bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 234: Expression of Activated PKCϵ Accelerates the Transition to Heart Failure in Response to Increased Hemodynamic Load
Authors: Yuzhakova Milana , Geenen David L , Goldspink Paul H
Published: July 2021

Expression of constitutively active PKCϵ in the myocardium promotes a steady progression to a dilated cardiomyopathy over time. Functional and biochemical studies demonstrate that the myofibrillar proteins are a target of PKCϵ mediated phosphorylation. In this model, changes in myofibrillar protein phosphorylation at 3-months are associated with compensatory mechanisms, whereas at 9 and 12-months are associated with the transition to failure. To test the hypothesis that PKCϵ phosphorylation alters the post-translational modification of the contractile proteins and that these changes predispose the heart to failure, we imposed a hemodynamic load on young mice (3-mice). Both transgenic (TG) and non-transgenic (NTG) mice were banded (AoB) for 10 weeks before assessment of cardiac function
in vivo
, gene expression and 2-D electrophoresis of myofibrillar extracts. Analysis of heart weight to body weight ratios (3mNTG=4.3±0.4 vs. 3mTG=4.2±0.3 vs. 3mNTG(AoB)= 5.9±0.3* vs. 3mT-G(AoB)=7.4±0.6** *p<0.05), shows hypertrophy in both groups of AoB mice, but to a greater degree in the TG (AoB). Gene expression analysis using quantitative qRT-PCR shows significant increases in both ANF and β-MHC mRNA in AoB mice compared with non-banded groups. Expression was significantly greater in the TG (AoB) along with a decrease in SERCA mRNA. Banding induced both diastolic and systolic dysfunction in TG compared to NTG hearts. Maximal −dP/dt in the TG (AoB) hearts was significantly lower compared to all other groups (3mNTG=9433±1000 vs. 3mTG=9474±857 vs. 3mNTG(AoB)=8620±1264 vs. 3mT-G(AoB)=4911±738* mmHg/sec, *p<0.05). Load-independent measurements revealed a significant decreases the time constant of isovolumic relaxation (3mNTG(AoB)=5.4±0.6 vs. 3mTG(AoB)=8.4±1.5* ms). In addition, maximal +dP/dt-end diastolic volume relation (3mNTG(AoB)=213±64 vs. 3mTG(AoB)=79±20*, p<0.05) was significantly depressed in banded TG compared to banded NTG. 2-D gel analysis of myofibrillar proteins revealed hyper-phosphorylation of troponin I in the PKCϵ TG mice. These data suggest that while the TG hearts have compensated to an initial insult (PKC activation) imposition of a second insult (hemodynamic load) accelerates the transition to failure.
Abstract 314: Inactivation-Resistant GSK3αβ Dual Isoform Expression Attenuates Isoproterenol-Induced Myocardial Hypertrophy and Preserves Cardiac Function
Authors: Webb Ian G , Nishino Yasuhiro , Clark James E , Murdoch Colin E , Shah Ajay M , Marber Michael S
Published: July 2021

Glycogen synthase kinase-3 (GSK-3) is implicated as an important signalling mediator in normal developmental myocardial growth and adverse hypertrophic remodelling. Upstream phosphorylation of isoform specific N-terminal serine residues - ser21(GSK-3α) and ser9(GSK-3β), respectively - inhibits kinase activity. Opposing effects of the two isoforms have recently been described in response to hypertrophic stimuli, but little is known of the overall effect of dual-isoform manipulation. We set out to characterise baseline and isoproterenol (ISO) stimulated phenotypes of mice with inactivation-resistant GSK-3αβ knockin (KI) alleles, in which ser21/9 are converted to non-phosphorylatable alanine. Between 5–15 weeks there was a non-significant trend towards higher heart weight (HW): body weight (BW) ratios in KI mice versus wild type (WT) mice. Mean left ventricular wall thickness (LVWT) at the level of the papillary muscle was comparable between genotypes (1.3±0.2mm
vs
1.6±0.2mm, ns, n=8). 8–10 week old weight-matched mice received 2 weeks subcutaneous ISO (30mg/kg/d) or control saline (Con) infusions. WT hearts hypertrophied with ISO (LVWT 1.9±0.1mm
vs
1.3±0.2mm Con, n=8; HW:BW ratio 6.7±0.6
vs
5.4±0.4 Con, n=10, both p< 0.05), but KI mice did not (1.4±0.1mm
vs
1.6±0.2mm KI Con; HW:BW 6.2±1.0
vs
6.1±0.6 KI Con, respectively, both ns). Hypertrophy in the WT hearts was associated with reduced LV ejection fraction (41±9%
vs
63±5% Con, n=6, p< 0.05) and greater interstitial fibrosis (2.1±0.5%
vs
0.8±0.6%, n=6, p< 0.05). These were both attenuated in the KI mice subjected to ISO (70±13%
vs
64±11% Con, and 1.3±0.7%
vs
0.8±0.3% Con, respectively, both ns). GSK-3 inhibition with daily intraperitoneal 6-bromoindirubin-3′-oxime restored hypertrophic sensitivity to ISO in the KI mice (LVWT 1.8±0.2mm; HW:BW 7.3±0.5, p< 0.05 for both groups
vs
KI + ISO alone) but had no effect in the absence of hypertrophic stimulus (1.4±0.2mm and 5.4±0.7, respectively, for KI + Con + Bio, ns vs KI + Con alone). In conclusion, inactivation-resistant GSK-3αβ dual isoform expression does not affect age-related (eutrophic) myocardial growth in the mouse. However, it protects against pathological growth, maintaining cardiac function and attenuating interstitial fibrosis.
Abstract 325: Administration Of Apelin Modulates Myocardial Expression Of Apelin-apj And Improves Right Ventricular Performance In Mct-induced Pulmonary Hypertension
Authors: Falcao-Pires Inês , Gonçalves Nadia , Henriques-Coelho Tiago , Roncon-Albuquerque Roberto , Daniel Moreira-Gonçalves , Leite-Moreira Adelino
Published: July 2021

Background:
Apelin (AP) is the endogenous vasoactive ligand of APJ receptor. It is widely expressed in the heart and lungs and has important cardiovascular functions such as endothelium-dependent vasodilatation and positive inotropic effect. However, the role of apelin-APJ system in pulmonary hypertension (PH) remains to be clarified.

