Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node.

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.

Nicotinamide for the treatment of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD+). Elevating NAD+ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD+ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.

Hypochlorite-Modified LDL Induces Arrhythmia and Contractile Dysfunction in Cardiomyocytes.

Neutrophil-derived myeloperoxidase (MPO) and its potent oxidant, hypochlorous acid (HOCl), gained attention as important oxidative mediators in cardiac damage and dysfunction. As cardiomyocytes generate low-density lipoprotein (LDL)-like particles, we aimed to identify the footprints of proatherogenic HOCl-LDL, which adversely affects cellular signalling cascades in various cell types, in the human infarcted myocardium. We performed immunohistochemistry for MPO and HOCl-LDL in human myocardial tissue, investigated the impact of HOCl-LDL on electrophysiology and contractility in primary cardiomyocytes, and explored underlying mechanisms in HL-1 cardiomyocytes and human atrial appendages using immunoblot analysis, qPCR, and silencing experiments. HOCl-LDL reduced ICa,L and IK1, and increased INaL, leading to altered action potential characteristics and arrhythmic events including early- and delayed-afterdepolarizations. HOCl-LDL altered the expression and function of CaV1.2, RyR2, NCX1, and SERCA2a, resulting in impaired contractility and Ca2+ homeostasis. Elevated superoxide anion levels and oxidation of CaMKII were mediated via LOX-1 signaling in HL-1 cardiomyocytes. Furthermore, HOCl-LDL-mediated alterations of cardiac contractility and electrophysiology, including arrhythmic events, were ameliorated by the CaMKII inhibitor KN93 and the INaL blocker, ranolazine. This study provides an explanatory framework for the detrimental effects of HOCl-LDL compared to native LDL and cardiac remodeling in patients with high MPO levels during the progression of cardiovascular disease.

ß-catenin regulates FOXP2 transcriptional activity via multiple binding sites.

The transcription factor forkhead box protein P2 (FOXP2) is a highly conserved key regulator of embryonal development. The molecular mechanisms of how FOXP2 regulates embryonal development, however, remain elusive. Using RNA sequencing, we identified the Wnt signaling pathway as key target of FOXP2-dependent transcriptional regulation. Using cell-based assays, we show that FOXP2 transcriptional activity is regulated by the Wnt coregulator ß-catenin and that ß-catenin contacts multiple regions within FOXP2. Using nuclear magnetic resonance spectroscopy, we uncovered the molecular details of these interactions. ß-catenin contacts a disordered FOXP2 region with α-helical propensity via its folded armadillo domain, whereas the intrinsically disordered ß-catenin N terminus and C terminus bind to the conserved FOXP2 DNA-binding domain. Using RNA sequencing, we confirmed that ß-catenin indeed regulates transcriptional activity of FOXP2 and that the FOXP2 α-helical motif acts as a key regulatory element of FOXP2 transcriptional activity. Taken together, our findings provide first insight into novel regulatory interactions and help to understand the intricate mechanisms of FOXP2 function and (mis)-regulation in embryonal development and human diseases. DATABASE: Expression data are available in the GEO database under the accession number GSE138938.

Myeloperoxidase-Derived 2-Chlorohexadecanal Is Generated in Mouse Heart during Endotoxemia and Induces Modification of Distinct Cardiomyocyte Protein Subsets In Vitro.

Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N3-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.

Empagliflozin protects heart from inflammation and energy depletion via AMPK activation.

