WT Heart Research Articles

Neutrophil NLRP3 promotes cardiac injury following acute myocardial infarction through IL-1β production, VWF release and NET deposition in the myocardium

NLRP3 inflammasome has been implicated in neutrophil polarization and extrusion of neutrophil extracellular traps (NETs) in vitro and facilitates secretion of Il1-beta (IL-1β). Permanent ligation of the left anterior descending artery was used to induce MI in WT and NLRP3−/− mice as well as in NLRP3−/− recipient mice transfused with either WT or NLRP3−/− neutrophils. NLRP3 deficiency reduced infarct size to roughly a third of WT heart injury and preserved left ventricular (LV) function at 12 h after MI as assessed by echocardiography and triphenyltetrazolium chloride staining of live tissue. Transfusion of WT but not NLRP3−/− neutrophils after MI increased infarct size in NLRP3−/− mice and significantly reduced LV function. The key features of myocardial tissue in WT neutrophil transfused recipients were increased H3Cit-positive deposits with NET-like morphology and increased tissue levels of IL-1β and plasma levels of von Willebrand Factor (VWF). Flow cytometry analysis also revealed that neutrophil NLRP3 increased the number of labeled and transfused neutrophils in the bone marrow of recipient mice following MI. Our data suggest a key role for neutrophil NLRP3 in the production of IL-1β and deposition of NETs in cardiac tissue exacerbating injury following MI. We provide evidence for a link between neutrophil NLRP3 and VWF release likely enhancing thromboinflammation in the heart. Neutrophil NLRP3 deficiency conferred similar cardioprotective effects to general NLRP3 deletion in MI rendering anti-neutrophil NLRP3 therapy a promising target for early cardioprotective treatment.

Modulation of cardiac cAMP signaling by AMPK and its adjustments in pressure overload-induced myocardial dysfunction in rat and mouse.

The beta-adrenergic system is a potent stimulus for enhancing cardiac output that may become deleterious when energy metabolism is compromised as in heart failure. We thus examined whether the AMP-activated protein kinase (AMPK) that is activated in response to energy depletion may control the beta-adrenergic pathway. We studied the cardiac response to beta-adrenergic stimulation of AMPKα2-/- mice or to pharmacological AMPK activation on contractile function, calcium current, cAMP content and expression of adenylyl cyclase 5 (AC5), a rate limiting step of the beta-adrenergic pathway. In AMPKα2-/- mice the expression of AC5 (+50%), the dose response curve of left ventricular developed pressure to isoprenaline (p<0.001) or the response to forskolin, an activator of AC (+25%), were significantly increased compared to WT heart. Similarly, the response of L-type calcium current to 3-isobutyl-l-methylxanthine (IBMX), a phosphodiesterase inhibitor was significantly higher in KO (+98%, p<0.01) than WT (+57%) isolated cardiomyocytes. Conversely, pharmacological activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) induced a 45% decrease in AC5 expression (p<0.001) and a 40% decrease of cAMP content (P<0.001) as measured by fluorescence resonance energy transfer (FRET) compared to unstimulated rat cardiomyocytes. Finally, in experimental pressure overload-induced cardiac dysfunction, AMPK activation was associated with a decreased expression of AC5 that was blunted in AMPKα2-/- mice. The results show that AMPK activation down-regulates AC5 expression and blunts the beta-adrenergic cascade. This crosstalk between AMPK and beta-adrenergic pathways may participate in a compensatory energy sparing mechanism in dysfunctional myocardium.

Stimulator of Interferon Genes (STING) regulates Re‐endothelialization Following Vascular Injury

