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Background NF-E2-related factor 2 (Nrf2) is a transcription factor playing cytoprotective effects in various pathological processes including oxidative stress and cardiac hypertrophy. Despite being a potential therapeutic target to treat several cardiomyopathies, the signaling underlying Nrf2-dependent cardioprotective action remains largely uncharacterized. Aim This study aimed to explore the signaling mediating the role of Nrf2 in the development of hypertensive cardiac pathogenesis by analyzing the response to Angiotensin II (Ang II) in the presence or absence of Nrf2 expression, both in vivo and in vitro. Results Our results indicated that Nrf2 deficiency exacerbated cardiac damage triggered by Ang II infusion. Mechanistically, our study shows that Ang II-triggered hypertrophy and inflammation is exacerbated in the absence of Nrf2 expression and points to the involvement of the IL-6/STAT3 signaling pathway in this event. Indeed, our results show that IL-6 abundance triggered by Ang II is increased in the absence of Nrf2 and demonstrate the requirement of IL-6 in STAT3 activation and cardiac inflammation induced by Ang II. Conclusion Our results show that Nrf2 is important for the protection of the heart against Ang II-induced cardiac hypertrophy and inflammation by mechanisms involving the regulation of IL-6/STAT3-dependent signaling.

Objective Due to limited immunological profiles of high‐grade serous ovarian cancer (HGSOC), we aimed to characterize its molecular features to determine whether a specific subset that can respond to immunotherapy exists. Materials and Methods A training cohort of 418 HGSOC samples from TCGA was analysed by consensus non‐negative matrix factorization. We correlated the expression patterns with the presence of immune cell infiltrates, immune regulatory molecules and other genomic or epigenetic features. Two independent cohorts containing 482 HGSOCs and in vitro experiments were used for validation. Results We identified immune and non‐immune groups where the former was enriched in signatures that reflect immune cells, infiltration and PD‐1 signalling (all, P, A comprehensive profiling of high‐grade serous ovarian cancer based on multifarious molecular features identified an immune group in high‐grade serous ovarian cancer that contains two robust microenvironment‐based subtypes with distinct likelihoods of response to immunotherapies that may also represent ideal immunotherapy candidates.

Interleukin-6 (IL-6) signaling is critical for cardiomyocyte hypertrophy, while the role of IL-6 in the pathogenesis of myocardium hypertrophy remains controversial. To determine the essential role of IL-6 signaling for the cardiac development during AngII-induced hypertension, and to elucidate the mechanisms, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were infused subcutaneously with either vehicle or AngII (1.5 μg/h/mouse) for 1 week. Immunohistological and serum studies revealed that the extents of cardiac fibrosis, inflammation and apoptosis were reduced in IL-6 KO heart during AngII-stimulation, while cardiac hypertrophy was obviously induced. To investigate the underlying mechanisms, by using myocardial tissue and neonatal cardiomyocytes, we observed that IL-6/STAT3 signaling was activated under the stimulation of AngII both in vivo and in vitro. Further investigation suggested that STAT3 activation enhances the inhibitory effect of EndoG on MEF2A and hampers cardiomyocyte hypertrophy. Our study is the first to show the important role of IL-6 in regulating cardiac pathogenesis via inflammation and apoptosis during AngII-induced hypertension. We also provide a novel link between IL-6/STAT3 and EndoG/MEF2A pathway that affects cardiac hypertrophy during AngII stimulation.

Protein kinase B2 (AKT2) is implicated in diverse process of cardiomyocyte signaling including survival and metabolism. However, the role of AKT2 in myocardium development and the signaling pathway is rarely understood. Therefore, we sought to determine the effect of AKT2 deletion on heart development and its downstream targets. By using experimental animal models and neonatal rat cardiomyocytes (NRCMs), we observed that AKT2 deficiency induces retardation of heart development and increased systemic blood pressure (BP) without affecting cardiac function. Further investigation suggested that deficiency of AKT2 in myocardium results in diminished MEF2A abundance, which induced decreased size of cardiomyocytes. We additionally confirmed that EndoG, which is also regulated by AKT2, is a suppressor of MEF2A in myocardium. Finally, our results proved that AKT2 deficiency impairs the response to β-adrenergic stimuli that normally causes hypertrophy in cardiomyocytes by downregulating MEF2A expression. Our data are the first to show the important role of AKT2 in determining the size of myocardium, its deficiency causes retardation of cardiomyocyte development. We also proved a novel pathway of heart development involving EndoG and MEF2A regulated by AKT2.

