bluuebunny/crossref_metadata_2025 huggingface dataset:
Time Trends in Simple Congenital Heart Disease Over 39 Years: A Danish Nationwide Study
Authors: El‐Chouli Mohamad , Mohr Grímur Høgnason , Bang Casper N. , Malmborg Morten , Ahlehoff Ole , Torp‐Pedersen Christian , Gerds Thomas A. , Idorn Lars , Raunsø Jakob , Gislason Gunnar
Published: July 2021

Background

We describe calendar time trends of patients with simple congenital heart disease.

Methods and Results

Using the nationwide Danish registries, we identified individuals diagnosed with isolated ventricular septal defect, atrial septal defect, patent ductus arteriosus, or pulmonary stenosis during 1977 to 2015, who were alive at 5 years of age. We reported incidence per 1 000 000 person‐years with 95% CIs, 1‐year invasive cardiac procedure probability and age at time of diagnosis stratified by diagnosis age (children ≤18 years, adults >18 years), and 1‐year all‐cause mortality stratified by diagnosis age groups (5–30, 30–60, 60+ years). We identified 15 900 individuals with simple congenital heart disease (ventricular septal defect, 35.2%; atrial septal defect, 35.0%; patent ductus arteriosus, 25.2%; pulmonary stenosis, 4.6%), of which 75.7% were children. From 1977 to 1986 and 2007 to 2015, the incidence rates increased for atrial septal defect in adults (8.8 [95% CI, 7.1–10.5] to 31.8 [95% CI, 29.2–34.5]) and in children (26.6 [95% CI, 20.9–32.3] to 150.8 [95% CI, 126.5–175.0]). An increase was only observed in children for ventricular septal defect (72.1 [95% CI, 60.3–83.9] to 115.4 [95% CI, 109.1–121.6]), patent ductus arteriosus (49.2 [95% CI, 39.8–58.5] to 102.2 [95% CI, 86.7–117.6]) and pulmonary stenosis (5.7 [95% CI, 3.0–8.3] to 21.5 [95% CI, 17.2–25.7]) while the incidence rates remained unchanged for adults. From 1977–1986 to 2007–2015, 1‐year mortality decreased for all age groups (>60 years, 30.1%–9.6%; 30–60 years, 9.5%–1.0%; 5–30 years, 1.9%–0.0%), and 1‐year procedure probability decreased for children (13.8%–6.6%) but increased for adults (13.3%–29.6%) were observed.

Conclusions

Increasing incidence and treatment and decreasing mortality among individuals with simple congenital heart disease point toward an aging and growing population. Broader screening methods for asymptomatic congenital heart disease are needed to initiate timely treatment and follow‐up.
Association of Psychological Distress, Contextual Factors, and Individual Differences Among Citizen Responders
Authors: Ries Emma Slebsager , Kragh Astrid Rolin , Dammeyer Jesper , Folke Fredrik , Andelius Linn , Malta Hansen Carolina
Published: July 2021

Background

Little is known about the psychological risks of dispatched citizen responders who have participated in resuscitation attempts.

Methods and Results

A cross‐sectional survey study was performed with 102 citizen responders who participated in a resuscitation attempt from July 23, 2018, to August 22, 2018, in the Capital Region of Denmark. Psychological distress, defined as symptoms of posttraumatic stress disorder, was assessed 3 weeks after the resuscitation attempt and measured with the Impact of Event Scale‐Revised. Perceived stress was measured with the Perceived Stress Scale. Individual differences were assessed as the personality traits of agreeableness, conscientiousness, extraversion, neuroticism, and openness to experience with the Big Five Inventory, general self‐efficacy, and coping mechanisms (Brief Coping Orientation to Problems Experienced Inventory). Associations between continuous variables were examined with the Pearson correlation. The associations between psychological distress levels and contextual factors and individual differences were analyzed in multivariable linear regression models to determine factors independently associated with psychological distress levels. The mean overall posttraumatic stress disorder score was 0.65 of 12; the mean perceived stress score was 7.61 of 40. The most common coping mechanisms were acceptance and emotional support. Low perceived stress was significantly associated with high general self‐efficacy, and high perceived stress was significantly associated with high scores on neuroticism and openness to experience. Non–healthcare professionals were less likely to report symptoms of posttraumatic stress disorder.

Conclusions

Citizen responders who participated in resuscitation reported low levels of psychological distress. Individual differences were significantly associated with levels of psychological distress and should be considered when engaging citizen responders in resuscitation.
Secondary Stroke Prevention Following Embolic Stroke of Unknown Source in the Absence of Documented Atrial Fibrillation: A Clinical Review
Authors: Kotadia Irum D. , Sim Iain , Mukherjee Rahul , O’Hare Daniel , Chiribiri Amedeo , Birns Jonathan , