Dominant Negative Mice Research Articles

Abstract P3008: Endothelial Stearoyl-CoA Desaturase-1 Catalyzes Fatty Acid Metabolites To Mitigate Arterial Inflammation And Stiffness

Introduction: Vascular metabolic plasticity is intimately linked with endothelial homeostasis. Stearoyl-CoA desaturase-1 (SCD1), the enzyme that catalyzes the saturated long-chain fatty acids (FA) into monounsaturated FA (MUFA), is responsive to pulsatile shear stress (PSS), and is associated with cancer cell metabolism. We assessed whether exercise-mediated PSS activates PPARγ-SCD1 to mediate anti-inflammatory metabolites for vascular protection. Methods and Results: Human aortic endothelial cells (HAEC) were subjected to PSS (τ avg = 23±8 dyne/cm 2 at 1 Hz) for 4 hours, followed by metabolomic analysis. SCD1-mediated fatty acid metabolites, azelaic acid (AZA) and oleic acid (OA), were identified to mitigate NFκB luciferase activity and monocyte binding ( p < 0.05, n= 5, vs. static condition). PSS upregulated PPARγ and SCD1 mRNA expression, whereas knockdown of PPARγ using siRNA downregulated SCD1. Wild type mice (C57BL/6J) undergoing 2-wk voluntary exercise developed increased perinuclear SCD1 expression in the aortic endothelium ( p < 0.05, n=6, vs. sedentary controls), whereas SCD1 expression was reduced in PPARγ -dominant negative mice following exercise. LDLR -/- mice with endothelial-specific SCD1 deletion (SCD1 EC-KO : Cdh5 Cre , Scd1 f/f ) on 4-wk high-fat diet (HFD) developed significantly increased VCAM-1 and Oil Red O positive lesions in the aortic arch, whereas exercise or selective overexpression (OE) of SCD1 in endothelial cells by pAd5/F35/CD144 adenoviral vector mitigated the lesions ( p < 0.05, n=5, HFD + exercise or HFD+Ad-SCD1 vs. HFD). Arterial stiffness as measured by Pulse Wave Velocity of the left common carotid was elevated in LDLR -/- SCD1 EC-KO on HF diet, but was decreased with exercise or SCD1 OE. Conclusion: PPARγ-SCD1 signaling is flow-responsive to mitigate arterial inflammation and stiffness. Despite SCD1-associated cancer cell metabolism, endothelial-specific SCD1 confers vascular protection, consistent with the reported skeletal muscle-specific model. Thus, we demonstrate that exercise activates PPARγ-SCD1 and SCD1 catalyzes AZA and OA as biomarkers with therapeutic potential.

Loss of protein kinase D activity demonstrates redundancy in cardiac glucose metabolism and preserves cardiac function in obesity

ObjectiveProtein kinase D (PKD) signaling has been implicated in stress-induced cardiac remodeling and function as well as metabolic processes including contraction-mediated cardiac glucose uptake. PKD has recently emerged as a nutrient-sensing kinase that is activated in high-lipid environments, such as in obesity. However, the role of PKD signaling in cardiac glucose metabolism and cardiac function in both normal and obese conditions remains unknown. MethodsA cardiac-specific and inducible dominant negative (DN) PKD mouse model was developed. Echocardiography was used to assess cardiac function, while metabolic phenotyping was performed, including stable isotope metabolomics on cardiac tissue in mice fed either regular chow or a high-fat diet (43% calories from fat). ResultsCardiac PKD activity declined by ∼90% following DN PKD induction in adult mice. The mice had diminished basal cardiac glucose clearance, suggesting impaired contraction-mediated glucose uptake, but normal cardiac function. In obesity studies, systolic function indices were reduced in control mice, but not in cardiac DN PKD mice. Using targeted stable isotope metabolomic analyses, no differences in glucose flux through glycolysis or the TCA cycle were observed between groups. ConclusionsThe data show that PKD contributes to cardiac dysfunction in obesity and highlight the redundancy in cardiac glucose metabolism that maintains cardiac glucose flux in vivo. The data suggest that impairments in contraction-mediated glucose uptake are unlikely to drive cardiac dysfunction in both normal and metabolic disease states.

Reducing hepatic PKD activity lowers circulating VLDL cholesterol.

Protein kinase D (PKD) is emerging as an important kinase regulating energy balance and glucose metabolism; however, whether hepatic PKD activity can be targeted to regulate these processes is currently unclear. In this study, hepatic PKD activity was reduced using adeno-associated virus vectors to express a dominant-negative (DN) version of PKD1, which impairs the action of all three PKD isoforms. In chow-fed mice, hepatic DN PKD expression increased whole-body glucose oxidation, but had only mild effects on glucose and insulin tolerance and no effects on glucose homeostasis following fasting and refeeding. However, circulating VLDL cholesterol was reduced under these conditions and was associated with hepatic fatty acid accumulation, but not lipids involved in lipoprotein synthesis. The limited effects on glucose homeostasis in DN PKD mice was despite reduced expression of gluconeogenic genes under both fasted and refed conditions, and enhanced pyruvate tolerance. The requirement for PKD for gluconeogenic capacity was supported by in vitro studies in cultured FAO hepatoma cells expressing DN PKD, which produced less glucose under basal conditions. Although these pathways are increased in obesity, the expression of DN PKD in the liver of mice fed a high-fat diet had no impact on glucose tolerance, insulin action, pyruvate tolerance or plasma VLDL. Together, these data suggest that PKD signalling in the liver regulates metabolic pathways involved in substrate redistribution under conditions of normal nutrient availability, but not under conditions of overnutrition such as in obesity.

Developmental abnormalities in the cornea of a mouse model for Marfan syndrome

Elastic fibres provide tissues with elasticity and flexibility. In the healthy human cornea, elastic fibres are limited to the posterior region of the peripheral stroma, but their specific functional role remains elusive.Here, we examine the physical and structural characteristics of the cornea during development in the mgΔloxPneo dominant-negative mouse model for Marfan syndrome, in which the physiological extracellular matrix of its elastic-fibre rich tissues is disrupted by the presence of a dysfunctional fibrillin-1 glycoprotein. Optical coherence tomography demonstrated a reduced corneal thickness in the mutant compared to wild type mice from embryonic day 16.5 until adulthood. X-ray scattering and electron microscopy revealed a disruption to both the elastic fibre and collagen fibril ultrastructure in the knockout mice, as well as abnormally low levels of the proteoglycan decorin. It is suggested that these alterations might be a result of increased transforming growth factor beta signalling. To conclude, this study has demonstrated corneal structure and ultrastructure to be altered when fibrillin-1 is disrupted and has provided insights into the role of fibrillin-1 in developing a functional cornea.

It's well and truly time to stop stating that AMPK regulates glucose uptake and fat oxidation during exercise.

None for Perspective.

PROP1-Dependent Retinoic Acid Signaling Regulates Developmental Pituitary Morphogenesis and Hormone Expression.

Dietary vitamin A is metabolized into bioactive retinoic acid (RA) in vivo and regulates the development of many embryonic tissues. RA signaling is active in the oral ectoderm-derived tissues of the neuroendocrine system, but its role there has not yet been fully explored. We show here that RA signaling is active during pituitary organogenesis and dependent on the pituitary transcription factor Prop1. Prop1-mutant mice show reduced expression of the aldehyde dehydrogenase gene Aldh1a2, which metabolizes the vitamin A-intermediate retinaldehyde into RA. To elucidate the specific function of RA signaling during neuroendocrine development, we studied a conditional deletion of Aldh1a2 and a dominant-negative mouse model of inhibited RA signaling during pituitary organogenesis. These models partially phenocopy Prop1-mutant mice by exhibiting embryonic pituitary dysmorphology and reduced hormone expression, especially thyrotropin. These findings establish the role of RA in embryonic pituitary stem cell progression to differentiated hormone cells and raise the question of gene-by-environment interactions as contributors to pituitary development and disease.