AIMS: Sodium-glucose co-transporter 2 (SGLT2) were originally developed as kidney-targeting anti-diabetic drugs. However, due to their beneficial cardiac off-target effects (as SGLT2 is not expressed in the heart), these antagonists currently receive intense clinical interest in the context of heart failure (HF) in patients with or without diabetes mellitus (DM). Since the mechanisms by which these beneficial effects are mediated are still unclear yet, inflammation that is present in DM and HF has been proposed as a potential pharmacological intervention strategy. Therefore, we tested the hypothesis that the SGLT2 inhibitor, empagliflozin, displays anti-inflammatory potential along with its glucose-lowering property. METHODS AND RESULTS: Lipopolysaccharide (LPS) was used to induce inflammation in vitro and in vivo. In cardiomyocytes and macrophages empagliflozin attenuated LPS-induced TNFα and iNOS expression. Analysis of intracellular signalling pathways suggested that empagliflozin activates AMP kinase (AMPK) in both cell types with or without LPS-treatment. Moreover, the SGLT2 inhibitor increased the expression of anti-inflammatory M2 marker proteins in LPS-treated macrophages. Additionally, empagliflozin-mediated AMPK activation prevented LPS-induced ATP/ADP depletion. In vivo administration of LPS in mice impaired cardiac contractility and aortic endothelial relaxation in response to acetylcholine, whereby co-administration of empagliflozin preserved cardiovascular function. These findings were accompanied by improved cardiac AMPK phosphorylation and ATP/ADP, reduced cardiac iNOS, plasma TNFα and creatine kinase MB levels. CONCLUSION: Our data identify a novel cardio protective mechanism of SGLT2 inhibitor, empagliflozin, suggesting that AMPK activation-mediated energy repletion and reduced inflammation contribute to the observed cardiovascular benefits of the drug in HF.

MAPK signaling determines lysophosphatidic acid (LPA)-induced inflammation in microglia.

BACKGROUND: In the extracellular environment, lysophosphatidic acid (LPA) species are generated via autotaxin (ATX)-mediated hydrolysis of lysophospholipid precursors. Members of the LPA family are potent lipid mediators transmitting signals via six different G protein-coupled LPA receptors (LPAR1-6). The LPA signaling axis is indispensable for brain development and function of the nervous system; however, during damage of the central nervous system, LPA levels can increase and aberrant signaling events counteract brain function. Here, we investigated regulation of the ATX/LPA/LPAR axis in response to lipopolysaccharide-induced systemic inflammation in mice and potential neurotoxic polarization programs in LPA-activated primary murine microglia. METHODS: In vivo, LPAR1-6 expression was established by qPCR in whole murine brain homogenates and in FACS-sorted microglia. ELISAs were used to quantitate LPA concentrations in the brain and cyto-/chemokine secretion from primary microglia in vitro. Transcription factor phosphorylation was analyzed by immunoblotting, and plasma membrane markers were analyzed by flow cytometry. We used MAPK inhibitors to study signal integration by the JNK, p38, and ERK1/2 branches in response to LPA-mediated activation of primary microglia. RESULTS: Under acute and chronic inflammatory conditions, we observed a significant increase in LPA concentrations and differential regulation of LPAR, ATX (encoded by ENPP2), and cytosolic phospholipase A2 (encoded by PLA2G4A) gene expression in the brain and FACS-sorted microglia. During pathway analyses in vitro, the use of specific MAPK antagonists (SP600125, SB203580, and PD98059) revealed that JNK and p38 inhibition most efficiently attenuated LPA-induced phosphorylation of proinflammatory transcription factors (STAT1 and -3, p65, and c-Jun) and secretion of IL-6 and TNFα. All three inhibitors decreased LPA-mediated secretion of IL-1ß, CXCL10, CXCL2, and CCL5. The plasma membrane marker CD40 was solely inhibited by SP600125 while all three inhibitors affected expression of CD86 and CD206. All MAPK antagonists reduced intracellular COX-2 and Arg1 as well as ROS and NO formation, and neurotoxicity of microglia-conditioned media. CONCLUSION: In the present study, we show that systemic inflammation induces aberrant ATX/LPA/LPAR homeostasis in the murine brain. LPA-mediated polarization of primary microglia via MAPK-dependent pathways induces features reminiscent of a neurotoxic phenotype.

Saxagliptin but Not Sitagliptin Inhibits CaMKII and PKC via DPP9 Inhibition in Cardiomyocytes.

Some oral anti-hyperglycemic drugs, including gliptins that inhibit dipeptidyl peptidase 4 (DPP4), have been linked to the increased risk of heart failure (HF) in type-2 diabetic patients. While the cardiovascular safety trial, TECOS, revealed no link between sitagliptin and the risk of HF, a substantial 27% increase in the hospitalization for HF was observed in type-2 diabetic patients treated with saxagliptin within the SAVOR-TIMI 53 trial. A previous in vitro study revealed that saxagliptin impairs the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-phospholamban (PLB)-sarcoplasmic reticulum Ca2+-ATPase 2a axis and protein kinase C (PKC) activity in cardiomyocytes leading to impaired cardiac contractility and electrophysiological function. However, the link between saxagliptin and its target proteins (CaMKII and PKC) remains to be explored. Since DPP8 and DPP9 (but not DPP4) are expressed by cardiomyocytes and saxagliptin is internalized by cardiomyocytes, we investigated whether DPP8/9 contribute to saxagliptin-mediated inhibition of CaMKII and PKC activity. Structural analysis revealed that the DPP4-saxagliptin interaction motif (S630, Y547) for the cyanopyrrolidine group is conserved in DPP8 (S755, Y669) and DPP9 (S730, Y644). Conversely, F357 that facilitates binding of the anchor lock domain of sitagliptin in the S2 extensive subsite of DPP4 is not conserved in DPP8/9. In parallel, unlike saxagliptin, sitagliptin did not affect phosphorylation of CaMKII/PLB or activity of PKC in HL-1 cardiomyocytes. These findings were recapitulated by pharmacological inhibition (TC-E-5007, a DPP8/9 antagonist) and knock-down of DPP9 (but not DPP8). In primary mouse ventricular cardiomyocytes, saxagliptin (but not sitagliptin) impaired Ca2+ transient relaxation and prolonged action potential duration (APD). These results suggest that saxagliptin-DPP9 interaction impairs the CaMKII-PLB and PKC signaling in cardiomyocytes. We reveal a novel and potential role of DPP9 in cardiac signaling. The interaction of saxagliptin with DPP9 may represent an underlying mechanism for the link between saxagliptin and HF. Elucidation of saxagliptin-DPP9 interaction and downstream events may foster a better understanding of the role of gliptins as modulators of cardiac signaling.

Dipeptidyl peptidase-4 independent cardiac dysfunction links saxagliptin to heart failure.

Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase-4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca2+/calmodulin-dependent protein kinase II-phospholamban-sarcoplasmic reticulum Ca2+-ATPase 2a axis and Na+-Ca2+ exchanger function in Ca2+ extrusion. This resulted in reduced sarcoplasmic reticulum Ca2+ content, diastolic Ca2+ overload, systolic dysfunction and impaired contractile force. Furthermore, saxagliptin reduced protein kinase C-mediated delayed rectifier K+ current that prolonged action potential duration and consequently QTc interval. Importantly, saxagliptin aggravated pre-existing cardiac dysfunction induced by ischemia/reperfusion injury. In conclusion, our novel results provide mechanisms for the off-target deleterious effects of saxagliptin on cardiac function and support the outcome of SAVOR-TIMI 53 trial that linked saxagliptin with the risk of heart failure.

Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death.

Despite considerable efforts to improve treatment modalities for osteosarcoma (OS), patient survival remains poor mainly due to pro-survival pathways in OS cells. Among others, prostaglandins (PGs) are the potent regulators of bone homoeostasis and OS pathophysiology. Therefore, the present study aimed to elucidate the impact of 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2, a stable PGD2 degradation product) on cell death/cell survival pathways in p53-deficient MG-63 OS cells. Our findings show that 15d-PGJ2 induces generation of reactive oxygen species that promote p38 MAPK activation and subsequent Akt phosphorylation. This pathway induced nuclear expression of Nrf2 and Egr1, and increased transcription of haem oxygenase-1 (HO-1) and the catalytic subunit of glutamate cysteine ligase (GCLc), catalysing the first step in GSH synthesis. Silencing of Nrf2, Egr1 and HO-1 significantly elevated 15d-PGJ2-mediated reduction of cellular metabolic activity. Activation of cell survival genes including HO-1 and GCLc inhibited 15d-PGJ2-induced cleavage of pro-caspase-3 and PARP. Annexin V/propidium iodide staining showed an increase in early/late apoptotic cells in response to 15d-PGJ2. The observed 15d-PGJ2-mediated signalling events are independent of PGD2 receptors (DP1 and DP2) and PPARγ. In addition, the electrophilic carbon atom C9 is a prerequisite for the observed activity of 15d-PGJ2. The present data show that the intracellular redox imbalance acted as a node and triggered both death and survival pathways in response to 15d-PGJ2. Pharmacological or genetic interference of the pro-survival pathway, the p38 MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis, sensitizes MG-63 cells towards 15d-PGJ2-mediated apoptosis.

