bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1178: Group X Secretory Phospholipase A
2
Negatively Regulates Adrenal Glucocorticoid Synthesis
Authors: Shridas Preetha , Bailey William , Oslund Rob C , Gelb Michael H , Webb Nancy R
Published: July 2021

Introduction:
Group X secretory phospholipase A
2
(GX sPLA
2
) has been implicated in a number of important biological processes, some of which appear to be independent of its lipolytic activity. These effects of GX sPLA
2
may be in part due to its high affinity binding to the M-type sPLA
2
receptor. To elucidate the physiological functions of this enzyme
in vivo
, we recently developed C57BL/6 mice with targeted deletion of GX sPLA
2
(GX KO mice).

Results
: Unexpectedly, GX KO mice have ~50% higher plasma corticosterone level compared to wild-type (WT) mice. Real-time RT-PCR and immunoblot analyses showed that expression of Steroidogenic Acute Regulatory Protein (StAR), a rate limiting protein in steroid production, is 2–5-fold higher in adrenals of GX KO mice compared to WT mice. GX KO mice responded normally to dexamethasone, indicating no impairment of the hypothalamic-pituitary axis. To provide additional evidence for a direct effect of Group X sPLA
2
on adrenal corticosteroid production, we performed studies in mouse adrenocortical tumor (Y-1) cells, which endogenously express GX sPLA
2
and the M-type receptor. Over-expression of GX sPLA
2
, or incubations with GX sPLA
2
, resulted in a significant reduction in progesterone production and StAR expression in Y-1 cells. Indoxam, an inhibitor of GX sPLA
2
activity and potent blocker of sPLA
2
binding to the M-type receptor, resulted in a significant increase in progesterone production and StAR expression in both control and GX sPLA
2
-overexpressing cells. Because several studies have implicated the extracellular signal-regulated kinase (ERK) cascade in the regulation of steroidogenesis, we investigated whether GX sPLA
2
altered adrenal ERK1/2 activation. Over-expression of GX sPLA
2
in Y-1 cells resulted in significantly increased ERK1/2 phosphorylation, whereas deficiency of GX sPLA
2
was accompanied by a significant decrease in phosphorylated ERK1/2 in mouse adrenals.

Conclusions:
We provide the novel finding that GX sPLA
2
has a direct effect on adrenal cells to regulate glucocorticoid production. Our
in vivo
and
in vitro
data indicate that Group X sPLA
2
down-regulates the expression of StAR, possibly through M-type receptor-mediated activation of ERK1/2.
Abstract 1184: Hdl Protects Macrophages From Oxidized Ldl-induced Apoptosis By Promoting Efflux Of 7-ketocholesterol Via Abcg1
Authors: TERASAKA NAOKI , Wang Nan , Yvan-Charvet Laurent , Tall Alan R
Published: July 2021

Oxidized sterols consumed in the diet or formed on low-density lipoprotein (LDL) are toxic to endothelial cells and macrophages and are thought to have a central role in promoting atherogenesis. The ATP-binding cassette transporter ABCG1 was recently shown to promote efflux of cholesterol from macrophages to HDL. We show that HDL protects macrophages from apoptosis induced by loading with free cholesterol or oxidized LDL (oxLDL). The protective effect of HDL was reduced in
Abcg1
−/−
macrophages, especially after loading with oxidized LDL. Similarly, HDL exerted a protective effect against apoptosis induced by 7-ketocholesterol, the major oxysterol present in oxLDL and atherosclerotic lesions, in
Abcg1
+/+
but not in
Abcg1
−/−
macrophages. In transfected 293 cells, efflux of 7-ketocholesterol was completely dependent on expression of ABCG1 and presence of HDL in media. In contrast, ABCA1 and apoA-1 did not stimulate efflux of 7-ketocholesterol into media. HDL stimulated efflux of 7-ketocholesterol from
Abcg1
+/+
but not from
Abcg1
−/−
macrophages, and
Abcg1
−/−
macrophages accumulated 7-ketocholesterol in mice fed the Western diet. The findings indicate a specific role of ABCG1 in promoting efflux of 7-ketocholesterol from macrophages onto HDL, and in protecting these cells from oxysterol-induced toxicity.
Abstract 1191: Elevated Concentration of Pentraxin 3 is Associated With Coronary Plaque Vulnerability
Authors: Soeki Takeshi , Niki Toshiyuki , Koshiba Kunihiko , Kusunose Kenya , Yamaguchi Koji , Kawano Tomohito , Yamada Hirotsugu , Wakatsuki Tetsuzo
Published: July 2021

Background:
Inflammation is a critical contributing factor to the development and the progression of atherosclerosis. Pentraxin 3 (PTX3), which has C-terminal sequence homology with the classic pentraxin C-reactive protein (CRP), is produced abundantly from atherosclerotic lesion while CRP is mainly produced in the liver. In this study, we investigated whether plasma level of PTX3 might be a sensitive marker for the severity of coronary artery disease by coronary angiography and vulnerable plaque by multidedector-row CT.

Methods:
We measured the concentration of PTX3 in peripheral and coronary sinus plasma of 36 patients with angina pectoris (AP) and 18 control subjects who had no coronary artery stenosis on angiography. Then, we determined the correlation between the plasma PTX3 concentration and the Gensini score as an index of severity of coronary atherosclerosis or the CT density of plaque as a quantitative index of plaque vulnerability. The plasma concentration of PTX3 was measured by enzyme-linked immunosorbent assay.

Results:
There was no significant difference of PTX3 concentrations in peripheral and coronary sinus plasma between patients with AP and control subjects (peripheral plasma, 2.6±1.1 vs 2.0±0.8 ng/ml; coronary sinus plasma, 2.8±1.2 vs 2.0±0.8 ng/ml). The concentration of PTX3 in coronary sinus but not peripheral plasma correlated with Gensini score (r=0.47, p<0.05 and r=0.34, NS, respectively). Interestingly, there was significantly negative correlation between plasma PTX3 concentration and the CT density (r=−0.71, p<0.01). On the other hand, plasma high-sensitive CRP concentration was not correlated with the CT density.

