bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 97: Genotype to Phenotype: Function of Common Noncoding and Rare Coding Variants In
ANGPTL3
Authors: Wang Xiao , Raghavan Avanthi , Vourakis A. Christina , Sperry Alexandra E , Li Wenjun , Lv Wenjian , Chadwick Alexandra C , Musunuru Kiran
Published: July 2021

Human genetics studies have demonstrated a strong link between
ANGPTL3
, which encodes lipoprotein lipase inhibitor Angiopoietin-like 3, and blood lipid phenotypes. Rare nonsense
ANGPTL3
mutations were identified in patients with familial combined hypolipidemia, while common variants at the
ANGPTL3
locus have been found by genome-wide association studies (GWASs) to associate with lower triglycerides (TGs) and low-density lipoprotein cholesterol. In light of the seemingly favorable clinical consequences of ANGPTL3 deficiency, we established an experimental framework to identify (1) causal common variants that regulate
ANGPTL3
expression and (2) rare missense mutations that disrupt
ANGPTL3
function. Using massively parallel reporter assays, we profiled the regulatory activity of all the common variants linked (
r
2
≥ 0.5) to the lead GWAS SNP in the
ANGPTL3
locus and found that rs10889356 demonstrated significant allele-specific enhancer activity. To validate this finding, we used CRISPR-Cas9 to alter the SNP in a human pluripotent stem cell line. When differentiated into hepatocytes, altered cells displayed a 67% increase in
ANGPTL3
expression (
n
= 4 wild-type and 4 mutant clones,
P
= 0.007). CRISPR interference using each of three guide RNAs targeting the SNP in HepG2 cells also substantially increased
ANGPTL3
expression. These findings support rs10889356-
ANGPTL3
as a causal SNP-gene set. Next, we examined the coding regions of
ANGPTL3
in 20,000 sequenced individuals and sought to experimentally define rare missense variants using a mouse model. We used CRISPR-Cas9 to generate
Angptl3
knockout mice, which exhibited decreased TG (61%,
P
< 0.001) and decreased cholesterol (31%,
P
< 0.002). We reconstituted the knockout mice to normal expression levels with adenoviruses expressing either wild-type
ANGPTL3
or missense variant
ANGPTL3
. So far we have assessed 28 rare missense variants computationally predicted to be deleterious, of which only 10—D42N, K58E, S117P, P264S, Q286H, L315S, L360Q, T383I, T383S, and Y417C—were validated as loss-of-function (conferring <25% of wild-type activity as assessed by changes in both TG and cholesterol levels), underscoring the need for functional characterization of variants of uncertain significance.
Outcome After Orthotopic Cardiac Transplantation in Adults With Congenital Heart Disease
Authors: Lamour Jacqueline M. , Addonizio Linda J. , Galantowicz Mark E. , Quaegebeur Jan M. , Mancini Donna M. , Kichuk Maryanne R. , Beniaminovitz Ainat , Michler Robert E. , Weinberg Alan , Hsu Daphne T.
Published: July 2021

Background
—Advances in surgical and medical management have greatly improved long-term survival rates in patients with congenital heart disease (CHD). As these patients reach adulthood, myocardial dysfunction can occur, leading to cardiac transplantation.

Methods and Results
—We reviewed the pretransplantation and posttransplantation courses of 24 patients >18 years old (mean age, 26 years; range, 18 to 56 years) with CHD who received a transplant between January 1985 and September 1998. The relation between preoperative and perioperative risk factors for complications and death was assessed. Single ventricle was the pretransplantation diagnosis for 12 patients (50%), and
d
-transposition of the great vessels was the diagnosis for 4 patients (16%). Twenty-two patients had a mean of 2 previous operations. At cardiac transplantation, additional surgical procedures were required to correct extracardiac lesions in 18 patients (75%). Refractory heart failure was present in 22 patients, significant cyanosis was present in 7, and protein-losing enteropathy was present in 4. There were 5 early deaths due to bleeding (n=3) and infection (n=2). The Kaplan-Meier survival rate after cardiac transplantation was 79% at 1 year and 60% at 5 years . No anatomic or surgical risk factor was predictive of death. The outcome of patients with CHD who received a transplant was compared with that for patients without CHD (n=788). Mean bypass and ischemic times were significantly longer in patients with CHD than in patients without CHD. Survival rates after transplantation did not differ significantly between patients with and those without CHD (
P
=0.83).

Conclusions
—Successful cardiac transplantation is obtainable in adults with complex CHD, with an outcome similar to that of patients without CHD. A detailed assessment of cardiac anatomy and careful surgical planning are essential to the pretransplantation and posttransplantation management of these patients.
Potential Role of Hepatocyte Growth Factor, a Novel Angiogenic Growth Factor, in Peripheral Arterial Disease
Authors: Hayashi Shin-ichiro , Morishita Ryuichi , Nakamura Shigefumi , Yamamoto Kei , Moriguchi Atsushi , Nagano Tomokazu , Taiji Mutsuo , Noguchi Hiroshi , Matsumoto Kunio , Nakamura Toshikazu , Higaki Jitsuo , Ogihara Toshio
Published: July 2021

Background
—Although hepatocyte growth factor (HGF), a novel angiogenic growth factor, plays an important role in angiogenesis, regulation of local HGF production under hypoxia has not yet been clarified in vascular smooth muscle cells (VSMC) and endothelial cells (EC). Thus, we have studied the role of HGF in hypoxia-induced endothelial injury and the regulation of local vascular HGF expression in response to hypoxia.

