bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P230: Orchestration of Cardiac Mitochondrial Biogenesis by Mitofusin2
Authors: Chen Yun , Matkovich Scot , Zhang Yan , Liu Yingqiu , Dorn Gerald
Published: March 2022

Mitochondria generate energy and play central roles governing cell signaling and programmed death. As an energy dependant tissue, the heart is more affected by mitochondrial dysfunction than other tissues. Mitochondrial fusion and fission are essential for normal morphology and function. Mitofusins Mfn1 and Mfn2 are the key components to process mitochondrial fusion. Previous data shows that Mfn1 and Mfn2 are functional redundant in skeletal muscle and the drosophila heart tube. But the role for the mitochondrial fusion proteins in mammalian cardiac myocytes is unknown. The functional role of mitofusins in the mammalian heart was investigated by conditional gene deletion of Mfn1 and Mfn2 in mice. Mice with myh6Cre-directed inactivation of Mfn1 exhibited normal cardiac structure and function. In contrast, ablation of Mfn2 in mouse heart caused progressive dilated cardiomyopathy. At 6 weeks old, the cardiac structure and function were normal, but at 16 weeks old, Mfn2 null mice developed severe cardiac dilation and impaired contractile performance at baseline and in response to β-adrenergic stimulation without induction of programmed cell death. Cardiomyocytes from young Mfn2 null mice had normal excitation-contraction coupling, whereas older cardiomyocytes showed decreased fractional shortening with normal calcium cycling. mRNA resequencing of Mfn2 null hearts revealed that cardiac dysfunction was associated with induction of nuclear-encoded mitochondrial genes. Mfn2 deficient cardiac mitochondria displayed normal function of the mitochondrial permeability transition, respiration and expression of respiration chain complexes, but increased mitochondrial oxidant stress. Remarkably, the development of cardiomyopathy paralleled dramatic (60-80%) loss of mitochondria, measured both as the ratio of mitochondrial DNA to nuclear DNA, and as μg protein/mg cardiac protein.

Conclusion:
1. Mfn1 and Mfn2 are not redundant in the heart.Mfn1 is dispensable for normal cardiac development and function. 2. Mfn2 deficiency reacts in a dilated cariomyopathy caused not by a failure of mitochondrial function, but by a crisis of mitochondrial biogenesis.
Abstract P232: Cardiac Excitation Contraction Coupling Stimulates Mitochondrial Respiration and Superoxide Flash Activity
Authors: Wang Wang , Liu Xiaoyun , Gong Guohua
Published: March 2022

Normal cardiac excitation-contraction (EC) coupling relies on highly coordinated intracellular calcium (Ca2+) handling and adequate mitochondrial energy metabolism. Whether mitochondria can sense the oscillating Ca2+ signals during EC coupling to adjust its respiration status and other functions is largely unknown. Recently, we discovered stochastic and transient mitochondrial superoxide production events, named superoxide flashes, in resting cardiac myocytes. Superoxide flashes are coupled with mitochondrial respiration and triggered by physiological relevant transient permeability transition pore (mPTP) openings. Using superoxide flash as the optical determination of mitochondrial respiration status in living myocytes, we found that EC coupling promotes mitochondrial respiration and reactive oxygen species (ROS) production as evidenced by the significantly increased superoxide flash activity by electrical stimulation (pacing frequency 0.5 - 2 Hz). Pre-incubation with Ru360 (50-100 uM), a mitochondrial Ca2+ uniporter inhibitor, or cyclosporine A (1 uM), an mPTP blocker, prevented this increase. Simultaneous monitoring of mitochondrial Ca2+ (by rhod-2) and superoxide (by mt-cpYFP, a mitochondrial targeted superoxide indicator) revealed step-wise accumulation of mitochondrial Ca2+ during cytosolic Ca2+ transients in individual mitochondria, which is followed by superoxide flash production and matrix Ca2+ release. Importantly, mitochondrial respiration only responds to transient Ca2+ signals (acute addition of ionophore or pacing) but not chronic manipulations of steady state Ca2+ (depleting or elevating resting cytosolic Ca2+). Through spatiotemporal mapping of superoxide flash and local sarcoplasmic reticulum (SR) Ca2+ releasing events, Ca2+ sparks, we found that significant amount of Ca2+ sparks clustered near the flashing mitochondria. Antioxidant pretreatment attenuated the superoxide flash associated Ca2+ sparks. In summary, mitochondrial Ca2+ uptake during cardiac EC coupling leads to stochastic acceleration of mitochondrial respiration, mPTP activity and free radical production, which in turn modulate local Ca2+ signaling through promoting SR Ca2+ release.
Abstract P242: Adenylate Kinase 2 Deficiency Disrupts Mitochondrial Energetics, AMP Metabolic Signaling, and Metabolomic Response to Myocardial Stress
Authors: Dzeja Petras , Zhang Song , Nemutlu Emirhan , Terzic Andre
Published: March 2022

Adenylate kinase 2 (AK2) isoform in mitochondrial intermembrane/intracristal space provides a conduit facilitating ATP export and delivery to cellular ATPases. AK2+/- transgenic mice is viable while homozygous AK2-/- deficiency results in midgestation embryonic lethality. AK2-/- mouse embryonic fibroblasts (MEFs) have severely disrupted mitochondrial cristae structure and display low growth and proliferation potential. Protein knockdown using siRNA indicates that AK2 is critical for cardiomyocyte mitochondrial biogenesis and network formation. Transgenic AK2+/- hearts display greater changes in AMP/ATP ratio and AMPK activation during ischemia compared to wild type suggesting deficient tuning of AMP signals. Metabolomic profile of AK2+/- hearts subjected to myocardial ischemia indicate lower ability to maintain adenine and guanine nucleotide pools. AK2+/- mice have abnormal metabolomic response to treadmill exercise compared to wild type. Metabolomic profiling revealed that most significant plasma metabolites critical in the discrimination between wild type and AK2+/- phenotypes were adenosine, tyrosine, tryptophan and hypoxanthine indicating lower energetic potential of AK2+/- mice. AK2+/- hearts have increased mitochondrial creatine kinase CKmit content which could be viewed as a compensatory mechanism. Thus, AK2 is the first phosphotransfer enzyme which deficiency causes embryonic lethality indicating critical significance of catalyzed nucleotide exchange and conductive transfer in the narrow and crowded mitochondrial intermembrane/intracristal space.
Abstract P247: APPL1 Transgenic Mice Are Protected from High-Fat Diet--Induced Lipotoxic Cardiomyopathy
Authors: Park Min , Park Tae-sik , Wu Donghai , Xu Aimin , Sweeney Gary
Published: March 2022