Foxp3 cooperates with Ikaros to control the suppressive function of regulatory T cells

Abstract Ikaros (encoded by Ikzf1) is a lymphocyte-specific transcription factor that controls a wide spectrum of T cell functions, including T helper differentiation, cytokine production, and tolerance in response to antigenic stimuli. Using heterozygous and dominant-negative mouse models, we have shown previously that Ikaros controls IL-2 production by conventional T cells, T helper differentiation, CD8+ effector differentiation, and anergy induction. While the role of Ikaros in many T cell subsets has been established, its role in Treg-mediated peripheral tolerance has not been examined. To examine the Treg-specific role of Ikaros, we generated Ikzf1-fl/fl-Foxp3-YFP-Cre+ mouse with conditional deletion of Ikaros in Tregs. Ikzf1-fl/fl-Foxp3-YFP-Cre+ mouse exhibited enlarged secondary lymphoid tissues, with increased frequencies of effector memory Tconv and Treg. We show that Ikaros expression by Tregs is required for immune tolerance in an allogenic cardiac transplant model. While CD28- and CD40L-blockade induced long-term cardiac allograft tolerance in control recipients, Ikzf1-fl/fl-Foxp3-YFP-Cre+ recipients rejected the allografts in a short duration (p<0.01). We show that Ikaros forms a complex with Foxp3, which is required for Foxp3 to bind to and repress inflammatory cytokine genes. Ikaros is also required for normal iTreg development in vitro, as TGF-B/IL-2-induced iTreg development is impaired in conventional CD4+ T cells with reduced Ikaros activity. These studies suggest that Ikaros interacts with Foxp3 and directs Foxp3 to exert its suppressive function in Tregs.

Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii

Little is known about strategies used by pathogens to facilitate CNS invasion. Toxoplasma gondii reaches the CNS by circulating in blood within leukocytes or as extracellular tachyzoites. T. gondii induces EGFR signaling in vitro during invasion of mammalian cells. We examined the effects of endothelial cell EGFR on CNS invasion. Transgenic mice whose endothelial cells expressed a dominant negative (DN) EGFR (inhibits EGFR signaling) exhibited diminished parasite load and histopathology in the brain and retina after T. gondii infection. I.V. administration of infected leukocytes or extracellular tachyzoites led to reduced parasite loads in mice with DN EGFR. This was not explained by enhanced immunity or reduced leukocyte recruitment. Endothelial cell infection is key for CNS invasion. Parasite foci in brain endothelial cells were reduced by DN EGFR. DN EGFR in these cells led to recruitment of the autophagy protein LC3 around T. gondii and spontaneous parasite killing dependent on the autophagy protein ULK1 and lysosomal enzymes. The autophagy inhibitor 3-MA prevented DN EGFR mice from exhibiting reduced CNS invasion. Altogether, EGFR is a novel regulator of T. gondii invasion of neural tissue, enhancing invasion likely by promoting survival of the parasite within endothelial cells.

Inducible and Reversible Dominant-negative (DN) Protein Inhibition.

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

The stress kinase JNK regulates gap junction Cx43 gene expression and promotes atrial fibrillation in the aged heart

BackgroundThe stress kinase c-jun N-terminal kinase (JNK) is critical in the pathogenesis of cardiac diseases associated with an increased incidence of atrial fibrillation (AF), the most common arrhythmia in the elderly. We recently discovered that JNK activation is linked to the loss of gap junction connexin43 (Cx43) and enhanced atrial arrhythmogenicity. However, direct evidence for JNK-mediated impairment of intercellular coupling (cell-cell communication) in the intact aged atrium is lacking, as is evidence for whether and how JNK suppresses Cx43 in the aged human atrium. Methods and resultsJNK activity in human atrial samples is correlated with both reduced Cx43 expression and increasing age. Using a unique technique of optical mapping space constant measurement, we found that impaired intercellular coupling and reduced Cx43 were linked to enhanced activation of JNK in intact aged rabbit atria. These JNK-associated alterations were further confirmed in naturally JNK activated aged mice and in cardiac-specific inducible MKK7D (JNK upstream activator) young mice. Moreover, JNK inhibition, using either JNK specific inhibitors in aged wild-type (WT) mice and JNK activator anisomycin-treated young WT mice or JNK1/2 dominant-negative mice with genetically inhibited cardiac JNK activity, completely eliminated these functional abnormalities. Furthermore, we discovered for the first time that long-term JNK activation downregulates Cx43 expression via c-jun suppressed transcriptional activity of the Cx43 gene promoter. ConclusionOur results demonstrate that JNK is a critical regulator of Cx43 expression, and that augmented JNK activation in aged atria downregulates Cx43 to impair cell-cell communication and promote the development of AF. JNK inhibition may represent a promising therapeutic approach to prevent or treat AF in the elderly.