Methods and Results
—HGF attenuated hypoxia-induced endothelial cell death. Importantly, hypoxic treatment of EC resulted in a significant decrease in local HGF production according to the severity of hypoxia and increased VEGF. Similarly, hypoxia significantly decreased in mRNA and protein of HGF and increased VEGF production in VSMC. In organ culture system, local HGF production was also significantly decreased by hypoxia (
P
<0.01). Downregulation of HGF by hypoxia is due to a significant decrease in cAMP, as hypoxic treatment decreased cAMP, a stimulator of HGF. Although active TGF-β, a suppressor of HGF, was increased at 72 hours after hypoxic treatment, treatment of anti-TGF-β antibody did not attenuate decreased HGF production. Finally, rHGF was intra-arterially administered into unilateral hind limb ischemia rabbits, to evaluate in vivo angiogenic activity. Of importance, a single intra-arterial administration of rHGF reduced severe necrosis due to ischemia in rabbit muscle, accompanied by a significant increase in angiographic score (
P
<0.01).

Conclusions
—Overall, these data demonstrated that hypoxic treatment of vascular cells significantly downregulated HGF production due to decreased cAMP, suggesting their potential roles in the pathophysiology of ischemic diseases. Moreover, administration of rHGF induced therapeutic angiogenesis, accompanied by improvement of necrotic changes in the ischemic hind limb model, as cytokine supplement therapy for peripheral arterial disease.
Prospectively Randomized Evaluation of Stentless Versus Conventional Biological Aortic Valves
Authors: Walther Thomas , Falk Volkmar , Langebartels Georg , Krüger Martin , Bernhardt Uta , Diegeler Anno , Gummert Jan , Autschbach Rüdiger , Mohr Friedrich W.
Published: July 2021

Background
—The aim of this prospectively randomized study was to evaluate left ventricular hypertrophy and its regression after stentless versus conventional biological aortic valve replacement.

Methods and Results
—From March 1996 through April 1998, 180 patients were prospectively selected; 106 patients received a stentless aortic valve (SAV), and 74 received a conventional stented bioprosthesis (CSB). Of these patients, 95% and 96%, respectively, had aortic stenosis. Their mean age was 72.3 and 74.8 years, and there were no significant differences in left ventricular function, preoperative pressure gradients, and NYHA functional status. Aortic annulus diameter indexes were comparable at 13.46 (SAV) versus 13.55 (CSB) mm (
P
=NS). Larger SAVs were implanted because of the oversizing technique. In-hospital mortality (n=3 and 1 for SAV and CSB) was not valve related. At follow-up, all patients were in NYHA class 1 or 2. Baseline end-diastolic left ventricular posterior wall thickness was 15.6 (SAV) and 14.8(CSB) mm (
P
=NS) and decreased to 11.8 (SAV) and 13.2 (CSB) mm (
P
<0.05) at 6 months. Left ventricular mass index was 213 and 202 g/m
2
at baseline (
P
=NS), whereas after 6 months, it was 141 (SAV) and 170 (CSB) g/m
2
(
P
<0.05).

Conclusions
—Regression of left ventricular hypertrophy occurs in all patients after aortic valve replacement but is significantly enhanced after SAV implantation. This possibly is due to improved transvalvular hemodynamics.
Alterations in Endogenous Nitric Oxide Production After Cardiopulmonary Bypass in Lambs With Normal and Increased Pulmonary Blood Flow
Authors: McMullan D. Michael , Bekker Janine M. , Parry Andrew J. , Johengen Michael J. , Kon Alexander , Heidersbach R. Scott , Black Stephen M. , Fineman Jeffrey R.
Published: July 2021

Background
—After cardiopulmonary bypass (CPB), altered vascular reactivity is a major source of complications, particularly for children with increased pulmonary blood flow. Although changes in agonist-induced NO activity are well described after CPB, potential changes in basal NO production and their role in post-CPB pulmonary hypertension remain unclear. By using aortopulmonary vascular graft placement in the fetal lamb (shunt lambs), we established a unique model of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. The objective of the present study was to investigate potential alterations in endogenous NO production after CPB in lambs with normal and increased pulmonary blood flow.

Methods and Results
—Vascular pressures and blood flows were monitored in 1-month-old lambs (n=7) with increased pulmonary blood flow and 6 age-matched control lambs. After shunt closure, hypothermic CPB (25°C) was performed for 2 hours. The hemodynamic variables were monitored for 4 hours after CPB. Before, during, and after CPB, peripheral lung biopsies were performed to determine tissue NO, nitrite, nitrate, and cGMP concentrations; total NO synthase (NOS) activity; and endothelial NOS protein levels. Hypothermic CPB increased both mean pulmonary arterial pressure and left pulmonary vascular resistance (
P
<0.05). The increase in pulmonary arterial pressure induced in shunt lambs was greater than that induced in control lambs (
P
<0.05). Four hours after CPB, tissue concentrations of NO, nitrite, nitrate, and cGMP were decreased to ≈70% of pre-CPB levels in both control and shunt lambs (
P
<0.05). Total NOS activity and endothelial NOS protein levels were unchanged.

Conclusions
—Modest decreases in basal NO production, the inability to increase NO production, or both may play a role in the altered pulmonary vascular reactivity after CPB. The decrease in NO is independent of gene expression. However, other mechanisms for this decrease, such as substrate or cofactor availability, warrant further study.
Intramyocardial Transplantation of Autologous Myoblasts
Authors: Pouzet Bruno , Vilquin Jean-Thomas , Hagège Albert A. , Scorsin Marcio , Messas Emmanuel , Fiszman Marc , Schwartz Ketty , Menasché Philippe
Published: July 2021

Background
—Autologous skeletal myoblast (SM) transplantation improves function of infarcted myocardium, but pretransplantation cultures remain a complex process. This study assessed whether it could be optimized by muscle preconditioning with the local anesthetic bupivacaine or even bypassed with the use of the so-called mince technique.