Increased circulating levels of free fatty acid (FA) in obesity and diabetes lead to the accumulation of FA in non-adipose tissue such as myocardium and this is known to be a major contributor to cardiomyopathy. In this present study, we investigated the role of APPL1 (Adaptor protein containing PH domain, PTB domain and Leucine zipper motif-1), an important regulator of insulin signaling, in a model of high fat diet (HFD) induced cardiomyopathy. We fed APPL1 transgenic (Tg) or wild type (wt) mice a high fat (60% kcal) or normal chow diet. After 16 weeks, in wt mice HFD induced hyperinsulinemia and hyperlipidemia, as well as cardiac dysfunction determined by echocardiography system (Vevo 2100). Furthermore, HFD induced cardiac insulin resistance and acetyl CoA carboxylase (ACC) phosphorylation. Lipid analysis using LC/MS/MS showed HFD significantly increased intracellular level of distinct ceramide and diacylglycerol (DAG) species in heart tissue. Interestingly, HFD fed APPL1 Tg mice showed a relatively normal circulating level of FA and improved insulin sensitivity in heart tissue, compared to the wt mice. Lipid analysis indicated that APPL1 Tg mice had approximately 20% reduced intra-myocardial accumulation of ceramides and DAGs after HFD compared to wt mice. APPL1 Tg mice also showed better cardiac performance in ejection fraction and cardiac output under these conditions. In summary, here we demonstrated that APPL1 Tg mice are protected from HFD-induced increases in circulating levels of FA, accumulation of lipotoxic fatty acid derivatives and insulin resistance in myocardial tissue and deterioration in cardiac function.
Abstract P258: Effect of MicroRNA-223 on Hypoxic Pulmonary Hypertension
Authors: Shi Lei , Kumar Bhola , Fisslthaler Beate , Schermuly Ralph T , Fleming Ingrid
Published: March 2022

Chronic hypoxia plays a key role in pulmonary hypertension and remodeling by inducing marked changes in gene expression. However, the regulatory effect of chronic hypoxia on microRNA (miR) expression in the lung has not yet been investigated. The purpose of this study was to determine which miRs are regulated by chronic hypoxia in the mouse lung and to determine their functional significance. We identified miRs altered in mouse lung after 3 weeks chronic hypoxia (10% O
2
(10 in 100) ) treatment by Microarray screening, verified miR target genes by reporter gene assays and Western blotting. Furthermore, we analyzed
in vitro
and
in vivo
functional significance for mouse pulmonary vascular artery smooth muscle cells. Among the miRs regulated, miR-223 was the most significantly decreased, a phenomenon verified by RT-qPCR. The insulin-like growth factor 1 receptor (IGF1R) is a known target of miR-223 and the binding of miR-223 to the 3’UTR of IGF1R was demonstrated. We found that IGF1R protein levels (but not RNA) were increased in lungs from hypoxic mice via a mechanism involving HIF1α/2α and reduced C/EBPα expression and transcription activity. Moreover, the expression of IGF1R was decreased following introduction of pre-mir-223 into pulmonary artery smooth muscle cells, an phenomenon coupled to attenuated IGF1-induced Akt phosphorylation, cell migration and proliferation. Decreasing endogenous miR-223 levels using a specific antagomir (3 weeks), increased pulmonary artery pressure and vessel muscularization. A similar phenomenon was observed in miR-223
-/y
mice, which also demonstrated a more severe response to hypoxia than wild-type mice. In lungs from primary pulmonary hypertension (PPH) patients we also detected a decrease in miR-223 and increase in IGF1R expression. These data indicate that the downregulation of miR-223 may contribute to the IGF1-induced pulmonary smooth muscle migration by regulating the expression of its receptor and thus contributing to pulmonary remodeling and the development of pulmonary hypertension.
Abstract P259: Functional Characterization of an ALOX15 Promoter Variant Associated with Coronary Artery Calcification: Regulation of ALOX15 by Vimentin
Authors: Samanta Susmita , Anderson Kurtis , Moran Sean , Gorenstein David , Fornage Myriam
Published: March 2022