Neutrophil proteolytic activation cascades: a possible mechanistic link between chronic periodontitis and coronary heart disease.

Cardiovascular diseases are chronic inflammatory diseases that affect a large segment of society. Coronary heart disease (CHD), the most common cardiovascular disease, progresses over several years and affects millions of people worldwide. Chronic infections may contribute to the systemic inflammation and enhance the risk for CHD. Periodontitis is one of the most common chronic infections that affects up to 50% of the adult population. Under inflammatory conditions the activation of endogenous degradation pathways mediated by immune responses leads to the release of destructive cellular molecules from both resident and immigrant cells. Matrix metalloproteinases (MMPs) and their regulators can activate each other and play an important role in immune response via degrading extracellular matrix components and modulating cytokines and chemokines. The action of MMPs is required for immigrant cell recruitment at the site of inflammation. Stimulated neutrophils represent the major pathogen-fighting immune cells that upregulate expression of several proteinases and oxidative enzymes, which can degrade extracellular matrix components (e.g. MMP-8, MMP-9 and neutrophil elastase). The activity of MMPs is regulated by endogenous inhibitors and/or candidate MMPs (e.g. MMP-7). The balance between MMPs and their inhibitors is thought to mirror the proteolytic burden. Thus, neutrophil-derived biomarkers, including myeloperoxidase, may activate proteolytic destructive cascades that are involved in subsequent immune-pathological events associated with both periodontitis and CHD. Here, we review the existing studies on the contribution of MMPs and their regulators to the infection-related pathology. Also, we discuss the possible proteolytic involvement and role of neutrophil-derived enzymes as an etiological link between chronic periodontitis and CHD.

Behavioral and molecular processing of visceral pain in the brain of mice: impact of colitis and psychological stress.

Gastrointestinal disorders with abdominal pain are associated with central sensitization and psychopathologies that are often exacerbated by stress. Here we investigated the impact of colitis induced by dextran sulfate sodium (DSS) and repeated water avoidance stress (WAS) on spontaneous and nociception-related behavior and molecular signaling in the mouse brain. DSS increased the mechanical pain sensitivity of the abdominal skin while both WAS and DSS enhanced the mechanical and thermal pain sensitivity of the plantar skin. These manifestations of central sensitization were associated with augmented c-Fos expression in spinal cord, thalamus, hypothalamus, amygdala and prefrontal cortex. While WAS stimulated phosphorylation of mitogen-activated protein kinase (MAPK) p42/44, DSS activated another signaling pathway, both of which converged on c-Fos. The DSS- and WAS-induced hyperalgesia in the abdominal and plantar skin and c-Fos expression in the brain disappeared when the mice were subjected to WAS+DSS treatment. Intrarectal allyl isothiocyanate (AITC) evoked aversive behavior (freezing, reduction of locomotion and exploration) in association with p42/44 MAPK and c-Fos activation in spinal cord and brain. These effects were inhibited by morphine, which attests to their relationship with nociception. DSS and WAS exerted opposite effects on AITC-evoked p42/44 MAPK and c-Fos activation, which indicates that these transduction pathways subserve different aspects of visceral pain processing in the brain. In summary, behavioral perturbations caused by colitis and psychological stress are associated with distinct alterations in cerebral signaling. These findings provide novel perspectives on central sensitization and the sensory and emotional processing of visceral pain stimuli in the brain.

Myeloperoxidase scavenges peroxynitrite: A novel anti-inflammatory action of the heme enzyme.

Peroxynitrite, a potent pro-inflammatory and cytotoxic species, interacts with a variety of heme containing proteins. We addressed the question whether (i) the interaction of myeloperoxidase (MPO, an enzyme generating hypochlorous acid from hydrogen peroxide and chloride ions) with peroxynitrite affects the clearance of peroxynitrite, and (ii) if peroxynitrite could modulate the chlorinating activity of MPO. Our results show that this interaction promotes the decomposition of the highly reactive pro-inflammatory oxidant, whereby MPO Compound II (but not Compound I) is formed. The efficiency of MPO to remove peroxynitrite was enhanced by L-tyrosine, nitrite and (-)-epicatechin, substances known to reduce Compound II with high reaction rate. Next, peroxynitrite (added as reagent) diminished the chlorinating activity of MPO in the presence of hydrogen peroxide. Alternatively, SIN-1, a peroxynitrite donor, reduced hypochlorous acid formation by MPO, as measured by aminophenyl fluorescein oxidation (time kinetics) and taurine chloramine formation (end point measurement). At inflammatory loci, scavenging of peroxynitrite by MPO may overcome the uncontrolled peroxynitrite decomposition and formation of reactive species, which lead to cell/tissue damage.