Methods and Results
—Muscle preconditioning consisted of intramuscular injections of the tibialis anterior of rats, 2 days before harvest. After 7 days of culture, the number of available myoblasts was significantly increased compared with nonconditioned controls (1 683 147 versus 85 300,
P
=0.0013). The mince technique was then assessed. A myocardial infarction was created in 66 rats by coronary artery ligation. One week later, rats were reoperated on and intramyocardially injected with culture medium alone (controls, n=23), autologous cultured SM (3.5×10
6
, n=21), or autologous muscle minced into a fine slurry, which was immediately transplanted (n=22). All muscles had been preconditioned. Left ventricular function was assessed by 2D echocardiography. Whereas end-diastolic volumes expanded over time in all groups, left ventricular ejection fraction (%, mean±SEM) was increased only in the cultured SM–transplanted group at 1 (
P
=0.0006) and 2 months (
P
=0.0008) versus baseline (37.52±1.92 and 40.92±2.17 versus 30.34±1.74), with a significant additional benefit between 1 and 2 months (
P
=0.0069).

Conclusions
—Cell culture remains mandatory for SM transplantation to be successful but, in a clinical perspective, this process can be made more expeditious by preharvest muscle conditioning with bupivacaine, which greatly enhances the baseline cell yield.
Reduction of Vein Graft Disease Using Photodynamic Therapy with Motexafin Lutetium in a Rodent Isograft Model
Authors: Yamaguchi Atsushi , Woodburn Kathryn W. , Hayase Motoya , Robbins Robert C.
Published: July 2021

Background
—Motexafin lutetium (Lu-Tex) is a photosensitizer that targets atheromatous plaque. Subsequent photoactivation (photodynamic therapy [PDT]) induces local cytotoxic effects. The aim of the present study was to investigate whether Lu-Tex targets vein graft intimal hyperplasia and whether subsequent photoactivation attenuates the disease process.

Methods and Results
—The subcellular localization of Lu-Tex and postillumination viability were studied in cultured human vein graft smooth muscle cells. Inferior vena cava–grafted rats were injected with Lu-Tex (10 mg/kg) 4 or 12 weeks after grafting. Biodistribution was assessed in a subgroup 24 hours after administration. Light therapy (742 nm) was performed 24 hours after Lu-Tex injection by illuminating intraperitoneally placed isografts using a laparotomy. Animals were divided into the following 4 groups: PDT (n=15), Lu-Tex injection and laparotomy (n=13), light treatment (n=14), and laparotomy only (n=13). Grafts were harvested 14 days after treatment for histochemical analysis. Lu-Tex localized within subcellular organelles of smooth muscle cells, and subsequent photoactivation induced cell death via apoptosis. The Lu-Tex concentrations present in the vein grafts were 9.3 times higher than those in the normal inferior vena cava. Postoperative PDT at 4 weeks after surgery significantly reduced the intima/media ratio, whereas treatment at 12 weeks did not reduce the intima/media ratio. Activated macrophages were observed 4 weeks after grafting; however, a significant reduction occurred in these cells by 12 weeks. The mechanism by which PDT works may be related to the presence of activated macrophages.

Conclusions
—PDT significantly reduces the intima/media ratio in the early phase of vein graft disease. Lu-Tex–mediated PDT may be a viable method for the attenuation of atherosclerotic disease in vein grafts.
Prosthesis-Patient Mismatch Affects Survival After Aortic Valve Replacement
Authors: Rao Vivek , Jamieson W. R. Eric , Ivanov Joan , Armstrong Susan , David Tirone E.
Published: July 2021

Background
—Surgeons traditionally avoid the use of “small” aortic prostheses because of the potential for residual left ventricular outflow tract obstruction and persistent transvalvular gradients. This study examines the ratio between prosthetic valve size and the body surface area (BSA) of patients undergoing aortic valve replacement (AVR). We sought to determine the effect of potential “prosthesis-patient” mismatch on long-term survival.

Methods and Results
—Follow-up was conducted on 2981 patients who underwent AVR with stented bioprostheses between 1976 and 1996. To account for differences between manufacturers’ labeled valve sizes, we calculated the ratio between the prosthetic valve effective orifice area (EOA) and the patient’s BSA (recorded for 2154 patients). The lowest decile in this cohort had a calculated EOA/BSA of <0.75 cm
2
/m
2
(Small group, n=227) compared with the control group (n=1927), in whom the EOA/BSA ratio was >0.75 cm
2
/m
2
. Operative mortality was higher in the Small group (8% versus 5%,
P
=0.03). Actuarial survival at 12 years was 50±5% in the Small group compared with 49±2% in the control group (
P
=0.27). However, freedom from valve-related mortality was significantly lower in the Small group (75±5% versus 84±2%,
P
=0.004). Cox regression analysis determined age and NYHA functional class to be the multivariate predictors of overall mortality, whereas advanced age and EOA/BSA <0.75 cm
2
/m
2
were found to be the predictors of valve-related mortality.

Conclusions
—Prosthesis-patient mismatch results in significantly higher early and late mortality after bioprosthetic AVR. We recommend careful selection of stented bioprostheses to ensure an adequate ratio of EOA to BSA. An EOA/BSA ratio of >0.75 cm
2
/m
2
may avoid residual left ventricular outflow tract obstruction and persistent transvalvular gradients. Careful prosthesis-patient matching will improve both early and late survival after AVR.
Complete Arterial Revascularization in Multivessel Coronary Artery Disease With 2 Conduits (Skeletonized Grafts and T Grafts)
Authors: Wendler Olaf , Hennen Benno , Demertzis Stefanos , Markwirth Torsten , Tscholl Dietmar , Lausberg Henning , Huang Qi , Dübener Lennard Friedrich , Langer Frank , Schäfers Hans-Joachim
Published: July 2021

Background
—Complete arterial CABG is a surgical option to improve long-term results in the treatment of coronary artery disease (CAD). Harvesting of multiple arterial grafts is commonly associated with prolonged operating times and increased trauma. By use of new operative techniques (skeletonized grafts and the T-graft approach), CABG in multivessel CAD is now possible with only 2 grafts. We present our experience in the use of these techniques on a routine basis.

