bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1354: Estrogen predisposes prepubertal Long QT Syndrome 2 (LQT2) rabbits to cardiac arrhythmias
Authors: Odening Katja E , Brunner Michael , Schofield Lorraine , Chaves Lenny , Peng Xuwen , Zehender Manfred , Kirk Malcolm , Koren Gideon
Published: July 2021

Introduction:
Adult women with LQT2 are at higher risk for clinical events and sudden cardiac death (SCD) than men. We have created transgenic LQT2 rabbits selectively over-expressing a pore mutant of the human HERG in the heart. All episodes of SCD in these LQT2 females occurred after sexual maturation. We hypothesize that sex hormones modulate cardiac repolarization and incidence of polymorphic VT in female LQT2 rabbits.

Methods:
In this ongoing study, prepubertally ovariectomized females were implanted with 90 day-release-pellets of estradiol (EST, 150mg), or dihydrotestosterone (DHT, 200mg), or placebo (OVX). A fourth group consisted of sham-operated females (SHAM). All groups underwent telemetric ECG monitoring, surface ECG after 30 days of hormone treatment, and
in vivo
electrophysiological studies (EPS) under isoflurane (2–5%) after 60 days.

Results:
EST steepened the QT/RR slope of prepubertal rabbits mimicking the adult LQT2 female phenotype (baseline: 0.608 ± 0.09, after 4 weeks: 0.69 ± 0.1, n=5, p<0.05; SHAM females: 0.699 ± 0.07, n=4). In contrast, DHT decreased the QT/RR slope (baseline: 0.739 ± 0.11, after 4 weeks: 0.521 ± 0.08, n=3, p=0.02). A smaller decrease was seen in OVX: 0.677 ± 0.01 vs. 0.599 ± 0.08, n=2. EST prolonged QT- and QTpeak-index compared to DHT rabbits. (EST, n=2: QT: 112.6% ± 1.5; QTp: 95.8% ± 1.6 vs. DHT, n=2: QT: 105.4% ± 4.1, p=0.07; QTp: 87.6% ± 0.9, p<0.05 vs. SHAM females, n=2: QT: 123.4% ± 9.5; QTp: 102.3% ± 2.5) EPS revealed a longer AERP and VERP in EST than in DHT rabbits - and a trend towards longer refractoriness than in OVX - reaching similarly prolonged refractoriness as in SHAM females (EST, n=2: AERP: 165.0 ± 35.4ms; VERP: 225.0 ± 25.0ms vs. DHT, n=3: AERP: 90.00 ± 5.8ms, p=0.03; VERP: 193.3 ± 8.8ms, p=0.07 vs. OVX, n=2: AERP: 125.0 ± 25.0ms, VERP: 205.0 ± 5.0ms, vs. SHAM females, n=2: AERP: 145.0 ± 5.0ms, VERP: 220.0 ± 20.0ms). One out of four EST-treated prepubertal rabbits died at the age of 96 days of spontaneous polymorphic VT.

Conclusion:
EST increases the QT/RR slope and prolongs QT and cardiac refractoriness mimicking the adult LQT2 female phenotype, whereas DHT decreases the QT/RR slope and shortens QT and refractoriness mimicking a male phenotype. EST predisposes prepubertal LQT2 rabbits to spontaneous polymorphic VT.
Abstract 1355: Determining Wavefront Size during Ventricular Fibrillation: Optical Mapping Study from Human Langendorff Hearts
Authors: Masse Stephane , Asta John , Sevaptsidis Elias , Cusimano Robert , Yau Terry , Ross Heather , Rao Vivek , Dhopeshwarkar Rajesh , Nanthakumar Kumar
Published: July 2021

Background:
We had recently established a model for the study of VF in humans using an optical mapping technique in Langendorff perfused human hearts. The optical mapping technique allows for the unequivocal evaluation of wavefront identification, the origin of wavefronts and their directional spread.

Objective:
The objective of this study was to measure wavefront sizes from different regions during human VF to determine the minimal spatial resolution required to study human VF in-vitro.

Methods:
The hearts were explanted from seven cardiomyopathic patients who underwent transplantation. The hearts were Langendorff perfused with Tyrode’s solution (95%O
2
+5%CO
2
) and the potentiometric dye (0.2 mg Di-4-ANEPPS dissolved in 20mg of perfusate) was injected as a bolus into the coronary circulation. The imaged region was physically immobilized to minimize motion. The fluorescence was excited at 530 nm with a HL 150 W halogen lamp, the emission signal was longpass filtered at 610 nm and recorded with a CCD and CMOS camera (MiCAM02, Ultima, BrainVision Inc., Japan). The spatial resolution depending on the mapping window was 0.65–0.85 mm and the temporal resolution was 1 kHz. The heart was paced at a cycle length of 600 ms and the VF was induced by briefly touching the heart with the two poles of a 9V battery. The changes in dye signal were recorded over 1887 ms and analyzed using BV-analyzer software (BrainVision Inc, Japan). Wavefronts were identified using high-pass filter (0.01 Hz) on the fluorescence data. Recordings were performed at 4 sites: LV Septum (LVS), Right Ventricle Endocardium (RV), Left Ventricle Endocardium (LVendo) and Left Ventricle Epicardium (LVepi).

Results:
16 episodes of VF were recorded and a total of 54 wavefronts were identified. Average wavefront size was 23.6 mm. By region, the average wavefront sizes were: LVS = 26.3 mm; RV = 22.4 mm; LVendo = 29.7 mm; LVepi = 19.3 mm.

Conclusion:
These results show that different regions on the human ventricle during VF show different wavefront size suggesting that propensity for wavebreak maybe spatially differentially distributed. To study human VF with multi-electrode mapping tools using inter-electrode spacing of less than 1–2 cm should provide sufficient resolution.
Abstract 1362: Degradation of Cardiac Troponins in Atrial Fibrillation is Calpain-dependent
Authors: Ke Lei , Dijkhuis Anne-Jan , Henning Robert H , Kampinga Harm H , Brundel Bianca J
Published: July 2021

Background:
The self-perpeptuation of atrial fibrillation (AF) is caused by myocyte remodeling including degradation of cardiac troponins. Additionally, we observed enhanced calpain activity in human AF and in the tachypaced cell model for AF. We assessed the hypothesis that calpain plays a role in the degradation of cardiac troponins during AF.