Hepatoma-Derived Growth Factor Secreted from Mesenchymal Stem Cells Reduces Myocardial Ischemia-Reperfusion Injury.

Objectives The present study aimed to explore the major factors that account for the beneficial effects of mesenchymal stem cells (MSCs). Methods Using isobaric tags for relative and absolute quantitation method, hepatoma-derived growth factor (HDGF) was identified as an important factor secreted by MSCs, but not by cardiac fibroblasts (CFs). The protective effects of conditioned medium (CdM) from MSCs or CFs were tested by using either H9C2 cells that were exposed by hypoxia-reoxygenation (H/R) insult or an in vivo mouse model of myocardial ischemia-reperfusion. Results Compared to CF-CdM, MSC-CdM conferred protection against reperfusion injury. CdM obtained from MSCs that were treated with HDGF-targeted shRNA failed to offer any protection in vitro. In addition, administration of recombinant HDGF alone recapitulated the beneficial effects of MSC-CdM, which was associated with increased protein kinase C epsilon (PKCε) phosphorylation, enhanced mitochondria aldehyde dehydrogenase family 2 activity, and decreased 4-hydroxy-2-nonenal accumulation. A significant decrease in infarct size and ameliorated cardiac dysfunction was achieved by administration of HDGF in wild-type mice, which was absent in PKCε dominant negative mice, indicating the essential roles of PKCε in HDGF-mediated protection. Conclusions HDGF secreted from MSCs plays a key role in the protection against reperfusion injury through PKCε activation.

Aged dominant negative p38α MAPK mice are resistant to age-dependent decline in adult-neurogenesis and context discrimination fear conditioning

A major aspect of mammalian aging is the decline in functional competence of many self-renewing cell types, including adult-born neuronal precursors. Since age-related senescence of self-renewal occurs simultaneously with chronic up-regulation of the p38MAPKalpha (p38α) signaling pathway, we used the dominant negative mouse model for attenuated p38α activity (DN-p38αAF/+) in which Thr180 and Tyr182 are mutated (T→A/Y→F) to prevent phosphorylation activation (DN-p38αAF/+) and kinase activity. As a result, aged DN-p38αAF/+ mice are resistant to age-dependent decline in proliferation and regeneration of several peripheral tissue progenitors when compared to wild-type littermates.Aging is the major risk factor for non-inherited forms of Alzheimer’s disease (AD); environmental and genetic risk factors that accelerate the senescence phenotype are thought to contribute to an individual’s relative risk. In the present study, we evaluated aged DN-p38αAF/+ and wildtype littermates in a series of behavioral paradigms to test if p38α mutant mice exhibit altered baseline abnormalities in neurological reflexes, locomotion, anxiety-like behavior, and age-dependent cognitive decline. While aged DN-p38αAF/+ and wildtype littermates appear equal in all tested baseline neurological and behavioral parameters, DN-p38αAF/+ exhibit superior context discrimination fear conditioning. Context discrimination is a cognitive task that is supported by proliferation and differentiation of adult-born neurons in the dentate gyrus of the hippocampus. Consistent with enhanced context discrimination in aged DN-p38αAF/+, we discovered enhanced production of adult-born neurons in the dentate gyrus of DN-p38αAF/+ mice compared to wildtype littermates. Our findings support the notion that p38α inhibition has therapeutic utility in aging diseases that affect cognition, such as AD.