Percutaneous coronary interventions (PCI) are commonly used to improve blood flow in Peripheral Artery Disease. Complications of PCI are delayed re-endothelialization and endothelial cell (EC) dysfunction leading to adverse clinical outcomes. Stimulator of Interferon Genes (STING), canonically activated downstream of the cGAS enzyme, is a signaling hub molecule modulating immune responses triggered by self-derived and/or foreign DNA. We hypothesized that EC-intrinsic STING regulates re-endothelialization in response to vascular injury. We modeled PCI in WT, STING-/-, and in a newly generated inducible vascular EC- specific mouse (VECad5Cre-ERT2STINGfl/fl mice: EC-STING-/-) using the wire induced carotid injury model. We used Evans Blue Dye injection post injury to identify the areas denuded of EC and quantified the % of non-blue (re-endothelialized) area. We found that WT mice had complete re-endothelialization five days post injury (95.16 ± 2.77 %). In contrast, injured carotid arteries in STING-/- mice exhibited a decrease in re-endothelialization at the same timepoint (43.57 ± 9.79%). Moreover, EC-STING-/- mice recapitulated the delayed re-endothelialization (10.45 ± 1.56% in Tamoxifen treated mice to induce excision of STING in EC, compared to 40 ± 1.28% observed in vehicle treated VECad5Cre-ERT2STINGfl/fl control mice 5 days post scratch) in the injured arteries, supporting a role of EC STING in re-endothelialization post injury. To further investigate this EC-specific effect of STING mechanistically in vitro, we performed scratch assays on WT and STING mouse heart EC (MHEC) monolayers, as well as on human EC monolayers lacking STING after CRISPR knock down and imaged wound healing in real time by IncuCyte. WT MHEC and HUVEC completely covered the scratch area within 24 hours, and 4 hours, respectively, whereas both EC lacking STING were delayed healing the scratch. Additionally, we found that the healing response was independent of cGAS, since cGAS KD HUVEC had similar post- scratch recovery time as control HUVEC. Ongoing experiments investigate the mechanisms involved in this novel STING dependent, cGAS-independent pathway of EC response to injury, as well as the consequences of EC-STING in adverse chronic vascular remodeling.

Stimulator of Interferon Genes (STING) Regulates Endothelial Healing and Reendothelialization Following Angioplasty‐Mediated Vascular Injury

Angioplasty and stent implantation are common clinical interventions to improve blood flow in Peripheral Artery Disease, which is rising in prevalence in America. However, these interventions result in vascular endothelial damage, leading to the activation of signaling pathways that can have a major effect on short‐ and long‐term vessel health. We hypothesized that endothelial cell‐intrinsic Stimulator of Interferon Genes (STING), a crucial regulator of immune responses triggered by pathogen or self‐derived DNA, regulates reendothelialization and healing in response to vascular injury. To study whether STING‐mediated reendothelialization was an endothelial cell specific effect, in vitro wound healing assay was performed on WT and STING deficient primary mouse heart endothelial cells (MHEC) using live imaging and quantification with Cytation and IncuCyte. We found that, while WT MHEC completely covered the scratch area within 24 hours of initial scratch, only 75.59% ± 5.8 of the initial wound was healed in STING deficient MHEC monolayers. Furthermore, our in vivo experiments using the wire injury‐mediated experimental model of angioplasty in mice followed by Evans Blue Dye staining to distinguish the areas with denuded endothelium, demonstrate a decrease in reendothelialization in injured carotid arteries in STING deficient mice as compared to WT mice five days post injury (43.57% ± 9.79 area reendothelialized vs 95.16% ± 2.77, respectively). Seven days post wire injury, WT carotid arteries were completely healed, in contrast to those of STING deficient mice that remained unhealed (67.74% ± 5.80 of reendothelialized vessel area). Additionally, multi‐parameter flow cytometry analysis of the enzymatically digested arteries from the seven days post wire injury group demonstrated an increase in myeloid cell infiltration in WT mice, which was not observed in either the injured STING deficient arteries or in the uninjured contralateral WT carotid arteries, indicating an early localized and differential immune response likely affecting long term vascular remodeling. Our results support that endothelial STING contributes to endothelial healing in vitro and in vivo, and that myeloid cell infiltration in the carotid arteries is required for reendothelialization and may be responsible for lack of healing in STING deficient mice. Ongoing experiments aim to definitively quantify temporal recruitment of immune cells over the course of reendothelialization and chronic vascular remodeling following wire injury and the mechanisms involved in short‐ and long‐term vascular health.Support or Funding InformationThis work was supported by the NIH‐RO1‐HL123658

All-trans retinoic acid attenuates isoproterenol-induced cardiac dysfunction through Crabp1 to dampen CaMKII activation

