhancing clonal expansion of existing cardiomyocytes with nuclear YES-associated protein expression. Stimulating cardiomyocyte proliferation and/or protection with TT-10 might complement current therapies for myocardial infarction.

Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes., The mechanisms underlying the transition from cardiac hypertrophy to heart failure following pressure overload are incompletely understood. Here the authors identify the gene programs encoding the morphological and functional characteristics of cardiomyocytes during the transition from early hypertrophy to heart failure via single-cell transcriptomics, establishing a key role for p53 signalling.

Metabolomics has recently emerged as a tool for understanding comprehensive tumor-associated metabolic dysregulation. However, only limited application of this technology has been introduced into the clinical setting of breast cancer. The aim of this study was to determine the feasibility of metabolome analysis using routine CNB/VAB samples from breast cancer patients and to elucidate metabolic signatures using metabolic profiling. After breast cancer screenings, 20 consecutive patients underwent CNB/VAB, and diagnosed with benign, DCIS and IDC by histology. Metabolome analysis was performed using CE–MS. Differential metabolites were then analyzed and evaluated with MetaboAnalyst 4.0. We measured 116-targeted metabolites involved in energy metabolism. Principal component analysis and unsupervised hierarchical analysis revealed a distinct metabolic signature unique to namely “pure” IDC samples, whereas that of DCIS was similar to benign samples. Pathway analysis unveiled the most affected pathways of the “pure” IDC metabotype, including “pyrimidine,” “alanine, aspartate, and glutamate” and “arginine and proline” pathways. Our proof-of-concept study demonstrated that CE–MS-based CNB/VAB metabolome analysis is feasible for implementation in routine clinical settings. The most affected pathways in this study may contribute to improved breast cancer stratification and precision medicine.

Background: Low carbohydrate diet (LCD) has been reported to reduce body weight and cardiovascular risk factors, including diabetic mellitus and dyslipidemia. Therefore, LCD was once expected to prevent cardiovascular events. However, recent meta-analysis reported that LCD was related to an increase in cardiovascular events. Moreover, other cohort studies reported that higher all-cause and cardiovascular mortality were observed only in LCD with animal derived-fat (LCD-A), not in LCD with plant derived-fat (LCD-P). Precise mechanisms of these effects have not been clarified, so we explored the potential roles of LCD-A and LCD-P in blood vessel remodeling by using two types of vascular disease model mice, atherosclerotic model and vascular injury model. Method: We generated two types of LCDs, beef tallow-based LCD (LCD-A) and cacao butter-based LCD (LCD-P). Six-week-old male ApoE Knockout (ApoE KO) mice (atherosclerosis model mice) and wild-type mice were subjected to LCD-A, LCD-P or normal chow for 14 weeks, and %plaque area of aortic root and serum lipid profile were measured. For vascular injury model, mice were inserted with a wire in a femoral artery of male 10-week-old wild-type mice and subjected to the three-types chow. After 4 weeks, femoral arteries were excised and embedded in paraffin and assessed with the extent of vascular injury severity. The femoral artery without injury on the other side was served as control. Results: In ApoE KO mice, serum levels of total cholesterol (TC) and triglyceride (TG) were decreased in LCD group (TC: p Conclusion: Our preliminary data showed that LCD had no effect on vascular remodeling despite the presence of significant alteration in lipid profiles. Additional experiments are needed to confirm the results and clarify the precise mechanisms.

Background: The association between low carbohydrate diet (LCD) and cardiovascular disease and mortality remains unknown. Results from meta-analyses have reported that cardiovascular mortality increased when the carbohydrate in LCD was replaced for animal-derived fat, whereas mortality decreased when the substitution was plant-derived fat. However, the molecular mechanism is yet to be elucidated. We aimed to assess the effects of the LCD under two conditions, LCD with animal-derived fat (LCD-A) and LCD with plant-derived fat (LCD-P), on mouse cardiac function. Methods: Using 10-week old male C57B/6J mice, we performed transverse aortic constriction surgery to generate a pressure overload model of heart failure and subjected them to either normal diet, LCD-A or LCD-P for 4 weeks. Cardiac function was measured by echocardiography at 4 weeks after surgery, and the extent of inflammation was assessed by immunohistological analysis. Expression levels of genes were assessed with RNA sequence. Results: LCD-A accelerated left ventricular dilatation and systolic dysfunction (PCpt1a, Slc25a20, Acadvl, Hadha and Acaa2 were upregulated only in LCD-P (P Conclusions: These results suggest that the effects of the LCD on cardiac function vary between the sources of fat alternatives to carbohydrate intake. LCD-P induces PPARα activation, an important regulator of mitochondrial lipid metabolism, that may be therapeutically relevant for heart failure treatment. Further studies are required to unveil the precise molecular mechanism.