Acute liver failure (ALF) is characterized by sudden large area of inflammation and extensive hepatocyte apoptosis. This study identified the natural product berberine as a potential agent for acute liver failure(ALF). First, in vitro, BBR pre-incubation (5, 10 and 20μM) alleviated L02 hepatocytes injury induced by D-GalN (5mM)/TNF-α (100ng/ml). Second, in vivo, BBR pre-treatment attenuated D-Galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver failure, as evidenced by the reduction of mortality, the alleviation of liver pathological changes and the inhibition of alanine aminotransferase (ALT)/aspartate aminotransferase (AST). Our results further illustrated that BBR inhibited the nuclear translocation of NF-κB p65 and subsequently suppressed the expressions of inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) at both mRNA and protein levels in ALF. Moreover, western blotting demonstrated that BBR effectively inhibited apoptosis via reducing cytochrome c release, Bax/Bcl-2 ratio and caspase-3/-9 cleavage in vitro and in vivo. In conclusion, our findings suggest that BBR serves as a potential agent for preventing or treating human ALF by inhibiting inflammation and mitochondria-dependent apoptosis.

Objective Obesity is associated with metabolic disorder and chronic inflammation that plays a crucial role in cardiovascular diseases. IL-6 is involved in regulating obesity-related lipid metabolism and inflammation. In this study, we sought to determine the role of IL-6 in high-fat diet (HFD)-induced cardiomyopathy and explore the signaling pathway. Methods Female, 5-week-old IL-6 knockout (KO) and littermate mice were fed a normal diet (ND, 10% fat) or HFD (45% fat) for 14 weeks. At the end of treatment, cardiac function was assessed by echocardiography. Adipose tissues and plasma were collected for further measurement. Immunohistology of CD68 was performed to detect inflammation in the heart. Masson's trichrome staining and Oil Red O staining was applied to evaluated cardiac fibrosis and lipid accumulation. Real-time PCR and Western immunoblotting analyses on heart tissue were used to explore the underlying mechanism. Results IL-6 KO mice displayed increased insulin resistance compared to WT mice at baseline. When fed HFD, IL-6 KO mice showed decreased gains in body weight and fat mass, increased insulin resistance relative to IL-6 KO mice feed ND. Furthermore, IL-6 KO mice developed cardiac dysfunction during HFD-induced obesity. Histological analysis suggested increased lipid accumulation, fibrosis and inflammation without affecting cardiac morphology during HFD treatment in the heart of IL-6 KO mice. Finally, IL-6 deficiency increased the phosphorylation of AMPK and ACC in the heart during HFD-induced obesity. Conclusion Our results suggest that IL-6 contributes to limit lipid metabolic disorder, cardiac hypertrophy, fibrosis, inflammation and myocardium lipotoxicity during HFD-induced obesity.

Polypyrimidine tract binding protein (PTB) regulates pre-mRNA splicing, having special relevance for determining gene expression in the differentiating muscle. We have previously shown that PTB protein abundance is progressively reduced during heart development without reduction of its own transcript. Simultaneous reduction of histone deacetylase (HDAC) expression prompted us to investigate the potential link between these events. HDAC5-deficient mice have reduced cardiac PTB protein abundance, and HDAC inhibition in myocytes causes a reduction in endogenous expression of cellular FLICE-like inhibitory protein (cFLIP) and caspase-dependent cleavage of PTB. In agreement with this, cardiac PTB expression is abnormally high in mice with cardiac-specific executioner caspase deficiency, and cFLIP overexpression prevents PTB cleavage in vitro. Caspase-dependent cleavage triggers further fragmentation of PTB, and these fragments accumulate in the presence of proteasome inhibitors. Experimental modification of the above processes in vivo and in vitro results in coherent changes in the alternative splicing of genes encoding tropomyosin-1 (TPM1), tropomyosin-2 (TPM2) and myocyte enhancer factor-2 (MEF2). Thus, we report a pathway connecting HDAC, cFLIP and caspases regulating the progressive disappearance of PTB, which enables the expression of the adult variants of proteins involved in the regulation of contraction and transcription during cardiac muscle development. This work was partially supported by the Ministerio de Ciencia e Innovación (MICINN) [grant number SAF2010_19125 to D.S.]; the 'Ramon y Cajal' Program from MICINN (to D.S.); the Agencia de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) [grant number 2009SGR-346 to J.X.C. and D.S.]; and the Wellcome Trust [programme grant number 092900 to C.W.J.S]. R.A.F. is an investigator of the Howard Hughes Medical Institute. Work done in the laboratory of E.N.O. was supported by grants from the National Institutes of Health, the Donald W. Reynolds Center for Clinical Cardiovascular Research, the Leducq Foundation and the Robert A. Welch Foundation. Deposited in PMC for release after 6 months.