Methods:
The tachypaced HL-1 atrial myocyte model was used to investigate degradation of endogenous cardiac troponin T, I and C (cTnT, cTnI, cTnC) as well as actin by Western-blotting. To study degradation of human cTnT, myocytes were transfected with V5-C-human cTnT. Inhibitors of calpain, caspase and proteasome were applied to study the underlying mechanism for degradation. In vivo cTn degradation was studied in atrial tissue from AF and sinus rhythm (SR) patients by Western-blotting.

Results:
Tachypacing (P, 3Hz) significantly and gradually induced the degradation of cTnT (Fig A

), cTnI and cTnC, compared to control (C, 1Hz), while actin were unaffected. The degradation was prevented by calpain inhibitor PD150606 (20μM), whereas caspase (CAS) and proteasome (MG) inhibition was not effective (Fig C

). Tachypacing of V5-C-human cTnT transfected HL-1 myocytes resulted in a specific degradation fragment of 25kDa, which was prevented by PD (Fig B

). In vivo, persistent AF was associated with specific degradation of cTnT, I and C, compared to patients with SR or paroxysmal AF. Actin levels were not changed.

Conclusions:
AF induces specific degradation of all cTn isoforms, which is mediated by calpain. Activation of calpain may represent a key component in linking cTn degradation, contractile dysfunction and the self-perpetuation of AF.
Abstract 243: Roles of TNF alpha and MCP-1 in Type II Diabetes-induced Endothelial Dysfunction
Authors: Yang Jiyeon , Louise Abbott C , Zhang Cuihua
Published: July 2021

Tumor necrosis factor alpha (TNF) is reported to underlie a component of vascular inflammation and ensuing endothelial dysfunction in diabetes, but the role of inflammatory cells in these events is unknown. Because TNF reportedly induces monocyte chemoattractant protein-1 (MCP-1) expression, we hypothesized that the interaction between TNF and MCP-1 contributes to the evolution of vascular inflammation in diabetes. To test this hypothesis, expression of TNF and MCP-1, and formation of nitrotyrosine (N-Tyr, an indicator of peroxynitrite production) were determined in isolated coronary arterioles (50–100um) from genetically modified mice with Type II diabetes (Lepr
db
) and the lean control heterozygotes (m Lepr
db
). Protein and mRNA expression of TNF, protein expression of MCP-1, and presence of N-Tyr were significantly increased in arterioles from Lepr
db
mice compared to those from m Lepr
db
. Immunofluorescence double labeling revealed the prominent localization of MCP-1 to endothelium (Von Willebrand factor) of arterioles and venules. MCP-1 also co-localized with macrophages (CD68) in the heart tissue. To determine if MCP-1 may be key to the vascular inflammation and ensuing endothelial dysfunction, we administered anti-MCP-1 (10 μg/day, 3 days, i.p., n=4; to block MCP-1 signaling) in the mice and then determined endothelial dilation in isolated, cannulated pressurized (60 cmH2O) arterioles. Anti-MCP-1 restored dilation to acetylcholine in Lepr
db
mice by 35±2% (P<0.05 vs Lepr
db
mice without treatment, 9.8±4%) but did not affect dilation in m Lepr
db
mice. To establish interactions between TNF and MCP-1, we administered anti-TNF (72hr treatment; 0.625mg/kg, i.p.) to block TNF signaling and determined effects on MCP-1 and oxidative stress. After anti-TNF administration, expression of MCP-1 was decreased and numbers of MCP-1 positive cells were markedly reduced in Lepr
db
mice. Moreover, anti-TNF prevented the formation of N-Tyr in Lepr
db
mice. These findings demonstrate that the endothelial dysfunction occurring in type II diabetes is the result of the effects of the inflammatory cytokine TNF and TNF related signaling, including the expression of MCP-1, which also furthers the oxidative stress.
Abstract 1382: AlbuBNP (Cardeva), a Novel Recombinant Human B-Type Natriuretic Peptide Serum Albumin Fusion Protein Has Prolonged Renal Enhancing Properties When Compared to Human BNP.
Authors: Chen Horng H , Matrin Fernando L , Cataliotti Alessandro , Schirger John A , Burnett John C
Published: July 2021

BACKGROUND:
AlbuBNP is a novel recombinant human BNP serum albumin fusion protein which possesses a significantly longer elimination half-life compared to BNP. To date, it remains unclear if this novel protein which represents a single molecule of BNP synthesized with the carrier protein albumin can enter the post glomerular/tubular system due to the fusion of BNP to albumin and mediate renal actions. We hypothesized that this fusion protein will have potent renal actions and possess prolonged renal hemodynamic enhancing and excretory properties based upon its unique structure as compared to native BNP.

METHODS:
We compared the cardiorenal and humoral actions of intravenous(IV) bolus of administration of AlbuBNP (Cardeva, CoGenesys, Rockville MD) (5 mg/kg, n=7) and Human BNP (Phoenix Pharmaceutical, Belmont CA) (25 μ g/Kg, n=5) in two groups of normal anesthetized dogs. * p<0.05

RESULTS:
Single IV bolus of AlbuBNP resulted in a sustained increase in plasma cGMP (5±1 to 9±2 pmol/ml*) and urinary cGMP excretion (1136±113 to 2556±417 pmol/min*), markers of the biological activity of BNP, which remained elevated at the termination of the experiment at 270 minutes. In contrast, with human BNP, both plasma and urinary cGMP peaked at 30 minutes and returned to baseline by 150 minutes. In a similar fashion, there was a sustained increase in natriuresis (39±12 to 159±38 μEq/min*), diuresis (0.2±0.1 to 1.1±0.3 ml/min*), renal blood flow (220±22 to 301±33 ml/min*), and glomerular filtration rate (34±2 to 62±13 ml/min*) with AlbuBNP while with human BNP, these renal effects peaked at 30 minutes and returned to baseline by 150 minutes. Furthermore, there was a gradual and sustained suppression of plasma aldosterone (7.8±2 to 3.6±1 ng/dL*) with AlbuBNP. Likewise, there was a more sustained reduction of cardiac filling pressures with AlbuBNP as compared to human BNP.

CONCLUSION:
We report for the first time that this newly developed BNP/albumin fusion protein has a more prolonged and sustained renal enhancing properties compared to human BNP. Thus, AlbuBNP (Cardeva) represents a novel long-acting renal enhancing and aldosterone suppressing drug which has therapeutic potential for the management of cardiovascular and renal diseases that should be defined in further studies.
Abstract 1407: Interferon Regulatory Factor-9 (irf-9) Mediates Short Term Host Protection, But Promotes Long Term Immune Injury In Evolution Of Myocarditis Leading To Dilated Cardiomyopathy
Authors: Konviser Michael , Liu Youan , Chen Manyin , Shi Yu , Sun Mei , Gramolini Anthony , Fish Eleanor , Opavsky Anne , Liu Peter
Published: July 2021

INTRODUCTION: Evolution of viral myocarditis to dilated cardiomyopathy (DCM) represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. However, the influence of innate immunity in general and IRF-9 in particular on acquired immunity and DCM is unknown.

HYPOTHESIS: IRF-9 signaling provides immediate host protection through interferon production, but stimulates acquired immunity leading to DCM in a coxsackievirus murine myocarditis model.

METHODS: Interferon-regulatory factor 9 (IRF-9) homozygous knockout mice were generated, and show impaired type I interferon production. Wild-type (WT, n=51) and IRF-9
−/−
(n=124) littermates were inoculated with 10^2 p.f.u. of coxsackievirus B3 as previously described. Survival, viral titers, cardiac hypertrophy, inflammation and fibrosis were evaluated on days 0, 4, 7, 10, 14, 28 and 42. Splenocyte subpopulations were cell sorted and quantitated by FACS.

RESULTS: IRF9−/− mice showed dramatically increased mortality compared to the wild-type littermates (0% WT vs 72% IRF-9
−/−
on day 14, P<0.0001). On day 42, there was less cardiac hypertrophy and inflammation in IRF-9
−/−
mice compared to WT controls (p<0.05). There was no difference in fibrosis. The mature T-lymphocyte population, defined as CD4 or CD8 single positive, was statistically identical in the two populations up until and including day 28 post-infection. However on day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9
−/−
spleens (p<0.05).

CONCLUSIONS: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.
Abstract 1413: Interleukin 13 Exerts Anti-appoptotic Effects On Hypoxic Cardiomyocytes Through Pi3 Kinase In The Absence Of Hypertrophic Effects
Authors: Poeling Jochen , Kubin Thomas , Rees Wolfgang , Hein Stefan , Shim Gil-Jin , Warnecke Henning , Braun Thomas
Published: July 2021

Objectives: Recent research has focussed on anti-inflammatory cytokines (a-ICs) as cardio-protective agents. A-ICs were mainly considered in the context of inhibition of secretion of pro-ICs such as tumour necrosis factor-α and with the exception of transforming growth factor β little is known about direct effects of a-ICs on cardiomyocytes (CMC).

Methods: To clarify a direct functional relationship of a-ICs we evaluated effects of interleukin-4, -10 and -13 on cultured CMC by planimetry, confocal microscopy (CF), protein synthesis determination (PS), 2DE-analysis (2DE), mass spectrometry and Western blotting. Intracellular pathways were analysed with specific chemical inhibitors. Anti-appoptotic effects were evaluated by determinating the cell number, cleaved caspase 3 (cC3) and PARP (cP) under 2% O
2
. Untreated cell lines (unCO) or leukemia inhibitory factor (LIF) treated CMC served as controls.

Findings: In contrast to Il-13 we did not detect effects of Il-4 and Il-10 on CMC. Il-13 treated CMC showed a 40% (p<0.01) increased viability over unCO under normoxic and 65% (p<0.01) under hypoxic conditions after 4d and 12h, respectively. Under hypoxia antiapoptotic effects were visible in 60% reduction of cC3 (60%; p<0.05) and cP (40%; p<0.05). Using a battery of more than 300 phospho-specific antibodies we found that phosphorylation of Akt (5-fold, p<0.01) was the predominant activated pathway but we found also mild increases in P-MEK1/2 (1.7-fold). The antiapoptotic effects and Akt activation were abolished in the presence of the PI3 kinase inhibitor (20μM) LY294002 (p<0.01) but not in the presence of the MEK1/2 inhibitor (5μM) UO126. In contrast we did not find any significant morphological changes of CMC in the presence of Il-13 neither in the protein composition of structural proteins (2DE, CF), nor increases in PS nor changes in cell surface area, width and length opposing hypertrophic responses visible after LIF treatment.

Conclusions: These findings indicate that Il-13 can directly support myocyte function in addition to its inactivation of immune cells. Our results and the existence of a Il-13 receptor in the heart altogether throws light on Il-13 as a promising therapeutical target.
Abstract 1418: Receptor For Advanced-glycation End Products Inhibits Myocardin Function And Induces Msx2-dependent Osteoblastic Differentiation Of Vascular Smooth Muscle Cells
Authors: Suga Toshihiro , Iso Tatsuya , Shimizu Takehisa , Tanaka Toru , Yamagishi Shoichi , Arai Masashi , Imaizumi Tutomu , Kurabayashi Masahiko
Published: July 2021

As an activator of diverse signaling pathways in the vascular cells, receptor for advanced-glycation end products (RAGE) has been related to atherosclerosis initiation and progression in the patients with diabetes and chronic renal failure (CRF). However, little is known about the efects of RAGE activation on the differentiation program of vascular smooth muscle cells (VSMC). We first examined effects of RAGE on expression of myosin heavy chain (MHC), a definitive VSMC marker. RT-PCR analyses showed that expression of MHC was diminished in VSMC transduced with adenovirus expressing RAGE. A RAGE ligand HMGB1, but not another ligand AGE2, enhanced RAGE-dependent MHC repression. Activation of HMGB1-RAGE pathway induced the endogenous RAGE expression, suggesting positive feedback loop in atherosclerotic lesion. In mesenchymal 10T1/2 cells, HMGB1-RAGE inhibited an induction of MHC gene expression by myocardin, a potent positive regulator of VSMC differentiation. We next examined the effects of RAGE on osteogenic conversion of VSMC by monitoring the expression and activity of alkaline phosphatase (ALP). RT-PCR analysis showed that expression of ALP was up-regulated in VSMC when RAGE was overexpressed by adenovirus. The degree of the ALP induction was dependent on the concentration of fetal bovine serum, suggesting that fetal bovine serum contains RAGE ligands. The induction of ALP was accompanied by ALP activity and an inducible expression of osteogenic transcription factors, Msx2 and Runx2 as well as an increase in osteopontin and bone morphogenic protein 2 (BMP2) expression. We further found that addition of BMP2 and RAGE synergistically enhanced expression of ALP mRNA and ALP activity. Immunohistochemistry revealed that RAGE antigen is present in calcified atherosclerotic plaque in a distribution overlapping with osteoblastic markers and calcium deposition detected by Kossa staining in human carotid artery. These findings suggest that RAGE induction in VSMC contributes to the development of calcifying atherosclerotic plaque characterizing the diabetes- and CRF-associated vascular lesion by repressing myocardin-induced VSMC differentiation and inducing Msx2-dependent osteogenic differentiation.
Abstract 1429: The Proline-Rich Tyrosine Kinase PYK2 Regulates NO Production by eNOS under Physiologic and Pathophysiologic Conditions.
Authors: Loot Annemarieke E , Fisslthaler Beate , Busse Rudi , Fleming Ingrid
Published: July 2021

In endothelial cells, the proline-rich tyrosine kinase (PYK2) is activated by fluid shear stress and phosphorylates the endothelial NO synthase (eNOS) on tyrosine residue 657. We have previously shown that eNOS phosphorylation on Tyr657 inhibits NO production in vitro and therefore addressed the consequences of PYK2 activation in a more physiological situation i.e., in perfused isolated carotid arteries as well as in response to other stimuli. Pressurised perfused murine carotid arteries subjected to step-wise increases in luminal flow responded with a graded endothelium-dependent vasodilatation that was partially sensitive to NOS inhibition. Carotid arteries in which the endothelium was infected with an adenovirus expressing a dominant-negative PYK2 mutant displayed normal flow-dependent vasodilatation (maximal response: R
max
49 ± 6 μm) that was sensitive to the NOS inhibitor L-NAME (R
max
28 ± 2 μm). However, flow-induced vasodilatation was markedly reduced in arteries overexpressing wild-type PYK2 and was insensitive to L-NAME (R
max
27 ± 4 μm and 28 ± 5 μm, respectively) indicating loss of the NO-dependent component of vasodilatation. Given that angiotensin II is known to stimulate PYK2 phosphorylation and activation in smooth muscle cells we assessed the effects of angiotensin II on endothelial PYK2 activation and NO-dependent vasodilatation. We observed a time-dependent phosphorylation of PYK2 immuno-precipitated from porcine aortic endothelial cells stimulated with angiotensin II for 30 min up to 24 hrs. This correlated with an angiotensin II-induced decrease in NO-dependent vasodilatation. These data indicate that under physiological conditions, the tyrosine phosphorylation of eNOS by PYK2 in response to fluid shear stress attenuates the activity of the enzyme and limits NO-mediated flow-dependent vasodilatation. This PYK2-dependent inhibition of NO production may at least partially limit the detrimental consequences of maintained high NO output e.g. the generation of peroxynitrite. Conversely, under pathophysiological conditions in which angiotensin II levels are elevated, the increased activity of PYK2 can contribute to endothelial dysfunction by reducing eNOS activity.
Abstract 1433: The Cardioregulatory Peptide Apelin is Preferentially Expressed by Endothelial Cells and Upregulated in Myocardial Disease States
Authors: Kundu Ramendra K , Sheikh Ahmad Y , Ho Michael Y , Chun Hyung J , Huynh Diem T , Ali Ziad A , Ransohoff Katherine J , Connolly Andrew J , Ashley Euan A , Robbins Robert C , Quertermous Thomas
Published: July 2021

Introduction:

The APJ receptor and its ligand, apelin, comprise a homeostatic, cardio-regulatory pathway. Although cardiac apelin expression levels are altered in humans with cardiac failure, the cell type responsible for apelin expression and modulation in disease states remains unknown.

Hypothesis:

Apelin production is restricted to the endothelial compartment and is upregulated in states of cardiovascular stress.

Methods:

Transgenic apelin-LacZ reporter mice (SVJ background) were created by insertion of the nuclear localizing bacterial LacZ gene immediately downstream of the apelin promoter. Mice (n=12) were randomized to left anterior descending coronary artery (LAD) ligation, thoracic aortic constriction (TAC) or sham groups. Hearts were harvested 3 and 8 weeks post-TAC or LAD ligation, respectively. Localization of apelin expression was determined by Xgal staining. Endothelial phenotype of lacZ positive cells was confirmed by CD31 co-staining. Apelin expressing cells were quantified by histology. Apelin reporter results were confirmed by quantitating apelin expression in WT animals following LAD ligation (n=11) or sham (n=11) procedure by RT-PCR.

Results:

Extensive immunohistochemistry studies of heart tissue revealed lacZ reporter gene expression to be restricted to the coronary venous and capillary endothelium, with no expression by cardiomyocytes. Following both LAD ligation and TAC, the number of LacZ-apelin (+) endothelial cells significantly increased (p<0.002) in all chambers of the heart (Table

), with no evidence of apelin expression by other cell types. Evaluation of WT hearts by RT-PCR for the apelin gene confirmed the reporter gene findings with 1.3±0.3 fold increase (p<0.05) of apelin expression induced by LAD ligation compared to sham.

Conclusions:

Apelin is primary expressed by endothelial cells within the heart and is upregulated in response to myocardial stress.

Apelin-LacZ Expressing Endothelial Cells are Increased Following Myocardial Injury
Abstract 1464: Cardiac Myosin Binding Protein-C (cMyBP-C) Directly Modulates Actomyosin Binding and Kinetics Independent of LMM and S2 Binding.
Authors: Saber Walid , Begin Kelly J , Warshaw David M , VanBuren Peter
Published: July 2021

BACKGROUND:
While mutations in cMyBP-C constitute a common cause of FHC, its role in sarcomere contraction remains unclear. cMyBP-C binds to actin, titin and the S2 and LMM proteolytic domains of myosin. Through its numerous binding interactions cMyBP-C may act as a tether, restricting myosin and/or actomyosin function. We directly tested this hypothesis in the in vitro motility assay using either whole myosin or the myosin subfragments, HMM and S1 (which lack LMM and LMM-S2, respectively).

METHODS AND RESULTS:
The motility assay is an in vitro model of muscle contraction in which thin filaments are propelled across a myosin coated surface. The addition of cMyBP-C to the motility assay resulted in a concentration dependent reduction in actin filament velocity when using either whole myosin, HMM or S1, demonstrating that cMyBP-C inhibits thin filament velocity independent of LMM or S2 binding. Using whole myosin and thin filaments reconstituted with troponin/tropomyosin, the addition of cMyBP-C resulted in a 29% reduction in maximal velocity (P=0.002) with no effect on maximal force. At sub-maximal calcium, the pCa50 for velocity was increased (6.64 ± 0.06 vs. control, 6.44 ± 0.03, P=0.003) whereas the pCa50 for force was decreased (6.25 ± 0.09 vs. control, 6.55 ± 0.02, P=0.008). Thin filament activation by myosin strong-binding demonstrated an increased amount of myosin required to half maximally activate the thin filament in the presence of cMyBP-C, indicating that myosin binding to the thin filament is reduced with cMyBP-C. These findings were supported by co-sedimentation experiments which demonstrate that cMyBP-C competes with S1 for actin binding in the presence of ATP, with no effect on S1/actin binding in the absence of ATP. Finally, while the number of cross-bridges interacting with the thin filament is rate limiting for velocity at shorter filament lengths, this was not observed at longer filament lengths indicating that cMyBP- C directly modulates the kinetics of actomyosin.

CONCLUSIONS:
The effects of cMyBP-C on velocity and force demonstrate that cMyBP-C does not simply act as a tether but likely affects both the kinetics and the recruitment of myosin cross-bridges through its direct interaction with the myosin head and/or the actin filament.
Abstract 1469: Diastolic dysfunction induced by Flecainide but not Amiodarone
Authors: Bisping Egbert , Pagel Christian , Wilken Andre , Toischer Karl , Pieske Burkert
Published: July 2021

Diastolic dysfunction is a significant risk factor for the development and progression of atrial fibrillation. Flecainide (Flec) and Amiodarone (AM) are frequently used in patients with atrial fibrillation but their impact on diastolic function has not been evaluated yet. We tested the effect of Flec and AM on systolic and diastolic performance in isolated muscle strips from failing human and nonfailing rabbit myocardium. Isolated ventricular trabeculae contracted isometrically at 1 Hz, Ca2+ 2.5 mmol/L, 37°C. Flec (0.01 – 100 μmol/L, dissolved in water) showed a concentration dependent negative inotropic effect in human myocardium (13 ± 2 vs. 3 ± 0.5 mN/mm
2
at base vs. 100 μmol/L; p< 0.05). This was associated with a significant prolongation of the relaxation time RT95 and an increase of diastolic tension (Dias) by 35 ± 9 % (at 100 μmol/L; p< 0.05). Water alone had no effect. Calcium transients measured by Aequorin technique declined proportionally to developed force after Flec. In contrast, AM (0.01 – 100 μmol/L, dissolved in 2% benzyl alcohol and 10% polsorbate) showed identical negative inotropic effects to solvent alone (maximally by 16 ± 8 %), and neither AM nor its solvent affected diastolic tension or relaxation times. Flec (3 μmol/L) resulted in a significant impairment of the Force frequency relationship (FFR) at 0.5–3.0 Hz in human myocardium. This was related to a decline in systolic force and a rise in Dias at high frequencies (at 3 Hz by 32 ± 12 % in control and 87 ± 25 % after Flec, p < 0.05 vs. control). In nonfailing rabbit myocardium (1.0–5.0 Hz) Dias decreased by 11 ± 10 % (n.s.) in control but raised by 65 ± 25 % after Flec, p < 0.05). AM (100 μmol/L) had no significant effect on FFR, whereas its solvent tended to impair the FFR by a decline in systolic performance.