Objective: Sustained inflammation is a key pathological process in the development of heart failure and thus suppression of inflammation possibly leads to improvement of cardiac dysfunction. It has been suggested that CC chemokine receptor 5 (CCR5) scavenges pro-inflammatory mediators and resolves inflammation. The aim of this study is to uncover the role of CCR5 in the pathogenesis of heart failure. Methods: Pressure overload heart failure was induced by transverse aortic constriction (TAC). Sham or TAC operation was performed in male 10-week-old CCR5 knockout (KO) mice and wild-type (WT) controls. Cardiac function and morphology were examined by echocardiography 1 week and 4 weeks after TAC surgery. After euthanization, the heart was extracted and embedded in paraffin for the measurements of cardiac fibrosis and cardiomyocyte hypertrophy with EVG stain and immunohistochemistry using WGA, respectively. The extent of inflammation was evaluated by the immunostaining against CD45 and F4/80 antigens, and by qRT-PCR for inflammatory cytokines and chemokines. Results: We found that heart weight was significantly larger and fractional shortening was significantly lower in CCR5 KO mice than those in WT 4 weeks after TAC, indicating CCR5-induced protective effects on heart failure development. Whilst there was no difference observed in the extent of fibrosis, the size of cardiomyocytes from CCR5 KO heart tended to be increased compared to WT. The number of CD45-positive inflammatory cells and F4/80-positive macrophage infiltration was increased 1 week after TAC in KO mice. The RNA expression levels of monocyte chemotactic protein-1, transforming growth factor-β and tumor necrosis factor-α were significantly higher and NFκB p65 was more phosphorylated in KO mice. Conclusions: We revealed that the inflammation in pressure overload-induced heart failure was exacerbated by the deletion of CCR5 and, therefore, CCR5 could contribute to resolution of inflammation and be a potential therapeutic target for the heart failure treatment.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

Increased transforming growth factor-β (TGF-β) signaling contributes to the pathophysiology of aortic aneurysm in Marfan syndrome (MFS). Recent reports indicate that a small but significant number of inflammatory cells are infiltrated into the aortic media and adventitia in MFS. However, little is known about the contribution of myeloid cells to aortic aneurysmal formation. In this study, we ablated the TGF-β type II receptor geneTgfbr2in myeloid cells ofFbn1C1039G/+MFS mice (Fbn1C1039G/+;LysM-Cre/+;Tgfbr2fl/flmice, hereinafter calledFbn1C1039G/+;Tgfbr2MyeKO) and evaluated macrophage infiltration and TGF-β signaling in the aorta. Aneurysmal formation with fragmentation and disarray of medial elastic fibers observed in MFS mice was significantly ameliorated inFbn1C1039G/+;Tgfbr2MyeKOmice. In the aorta ofFbn1C1039G/+;Tgfbr2MyeKOmice, both canonical and noncanonical TGF-β signals were attenuated and the number of infiltrated F4/80-positive macrophages was significantly reduced.In vitro, TGF-β enhanced the migration capacity of RAW264.7 macrophages. These findings suggest that TGF-β signaling in myeloid cells promotes aortic aneurysmal formation and its inhibition might be a novel therapeutic target in MFS.