Inhibiting Ca2+/calmodulin-dependent protein kinase II (CaMKII) over activation can decrease detrimental cardiac remodeling that leads to dilated cardiomyopathy, cell death, and heart failure. We previously showed that cellular retinoic acid binding protein 1 (Crabp1) knockout mice (CKO) exhibited a more severe isoproterenol (ISO)-induced heart failure and cardiac remodeling phenotype with elevated CaMKII activity in the heart, suggesting a cardiac-protective function of Crabp1 through modulating CaMKII activity. Here we examine whether the highly selective, endogenous ligand of Crabp1, all-trans retinoic acid (RA), can attenuate ISO-induced cardiac dysfunction. We also examine if this attenuation involves Crabp1 and the inhibition of CaMKII. RA pre-treatment followed by ISO challenge effectively restores ejection fraction in wild type, but not in CKO mice. This is correlated with reduced CaMKII auto-phosphorylation at T287 and phospholamban phosphorylation at T17, a substrate of CaMKII. RA pretreatment also reduces ISO-induced apoptosis in WT heart. Cell culture experiments confirm that RA inhibits CaMKII phosphorylation, which requires Crabp1. Molecular data reveal interaction of Crabp1 with the kinase and regulatory domains of CaMKII, and that RA selectively enhances Crabp1 interaction with the regulatory domain, suggesting a potential regulatory role for holo-Crabp1 in CaMKII activation. Together, these data demonstrate that RA bound Crabp1 plays a protective role in β-adrenergic stimulated cardiac remodeling, which is partially attributed to its dampening CaMKII activation. Targeting Crabp1 provides a potentially new therapeutic strategy for managing heart diseases.

GSK3β inhibition and canonical Wnt signaling in mice hearts after myocardial ischemic damage.

AimsMyocardial infarction induces myocardial injury and tissue damage. During myocardial infarction strong cellular response is initiated to salvage the damaged tissues. This response is associated with the induction of different signaling pathways. Of these, the canonical Wnt signaling is increasingly important for its prosurvival cellular role, making it a good candidate for the search of new molecular targets to develop therapies to prevent heart failure in infarcted patients.MethodsHerein we report that GSK3β regulates the canonical Wnt signaling in C57Bl6 mice hearts. GSK3β is a canonical Wnt pathway inhibitor. Using GSK3β inhibitors and inducing myocardial injury (MI) in Lrp5-/- mice model we show that GSK3β phosphorylation levels regulate downstream canonical Wnt pathway genes in the ischemic heart. In the setting of MI, myocardial damage assessment usually correlates with functional and clinical outcomes. Therefore, we measured myocardial injury size in Wt and Lrp5-/- mice in the presence and absence of two different GSK3 inhibitors prior to MI. Myocardial injury was independent of GSK3 inhibitor treatments and GSK3β expression levels.ResultsThese studies support a central role for GSK3β in the activation of the canonical Wnt pathway in the Wt heart. Although LRP5 is protective against myocardial injury, GSK3β expression levels do not regulate heart damage.

Exploration of the mechanistic role of Cyclophilin D in the mitochondria

IntroductionCyclophilin D (CypD) is an 18 kda protein encoded by the nuclear ppif gene and transported to the mitochondria. Being the only member of the cyclophilin family localized in the mitochondria, its known function is the regulation of the mitochondrial permeability transition pore (MPTP) which usually precedes mitochondrial dysfunction during calcium overload and oxidative stress. However, the mechanism of MPTP induction by CypD is still unknown, therefore, this study is aimed at understanding the generic role of CypD in the mitochondria.MethodCypD of murine liver mitochondria was quantitatively determined from recombinant CypD standard curve using densitometric analysis programme, image J. Difference in CypD level between liver and heart tissue mitochondria was quantified. Modulation of the binding stability of recombinant CypD by some mitochondrial metabolites (in vitro) was carried out by dynamic scanning fluorimetry (DSF). Mitochondrial swelling and NAD(P)H fluorescence were monitored spectrophotometrically at 540 nm and 355/450 (Ex/Em) respectively. Metabolic differences between CypD WT and KO murine tissues were carried out using HNMR spectroscopy.ResultsCypD was observed to constitute about 0.1% of the total mitochondrial protein. It was also observed to be more than 2‐fold higher in the liver than heart tissue mitochondria. MPTP activation experiments revealed 2‐fold higher calcium induced swelling in liver than heart tissue mitochondria. Mitochondrial NAD(P)H fluorescence was observed to be highest at 100 uM calcium in the presence of CsA while a massive loss of NAD(P)H was observed when CypD was uninhibited at this concentration. Succinyl CoA and alphaketoglutarate, TCA cycle intermediates enhanced calcium induced mitochondrial swelling however, DSF reveals that Succinyl CoA negatively regulates CypD binding interaction with CsA while alphaketoglutarate had no effect. Global metabolite differences were observed in CypD genotypes and increased level of succinate and fumarate in were observed in ppif KO when compared with WT heart tissues.ConclusionThese findings give further insight into the role of CypD in the mitochondria as it revealed differences in MPTP between tissues and impact on cellular reductive equivalents which are associated with the differential amount of CypD in the mitochondria of these tissues. It also reveals the influence of CypD on global metabolism especially in the liver which is the majour site of metabolism and that some of these may play direct or indirect role in MPTP activation.Support or Funding InformationOSA acknowledge Osun State University, Nigeria for financial support.YIA is grateful to University of Liverpool for a Postgraduate BursaryThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping transcriptome profiles of in vitro iPSC-derived cardiac differentiation to in utero heart development.