Conclusion: Flec exerts calcium dependent negative inotropic effects in human myocardium and significantly impairs diastolic function. The latter is observable not only in human failing myocardium with preexisting diastolic dysfunction but also in nonfailing animal myocardium. In contrast AM shows no compound specific negative inotropic effect and no change in diastolic function. In patients treated with Flec attention should be turned to the potential of the drug to deteriorate diastolic function.
Abstract 1472: Genetic Deficiency in Caveolin-3 Alters Stress Hormone-activated Membrane Signaling in Mouse Hearts
Authors: Doi Masahiro , Zhao Youyang , Hayashi Takeharu , Dalton Nancy , Gu Yusu , Peterson Kirk L , Hoshijima Masahiko
Published: July 2021

Caveolin-3 is a primary molecular component that forms caveolae, flask-shaped plasma membrane domains in cardiac and skeletal myocytes. Caveolae provide a critical micro-environment to receptors, ion channels and other membrane-bound molecules. In this study, we established a new caveolin-3 knockout (cav3−/−) mouse line and tested whether disruption of caveolin-3 alters the regulation of cardiac excitation-contraction coupling (ECC) by G-protein coupled receptor (GPCR) agonists, including beta-adrenergic agonists and urocortin 2 (Ucn2), a member of the corticotropin-releasing factor, which we have shown to have strong inotropic and lusitropic actions in normal and failing hearts (Bale TL. PNAS. 2004, 101: 3697–3702). We tested baseline phenotypes of our cav3−/− mice by echocardiography at 8, 20, 35 weeks old, as well as challenged with trans-aortic banding for 1 week and confirmed there is no overt cardiac dysfunction developed in these mice, in which missing of caveolae in cardiomyocytes was confirmed by electron microscopy. Cav3−/− mice (10–16 weeks old) and age-matched control C57BL/6 mice (wild type; WT) were first tested in in vivo hemodynamic studies. No obvious difference was observed at baseline between 2 groups. Ucn2 strongly enhanced cardiac contractility in WT. However, the response to Ucn2 was attenuated in cav3−/− (max dP/dt: 18869+−851 mmHg/sec in WT vs. 16797+−1891 in cav3−/−, p=0.01, min dP/dt: −10023+−1023 in WT vs. −8376+−716 in cav3−/−, p<0.01 (ANOVA), n=3). We also tested single adult cardiomyocytes isolated from both animal groups. Consistently with hemodynamic results, Ucn2-dependent enhancement of cell shortening was significantly suppressed in cav3−/− (% sarcomere length shortening, %SLS: 15.17+−3.35% in WT vs. 9.78+−3.41% in cav3−/−, p<0.01, n=26). In contrast, beta2 stimulant (combination of isoproterenol and CGP20712A) response was augmented in cav3−/− (%SLS: 7.44+−2.66% in WT vs. 9.65+−2.92% in cav3−/−, p=0.011, n=21). Calcium transient analyses with Fura2 supported these data. We conclude that caveolin-3 associated membrane micro-environment is critical for normal GPCR signaling and speculate their disease-related alterations affect ECC regulation by various stress-hormones.
Abstract 1476: Angiotensin II Induced Hypertrophic Response And Oxidative Stress Is Attenuated In Mice Lacking The Gene For Tnf-α
Authors: Sriramula Srinivas , Mariappan Nithya , McILwain Elizabeth , Francis Joseph
Published: July 2021

Tumor necrosis factor-alpha (TNF-α) and angiotensin II (Ang II) play an important role in the pathophysiology of cardiovascular disease in part by inducing the cardiac hypertrophic response and oxidative stress. Recently we demonstrated that angiotensin induced hypertensive response is attenuated in mice lacking the gene for TNF-α. In this study, we examined whether Ang II induced cardiac hypertrophy and increased oxidative stress is mediated through TNF-α.

Methods and results:
Male TNF-α (−/−) and age matched control (WT) mice were subcutaneously implanted with osmotic minipumps containing Ang II (1 μg/kg/min) or saline for 14 days. Human recombinant TNF-α was injected in one group of TNF-α (−/−) mice (10 μg/kg/day) for 14 days. In WT+Ang mice, a temporal increase in blood pressure was observed during the study as measured by radio telemetry transmitters. At the end of the study, echocardiography revealed an increase in thickness and dimensions of left ventricle (LV) and decreased fractional shortening (%FS) in WT+Ang mice. Real time RT-PCR showed that Ang II- infusion resulted in an increase in heart/bodyweight ratio and of cardiac hypertrophy markers ANP and BNP, and profibrotic genes Collagen Type I, Collagen Type II, and TGF-β in WT mice. Electron Spin resonance spectroscopy revealed an increase in total ROS, superoxide and peroxynitrite in the WT+ANG mice when compared to control WT mice. However, these changes were all attenuated in TNF-α (−/−)+Ang mice. Ang II infusion also increased significantly the mRNA expression of gp91Phox, NOX-1, NOX-4 and AT1R in the LV of WT mice, but not in TNF-α (−/−) mice. Interestingly, injection of TNF-α in the TNF-α (−/−) mice, treated with Ang II resulted in increased cardiac hypertrophy and oxidative stress.

Conclusions:
Findings from the present study suggest that TNF-α plays an important role in the development of cardiac hypertrophy and oxidative stress in Ang II-induced hypertension.
Abstract 1497: Differential Modulation of Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathways by Tetrahydrobiopterin Synthesis vs. Salvage
Authors: Sugiyama Toru , Kou Ruqin , Michel Thomas
Published: July 2021