Objective:
To evaluate the chronic effects of apelin in:

right ventricular (RV) myocardial function;

cardiac expression of apelin and APJ in healthy and MCT-PH rats

Methods:
Male Wistar rats were randomly injected with MCT (60mg/Kg, sc) or vehicle (CTR, day 0). One week later, half of these animals were randomly treated with Pyr-apelin-13 (200μg/Kg/day, ip) or a similar volume of vehicle. The study resulted in 4 groups: CTR; AP; MCT and MCT+ AP. At day 24, animals were instrumented to record RV peak systolic (Pmax) pressures, dP/dtmax, dP/dtmin and time constant Tau. Also, heart and lungs were weighted and transmural RV samples were collected for relative quantification of mRNA by real-time RT-PCR. Results are on table

.

Conclusions:
In healthy rats, exogenous apelin potentiates its endogenous production with no significant changes in APJ. MCT-induced PH resulted in a significant down-regulation of apelin-APJ system, which might play a role in the systolic and diastolic dysfunction observed in this model. This down-regulation was completely reverted by chronic administration of exogenous apelin. Therefore, apelin upregulates the cardiac apelin-APJ system exerting beneficial effects on cardiac remodeling and function, namely, by decreasing PH, RV diastolic function and hypertrophy. These results reveal the importance of apelin-APJ system in the pathophysiology of PH, suggesting that apelin is a potential therapeutic target in this disease.

Results table
Abstract 359: Disordered Chemokine Expression in Diabetes Following Experimental Myocardial Infarction: Potential Role in Impaired Progenitor Cell Recruitment
Authors: Desjardins Jean-Francois , Connelly Kim A , Thai Kerri , Johnson Cory A , Parker Thomas G , Gilbert Richard E
Published: July 2021

BACKGROUND: Impaired collateral vessel formation is thought to contribute to the excessive morbidity in diabetic subjects with ischaemic heart disease where following myocardial infarction (MI) patients with diabetes are twice more likely to develop heart failure than non-diabetic subjects. Since the trafficking of bone marrow derived endothelial progenitor cells is thought to contribute to new vessel formation in ischemia, we postulated that altered chemokine expression may contribute to the impaired angiogenic response in diabetes.

METHODS: Six week old F344 rats were randomized to vehicle or streptozotocin (35mg/kg i.p.) in order to induce diabetes. Once diabetes was confirmed (plasma glucose >15 mM), a total of 40 animals then underwent sham procedure or LAD ligation (MI) at age 10 weeks, and sacrificed at day 0 (sham) and days 1, 2, 4 and 7 post-MI (4 per group). Infarct size was determined by echocardiography 24 hours after ligation. RT q PCR was used to assess mRNA expression levels of SDF-1 and its receptor CXCR4 at each time point, from tissue obtained from the peri-MI zone.

RESULTS: There was no difference in mortality or infarct size between the groups. In control rats an increase in SDF-1 gene expression was apparent at Day 2 post-MI, persisting through Day 7 (p<0.05). In contrast, diabetic rats showed high basal expression (6-fold vs. control, p<0.01) that fell abruptly after MI and remained persistently low throughout the 7-day time course. Diabetes was also associated with increased basal gene expression of CXCR4 (2-fold vs. control, p<0.05). Post-MI, CXCR4 gene expression increased in control rats at each time point, with fold differences up to maximally 3 times at Day 2 (p<0.05 vs. Day 0). In diabetic rats, the level of expression remained high at Day 1, significantly decreased at Day 2 (3-fold vs. non-diabetic, p<0.01) and was comparable to that of non-diabetic rats at days 4 and 7.

CONCLUSIONS: Diabetes was associated with paradoxical alterations in gene expression of both SDF-1 and its receptor CXCR4 with high baseline expression and diminution following an ischemic insult. These findings suggest that disordered chemokine expression may reduce the diabetic heart’s ability to recruit endothelial progenitor cells to sites of ischemic injury.
Abstract 370: Type 2 Diabetes Is Associated with the Exacerbation of Heart Failure via Increasing Myocardial NAD(P)H Oxidase-Derived Superoxide Production
Authors: Kinugawa Shintaro , Matsushima Shouji , Yokota Takashi , Ohta Yukihiro , Inoue Naoki , Tsutsuin Hiroyuki
Published: July 2021

