Search

The present study aims to investigate the role of AKT2 in the pathogenesis of hepatic and cardiac lipotoxicity induced by lipid overload-induced obesity and identify its downstream targets. WT and Akt2 KO mice were fed either normal diet, or high-fat diet (HFD) to induce obesity model in vivo. Human hepatic cell line (L02 cells) and neonatal rat cardiomyocytes (NRCMs) were used as in vitro models. We observed that during HFD-induced obesity, Akt2 loss-of-function mitigated lipid accumulation and oxidative stress in the liver and heart tissue. Mechanistically, down-regulation of Akt2 promotes SIRT6 expression in L02 cells and NRCMs, the latter deacetylates SOD2, which promotes SOD2 activity and therefore alleviates oxidative stress-induced injury of hepatocytes and cardiomyocytes. Furthermore, we also proved that AKT2 inhibitor protects hepatocytes and cardiomyocytes from HFD-induced oxidative stress. Therefore, our work prove that AKT2 plays an important role in the regulation of obesity-induced lipid metabolic disorder in the liver and heart. Our study also indicates AKT2 inhibitor as a potential therapy for obesity-induced hepatic and cardiac injury.

Metformin is accepted as a first-line drug for the therapy of Type 2 diabetes (T2D), while its mechanism is still controversial. In the present study, by taking advantage of mouse model of high-fat-diet (HFD)-induced obesity and primary mouse hepatocytes (PMHCs) as well as human hepatocyte L02 cell line, we aimed to investigate the involvement of SIRTs during the application of metformin for the therapy of T2D. Our data evidenced that during HFD-induced obesity, there was elevation of nucleus protein acetylation. Analysis of liver tissue showed that among all SIRT members, SIRT6 expression was significantly down-regulated during HFD feeding, which was sustained to regular level with metformin administration. Our result also showed that SIRT6 suppressed intracellular oxidative stress upon FAs stimulation in PMHCs and L02 cells. Mechanistically, SIRT6, but not SIRT1 promoted PGC-1α expression. We further prove that ENDOG is downstream of PGC-1α. In addition, we evidenced that ENDOG protects hepatocytes from lipid-induced oxidative stress, and down-regulation of Endog blunted the protective role of metformin in defending against FAs-induced oxidative stress. Our study established a novel mechanism of metformin in counteracting lipid-induced hepatic injury via activating SIRT6/PGC-1α/ENDOG signaling, thus providing novel targets of metformin in the therapy of T2D.

Pro-inflammatory cytokines play important roles in sepsis-induced cardiac injury. Among various cytokines, the function of Interleukin-6 (IL-6) in the regulation of cardiomyocyte injury remains to be elucidated. This study aimed to investigate whether IL-6 plays a key role in the sepsis-induced cardiomyocyte injury and the possible mechanism. Mice deficient for Il-6 exhibited impaired heart rhythm after LPS stimulation. Histological analysis revealed significantly increased oxidative stress after LPS stimulation in the heart with Il-6 knockout. On the contrary, IL-6 supplementation alleviated LPS-induced oxidative stress. Mechanically, IL-6 facilitates Nrf2 expression and its nucleus translocation, which subsequently promotes the expression of antioxidant genes and sustains redox homeostasis in cardiomyocytes, and Nrf2 deletion results in elevated oxidative stress during LPS stimulation and cannot be inverted by IL-6 supplement. Our study presents a new sight for the protective role of IL-6 during the pathological development of LPS-induced cardiac injury, which functions as an anti-oxidant molecule via activating Nrf2 signaling.

The AKT family plays key roles in global metabolism. However, the function of different AKT isoforms during obesity-associated hepatic and cardiac injury remained unclear. This study aims to elucidate the role of AKT2 in the pathogenesis of hepatic and cardiac lipotoxicity due to nutrition overload-induced obesity and explore the signaling pathway that it involves. Akt2 KO mice were fed with high fat diet (HFD) to induce obesity model in vivo. The oxidative stress of the normal human hepatic cell line (L02 cells) and neonatal rat cardiomyocytes (NRCMs) were measured by using Oil Red O and Rho123 staining as well as Flow cytometry. Protein levels were determined by using western blot technique. We observed that during HFD-induced obesity, Akt2 loss-of-function mitigated lipid accumulation and oxidative stress in the liver and heart tissue. Mechanistically, during HFD-induced obesity, Akt2 deficiency promotes SIRT6 expression in the hepatocytes and cardiomyocytes. Upregulation of SIRT6 deacetylates SOD2, which promotes SOD2 activity and thus alleviating oxidative stress-induced injury of hepatocytes and cardiomyocytes. Furthermore, We also proved that AKT2 inhibitor protects hepatocytes and cardiomyocytes from HFD-induced oxidative stress. This study demonstrates that Akt2 deficiency plays a beneficial role in protecting hepatocytes and cardiomyocytes from oxidative stress via SIRT6-dependent SOD2 deacetylation during lipid overload-induced obesity, indicating an important function of AKT2 in the regulation of obesity-induced lipid metabolic disorder in the liver and heart. Our study also indicates AKT2 inhibitor as a potential therapy for obesity-induced hepatic and cardiac injury.