Conclusion:
PTX3 might reflect coronary plaque vulnerability that leads to plaque rupture more specifically than CRP.
Abstract 1207: In Vivo Molecular Imaging Links Macrophage Burden and Microcalcifications in Early Atherosclerosis
Authors: Aikawa Elena , Nahrendorf Matthias , Figueiredo Jose-Luiz , Swirski Filip K , Shtatland Timur , Kohler Rainer , Jaffer Farouc A , Aikawa Masanori , Weissleder Ralph
Published: July 2021

Backround
Microcalcifications may provoke plaque rupture leading to acute cardiovascular events. However, the mechanisms that initiate arterial calcification remain obscure.

Methods and Results
We tested the hypothesis that inflammation promotes osteogenesis in atherosclerotic plaques using
in vivo
molecular imaging in apoE
−/−
mice (n=40). A bisphosphonate-derivatized near-infrared fluorescent (NIRF) imaging agent (750 nm) visualized osteogenic activity that was otherwise undetectable by X-ray computed tomography. Flow cytometry validated target cells as osteoblasts. A spectrally distinct NIRF nanoparticle (680 nm) was coinjected to simultaneously image macrophages. Fluorescence reflectance mapping associated osteogenic activity with inflammation in aortas of apoE
−/−
mice. Intravital dual-channel fluorescent microscopy further monitored osteogenic changes in carotid plaques at 20 and 30 weeks of age and revealed that both macrophage burden and osteogenesis concomitantly increased during plaque progression and decreased after statin treatment

(Figure

)

. Fluorescence microscopy on cryosections colocalized NIRF osteogenic signals with alkaline phosphatase activity, bone-regulating proteins and hydroxyapatite nanocrystals detected by electron microscopy, while von Kossa staining showed no evidence of calcification. Real-time RT-PCR revealed that macrophage-conditioned media induced alkaline phosphatase mRNA expression in vascular smooth muscle cells.

Conclusion
This serial
in vivo
study detects osteogenic activity within macrophage-rich plaques, offering a cellular resolution tool to identify preclinical microcalcifications.
Abstract 1208: Deficiency of Interleukin-1 receptor antagonist in T cells promotes Aortic Valve Stenosis by releasing Tumor Necrosis Factor-α
Authors: Isoda Kikuo , Matsuki Taizo , Niida Tomiharu , Ishigami Norio , Kondo Harumi , Kusuhara Masatoshi , Iwakura Yoichiro , Ohsuzu Fumitaka
Published: July 2021

The cytokine interleukin (IL)-1 is an important mediator of inflammation and cardiovascular disease. Activity of this cytokine is modulated endogenously via IL-1 receptor antagonist (IL-1Ra). We have shown that IL-1Ra-deficient (IL-1Ra
−/−
) mice develop degenerative aortic valve stenosis (AS) that is the most common valvular disease in Western countries. The objective of this study is to determine the mechanisms by which deficiency of IL-1Ra may promote AS.

Method and Results
: IL-1Ra
−/−
mice (backcrossed 8 generations to the BALB/c background) and wild-type (WT) mice did not differ with regard to body weight, heart weight, and systolic arterial pressure. Furthermore, no significant differences in plasma lipid levels were observed between IL-1Ra
−/−
and WT mice. However histological analysis revealed that IL-1Ra
−/−
mice developed aortitis and showed increased aortic valve leaflet thickness compared to WT mice at the age of 12 weeks (p<0.001). Echo cardiograms also revealed that IL-1Ra
−/−
mice displayed higher transvalvular velocities than WT mice at the age of 40 weeks (p<0.01). These findings showed that IL-1Ra−/− mice spontaneously developed AS. In this study, we examined the role of T cells in development of AS by peripheral T cell transplantation. Transplantation of T cells from WT mice induced mild swelling of aortic valve leaflet in nu/nu mice. In contrast, T cells from IL-1Ra
−/−
mice induced an increase in aortic valve thickness in nu/nu mice. We next checked T cell function. After anti-CD3 antibody stimulation, IL-1Ra
−/−
T cells produced much higher levels of tumor necrosis factor (TNF)-α in culture supernatants compared to those from WT mice (p<0.001). Furthermore, in IL-1Ra
−/−
mice, TNF-α protein levels were higher than in WT mice (106.2±12.5 vs. 216.5±12.5 pg/mL, p<0.001) in vivo. Finally, we studied the roles of TNF-α in the development of AS in IL-1Ra
−/−
mice by generating double gene deficient (TNF-α
−/−
/
IL-1Ra
−/−
) mice. Interestingly, TNF-α
−/−
/IL-1Ra
−/−
mice did not develop AS.

Conclusions
: These findings suggest that IL-1Ra deficiency in T cells disrupts homeostasis of the immune system and TNF-α plays an important role in the development of AS in IL-1Ra
−/−
mice. These novel findings provide a clue for the development of new therapies for AS.
Abstract 1211: Hif-1α In T Cells Pathway Plays A Crucial Role In The Progression Of Arteriosclerosis And Artery Intimal Thickening
Authors: Kurobe Hirotsugu , Urata Masahisa , Izawa Yuki , Fukuhara Yayoi , Kanbara Tamotsu , Aihara Kenichi , Akaike Masashi , Gonzalez Frank , Matsumoto Toshio , Tamaki Toshiaki , Kitagawa Tetsuya , Yoshizumi Masanori , Tomita Shuhei
Published: July 2021

Background and Objective

T cell-mediated inflammatory process is involved in arteriosclerosis and vascular remodeling. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor and regulates the gene expressions in response to hypoxia in order to maintain physiological oxygen homeostasis. In the previous annual meeting, we showed that arterial cuff injury caused prominent neointimal and adventitial hyperplasia in Hif-1α-deficient mice compared to that of the control mice. The object of the present study is to reveal how the function of HIF-1α in T cells contributes to the vascular remodeling.

