bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5302: Control of Reversible Phosphorylation of Ryanodine Receptor by
MiR-1
: Mechanism of Cardiac Arrhythmia
Authors: Terentyev Dmitry , Belevych Andriy , Terentyeva Radmila , Kuhn Donald E , Malana Geraldine E , Martin Mickey M , Abdellatif Maha , Feldman David S , Elton Terry S , Gyorke Sandor
Published: July 2021

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA,
miR-1
, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of
miR-1
in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of
miR-1
on excitation-contraction coupling and Ca cycling in adult rat ventricular myocytes by using methods of electrophysiology, confocal Ca imaging and quantitative immunoblotting. Adenoviral-mediated overexpressions of
miR-1
in myocytes resulted in a marked increase in the amplitude of the inward Ca current and flatten cytosolic Ca transients voltage dependency. The frequency of spontaneous Ca sparks recorded in intact resting cells was enhanced, while the sarcoplasmic reticulum Ca content was reduced in
miR-1
- overexpressing myocytes as compared with controls. In the presence of beta-adrenergic receptor agonist isoproterenol, rhythmically paced
miR-1
-overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca, events that occurred only rarely in control myocytes. The effects of
miR-1
were completely reversed by the CaMKII inhibitor KN93. Immunological analysis with phospho-specific antibodies showed that while phosphorylation of phospholamban was not altered,
miR-1
overexpression increased phosphorylation of the ryanodine receptor at Ser-2814 (CaMKII) but not at Ser-2808 (PKA). Overexpression of
miR-1
was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that
miR-1
through translational inhibition of this mRNA target, causes CaMKII-dependent hyperphosphorylation of RyR2 via disrupting localization of PP2A activity to this channel, enhances RyR2 activity, and promotes arrhythmogenic SR Ca release. This mechanism could contribute to induction of arrhythmia in disease states accompanied by elevated
miR-1
.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5303: Decreased Affinity of Calmodulin Binding to RyR2 May Cause Leaky Channel in CPVT-Associated Mutation: Insight from RyR2
R2474s/+
Knock-In Mouse Model
Authors: Xu Xiaojuan , Yano Masafumi , Suetomi Takeshi , Ono Makoto , Uchinoumi Hitoshi , Tateishi Hiroki , Okuda Shinichi , Kobayashi Shigeki , Yamamoto Takeshi , Ikeda Yasuhiro , Ohkusa Tomoko , Matsuzaki Masunori
Published: July 2021

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), has been shown to play a significant role in regulating the channel gating in RyR2. However, the role of calmodulin on the channel gating in diseased hearts is unclear. Here, using knock-in (KI) mice model with CPVT-associated RyR2 mutation (R2474S), we investigated whether CaM plays a role in the pathogenesis of CPVT. Sarcoplasmic reticulum (SR) vesicles were isolated from mouse hearts (n=4). To evaluate the characteristics of CaM binding to RyR2, SR was mixed with various concentrations of CaM-SANPAH conjugate (16nM-256nM) , followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. There was no significant difference in the affinity of CaM binding to RyR2 at baseline between KI (Kd=26nM) and WT (Kd=16nM). However, compared to WT, CaM binding was markedly decreased in response to cAMP (1 μM) in KI (18±1.9 % decrease, at 128nM CaM), although 2808Ser phosphorylation in RyR2 was similarly increased by cAMP in WT and KI. This decrease of CaM binding in KI was, however, markedly diminished by co-addition of dantrolene which was previously found to prevent the abnormal Ca
2+
leak by the correction of the defective inter-domain interaction between N-terminal (1–600) and central (2000–2500) domains in both MH-type and CPVT-type RyR2. Moreover, in the presence of FK506 (10μM), which was found to dissociate FKBP12.6 from RyR2 and subsequently to induce domain unzipping, CaM binding was indeed decreased in WT, whereas unchanged by FK506 in KI. In saponin-permeabilized, isolated cardiomyocytes, line scan images in cardiomyocytes were obtained to measure local Ca
2+
release events using a confocal microscopy with Rhod-2 as a Ca
2+
indicator; [Ca
2+
] was buffered at 30 nM by 0.5 mM EGTA. In the KI cardiomyocytes, the frequency of Ca
2+
sparks (SpF:s
−1
·100μm
−1
) was more increased than in WT, in response to cAMP (at 1μM; +33%, p<0.01 vs WT). In conclusion, CaM binding ability to RyR2 seems to be markedly decreased in response to PKA phosphorylation in KI mice, perhaps via defective inter-domain interaction, causing spontaneous diastolic Ca2+ sparks that may lead to triggered activity and hence lethal arrhythmia.
Abstract 5341: Spatial Heterogeneity Of The Mitochondrial Membrane Potential Underlying Arrhythmias In Pressure Overload Hypertrophy
Authors: Jin Hongwei , Chemaly Elie R , Lyon Alex , Stocks Joanne , Akar Fadi G
Published: July 2021

Increased production and/or impaired scavenging of reactive oxygen species (ROS) are a hallmark of mitochondrial dysfunction in structural heart disease. Previously, we demonstrated a mechanism by which depolarization of the mitochondrial membrane potential (mitoVm) caused by ROS release through the mitochondrial benzodiazepine receptor (mBZR) promotes conduction failure. Since pressure overload hypertrophy (PoH) is a major risk factor for sudden death, we hypothesized that instability of mitoVm in PoH predisposes to arrhythmias.