Type 2 diabetes mellitus (DM) adversely affects the outcomes in patients with myocardial infarction (MI), which is associated with the development of left ventricular (LV) remodeling and failure. NAD(P)H oxidase-derived superoxide (O
2
−
) production is increased in DM. However, its pathophysiological significance in advanced post-MI LV failure associated with DM remains unestablished. We thus determined whether an inhibitor of NAD(P)H oxidase activation, apocynin, could attenuate the exacerbated LV remodeling and heart failure after MI in high-fat diet (HFD)-induced obese mice with DM. Male C57BL/6J mice were fed on either HFD or normal diet (ND) for 8 weeks. At 4 weeks of feeding, MI was created in all mice by ligating left coronary artery. MI mice were treated with either apocynin (10 mmol/l in drinking water; n = 10 for ND and n = 11 for HFD) or vehicle (n = 15 for ND and n = 13 for HFD). HFD significantly increased body weight (BW), adipose tissue mass, fasting plasma glucose and insulin levels compared to ND after 4 and 8 weeks. HFD + MI had significantly greater LV end-diastolic diameter (LVEDD; 5.7 ± 0.1 vs. 5.3 ± 0.2 mm) by echocardiography, end-diastolic pressure (EDP; 12 ± 2 vs. 8 ± 1 mmHg) and lung weight/tibial length (10.1 ± 0.3 vs. 8.7 ± 0.7 mg/mm) than ND + MI, which was accompanied by an increased interstitial fibrosis of non-infarcted LV. Treatment of HFD + MI with apocynin significantly decreased LVEDD (5.4 ± 0.1 mm), LVEDP (9.7 ± 0.8 mmHg), lung weight/tibial length (9.0 ± 0.3 mg/mm), and concomitantly interstitial fibrosis of non-infarcted LV to ND + MI level without affecting BW, glucose metabolism, infarct size and aortic pressure. On the other hand, treatment of ND + MI with apocynin did not affect LV remodeling and failure. NAD(P)H oxidase activity, O
2
−
production measured by lucigenin chemiluminescence, and thiobarbituric acid-reactive substances were increased in non-infarcted LV tissues from HFD + MI, all of which were also attenuated by apocynin to ND + MI level. Type 2 DM was associated with the exacerbation of LV remodeling and failure after MI via increasing NAD(P)H oxidase derived O
2
−
production, which may be a novel important therapeutic target in advanced heart failure with DM.
Abstract 376: Ischemic Preconditioning Regulates Myocardial Viability Through The Proteasomal Regulation Of Two Opposing PKC Isozymes
Authors: Churchill Eric , Ferreira Julio , Brum Patricia , Szweda Luke , Mochly-Rosen Daria
Published: July 2021

The response of the myocardium to an ischemic insult is regulated by two opposing PKC isozymes, δ and ϵPKC. We therefore wanted to determine how two highly homologous kinases could play such distinct roles in cardiac ischemia/reperfusion (I/R) injury. Using an
ex vivo
rat model of myocardial infarction, we determined that short bouts of ischemia and reperfusion prior to the prolonged ischemic event (IPC) diminished δPKC and enhanced ϵPKC translocation to the mitochondria. In addition, cellular levels of δPKC decreased by 60% in response to IPC, whereas the levels of ϵPKC remained unchanged. Prolonged ischemia induced a 50% decline in the ATP-dependent proteasome activity and increased the accumulation of unfolded proteins during reperfusion; both of which were completely prevented by IPC. Pharmacological inhibition of the proteasome and selective inhibition of ϵPKC during IPC restored δPKC translocation to the mitochondria and elevated the levels of unfolded proteins while increasing myocardial injury associated with I/R. (Note that none of these treatments had any effect on viability under normoxic conditions.) Interestingly, administration of a specific peptide activator of ϵPKC during I/R completely prevented the loss in proteasome activity seen during ischemia. Importantly, increased myocardial injury was the result of restoring the δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signaling and increasing cytochrome
c
release into the cytosol. Taken together, this study shows that IPC prevents I/R injury by protecting ATP-dependent 26S proteasome function, which in turn decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria resulting in the accumulation of the pro-survival kinase, ϵPKC. Additionally, ϵPKC seems to be a novel regulator of proteasome function as it relates to I/R injury. The proteasome can therefore be viewed as a sensor of cellular viability, determining the ratio of pro-apoptotic δPKC and pro-survival ϵPKC at the mitochondria and thus the ultimate fate of the cell.
Abstract 502: Long-Term Effects of a Rapid Response Team on Hospital-Wide Code Rates and Mortality
Authors: Chan Paul S , Khalid Adnan , Longmore Lance , Berg Robert , Peberdy Mary Ann , Kosiborod Mikhail , Spertus John A
Published: July 2021

Background:
Rapid response teams (RRTs) have been shown to decrease cardiopulmonary arrest (code) rates outside of the intensive care unit (ICU). Because a primary action of RRTs is to transfer patients to the ICU, their impact on hospital-wide code rates and mortality remains poorly understood.

Methods:
Data on RRT activations and codes were prospectively collected at a 404-bed tertiary care academic hospital. RRT education and program rollout occurred from September to December, 2005. A total of 24,193 patient admissions were evaluated pre-intervention (January 2004 to August 2005), and 24,978 admissions were evaluated post-intervention (January 2006 to August 2007). Main outcome measures were hospital-wide code rates and mortality; these were compared between the pre- and post-intervention periods using multivariable regression models that adjusted for pre-intervention trends and differences in demographics and hospital case mix.

Results:
After RRT implementation, mean hospital-wide code rates decreased from 11.2 to 7.5 per 1000 admissions. This was associated with a trend toward lower code rates (Odds Ratio [OR]=0.76; 95% CI: 0.57 to 1.01; p=0.06) and was mediated through lower rates of non-ICU codes (non-ICU: OR=0.59; 95% CI: 0.40 – 0.89 vs. ICU: OR=0.95; 95% CI: 0.64–1.43; p-value for interaction of 0.03). The effect of RRTs on code rates was similar for codes due to respiratory arrests, shockable cardiac arrests (ventricular fibrillation and pulseless ventricular tachycardia), and non-shockable cardiac arrests (pulseless electrical activity and asystole) (p-value for interaction of 0.42). However, hospital-wide mortality did not differ between the pre- and post-intervention periods (3.22 vs. 3.09 per 100 admissions; OR=0.95; 95% CI: 0.81 to 1.11; p=0.52). Post-hoc analyses revealed 18 instances of RRT under-treatment or underutilization that would not have significantly affected the mortality findings had these deaths been avoided (OR of 0.93; 95% CI: 0.79 to 1.09; p=0.35).