Methods and Results

T cell-specific Hif-1α-deficient mice were generated using a Cre-LoxP technology. Vascular remodeling was characterized 4 weeks after femoral arterial injury induced by an external vascular polyethylene cuff model. Morphological and histological studies showed that the cuff placement caused prominent neointimal and adventitial hyperplasia in Hif-1α-deficient mice compared to that of the control mice, and that infiltration of mononuclear cells at the adventitia was remarkably increased in the mutant mice. Hif-1α-deficient T cells were normally generated in the thymus and their distribution in the spleen and lymph nodes was unimpaired. However, number of peripheral Hif-1α-deficient T cells was significantly increased compared to the control. In the in vitro culture condition, Hif-1α-deficient T cells exhibited significant more increases of IL2 production and proliferation upon stimulation with anti-CD3/anti-CD28 antibodies compared to the controls. In addition, productions of 2,4,6-trinitrophenol (TNP)-KLH antigen-specific antibodies, including IgG1, IgG2b and IgG2c, were more enhanced in the mutant mice after stimulation of the antigen.

Conclusions

HIF-1α in T cells plays a crucial role in vascular inflammation and remodeling in response to cuff injury, serving as a negative regulator of T cell-mediated immune response.
Abstract 1213: Cardiac Overexpression of Epac1 in Transgenic Mice Protects Heart from Lipopolysaccharide-induced Cardiac Dysfunction and Inhibits JAK-STAT Pathway
Authors: Okumura Satoshi , Bai Yunzhe , Jin Meihua , Suzuki Sayaka , Kuwae Akiko , Kurotani Reiko , Ishikawa Yoshihiro
Published: July 2021

The sympathetic nervous system and proinflammatory cytokines are believed to play independent roles in the pathophysiology of heart failure. However, the recent identification of Epac (exchange protein activated by cyclic AMP), a new cyclic AMP-binding protein that directly activates Rap1, have implicated that there may be a potential cross talk between the sympathetic and cytokine signals. In order to examine the role of Epac in cytokine signal to regulate cardiac function, we have generated transgenic mice expressing the human Epac1 gene under the control of alpha-cardiac myosin heavy chain promoter (Epac1-TG), and examined their response in lipopolysaccharide (LPS)-induced cardiac dysfunction, a well established model for sepsis-induced cardiac dysfunction. Sepsis-induced cardiac dysfunction results from the production of proinflammatory cytokines. At baseline, left ventricular ejection fraction (LVEF) was similar (TG vs. NTG, 67±1.7 vs. 69±2.1%,
n
=7–9). The degree of cardiac hypertrophy (LV(mg)/tibia(mm)) was also similar at 3 months old (TG vs. NTG 4.0±0.1 vs. 4.2±0.1,
n
=5–6), but it became slightly but significantly greater in Epac1-TG at 5 month old (TG vs. NTG 4.9±0.1 vs. 4.4±0.1,
p<
0.05,
n
=5–7). LPS (5mg/kg) elicited a significant and robust reduction of LVEF in both Epac1-TG and NTG, but the magnitude of this decrease was much less in Epac1-TG at 6 hr after injection (TG vs. NTG 48±2.4 vs. 57±1.8%,
p<
0.01,
n
=6–9). At 24 hr after injection, cardiac function was restored to the baseline in both Epac1-TG and NTG. We also examined the activation of JAK-STAT pathway at 24 hr after injection. The tyrosine phosphorylation of STAT1 (Tyr701) and STAT3 (Tyr705) in LV, which is an indicator of STAT activation, was reduced to a greater degree in Epac1-TG by 31±8.8% (
p<
0.05,
n
=4) and 29±5.9% (
p<
0.05,
n
=7), respectively, relative to that in NTG. Taken together, Epac1 protects the heart from the cytokine-induced cardiac dysfunction, at least in part, through the inhibition of the JAK-STAT pathway, suggesting the beneficial role played by sympathetic signal to antagonize proinflammatory cytokine signal in heart failure.
Abstract 1224: Rock1 Deletion Prevents Cardiac Dilation And Improves Contractile Function In Pathological Cardiac Hypertrophy
Authors: Shi Jianjian , Zhang Yi-Wei , Dorn Gerald W , Wei Lei
Published: July 2021

The development of left ventricular cardiomyocyte hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response. However, persistent stress eventually leads to dilated heart failure, which is a common cause of heart failure in human hypertensive and valvular heart disease. We have recently reported that ROCK1 homozygous knockout mice exhibited reduced cardiac fibrosis and cardiomyocyte apoptosis, while displayed a preserved compensatory hypertrophic response to pressure overload. Here, we tested effects of ROCK1 deficiency on cardiac hypertrophy, dilation, and dysfunction by using the transgenic Gαq mice which represent a well-characterized and highly relevant genetic mouse model of pathological hypertrophy and heart failure. We have shown that ROCK1 deletion prevented left ventricular dilation and contractile dysfunction in Gαq mice under basal condition. ROCK1 deletion also partially rescued bradycardia and improved contractile response to β-adrenergic stimulation in Gαq mice. Although the development of cardiomyocyte hypertrophy was not affected, ROCK1 deletion in Gαq mice resulted in a concentric hypertrophic phenotype associated with reduced induction of hypertrophic markers. Finally, ROCK1 deletion prevented down-regulation of type V adenylyl cyclase expression, which is a critical molecular defect contributing to the impaired β-adrenergic signaling in Gαq mice. The present study establishes for the first time a role for ROCK1 in cardiac dilation and contractile dysfunction.
Abstract 227: Inhibition Of Protein Tyrosine Phosphatase Improves Hyperglycemic Impaired Angiogenesis
Authors: Chen Jian Xiong , Yu Heidi , Tuo Qinhui
Published: July 2021