Methods: PoH was produced in rats by ascending aortic banding for 4 weeks. LV dysfunction characteristic of PoH was confirmed in vivo using M mode 2D echocardiography. Ultrahigh resolution optical imaging was performed in ex vivo perfused hearts from PoH (n=6) and normal (n=3) rats. Normalized mitoVm was measured using a novel quantitative technique of TMRM imaging with subcellular resolution during a protocol of ischemia (for 7min) and reperfusion. Mean and SD mitoVm across 6400 sites were compared as was the expression of the mBZR gene (PKBS) using western blot analysis.

Results: Aortic banding for 4 weeks resulted in a significant increase in LV anteroseptal and posterior wall dimensions consistent with structural remodeling in PoH. Ischemia reperfusion related ventricular fibrillation (VF) was observed in 5/6 PoH and 1/3 normal hearts. Average mitoVm was surprisingly greater in PoH compared to normal hearts during both ischemia (17%, p=0.012) and reperfusion (24%, p=0.004). However, spatial heterogeneity of mitoVm indexed by SD was markedly greater in PoH during ischemia (0.42 vs 0.11, p=0.00007) but not reperfusion (p=0.15). Analysis of mitoVm in susceptible (VF+) versus resistant (VF−) rats revealed a 4X increase in mitoVm heterogeneity in ischemia (p=0.0004) but not reperfusion (p=0.12) in the VF+ group. There were no differences in overall protein content of PKBS between groups indicating that altered mitoVm dynamics are independent of PKBS expression.

Conclusions: These data demonstrate that increased spatial heterogeneity of mitoVm during ischemia in PoH promotes VF. Strategies aimed at preventing the desynchronization of mitoVm may form a novel therapeutic approach for combating malignant arrhythmias.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5349: Dual Effects of Nifekalant on I
Kr
Authors: Morishima Mikio , Niwa Ryoko , Yamaguchi Takahiro , Kato Sara , Tsuji Yukiomi , Honjo Haruo , Sakuma Ichiro , Kamiya Kaichiro
Published: July 2021

Purpose: Nifekalant, a potent and common antiarrhythmic drug, has been regarded to treat reentry arrhythmias through I
Kr
block (antagonist action). But, recently nifekalant was reported to increase I
Kr
at slight depolarizing level (agonist action) and details are still not known. In this study, profiles and molecular mechanisms of I
Kr
antagonist and agonist actions by nifekalant were comparatively investigated.

Methods: I
Kr
was measured in rabbit ventricular myocytes. hERG currents were expressed in
Xenopus oocytes
. An Ala-scanning mutagenesis of hERG channel was performed in S4-S5 linker regions and S6 domain to seek for important amino acid residues to develop agonist action.

Results: In I
Kr
of myocytes, nifekalant (0.3 μM) suppressed currents at all test voltages ranging from −60 mV to +40 mV and showed antagonist action. However, when a preceding depolarizing pulse (+20 mV for 2 sec) was applied, current amplitudes below −20 mV were transiently increased and showed agonist action. When repetitive short depolarizing pulses, mimic action potentials, were applied, agonist action was enhanced at higher frequency. When a preceding depolarizing pulses was prolonged, agonist action rapidly increased (+20 mV, time constant: 428±31 ms, n=4). When an interval at −90 mV between a preceding pulse and a tested depolarization to −50 mV was prolonged, currents were slowly decreased with time constant of 306±55 sec (n=4). These time constants were relatively similar to onset and offset time constant of current blocking by antagonist action (onset at 20 mV: 769±33 ms, offset at −90 mV: 218±44 sec, n=4). Nifekalant shifted activation curve to a hyperpolarizing direction by 28.0±2.5 mV (n=4), but did not affect inactivation curve. In 2 mutations made in the S4–S5 linker, which could affect activation gate, agonist action was abolished but antagonist action was maintained.

Conclusions: Our results indicated that both antagonist and agonist actions on I
Kr
by nifekalant may exhibit continuously within normal range of heart rate, thereby contributing to antiarrhythmic effects. Agonist action may be derived by two factors; first, a binding to amino acid residues same as antagonist action and, second, an interaction with activation gate.
Abstract 5360: A Nox-2 Containing NADPH Oxidase Plays a Critical Role in Cardiac Remodeling Associated with Doxorubicin Chemotherapy
Authors: Zhao Youyou , McDermott Barbara J , Grieve David J
Published: July 2021

Anthracycline antibiotics, such as doxorubicin (DOX), are effective anti-cancer drugs, although their use is limited by a dose-dependent cardiotoxicity. Chronic anthracycline treatment is known to increase myocardial production of reactive oxygen species (ROS), which are implicated in the processes underlying cardiac remodeling. A Nox2-containing NADPH oxidase is an important source of ROS in the heart. Here, we investigated its role in DOX-induced cardiac remodeling using gene-modified mice lacking Nox2 (KO) and wild-type (WT) littermate controls. Adult male mice (8–10 weeks) were injected with DOX (12 mg/kg cumulative dose) or vehicle control and studied 8 weeks later (
n
≥ 7 per group). Echocardiography indicated that DOX caused significant systolic and diastolic dysfunction in WT mice compared to controls (fractional shortening: 25.3 ± 0.6 vs 34.3 ± 1.2 %; E/A ratio: 1.27 ± 0.05 vs 1.67 ± 0.05; P<0.05). In contrast, contractile function was preserved in KO mice subjected to DOX (fractional shortening: 34.2 ± 1.7 vs 34.8 ± 1.4 %; E/A ratio: 1.39 ± 0.04 vs 1.60 ± 0.09; P=NS). Furthermore, mRNA expression (real-time RT-PCR) of several pro-hypertrophic and pro-fibrotic genes was significantly increased by DOX in WT (eg ANP: 326 ± 63; β-MHC: 266 ± 68; procollagen III: 56 ± 18; MMP-9: 46 ± 11 %, P<0.05) but not KO mice (eg ANP: −6 ± 27; β-MHC: 23 ± 22; procollagen III: 4 ± 11; MMP-9: −11 ± 10 %, P=NS) compared to controls. Myocardial NADPH oxidase activity (lucigenin-enhanced chemiluminescence) was also significantly increased by DOX in WT but not KO mice (88 ± 28 vs −6 ± 13 %, P<0.05). This was associated with increased mRNA expression of Nox2 and Nox4 in WT DOX-treated mice (74 ± 33 and 106 ± 35 %, respectively; P<0.05), whereas Nox4 mRNA remained unchanged in KO mice (0.6 ± 15 %, P=NS). Isolated ventricular cardiomyocytes subjected to acute DOX treatment (0.5 μM for 3 h) demonstrated similar changes in superoxide production and Nox isoform and pro-hypertrophic/fibrotic gene expression, which were attenuated by the AT
1
antagonist, losartan (10 μM) in WT cells.