Methods and Results
—Between March 1996 and September 1999, 490 patients (aged 61±9 years, 20% female) underwent complete arterial CABG. Left ventricular ejection fraction ranged from 15% to 85% (mean 59±15%). Triple-vessel disease was present in 88% of the patients. The incidence of diabetes mellitus was 32% (14% insulin dependent). Either both internal thoracic arteries (ITAs) (23%) or the left ITA and radial artery (77%) were used as conduits. In 85% of the patients, a T graft was created. Mean operating time was 198±46 minutes; bypass time, 82±25 minutes; and ischemic time, 58±22 minutes. Two to 7 (mean 4.1±0.9) anastomoses were performed per patient. Perioperative intra-aortic balloon pump was necessary in 12 patients (2.4%). The rate of perioperative myocardial infarction was 1.2%. Sternal complications occurred in 1.0%, and in-hospital mortality was 2.2%. Postoperative coronary angiography in 172 patients (35%) documented excellent patency rates (left ITA 98.3%, right ITA 96.5%, and radial artery 96.6%).

Conclusions
—Complete arterial revascularization in multivessel CAD is possible with the use of only 2 grafts with good perioperative results. This approach allows for complete arterial CABG on a routine basis.
Autologous Endothelialized Vein Allograft
Authors: Lamm Peter , Juchem Gerd , Milz Stefan , Schuffenhauer Matthias , Reichart Bruno
Published: July 2021

Background

Early graft failure after the use of less satisfactory autologous grafts (30% of all vein grafts) is caused primarily by the following problems: (1) suitable autologous transplants are limited; (2) biotechnology has not yet been able to produce reliable graft substitutes that are legally and ethically approved; and (3) current prosthetic materials are prothrombotic. To overcome these problems, we developed an easily accessible, quality-controlled graft.

Methods and Results

Human autologous venous endothelial cells (HAVECs) were isolated from short segments of peripheral veins unsuitable for bypass grafting. After mechanical deendothelialization of cryopreserved allograft veins (CAVs) and precoating with recipient autologous serum, these homologous cells were seeded by use of a rotating device. Growth of a confluent HAVEC layer within 1 week in a special incubator was observed. After histological and mechanical tests, 12 patients received 15 grafts in total. Bypass operation was followed by clinical and angiographic follow-up. Production period was 22±8 days. HAVEC-coated CAVs showed normal connective tissue wall structure and a tight endothelial monolayer (burst pressure >2000 mm Hg). To date, 12 CABG patients lacking suitable autologous graft material have been treated. One patient died of a cause unrelated to the grafts, which were found morphologically normal and patent during autopsy. Of 15 grafts, 2 were occluded at the first angiographic follow-up. The oldest graft has now been in place for ≈3 years. Immune suppression was not administered.

Conclusions

At present, autologous endothelialized CAVs present good alternative small-caliber grafts for patients lacking suitable autologous vessels.
General Health Status of Children With
d
-Transposition of the Great Arteries After the Arterial Switch Operation
Authors: Dunbar-Masterson Carolyn , Wypij David , Bellinger David C. , Rappaport Leonard A. , Baker Annette L. , Jonas Richard A. , Newburger Jane W.
Published: July 2021

Background

To study the long-term impact on general health status of
d
-transposition of the great arteries (D-TGA) after the arterial switch operation (ASO) during infancy, we asked parents to complete the Child Health Questionnaire, Parent Form-50 when their children were 8 years old.

Methods and Results

Of 160 eligible patients, questionnaires were completed for 155 subjects (96%). Median age at surgery was 6 days (range 1 to 67 days), and median age at completion of the Child Health Questionnaire was 8.1 years (7.6 to 10.0 years). Subsequent to questionnaire completion, children underwent psychometric testing. Mean Physical Health Summary and Psychosocial Summary scores were 54.0±6.1 and 49.7±9.9, respectively, which were similar to those of normal subjects. Compared with the normative sample, parents of D-TGA patients reported more problems with attention, learning, and speech, as well as greater frequency of developmental delay (
P
<0.001 for each). Worse Psychosocial Summary scores were significantly associated with lower full-scale IQ (
P
=0.001) and lower achievement in reading (
P
=0.005) and math (
P
=0.007). Worse Physical Health Summary scores were associated with longer hospital stay after the ASO (
P
=0.02). General health status scores were not significantly related to presence of ventricular septal defect, age at surgery, perfusion variables during the ASO, sex, or history of cardiac reoperation.

Conclusions

At age 8 years, children with D-TGA after ASO have an overall physical and psychosocial health status similar to that of the general population. Lower IQ and academic achievement are associated with worse psychosocial health status, whereas longer hospital course after initial surgery is associated with worse physical health status.
Increased Prevalence of Autoimmune Phenomena and Greater Risk for Alloreactivity in Female Heart Transplant Recipients
Authors: Lietz Katherine , John Ranjit , Kocher Alfred , Schuster Michael , Mancini Donna M. , Edwards Niloo M. , Itescu Silviu
Published: July 2021

Background

The influence of sex on alloreactivity and graft outcome after heart transplantation was evaluated.