Human 12/15-lipoxygenase (ALOX15) catalyzes the oxidation of polyunsaturated fatty acids and has been implicated in the pathogenesis of atherosclerosis. We previously reported that a common haplotype of the ALOX15 gene is associated with higher prevalence of coronary artery calcification in a cohort of middle-aged African-Americans. This haplotype was uniquely tagged by a promoter variant (rs2255888). We carried out an in vitro characterization of this promoter variant to further investigate regulatory mechanisms of the ALOX15 gene. We evaluated the activity of ALOX15 variant A-carrying and wild type G-carrying promoter haplotypes using a luciferase assay. We demonstrated a 65% higher activity of the A-carrying promoter haplotype as compared to the G-carrying promoter haplotype. Using mass-spectrometry and electrophoretic mobility shift assay we showed that vimentin, a structural protein, specifically binds to both A-carrying and G-carrying promoter haplotypes in vitro. However, the A allele-carrying promoter haplotype had an 83% increased in vitro binding of vimentin compared to the G allele-carrying promoter haplotype. In vivo binding of vimentin to this particular promoter region has also been detected by chromatin immunoprecipitation. Using non-denaturing polyacrylamide gel electrophoresis and UV thermal melting we showed evidence of the formation of intramolecular G-quadruplex structure in the DNA sequence of the G-carrying promoter haplotype. However, for the A-carrying promoter haplotype, formation of intramolecular G-quadruplex structure was reduced and intermolecular G-quadruplex was increased. We also observed that vimentin increased ALOX15 gene expression. The current studies indicate that sequence variant of the rs2255888 polymorphism is associated with a structural change of ALOX15 promoter region. This change may be responsible for the increased binding of vimentin to the promoter region. Our study is the first to show that vimentin may act as a transcription regulator of ALOX15. It has been reported that vimentin is secreted by activated macrophages. Thus, the novel function identified here may be relevant to ALOX15 pathogenesis mechanisms in atherosclerosis.
Abstract P262: Receptor-Ligand Functional Interaction Between CD44 and Osteopontin During Calcific Aortic Stenosis: A Molecular Mechanism for Accelerated Biomineralization of Bicuspid Aortic Valve
Authors: Ferrari Giovanni , Grau Juan B , Sainger Rachana , Poggio Paolo , Field Benjamin C , Branchetti Emanuela , Seefried William F , Bavaria Joseph E
Published: March 2022

Introduction:
Calcific aortic valve degeneration (CAVD) is the most common indication for surgical valve replacement in the US. The calcification starts with a Tricuspid Aortic Valve (TAV) or with a Bicuspid Aortic Valve (BAV). Initial phases of the disease, include thickening of the valve, a condition known as aortic valve sclerosis (AVSc), whereas advanced stages, aortic valve stenosis (AVS), are associated with leaflets biomineralization. The biomineralization process, occurring in an accelerated manner in BAV, has not been described in its molecular mechanisms. We reported that Osteopontin (OPN) is implicated in the calcification of dystrophic and ectopic sites. Here we investigate the interaction between OPN, and one of its functional regulators, CD44, during valve degeneration in TAV and BAV patients.

Methods:
Five groups of 10 subjects each (TAV: control, AVSc, AVS; BAV: control and AVS) were selected from our tissue biorepository according to echocardiographic analysis, inclusion/exclusion criteria, and/or intra-op observations. Histological studies, cellular and molecular analysis, and in situ proximity-ligation-assay (PLA) on surgically resected tissues were used to investigate the functional interaction between OPN and CD44.

Results:
1) In vitro calcification assays shows that OPN protects against calcification through CD44 interaction. 2) OPN and CD44 levels are elevated in the leafelts of BAV patients, both stenotic patients and controls. In TAV, AVSc and AVS patients shows elevated levels of OPN and CD44 when compared to controls. 3) In situ PLA assay displayed a direct interaction between CD44 and OPN in BAV patients, in both stenotic patients and controls, and in TAV AVSc and AVS patients. Healthy, non-calcified TAV subjects, shows barely detectible OPN-CD44 interaction.

Conclusion:
OPN-CD44 direct interaction correlate with the severity of biomineralization in TAV patients. Conversely, in BAV we demonstrate a direct interaction between OPN and CD44 in both stenotic and non-calcified tissues. These results are aims to elucidate the mechanisms behind the accelerated biomineralization of BAV compared to TAV, and generate important insights into the development of diagnostic and therapeutic approached for CAVD.
Abstract P263: Mechanism for Reductive Stress in Mutant Protein Aggregation Cardiomyopathy
Authors: Namakkal Soorappan Rajasekaran , Khanderao Gayatri D , Varadharaj Saradhadevi , Miller Corey J , Kannan Sankarnarayanan , Ramachandran Kalavathy , Olson Curtis , Davidson Christopher J , Zweier Jay L , Litwin Sheldon E , Benjamin Ivor J
Published: March 2022

Background:
We recently demonstrated that the hR120GCryAB associated with protein aggregation cardiomyopathy (PAC) causes reductive stress (RS) linked to dysregulation of G6PD in mice (Rajasekaran et.al Cell, 2007). Nrf2 (nuclear erythroid related factor -2) is the master transcription factor that regulates induction of genes encoding antioxidant responsive element (ARE) dependent pathways. However, the temporal effects of reductive stress on the molecular mechanisms of transcriptional regulation of Nrf2 - Keap1 pathway are unknown.

Methods:
Age-matched control and mutant TG mice at 3 and 6 months with RS were used to assess activation and nuclear translocation of Nrf2 and ARE-dependent transcriptional mechanisms. Electron paramagnetic resonance (EPR) analysis was used to measure the ROS and RNS in heart tissues.

Results:
Nuclear translocation of Nrf2 was significantly increased in compensated hR120GCryAB transgenic mice at 3 months compared with controls. EPR analysis revealed significantly increased ROS (superoxide, hydrogen peroxide and nitroxide) in 3-month old hR120GCryAB TG compared with controls. In contrast, hR120GCryAB-TG mice at 6-months exhibiting cardiac hypertrophy and RS (i.e., increased GSH/GSSG ratio) had attenuated ROS/RNS levels that were indistinguishable from age-matched controls.