The dataset includes microarray data (Affymetrix Mouse Genome 430 2.0 Array) from WT and Nos3−/− mouse embryonic heart ventricular tissues at 14.5 days post coitum (E14.5), induced pluripotent stem cells (iPSCs) derived from WT and Nos3−/− mouse tail tip fibroblasts, iPSC-differentiated cardiomyocytes at Day 11, and mouse embryonic stem cells (mESCs) and differentiated cardiomyocytes as positive controls for mouse iPSC differentiation. Both in utero (using embryonic heart tissues) and in vitro (using iPSCs and differentiated cells) microarray datasets were deposited to the NCBI Gene Expression Omnibus (GEO) database. The deposited data in GEO include raw microarray data, metadata for sample source information, experimental design, sample and data processing, and gene expression matrix. The data are available under GEO Access Number GSE69317 (GSE69315 for tissue sample microarray data, GSE69316 for iPSCs microarray data, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE69317).

Abstract 14317: Kvβ2 Mediates Cardiac Repolarization and Injury: Role of Pyridine Nucleotides

Background: Voltage-gated potassium channel (Kv) β2 subunits (Kvβ2) belong to the aldoketo-reductase superfamily and associate with the Kv channel modulating its function. Also, Kvβ2 binds pyridine nucleotides (NAD[P] + /NAD[P]H) with high affinity altering Kv channel gating. However, the physiological relevance of Kvβ2 in cardiac health and disease is unknown. Hypothesis: We tested the hypothesis that Kvβ2 is essential to both basal and pyridine nucleotide mediated regulation of cardiac repolarization. Methods and Results: Kvβ2 knockout (KO) and wild type (Wt) mice were utilized for this study. ECG analysis showed prolonged QTc interval (74.7± 3.7 vs. 65.6 ± 3, n=3 , p&lt;0.05 ) in KO mice when compared to Wt. Monophasic action potential (MAP) data also showed prolonged duration (ms) at 50% repolarization level in KO mouse hearts vs. Wt (MAPD50; 13.2 ± 0.3 vs. 9.7 ± 0.6, n=5-6 , p&lt;0.05 ). To test the obligatory role of Kvβ2 in pyridine nucleotide modulation of cardiac repolarization, we exposed ex vivo perfused hearts or isolated cardiomyocytes to higher lactate levels. These data revealed that acute lactate exposure leads to significant but similar raise in NADH concentration [NADH] i in both KO and Wt heart tissue. However, marked repolarization deficits appeared only in Wt group when tested utilizing ex vivo perfused hearts (MAPD50 - Wt:11.5 ± 0.7 vs. 8.3 ± 0.5, p&lt;0.05 ; KO:12.6 ± 0.7 vs. 12± 0.5, p=0.5 , ms) or isolated myocytes (ΔAPD50 – Wt: 4.7±1.6 ms, p&lt;0.05 ; KO:1.5 ± 0.7 ms, p=0.1 ). Perfusion with high pyruvate levels returned both [NADH] i and MAPD50 to basal levels in Wt mouse hearts. Similarly, we observed that a 14 day isoproterenol treatment also leads to significant increase in cardiac [NADH] i , but only the Wt mouse hearts showed significant repolarization deficits; ex-MAPD50 (5.5 ± 1.6, p&lt; 0.01 ), when compared to no changes in KO (1.9 ± 0.9, p&lt; 0.1) . Conclusions: Our results suggest that Kvβ2 regulates cardiac repolarization. Further, the differential repolarization changes despite similar increases in cardiac [NADH] i indicates an obligatory role of Kvβ2 in the pyridine nucleotide modulation of cardiac repolarization.