15-deoxy-Δ¹²,¹4-PGJ2 promotes inflammation and apoptosis in cardiomyocytes via the DP2/MAPK/TNFα axis.

BACKGROUND: Prostaglandins (PGs), lipid autacoids derived from arachidonic acid, play a pivotal role during inflammation. PGD2 synthase is abundantly expressed in heart tissue and PGD2 has recently been found to induce cardiomyocyte apoptosis. PGD2 is an unstable prostanoid metabolite; therefore the objective of the present study was to elucidate whether its final dehydration product, 15-deoxy-Δ¹²,¹4-PGJ2 (15d-PGJ2, present at high levels in ischemic myocardium) might cause cardiomyocyte damage. METHODS AND RESULTS: Using specific (ant)agonists we show that 15d-PGJ2 induced formation of intracellular reactive oxygen species (ROS) and phosphorylation of p38 and p42/44 MAPKs via the PGD2 receptor DP2 (but not DP1 or PPARγ) in the murine atrial cardiomyocyte HL-1 cell line. Activation of the DP2-ROS-MAPK axis by 15d-PGJ2 enhanced transcription and translation of TNFα and induced apoptosis in HL-1 cardiomyocytes. Silencing of TNFα significantly attenuated the extrinsic (caspase-8) and intrinsic apoptotic pathways (bax and caspase-9), caspase-3 activation and downstream PARP cleavage and γH2AX activation. The apoptotic machinery was unaffected by intracellular calcium, transcription factor NF-κB and its downstream target p53. Of note, 9,10-dihydro-15d-PGJ2 (lacking the electrophilic carbon atom in the cyclopentenone ring) did not activate cellular responses. Selected experiments performed in primary murine cardiomyocytes confirmed data obtained in HL-1 cells namely that the intrinsic and extrinsic apoptotic cascades are activated via DP2/MAPK/TNFα signaling. CONCLUSIONS: We conclude that the reactive α,ß-unsaturated carbonyl group of 15d-PGJ2 is responsible for the pronounced upregulation of TNFα promoting cardiomyocyte apoptosis. We propose that inhibition of DP2 receptors could provide a possibility to modulate 15d-PGJ2-induced myocardial injury.

I(f) blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes.

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability.

We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development.

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100.

Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.

Molecular targets and regulators of cardiac hypertrophy.

Cardiac hypertrophy is one of the main ways in which cardiomyocytes respond to mechanical and neurohormonal stimuli. It enables myocytes to increase their work output, which improves cardiac pump function. Although cardiac hypertrophy may initially represent an adaptive response of the myocardium, ultimately, it often progresses to ventricular dilatation and heart failure which is one of the leading causes of mortality in the western world. A number of signaling modulators that influence gene expression, apoptosis, cytokine release and growth factor signaling, etc. are known to regulate heart. By using genetic and cellular models of cardiac hypertrophy it has been proved that pathological hypertrophy can be prevented or reversed. This finding has promoted an enormous drive to identify novel and specific regulators of hypertrophy. In this review, we have discussed the various molecular signal transduction pathways and the regulators of hypertrophic response which includes calcineurin, cGMP, NFAT, natriuretic peptides, histone deacetylase, IL-6 cytokine family, Gq/G11 signaling, PI3K, MAPK pathways, Na/H exchanger, RAS, polypeptide growth factors, ANP, NO, TNF-alpha, PPAR and JAK/STAT pathway, microRNA, Cardiac angiogenesis and gene mutations in adult heart. Augmented knowledge of these signaling pathways and their interactions may potentially be translated into pharmacological therapies for the treatment of various cardiac diseases that are adversely affected by hypertrophy. The purpose of this review is to provide the current knowledge about the molecular pathogenesis of cardiac hypertrophy, with special emphasis on novel researches and investigations.