PGC-1α-mediated angiogenesis prevents pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a life-threatening disease characterized by a progressive narrowing of pulmonary arterioles. Although VEGF is highly expressed in lung of patients with PH and in animal PH models, the involvement of angiogenesis remains elusive. To clarify the pathophysiological function of angiogenesis in PH, we compared the angiogenic response in hypoxia (Hx) and SU5416 (a VEGFR2 inhibitor) plus Hx (SuHx) mouse PH models using 3D imaging. The 3D imaging analysis revealed an angiogenic response in the lung of the Hx-PH, but not of the severer SuHx-PH model. Selective VEGFR2 inhibition with cabozantinib plus Hx in mice also suppressed angiogenic response and exacerbated Hx-PH to the same extent as SuHx. Expression of endothelial proliferator-activated receptor γ coactivator 1α (PGC-1α) increased along with angiogenesis in lung of Hx-PH but not SuHx mice. In pulmonary endothelial cell-specific Ppargc1a-KO mice, the Hx-induced angiogenesis was suppressed, and PH was exacerbated along with increased oxidative stress, cellular senescence, and DNA damage. By contrast, treatment with baicalin, a flavonoid enhancing PGC-1α activity in endothelial cells, ameliorated Hx-PH with increased Vegfa expression and angiogenesis. Pulmonary endothelial PGC-1α-mediated angiogenesis is essential for adaptive responses to Hx and might represent a potential therapeutic target for PH.

Low-carbohydrate diets containing plant-derived fat but not animal-derived fat ameliorate heart failure.

Cardiovascular disease (CVD) is a global health burden in the world. Although low-carbohydrate diets (LCDs) have beneficial effects on CVD risk, their preventive effects remain elusive. We investigated whether LCDs ameliorate heart failure (HF) using a murine model of pressure overload. LCD with plant-derived fat (LCD-P) ameliorated HF progression, whereas LCD with animal-derived fat (LCD-A) aggravated inflammation and cardiac dysfunction. In the hearts of LCD-P-fed mice but not LCD-A, fatty acid oxidation-related genes were highly expressed, and peroxisome proliferator-activated receptor α (PPARα), which regulates lipid metabolism and inflammation, was activated. Loss- and gain-of-function experiments indicated the critical roles of PPARα in preventing HF progression. Stearic acid, which was more abundant in the serum and heart of LCD-P-fed mice, activated PPARα in cultured cardiomyocytes. We highlight the importance of fat sources substituted for reduced carbohydrates in LCDs and suggest that the LCD-P-stearic acid-PPARα pathway as a therapeutic target for HF.

Tie2-Cre-Induced Inactivation of Non-Nuclear Estrogen Receptor-α Signaling Abrogates Estrogen Protection Against Vascular Injury.

Using the Cre-loxP system, we generated the first mouse model in which estrogen receptor-α non-nuclear signaling was inactivated in endothelial cells. Estrogen protection against mechanical vascular injury was impaired in this model. This result indicates the pivotal role of endothelial estrogen receptor-α non-nuclear signaling in the vasculoprotective effects of estrogen.

Cardiac fibroblasts regulate the development of heart failure via Htra3-TGF-ß-IGFBP7 axis.

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-ß (TGF-ß). Pressure overload downregulates expression of Htra3 in cardiac fibroblasts and activated TGF-ß 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-ß 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-ß 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-ß-IGFBP7 pathway, which would be a therapeutic target for heart failure.

CXCR7 ameliorates myocardial infarction as a ß-arrestin-biased receptor.

Most seven transmembrane receptors (7TMRs) are G protein-coupled receptors; however, some 7TMRs evoke intracellular signals through ß-arrestin as a biased receptor. As several ß-arrestin-biased agonists have been reported to be cardioprotective, we examined the role of the chemokine receptor CXCR7 as a ß-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 ß-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 ß-arrestin-biased receptor and that CXCR7 may be a novel therapeutic target for myocardial infarction.

Factors associated with left ventricular reverse remodelling after percutaneous coronary intervention in patients with left ventricular systolic dysfunction.

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 < 50% at initial left ventriculography) were included in the analysis. LVRR was defined as LV end-systolic volume reduction ≥ 15% and improvement of LVEF ≥ 10% at 6 months follow-up left ventriculography. Patients were divided into LVRR (n = 63) and non-LVRR (n = 223) groups. Multivariate logistic regression analysis revealed that unprotected left main coronary artery (LMCA) intervention was significantly associated with LVRR (P = 0.007, odds ratios [OR] 4.70, 95% confidence interval [CI] 1.54-14.38), while prior PCI (P = 0.001, OR 0.35, 95% CI 0.19-0.66), presence of in-stent restenosis (P = 0.016, OR 0.32, 95% CI 0.12-0.81), and presence of de-novo stenosis (P = 0.038, OR 0.36, 95% CI 0.14-0.95) were negatively associated with LVRR. These data suggest the potential prognostic benefit of unprotected LMCA intervention for LVRR and importance of angiographic follow-up in patients with CAD and LV systolic dysfunction.

Inhibition of transforming growth factor-ß signaling in myeloid cells ameliorates aortic aneurysmal formation in Marfan syndrome.

Increased transforming growth factor-ß (TGF-ß) 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-ß type II receptor gene Tgfbr2 in myeloid cells of Fbn1C1039G/+ MFS mice (Fbn1C1039G/+;LysM-Cre/+;Tgfbr2fl/fl mice, hereinafter called Fbn1C1039G/+;Tgfbr2MyeKO) and evaluated macrophage infiltration and TGF-ß signaling in the aorta. Aneurysmal formation with fragmentation and disarray of medial elastic fibers observed in MFS mice was significantly ameliorated in Fbn1C1039G/+;Tgfbr2MyeKO mice. In the aorta of Fbn1C1039G/+;Tgfbr2MyeKO mice, both canonical and noncanonical TGF-ß signals were attenuated and the number of infiltrated F4/80-positive macrophages was significantly reduced. In vitro, TGF-ß enhanced the migration capacity of RAW264.7 macrophages. These findings suggest that TGF-ß signaling in myeloid cells promotes aortic aneurysmal formation and its inhibition might be a novel therapeutic target in MFS.

Omega-3 fatty acid prevents the development of heart failure by changing fatty acid composition in the heart.

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.

Estrogen Receptor-α Non-Nuclear Signaling Confers Cardioprotection and Is Essential to cGMP-PDE5 Inhibition Efficacy.

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.

A metabolic profile of routine needle biopsies identified tumor type specific metabolic signatures for breast cancer stratification: a pilot study.

INTRODUCTION: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Overview of the 83rd Annual Scientific Meeting of the Japanese Circulation Society　- Renaissance of Cardiology for the Creation of Future Medicine.

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.

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo.

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.

High-throughput single-molecule RNA imaging analysis reveals heterogeneous responses of cardiomyocytes to hemodynamic overload.

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 ß (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.

Discovery of a Small Molecule to Increase Cardiomyocytes and Protect the Heart After Ischemic Injury.

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.

Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure.

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.

Author Correction: Activation of endothelial ß-catenin signaling induces heart failure.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.