Our previous study shows that hyperglycemia impairs myocardial angiogenesis via impairment of angiopoietin-1(Ang-1)/Akt/eNOS phosphorylation. Protein tyrosine phosphatase (PTP) plays an essential role in intracellular signal transduction by regulating tyrosine phosphorylation. PTP inhibitor has been shown to modulate Akt/eNOS phosphorylation, but little is known about the inhibition of PTP on angiogenesis under hyperglycemic conditions. We investigated the role of inhibition of PTP on hyperglycemia-induced impairment of angiogenic signaling pathways and angiogenesis. Our data demonstrate that exposure of mouse heart microvascular endothelial cells (MHMECs) to hyperglycemia leads to an increase in caspase-3 activity. Pretreatment of cells with the protein tyrosine phosphatase inhibitor, sodium orthovanadate (OV, 5 μM), significantly enhances the Ang-1-inhibitory effect on caspase-3 activity both under normal or hyperglycemic conditions. Pretreatment of MHMEC with the PTP inhibitor OV (0.5–20 μM) significantly enhances cell survival in a dose dependent manner under hyperglycemic conditions as measured by MTT assay. Pretreatment of MHMEC with OV (5 μM) enhances the effect of Ang-1 on endothelial cell survival under hyperglycemic conditions. As expected, Ang-1-induced Akt and eNOS phosphorylation is minimal under hyperglycemic conditions. Pretreatment of MHMEC with OV (5 μM) for 30 minutes leads to an increase in Akt phosphorylation at baseline and augments Ang-1-induced Akt and eNOS phosphorylation under hyperglycemic conditions. Additionally, systemic administration of bis(maltolato) oxovanadium(IV) (BMOV, 0.2g/L) in db/db mice results in a dramatic decrease in plasma glucose levels and an increase in vessel outgrowth in response to Ang-1 (250 ng/ml) stimulation. Treatment of db/db mice with BMOV significantly increases myocardial ischemia-induced HIF expression. Furthermore, bone marrow differentiation into endothelial cell progenitor cell is also significantly improved in the BMOV treated db/db mice. Our data implicate that upregulation of Ang-1/Akt/eNOS phosphorylation by PTP inhibitors should be considered as a new therapeutic strategy for the treatment or prevention of diabetic impaired angiogenesis.
Abstract 231: Cloning, Expression, Charecterization and Redox Regulation of Human DDAH-1: Implications in NO Regulation
Authors: Forbes Scott P , Druhan Lawrence , Pope Arthur , Cardounel Arturo
Published: July 2021

Altered NO biosynthesis is known to play a central role in the pathogenesis of endothelial dysfunction. It is hypothesized that reduced NO bioavailability may result from an increase in endogenous NOS inhibitors, ADMA and NMMA, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). DDAH hydrolyzes side-chain methylated L-arg and thus regulates the activity of NOS. To date, the few studies published on DDAH have been carried out using enzyme purified from pseudomonas or rat kidney homogenates. Herein we report the cloning, expression and kinetic properties of hDDAH-1 and its regulation by redox environment. Human DDAH-1 was cloned into an e. coli expression vector and recombinant hDDAH-1 purifed to > 95% purity. Kinetic studies from the enzyme demonstrated Km values of 18.2 and 27.1 μM and Vmax values of 303 and 182 nmols/mg/min for ADMA and L-NMMA, respectively. Oxidant exposure studies were performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity. Results demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching 1 mM were needed to confer significant enzyme inhibition. However, exposure of hDDAH-1 to lipid oxidation products, such as 4-HNE, dose dependently inhibited DDAH activity with 15% inhibition observed at 10 μM, 50% inhibition at 50 μM and near complete inhibition at 100 μM. These levels represent pathophysiological concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Proteomic analysis revealed that exposure of hDDAH-1 to 4-HNE results in Schiff base adduct formation of critical amino acid residues located within the enzyme active site. Thus, 4-HNE directly modifies hDDAH-1 with subsequent loss of enzyme activity. To determine whether this loss of DDAH activity results in NOS impairment, studies were performed using cultured endothelial cells. Results demonstrated that exposure of cells to 4-HNE (50 μM) inhibited eNOS derived NO production by 45% and reduced cellular DDAH activity by 41%. These results demonstrate a critical role for DDAH in the pathogenesis of endothelial dysfunction under conditions of oxidative stress.
Abstract 1235: Molecular Basis for Sympathetic Remodeling in the Left Atrium in the Ventricular Tachypacing Canine Model of Congestive Heart Failure
Authors: Belin Rashad J , Mottl Steve , Goldstein Jacob , Angulo Abigail , Salwan Ashima , Villuendas Roger , Aistrup Gary , Goldberger Jeffrey , Kadish Alan , Arora Rishi
Published: July 2021

Background:

The sympathetic nervous system (SNS) is thought to play an important role in promoting atrial fibrillation (AF) initiation and maintenance, especially in the setting of congestive heart failure (CHF). However, it is unclear as to which components of the SNS are altered in atria isolated from failing hearts. These studies are essential, as they will delineate potential molecular targets that may be probed for therapeutic intervention in order to reduce AF pathogenesis.

Methods

: CHF was induced by ventricular tachypacing (240 beats/minute) for 4 weeks in hound dogs. A total of 4 CHF and 5 control dogs were used. Immunoblot analysis was performed on left atrial appendage (LAA) homogenates to examine the protein expression of Gsα, Gi2α, and the catalytic subunit of protein kinase A (PKA). Catalytic activities of PKA and protein phosphatase type 2a (PP2a) were determined using non-radioactive enzyme activity assay systems. Cyclic AMP (cAMP) accumulation was assessed by an enzyme linked immunoabsorbant assay and phosphorylation of cardiac troponin I (cTnI) was determined using a phosphospecific antibody against cTnI.