Abstract 228: CaMKII as a Link Between Inflammation and Fibrosis Induced by Chronic Isoproterenol and Angiotensin II Treatment

The Ca2+/calmodulin-dependent kinase, CaMKIIδ, is an established mediator of the development of heart failure and myocardial injury. Cardiac inflammation has been increasingly recognized as an important player in these cardiac pathophysiological changes. We previously demonstrated that CaMKIIδ contributes to cardiac inflammation induced by ischemia/reperfusion through activation of cardiomyocyte NF-kB. In the current study we ask whether angiotensin II (Ang II) and isoproterenol (ISO), both known to activate CaMKII, promote cardiac inflammation through this protein kinase and its effects on NF-kB activation. In addition, chronic ISO and Ang II treatment promote cardiac fibrosis and we hypothesize that this response is initiated through activation of CaMKIIδ and subsequent inflammatory responses. We report on our recent findings that show attenuated inflammatory cytokine expression (e.g. IL-6, MCP1, and TNFα) in response to 7 days Ang II infusion in mice in which CaMKIIδ is specifically deleted in cardiomyocytes (cardiac specific knockout; CKO). In addition the expression of fibrotic markers (e.g. col1a1, col3a1, and CTGF) in response to Ang II infusion is decreased in the CKO mice. This is associated with attenuated fibrosis as evident in histological analysis of CKO vs WT heart sections. Ongoing studies will compare the effects of chronic ISO and Ang II in CKO and WT mice to determine whether inflammation precedes fibrosis and assess the extent to which apoptosis induced by CaMKII activation plays a part in these responses. Currently, we are determining whether Ang II and ISO act through CaMKII to activate NF-kB in the cardiomyocyte compartment to induce proinflammatory and profibrotic factors and whether preventing the expression of these factors block development of further inflammatory and fibrotic responses. Findings from these studies may implicate CaMKII as a promising therapeutic target for attenuating cardiac fibrosis.

Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B(-/-) mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B(-/-) mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca(2+)-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B(-/-) heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3-enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.

Abstract 89: Mitochondrial Protein Acetylation in Aged Hearts in the Context of Ischemia-Reperfusion Injury

Introduction: SIRT3 deficiency leads to increased mitochondrial acetylation, and enhances sensitivity of the heart to stress. Recently we demonstrated that a worse outcome of ischemia-reperfusion (IR) injury in aged hearts was associated with increased protein acetylation. The aim of this study was to investigate the mechanism of acetylation and the identity of acetylated targets in aged hearts in the context of IR injury. Methods: Mitochondria were isolated from WT adult (7 mo.), SIRT3 +/- adult (7 mo.) and WT aged (18 mo.) hearts. SIRT3 protein expression and identification of acetylated proteins from SIRT3 +/- adult and WT aged samples were investigated using proteomics (2D gel, western blot, peptide mass fingerprinting). Results: Mitochondrial acetylome analysis revealed that many hyperacetylated patterns were identical between adult SIRT3 +/- and aged WT hearts, suggesting that increased protein acetylation in the aged WT heart might be due to SIRT3 inhibition. Several targets were indentified, including complex I NADH dehydrogenase flavoprotein 1, which is known to interact with SIRT3. In this regard, we demonstrated that respiratory complex I (CxI) activity was significantly inhibited in both SIRT3 +/- adult and WT aged hearts. Analysis of SIRT3 protein revealed that negatively charged species of SIRT3 (relative to the bulk SIRT3 population), were lost in aged hearts. These alterations suggest post-translational modification of SIRT3 which may lead to loss of its deacetylase activity. Alternatively, we found that upregulation of the mitochondrial acetyltransferase GCN5L1 in aged hearts (examined by western blot) may also contribute to enhanced protein acetylation. Conclusions: These data support a connection between SIRT3 downregulation, mitochondrial protein acetylation, and exacerbation of IR injury in aging hearts. Furthermore, it is known that functional CxI is required for several modes of cardioprotection suggesting that downregulation of CxI in SIRT3 +/- or WT aged hearts may underlie their poor recovery from IR injury.