Some clinical trials showed that omega-3 fatty acid (FA) reduced cardiovascular events, but it remains unknown whether omega-3 FA supplementation changes the composition of FAs and their metabolites in the heart and how the changes, if any, exert beneficial effects on cardiac structure and function. To clarify these issues, we supplied omega-3 FA to mice exposed to pressure overload, and examined cardiac structure and function by echocardiography and a proportion of FAs and their metabolites by gas chromatography and liquid chromatography-tandem mass spectrometry, respectively. Pressure overload induced cardiac hypertrophy and dysfunction, and reduced concentration of all FAs’ components and increased free form arachidonic acid and its metabolites, precursors of pro-inflammatory mediators in the heart. Omega-3 FA supplementation increased both total and free form of eicosapentaenoic acid, a precursor of pro-resolution mediators and reduced free form arachidonic acid in the heart. Omega-3 FA supplementation suppressed expressions of pro-inflammatory cytokines and the infiltration of inflammatory cells into the heart and ameliorated cardiac dysfunction and fibrosis. These results suggest that omega-3 FA-induced changes of FAs composition in the heart have beneficial effects on cardiac function via regulating inflammation.

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Background The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. Methods and results We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. Conclusions We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants.

Variants in TGFBR1 have been reported to induce two completely distinct diseases, namely Loeys–Dietz syndrome (LDS) and multiple self-healing squamous epithelioma (MSSE). However, detailed mechanisms underlying this effect remain unknown. We report a Japanese familial case of LDS with a novel splice donor site variant in TGFBR1 gene (c.973 + 1 G > A; NG_007461.1). The intronic variant was predicted to mediate in-frame exon 5 skipping within the serine/threonine kinase (STK) domain, which may also be mediated by a similar TGFBR1 variant of a splice acceptor site in intron 4 (c.806-2 A > C), identified in a British familial case of MSSE. Therefore, ex vivo splicing and functional assays were performed in mammalian cells to evaluate the effect of these sequence variants. The MSSE variant activated a cryptic acceptor site at 76 bp downstream of the 3′ natural splice acceptor site, which produced an out-of-frame transcript (r.807_882del, p.Asn270Thrfs*8). In contrast, the LDS variant generated two types of in-frame transcription products, r.[806_973del, 965_973 del], and produced two functionally inactivated proteins, p.[Asp269_Gln324del, Thr323_Gly325del], as a result of exon 5 skipping and the activation of a cryptic donor splice site at 9 bp upstream of the 5′ natural splice donor site, respectively. Our results support the previously proposed but not yet approved mechanism that dominant-negative and truncating variants in STK domain induce LDS and MSSE, respectively.

Marfan syndrome (MFS) is an autosomal dominant heritable disorder of connective tissue that affects the cardiovascular, skeletal, ocular, pulmonary, and nervous systems and is usually caused by mutations in the FBN1 gene, which encodes fibrillin-1. MFS is traditionally considered to result from the structural weakness of connective tissue. However, recent investigations on molecular mechanisms indicate that increased transforming growth factor-β (TGF-β) activity plays a crucial role in the pathogenesis of MFS and related disorders, such as Loeys-Dietz syndrome (LDS), which is caused by mutation in TGF-β signaling-related genes. In addition, recent studies show that angiotensin II type 1 receptor (AT1R) signaling enhances cardiovascular pathologies in MFS, and the angiotensin II receptor blocker losartan has the potential to inhibit aortic aneurysm formation. However, the relationship between TGF-β and AT1R signaling pathways remains poorly characterized. In this review, we discuss the recent studies on the molecular mechanisms underlying cardiovascular manifestations of MFS and LDS and the ensuing strategies for management.

Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.