Conclusions:
RRT implementation was associated with a strong trend toward lower hospital-wide code rates, especially in non-ICU settings. However, further studies are required to demonstrate a mortality benefit before RRTs become the standard of care.
Abstract 519: Pathogenic Mechanisms of Il-17 In Atherogenesis - In Vivo and In Vitro Studies
Authors: Erbel Christian , Chen Lili , Celik Sultan , Wambsganss Nadine , Lasitschka Felix , Boeckler Dietmar , Katus Hugo A , Dengler Thomas J
Published: July 2021

Atherosclerotic plaque typically contains infiltrates of activated macrophages and T cells. In human carotid artery plaques, we previously showed the presence of IL-17 producing T cells (Th17) and IL-23. This study investigated possible pathogenic mechanisms of IL-17 on atherogenesis in ApoE
−/−
mice and its effects on vascular cells in vitro. 25 female ApoE
−/−
mice fed a normal diet were treated with a specific blocking antibody against IL-17 for 12 weeks (100 μg intraperitoneally once per week) compared to unteated mice (reveiving a control IgG). In vitro endothelial cells and RAW cells were either treated with IL-17 1000ng/ml alone as well as in addition to TNF-α or LPS. SMCs were incubated with IL-17 or additional IFN-γ to induce apoptosis. IL-17 mAb treatment resulted in a > 60% reduction of plaque volume and fractional luminal stenosis in the aortic root. IL-17 mAb treated mice showed higher percentage of collagen content in the lesion, especially in the area of the fibrous cap. In addition, the amount of SMCs was also significantly higher in the area of the fibrous cap in IL-17 mAb treated mice. RT-PCR results showed signifcantly reduced caspase 3 expression in IL-17 mAb treated mice. The possible stabilizing effects in IL-17 mAb treated mice was in line with findings that IL-17 was found to induce apoptosis in SMCs in vitro. In vitro studies further showed that IL-17 itself induced pro-inflammatory changes in macrophages as well as endothelial cells and also acted synergistically with TNF-α or LPS. Functional blockade of Th1 cytokine IL-17 reduces atherosclerotic plaque, stabilizes the atherosclerotic lesion by higher amount of SMC and collagen content in the area of the fibrous cap as well as reduced apoptosis in the lesion. Inflammatory changes in ECs, Ms and SMCs leading to cell death and increased immune cell infiltration may represent possible pathogenic mechanisms of IL-17 in atherogenesis.
Abstract 1376: The Nadph Oxidase Nox4 Controls Mkp1 Expression and Thereby the Development of White Adipose Tissue
Authors: Schröder Katrin , Schreiber Judith G , Brandes Ralf P
Published: July 2021

Obesity is a cardiovascular risk factor. Insulin promotes the formation of adipocytes by promoting fibroblast into adipocyte differentiation. Insulin however also acts as a mitogen for fibroblasts and the balance of insulin-induced proliferation and differentiation is incompletely understood. We hypothesize that the NADPH oxidase Nox4 acts as a central switch between these two processes. In 3T3-fibroblasts Nox4 siRNA reduced the cellular radical formation and attenuated the insulin-stimulated differentiation from fibroblasts into adipocytes. Importantly, insulin-induced proliferation was enhanced by Nox4 siRNA. Accordingly, Nox4 overexpression enhanced differentiation and reduced insulin-stimulated proliferation. We generated Nox4 knockout mice to study the in vivo relevance of this observation: The amount of adipose tissue was reduced in the Nox4−/− animals as compared to their wildtype littermates suggesting indeed an attenuated adipocyte differentiation, whereas fibroblasts obtained from Nox4−/− mice presented with enhanced proliferation. To uncover the underlying mechanism, we focused on the Erk1/2 pathway. Erk1/2 signaling is prevented by dephosphorylation through the dualspecific phosphatase MKP1. Luciferase reportergene assays using the full-length MKP1 promotor revealed that Nox4 siRNA reduced, and overexpression of Nox4 as well as treatment of the cells with H
2
O
2
induced MKP1 promotor activity. Indeed, Nox4 siRNA reduced MKP1 protein expression and thus enhanced basal and insulin-induced activation of Erk1/2 in fibroblasts. Nox4 overexpression had the opposite effect. Importantly, MKP1 expression was also reduced in adipose tissue from Nox4−/− mice. Overexpression of MKP1 in fibroblastes increased insulin-induced differentiation and attenuated proliferation, whereas MKP1 siRNA had the opposite effect. We conclude that Nox4, by regulating MKP1, is essentially involved in adipocyte differentiation and development of obesity.
Abstract 1428: Cyclophilin a Promotes Vascular Oxidative Stress and Accelerates Development of Angiotensin II-Induced Aortic Aneurysms
Authors: Satoh Kimio , Nigro Patrizia , Matoba Tetsuya , O’Dell Michael R , Cui Zhaoqiang , Shi Xi , Mohan Amy , Yan Chen , Abe Jun-ichi , Berk Bradford C
Published: July 2021

Cyclophilin A (CyPA) is a chaperone protein secreted from vascular smooth muscle cells (VSMC) in response to reactive oxygen species (ROS), that stimulates VSMC migration and inflammatory cell migration in vitro. Abdominal aortic aneurysm (AAA) formation involves inflammatory cytokine release, leukocyte recruitment, aortic wall degradation, and neovascularization. We hypothesized that VSMC-derived CyPA contributes to AAA pathogenesis due to its proinflammatory properties. To determine the role of CyPA in AAA formation, ApoE
−/−
and ApoE
−/−
CyPA
−/−
mice were infused with angiotensin II (AngII, 1000 ng/min/kg) for 4 weeks. There were no differences in blood pressure and cholesterol between ApoE
−/−
and ApoE
−/−
CyPA
−/−
mice before and after Ang II treatment. AngII-induced AAA formation and aortic rupture was frequently observed in ApoE
−/−
mice (89% and 40%). In contrast, ApoE
−/−
CyPA
−/−
mice were completely protected from AngII-induced AAA formation and aortic rupture (0% and 0%). ApoE
−/−
CyPA
−/−
mice showed decreased levels of monocyte chemoattractant protein (MCP)-1 in the aorta and lacked elastic lamina degradation, microvessel formation, and aortic expansion. In response to AngII, recruitment of leukocytes to the aortic wall was markedly impaired in ApoE
−/−
CyPA
−/−
mice compared with ApoE
−/−
mice (counts/area; 8.6±4.3 vs. 60.0±13.8,
P
<0.01). The incidence of AAA was 63% in CyPA
+/+
marrow-transplanted ApoE
−/−
mice, while the incidence of AAA in ApoE
−/−
CyPA
−/−
mice remained 0% after transplantation of CyPA
+/+
bone marrow cells. In situ and gelatin zymography demonstrated that CyPA was required for matrix metalloproteinase (MMP) activation in aortic wall. Treatment of mouse aortic wild-type VSMC with AngII augmented MMP activity, which was significantly less in CyPA
−/−
VSMC. Treatment of VSMC with 100 nM CyPA augmented MMP activity, suggesting the importance of extracellular CyPA as well as intracellular CyPA. Finally, VSMC-specific CyPA overexpressing mice revealed augmented AngII-induced MMP activity in the vascular wall. Vascular-derived CyPA contributes to AAA pathogenesis in mice by increasing proinflammatory cytokine expression, inflammatory cell migration, and MMP activation.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 1436: Gene Dosage of the Common Variant 9p21 Predicts Severity of Coronary Atheromatous Burden
Authors: Dandona Sonny , Stewart Alexandre F , Chen Li , Williams Kathryn , So Derek , O’Brien Edward , Glover Christopher , LeMay Michel , Vo Lan , Wang Yan Q , Labinaz Marino , Wells George A , McPherson Ruth , Roberts Robert
Published: July 2021