Abstract 12746: Impaired Autophagic Flux in Ischemic/Reperfused Diabetic Heart and Its Reversal by a Novel Orally Active Adiponectin Receptor Agonist via AMPK-Mediated Autophagosome Formation and AMPK-Independent Autophagosome Clearance

Autophagy is increasingly recognized as an important regulator of myocardial ischemia/reperfusion (MI/R) injury. However, whether and how autophagy is altered in the ischemic/reperfused diabetic heart remains unknown. Normal control (NC), high-fat-diet-induced diabetic (HFD), and adiponectin (APN) knockout (APNKO) mice were subjected to MI/R (30 minutes/3-24 hours) and treated with vehicle or AdipoRon (50 mg/kg gavage, 10 minutes before MI; or 10 mg/kg IP, 10 min before R). Compared to NC mice, autophagosome formation was significantly inhibited and autophagosome clearance was nearly completely blocked in the HFD heart after MI/R (P<0.01). APNKO largely reproduced the phenotypic alterations observed in the HFD heart, with both autophagosome formation and clearance inhibited after MI/R (P<0.01 vs. WT). Treatment of HFD and APKNO mice with AdipoRon stimulated autophagosome formation, increased autophagosome clearance, reduced infarct size, and improved cardiac function (P<0.01). Mechanistically, AdipoRon caused significant phosphorylation of AMPK, BeclineT119, and VPS34S16, and enhanced expression of lysosome protein LAMP2. Compound C pretreatment abolished AdipoRon-induced BeclineT119 and VPS34S164 phosphorylation without altering AdipoRon-induced LAMP2 expression. In vitro AdipoRon treatment of adult wild type cardiomyocytes enhanced phosphorylation of AMPK, BeclineT119, and VPS34S164. These effects are lost in cardiomyocytes isolated from cardiomyocyte-specific AMPK dominant negative mice (AMPK-DN). In contrast, AdipoRon-stimulated LAMP2 expression was preserved in AMPK-DN cardiomyocytes. Collectively, these results demonstrated for the first time that hypoadiponectinemia during diabetic conditions impairs autophagic flux in cardiomyocytes after MI/R via marked inhibition of both autophagosome clearance and formation. Administration of an orally active APN receptor agonist increased autophagosome formation via AMPK activation, and stimulated autophagosome clearance in an AMPK-independent fashion. Improving autophagic flux by restoring APN signaling systems might be a novel intervention against MI/R injury in diabetic conditions.

AMPK-α2is involved in exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following high-fat diet

AMP-activated protein kinase (AMPK) has been studied extensively and postulated to be a target for the treatment and/or prevention of metabolic disorders such as insulin resistance. Exercise training has been deemed a beneficial treatment for obesity and insulin resistance. Furthermore, exercise is a feasible method to combat high-fat diet (HFD)-induced alterations in insulin sensitivity. The purpose of this study was to determine whether AMPK-α2 activity is required to gain beneficial effects of exercise training with high-fat feeding. Wild-type (WT) and AMPK-α2 dominant-negative (DN) male mice were fed standard diet (SD), underwent voluntary wheel running (TR), fed HFD, or trained with HFD (TR + HFD). By week 6, TR, irrespective of genotype, decreased blood glucose and increased citrate synthase activity in both diet groups and decreased insulin levels in HFD groups. Hindlimb perfusions were performed, and, in WT mice with SD, TR increased insulin-mediated palmitate uptake (76.7%) and oxidation (>2-fold). These training-induced changes were not observed in the DN mice. With HFD, TR decreased palmitate oxidation (61-64%) in both WT and DN and increased palmitate uptake (112%) in the WT with no effects on palmitate uptake in the DN. With SD, TR increased ERK1/2 and JNK1/2 phosphorylation, regardless of genotype. With HFD, TR reduced JNK1/2 phosphorylation, regardless of genotype, carnitine palmitoyltransferase 1 expression in WT, and CD36 expression in both DN and WT. These data suggest that low AMPK-α2 signaling disrupts, in part, the exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following HFD.

Abstract 81: Novel Adult Endothelial Hierarchy Defined by Endovascular Progenitor Reexpression of SOX18