Heart-type fatty acid-binding protein (H-FABP) is a small cytosolic protein and released into the circulation when the myocardium is injured. Previous studies have demonstrated that both H-FABP and troponin T (TnT) are detectable in venous blood samples in chronic heart failure (CHF) patients, suggesting the presence of ongoing myocardial damage (OMD). We hypothesized that a cytosolic marker (H-FABP) is more sensitive than a myofibrillar component (TnT) in the detection of OMD in CHF.We measured serum H-FABP and TnT levels in 126 consecutive CHF patients at admission, and patients were followed-up with a mean period of 474 +/- 328 days. Cutoff values for H-FABP (4.3 ng/mL) and TnT (0.01 ng/mL) were determined from previous studies. Positive rate of H-FABP was higher than that of TnT in all CHF patients (46% [58/126] versus 26% [33/126], P.0001), and in severe CHF (New York Heart Association III/IV) patients (69% [34/49] versus 47% [23/49], P = .0121). There were 27 cardiac events during a follow-up period. In patients with cardiac events, H-FABP was more frequently detected than TnT (88% [24/27] versus 44% [12/27], P = .0103). There were 33 patients with positive H-FABP among 93 patients with negative TnT. Those patients had more severe New York Heart Association class, higher levels of brain natriuretic peptide, and higher rates of cardiac events (36% versus 5%, P.0001) compared with those both H-FABP and TnT were negative. Kaplan-Meier analysis demonstrated that in patients with negative TnT, positive H-FABP group had higher risk for cardiac events than negative H-FABP group (P.0001). A multivariate analysis with Cox proportional hazard model showed that H-FABP was the only independent predictor of cardiac events (hazard ratio 15.677, P = .0001). The area under the receiver operating characteristic curve was larger for H-FABP than for TnT (0.779 versus 0.581; P = .009), suggesting that H-FABP had greater predictive capacity for cardiac events than TnT.H-FABP was more sensitive to detect OMD and could identify patients at high risk more effectively than TnT.

Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Azelnidipine is a novel dihydropyridine calcium channel blocker. Several studies have demonstrated that some dihydropyridine calcium channel blockers have antioxidant effects. We evaluated the antioxidant effects of azelnidipine compared to another dihyropyridine calcium channel blocker, nifedipine, in neonatal rat cardiomyocytes.We examined effects of azelnidipine and nifedipine on the H(2)O(2)-induced mitogen-activated protein kinase (MAPK) activity and cell death in neonatal rat cardiomyocytes.Extracellular signal-regulated protein kinases (ERK), p38 MAPK and c-Jun NH(2)-terminal kinases (JNK) were activated by H(2)O(2) stimulation. Azelnidipine and nifedipine did not affect the H(2)O(2)-induced activation of ERK and p38 MAPK. In contrast, azelnidipine, but not nifedipine, inhibited the H(2)O(2)-induced JNK activation. The numbers of viable cell were significantly decreased by H(2)O(2) treatments (65.8 +/- 4.11% of control, P0.0001). Azelnidipine, but not nifedipine, inhibited the H(2)O(2)-induced cell death (azelnidipine: 76.0 +/- 4.66% of control, P0.05; nifedipine: 70.7 +/- 4.01% of control, P = 0.32).Azelnidipine inhibited the H(2)O(2)-induced JNK activation and cardiac cell death. Azelnidipine may have cardioprotective effects against oxidative stress.

Background The Gaq protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in the development of cardiac hypertrophy and heart failure. DAG kinase (DGK) phosphorylates DAG and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been previously reported that DGK inhibited GPCR agonist-induced activation of the DAG-PKC signaling and subsequent cardiomyocyte hypertrophy, so the purpose of this study was to examine whether DGK modifies the development of cardiac hypertrophy induced by pressure overload. Methods and Results Thoracic transverse aortic constriction (TAC) was created in transgenic mice with cardiac-specific overexpression of DGKξ (DGKξ-TG) and wild-type (WT) mice. Increases in heart weight at 4 weeks after TAC were attenuated in DGKξ-TG mice compared with WT mice. Increases in interventricular septal thickness, dilatation of the left ventricular cavity, and decreases in left ventricular systolic function in WT mice were observed with echocardiography at 4 weeks after TAC surgery. However, these structural and functional changes after TAC were attenuated in DGKξ-TG mice. In WT mice, cardiac fibrosis and up-regulation of profibrotic genes, such as transforming growth factor-β1, collagen type I, and collagen type III, were observed at 4 weeks after TAC. However, cardiac fibrosis and gene induction of type I and type III collagens, but not transforming growth factor-β1, were blocked in DGKξ-TG mice. Conclusion These results are the first in vivo evidence that DGKξ suppresses cardiac hypertrophy and fibrosis and prevents impaired left ventricular systolic function caused by pressure overload. (Circ J 2007; 71: 276 - 282)