Conclusions:
Oxidative stress triggers the activation of redox sensitive Nrf2-Keap1 pathway, which might be targeted therapeutically to prevent pathogenic transition and reductive stress-induced hR120GCryAB cardiomyopathy and heart failure.
Abstract P275: Time-of-Day--Dependent Variation in Myocardial Ischemia/Reperfusion Injury Is Lost in Mice Lacking the Regulator of Calcineurin 1 Gene
Authors: Grinsfelder D B , Rotter David , Sachan Nita , Rothermel Beverly A
Published: March 2022

Many important components of the cardiovascular system display circadian rhythmicity. In humans, the incidence of myocardial infarction peaks in the morning. Furthermore, studies in mouse models have demonstrated that damage from ischemia/reperfusion (I/R) occurring during the transition to waking is greater than at the end of the active period. In the hearts of wild type mice we have observed large circadian oscillations in the transcript and protein levels of the exon 4 isoform of the calcineurin regulatory protein (
Rcan1.4
). This 15 to 20-fold oscillation peaks around circadian time 0 (CT:0) when mice enter a period of rest. Using an
in vivo
murine model of myocardial I/R injury we subjected wild type and
Rcan1
KO mice to 45 minutes of myocardial ischemia followed by reperfusion for 24 hrs. Mice were housed in hermetic light boxes with opposing light cycles such that the surgeon was blinded as to the light entrainment of individual mice. I/R was performed at the beginning (CT:0 to CT:2) and the end (CT:10 to CT:12) of the mouse's light entrainment cycle. Twenty-four hours after reperfusion Evan's Blue and 2,3,5-Triphenyltetrazolium chloride staining were used to quantify the area-at-risk per left ventricle (AAR/LV) and the extent of myocardial infarction per area-at-risk (INF/AAR). Serum troponin-I levels were quantified as an indication of muscle damage. AAR/LV was the same in all animals, however, I/R injury in wild-type mice at CT:12 was 30% higher than in mice subjected to I/R at CT:0. Plasma troponin-I levels were also higher. Remarkably, there was no circadian difference in the extent of I/R injury or troponin-I levels in the
Rcan1 KO
mice. Damage in the
Rcan1 KO
mice at both time points was comparable to the level of damage observed in wild type mice at CT:12, when
Rcan1.4
expression is low. Importantly, expression of all central clock genes remained circadian in the hearts of
Rcan1 KO
mice suggesting that circadian changes in the vulnerability of the myocardium to I/R damage is directly dependent upon
Rcan1
function.
Abstract P284: Inhibition of the Rheb/mTORC1 Pathway During Prolonged Ischemia Is Protective Through Autophagy Activation in Cardiomyocytes
Authors: Sciarretta Sebastiano , Zhai Peiyong , Sadoshima Junichi
Published: March 2022

The mTOR complex 1 (mTORC1) mediates the cellular response to energy stress. However, the role of mTORC1 signaling in mediating cardiomyocyte (CM) tolerance of nutrient starvation and ischemia remains unclear. mTORC1 activity, as evaluated by the phosphorylation status of p70S6K and 4E-BP1, was decreased in CMs during glucose deprivation (GD)
in vitro
, and was accompanied by decreases in the activity of Rheb, a positive regulator of mTORC1 (31.7% reduction in Rheb-bound GTP, p<0.05). Exogenous Rheb was sufficient to restore mTORC1 activity, suggesting that Rheb regulates the activity of mTOR during GD. Rheb overexpression reduced CM survival during GD (42.6% reduction at 18 hours, p<0.01), whereas knock-down of Rheb increased CM survival (158.6% increase at 18 hours, p<0.05). Rheb overexpression significantly inhibited autophagy, reduced expression of autophagic genes, including Atg7, reduced the ATP content, and increased ER stress in CMs during GD. Under these conditions, restoration of autophagy through Atg7 overexpression or trehalose inhibited cell death, increased ATP content, and reduced ER stress. The activity of Rheb/mTORC1 is also inhibited during myocardial ischemia
in vivo
. After 3 hours of ischemia, transgenic mice with cardiac-specific and inducible Rheb overexpression (Tg-Rheb) exhibited a larger myocardial infarction (MI) (59.7% vs. 37.8%, p<0.05) and a significant reduction in autophagy compared to control mice, both of which were reversed by rapamycin, a selective mTORC1 inhibitor. Interestingly, both Rheb (+135% in Rheb-bound GTP, p<0.05) and mTORC1 (+158% in phospho-p70S6K, p<0.05) were activated in the hearts of mice with high fat diet-induced obesity. Prolonged ischemia induced less autophagy and a larger MI (60.6% vs. 37.2%, p<0.05) in the obese mice, effects which were reversed in the presence of rapamycin. However, rapamycin failed to protect the obese mice in the presence of genetic downregulation of Beclin 1 (
beclin1
+/− mice fed with high fat diet). In summary, inhibition of the Rheb/mTORC1 pathway during energy deprivation is protective through activation of autophagy. Rheb and mTORC1 represent potential therapeutic targets to reduce myocardial damage during prolonged myocardial ischemia in obese patients. .
Abstract P285: Impaired Recovery of Left Ventricular Function After Acute Myocardial Reperfusion Injury Is Rescued by the Anti-inflammatory Peptide Annexin-A1 ex Vivo
Authors: Ritchie Rebecca H , Buxton Keith , Pepe Salvatore , Cao Anh H , Venardos Kylie , Love Jane , Yang Yuan , Morand Eric , Kaye David
Published: March 2022