Impaired local regulation of ryanodine receptor type-2 by protein phosphatase 1 promotes atrial fibrillation

Purpose: Atrial fibrillation (AF) is the most common cardiac arrhythmia with a growing prevalence yet current pharmaceutical treatments are ineffective. Previous studies suggest that protein phosphatase 1 (PP1) is dysregulated globally in AF patients but its local regulation on key Ca2+-handling proteins such as ryanodine receptor type-2 (RyR2) is not known. In this study we hypothesize that decreased PP1 regulation of RyR2 promotes AF through increasing Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2. Methods: To study the local regulation of RyR2 by PP1, we used spinophilin knockout mice (KO), since PP1 targets RyR2 via spinophilin. The interaction between RyR2 and PP1 was measured using co-immunoprecipitation while RyR2 phosphorylation level was quantified using Western blot. RyR2 activity was determined by measuring 1) single RyR2 channel activity in a lipid bilayer system and 2) Ca2+ sparks frequency in atrial myocytes loaded with Fluo4-AM. Baseline cardiac characteristics were determined using echocardiography and histological staining while AF inducibility was assessed by intracardiac electrophysiology study. Results: The interaction of PP1 with RyR2 was reduced by 55% in the KO hearts compared to WT (P<0.001). RyR2 phosphorylation at Serine 2814 was increased by 43% in the KO atria (P<0.05 vs. WT) while unchanged at Serine 2808. Single RyR2 channels isolated from KO hearts had a higher open probability (0.10±0.02) compared to that from WT (0.02±0.01; P<0.01). Ca2+ spark frequency normalized to sarcoplasmic reticulum (SR) Ca2+ content was increased in atrial myocytes isolated from KO mice (1.72±0.26 a.u.) compared to WT (0.77±0.20 a.u.; P<0.05). Furthermore, this increase was rescued by CaMKII-inhibitor KN93 but not by protein kinase A (PKA) inhibitor H89. End diastolic diameter of hearts from KO mice was 10% smaller than that from WT (P<0.01) but both the heart weight to body weight ratio and ejection fraction were unchanged. Also, there was no difference in fibrosis between KO and WT heart based on histological sections stained with Masson's trichome. However, when subjected to intracardiac electrical stimulation, KO mice had a higher incidence of pacing-induced AF (100%) compared to WT (33%; P<0.01). Conclusions: PP1 regulates RyR2 by counteracting CaMKII but not PKA phosphorylation of RyR2. Spinophilin deficiency leads to decreased PP1 regulation of RyR2 which may promote AF. This regulation may represent a novel therapeutic target for treating AF.

Effect of Ischemia Reperfusion Injury and Epoxyeicosatrienoic Acids on Caveolin Expression in Mouse Myocardium

Caveolins (Cav) are structural proteins that insert into the plasma membrane to form caveolae that can bind molecules important in cardiac signal transduction and function. Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase reduces the protective effect. (1) To assess the effect of ischemia-reperfusion injury on expression and subcellular localization of caveolins. (2) To study the effect of EETs on caveolins. Hearts from soluble epoxide hydrolase null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20 minutes ischemia followed by 40 minutes reperfusion. Immunohistochemistry, immunoblot, and electron microscopy were performed to study localization of caveolins and changes in ultrastructure. In WT heart, Cav-1 and Cav-3 were present in cardiomyocyte and capillary endothelial cell at baseline. After ischemia, Cav-1 but not Cav-3, disappeared from cardiomyocyte; moreover, caveolae were absent and mitochondrial cristae were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded to higher Cav-1 expression and maintained caveolae structure. In addition, KO mice preserved the Cav-1 signaling after ischemia that lost in WT mice. Taken together, our data suggest that ischemia-reperfusion injury causes loss of Cav-1 and caveolins, and EETs-mediated cardioprotection involves preservation of Cav-1.

Cognate Antigen Is Necessary and Sufficient for the Migration of Effector and Memory T Cells to Vascularized Allografts