Left ventricular mass (LVM) is a highly heritable trait1 and an independent risk factor for all-cause mortality2. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation3, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood4, 5. Unbiased systems genetics approaches in the rat6, 7 now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis8 but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function)9, 10, 11, interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy. We acknowledge funding from the Medical Research Council (UK), theNational Institute for Health Research (UK), the Royal Brompton andHarefield Cardiovascular Biomedical Research Unit, the Imperial College Healthcare Biomedical Research Centre, the British Heart Foundation, Fondation Leducq, the Wellcome Trust, the Grant Agency of the Czech Republic (301/08/0166), the Ministry of Education of the Czech Republic (1M0520), the Ministerio de Ciencia e Innovacion (Spain; PTQ-08-03-07880, SAF2008-02271, SAF2008-03067 and SAF2010-19125), the Agència de Gestió d’AjutsUniversitaris i Recerca (Spain; 2009-SGR-346), the Fondo de Investigaciones Sanitarias (Spain; PS09/02034, PS09/01602 and PS09/01591), the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. HEALTH-F4-2010-241504 (EURATRANS), and the German National Genome Research Network (NGFN-Plus) Heart Failure. We thank M. R. Lieber for providing the Endog deleted mice and E. Wahle for providing the CG4930 expression plasmid. We thank the National BioResource Project for the Rat (http:// www.anim.med.kyoto-u.ac.jp/NBR/) for providing rat strains.

The Myocyte Enhancer Factor-2 (MEF2) family of transcription factors regulates gene expression during cardiomyocyte differentiation and adaptation of the myocardium to stress. MEF2 activity is enhanced by increasing its transcription and by MAPK-dependent phosphorylation, and is reduced by binding to class-II Histone Deacetylases and by miR-1-mediated degradation of its transcript. Here we show that MEF2 protein abundance is regulated at the translational level, determining myocyte size, during hypertrophy. In order to reduce MEF2 protein expression, its silencing through RNA interference required serum deprivation and, even in this condition, MEF2 protein abundance recovered to basal levels in presence of phenylephrine. Hypertrophic agonist stimulation of neonatal ventricular cardiomyocytes increased Mef2 expression by enhancing its translation, without changing its transcription or blocking degradation of the protein. MEF2 abundance was increased by Calcineurin overexpression in vivo and was reduced by Calcineurin inhibition in vitro, without affecting Mef2 mRNA levels. Calcineurin activity influenced expression of Polypyrimidine Tract Protein (PTB), contributing to MEF2 translation. Thus, our results show a previously unrecognized but relevant level of MEF2 activity regulation through the control of its translation that involves Calcineurin and PTB.

The AKT (protein kinase B, PKB) family has been shown to participate in diverse cellular processes, including apoptosis. Previous studies demonstrated that protein kinase B2 (AKT2 − / − ) mice heart was sensitized to apoptosis in response to ischemic injury. However, little is known about the mechanism and apoptotic signaling pathway. Here, we show that AKT2 inhibition does not affect the development of cardiomyocytes but increases cell death during cardiomyocyte ischemia. Caspase-dependent apoptosis of both the extrinsic and intrinsic pathway was inactivated in cardiomyocytes with AKT2 inhibition during ischemia, while significant mitochondrial disruption was observed as well as intracytosolic translocation of cytochrome C (Cyto C) together with apoptosis-inducing factor (AIF) and endonuclease G (EndoG), both of which are proven to conduct DNA degradation in a range of cell death stimuli. Therefore, mitochondria-dependent cell death was investigated and the results suggested that AIF and EndoG nucleus translocation causes cardiomyocyte DNA degradation during ischemia when AKT2 is blocked. These data are the first to show a previous unrecognized function and mechanism of AKT2 in regulating cardiomyocyte survival during ischemia by inducing a unique mitochondrial-dependent DNA degradation pathway when it is inhibited. This work was supported by the National Natural Science Foundation of China, (Grant No. 81500179); the Natural Science Foundation of Jiangsu Province (Grant No. BK20150696); the Fundamental Research Funds for the Central Universities (Grant No. 2015PY005); the National Found for Fostering Talents of Basic Science (NFFTBS) (Grant No. J1310032); the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); the National High Technology Research and Development Program of China (863 Program, No.2015AA020314); and the National Natural Science Foundation of China (Grant No. 81570696 and No. 31270985); this work is also sponsored by Qing Lan Project.

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-xL. These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells. The work was supported by the Ministry of Science and Innovation of Spain (SAF2005-02197 and SAF2008-02271) to DS and Programa de Suport a Grups de Recerca de Catalunya from the Government of Catalonia (AGAUR) (SGR2005-00628) and Ciberned from the Spanish Government to JXC, Instituto de Salud Carlos III -Ministerio de Sanidad y Consumo (FIS) (PI04/2537 and PS09/00140) to ML. JZ was recipient of a pre-doctoral fellowship (FPI program) from the Ministry of Education and Science of Spain and JY is recipient of a fellowship from the Universitat de Lleida. URLs: Ministry of Science and Innovation of Spain: http://www.micinn.es; AGAUR: http://www10.gencat.cat/agaur_web/AppJava/catala/index.jsp; Ministry of Education: http://www.educacion.es/portada.html; Instituto de Salud Carlos III: http://www.isciii.es/htdocs/index.jsp. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.