Conclusion:
These data suggest that ROS derived specifically from a Nox2-containing NADPH oxidase make a significant contribution to the processes underlying cardiac remodeling associated with doxorubicin chemotherapy.
Abstract 5405: Real Time High-Resolution Dynamic Imaging Provides New Insights for Understanding Cardiac Function
Authors: Ashraf Muhammad , Li Xiaokui , Mahajan Aman , Sahn David J
Published: July 2021

We used high frame rate echocardiography to define the relationship of strain and rotation parameters with dynamic cardiac function. We studied 15 open chest pigs scanned with a 10S probe on a GE Vivid 7 ultrasound system directly from the surface of heart to allow acquisition of high-resolution apical and basal short axis views (>150 frames/sec). Images were analyzed offline in EchoPac PC using a speckle tracking based strain and motion calculation program. We divided the cardiac cycle into 3 phases to describe results. Phase 1 (37% ± 4.5% of RR’ interval; red in figure

) ended when septal and global circumferential strain reached peak. After initial clockwise motion, the LV showed counterclockwise rotation and 55% ± 5.5% decrease in short axis area during this phase. Phase 2 (marked blue; 18% ± 3.75% of RR’ interval) ended when lateral wall strain reached peak. Global circumferential strain (1–3% decrease) and short axis area (4–6% increase) showed minimal changes. The septum showed 4–7% decrease and lateral wall showed 3–5% increase in strain. All segments continued to twist and reached peak twist coinciding with peak strain in the LV lateral wall. During Phase 3 (33% ± 2.65% of the RR’ interval; red), all segments showed untwisting, 16–18% reduction in circumferential strain, and 52–59% increase in short axis area. Coordinated and sequential contraction results in LV twisting that continues into very early diastole. The isovolumic phase demonstrates minimal contraction in the lateral wall and relaxation in the septum, resulting in continued early isovolumic twisting (torsion), which is then rapidly released.
Abstract 5408: PKA and PKC Phosphorylation of Troponin I Canonical Sites Regulate Calcium Sensitivity During Force Frequency Response
Authors: Correa Genaro A Ramirez , Xin Zhong , Robinson John C , Gao Wei D , Murphy Anne M
Published: July 2021

In failing hearts, the force-frequency response (FFR) is blunted, flat or negative. A positive FFR is crucial for healthy myocardium to respond to an increased working demand. There is no consensus in weather a positive FFR relies on myofilament Ca
2+
sensitization or desensitization and weather this is modulated by cTnI phosphorylation. In the present work we aimed to address the FFR and Ca
2+
cycling in intact mouse trabeculae loaded with Fura-2. To achieve this we used two transgenic models with pseudo phosphorylation mutants of troponin I (TnI), TnIDD
22,23
mice, which mimic increased phosphorylation at PKA sites of TnI at Ser 22 and 23 and TnI PKA/PKC mice, which mimic dephosphorylation at same PKA sites and increased phosphorylation at PKC sites of TnI at Ser 42 and 44. We hypothesized that controlling for cTnI phosphorylation will clarify the contribution of cTnI to the differences in force and Ca
2+
dynamics during FFR. When we examined the isometric contraction and Ca
2+
dynamics in each of these lines (TnIDD
22,23
, n= 8; TnI PKA/PKC, n=6) and non transgenic controls (NTG, n=7) we found that all three groups showed a positive FFR, although peak Ca
2+
increased with frequency rate in all three a less steep Ca
2+
transient increase (myofilament Ca
2+
sensitization) was observed in both transgenic lines compared to NTG (TnIDD
22,23
, p= 0.001; TnI PKA/PKC, p=0.03). Additionally, the peak force during the FFR was greater in the TnIDD
22,23
mice compared to NTG (p < 0.0001), suggesting that TnIDD
22,23
mice posses an enhanced frequency rate-related myofilament Ca
2+
sensitivity. WB analysis of Ca
2+
handling proteins including PLB, pPLB, SERCA2a and Ryanodine receptor normalized levels showed no major differences among all three groups, suggesting the differences observed in TnIDD
22,23
mice were not due to altered Ca
2+
handling but rather to myofilament Ca
2+
sensitivity. We conclude that a positive systolic peak FFR is followed by increasing myofilament Ca
2+
esensitization but mimicking increased phosphorylation at PKA sites of TnI Ser 22,23 enhances FFR and Ca
2+
responsiveness. Overall, our results support the concept that myofilament alterations feedback onto Ca
2+
handling mechanisms and these findings have important implications for human heart failure.
Abstract 5420: Dephosphorylation of the Cardiac Mitochondrial Voltage-Dependent Anion Channel Prevents Inhibition by Hexokinase
Authors: Cheng Qunli , Bosnjak Zeljko J , Kwok Wai-Meng
Published: July 2021