Background: A key step in the rejection process is the transendothelial migration of Ag-specific effector and memory T cells into the graft. It is generally assumed that this step is mediated by Gai-coupled chemokine receptor signaling. Here, we revisited this paradigm by investigating the migration of polyclonal and monoclonal effector and memory T cells to vascularized transplants in the presence or absence of Gai signaling. Methods: B6 CD4 and CD8 polyclonal effector and memory T cells primed against BALB/c were treated with pertussis toxin (PTx) to block Gai signaling or not and co-transferred to B6 wt or alymphoplastic (aly/aly) mice 2 days after transplanting BALB/c hearts. Ag(OVA)-specific (OT-I) and non-specific (P14) CD8 effector/memory T cells were transferred to B6 recipients of B6 Act-OVA or wt heart or kidney grafts to investigate the role of cognate Ag in T cell migration. Migration of transferred T cells to the grafts was quantified by flow cytometry and 2-photon intravital microscopy using congenic markers, MHC-tetramers and cell tracker reagents. Results: PTx-treated and untreated polyclonal effector and memory T cells migrated to and rejected heart allografts in equal numbers and with the same tempo. Ag(OVA)-specific (OT-I) effector/memory T cells migrated only to heart grafts that expressed the cognate Ag (OVA) and their migration was not inhibited by PTx. Non-specific (P14) T cells, on the other hand, did not migrate to Act-OVA grafts unless Ag-specific (OT-I) T cells were present, and their migration was abrogated by PTx. Intravital imaging of transplanted Act-OVA kidneys established that PTx did not inhibit the firm adhesion or transendothelial migration of Ag-specific (OT-I) effector/memory T cells. In contrast, adhesion and transmigration of non-specific (P14) T cells occurred only if their Gai signaling was intact. Conclusion: In contrast to the chemokine-dependent paradigm, the migration of Ag-specific effector/memory T cells to vascularized allografts requires interaction with cognate Ag and is independent of chemokine signaling. Entry of non-specific effector/memory T cells, on the other hand, requires chemokines but occurs only if Ag-specific T cells are present. These findings underscore the critical role of cognate Ag in directing the entry of primed T cells to non-lymphoid tissues and suggest novel mechanisms for their transmigration across the endothelium.

Abstract P276: Cardiac-Specific Knockout NF-kB Mice Resist Dysfunction Induced by Global Ischemia-Reperfusion

Subjective: Nuclear factor Kappa B (NF-kB) is a pluripotent transcriptional factor that has been implicated as mediator of cardiac injury after ischemia-reperfusion (IR). Multiple approaches have been used to target inhibition of NF-kB, yet these strategies are often non-specific. P65-NF-kB exists as a heterodimer with its p50 subunit and is the dominant transcriptional subunit of NF-kB. In order to better understand its role in the heart, we have recently generated mice that have cardiomyocyte (CMC) specific deletion of p65 NF-kB and hypothesized that these animals would be protected from ischemic stress. Methods: Transgenic mice were generated with (WT) or without CMC p65 (KO). NF-kB p65 expression was assessed with Western Blots. We subjected the mice to global IR using Langendorf system with baseline 20 min, ischemia 45 min and reperfusion 60 min. Cardiac contractile functional recovery was analyzed using rate pressure product (RPP), contractility (+dp/dt), and relaxation (-dp/dt). Infarct size was assessed with TTC staining. LDH and CK were measured in reperfused solutions. Results: 1. No differences were found between groups of WT (n=7) and KO (n=8) in body weight and heart weight. 2. KO mice express significant less cardiac-specific NF-kB p65. 3. During reperfusion, RPP, +dp/dt, -dp/dt recovery to base line were all better in p65 KO mice (RPP: 0.841±0.074 in KO vs 0.635±0.080 in WT; +dp/dt: 0.997±0.096 in KO vs 0.731±0.108 in WT; -dp/dt: 0.869±0.096 in KO vs 0.630±0.090 in WT; p&lt;0.05 respectively). 4. After ischemia, both LDH and CK release were markedly less in solutions collected from p65 KO heart (LDH: 45.2±28.0 u/L; CK: 28.1±23.6 u/L) than those in p65 WT heart (LDH: 143.2±25.9 u/L; CK: 107.2±26.2 u/L; p,0.01 respectively). 5. TTC staining shown significant less necrotic tissue in KO heart vs WT heart (0.314±0.052 vs 0.653±0.061, p&lt;0.05). Conclusion: CMC-specific knockout NF-kB p65 mice have improved cardiac recovery function, less heart necrosis, and decreased CMC injury compared to WT after global heart ischemia. These studies confirm many of the previous studies suggesting a protective role for anti-NF-kB strategies for ischemia. It furthermore provides a template to further define the mechanisms of p65-NF-kB afforded cardioprotection.

06 Epoxyeicosatrienoic acid mediated cardioprotection involves mitochondrial caveolin-1 and PKCe

Cytochrome P450 epoxygenases can metabolise arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase (sEH) reduces the protective effect. Caveolins...