Objective—Angiotensin II is critically involved in left ventricular remodeling after myocardial infarction. Neovascularization has been thought to prevent the development of left ventricular remodeling and deterioration to heart failure. To elucidate the role of angiotensin II in neovascularization during cardiac remodeling, we induced myocardial infarction in angiotensin II type1a receptor (AT1) knockout (KO) mice.Methods and Results—There were more vessels in the border zone of infarcted hearts of wild-type (WT) mice and AT1KO mice at 14 days after operation, compared with in the left ventricle of sham-operated mice, and the number was larger in WT mice than in AT1KO mice. Consistent with these observations, the infarcted heart of AT1KO mice expressed lower levels of matrix metalloproteinase and endothelial nitric oxide synthase activity. More inflammatory cells such as granulocytes and macrophages were infiltrated in the infarcted hearts of WT mice than AT1KO mice at 4 days. A variety of cytokines and chemokines were increased in infarcted hearts of WT and AT1KO mice, and many of them were more remarkable in WT mice than in AT1KO mice at 14 days.Conclusions—AT1 plays a critical role in inflammatory cell infiltration, cytokine production, and neovascularization in infarcted hearts.

Many cytokines have been reported to be increased in human and animal models with cardiovascular diseases. Myocardial infarction (MI) is accompanied with an inflammatory reaction which induces cardiac dysfunction and remodeling. The inflammatory reaction has been investigated in animal models of MI or myocardial ischemia-reperfusion injury. The mechanisms by which cytokine cascade is activated in the infarcted myocardium have been recently elucidated. Several hematopoietic growth factors including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factors (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) have been reported to be positive regulators of granulopoiesis and act at different stages of myeloid cell development. G-CSF plays a critical role in regulation of proliferation, differentiation, and survival of myeloid progenitor cells. G-CSF also causes a marked increase in the release of hematopoietic stem cells (HSCs) into the peripheral blood circulation, a process termed mobilization. Although cardiac myocytes have been considered as terminally differentiated cells, it has been recently reported that there are many proliferating cardiac myocytes after MI in human heart. After it was demonstrated that bone marrow stem cells (BMSCs) can differentiate into cardiac myocytes, myocardial regeneration has been widely investigated. Recently, G-CSF has been reported to improve cardiac function and reduces mortality after acute MI. Although the mechanism by which G-CSF ameliorates cardiac dysfunction is not fully understood, there is the possibility that G-CSF may regenerate cardiac myocytes and blood vessels through mobilization of BMSCs. In the future, cytokine-mediated regeneration therapy may become to be a novel therapeutic strategy for MI.

Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT–PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα+ cells. Clonal progeny of single Sca1+ SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα− cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα+/CD31− cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα−/CD31+ cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1+ stem/progenitor cell., Adult cardiac progenitor/stem cells (CPSCs) possess valuable potential for heart repair that is limited by the elusiveness of these cells. Here Noseda et al. refine the definition of murine CPSCs producing stem cell antigen 1 (Sca1), mapping the cardiogenic signature and clonogenicity to the subgroup of Sca1+ cells expressing PDGFRα.

Emerging evidence has suggested a potential impact of gut microbiota on the pathophysiology of heart failure (HF). However, it is still unknown whether HF is associated with dysbiosis in gut microbiota. We investigated the composition of gut microbiota in patients with HF to elucidate whether gut microbial dysbiosis is associated with HF. We performed 16S ribosomal RNA gene sequencing of fecal samples obtained from 12 HF patients and 12 age-matched healthy control (HC) subjects, and analyzed the differences in gut microbiota. We further compared the composition of gut microbiota of 12 HF patients younger than 60 years of age with that of 10 HF patients 60 years of age or older. The composition of gut microbial communities of HF patients was distinct from that of HC subjects in both unweighted and weighted UniFrac analyses. Eubacterium rectale and Dorea longicatena were less abundant in the gut microbiota of HF patients than in that of HC subjects. Compared to younger HF patients, older HF patients had diminished proportions of Bacteroidetes and larger quantities of Proteobacteria. The genus Faecalibacterium was depleted, while Lactobacillus was enriched in the gut microbiota of older HF patients. These results suggest that patients with HF harbor significantly altered gut microbiota, which varies further according to age. New concept of heart-gut axis has a great potential for breakthroughs in the development of novel diagnostic and therapeutic approach for HF.