The glucocorticoid-regulated protein annexin-A1 (ANX-A1) is a key effector molecule of anti-inflammatory glucocorticoid actions. We have shown that the N-terminal derived peptide ANX-A1(2–26) preserves cardiomyocyte viability after metabolic inhibition in vitro. Our hypothesis was that ANX-A1 preserves the myocardium post-ischemic injury. ANX-A1(2–26)(0.3μM) prevented adult rat cardiomyocyte injury, whether present for hypoxia-reoxygenation (LDH release reduced from 4.1±0.7- to 1.1±0.2-fold n=11 P<0.001) or only on reoxygenation (LDH release reduced to 1.4±0.4-fold n=11, P<0.001). Similar protection at both time points was also evident on cardiomyocyte viability (trypan blue exclusion). The recovery of post-ischemic left ventricular (LV) function in intact rodent hearts was also protected. After 20mins reperfusion, the recovery of LV developed pressure (LVDP) remained significantly impaired in untreated rat hearts (47±5% baseline, n=11) whereas addition of ANX-A1(2–26)(0.3μM) on reperfusion improved LVDP recovery (to 66±7% baseline, n=13, p<0.05). Phosphorylation of both Akt (1.9±0.2-fold, P<0.05) and phospholamban (3.4±0.4-fold, P<0.05) was also enhanced by ANX-A1(2–26). Similar ANX-A1(2–26)-induced rescue of LV function was observed in mouse hearts after acute myocardial reperfusion injury (57±7% of baseline, n=11 untreated hearts vs 78±8% baseline, n=6 ANX-A1(2–26)-treated hearts, p<0.05). ANX-A1(2–26) cardioprotection was associated with attenuated cardiac enzyme release (LDH, CK) and Akt phosphorylation (3.1±0.8-fold, P<0.005). In contrast, deficiency of endogenous ANX-A1 further exacerbated recovery of post-ischemic LV function, across LVDP (to 29±4% baseline, P<0.001), LV end-diastolic pressure (to 5.5±1.0-fold baseline, P<0.001), LV+dP/dt (to 33±4% baseline, P<0.001), LV-dP/dt (to 36±4% baseline, P<0.001) and rate-pressure product (to 24±4% baseline, P<0.001). This is the first evidence that ANX-A1 or its mimetic ANX-A1(2-26) preserve post-ischemic recovery of LV function. ANX-A1 is thus an endogenous regulator of LV function. Furthermore, ANX-A1-based therapies may thus represent a novel clinical approach for the prevention and treatment of reperfusion injury.
Abstract P298: Probucol Inhibits Oxidized Low-Density Lipoprotein--Induced Immune Maturation of Dendritic Cells via Heme Oxygenase 1
Authors: Sun Aijun , Jin Xueting , Wang Keqiang , Zou Yunzeng , Ge Junbo
Published: March 2022

The anti-atherosclerotic mechanism of Probucol remains to be elucidated. Studies confirmed the key role of Dendritic Cells (DCs) and cytoprotective role of heme oxygenase 1(HO1) in the development of atherosclerosis. Here, we tested the hypothesis that probucol exerts is anti-atherosclerotic effect by inhibiting maturation of DCs via HO1.

[Method]Immature DCs were derived from purified human monocytes; HO1 siRNA, probucol ,ox-LDL or LDL was added to different groups of medium. The maturation markers of DCs were analyzed by Flow cytometric analysis, including CD1a, CD40, CD86, HLA-DR, endocytosis function and the cytokines secretions of culture supernatants (IL-4,TNF-α) by ELISA. The expression of HO1, STAT1 (signal transducers and activators of transcription 1) and STAT1(Tyr 701) phosphorylation were analyzed by Westernblot and the expression of CIITA(MHC II transactivator) by Real Time PCR.

[Results] Ox-LDL promoted immune maturation of DCs : MIF of CD86,CD 40, CD1a and HLA-DR was significantly increased after ox-LDL stimulation (7697±672 vs 2120±662, 7844±754 vs 843±109 ,323±31 vs 272±14,2636±173 vs 1443±45,respectively); decreased endocytosis function (26256±354 vs 34511±3078); increased TNF-α(43±5 vs 30±0.3 pg/ml), suppressed IL-4 (852±9 vs 793±11 pg/ml)2. Probucol inhibited immune maturation of DCs ,which was most obviously in ox-LDL treated group : MIF of CD 86 CD 40, CD1a and HLA-DR was significantly decreased after probucol treatment (7697±672 vs 3202±660,7844±754 vs 843±109,323±31 vs 56±31, 2636±173 vs 304±60), endocytosis (26256±1689 vs 109568±4195), TNF-α(43±5 vs 14±0.6 pg/ml), IL-4(793±11 vs 1037±17 pg/ml) (P<0.05). 3. After silencing of the expression of HO-1 by siRNA, immune maturation markers of DCs were increased than respective non-silencing group ,such as CD86, CD 40, HLA-DR (549±19 vs 353±23, 2645±307vs 524±119,1179±66 vs 766±23,respectivly), endocytosis function were suppressed (7408±229 vs 10860±706), TNF-αwas increased (22±0.2 vs 14±0.6 pg/ml), IL-4 was decreased (926±2 vs 1037±17 pg/ml) (P<0.05), STAT1(701) phosphorylation and CIITA expression level were increased.

[Conclusion]Probucol significantly inhibited immune maturation of DCs and is associated with HO1, STAT1/CIITA signaling pathway.
Abstract P301: Investigation of the Mechanism of the Cardioprotective Effects of Flavonols in Langendorff Perfused Rat Hearts
Authors: Woodman Owen L , Qin Cheng X , Williams Spencer J
Published: March 2022

We investigated the mechanism(s) of flavonol-induced cardioprotection using a flavonol which inhibits calcium utilization and has antioxidant activity 3’,4’-dihydroxylflavonol, a flavonol which only affects calcium activity 4’-OH-3’-OCH3 flavonol and a water-soluble flavonol with selective antioxidant activity DiOHF-6-succinamic acid in rat isolated hearts. Hearts were perfused with physiological solution using a Langendorff apparatus and subjected to global, no-flow 20-min ischaemia followed by 30-min reperfusion. Hearts (n = 6–8 per group) were randomly treated with vehicle (0.05% DMSO), DiOHF, 4’-OH-3’-OCH3 flavonol or DiOHF-6-succinamic acid (all 10 µM). Flavonols were included in the perfusate for 10 min before ischaemia and during the entire duration of reperfusion. In vehicle treated hearts, LV+dP/dt was significantly reduced at the end of reperfusion (60±8% decrease in LV+dP/dt) compared to the pre-ischaemic level. Furthermore, LDH release (895±121 U/L) was significantly elevated during reperfusion compared to shams (70±10 U/L) and total eNOS expression was significantly lower in vehicle-treated hearts. In comparison, DiOHF treatment significantly improved LV function upon reperfusion (35±6% reduction in LV+dP/dt), significantly decreased the level of LDH (265±66 U/L) and preserved total eNOS expression. Hearts treated with the antioxidant DiOHF-6-succinamic acid had a similar preservation of contractility, resulting in a 36±5% reduction in LV+dP/dt, and a significantly lower release of LDH (388±57 U/L) and preserved total eNOS expression. In contrast, hearts treated with 4’-OH-3’-OCH3 showed similar impairment of contractility to the vehicle group. DiOHF-6-succinamic acid also exerted cardioprotection when given only during reperfusion but not when administered only prior to ischaemia. This study demonstrated that flavonol-induced cardioprotection relies on the antioxidant activity and is mainly exerted during the reperfusion phase. The water-soluble DiOHF-6-succinamic acid with selective antioxidant activity may lead to the development of a new cardioprotective agent effective when applied at reperfusion and therefore appropriate for clinical use.
Abstract P302: CaMKII Is Not Critical for Ischemia/Reperfusion Injury
Authors: Weinreuter Martin , Lehmann Lorenz H , Kreußer Michael , Gröne Hermann-Josef , Müller Oliver J , Olson Eric N , Katus Hugo A , Backs Johannes
Published: March 2022