Introduction: During adult life, formation of new blood vessels in response to insults or physiological signals is thought almost exclusively to result from the sprouting and extension of existing vessels defined as angiogenesis. An alternate proposal for de novo vascularization relates to endothelial progenitors and their ability to assemble the intimal layers of mature vessels. However, the cell lineage and possible hierarchy of such precursors remains controversial. We here aimed to define endothelial progenitors in vivo in situations of neo-vessel formation in adult mice. Methods and Results: Based on protein expression levels of markers CD31 and VEGFR2 using flow cytometry, three populations of endothelial cells could be identified among VECAD+CD34+CD45- cells in a variety of angiogenic situations such as wound healing, tumours, placental development, or in subcutaneous Matrigel plugs. Lineage tracing based on sox18 re-expression using a sox18CreER/Rosa-YFP reporter strategy demonstrated that the CD31loVEGFR2- endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2- transit amplifying and a definitive differentiated CD31hiVEGFR2hi population. Loss of SOX18, in a dominant-negative mouse model, did not affect levels of EVP populations in vivo, but depleted both the intermediate and definitive populations. Only the sorted CD31loVEGFR2- as opposed to the other two endothelial populations could form colonies in vitro and engraft in vivo in a Matrigel plug assay. Conclusions: These results define and characterize an in vivo endothelial hierarchy and suggest a prominent role for vessel resident EVP populations in situations of neo-vessel formation. This data opens the medium for new perspectives in modulating blood vessel formation.

AMPKα2 skeletal muscle activity is required to induce the physiological effects of insulin on fatty acid uptake (1162.3)

Exercise training has been deemed a beneficial treatment for obesity and insulin resistance induced by a high fat diet. Further, exercise training may modulate its metabolic effects via AMPK activation, a key signaling molecule in metabolic regulation. It is widely accepted that insulin stimulation (IS) results in an increase in fatty acid uptake (FAU) in skeletal muscle. The purpose of this study was to determine whether AMPKα2 activity in skeletal muscle is required for the benefits of exercise training in regards to insulin sensitivity while under high fat‐fed conditions. Wild type (WT) and AMPKα2 dominant negative (DN) male C57BL/6 mice were divided into control diet ± voluntary wheel running (CD, n=10; VWR, n=10) or high fat diet (60% fat Bio‐Serv) ± VWR (HFD, n=10; VWR+HFD, n=10). After 6 wks, hindlimbs were perfused (550μM palmitate, 6mM glucose, [1‐14C]palmitate, and 450μU/ml insulin). As expected, IS increased FAU (45%) in CD WT mice (P<0.05). However, IS decreased FAU by 35% (P<0.05) in CD DN mice. With VWR, insulin maintained its ability to increase FAU (26%) in WT mice (P<0.05). In DN mice, VWR did not improve the actions of insulin on FAU (39% reduction; P<0.05). When VWR was performed under HFD conditions, the added metabolic stress reduced the ability of insulin to increase FAU (P>0.05) in WT mice. In DN mice, VWR combined with HFD did not change the actions of insulin on FAU (32% reduction; P<0.05). AMPKα2 signaling appears to be required to induce the physiological effects of insulin on FA metabolism. VWR did not improve the aberrant FAU responses to IS in mice lacking skeletal muscle AMPKα2. Further, it appears that VWR is not sufficient to rescue or alter the effects of HFD on insulin‐mediated FA metabolism. Overall, AMPKα2 activity is a necessary component in the regulation of skeletal muscle FA metabolism.

Mitochondria Oxidative Stress, Connexin43 Remodeling, and Sudden Arrhythmic Death

Previously, we showed that a mouse model (ACE8/8) of cardiac renin-angiotensin system activation has a high rate of spontaneous ventricular tachycardia and sudden cardiac death secondary to a reduction in connexin43 level. Angiotensin-II activation increases reactive oxygen species (ROS) production, and ACE8/8 mice show increased cardiac ROS. We sought to determine the source of ROS and whether ROS played a role in the arrhythmogenesis. Wild-type and ACE8/8 mice with and without 2 weeks of treatment with L-NIO (NO synthase inhibitor), sepiapterin (precursor of tetrahydrobiopterin), MitoTEMPO (mitochondria-targeted antioxidant), TEMPOL (a general antioxidant), apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), allopurinol (xanthine oxidase inhibitor), and ACE8/8 crossed with P67 dominant negative mice to inhibit the nicotinamide adenine dinucleotide phosphate oxidase were studied. Western blotting, detection of mitochondrial ROS by MitoSOX Red, electron microscopy, immunohistochemistry, fluorescent dye diffusion technique for functional assessment of connexin43, telemetry monitoring, and in vivo electrophysiology studies were performed. Treatment with MitoTEMPO reduced sudden cardiac death in ACE8/8 mice (from 74% to 18%; P<0.005), decreased spontaneous ventricular premature beats, decreased ventricular tachycardia inducibility (from 90% to 17%; P<0.05), diminished elevated mitochondrial ROS to the control level, prevented structural damage to mitochondria, resulted in 2.6-fold increase in connexin43 level at the gap junctions, and corrected gap junction conduction. None of the other antioxidant therapies prevented ventricular tachycardia and sudden cardiac death in ACE8/8 mice. Mitochondrial oxidative stress plays a central role in angiotensin II-induced gap junction remodeling and arrhythmia. Mitochondria-targeted antioxidants may be effective antiarrhythmic drugs in cases of renin-angiotensin system activation.