The mitochondrial permeability transition pore (mPTP) has been implicated as the end effector in ischemic and pharmacological preconditioning. Though the molecular composition of the mPTP is thought to consist of cyclophilin D located in the mitochondrial matrix, adenine nucleotide translocase on the inner mitochondrial membrane, and the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane, recent studies have raised the possibility that VDAC may be a regulatory, rather than a major, component of mPTP. Nevertheless, VDAC is likely to be a critical component of the preconditioning signaling pathway since it is the main conduit for metabolite diffusion across the mitochondrial outer membrane. Yet, the direct measurements of cardiac VDAC activity and modulation have been limited. In the present study, we purified VDAC from rat hearts using standard procedure and investigated its modulation by phosphatase and hexokinase. VDAC was incorporated into planar lipid bilayer for measurements of channel activities. The channel exhibited the reported voltage-dependent gating. Several conductance states were identified, with the most prevalent between 1.5 to 2 nS in 0.5 M NaCl. Koenig’s polyanion, a VDAC blocker, triggered channel flickering and decreased the mean current by 78±6%. In the presence of phosphatase (1 unit/ml), the mean conductance significantly increased from 1.81±0.03 to 3.68±0.61 nS (n=9; mean±SEM). However, the addition of a recombinant hexokinase (5 units/ml; GenWay Biotech) had no significant effect on the phosphatase-enhanced VDAC current (n=4). In contrast, recombinant hexokinase alone significantly decreased the mean conductance from 1.75±0.05 to 0.79±0.19 nS (n=4). The addition of phosphatase reversed the inhibitory effect of hexokinase and further enhanced VDAC activity, increasing the mean conductance to 2.69±0.19 nS (n=4). Our results suggest that the dephosphorylation of VDAC prevents the inhibitory effects of hexokinase. Furthermore, VDAC activity suppressed by hexokinase can be reversed by dephosphorylation of the channel. In conclusion, we have reported on a novel observation at the functional level that basal phosphorylation of the cardiac VDAC may be required for its modulation by hexokinase.
Abstract 5493: Heparan-Sulfate Deficient Perlecan Mice Exhibit Increased Vascular Smooth Muscle Cell Activity and Response to Arterial Injury
Authors: Gotha Lara , Abdulla Parween , Lockwood Julia , Strauss Bradley
Published: July 2021

Percutaneous coronary interventions (PCI) are limited by restenosis, a process that involves smooth muscle cell (SMC) proliferation and migration. Perlecan, a heparan-sulfate proteoglycan present in the vessel wall has been shown to inhibit SMC activity in vitro, and decrease intimal hyperplasia in animal models of arterial injury. As well, heparinase treatment of perlecan results in a loss of these inhibitory effects in vitro. This study examined the hypothesis that heparan sulfate (HS) has an inhibitory role in perlecan’s regulation of SMC activity. Aortic SMC from transgenic mice expressing HS-deficient Perlecan (M
Δ3/Δ3
) and control (WT) mice were isolated and cultured to characterize SMC activity. Additionally, M
Δ3/Δ3
and control mice underwent a carotid artery wire injury and carotid arteries were harvested 1 week (n=6) and 3 weeks (n=12) post-surgery for histological analysis. Over 7 days, M
Δ3/Δ3
cells showed significantly higher proliferation during the log phase (days 2–4), with almost a 2-fold increase in cell density during the plateau phase (days 5–7), compared to WT SMC (p<0.001). Incubation with FGF-2 or PDGF-BB resulted in significantly greater M
Δ3/Δ3
cell density versus WT (p<0.001). PDGF-BB induced migration was also significantly greater in the M
Δ3/Δ3
cells (216.2 ± 2.81) than the WT cells (151.9 ± 2.97) (p<0.01). Following arterial injury, significant medial thickening was seen at 1 and 3 weeks, in WT and M
Δ3/Δ3
arteries. Increases in medial thickness of M
Δ3/Δ3
arteries were significantly greater than those seen in WT arteries at all respective time points (p<0.001). In conclusion, the differences seen in SMC activity and response to arterial injury between WT and M
Δ3/Δ3
populations demonstrate that the HS side chains play a critical role in perlecan’s inhibitory effects of SMC activity, and may indicate a promising therapeutic target for the prevention of restenosis following PCI.
Abstract 5510: Arginine Methylation Index as Integrated Quantification of Arginine Methylation Provides Stronger Independent Risk Prediction of Long-term Cardiovascular Events than Individual Methylated Arginine Metabolites
Authors: Tang W. H. Wilson , Wang Zeneng , Cho Leslie , Brennan Danielle M , Hazen Stanley L
Published: July 2021

Background:
The prognostic value of asymmetric dimethylarginine (ADMA) is attributed to its role as an endogenous inhibitor of nitric oxide synthases (NOS). The relationship of related post-translational modification products of arginine methylation and cardiovascular disease (CVD) risks is uncertain.