Results:

Gsαconcentrations were increased approximately 4-fold in failing left atrial preparations relative to control. In contrast, expression of Gi2αwas modestly increased ~1-fold in failing LAA relative to non-failing atrial muscles. PKA activity was significantly increased in failing LAA preparations compared to control (124.7 vs. 42.1 pmole phosphate/minute,
P
<0.05); while, PP2a activity was substantially decreased in failing LAA (4.60 vs. 7.06 pmole phosphate/μg protein/minute,
P
<0.05). cAMP concentrations were increased and expression of the catalytic subunit of PKA was upregulated (~1.5-fold increase) in failing LAA. Lastly, phosphorylation of cardiac troponin I (cTnI) was enhanced in failing LAA preparations.

Conclusions:

Our data are the first to characterize sympathetic remodeling in left atrial muscles isolated from an experimental model of CHF. Additionally, our results support the hypothesis that augmented sympathetic signaling and activation may contribute to the AF substrate in a canine model of CHF characterized by increased AF susceptibility.
Abstract 1238: Myofilament Calcium Sensitization Creates Arrhythmia Susceptibility in Familial Hypertrophic Cardiomyopathy
Authors: Baudenbacher Franz , Sidorov Veniamin Y , Schober Tilmann , Chopra Nagesh , Venkataraman Raghav , Solaro R J , Potter James D , Knollmann Bjorn C
Published: July 2021

Although sudden cardiac death is a common feature in hypertrophic cardiomyopathy (HCM), the underlying mechanisms are unclear. We recently reported that some but not all mice expressing troponin mutations associated with HCM display increased myofilament calcium sensitivity. Here, we used these mice as well as acute challenge with the Ca
2+
sensitizing agent
EMD57033
(EMD) in isolated mouse and rabbit hearts to test the hypothesis that myofilament sensitization leads directly to arrhythmia susceptibility. HCM mice with Ca
2+
sensitizing troponin mutations displayed significantly higher rate of ventricular ectopy (TnT-I79N 10±3, TnT-F110I 11±5, ssTnI 15±4 PVC/h) than mice expressing non-sensitizing troponins (TnT-R278C 1±1, TnT-WT 1±1, non-Tg 2±1 PVC/h, n=5–14 per group, p<0.01). Myofilament sensitization by troponin mutations or EMD shortened the effective refractory period and the ventricular action potential of isolated perfused mouse and rabbit hearts, and caused early afterdepolarizations and triggered beats after fast pacing trains. The action potential shortening was attributable to increased cytosolic Ca
2+
buffering by the Ca
2+
sensitized myofilaments, which resulted in decreased systolic and increased end-diastolic Ca
2+
during fast pacing. Optical mapping demonstrated that Ca
2+
sensitization slowed the ventricular conduction velocity and increased the size of the vulnerable window for ventricular tachycardia both in mouse and rabbit hearts, resulting in a significantly higher incidence of sustained ventricular tachycardia in Ca
2+
sensitized compared with control hearts (5/6
vs
0/6, p<0.05).

Conclusion:

Myofilament Ca
2+
sensitization alters Ca
2+
buffering to render hearts susceptible to ventricular arrhythmias by creating both an arrhythmogenic substrate and trigger. These results identify a novel mechanism linking sarcomeric mutations to susceptibility to ventricular arrhythmias and raise the prospect of a molecular approach to stratifying arrhythmia risk in HCM.
Abstract 1247: Heart Function is Transiently Sustained Despite a Near Loss of SERCA2 Protein in Cardiomyocyte-specific
Serca2
null
Mice
Authors: Andersson Kristin B , Finsen Alexandra V , Sjaastad Ivar , Wang Yibin , Chen Ju , Molkentin Jeffery D , Chien Kenneth R , Sejersted Ole M , Christensen Geir
Published: July 2021

The SERCA2 Ca
2+
ATPase is of central importance for refilling of the sarcoplasmic reticulum (SR) Ca
2+
store and cardiac contractility. Reduced SERCA2 function is associated with heart failure. We hypothesized that loss of SERCA2 would result in immediate severe myocardial contractile dysfunction and death. Transgenic mice were generated with a Cre-loxP strategy in which tamoxifen induces
Serca2
(
Atp2a2
) gene excision in the cardiomyocytes (SERCA2KO) of adult mice. In SERCA2KO mice, SERCA2 protein was rapidly reduced in left ventricular myocardium with a half-life < 3 days. After 4 weeks, SERCA2 protein was reduced to < 5% of control values. In isolated cardiomyocytes, SERCA2a, SERCA2b, SERCA1 and SERCA3 proteins were not detectable. Strikingly, SERCA2KO mice did not present clinical signs of circulatory failure at 4 weeks. Fractional shortening was preserved, and cardiac output was reduced to 80% of control values. The left atrial diameter, lung weight and left ventricular end-diastolic pressure (LVEDP) were slightly increased in SERCA2KO mice compared with controls, and the maximal rates of pressure development and decline in the left ventricle were affected with a prolongation of the ventricular relaxation time. After seven weeks, SERCA2KO mice developed severe congestive heart failure with dilated chambers, elevated LVEDP and pronounced increases in lung and atrial weights. Cardiac output was reduced to 70% of control values. There were no indications of major cardiomyocyte disarray in the myocardium at the 4 or 7 week timepoints. The abundance of Na
+
,Ca
2+
exchanger, L-type Ca
2+
channel 1c and alpha2delta1 subunit proteins and
Pmca1
mRNA were all increased at 4 and 7 weeks. The expression of calsequestrin protein and
Ryr2
mRNA were unchanged. L-type Ca
2+
channel alpha2delta1 subunit and PMCA1 expression were further enhanced at 7 weeks in SERCA2KO mice. Thus, cardiac function is supported in SERCA2KO mice for several weeks despite the near absence of SERCA2 protein. Alterations in the expression of Ca
2+
transporting proteins suggest that Ca
2+
transients are generated over the plasma membrane rather than the SR. However, the adaptations induced by loss of SERCA2 are not sufficient for long-term support of heart function in adult mice.
Abstract 1252: A Mouse Model of a Familial Dilated Cardiomyopathy Mutation in Titin Recapitulates the Human Phenotype
Authors: Gramlich Michael , Michely Beate , Gerull Brenda , Krohne Christian , Morano Ingo , Labeit Siegfried , Thierfelder Ludwig
Published: July 2021