Objectives:
The role of the Calcium/calmodulin-dependent protein kinase II (CaMKII) in Ischemia/Reperfusion of the heart (I/R) has not completely been clarified yet - partly due to lacking evidence from animal studies with specific gene knockout (KO) strategies. Moreover, the contribution of the nuclear and cytosolic CaMKIIδ splice variants δB and δC to I/R-induced myocardial damage have been controversially discussed. To investigate the role of CaMKII in myocardial I/R we used several KO mice with genetic deletion of the two major isoforms expressed in the heart, CaMKIIδ and CaMKIIγ, respectively. Both single and double KO models were examined. In addition, we developed a reliable approach to express CaMKII δB and δC splice variants in CaMKIIδ-null mice using adeno-associated-virus serotype 9 (AAV9) to investigate their specific roles in I/R.

Methods and Results:
A surgical in vivo I/R model was used following two protocols: 30 minutes ischemia with 48 hours of subsequent reperfusion (30/48) and 60 minutes ischemia followed by a 24 hour reperfusion period (60/24). Planimetric measurements of infarct size after staining with Evans Blue and Triphenyl tetrazolium chloride showed no significant difference between CaMKII KO and control groups (infarct-zone/area-at-risk ratio for 30/48-protocol: CaMKIIγ
−/−
vs. C57/Bl6: 0.49 ± 0.04 vs. 0.43 ± 0.05, p=0.43, n≥6; CaMKIIδ
−/−
vs. C57/Bl6: 0.42 ± 0.04 vs. 0.44 ± 0.04, p=0.69, n≥19; infarct-zone/area-atrisk ratio for 60/24-protocol: CaMKIIδ
−/−
vs. C57/Bl6: 0.52 ± 0.02 vs. 0.56 ± 0.03, p=0.33, n≥8; CaMKIIγ
loxP/loxP
δ
loxP/loxP
; alphaMHC-Cre vs. CaMKIIγ
loxP/loxP
δ
loxP/loxP
: 0.52 ± 0.03 vs. 0.52 ± 0.04, p=0.86, n≥9). Consistently, no differences in high-sensitive serum troponin T levels 24 hours after onset of ischemia could be detected. Neither histologic TUNEL-staining nor Caspase3/7-acivity assays revealed any significant differences in apoptotic cell death processes. Importantly, specific expression of CaMKII δB or δC splice variants via AAV9 gene transfer - at approximate endogenous protein levels - in CaMKIIδ
−/−
mice had no effect on infarct size and necrotic or apoptotic markers.

Conclusion:
Our findings indicate that CaMKII is not critical for myocardial infarct size and apoptosis in response to I/R.
Abstract P304: Salvianolic Acid B Suppresses Human Monocyte-Derived Dendritic Cell Maturation via PPARγ Activation
Authors: Sun Aijun , Liu Hongying , Wang Shijun , Shi Dazhuo , Xu Lei , Cheng Yong , Wang Keqiang , Chen Keji , Zou Yunzeng , Ge Gunbo
Published: March 2022

Background and purpose: Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing human monocyte-derived dendritic cells (DCs) maturation.

Experimental approach: DCs was derived by incubating purified human monocytes with GM-CSF and IL-4. DCs was pre-incubated with or without Sal B and stimulated by oxidized low density lipoprotein (ox-LDL) in the presence or absence of PPAR-γ siRNA. Expression of DCs membrane molecules were analyzed by FACS, cytokines were measured by ELISA, and TLR4 associated signaling pathway was determined by Western blotting. Key results: Ox-LDL promoted DCs maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production and upregulated TLR4 signaling. These effects could be significantly inhibited by Sal B. Sal B also triggered PPAR-γ activation and promoted PPAR-γ nuclear translocation, attenuated ox-LDL-induced upregulation of TLR4 and myeloid differentiation primary-response protein 88 (MyD88), and inhibited downstream p38-MAPK signaling cascade. Knocking down PPAR-γ through siRNA transfection significantly blocked above mentioned effects of Sal B. Conclusions and implications: Our data suggested that Sal B effectively suppressed ox-LDL-induced DCs maturation through PPAR-γ activation.
Abstract P307: A Novel Protective Effect of the Antidiabetic Drug Voglibose via GLP-1 Receptor in the Infarcted Heart
Authors: Iwasa Masamitsu , Yamada Yoshihisa , Yasuda Shinji , Kobayashi Hiroyuki , Ushikoshi Hiroaki , Aoyama Takuma , Nishigaki Kazuhiko , Takemura Genzou , Fujiwara Takako , Fujiwara Hisayoshi , Minatoguchi Shinya
Published: March 2022