Methods:
Plasma was isolated from 1,011 subjects undergoing diagnostic cardiac catheterization, and future major adverse cardiac events (MACE, including myocardial infarction, stroke, and death) were investigated. Levels of ADMA, symmetric dimethylarginine (SDMA), and N-mono-methylarginine (MMA) were quantified by mass spectrometry, and their ratios [(ADMA+SDMA)/MMA], the arginine methylation index (Methy-I
Arg
), were determined.

Results:
After adjusting for Framingham risk factors, CRP, and creatinine clearance, elevated (4th vs. 1st quartile) plasma levels of both SDMA [2.0-fold (95%CI, 1.3–3.1)] and ADMA [1.5-fold (95%CI, 1.00–2.4)] remained associated with higher prevalence of CVD, whereas elevated levels of MMA [0.56-fold (95%CI, 0.36–0.87)] were paradoxically associated with lower CVD. While elevated ADMA and SDMA levels remained significant predictors, MethyI
Arg
was the strongest independent predictor of incident MACE[2.4-fold (95%CI, 1.3 to 4.5)].

Conclusions:
An integrated quantification of arginine methylation in the form of an “Arginine Methylation Index” provided the strongest independent risk prediction for incident major adverse cardiac events in stable patients undergoing elective cardiac evaluation, and significantly added to the prognostic utility of traditional risk factors.
Abstract 5519: High Resolution Genetic Mapping Strategies for Metabolic Disease in Mice: Towards Association Based Studies
Authors: Bennett Brian J , Farber Charles R , Orozco Luz , Lusis Aldons J
Published: July 2021

Background: Common complex diseases, such as atherosclerosis, diabetes and obesity, are the result of genetic, environmental and gene/environmental interactions. Genetic studies using mice have relied on linkage studies where two mouse strains with different disease phenotypes are crossed to identify a genetic loci conferring susceptibility or resistance to the disease. One limitation with such an approach is that linkage areas are many megabases in size and thus gene resolution is low. Recently, several whole genome association studies in large human cohorts have reported single nucleotide polymorphisms (SNPs) associated with common forms of disease. We have begun similar association based approaches to common diseases using mice. One important advantage of murine association studies is that association results can be validated biologically with knockout or transgenic approaches.

Methods and Results: Our initial studies have focused on the metabolic syndrome and we have collected traits related to diabetes, adiposity and cholesterol metabolism. We have phenotyped 100 strains of mice including a panel of classic inbred mice and several panels of recombinant inbred mice (BxH, AxB, BxA and BxD). We report here our initial strategy for association in mice, power calculations and describe strain selection along with initial phenotypes and associations. Our laboratory has previously identified linkage peaks on chromosome 1 for HDL and Total Cholesterol. Studies from our laboratory and others have identified apolipoprotein A2 as a major determinate of circulating lipoproteins in mice. As a proof of principle we have focused on associations of total cholesterol and HDL cholesterol at the A2 locus on chromosome 1 and demonstrate the increased resolution of this approach. We also report loci contributing to HDL and total cholesterol levels from independent F2 crosses and coincident lipoprotein associations in the mouse diversity panel.

Conclusion: Association based studies in mice have high resolution can successfully identify previously validated genes. Studies are currently ongoing to validate novel SNPs associated with other metabolic phenotypes.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5551: Double Deficiency of Tumor Necrosis Factor-α and Interferon-γ in Bone Marrow-Derived Cells Prevents Neointimal Formation in a Murine Model of Vascular Injury
Authors: Murayama Hideki , Takahashi Masafumi , Shiba Yuji , Takamoto Masaya , Ise Hirohiko , Tagawa Youichi , Ikeda Uichi
Published: July 2021

Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Recent evidence indicates that inflammatory responses induced by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), are involved in the progression of neointimal formation. However, the role of TNF-α and IFN-γ in the restenosis after PCI has not been fully understood. The purpose of this study is to examine the impact of TNF-α and IFN-γ in bone marrow-derived cells in the development of neointimal formation after vascular injury in mice. Wild-type (WT), TNF-α-deficient (TNF-α
−/−
), IFN-γ-deficient (IFN-γ
−/−
), and TNF-α/IFN-γ double-deficient (DKO) mice were subjected to wire-mediated vascular injury of the right femoral artery. Immunohistochemical analysis showed the expression of TNF-α and IFN-γ was detected in the neointimal lesion of WT mice, but these cytokines were not detected in the lesion of the corresponding deficient mice. Neointimal formation was significantly reduced after the injury in the DKO mice, compared to that in the WT, TNF-α
−/−
, and IFN-γ
−/−
mice (I/M ratio, WT: 2.28±0.17, TNF-α
−/−
: 2.13±0.20, IFN-γ
−/−
: 2.37±0.16, DKO: 1.32±0.10, p<0.05, each n=14–17). No significant difference in reendothelialization (CD31 staining) was observed among these groups. Further, vascular smooth muscle cell (α-SMA) and macrophage (F4/80) contents in the neointimal area also did not differ among the groups. The number of proliferating cell nuclear antigen (PCNA) and Ki-67 positive cells in the neointimal lesion was significantly decreased in DKO mice. To determine the contribution of bone marrow cells, we developed 3 types of bone marrow chimeric (BMT
Wild→Wild
, BMT
DKO→Wild
, and BMT
Wild→DKO
) mice. The neointimal formation in BMT
DKO→Wild
mice was significantly reduced as compared to that in BMT
Wild→Wild
(I/M ratio, p<0.05, each n=7) and BMT
Wild→DKO
mice (p<0.05). These results suggest that the lack of TNF-α and IFN-γ in bone marrow-derived cells synergistically prevents neointimal formation after vascular injury and provide new insights into the mechanisms underlying the restenosis after PCI.
Abstract 5559: Role of AMP-activated Protein Kinase in Ischemia-Reperfusion-Induced Barrier Failure in Endothelial Monolayers
Authors: Assad Muhammad , Arshad Muhammad , Haertel Frauke V , Aslam Muhammad , Fleming Ingrid , Piper H M , Noll Thomas
Published: July 2021

Background:
Ischemia-reperfusion provokes endothelial barrier dysfunction leading to edema formation and organ failure. The AMP-activated protein kinase (AMPK) is a fuel sensor which becomes activated under ischemia. It is now apparent that AMPK may also play important roles in stabilization of cell-adhesion. Here, it is
hypothesized
that AMPK may play an important role in protection barrier function under metabolic stress.