Dilated cardiomyopathy (DCM) is the most common form of primary myocardial diseases and the third most common cause of heart failure. Familial occurrence, mostly as an autosomal dominant trait, is responsible for 20 –30% of all DCM cases. We have previously shown that mutations in the giant muscle filament titin (
TTN
) cause dilated cardiomyopathy. In a large DCM kindred (A1) with autosomal dominant inherited DCM, we could identify a 2 bp insertion mutation in exon 326 of
TTN
. This heterozygous nonsense mutation leads to a framshift generating a premature stop codon after the addition of 4 novel amino acid residues. We have recently evaluated a cardiac biopsy sample from an affected family member of kindred A1 showing that no truncated protein is observed in a western blot analysis. To further investigate the functional consequences of the identified human
TTN
mutation, we now generated a mouse model that includes the 2bp insertion at the corresponding site in the mouse genome. Heterozygous mice are viable and fertile. As in the human situation, the truncated titin is not detectable in western blot analysis of cardiac tissue indicating haploinsufficiency. The ventricles of the heterozygous animals show a decrease in ventricular stiffness as seen in isolated working heart pressure measurements and transmitral Doppler echocardiography (E:A 1.34 vs. 1.075, p<0.01; IVRT 13.57ms vs. 17.01ms, p<0.05). When exposed to angiotensin II (1.4 mg/kg/d for 14d) as a cardiac stressor, heterozygous animals develop dilatation of the left ventricles (4.45mm vs. 3.77 mm, p<0.05) with impaired fractional shortening (25.12% vs. 32.86%, p<0.01) and a diffuse myocardial fibrosis. Homozygous mice die in utero before E8.0. Whether a defect in the formation of sarcomeres or, alternatively, a defect in yet unknown non-muscle functions of titin account for this early embryonic lethality remains to be determined.

Conclusion: Our mouse model shows that a mutation in
TTN
leads to impaired biomechanical properties of the heart, resulting in left ventricular dilatation and decreased systolic function, thereby recapitulating the human phenotype.
Abstract 1263: Midkine Prevents Ventricular Remodeling After Myocardial Infarction Through Enhancement Of Angiogenesis Via PI3k/akt Pathway
Authors: Takenaka Hiroharu , ishiguro Hisayuki , Simida Arihiro , Usui Akihiko , Kadomatu Kenji , Kodama Itsuo , Ueda Yuichi , Horiba Misturu
Published: July 2021

Background:
Midkine (MK) protein is a heparin-binding growth factor. We reported recently endogenous MK ameliorates not only acute myocardial infarction (MI) but also long-term survival after MI using MK knockout mice (MKKO). We aim to assess the therapeutic potential of MK protein after MI by exogenous application.

Method and results:
MI was created in C57BL/6 mice (n=29) by ligation of the left coronary artery. In 11/29 mice, human recombinant MK protein was administrated for 28 days by osmotic pump intraperitoneally (MK-T). Saline was administrated in the same manner for control group (Control) (n=18). Survival rate 28 days after MI in MK-T was significantly higher than Control (82% vs. 44%, p<0.05). In echocardiography 28 days after MI, MK-T showed significantly better LV function compared to Control ( LVEDd; 4.3±0.5 vs. 6.0±0.4 mm, LVESd; 3.6±1.0 vs. 5.3±0.4 mm, EF; 44.3±10.1, vs. 32.1±0.6%, FS; 21.2±5.4 vs.15.3±0.3%, P<0.05). The serum BNP levels 28 days after MI in MK-T was significantly lower than that of Control (129 ± 5 pg/dl vs. 370 ± 98 pg/dl, P<0.05). Size of scar volume quantified by fibrosis area (picrosirius red) in MK-T was significantly smaller than Control (30.3 ± 5 vs. 52.3± 5%, p<0.05). The number of CD31-immunopositive cells in peri-infarct area of LV at 28 days in MK-T was significantly higher than those of Control (45.2 ± 5 V.S. 27.3 ± 5, p<0.05). These changes were associated with an activation of Akt phosphorylation (1.61 fold higher than control, p<0.05) in LV tissue. In-vitro experiments using human umbilical vein endothelial cells (HUVEC) with 100 ng/ml MK caused a significant increase of tube formation with enhacement of Akt phosphorylation (1.68 fold higher than control, P<0.05), and this upregulation was reversed completely by wortmannin, a specific inhibitor of PI3K.

Conclusion:
MK protein prevents the cardiac remodeling after MI and improves the survival through enhancement of angiogenesis via PI3K/Akt pathway. Exogenous application of MK protein might be a new therapeutic strategy for treatment of heart failure.
Abstract 1272: Inhibition of Nuclear Factor-Kappa B Improves Endoplasmic Reticulum Stress-Induced Apoptosis and LV Remodeling in Heart Failure
Authors: Hamid Tariq , Xiang Xilin , Marshall James P , Cardwell Ernest M , Dawn Buddhadeb , Prabhu Sumanth D
Published: July 2021

The endoplasmic reticulum (ER) normally participates in protein folding, calcium storage, and cell signaling. Cellular insult, however, induces the ER-stress response that can produce apoptosis and cell death with persistent activation. Nuclear factor (NF)-κB, a transcriptional regulator of cell survival responses is implicated in the maladaptive responses of ER-stress. However, its role in modulating ER stress-responses in the failing heart is unknown.