Methods and Results

A retrospective review of 520 consecutive recipients of a primary cardiac allograft between 1992 and 2000 at a single center was performed. The influence of sex on alloreactivity, acute rejection, transplant-related coronary artery disease, and survival was determined. Statistical methods included logistic regression analysis and Kaplan-Meier actuarial survival analysis. Female recipients had an increased prevalence before transplant of idiopathic cardiomyopathy, antinuclear antibodies, and HLA-B8, DR3 haplotypes. After transplant, female sex predicted shorter duration to a first rejection, higher cumulative rejection frequency, and earlier posttransplant production of anti-HLA antibodies. Female recipients had higher early mortality rates (<6 months) that were due to infection. Fatal infections correlated with 2-fold higher cyclosporine levels in female recipients. However, the incidence of transplant-related coronary artery disease developing beyond 1 year after transplant was lower in female than in male recipients.

Conclusions

Females undergoing cardiac transplantation are more likely to manifest features of an underlying autoimmune state. This may predispose to a higher posttransplant risk of allograft rejection and requirement for increased immunosuppression. Earlier diagnosis and management of alloreactivity in female recipients before development of acute rejection and the use of more focused and less globally immunosuppressive agents during established rejections may have a significant effect on the clinical outcome of female cardiac allograft recipients.
Abstract 165: Diacylglycerol kinase α Exacerbates Cardiac Injury After Ischemia/reperfusion In Transgenic Mouse Hearts
Authors: Sasaki Toshiki , Takeishi Yasuchika , Kitahara Tatsuro , Koyama Yo , Niizeki Takeshi , Suzuki Satoshi , Kubota Isao
Published: July 2021

Background: Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade has been implicated in cardiac dysfunction after ischemia/reperfusion (I/R). DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid, and thus may act as a regulator of DAG-PKC signaling. It has been reported that α, ϵ, and B6; isoforms of DGK have been identified in the heart. We examined the functional role of DGKα in cardiac injury after I/R.

Methods and Results: We generated transgenic mice with cardiac specific overexpression of DGKα (DGKα-TG) using α-myosin heavy chain promoter. Left anterior descending coronary artery was transiently occluded for 30 min and reperfused for 24 hr in DGKα-TG and wild type littermate (WT) mice. After 24 hr, echocardiography was performed, mice were sacrificed, and the hearts were removed. Areas at risk and infarct size were assessed by Evans blue/TTC double staining. Left ventricular chamber dilatation after I/R was more pronounced in DGKα-TG mice than in WT mice (left ventricular end-diastolic dimension: 3.44 +/− 0.41 mm vs. 3.05 +/−0.24 mm, P < 0.01). Left ventricular systolic function was more severely depressed in DGKα-TG mice than in WT mice (left ventricular fractional shortening: 34.3 +/− 3.6% vs. 42.2 +/− 4.0%, P < 0.01). The ratio of infarct size/area at risk at 24 hr after I/R was larger in DGKα-TG mice than in WT mice (17.9 +/− 4.2% vs. 11.3 +/− 3.3%, P < 0.01). The phosphorylation activity of extracellular-signal regulated kinase (ERK) was increased after I/R in WT mouse hearts (2.58 +/−0.52-fold increase, P<0.0001). However in DGKα-TG mice, activation of ERK after I/R was abolished (1.77+/−0.67-fold increase, P<0.01 vs. WT).

Conclusion: DGKα exacerbates cardiac injury by inhibiting cardio-protective effect of ERK activation.
Abstract 1029: Stromelysin-1 is Critical For Intracranial Bleeding After Tissue-type plasminogen activator Treatment Of Stroke In Mice
Authors: Suzuki Yasuhiro , Nagai Nobuo , Collen Desire , Lijnen Roger , Umemura Kazuo
Published: July 2021

Background:
Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it may increase the risk of intracranial bleeding (ICB). Matrix metalloproteinases (MMPs), which can be activated through the plasminogen/plasmin system, may contribute to ICB after ischemic stroke.

Objectives:
To explore the contribution of plasminogen, MMP-3 and MMP-9 to ICB associated with t-PA treatment after ischemic stroke.

Methods:
Using a thrombotic middle cerebral artery occlusion (MCA-O) model, ICB was studied in mice with genetic deficiencies of plasminogen (Plg
−/ −
), stromelysin-1 (MMP-3
−/ −
) or gelatinase B (MMP-9
−/ −
) and their corresponding wild-type (WT) littermates. t-PA (10 mg/kg) or its equivalent volume of solvent was administered intravenously 4 hours after MCA-O. The induction of MMP-3 and MMP-9 was also studied in C57BL/6 WT mice.

Results:
In MMP-3
+/+
WT mice given solvent, ICB was 4.3 ± 2.9 mm
3
(mean ± SD), which was significantly increased with tPA treatment to 9.7 ± 4.7 mm
3
(P<0.05), whereas ICB in MMP-3
−/ −
mice was not altered by t-PA treatment (5.7 ± 2.7 mm
3
, as compared to 5.1 ± 1.8 mm
3
without tPA; n = 7–9 in each group). ICB induced by t-PA was significantly less in Plg
−/ −
(5.7 ± 3.9 mm
3
) than in WT mice (8.8 ± 3.2 mm
3
, p<0.05) but ICB by t-PA in MMP-9
−/ −
(8.3 ± 2.3 mm
3
) was comparable with that in WT (8.3 ± 3.1 mm
3
; n=8 –12 in each group). Administration of the broad-spectrum MMP inhibitor GM6001 after t-PA treatment reduced ICB significantly in MMP-3
+/+
(from 6.4 ± 1.9 mm
3
to 4.1 ± 1.9 mm
3
, p<0.05) but not in MMP-3
−/ −
mice (2.2 ± 0.6 mm
3
without versus 2.9 ± 1.5 mm
3
with GM6001; n=6 – 8 in each group). MMP-3 expression was significantly enhanced at the ischemic hemisphere; with placebo treatment, it was expressed only in neurons, whereas it was upregulated in endothelial cells with t-PA treatment. Although MMP-9 expression was also significantly enhanced at the ischemic brain, the amount and the distribution were comparable in mice with and without t-PA treatment.