Methods and Results:
Overexpression of a dominant negative AMPK mutant in endothelial monolayers from human umbilical veins (EC) caused a 2-fold increase in basal permeability (albumin flux across the monolayers). In accordance, downregulation of AMPK by siRNA (~60%) leads to gap formation between adjacent EC, disintegration of cell adhesion structures and alterations of the cytoskeleton (loss of VE-cadherin at cell borders, actin stress fiber formation; immunocytochemistry), indicating that AMPK plays an important role in maintenance of barrier integrity. To analyse the role of AMPK on barrier function EC were exposed to ischemia (40 min, Po
2
<5 mm Hg; pH 6.4) followed by reperfusion (40 min, Po
2
=140 mm Hg; pH 7.4). Ischemia caused an immediate increase in permeability (gap formation, video-imaging technique) and a 3-fold increase in AMPK activity (AMPK phosphorylation; western blot) after 40 min. During reperfusion gap formation was further increased by 307± 9 % (P<0.05, n=5) within the ongoing 40 min. In contrast, AMPK phosphorylation rapidly declined to basal level within the first 10 minutes of reperfusion. However, addition of AICAR (AMPK activator; 5-aminoimidizole-4-carboxamide riboside), at the onset of reperfusion caused a rapid increase in AMPK phosphorylation and abolished the reperfusion-induced gap formation. Pretreatment of the cells with AICAR before the onset of ischemia reduced reperfusion-induced gap formation by 50 %.

Conclusion:
AMPK is involved in maintenance of cell-cell contacts and stabilization of basal barrier function. Pharmacological activation of AMPK within the first minutes of reperfusion can prevent hyperpermeability induced by reperfusion stress. Hence, activation of AMPK may provide a new therapeutic option to prevent ischemia-reperfusion barrier failure.
Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals
Authors: Ren Bin , Mukhopadhyay* Arpita , Lanahan Anthony A , Zhuang Zhen W , Moodie Karen L , Peterson Randall T , Simons Michael
Published: July 2021

Background
: Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct.

Methods
: In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos.

Results:
Exposure of synectin
−/−
arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and
in vitro
branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct
in vitro
or in a Matrigel model
in vivo
had similar effect. Systemic treatment of synectin
−/−
mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino.

Conclusions:
ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.
Abstract 555: The Universal Clinical Classification of Acute Myocardial Infarction is a Better Predictor of Long Term Outcomes than ST Segment Classification
Authors: Gonzalez Manuel A , Eilen Dana , Marzouq Rana A , Awadallah Saed , Patel Hiren R , Bloomfield Ricky , Bennett Justin M , Lim Heang M , Porterfield Christopher , Patel Amit A , Gilmore Laura C , Nasir Summiyah , Babb Joseph D , Rose John D , Cascio Wayne E
Published: July 2021

Introduction:

The universal classification (UC) of AMI aims to facilitate cross-study analysis, yet the long-term outcomes using UC are largely unknown.

Hypothesis:

We tested the hypothesis that the long-term outcome of patients with AMI is better predicted by UC than ST segment classification.

Methods:

We conducted a prospective study of 348 consecutive patients with AMI with mean follow-up of 30.6 months. The primary outcome was the major adverse cardiovascular events (MACE) [composite of all causes of mortality, recurrent AMI, and stroke]. Multivariate and survival analysis of MACE was performed.

Results:

The study population was STEMI=168, NSTEMI=180, Type 1=278, Type 2=55, Type 3=5, Type 4a=2, Type 4b=5, and Type 5=3. During follow-up 80 patients died, 31 had an AMI, and 7 had a stroke. UC correlates with the ST segment classification (p<0.005). MACE free survival was different for Type 1 and Type 2 (p=0.043), but not for STEMI and NSTEMI. There was a positive association between MACE and the quartile of peak Troponin, number of cardiovascular risk factors, and number of vascular beds affected, and an inverse relationship with the utilization of discharge cardiovascular protective medications (all p≤0.01). No such inverse relationship existed for Type 2.

Conclusions:

UC of AMI is a better long-term predictor of MACE. The quartile of peak Troponin levels, cardiovascular risk factors, and number of vascular beds affected are independent predictors of MACE, while cardiac medications protect against MACE, except in Type 2 patients.
Abstract 568: Coronary Artery Plaque Spatial Distribution Matches that of Thrombosis in STEMI, and is Underestimated by Calcified Plaque Segments Alone
Authors: Pastorius Catherine A , Merkel-Kraus Stacia , Schwartz Jonathan G , Schwartz Robert S , Pink Vicki , Peichel Gretchen , Lesser John R , Knickelbine Thomas , Flygenring Bjorn , Longe Terry , Duval sue
Published: July 2021

Angiographic studies of ST-elevation myocardial infarction (STEMI) show that occlusive thrombi occur in coronary arteries within the proximal third of each major vessel, within about 35 mm from the ostium. These findings suggest that these high-risk regions are sites of plaque vulnerability. Multidetector CTA (MDCTA) visualizes not only lumen but also calcified and uncalcified coronary plaque. We therefore studied the distribution of coronary plaque in patients to determine plaque distribution to compare result with location of angiographic myocardial infarction thrombus.