Tetrahydrobiopterin (BH4) is an essential redox-active cofactor for eNOS that can be both synthesized de novo or salvaged by enzymatic reduction of the oxidized compound. The endothelial dysfunction associated with diabetes is accompanied a decrease in the abundance of bioactive BH4. De novo biosynthesis of BH4 is catalyzed by GTP cyclohydrolase-1 (GTPCH1); recycling of BH4 is catalyzed by dihydrofolate reductase (DHFR). The relative roles of de novo BH4 synthesis and BH4 redox recycling in regulation of eNOS bioactivity remain incompletely defined. In the present study, we have used siRNA methods to investigate the effects of BH4 “knockdown” on eNOS regulation and endothelial signal transduction pathways in bovine aortic endothelial cells. We transfected duplex siRNA constructs designed to specifically target DHFR and GTPCH1, and suppressed levels of these proteins by ~90% (n = 37) relative to control siRNA-transfected cells. Transfection of siRNA constructs targeting DHFR or GTPCH1 suppressed VEGF-induced eNOS activity (using [
3
H]-citrulline assay) or NO production (using an electrochemical NO sensor) by 90 ± 9% (n = 8, p < 0.01). siRNA-mediated knockdown of either DHFR or GTPCH1 had no effect on the abundance of stability of eNOS dimers, assessed using low-temperature SDS-PAGE (n = 4). DHFR knockdown completely blocked VEGF-induced eNOS dephosphorylation at the inhibitory phosphoserine residue 116 (n = 4, p < 0.01), but had no effect on agonist-modulated eNOS phosphorylation at the activating phosphoserine residue 1179. GTPCH1 knockdown had no effect either on phosphorylation or dephosphorylation of eNOS at these residues. Phosphorylation of Akt was decreased by 85 ± 4% by DHFR knockdown (p < 0.001, n = 4) but Akt phosphorylation was unaffected by GTPCH1 knockdown. These studies demonstrate for the first time a striking contrast in the consequences for eNOS signaling pathways from the suppression of BH4 salvage/reduction vs. de novo BH4 synthetic pathways. The abrogation of VEGF-mediated Akt activation by siRNA-mediated DHFR knockdown indicates that alterations in BH4 recycling may have broad effects on cell signaling pathways, with important consequences for the development of endothelial dysfunction in vascular disease states.
Abstract 260: Autophagy And Necrosis Underlie Polyglutamine Amyloid Oligomer Induced Heart Failure.
Authors: Pattison Scott , Sanbe Atsushi , Klevitsky Raisa , Osinska Hanna , Robbins Jeffrey
Published: July 2021

Introduction:
Amyloid oligomers, the entities believed to cause toxicity in many neurodegenerative diseases, have been observed in mouse and human heart failure samples. Amyloid oligomers are a diverse group of proteins that differ in sequence, but share a common conformational structure, and may impart a shared pathogenic mechanism. The purpose of this study was to test the hypothesis that expression and accumulation of amyloid oligomers are cytotoxic and sufficient to directly cause heart failure. Polyglutamine (PQ) repeats (>50) are known to form amyloid oligomers and induce toxicity in neural cells, causing Huntington′s and other neurodegenerative diseases, while shorter PQ peptides are benign.

Hypothesis:
Cardiomyocyte-autonomous expression of an exogenous PQ amyloid oligomer will be toxic to cardiomyocytes and result in heart failure.

Methods:
Transgenic mice were created with cardiomyocyte-specific expression of an amyloid oligomer forming peptide of 83 PQ repeats (PQ83) or a non-amyloid forming peptide of 19 PQ repeats (PQ19) as a non-pathological control. Both constructs were HA tagged.

Results:
A PQ83 line with relatively low levels of expression was generated, along with a PQ19 line that expressed at approximately 9-fold the levels observed in the PQ83 line. Hearts expressing PQ83 develop cardiac dysfunction and dilation by 5 months and exhibit 100% mortality by 8 months of age, whereas PQ19 mice have normal cardiac function, morphology and lifespan. PQ83 protein accumulates in cardiomyocytes as SDS-insoluble aggresomes with amyloid oligomer-positive staining. PQ83 hearts exhibited no signs of apoptosis. Ultrastructural analysis revealed that PQ83 hearts undergo autophagy, as evidenced by increased autophagosomal and lysosomal content. PQ83 hearts also show characteristics of necrotic death, including infiltration of inflammatory cells, cardiomyocyte vacuolization and increased staining for the membrane attack complex that causes sarcolemmal permeabilization. The data confirm the hypothesis that expression of an exogenous amyloid oligomer is toxic to cardiomyocytes and is sufficient to cause heart failure. The pathogenic mechanism appears to lead to cardiomyocyte death through autophagy and necrosis.
Abstract 1526: Presence of Chronic Kidney Disease is Associated with Enhanced Inflammatory Response, Oxidative Stress and Adverse Left Ventricular Remodeling after Reperfused Acute Myocardial Infarction
Authors: Naito Kotaro , Anzai Toshihisa , Yoshikawa Tsutomu , Kaneko Hidehiro , Jo Yusuke , Ueno Koji , Kohno Takashi , Takahashi Toshiyuki , Kawamura Akio , Ogawa Satoshi
Published: July 2021

Background:
Patients with chronic kidney disease (CKD) have worse clinical outcome after acute myocardial infarction (AMI). We sought to determine the prognostic significance of CKD in patients with reperfused AMI in relation to left ventricular (LV) remodeling.

Methods:
We examined 120 consecutive patients with reperfused first anterior AMI, admitted within 24 hours of the onset. Glomerular filtration rate (GFR) was estimated by MDRD equation using serum creatinine level on admission. Patients with hemodialysis were excluded. Patients were divided into 2 groups according to the presence or absence of CKD, defined as GFR < 60 ml/min/1.73m
2
. Serum creatine kinase (CK) and C-reactive protein (CRP) levels were serially measured (q. 6, q. 24 hours). Left ventriculography along with plasma interleukin-6 (IL-6) and oxidized low-density lipoprotein (oxLDL) measurements were performed on admission and 2 weeks after AMI. Patients were followed for 27 ± 23 months.

Results:
Mean GFR was 74 ± 24 ml/min/1.73m
2
and CKD was observed in 25% (n = 30). The prevalence of cardiovascular risk factors, medication and coronary angiographic findings did not differ between the 2 groups. Peak CRP was higher in patients with CKD than those without (p = 0.011), despite similar peak CK. Patients with CKD had higher incidence of in-hospital cardiac death (p = 0.048) and major adverse cardiac events (MACE; p = 0.021), including in- and out-of-hospital cardiac death, non-fatal MI, reintervention, coronary artery bypass grafting and readmission for heart failure, than those without. CKD was associated with greater LV end-diastolic (p = 0.0002) and end-systolic volume (p = 0.0003) and lower LV ejection fraction (p = 0.013) 2 weeks after AMI. Plasma IL-6 (p = 0.037) on admission, and brain natriuretic peptide (p = 0.019) and oxLDL (p = 0.029) 2 weeks after AMI were higher in patients with CKD than in those without. CKD, as well as diabetes mellitus, was an independent predictor of MACE by Cox proportional hazards model analysis (relative risk = 3.13, p = 0.001).