Background:
The first common genetic risk variant for coronary artery disease (CAD), 9p21, was recently identified and shown to occur in 75% of Caucasian and Asian populations. However, 9p21 does not contain a protein coding gene and its function is unknown. Given the phenotypes of atherosclerosis and thrombosis, pursuit of the molecular mechanisms mediating 9p21 risk requires elucidation of the 9p21 phenotype. Accordingly, we tested the hypothesis that 9p21 is directly related to coronary atherosclerosis.

Methods:
We studied 709 patients who were prospectively enrolled from the coronary catheterization laboratory at the University of Ottawa Heart institute from April 15
th
2006 to 4
th
January 2008. Each subject was genotyped for the single nucleotide polymorphism rs1333049 (risk allele located at 9p21). Patients were designated AA (risk/risk), AB (risk/non-risk) and BB (non-risk/non-risk). Angiograms were reviewed by two independent parties blinded to genotype. Lesions with 50% or more stenosis were considered significant for the purposes of this study. Odds ratios were generated for proportion of patients with 3VD, significant left main disease and CABG relative to the non-risk genotype, BB.

Results:
We demonstrated a strong direct association between 9p21 gene dosage and 3VD (Fig. 1A

). Conversely, a strong inverse association between 9p21 gene dosage and 1VD was observed (Fig. 1B

). A strong direct association between gene dosage and LM disease (Fig. 1C

) and CABG (Fig. 1D

) was seen.

Conclusions:
The 9p21 variant risk is mediated through deposition of coronary atheroma. Therefore, elucidating 9p21 function should target atherogenesis.
Abstract 1482: Curcumin Attenuates Cardiac Hypertrophy and Left Ventricular Remodeling Secondary to Experimental Chronic Renal Failure Independent of Blood Pressure in Nephrectomized Rats
Authors: Ghosh Siddartha , Salloum Fadi , Abbate Antonio , Toldo Stefano , Kukreja Rakesh C
Published: July 2021

Background:
The prevalence of heart failure (HF) is frequent in patients with chronic renal failure (CRF). One common link between CRF and HF is inflammation. Curcumin, the principal curcuminoid of the Indian curry spice turmeric, is known for its antitumor anti-inflammatory properties. We therefore hypothesized that curcumin would attenuate cardiomyopathy in nephrectomized rats.

Methods and Results:
Adult Sprague-Dawley rats underwent nephrectomy (Nx) by removal of 5/6 of the kidneys. Four groups were studied for 7 weeks: 1- Control (sham), 2- Nx, 3- Nx + curcumin (75mg/kg; bid), 4- Nx + enalapril (15 mg/kg; bid) as positive control. The systolic blood pressure (SBP, mmHg) of Nx rats was 152±5, significantly higher than control (115±2) and enalapril-treated rats (125±3). Curcumin did not reduce SBP (141±4). BUN (mg/dl) and creatinine (mg/dl) levels of Nx rats (67±7 and 1.92±0.08, respectively) were higher than control (P<0.001). Both curcumin and enalapril significantly reduced BUN (40±2 and 43±4, respectively) and creatinine (0.95±0.05 and 0.77±0.06, respectively) (P<0.001 vs. Nx). Moreover, Nx rats exhibited severe proteinurea (mg/18 hours) (316±36) as compared to controls (34±5) which was blunted by curcumin (183±40, P<0.001) and enalapril (180±17, P<0.001). Right and left cardiac hypertrophy was assessed by weighing the right (RV) and left ventricles (LV) and normalizing with body weight. Nx rats had significant RV and LV hypertrophy (P<0.01). Curcumin and enalapril effectively decreased LV hypertrophy (P<0.05), however, RV hypertrophy was only decreased by curcumin (P<0.05). Echocardiography data revealed a significant increase in LV mass in Nx rats (1.47±0.19) as compared with controls (0.98±0.04, P<0.01). Curcumin and enalapril-treated rats had LV mass of 0.95±0.1 (P<0.01) and 0.93±0.3 (P<0.05), respectively. LV end-diastolic diameter (EDD, mm) was higher in Nx rats (7.2±0.2) as compared to controls (6.4±0.2, P=0.01). Curcumin and enalapril attenuated LV dilatation as shown by EDD of 6.3±0.5 and 6.2±0.5, respectively (P=0.01).