Methods: 48 outpatients (mean age 57 +− 5) undergoing Dual Source MDCTA (asymptomatic or with atypical symptoms) were studied. Most distal plaque location was measured for ostial-termination distance and composition (calcified/noncalcified) determined by one expert observer using validated, commercial plaque characterization software for lesion location and lesion length.

Results: Location of the most distal plaque was as follows, LAD (n=35) 31.8 mm, LCX (n=21) 33.0 mm, and RCA (n=24) 56.0 mm. Mean lesion lengths were LAD 19.4 +− 13.4 mm, LCX 15.1 +− 9.6 and RCA 18.4 +− 9.8 mm. Mean volume (cu mm) for calcified and noncalcified plaque was 130 +− 176 and 204 +− 249 respectively, for a ratio of 0.64. There was no propensity of either calcified or uncalcified plaque to aggregate longitudinally in the arteries.

Conclusions:

The distribution and location of coronary artery plaque by MDCTA identically matches the known location of coronary artery vulnerability determined by angiographic studies. This finding suggests that STEMI does not occur in distal coronary locations simply because little plaque occurs in these areas.

Calcified plaque volume measured in 3-dimensions underestimates total plaque volume by a factor of 2.6. Calcific plaque is thus the ‘tip of the iceberg’.
Abstract 576: The Prognostic Value of the Long Pentraxin 3 as Compared to B-Type Natriuretic Peptide and High-Sensitive C-Reactive Protein, and their Combination, as a Prognostic Marker of Mortality in Acute Chest Pain Patients
Authors: Brügger-Andersen Trygve , Pönitz Volker , Kontny Frederic , Staines Harry , Grundt Heidi , Miyamoto Kyoko , Miyazawa Chihiro , Matsuura Tadashi , Sagara Mina , Nilsen Dennis W.T.
Published: July 2021

Elevated plasma levels of long pentraxin 3 (PTX3), high-sensitive C-reactive protein (hsCRP) and B-type natriuretic peptide (BNP) are found in acute coronary syndromes (ACS). The aim of this study was to assess the prognostic value of PTX3 as compared to BNP and hsCRP, and their combination, as a prognostic marker of mortality in acute chest pain patients. PTX3 was measured in EDTA plasma with a new, high-sensitive ELISA method (PPMX, Tokyo, Japan). BNP was analysed in EDTA plasma using the Microparticle Enzyme Immunoassay (MEIA) Abbott AxSYM®. HsCRP was measured with the use of an immunoturbidimetric assay (Tinaquant® C-reactive protein (latex) high sensitive assay, Roche Diagnostics). The blood samples were taken on admission in 795 patients. The patients were followed for 24 months concerning mortality. For statistical analysis, the study cohort was divided into quartiles (Q1–4) according to PTX3 levels. A multiple logistic regression was fitted which included standard risk measures. At 24 months follow-up, 121 of the 784 patients included in the model had died. The odds ratio for comparing Q4 versus Q1 for PTX3, BNP and hsCRP were 4.34, 3.35 and 0.52 (p=0.001, p=0.024 and p=0.096), respectively, and the combination of PTX3 and BNP showed an incremental prognostic value (figure

). PTX3 is a new independent marker that strongly predicts long-term all-cause mortality in patients with acute chest pain and the combination of this marker with BNP adds substantially to the prognostic value as compared to either marker alone.

Figure

The number of biomakers greater than the median for % death in 24 months (p<0.001 for comparing the groups)
Abstract 596: Therapeutic Hypothermia After Cardiac Arrest: A Cost-Effectiveness Analysis
Authors: Mader Timothy J , Bowdoin Jennifer J , Nobles Carrie J
Published: July 2021

Background: Mild induced therapeutic hypothermia (TH) is currently recommended for survivors of out-of-hospital cardiac arrest (OHCA) who meet specified criteria. If TH were fully implemented in the US, 2298 additional OHCA victims every year could be expected to survive. Despite a 2006 meta-analysis clearly demonstrating a decrease in mortality and an improvement in neurological outcome (with no treatment-limiting side effects) for TH, clinicians have been reluctant to adopt the therapy, often citing cost as a reason. The aim of this study was to determine the cost-effectiveness (CE) of TH after OHCA relative to supportive care.

Methods: Monte Carlo simulation was used to approximate the cost per in-hospital death prevented, the cost per poor neurological outcome averted, and annual hospital costs. Estimates of effectiveness and resource use were extracted from secondary data sources. Included variables were: in-hospital mortality, neurological status, ICU and hospital ward length-of-stay (LOS), ICU and hospital ward cost/day, equipment costs, physician time, and medications. For our base case, we calculated the CE of treating 60 patients/year using a moderate cost method. We did sensitivity analyses through systematic bidirectional adjustments of selected variables’ costs, by reducing the treated patients/year to 12, and by excluding the cost of medications and physician time. All analyses were done from a US hospital perspective and reported in 2008 US$.