Glucagon-like peptide 1 (GLP-1) reportedly exerts a protective effect against cardiac ischemia. We hypothesized that the α-glucosidase inhibitor voglibose, an unabsorbable antidiabetic drug with cardioprotective effects, may act via stimulation of GLP-1 receptors. The results of the present study suggest oral administration of voglibose reduces myocardial infarct size and mitigates cardiac dysfunction in rabbits following 30 min of coronary occlusion and 48 h of reperfusion. Voglibose increased basal and postprandial plasma GLP-1 levels and reduced postprandial plasma glucose levels. The infarct size-reducing effect of voglibose was abolished by treatment with exendin(9–39), wortmannin, L-NAME or 5-HD, which inhibit GLP-1 receptors, PI3K, NOS and K
ATP
channels, respectively. Western blot analysis showed that treatment with voglibose upregulated myocardial expression of phospho-Akt, phospho-eNOS, phospho-ERK and HSP27 following myocardial infarction. The upregulation of phospho-Akt was inhibited by exendin(9–39) and wortmannin, while the upregulation of phospho-ERK and HSP27 was inhibited by wortmannin but not by exendin(9–39). These findings suggest that voglibose reduces myocardial infarct size through stimulation of GLP-1 receptors, activation of PI3K-Akt-eNOS and ERK pathways, and the opening of mitochondrial K
ATP
channels. These findings may provide new insight into therapeutic strategies for the treatment of diabetic patients with coronary artery disease.
Abstract P308: Detection of Transient Regional Myocardial Ischemia with 80-Electrode Body Surface Delta Map and Its Comparison with Myocardial Perfusion Imaging and 12-Lead ECG
Authors: Zeb Mehmood , Garty Florence , Banister Wendy , Nagaraj Nirmala , Curzen Nick
Published: March 2022

Background
The diagnosis of transient regional myocardial ischaemia (TRMI) in patients presenting to the Chest Pain Clinics is a challenge. Traditionally EET has been most commonly employed test for diagnosing TRMI, however due to the limitations of 12-Lead ECG this is no longer recommended. Thus the clinicians have to rely on more expensive and time consuming test including Myocardial Perfusion Imaging (MPI) and Cardiac MRI. Delta Map was described by this group and is an intuitive colour display of digitally subtracted ST segment shift derived from two 80-electrode BSM recordings, one at baseline and one at peak stress.

Objectives
The purpose of this pilot study was to asses the diagnostic ability of Delta-Map & 12-Lead ECG in detection of TRMI, and to compare the results with MPI.

Method
Forty consecutive patients who were referred by two consultant cardiologist for MPI with a history of angina-like symptoms were consented and recruited in to this study. The Delta Map was derived from two 80-electrode PRIME BSM carried out simultaneously with MPI at rest and peak stress, using dobutamine.12-Lead ECGs were also recorded at the same time.

Results
The mean age was 68±7.1, 50% (20/20) were female, 70% (28/40) hyperlipidaemic, 64% (25/40) had history of hypertension and 60% (24/40) were smoker, 27% (11/40) patients had previous PCI. Using MPI as the reference, PRIME Delta Map was more sensitive for the detection of TRMI than the 12-Lead ECG 77% vs. 15%, p 0.0001 and was also more specific for TRMI 96% vs. 84%, p 0.0001. The PPV & NPV for Delta Map was 91% & 89% respectively, where as 33.3% & 66.7% for 12-Lead ECG.

Conclusion
The PRIME ECG Delta map is a near patient, rapid, accurate, objective and intuitive method for detecting TRMI and is superior to the 12-Lead ECG. The Delta Map could potentially facilitate more rapid identification and appropriate treatment in patients with coronary artery disease. A larger clinical study is now required.
Abstract P319: Protein Modulation in Mouse Heart Under Acute and Chronic Hypoxia
Authors: Gelfi Cecilia , Viganò Agnese , Vasso Michele , Capitanio Daniele , Leone Roberta , Caretti Anna , Samaja Michele
Published: March 2022

Exploring cellular mechanisms underlying beneficial adaptive processes and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1α), energy balance (AMPK), mitochondrial biogenesis, (PGC-1α) and the regulation of autophagic/apoptotic processes, combined with proteomic dysregulation were assessed. Responses to normobaric acute (48 hrs FiO
2
= 0.08) and chronic (10 days FiO
2
= 0.08) hypoxia in mouse heart proteome were detected applying 2-D DIGE, mass spectrometry and antigen antibody reactions. Both in acute (AH) and chronic (CH) hypoxia, results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH HIF-1α stabilization was detected. In AH, the metabolic adaptation to oxygen lack is controlled by AMPK activation and sustained by the massive up-regulation of adenosylhomocysteinase and of acetyl coenzyme A synthetase. AH is also characterized by an up-regulation of Bnip 3 which is known to induce mitophagy and decreasing oxidative damage. PGC-1α, a master regulator of mitochondrial biogenesis, was down- regulated. CH was characterized by up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a massive down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic signals were correlated with the increment of apoptotic program and sustained by the down-regulation of specific target proteins. In conclusion, differential analysis provided new insight into target molecules sensing lack of oxygen in vivo and could open new possibilities for protection from damages induced by long term hypoxia exposure.
Abstract P326: An Innovative Peptide Spectral Library Search Engine for Cardiovascular Proteomics
Authors: Li Haomin , Deng Ning , Zong Nobel , Zolyan Anna , Chan Melissa , Kim Jenny , Apweiler Rolf , Duan Huilong , Ping Peipei
Published: March 2022