Role of caveolins in epoxyeicosatrienoic acid mediated protective effects against ischemia reperfusion injury

Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase (sEH) reduces the protective effect. Caveolins (Cav-1, -2, and -3) are structural proteins that insert into the plasma membrane to form caveolae, which can bind molecules important in cardiac signal transduction and function. The objective of this study was to assess the role of Cav in EET mediated cardioprotection. Hearts from sEH null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20min ischemia followed by 40min reperfusion. Immunohistochemistry, immunoblot and electron microscopy were performed to study localization of Cav and changes in ultrastructure. In WT heart, cav-1 and cav-3 were present in cardiomyocyte (CM) and capillary endothelial cell at baseline. Following ischemia, cav-1 but not cav-3 disappeared from CM; moreover, caveolae were absent in WT hearts. Mitochondria and T-tubules were swollen, and cristae of mitochondria were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded with higher cav-1 expression in plasma membrane and mitochondrial fractions. Taken together, our data suggest EETs prevent the loss of cav-1, resulting in increased mitochondrial targeting and cardioprotection.

H030 Loss of Ephrin-B1 disrupts myocardial architecture and leads to abnormal sympathetic heart rate variability

Ephrin-B1 is a ligand from Eph/Ephrin family involved in cell-cell interactions. If the role of ephrine molecules is well known in embryonic tissue, their expressions/functions in adults remain unclear and to date, no study have paid attention to their potential implication in heart physiopathology. To characterize the cardiac phenotype of ephrin-B1 knockout mice. Two months old-mice were analyzed. Heart tissue was fixed (formaldehyde 10 %) and embedded in paraffin for immunohistochemistry or frozen for western-blot (WB) analysis. Immunofluorescence (IF) studies were performed on heart cryosections. ECG was recorded (PowerLab, DSI) under isoflurane anaesthesia and heart rate spectral variability (HRV) was performed (FFT) in low frequency (LF: 0.15-1.5 Hz) and high frequency (HF: 1.5-5 Hz) ranges ; LH/HF ratio was also calculated. We first assessed expression of Ephrin-B1 by WB in heart from WT animals. A specific band around 47 kDa was detected in WT heart total protein extracts that was lost in KO mice. Further IF studies demonstrated broad expression of Ephrin-B1 protein throughout all heart compartments with different cellular localizations (cardiomyocytes and micro/macrocirculation). Hematoxylin-eosin (HE) staining of paraffinembedded heart sections from KO mice revealed loss of organized cardiac tissue characterized by the presence of wavy cardiomyocytes in both septum and ventricles. Myocytes intersected at various angles with bundles wavy appearance. No inflammation, interstitial fibrosis or necrosis were noticed. These pathological observations correlated well with the lack of stiffness of hearts from KO mice compared with controls. When we examined ANS-dependent heart rate variability, LFHRV was significantly reduced in KO mice (16.3±1.2 %) when compared to controls (48.5±6.2 %) without any change in HF, suggesting a specific loss of cardiac sympathetic innervation in these animals. LF/HF ratio was lower in KO mice (0.6±0.2 vs 1.3±0.2 in controls). This study provides the first evidence for the presence of ephrin molecules in the adult heart tissue with a specific expression of Ephrin-B1 ligand. The use of ephrin-B1 genetic mouse model highly suggests a role for ephrin-B1 in heart tissue architecture and in sympathetic control of heart rate variability.

Role of PAR2 in Type 2 Diabetes‐induced Endothelial Dysfunction

Previously we have found a critical role of tumor necrosis factor‐α (TNF) in endothelial dysfunction resulting from type 2 diabetes. Because TNF can influence many pathways of inducing oxidative stress and cell injury, our goal was to identify one of the contributing mechanisms. Protease‐activated receptors (PARs), particularly endothelial PAR2 are thought to play a role in inflammatory cardiovasculardisease. We investigated the role of PAR2 in regulating TNF‐induced endothelial dysfunction in Type 2 diabetes. Coronary arterioles of WT (normal) and db/db (diabetic) mice (12–16 weeks) were isolated and pressurized (60 cmH2O). Although vasodilation to endothelium‐independent vasodilator sodium nitroprusside was not different among WT, db/db and dbTNF‐/dbTNF‐ (db/db null for TNF) mice, dilation to endothelium‐dependent agonist ACh was impaired in db/db mice but was greater in dbTNF‐/dbTNF‐compared to db/db (P&lt;0.05). Administration of PAR2 antagonist (FSLLRY‐NH2) partially enhanced ACh‐induced vasodilation in db/db mice. Flow‐induced vasodilation showed similar response to ACh‐induced vasodilation. Both mRNA and protein expression of PAR2 were higher in db/db heart compared to WT heart whereas mRNA and protein expression were reduced in dbTNF‐/dbTNF‐ vs db/db. These results indicate that PAR2 plays a pivotal role in type 2 diabetes‐induced endothelial dysfunction through TNF mechanism.