Conclusion:
CKD was associated with a worse clinical outcome and infarct expansion through modification of inflammatory response and oxidative stress, suggesting an important role of CKD in the development of LV remodeling after reperfused AMI.
Abstract 1528: Gender Differences In The Effect Of Traditional Cardiac Risk Factors On Age At Presentation With Stemi
Authors: Herzog William , Aversano Thomas
Published: July 2021

For coronary artery disease (CAD), female gender is ’protective’, so that women typically present with clinically apparent CAD a decade later than men. We examined the extent to which traditional cardiovascular risk factor influence the age at presentation with STEMI in men and women. The Cardiovascular Patient Outcomes Research Team (C-PORT) primary PCI registry includes 7197 patients (5070 males and 2109 females) who presented with STEMI at 33 participating hospitals. The table

below depicts the average age at presentation with STEMI in males and females with and without diabetes, hypercholesterolemia, hypertension, a family history of coronary artery disease and smoking history (current or former). The effect of smoking, family history and hypertension on age at presentation remained significant in multivariate analysis in both men and women. In both males and females, a family history of CAD and a positive smoking history are associated with presentation with STEMI at a younger age. Both have a greater effect in females. This is particularly true of smoking with lowers the age of presentation by 9 years in women, compared with 3.8 years in men. Male and female patients with a history of hypertension are older at presentation with STEMI, perhaps because the anti-ischemic effects of anti-hypertensive medications. We conclude that while the effect of most traditional risk factors for CAD on age at presentation with STEMI are similar in men and women, smoking lowers the age at presentation to a much greater degree in women. In women who do not smoke, STEMI is delayed for a decade or more compared to men; for women who do, the protective effect of female gender is nearly obliterated.
Abstract 1531: Altered Brain Fiber Tracts in Heart Failure
Authors: Woo Mary A , Macey Paul M , Kumar Rajesh , Serber Stacy L , Cross Rebecca , Fonarow Gregg C , Harper Ronald M
Published: July 2021

Introduction:
Heart failure (HF) patients show neuronal brain injury which contributes to cognitive, affective, and autonomic symptoms. However, earlier studies did not consistently report injury within cognitive sites (e.g., frontal cortex) or limbic structures linked to affective and autonomic function. The inconsistent signs of injury in these neuronal structures may result from compromised communication between cognitive and limbic regions from damaged axons.

Methods:
We studied axon integrity between these regions using tract-based spatial statistics (TBSS; allows study of white matter tracts) in 17 HF (age 54 ± 8 years; 12 males; LVEF 0.28 ± 0.07) and 51 control subjects (age 50 ± 7 years, 29 males). We collected diffusion tensor imaging data using a 3T MRI scanner, from which we calculated fractional anisotropy (FA), an index of axonal integrity. Whole-brain FA “maps” for each subject were processed using TBSS, giving estimated axonal tracts co-localized across subjects. FA values throughout the tracts were compared between groups using 2-sample t-tests, controlling for multiple comparisons by permutation testing (form of randomization test).

Results:
Fiber tracts in HF showed reduced FA values across multiple brain regions (Figure

), including the anterior corona radiata (projecting to and from frontal cortex) and the superior and inferior cingulum bundle (communicating to and from the cingulate, hippocampus and other limbic regions), and cerebellum (dampens blood pressure changes).

Discussion:
Cognitive and affective symptoms in HF patients may arise from a combination of damaged structural areas and poor communication between brain regions.

Regions of reduced fiber tract integrity (lower FA) in 17 HF versus 51 control subjects indicated in white (
p
< 0.05, false discovery rate correction for multiple comparisons). Background image is a reference anatomical scan, with detected fiber tracts as found with TBSS in black. Orientation: images left of image is left of person; locations relative to standard reference (anterior commusure is origin): from left to right, 48 mm anterior, 7 mm left, 39 mm posterior.
Abstract 1534: Diabetes Mellitus Is An Independent Predictor Of Mortality In Defibrillator Recipients
Authors: Chen-Scarabelli Carol , Scarabelli Tiziano M
Published: July 2021

Background: Previous studies have shown an increased morbidity and mortality risk in patients with diabetes mellitus (DM) undergoing percutaneous coronary interventions and cardiac surgery. However, the impact of DM on mortality in patients with implantable cardioverter defibrillators (ICDs) is unknown.

Aim: to investigate whether DM was associated with increased mortality risk in patients with ICDs

Methods: A retrospective review of all ICD patients followed within a consecutive 2.5 year period was performed. Baseline characteristics and mortality data were compared between DM and non-DM patients. Mann Whitney test was used to analyze the differences between the means, and multivariate logistic regression analysis was performed to identify independent predictors of mortality.

Results: A total of 336 patients with ICDs (including 141 patients with DM and 195 without DM) were studied. The mean age was 66.8 ± 9.6 years in the overall population, with no significant difference in baseline characteristics between the 2 groups, except for beta-blocker therapy, creatinine, and estimated glomerular infiltration rate (EGFR). Use of beta-blockers was greater in the non-DM group (92.8% vs 84.4%, p=0.02). Renal function was worse in the DM group, as documented by a higher serum creatinine (1.6 ± 0.8 mg/dl in DM vs 1.38 ± 0.74 mg/dl in non-DM, p = 0.003) and a lower EGFR (55.9 ± 22.9 ml/min/1.73m
2
vs 63.5 ± 19.9 ml/min/1.73m
2
, p=0.0005). Although the incidence of VT was not significantly different between the two groups, there was a significantly higher mortality in the DM group (27.7% vs 11.3% in non-DM, p=0.0002). On multivariate stepwise logistic regression analysis, DM was a significant independent predictor of mortality (OR 2.38, 95% CI 1.3– 4.36, p =0.005).

Conclusion: DM is an independent predictor of mortality in ICD patients. Prevention of DM, as well as early diagnosis and intervention, should be emphasized in order to reduce the growing epidemic of type 2 DM and its associated complications.