Conclusion:
These results show that curcumin attenuates cardiac hypertrophy and remodeling in nephrectomized rats independent of BP. Therefore, curcumin may be useful in suppressing HF in patients with CRF.
Abstract 1494: Hepatocyte Growth Factor Attenuates Angiotensin II Induced Renal Fibrosis through TGF-β1 Suppression by Anoikis of Myofibroblasts
Authors: Iekushi Kazuma , Taniyama Yoshiaki , Azuma Junya , Sanada Fumihiro , Dosaka Norio , kusunoki Hiroshi , Koibuchi Nobutaka , Rakugi Hiromi , Morishita Ryuichi
Published: July 2021

Progression of chronic kidney disease (CKD) is characterized by the persistent accumulation of extracellular matrix. Especially, α-SMA positive myofibroblast which produce high amounts of TGF-β1 are considered to play a key role in interstitial fibrosis. Previous studies demonstrated that hepatocyte growth factor (HGF) improved kidney fibrosis in murine models, where direct molecular mechanisms of myofibroblasts have not yet been understood. We tested the hypothesis in vivo using cardiac specific overexpression HGF mice (HGF-Tg), which showed a significant increase in serum HGF concentration. Angiotensin II (Ang II) infusion significantly induced renal fibrosis in wild type mice, while renal fibrosis was significantly decreased in HGF-Tg mice accompanied by the degrease in interstitial myofibroblasts (P<0.05). Quantitative analysis demonstrated 1.69-folds induction of profibrtic cytokine, TGF-β1 mRNA in HGF-Tg with Ang II group compared with wild type with Ang II, and Collagen type I and IV mRNA expression was significantly decreased in HGF-Tg mice with Ang II. The antifibotic action of HGF-Tg mice was concordant with an increase in MMP-2, MMP-9 expression (1.32-fold, 1.33-fold vs wild type with Ang II infusion, P<0.05, respectively), and decreased TIMP-1, TIMP-2 expression (1.5-fold, 1.28-fold vs wild type with Ang II infusion, P<0.05, respectively). To further investigate the anti-fibrotic effect of HGF, we used cultured human mesangial cells (HMC). When HMC were treated with TGF-β1, cells underwent to phenotypic change similar to myofibroblasts, accompanied by the significant increase in c-Met/HGF receptor (P<0.05). Under such conditions, HGF induced anoikis-induced apoptosis of myofibroblasts. It also linked with FAK phosphorylation especially p-FAK (Y925) (P<0.05). When GM6001 (a broad-spectrum MMP inhibitor) was added with HGF, HGF-induced apptosis was significantly decreased. It was suggested that increased activities of MMPs underlie the major mechanism of HGF mediated anoikis induced apoptosis. The present study demonstrated that HGF elicited myofibroblast anoikis. Activation of MMPs in fibrotic kidney might be considered as a target to attenuate the progression of CKD.
Abstract 1573: Phosphorylation of Cardiac Troponin I by Mammalian Sterile20-like Kinase Mst1
Authors: You Bei , Yan Guijun , Zhang Zhiling , Jin Jian-Ping , Sun Jianxin
Published: July 2021

Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of human heart cDNA library with a dominant negative Mst1 (K59R) as bait, cardiac troponin I (cTnI) was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiac myocytes, in which cTnI interacted with full length Mst1, but not with its N-terminal kinase fragment.
In vitro
phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1 in its free form or in reconstituted troponin complex. In contrast, cardiac TnT was phosphorylated by Mst1 only when incorporated in the troponin complex. Mass spectrometric analysis indicated that Mst1 phosphorylates cTnI at Thr
31
, Thr
51
, Thr
129
, and Thr
143
. Substitution of Thr
31
with Ala substantially reduced Mst1-mediated cTnI phosphorylation by approximately 90%, while replacement of either Thr
51
orThr
129
, or Thr
143
with Ala reduced Mst1-catalyzed cTnI phosphorylation by approximately 60%, suggesting that Thr
31
is a preferential phosphorylation site for Mst1. Protein epitope analysis and binding assays showed that Mst1 mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. Our results suggest that Mst1 is a novel mediator of cTnI and/or cTnT phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1577: Transmural Dyssynchrony of Myofiber Shortening is Determined by Depolarization Sequence within Myocardial Layers
Authors: Gurev Viatcheslav , Constantino Jason , Trayanova Natalia A
Published: July 2021

The delay in myofiber shortening (MS) onset between the endocardial and epicardial layers is greater than the electrical activation delay during sinus rhythm (SR) and decreases at the location of the pacing site during epicardial pacing (EP). The goal of this study is to demonstrate that increased transmural mechanical delay during SR is determined by the electrical activation sequence within the myocardial layers, rather than the transmural activation pattern. We employed a novel 3D anatomically-accurate electromechanical model of the rabbit ventricles that incorporated a detailed model of cardiac myofilament dynamics. MS was calculated throughout the ventricular volume during SR and for EP at the left ventricular anterior wall. Onset of MS followed the direction of electrical activation for both SR and EP, as shown in Figure

. However, transmural delay in MS onset was greater during SR, consistent with experiments (28ms-SR vs. 19ms-EP). Difference in the MS delay could not be explained by changes in transmural electrical activation delay (19ms-SR vs. 25ms-EP). Examination of the mechanisms revealed a longer apex-to-base electrical activation delay on the epicardium as compared to that on the endocardium during SR. “Prestretching” of the late-activated epicardial myofibers at the base by the shortening of the early-activated apical region increased MS delay. No prestretching was observed following EP. This finding indicates that transmural MS delay strongly depends on the electrical and mechanical activation sequence within the myocardial layers.