Conclusions:
Our data with gene deficient mice suggest that plasminogen and MMP-3 are relatively more important than MMP-9 for the increased ICB induced by t-PA treatment of ischemic stroke.
Abstract 1358: Reentry within the Working Myocardium is not the Sole Mechanisms by which Ventricular Fibrillation is Maintained
Authors: Li Li , Jin Qi , Huang Jian , Smith William M , Ideker Raymond E
Published: July 2021

Background: For well over 50 years, it has been assumed that ventricular fibrillation (VF) is maintained solely by reentry in the working myocardium. This hypothesis has never been tested by recording VF with electrodes spaced sufficiently close together to map activation sequences in 3-dimensions.

Methods and Results: We recorded for 10 min during VF from the region of insertion of the anterior papillary muscle in the left ventricle of 4 open-chest pigs. A 3-D transmural unipolar electrode array consisting of a 9×9 array of needles with 2-mm spacing and 6 electrodes 2 mm apart on each needle was used to record from the working myocardium. The numbers of wavefronts, foci and reentrant circuits per sec were counted. A focus was identified when a wavefront appeared de novo within the central portion of the mapped region, insuring that it did not arise from another wavefront nor propagate into the mapped region from outside it. While intramural reentry was present early but not late during VF in the mapped region, foci were numerous and increased as VF continued (Figure

).

Conclusion: Intramural foci are present during VF in pigs and, as VF continues, increase in incidence while the incidence of reentry in working myocardium decreases. These results suggest that, particularly after the first 2 min of VF, mechanisms other than reentry in the working myocardium maintain VF. It remains to be determined if these mechanisms consist of abnormal automaticity, afterdepolarizations, or reentry in which Purkinje fibers form all or part of the circuit.
Abstract 1375: Brain Renin Angiotensin Blockade Attenuates Cytokines and Oxidative Stress in the Paraventricular Nucleus of Hypothalamus and Decreases Sympathoexcitation in Heart Failure Rats
Authors: Ma Ying , Kang* Yu-Ming , Yang Zhi-Ming , Francis* Joseph
Published: July 2021

Introduction:
Neurohumoral mechanisms play an important role in the pathophysiology of congestive heart failure (HF). Recent studies suggest that the brain renin angiotensin system (RAS) plays an important role in regulating body fluids and sympathetic drive in HF. In addition, it has been shown that there is cross talk between cytokines and RAS in cardiovascular disease. In this study we determined whether blockade of brain RAS attenuate inflammatory cytokines and oxidative stress in HF rats.

Methods and Results:
Adult male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannulae and subjected to coronary artery ligation to induce HF and confirmed by echocardiography. Rats were treated with an angiotensin type 1 receptors (AT1-R) antagonist losartan (LOS, 20 μg/hr, ICV) or vehicle (VEH) for 4 weeks. At the end of the study, left ventricular (LV) function was measured by echocardiography and rats were sacrificed, and brain and plasma samples were collected for measurements of cytokines and superoxide using immunohistochemistry, Western blot and real time RT-PCR. HF rats induced significant increases in Nuclear Factor-kappaB (NF-κB) p50-positive neurons and activated microglia in the paraventricular nucleus (PVN) of hypothalamus, and TNF-α, IL-1β, IL-6 and NF-κB p50 in hypothalamus when compared with sham rats. These animals also had increased staining for dihydroethidium (DHE) and plasma levels of norepinephrine (NE), an indirect indicator of sympathetic activity. In contrast, ICV treatment with LOS attenuated cytokine expression and oxidative stress in the PVN and hypothalamus when compared with VEH treated HF rats. ICV treatment with LOS also reduced plasma NE levels, and proinflammatory cytokine, heart weight to body weight ratio with decreased LV end-diastolic pressure.

Conclusions
: These findings suggest the cross talk between the cytokines and renin angiotensin system within the brain contribute to sympatho-excitation in HF.
Abstract 255: Laminar Shear Stress Enhances the Activity and Localization of RNA Polymerase II on the eNOS Gene
Authors: Weber Martina , Moore Jeffrey P , Searles Charles D
Published: July 2021

We have previously found that laminar shear enhances eNOS mRNA stability and translation by altering endothelial NO synthase (eNOS) mRNA 3′ polyadenylation. Transcription is tightly coupled to pre-mRNA processing, and is coordinated by RNA polymerase (RNAP) II. We assessed whether laminar shear stress alters the activity and localization of RNAP II on the eNOS gene. We found that endothelial cells exposed to laminar shear stress had a 2-fold (n=3, p < 0.05) increase in total RNAP II protein levels compared to control; this effect was dose-dependent (0–15 dynes/cm2). Since three different RNAP II phosphoisoforms are associated with different stages of mRNA synthesis, we examined whether shear stress affected protein expression of these particular phosphoisoforms. We found that cells exposed to laminar shear had a 3-fold increase (p<0.05) in expression of RNAP II phosphorylated at serine 2, which is associated with transcription elongation and 3′ polyadenylation. In contrast, shear stress did not alter expression of the other phosphoisoforms. We performed chromatin immunoprecipitation (ChIP) analysis to examine shear-induced changes in RNAP II localization on the eNOS gene. Using antibody against total RNAP II, eNOS sequences along the entire gene were amplified. Using the phosphoserine 2 specific RNAP II antibody, we found active RNAP II predominantly bound to the 3′UTR (exon 26) and downstream sequence of eNOS in sheared cells. These findings were confirmed by quantitative ChIP (3 fold increase, n = 8, p = 0.0378). This suggests that shear-induced changes in RNAP II phosphorylation enhance its ability to polyadenylate eNOS mRNA. Serine 2 phosphorylation is dependent on cyclin-dependent kinase 9 (CDK 9), and shear-induced recruitment of CDK 9 to the eNOS gene was examined. We found that endothelial cells exposed to laminar shear had enhanced localization of CDK 9 to the eNOS promoter and exons 8 and 22, and diminished localizationto exon 26 and in the downstream sequence. This indicates that shear recruits CDK 9 while RNAP II is bound to the promoter and early exons to activate RNAP by phosphorylating serine 2. In conclusion, laminar shear stress enhances eNOS mRNA processing and increases gene expression through recruitment of CDK 9 and activation of RNAP II.
Abstract 1835: Magnetic Resonance Imaging Detects Chronic Left Atrial Wall Injury Post Ablation of Atrial Fibrillation
Authors: McGann Christopher J , Khoumovski Eugene , Oakes Robert , DiBella Edward , MacLeod Robert , Parker Dennis , Fish Brenda , Daccarett Marcos , Segerson Nathan , Marrouche Nassir F
Published: July 2021