Abstract 19711: Mitochondria-Targeted Antioxidant Therapy Prevents Connexin43 Remodeling and Sudden Death Caused by Renin-Angiotensin System Activation

Introduction: Previously, we showed a mouse model (ACE8/8) of cardiac renin-angiotensin system (RAS) activation has a high rate of spontaneous ventricular tachycardia (VT) and sudden cardiac death (SCD) secondary to a reduction in connexin43 (Cx43) level. Angiotensin-II activation increases reactive oxygen species (ROS) production, and these mice show cardiac oxidation. We sought to determine the source of ROS and if ROS played a role in the arrhythmogenesis. Methods and Results: Wild-type and ACE8/8 mice with and without two weeks of treatment with L-NIO (nitric oxide synthase inhibitor), sepiapterin (precursor of tetrahydrobiopterin), MitoTEMPO (mitochondria-targeted antioxidant), TEMPOL (a general antioxidant), apocynin (NADPH oxidase inhibitor), allopurinol (xanthine oxidase inhibitor), and ACE8/8 crossed with P67 dominant negative mice to inhibit the NADPH oxidase were studied. Western blotting, detection of mitochondrial ROS by MitoSOX Red, electron microscopy, immunohistochemistry, fluorescent dye diffusion technique for functional assessment of Cx43, and in-vivo electrophysiology study were performed. Treatment with MitoTEMPO reduced SCD in ACE8/8 mice (from 74% to 18%, P=0.0005), decreased VT inducibility (from 90% to 17%, P&lt;0.05), diminished elevated mitochondrial ROS to the control level, prevented structural damage to mitochondria, resulted in 2.6 fold increase in Cx43 level at the gap junctions, and corrected gap junction conduction. None of the other antioxidant therapies prevented VT and SCD in ACE8/8 mice. Conclusion: Mitochondrial oxidative stress plays a central role in angiotensin II-induced gap junction remodeling and arrhythmia. Mitochondria-targeted antioxidants may be effective antiarrhythmic drugs in cases of RAS activation.

Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3'-UTR. This shortening of the 3'-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3'-UTR relieves translational suppression observed in the long 3'-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

T cell specific deletion of the phosphatase SHP1 decreases incidence and severity of experimental autoimmune encephalomyelitis (101.41)

Abstract T cell activation is regulated by a dynamic balance between kinase and phosphatase activity. The tyrosine phosphatase SHP1 is a critical negative regulator of the immune system, and a proximal regulator of T cell receptor signaling. Here, we have utilized two novel models to study the deletion of SHP1 specifically in mature T cells. We have bred mice bearing a floxed SHP1 allele that deletes the majority of the gene with mice bearing an inducible Cre (Mx1 promoter). We also utilized a dominant negative allele of SHP1 whose expression requires Cre-driven recombination. By transferring mature T cells from these mice into T cell deficient hosts and subsequently inducing Cre expression we have isolated the effect of loss of SHP1 activity to T cells. We have determined that selective loss of this phosphatase in mature T cells dramatically attenuates experimental autoimmune encephalomyelitis (EAE). Both severity and incidence were decreased significantly in the conditional knockout and dominant negative mice relative to controls (mean max score 1.0, 1.7, and 2.7, respectively and mean incidence 29, 37 and 79%). These data differ from results in mice heterozygous for the motheaten allele, a naturally occurring SHP1 mutant, which exhibit EAE severity that is similar to or slightly worse than controls. Collectively, this suggests that lack of SHP1 activity in T cells may protect from disease, while deletion in other cell types may exacerbate susceptibility or symptoms.