The sirtuin 6 (SIRT6) participates in regulating glucose and lipid homeostasis. However, the function of SIRT6 in the process of cardiac pathogenesis caused by obesity-associated lipotoxicity remains to be unveiled. This study was designed to elucidate the role of SIRT6 in the pathogenesis of cardiac injury due to nutrition overload-induced obesity and explore the downstream signaling pathways affecting oxidative stress in the heart. In this study, we used Sirt6 cardiac-specific knockout murine models treated with a high-fat diet (HFD) feeding to explore the function and mechanism of SIRT6 in the heart tissue during HFD-induced obesity. We also took advantage of neonatal cardiomyocytes to study the role and downstream molecules of SIRT6 during HFD-induced injury in vitro, in which intracellular oxidative stress and mitochondrial content were assessed. We observed that during HFD-induced obesity, Sirt6 loss-of-function aggravated cardiac injury including left ventricular hypertrophy and lipid accumulation. Our results evidenced that upon increased fatty acid uptake, SIRT6 positively regulated the expression of endonuclease G (ENDOG), which is a mitochondrial-resident molecule that plays an important role in mitochondrial biogenesis and redox homeostasis. Our results also showed that SIRT6 positively regulated superoxide dismutase 2 (SOD2) expression post-transcriptionally via ENDOG. Our study gives a new sight into SIRT6 beneficial role in mitochondrial biogenesis of cardiomyocytes. Our data also show that SIRT6 is required to reduce intracellular oxidative stress in the heart triggered by high-fat diet-induced obesity, involving the control of ENDOG/SOD2.

Skeletal muscle is responsible for the majority of glucose disposal in the body. Insulin resistance in the skeletal muscle accounts for 85–90% of the impairment of total glucose disposal in patients with type 2 diabetes (T2D). However, the mechanism remains controversial. The present study aims to investigate whether AKT2 deficiency causes deficits in skeletal muscle development and metabolism, we analyzed the expression of molecules related to skeletal muscle development, glucose uptake and metabolism in mice of 3- and 8-months old. We found that AMP-activated protein kinase (AMPK) phosphorylation and myocyte enhancer factor 2 (MEF2) A (MEF2A) expression were down-regulated in AKT2 knockout (KO) mice, which can be inverted by AMPK activation. We also observed reduced mitochondrial DNA (mtDNA) abundance and reduced expression of genes involved in mitochondrial biogenesis in the skeletal muscle of AKT2 KO mice, which was prevented by AMPK activation. Moreover, AKT2 KO mice exhibited impaired AMPK signaling in response to insulin stimulation compared with WT mice. Our study establishes a new and important function of AKT2 in regulating skeletal muscle development and glucose metabolism via AMPK-dependent signaling.

Four pillar[5]arene based [3]rotaxanes (1-4) involving two 1,4-diethoxypillar[5]arene (DEP5) rings and a dumbbell-shaped component were successfully synthesized. The dumbbell-shape molecules contain one longer bridge, two triazole sites and two multicomponent stoppers. After threading DEP5 rings with linear guests (G1-G4) which contain two benzaldehyde units, the base catalyzed three-component reaction of dimedone, malononitrile and benzaldehyde was performed to construct the stoppers and connected the pseudorotaxanes with stoppers to generate 1–4. The structures of [3]rotaxanes and their self-assembly behaviors were characterized by 1H NMR, 13C NMR, NOESY, HR-ESI-MS, DLS and TEM technologies. We hope that pillar[5]arene based [3]rotaxanes may have potential applications in drug delivery systems and molecular devices.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder with intricate etiology. It is closely associated with metabolic syndrome, insulin resistance and endoplasmic reticulum (ER) stress. Exostosin1 (Ext1) is an ER-resident transmembrane glycosyltransferase, which plays an important role in ER homeostasis. Loss-of-function mutations in Ext1 link to hereditary multiple exostosis (HME). The present research was undertaken to identify the effect of Ext1 in the progress of NAFLD. High-fat-diet induced mice obesity, hepatic steatosis and decreased hepatic Ext1 expression. In consistent with evaluation of NAFLD mice possessing down-regulated Ext1 expression, free fatty acid (FFA) treatment blunted Ext1 expression in hepatocytes. In human subjects, HME patients presented elevated fasting blood glucose-one of the criteria that define insulin resistance. In vitro experiments, Ext1 deficiency promoted FFA-induced insulin resistance in hepatocytes by analysis of glycogen storage and hallmarks of gluconeogenesis, ascertaining its association with insulin resistance. Mechanically, Ext1 silencing exacerbated ER stress triggered by FFA, which severely disrupted autophagy in hepatocytes, and thereby accelerated the progression of NAFLD. In conclusion, our study demonstrates a beneficial role for Ext1 during the development of NAFLD, which establishes a novel correlation between Ext1 and ER stress-induced perturbations of autophagy during NAFLD progression.

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.

Mechanically interlocked molecules are a class of smart supramolecular species because of their interesting topological structure and application in various areas, such as biology and nanoscience. In this work, we used "multicomponent reaction" to fabricate a new [2]rotaxane based on pillar[5]arene from different small-sized molecules. The molecular structure of the obtained [2]rotaxane

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.

Background: Due to limited immunological profiles of high-grade serous ovarian cancer (HGS-OvCa), we aimed to characterise its molecular features to determine whether a specific subset that can respond to immunotherapy exists. Methods: A training cohort of 418 HGS-OvCa 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 HGS-OvCa samples and in vitro experiments were used for validation. Findings: We identified immune and normal groups where the former was enriched in signatures that reflect immune cells, infiltration, and PD-1 signalling (all, P

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.