Hypothesis:
NF-κB activation in heart failure (HF) exacerbates LV remodeling in part by augmenting maladaptive ER-stress responses. Coronary ligation (or sham operation) was performed in male transgenic (TG) mice with cardiac-specific overexpression of a dominant-negative (DN) IκBα (n=16) and in non-transgenic (NTG) littermates (n=16). Compared to NTG sham, 4 weeks after surgery, NTG HF hearts exhibited significant (p < 0.05):

LV dilatation and systolic dysfunction;

myocyte hypertrophy and fibrosis; and

NF-κB activation (~2-fold).

Immunoblotting also revealed reduced expression (p < 0.05) of ER resident chaperones Grp78 and Grp94 and increased expression (p < 0.001) of CHOP, a pro-apoptotic transcription factor induced by ER stress, that correlated with increased (p < 0.05) apoptosis by TUNEL staining (1.1 ± 0.5 vs. 0.03 ± 0.03%, p < 0.05)). In contrast, compared to NTG HF, TG HF hearts had markedly reduced (p < 0.01) NF-κB activation, significantly less (p <0.05) LV dilatation, systolic dysfunction, and fibrosis but similar degrees of myocyte hypertrophy. Importantly, NF-κB abrogation also resulted in significantly increased expression of Grp78 and Grp94 and complete prevention of CHOP induction. Also, compared to TG sham, TG HF showed a mild but non-significant (p <0.45) increase in apoptotic rates (0.4 ± 0.3 %) by TUNEL. Parallel H9c2 cell studies indicated that induction of ER stress with tunicamycin activated NF-κB and increased apoptosis as measured by caspase-3, -12, and PARP cleavage. In contrast, DN IκBα adult cardiomyocytes maintained viability upon tunicamycin exposure suggesting a pro-apoptotic role for NF-κB activation during ER stress.

Conclusion:
Persistent NF-κB activation in post-infarction HF exacerbates cardiac ER stress and apoptosis and worsens LV remodeling.
Abstract 238: Oxygen Regulates the Flux of Nitric Oxide Diffusion across the Vascular Wall
Authors: Liu Xiaoping , Srinivasan Parthasarathy , Collard Eric , Grajdeanu Paula , Zweier Jay L , Friedman Avner
Published: July 2021

Endothelium-derived nitric oxide (NO) plays an important role in maintaining vascular tone. It is known that NO may be consumed by heme proteins, superoxide and oxygen during diffusion from the endothelium to smooth muscle cells in the vascular wall. Due to the limitation of available techniques, it is unclear to what extent these consumptions can affect the diffusion distance of NO, and if the vascular NO consumption could serve as a “sensor” of oxygen concentrations in the blood vessels. In this study, rat aortas were used as an experimental model for studying NO diffusion process in the vascular system. A Clark-type NO electrode was used to directly measure the flux of NO diffusion across the vascular wall at 37 °C. A segment of aorta was isolated from a 12-week old WKY rat. After the aorta was cleaned and surrounding tissue was removed, it was longitudinally opened. A specifically-designed aorta holder was attached on the tip of the Clark-type NO electrode. The aorta holder surface and the electrode tip surface were aligned in the same plane so that the opened aorta segment could be placed flat on the electrode tip surface and pinned to the aorta holder. Using this technique, we measured the flux of NO diffusion across the aortic wall at different oxygen concentration. It was observed that the NO flux increased 6 to 10 fold when oxygen concentrations dropped from 200 μM to zero. A mathematical model describing the steady-state diffusion-reaction was used in analyzing the experimental data. It was found that the rate of NO decay is first order with respect to [O
2
] and first order with respect to [NO], and hence of the form k[O
2
][NO]. The rate constant k was determined as (3.8±0.4)x10
−3
μM
−1
s
−1
(n=6). With this rate constant, the half-life of NO in the aortic wall in the presence of 200 μM O
2
(equilibrium with room air) will be 0.9 seconds. Our results show that the flux and diffusion distance of NO in the aortic wall is largely regulated by oxygen concentration. When oxygen concentrations drop, NO diffusion distance will significantly increase. As a result, the blood vessel will dilate to a larger extent to allow more blood to be delivered to the hypoxic tissues. Therefore this vascular NO consumption appears to play the role of an oxygen sensor in the regulation of blood flow in the body.
Abstract 1331: Myocardial Function Alterations Correlate to Cardiac Fibrosis and Upregulation of Profibrotic Signaling in Plasminogen Activator Inhibitor-1 Deficient Mice
Authors: Bradham William , Gleaves Linda , Medda Mousumi , Vaughan Douglas
Published: July 2021

Cardiac fibrosis is a common sequelae of cardiac injury and has deleterious functional consequences impacting cardiac filling, function and rhythm. Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of tissue fibrosis in mice. To investigate the longitudinal effect of PAI-1 on cardiac structure and function, M-mode echocardiography was employed to examine cardiac function in PAI-1 deficient (PAI-1
−/−
) and wild-type (WT) control mice in four age groups (6,12,18, 24 months). Eighteen month old PAI-1
−/−
mice exhibited reduced left ventricular (LV) diastolic internal dimension (
p
=0.0118) and a trend towards increased LV posterior wall (LVPW) thickness, compared to WT. Two year old PAI-1
−/−
mice showed increased diastolic and systolic LVPW thickness (
p
=0.0127 and
p
=0.0212, respectively), reduced diastolic and systolic LV internal dimension (
p
=0.0486 and
p
=0.0124), but with preserved LV fractional shortening compared to WT. Histological examination of cardiac sections revealed fibrosis on the anterior epicardial surface of the hearts in 18 month old PKO, which in 26 month old mice had become confluent with extensive (10 –17% by area) epicardial, perivascular, and interstitial distribution (compared to none in WT). Real time polymerase chain reaction (RT-PCR) revealed upregulation of transforming growth factor beta (TGF-β) and fibroblast growth factor 2 in PAI-1
−/−
compared to WT (
p
=0.0234 and
p
=0.037, respectively). Immunofluoresence confirmed this finding with bright TGF-β staining localized in the media of intra-myocardial arterioles, and phosphorylated SMAD2/3, the downstream TGF-β signaling mediator, in areas of fibrosis. Thoracic aortic cells from aged (18 –24 month) PKO and WT mice were grown in culture, with RT-PCR revealing 4 fold increased TGF-β and 17 fold increased SMAD3 (
p
<0.05 for both) RNA levels in PAI-1
−/−
, supplying additional evidence for upregulation of a profibrotic TGF-β/SMAD tissue signaling pathway. The present study is one of the first to elucidate some of the functional consequences and relevant molecular signaling pathways related to aging and PAI-1 deficiency mediated cardiac fibrosis.
Abstract 1336: Increased Enzyme Activity and Increased Beta-adrenergic-mediated Vasodilation in Subjects Expressing a Single Nucleotide Variant of Human Adenylyl Cyclase 6
Authors: Gros Robert , Van Uum Stan , Hutchinson-Jaffe Adam , Ding Qingming , Pickering J. Geoffrey , Hegele Robert A , Feldman Ross D
Published: July 2021