Results: LOS dominated the difference in cost. The median cost per death prevented was $280,092 [IQR $202,279-$386,215] and per poor neurological outcome averted it was $147,314 [IQR $118,009-$182,251]. The median annual cost to treat 60 patients/year using a moderate cost method was $1,588,730 [IQR $1,322,343-$1,865,462]. Reducing the number of patients treated per year to 12 lowered the annual cost to $319,759 [IQR $268,122-$373,670]. The results of all other sensitivity analyses were similar to the base case. Assuming a mean duration of survival following hospital discharge of 6.13 years, TH had a cost/life-year saved of $45,692, which compares favorably to other life-saving health and social interventions.

Conclusion: TH after OHCA is a relatively cost-effective treatment modality.
Abstract 636: Conduction Delay in Right Ventricle as a Marker for Identifying High-Risk Patients in Brugada Syndrome
Authors: Doi Atsushi , Takagi Masahiko , Maeda Keiko , Tatsumi Hiroaki , Shimeno Kenji , Yoshiyama Minoru , Nakagawa Eiichi
Published: July 2021

BACKGROUND

In Brugada syndrome (BS), abnormal conduction delay in right ventricle has been reported. However, the meaning of the conduction delay for risk stratification in BS is still unclear.

OBJECTIVES

To evaluate the significance of conduction delay in patients with BS as a marker for risk stratification.

METHODS

Twenty-four patients with BS in whom pilsicainide challenge test was performed (documented VF: N = 7, syncope: N = 7, and asymptomatic: N = 10) were paced from right ventricular apex (RVA), using a basic cycle length of 500ms (8 beats) and a single extrastimulus. A 2.5-French 16-electrode catheter was positioned into the coronary sinus and the great cardiac vein to record intracardiac electrograms on the epicardial sites in right ventricular outflow tract area (RVOT) and lateral left ventricle (l-LV). We measured the conduction time from the stimulus artifact at RVA to the epicardial ventricular electrogram at RVOT or l-LV. The conduction delay between RVA and RVOT (CD-RV) or between RVA and l-LV (CD-LV) was defined as the time interval between the ventricular response at RVOT (RV-V1V2) or at l-LV (LV-V1V2) and the stimulus coupling interval (S1S2) at RVA, respectively (CD-RV; RV-V1V2 minus S1S2, and CD-LV; LV-V1V2 minus S1S2). We also measured 12-lead ECG parameters at baseline and after pilsicainide challenge test, and evaluated the differences of the ECG parameters before and after pilsicainide challenge test.

RESULTS

Max CD-RV was significantly larger than max CD-LV in all patients (28±9 vs 19±7 ms, p<0.05). Max CD-RV in patients with documented VF was significantly larger than that in patients without (35±10 vs 25±7 ms, p<0.05). However, there was no significant difference in max CD-RV and CD-LV between patients with induced VF and those without (30±9 and 20±5 vs 26±7 and 21±7 ms, p=NS). There was significant positive correlation between max CD-RV and the differences in QRS duration in leads V1, V2, and V6 after pilsicainide administration (r = 0.51, 0.53, and 0.48, respectively, p<0.05).

CONCLUSIONS

The conduction delay in right ventricle (RV) was a useful marker for identifying high-risk patients in BS. The conduction delay at RV may be related to depolarization abnormality due to sodium channel dysfunction in BS.
Abstract 642: Effect of Acute and Chronic Stretch on the Pulmonary Veins in Humans: Implications for Arrhythmogenic Triggers
Authors: John Bobby , Brooks Anthony G , Kuklik Pawel , Chandy Sunil T , Sanders Prashanthan
Published: July 2021

Introduction
Stretch is characteristic of most conditions predisposed to atrial fibrillation (AF). However, there is no human data on effect of atrial stretch on pulmonary veins (PV), the dominant trigger of AF

Method
Two groups were studied; Group A (n=12; 31±7 yrs) undergoing left sided accessory pathway ablation and Group B (n=12; 29±7 yrs) undergoing percutaneous valvuloplasty for mitral stenosis (MVA 0.9±0.3 cm
2
). Multi-electrode basket catheter was positioned in the PVs to perform high-density mapping using NavX system to determine: proximal/ distal effective refractory period (ERP); conduction time (CT); intra-PV conduction block (CB; ≥30ms between adjacent poles) resulting in circuitous conduction during the drive (S
1
) and pre-excited stimulus (S
2
); and intra-PV voltage. Effect of acute stretch was determined in Group A by simultaneous RV and PV pacing and chronic stretch in Group B

Results
Twenty-four PVs were studied in each group. Acute stretch (Group A) resulted in ERP shortening (240±33 to 225±39 ms; p=0.03), prolonged CT with S
2
, which increased further with stretch (77±30 to 88±37ms; p=0.02). Intra-PV CB became prominent with S
2
and accentuated with stretch (40±23 to 59±32ms; p<0.0001). The changes were not site specific. Left atrial volume (p=0.001) and pressure (p=0.04) were elevated in Group B. Unlike acute stretch, ERP was prolonged, conduction delayed in sinus rhythm (p=0.005) and S
1
which further accentuated with S
2
(refer Table

). Intra-PV CB was seen at S
2
. Additionally, mean bipolar voltage was reduced (0.8[0.3–3.5] mV vs 1.4[0.8–3.7] mV; p=0.005)

Conclusion
Acute stretch results in abbreviation of PV ERP while chronic prolongs it. Nevertheless, both cause marked PV conduction abnormalities that are pronounced with chronic stretch and extra-stimuli. Additionally, structural remodeling was seen with chronic stretch. These abnormalities implicate stretch in the milieu for re-entry and PV arrhythmogenesis in conditions predisposed to AF

Electrophysiological characteristics