This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).
Abstract 1582: Adenosine A3 Receptor Deficiency Exerts Unanticipated Protective Effects on Pressure Overloaded-Induced Left Ventricular Hypertrophy and Dysfunction in Mice
Authors: Lu Zhongbing , Fassett John , Xu Xin , Hu Xinli , Zhu Guangshuo , Schnermann Jurgen , Bache Robert J , Chen Yingjie
Published: July 2021

Endogenous adenosine can protect the overloaded heart against the development of hypertrophy and heart failure, but the contribution of A
1
receptors (A
1
R) and A
3
receptors(A
3
R) is not known. To test the hypothesis A
1
R and A
3
R can protect the heart against systolic overload, we exposed A
3
R gene deficient (A
3
R KO) mice and A
1
R KO mice to transverse aortic constriction (TAC). Contrary to our hypothesis, A
3
R KO attenuated 5 weeks TAC-induced left ventricular (LV) hypertrophy (ratio of ventricular mass/body weight increased to 7.6 ±0.3 mg/g in wild type (Wt) mice as compared with 6.3±0.4 mg/g in KO), fibrosis and dysfunction (LV ejection fraction decreased to 43±2.5% and 55±4.2% in Wt and KO mice, respectively). A
3
R KO also attenuated the TAC-induced increases of myocardial ANP and the oxidative stress markers 3-nitrotyrosine(3-NT ) and 4-hydroxynonenal. In addition, A
3
R KO significantly attenuated TAC-induced activation of multiple MAP kinase pathways, and the activation of Akt-GSK signaling pathway. In contrast, A
1
R-KO increased TAC-induced mortality, but did not alter ventricular hypertrophy or dysfunction compared to Wt mice. In mice in which extracellular adenosine production was impaired by CD73 KO, TAC caused greater hypertrophy and dysfunction, and increased myocardial 3-NT, indicates that extracellular adenosine protects heart against TAC-induced ventricular oxidative stress and hypertrophy. In neonatal rat cardiomyocytes induced to hypertrophy with phenylephrine, the adenosine analogue 2-chloroadenosine (CADO) reduced cell area, protein synthesis, ANP and 3-NT. Antagonism of A3R significantly potentiated the anti-hypertrophic effects of CADO. Our data demonstrated that extracellular adenosine exerts protective effects on the overloaded heart, but A
3
R act counter to the protective effect of adenosine. The data suggest that selective attenuation of A
3
R activity might be a novel approach to attenuate pressure overload-induced myocardial oxidative stress, LV hypertrophy and dysfunction.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 1611: Remote Ischemic Preconditioning Improves 6-month Outcome after Elective Percutaneous Coronary Intervention: A Single-Center Randomized Controlled Trial
Authors: Hoole Stephen , Heck Patrick , Khan Sadia , Sharples Linda , O’Sullivan Michael , Dutka David
Published: July 2021

Endogenous “early” protection by remote ischemic preconditioning (rIPC) attenuates infarct size in animals but improvement in infarct size and clinical outcome in humans undergoing percutaneous coronary intervention (PCI) has not been documented. We hypothesized that rIPC would attenuate procedure-related cardiac troponin (cTnI) release during elective PCI and improve clinical outcome at 6-months. We randomized 242 patients undergoing elective PCI to either rIPC (n = 125): three 5-minute blood pressure cuff inflations to 200mmHg around the upper arm with 5-minutes of cuff deflation between, or control (n = 117): a deflated cuff throughout, before PCI. Patients taking nicorandil or glibenclamide and those with raised cTnI at baseline were excluded. Interventional cardiologists were blinded to the pretreatment protocol. Post-PCI serum cTnI level at 24-hours was recorded and major adverse cardiac and cerebral event (MACCE) rate determined at 6-months. The two groups were matched demographically. Interval between rIPC and first coronary balloon inflation was 66 ± 30 minutes. Median 24-hour cTnI was significantly lower in the rIPC group (0.06ng/mL vs. 0.16ng/mL, p=0.04). Subjects in the rIPC group experienced less chest pain (p=0.0006) and ST-segment deviation (p=0.005) during PCI than control subjects. MACCE free survival at 6-months was significantly higher in the rIPC group (Figure

), predominantly due to less acute coronary syndromes in the rIPC group (Hazard Ratio (95%CI): 0.28 (0.12– 0.82)). rIPC attenuates procedure-related cTnI release after elective PCI, and reduces 6-month MACCE rate.
Abstract 1653: The Role of the Fibrinogen Alpha Chain in Controlling the Rate of Factor XIII Activation: Implications for Coronary Artery Thrombus Formation
Authors: Smith Kerrie A , Adamson Penelope J , Ariens Robert A , Philippou Helen , Grant Peter J
Published: July 2021

Background:
The development of an obstructive arterial thrombus is dependent on the generation of a platelet rich fibrin mesh secondary to plaque rupture. Fibrin itself is formed by the cleavage of fibrinogen by thrombin with subsequent fibrin cross-linking by activated Factor XIII. Critical to these processes, fibrinogen α-chain residues 242– 424 have a major regulatory role in the dissociation of coagulation Factor XIII A subunit (FXIIIA) from the Factor XIII B subunit (FXIIIB), however the mechanisms for this enhancing effect have not been determined.

Methods:
α-Chain residues 242– 424 were expressed as a GST-fusion protein in E
.coli,
along with six further truncations; αC fragments α242– 402, α242–387, α242–374, α242–341, α242–289 and α242–265. FXIII-A and an inactive double thrombin cleavage mutant (R37A/K513A) were also expressed as GST-fusion proteins in E
. coli
.

Results:
Using surface plasmon resonance (SPR) and ELISA we can confirm the presence of at least two binding sites on the αC for activated FXIII-A within residues α388 – 424 and α242–265 with an overall KD of 4.9 ± 2.29 μM. This interaction was specific for activated FXIII-A, as the inactive FXIII-A double thrombin cleavage mutant did not bind to the αC as indicated by SPR and ELISA. We demonstrated that the binding of the αC is dependent on a conformational change which occurs during activation of FXIII-A with calcium and thrombin. FXIII-A activated with thrombin alone did not bind αC as determined by SPR and ELISA. Competition studies showed that the αC competitively inhibits binding with a K
I
of 1.2 μM confirming the specificity of this interaction. Iodoacetamide blocking of FXIII-A active site cysteine did not prevent binding of the αC suggesting this interaction is independent of FXIII-A cross-linking. Furthermore we have identified a novel interaction between FXIIIB and α-chain by ELISA. Utilising the αC fragments we have localised two binding regions between α290–341 and α242–265 with a K
D
of 123nM ± 11 and 286nM ± 24.7 respectively.