Radiofrequency (RF) based ablation of atrial fibrillation (AF) through pulmonary vein antrum isolation (PVAI) is a promising treatment for this arrhythmia. Success of the procedure relies in part on the transmural extent of the RF lesions delivered to the left atrium (LA). We describe a novel technique using a 3D MRI to assess sites and extent of LA injury resulting from RF lesions after PVAI. Twelve patients (7 men, mean age 56±11 years) undergoing PVAI for AF were imaged pre and three months post procedure to detect LA scarring with delayed contrast-enhanced MRI using a 3D inversion recovery, navigated, gradient echo pulse sequence. LA injury post procedure was seen in all patients on MRI as areas of bright, signal enhancement, which correlated with the RF energy delivery map during PVAI (figure 1

).

Conclusion: This is the first study to show LA wall injury post PVAI using 3D MRI. These findings may prove important for tracking and monitoring outcomes of AF ablation.

Figure 1.

Posterior views of the left atrial wall in a patient one day pre (left) and three months post (right) ablation by delayed contrast-enhanced, navigated, 3D MRI. Regions of bright signal show injury resulting from RF ablation.
Abstract 2113: Early End-organ Alterations Due To High Blood Pressure In Children
Authors: Ketelhut Reinhard G , Rode Ulrich , Schröter Jörg
Published: July 2021

Introduction
: Increased left ventricular (LV) mass is known to be an important risk for future cardiovascular morbidity and mortality. Since high blood pressure can be observed more often in early childhood a study was designed to evaluate the influence of blood pressure (BP) on LV mass and function in children.

Methods:
2-D-guided-echocardiography and Doppler-echocardiography were performed in 103 children (aged 14.1±0.9 years, 58 girls). BP, LV-mass-index (LVMI) and diastolic function (E/A ratio) were measured and calculated by standardized formula and procedure.

Results:
Systolic BP (SBP) was significantly higher in boys (124.8±10.6 mmHg) when compared with girls (120.5±8.4 mmHg) (p<0.01). There were no significant differences in diastolic pressure. LVMI was significantly lower in girls (57.6±8.9 g/m
2
) than in boys (68.3±11.2 g/m
2
) (p<0.01). There was a negative correlation between SBP and E/A ratio as a measure of LV diastolic function (p<0.01). Hypertensives had a 17% higher LVMI (78±11g/m
2
versus 67±11g/m
2
; p<0.05), and a 20% lower E/A ratio (1.58±0.3 versus 1.97±0.42) than their normotensive counterparts (p<0.01).

Conclusion:
Despite the young age of participants with higher blood pressure they had prognostically adverse preclinical cardiovascular disease, including LV hypertrophy and evidence of impaired LV function. Therefore children should be encouraged to enter preventive programs as a primary and early strategy against future cardiovascular morbidity and mortality.
Abstract 2220: The Relative Safety and Efficacy of Clopidogrel in Women and Men: Meta-Analysis of 79,613 Patients Enrolled in CREDO, CURE, CLARITY-TIMI 28, COMMIT, and CHARISMA
Authors: Berger Jeffrey S , Bhatt Deepak L , Cannon Christopher P , Chen Zhengming , Jones J B , Mehta Shamir R , Sabatine Marc S , Steinhubl Steven R , Topol Eric J , Berger Peter B
Published: July 2021

Background
: Previous analyses have demonstrated sex-based differences in the efficacy of several anti-platelet medications. Little is known about the safety and efficacy of clopidogrel in women and men.

Methods
: We performed a meta-analysis of all blinded randomized clinical trials comparing clopidogrel and placebo (CREDO, CURE, CLARITY-TIMI 28, COMMIT, and CHARISMA). The relative safety and efficacy of clopidogrel at reducing cardiovascular events (death, MI or stroke) in women and men was estimated using random-effects modeling.

Results
: In all 79,613 patients, there was a reduction of cardiovascular events with clopidogrel vs placebo (9.7% vs 8.7%; OR 0.88 [0.84–0.93], P<0.01). Among the 23,522 women enrolled, there were 1289/11757 (11.0%) cardiovascular events in the clopidogrel group vs. 1372/11766 (11.7%) in the placebo group (OR 0.93 [0.86–1.01], P=0.09). Clopidogrel had its greatest effect in reducing the risk of MI (OR 0.82 [0.70–0.95], P=0.01) in women. No significant reduction was noted for stroke (OR 0.91 [0.67–1.24], P=0.56) or death (OR 0.99 [0.90–1.08], P=0.78). Among the 56,091 men enrolled, there were 2166/28064 (7.7%) cardiovascular events in the clopidogrel group vs 2492/28027 (8.9%) in the placebo group (OR 0.85 [0.79–0.91], P<0.01). Clopidogrel significantly reduced the risk of MI (OR 0.84 [0.76–0.92], P<0.01), stroke (OR 0.83 [0.71–0.96], P=0.01), and death (OR 0.91 [0.84–0.98], P<0.01). Clopidogrel increased the risk of major bleeding in both women (1.6% vs 1.1%, OR 1.43 [1.14–1.79]) and men (1.2% vs 1.0%, OR 1.21 [1.03–1.42]). The safety and efficacy were similar in patients with an acute coronary syndrome and established cardiovascular disease. There was no evidence of statistical heterogeneity (as measured by the Q-test and I
2
) in pooled analyses comparing women and men for the composite endpoint, its individual components, or bleeding.