Background Increasing evidence suggests a critical role for mitochondrial aldehyde dehydrogenase 2 ( ALDH 2) in protection against cardiac injuries; however, the downstream cytosolic actions of this enzyme are largely undefined. Methods and Results Proteomic analysis identified a significant downregulation of mitochondrial ALDH 2 in the heart of a rat heart failure model after myocardial infarction. The mechanistic insights underlying ALDH 2 action were elucidated using murine models overexpressing ALDH 2 or its mutant or with the ablation of the ALDH 2 gene ( ALDH 2 knockout) and neonatal cardiomyocytes undergoing altered expression and activity of ALDH 2. Left ventricle dilation and dysfunction and cardiomyocyte death after myocardial infarction were exacerbated in ALDH 2 ‐knockout or ALDH 2 mutant‐overexpressing mice but were significantly attenuated in ALDH 2‐overexpressing mice. Using an anoxia model of cardiomyocytes with deficiency in ALDH 2 activities, we observed prominent cardiomyocyte apoptosis and increased accumulation of the reactive aldehyde 4‐hydroxy‐2‐nonenal (4‐ HNE ). We subsequently examined the impacts of mitochondrial ALDH 2 and 4‐ HNE on the relevant cytosolic protective pathways. Our data documented 4‐ HNE ‐stimulated p53 upregulation via the phosphorylation of JNK , accompanying increased cardiomyocyte apoptosis that was attenuated by inhibition of p53. Importantly, elevation of 4‐ HNE also triggered a reduction of the cytosolic HSP 70, further corroborating cytosolic action of the 4‐ HNE instigated by downregulation of mitochondrial ALDH 2. Conclusions Downregulation of ALDH 2 in the mitochondria induced an elevation of 4‐ HNE , leading to cardiomyocyte apoptosis by subsequent inhibition of HSP 70, phosphorylation of JNK , and activation of p53. This chain of molecular events took place in both the mitochondria and the cytosol, contributing to the mechanism underlying heart failure.

Background For assessment of cardiac sympathetic nervous activity, the conventional protocol for iodine-123-metaiodobenzylguanidine (123I-MIBG) imaging requires several hours. Methods and Results To establish whether it is possible to shorten the conventional 123I-MIBG imaging protocol, anterior planar imaging was performed in 42 heart failure (HF) patients at 5, 15 and 180 min. The washout rate of 123I-MIBG from 5 to 15 min (WR5-15 min) was calculated as a novel index. WR5-15 min closely correlated with the conventional washout rate and inversely correlated with the heart to mediastinum ratio. Univariate Cox analysis revealed that rapid WR5-15 min, augmented plasma B-type natriuretic peptide level, and decreased left ventricular ejection fraction (LVEF) were predictors for cardiac events. Multivariate analysis showed WR5-15 min and LVEF were independent predictors. The cardiac event rate was markedly higher (73%) in patients when both WR5-15 min and LVEF were abnormal. Conclusions WR5-15 min obtained from anterior planar imaging is useful for evaluating the severity of HF and clinical outcome, and may shorten the cardiac 123I-MIBG scintigraphy protocol. (Circ J 2008; 72: 1106 - 1111)

Aims High-mobility group box 1 (HMGB1) is a nuclear DNA-binding protein and is released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis. To test the hypothesis that HMGB1 enhances angiogenesis and restores cardiac function after myocardial infarction (MI), we generated transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) using α-myosin heavy chain promoter. Methods and results The left anterior descending coronary artery was ligated in HMGB1-Tg and wild-type littermate (Wt) mice. After coronary artery ligation, HMGB1 was released into circulation from the necrotic cardiomyocytes of HMGB1-overexpressing hearts. The size of MI was smaller in HMGB1-Tg than in Wt mice. Echocardiography and cardiac catheterization demonstrated that cardiac remodelling and dysfunction after MI were prevented in HMGB1-Tg mice compared with Wt mice. Furthermore, the survival rate after MI of HMGB1-Tg mice was higher than that of Wt mice. Immunohistochemical staining revealed that capillary and arteriole formation after MI was enhanced in HMGB1-Tg mice. Conclusion We report the first in vivo evidence that HMGB1 enhances angiogenesis, restores cardiac function, and improves survival after MI. These results may provide a novel therapeutic approach for left ventricular dysfunction after MI.

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.