We developed a peptide spectral library search engine for the cardiovascular community. Over 50,000,000 spectra obtained with LTQ-Orbitrap instrument on cardiac mitochondria and proteasome were analyzed, and 108,268 representative spectra were included in this organelle-based library. An improved dot product algorithm, slide dot product, was coded to query user spectra against spectra in the library. This procedure provides a solution to optimally balance the speed and sensitivity in peptide identification. In addition, an innovative noise decoy protocol was engineered to distinguish spectra correlation attributed to noise signals of the analytical instruments. The immediate advantages include an effective improvement of both specificity and accuracy in protein identification, overcoming a long-lasting concern in experimental spectra library, the possibility of propagating spectra with inaccuracy and error. With an independent test dataset collected by LTQ-Orbitrap (∼10,000 spectra), this novel search engine identified 20% more spectra matches within 10% analytical time compared to other existing theoretical database search route. Furthermore, the broad utility of this search engine was also demonstrated in characterizing spectra collected by LCQ and Q-TOF instrumentation. This peptide spectral library search engine has been incorporated into the Cardiac Organellar Peptide Atals Library (COPa Library) of NHLBI Proteomics Center at UCLA and will be made accessible at
www.HeartProteome.org
.
Abstract P335: Is GSK-3α a Regulator of Aging?
Authors: Zhou Jibin , Lal Hind , Shang Xiying , Vagnozzi Ronald , Farber John , DeCaul Morgan , Woodgett James R , Gao Erhe , Force Thomas
Published: March 2022

The glycogen synthase kinase-3 (GSK-3) family of protein kinases consists of two highly related isoforms, α and β. The GSK-3 family has been reported to regulate an astonishing variety of physiological and pathophysiological processes, whereas virtually nothing is known as for whether they regulate aging. Aging, defined as the progressive loss of function accompanied by decreasing fertility and increasing mortality with advancing age, is a complex biological process, controlled by multiple genetic, epigenetic and environmental factors. We now present the first studies defining the role of GSK-3α in the aging process using germline GSK-3α knockout (KO) mice at age of 3, 6, 12, and 24 mos. Age-matched wildtype (WT) mice served as control. Kaplan-Meier analysis shows that percent survivals (WT vs. KO) was 73.33 vs. 42.11
‡
at 24 mos of age.

In the heart we find cardiac hypertrophy, marked cell loss with replacement fibrosis, contractile dysfunction with profound abnormalities of mitochondrial structure, and impaired autophagy (assessed by Beclin1/ATG6), all becoming evident after 6 mos of age. Echocardiographic LV mass (in mg) is as follows (4 time points): 124.0±3.15 vs. 132.4±3.93, 136.2±4.75 vs. 171.5±7.57
‡
, 169.2±8.53 vs. 203.5±8.18
‡
, 195.4±9.82 vs. 257.4±7.24
‡
; ejection factor: 60.6±1.08 vs. 62.5±1.52, 64.3±1.10 vs. 60.7±1.61*, 58.5±1.74 vs. 52.2±1.41
‡
, 55.5±0.96 vs. 49.4±1.02
‡
; invasive hemodynamic dP/dt+: 6863.8±673.8 vs. 6999.4±473.2, 7115.4±392.9 vs. 6012.6±407.3*, 7509.6±419.6 vs. 4291.6±473.5*, 6889.0±460.9 vs. 3955.5±306.9
‡
. We also observed increased BrdU positive nuclei in isolated cardiomyocytes from the KO at 6 mos of age, and increased stem cells at every time-point, compared with age-matched WT. In the small intestine, we find marked senescence-associated β-galactosidase (SA-β-GAL) activity associated with thinning of the crypts, consistent with reduced cell cycling in this tissue. The results imply accelerated development of age-related pathologies in the KO heart and small bowel. These phenotypes are associated with a reduced lifespan in the mouse. Thus GSK-3α is a novel regulator of aging which retards age-related pathologies and prolongs lifespan of the organism. (*, P<0.05; ‡, P<0.01 KO vs. WT)
Abstract P340: The Role of Osteoclast-like Cells in Abdominal Aortic Aneurysm
Authors: Yamanouchi Dai , Stair Colin , Morgan Stephanie , Kent K C
Published: March 2022

Increasing evidence suggests that arterial calcification is the result of highly organized processes resembling those seen in bone which relies upon a delicate balance between mineral deposition and resorption by osteoblasts and osteoclasts, respectively. Osteoclast-like cells (OLCs) are derived from the monocyte/macrophage lineage and share osteoclast features such as the ability to dissolve extracellular matrix. Although osteoclastogenesis, the development of OLCs, has been reported to occur in calcified arteries, its potential role in the development of abdominal aortic aneurysm (AAA) has yet to be explored. Firstly, we obtained aortic tissues from patients undergoing surgical repair for AAA (N = 5) and aortic occlusive disease (N = 5). Alizarin-Red staining showed calcification in both aneurysmal and occlusive aorta. In contrast, OLCs, recognized as multi-nucleated cells positive for enzymatic TRAP staining, were identified only in aneurysmal aorta (100%, 5 of 5). A time course experiment in mouse calcium chloride-induced AAA (CaCl
2
AAA) showed similarities in both spatial and temporal patterns of calcium deposition and monocyte/macrophage accumulation starting 48 hours followed by OLCs formation peaked at 7 days after CaCl
2
injury. Secondly, we administered bisphosphonate (pamidronate disodium, 1.25 mg/kg, weekly), an inhibitor of osteoclasts, or normal saline as a control, intravenously to mice after CaCl
2
injury (N = 5). Bisphosphonate-treated mice showed a significant reduction in aortic dilation compared to control mice after 7 and 42 days with mean fold change of 1.16 ± 0.04 (vs. 1.65 ± 0.05, P < 0.01) and 1.37 ± 0.12 (vs. 2.17 ± 0.08, P<0.01) respectively. We also studied the effect of bisphosphonate on “developed aneurysm” by delaying the injection of bisphosphonate until one week after the injury to allow the aneurysm to develop prior to injection (N = 5). The mice with delayed bisphosphonate injection also showed significant inhibition compared to the saline control at 42 days (1.56 ± 0.13 vs. 2.42 ± 0.06, P < 0.01). In conclusion, we have demonstrated the existence of OLCs in AAA. We also have demonstrated the inhibitory effect of bisphosphonate on aneurysm. These results indicate a novel approach for the pathogenesis and therapy of AAA.