Conclusions
: Clopidogrel reduces the risk of cardiovascular events in both women and men. While the directionality and proportionality of the reductions are roughly similar, the effect was driven by a reduction of MI in women. The reduction of MI, stroke and death by clopidogrel in men were all significant. Clopidogrel increased the risk of major bleeding by 43% in women and 21% in men.
Abstract 2636: Age-Related Triggers for Life-Threatning Arrhythmia in the Genotyped Long QT Syndrome
Authors: Sakaguchi Tomoko , Shimizu Wataru , Ito Hideki , Noda Takashi , Miyamoto Yoshihiro , Nagaoka Iori , Ashihara Takashi , Ito Makoto , Tsuji Keiko , Ohno Seiko , Makiyama Takeru , Kamakura Shiro , Horie Minoru
Published: July 2021

OBJECTIVES
We aimed to examine whether triggers for cardiac events are different depending on the age in genotyped and symptomatic long QT syndrome (LQTS) patients.

BACKGROUND
Patients with LQTS become symptomatic in adolescence, but some at age ≥20 y/o. It remains unknown whether clinical features of these symptomatic LQTS patients differ depending on the onset of age.

METHODS
Study cohort consisted of consecutive 108 symptomatic LQTS patients (34 males and 74 females from 88 families) who were identified to carry the following genotypes; LQT1 (n = 48), LQT2 (n = 52), LQT3 (n = 8). They were divided into 2 groups according to the age of their first onset of symptoms (low age <20 y/o, n = 75, average age = 10 y/o, and high age ≥20 y/o, n = 33, average age = 43 y/o), and triggers of their cardiac events (ventricular tachycardia, syncope or cardiac arrest) were analyzed. We divided the triggers into 3 categories:

adrenergically-mediated factors; exercise, emotional stress, loud noise and arousal,

vagally-mediated factors; rest/sleep, and

secondary factors; drugs, hypokalemia, and bradycardia.

RESULTS
Percentage of patients with family history was significantly higher in the low age group (68% vs. 39%; p = 0.005), and prevalence of females and probands were significantly higher in the high age group (60% vs. 88%; p = 0.004, 75% vs. 97%; p = 0.006, respectively). There were no significant differences in both QTc intervals (523 ± 7 ms vs. 505 ± 10; p = 0.15) and Schwartz scores (6.3 ± 0.2 vs. 5.6 ± 0.3; p = 0.06) between the 2 groups. In the low age group, 76% of the cardiac events were initiated by adrenergically-mediated factors, and 20% were vagally mediated, but none was triggered by secondary factors. In contrast, in the high age group, 48% of the cardiac events were triggered by secondary factors, 12% adrenergically-mediated, and 18% vagally-mediated factors. The prevalence of secondary factors were as follows; 0/75 [0%] in low age vs. 16/33 [48%] in high age; p < 0.001. About the subdivision of secondary triggers on the genotype, hypokalemia was only observed in LQT1, drugs mainly in LQT2, and bradycardia only in LQT2.

CONCLUSION
Arrhythmic triggers in LQTS differ depending on the age of the patients, stressing the importance of age-related therapy for genotyped LQTS patients.
Abstract 2793: Comparison of Midregional Pro-atrial Natriuretic Peptide, B-type Natriuretic Peptide, and Amino-terminal Pro-B-type Natriuretic Peptide in Patients with Chronic Heart Failure: Influencing Factors, Detection of Left Ventricular Systolic Dysfunction, and Prediction of Death
Authors: Moertl Deddo , Huelsmann Martin , Struck Joachim , Gleiss Andreas , Hammer Alexandra , Morgenthaler Nils G , Bergmann Andreas , Berger Rudolf , Pacher Richard
Published: July 2021

Background: Although natriuretic peptides are increasingly used for the management of chronic heart failure (CHF), there are sparse comparative data. Therefore, we compared the importance of influencing factors, the ability to detect left ventricular systolic dysfunction, and the prognostic power of midregional pro-atrial natriuretic peptide (MR-proANP), B-type natriuretic peptide (BNP), and aminoterminal pro-B-type natriuretic peptide (NT-proBNP) in patients with chronic heart failure.

Methods and Results: MR-proANP, using a new assay directed at the midregion of aminoterminal-proANP, was compared with BNP and NT-proBNP, using conventional assays, in 797 patients with CHF. All three natriuretic peptides were independently influenced by left ventricular ejection fraction (LVEF), glomerular filtration rate (GFR), and the presence of ankle edema. Area under receiver-operator characteristic curves for detection of an LVEF <40% were similar between MR-proANP (0.799 [0.753– 0.844]) and BNP (0.803 [0.757– 0.849]), and NT-proBNP (0.730 [0.681– 0.778]. During a median observation time of 68 months, 492 patients died. In multiple Cox regression analysis each natriuretic peptide was the strongest prognostic parameter among various clinical variables, but proportion of explained variation showed that NT-proANP was a significantly stronger predictor of death than NT-proBNP and BNP (Figure

).

Conclusions: Despite similarities in influencing factors and detection of reduced LVEF, MR-proANP outperformed BNP and NT-proBNP in the prediction of death. A new assay technology and the high biological stability of MR-proANP are potential explanations for these findings.