Conclusions:
The binding of both FXIII A- and B-subunits to this area of the fibrinogen α-chain suggests a crucial role in controlling the amount of activated FXIII generated for clot stabilisation and presents a potential target for therapeutic intervention.
Abstract 1701: Deletion of β PDGF Receptor Dependent PI-3 Kinase and PLCγ Signaling Attenuates Vascular Smooth Muscle Cell Proliferation and Inhibits Neointima Formation in Mice
Authors: Caglayan Evren , Vantler Marius , Leppanen Olli , Mustafov Lenard , Gerhardt Felix , Odenthal Margarete , Kappert Kai , Rosenkranz Stephan
Published: July 2021

Platelet-derived growth factor β-receptor (βPDGFR)-mediated proliferation of vascular smooth muscle cells (VSMC) plays a pivotal role in the development of restenosis. The βPDGFR binds and activates cytoplasmic signaling molecules such as Src, PI 3-kinase (PI3K), RasGAP, SHP-2 and phospholipase C-gamma (PLCγ). However, the βPDGFR-induced signaling pathways leading to cell cycle progression are largely unknown. In order to characterize the signaling molecules, which are important for βPDGFR-induced VSMC proliferation, we generated several stable VSMC lines with mutated βPDGFRs which were unable to bind/activate each individual signaling molecule. BrdU incorporation assays revealed PI3K and PLCγ as the main mediators of βPDGFR-mediated VSMC proliferation. Deletion of the binding sites for PI3K or PLCγ diminished DNA-synthesis about 47 ± 6% and 54 ± 5%, respectively, whereas the deletion of Src, RasGAP, or SHP-2 binding to the βPDGFR had no influence on the mitogenic response. Mutant cell lines which were only able to bind/activate PI3K or PLCγ induced 43 ± 4% and 52 ± 5% of the WT response, respectively. We further investigated the influence of PI3K and PLCγ on the distinct expression of the key players of cell cycle progression. Western blot analysis revealed that βPDGFR-activated PI3K mainly mediated the up-regulation of cyclin D1, whereas activation of PLCγ mediated the down-regulation of the cyclin-dependent kinase-inhibitor p27
kip1
. Consistently, βPDGFR-dependent phosphorylation of the retinoblastoma protein which is important for the G1/S transition was attenuated when either the binding of PI3K or PLCγ to the βPDGFR was abolished. Moreover, we confirmed our in vitro results in an in vivo model of balloon-induced carotid artery neointima formation. Mice lacking binding of PI3K and PLCγ to the βPDGFR (F3 mice) developed ~50% less neointima formation after balloon angioplasty compared to control WT mice after 3 weeks (p < 0.05). These results indicate that the mitogenic signal of the β PDGFR is mediated by PI3K and PLCγ affecting distinct targets of cell cycle progression. Targeting specific downstream molecules of the βPDGFR in VSMCs might provide a novel approach to reduce neointima formation in patients undergoing balloon angioplasty.
Abstract 1732: Deletion of Fc Receptor Ameliorates Progression of Atherosclerotic Lesions in LDL Receptor Knockout Mice: Protective Role against Vascular Inflammation and Oxidative Stress
Authors: Sumiyoshi Katsuhiko , Shimada Kazunori , Iesaki Takafumi , Miyazaki Tetsuro , Kume Atsumi , Kiyanagi Takashi , Hiki Makoto , Fukao Kosuke , Hirose Kuniaki , Matsumori Rie , Tada Norihiro , Mokuno Hiroshi , Daida Hiroyuki
Published: July 2021

Background
: Vascular endothelial dysfunction through the production of reactive oxygen species (ROS) plays an important role in the initiation and progression of atherosclerosis. Immunoglobulin Fc receptor (FcR) involves the activation of immune and inflammatory reaction, however, the role of FcR in endothelial function, ROS production, and atherogenesis, has not clearly elucidated. To examine the hypothesis that FcR deletion blunts the progression of atherosclerotic lesions via improvements of superoxide production and endothelial dysfunction, we generated FcR/LDL receptor double-knockout (DKO) mice.

Methods
: Mice (8 weeks) with LDL receptor knockout (LDLR-KO) and DKO were fed a high cholesterol diet for 10 weeks. We examined the endothelium-dependent relaxation of isolated thoracic aorta, superoxide production by dihydroethidium-derived fluorescence (DHE) staining before and after high cholesterol feeding. We also assessed atherosclerotic lesions by Oil Red O-staining, macrophage infiltration, and p22phox expression by immnohistochemical analysis.

Results
: Vascular endothelial-dependent relaxation induced by high cholesterol diet was significantly higher in DKO mice than in LDLR-KO mice (−53 ± 7% vs −22 ± 9, p < 0.05). Superoxide production was significantly lower in DKO mice than in LDLR-KO mice (13.6 ± 10 vs 38.8 ± 20 ×10
3
μ m
2
p < 0.05). The expression of p22phox in DKO mice was significantly lower than in LDLR-KO mice (90 ± 19 vs 141 ± 42 × 10
3
μm
2
p < 0.05). The area of macrophage infiltration of the vascular wall was significantly lower in DKO mice than LDLR-KO mice (34 ± 5 vs 59 ± 17 × 10
3
μ m
2
p < 0.05). The Oil -Red O staining showed the significant reduction of atherosclerotic lesions in DKO mice than in LDLR-KO mice (73 ± 24 vs 133 ± 31 × 10
4
μ m
2
p < 0.005).

Conclusion
: Deletion of FcR attenuated the production of ROS, vascular inflammation, and endothelial dysfunction, resulting in prevention of atherosclerotic formation in LDLR-KO mice. These data suggested that FcR might be involved in atherosclerotic process through not only vascular inflammation, but also oxidative stress in vasculature.