Cyclic AMP is a critical regulator of metabolic and cardiovascular function. Alterations in adenylyl cyclase (AC) regulation have been implicated in the pathogenesis and/or maintenance of insulin resistance, obesity, hypertension and heart failure. However, the role of genetic variability in the regulation of cyclic AMP-mediated effects is unclear. Therefore, we examined the role of a recently identified missense genetic variant of human AC isoform 6 (AC6 S674) in a sample of Caucasian subjects. We assessed the effect of AC6 S674 expression on AC-mediated function in adherent mononuclear leukocyte fractions and on beta-adrenergic-mediated vasodilation in human subjects. In addition, we examined the role of AC6 S674 on AC activity and cytoskeletal reorganization of rat vascular smooth muscle cells (VSMCs) following AC6 S674 gene transfer. In adherent mononuclear leukocytes isolated from AC6 S674-expressing individuals, forskolin-stimulated AC activity was significantly increased as compared to wild-type AC6 (WT AC6)-expressing subjects (WT AC6: 104±19 vs. AC6 S674: 193±33 pmol/min/mg protein, n=12, p<0.05). Similarly, forskolin-mediated cytoskeletal reorganization of adherent mononuclear leukocytes from AC6 S674 expressing subjects was significantly increased as compared to WT AC6 control subjects (154±18% of WT AC6, p<0.05). In dorsal hand vein vascular reactivity studies, subjects expressing the AC6 S674 variant had significantly greater vascular sensitivity to the beta-adrenergic agonist isoproterenol as assessed by both a greater potency and greater maximal effect than in subjects expressing WT AC6. In rat VSMCs, gene transfer of AC6 S674 increased forskolin-stimulated AC activity as compared to WT AC6-infected rat VSMCs (141±18% of AC6 WT, n=8, p<0.05). Similarly, forskolin-induced cytoskeletal reorganization was significantly greater in AC6 S674 expressing VSMCs than in WT AC6 expressing VSMCs (139±15% of WT AC6, n=6, p<0.05). In summary, these data indicate that the expression of a novel, relatively common variant of human AC6 parallels an increase in adenylyl cyclase activity and AC-mediated function both in humans.
Abstract 1342: The NF-kappaB P50 Subunit Protects Against Remodeling and Cardiac Dysfunction Following Myocardial Infarction
Authors: Timmers Leo , van Keulen J Karlijn , Hoefer Imo I , Sluijter Joost P , Goumans Marie Jose , Doevendans Pieter A , van Echteld Cees J , Piek Jan J , Pasterkamp Gerard , de Kleijn Dominique P
Published: July 2021

Introduction
Left ventricular (LV) remodeling leads to congestive heart failure and is a main determinant of morbidity and mortality following myocardial infarction (MI). To further improve the treatment of post infarct LV remodeling, a better understanding of the molecular mechanisms involved in this complicated process is required. The nuclear factor (NF)- κB family (p50, p52, p65) usually forms dimers that regulate DNA transcription in response to a variety of stimuli including pro-inflammatory cytokines, oxidative stress and also ischemia. Inhibition of NF- κB has been shown to reduce heart failure following MI in rats. The specific role of the different NF- κB subunits during LV remodeling, however, has not been clarified thus far. In this study, we elucidate the role of the NF- κB p50 subunit in post infarct LV remodeling.

Methods and Results
MI was induced in wild type C57Bl6 mice and NF- κB p50 KO mice. Without affecting infarct size (45.4 ± 4.3 vs. 42.5 ± 4.6%; p=0.461), the absence of NF- κB p50 increased the extent of LV remodeling (EDV: 175 ± 13 vs. 107 ± 11 μl; p=0.005) and aggravated systolic dysfunction (LVEF: 16.1 ± 1.5 % vs. 24.7 ± 3.7%; p=0.045) 28 days following MI as assessed by magnetic resonance imaging (9.4 T). In the non-infarcted myocardium, interstitial fibrosis (1.53 ± 0.28 vs. 1.05 ± 0.15 grayvalue/μm
2
; p=0.042) and hypertrophy (426 ± 51 vs. 251 ± 12 μm
2
/cardiomyocyte; p=0.018) were increased in NF-κB p50 KO mice. In the infarct area, however, collagen density was decreased (15.11 ± 1.16 vs. 27.28 ± 4.93 grayvalue/μm
2
; p=0.028), which was accompanied by increased TNF-alpha mRNA expression (0.086 ± 0.04 vs. 0.026 ± 0.015; p=0.046) and increased MMP9 activity (0.31 ± 0.03 vs. 0.19 ± 0.03; p=0.049)

Conclusion
These data provide evidence for a protective role of NF- κB p50 in post infarct maladaptive LV remodeling, most likely by reducing inflammatory cytokine production and matrix degradation.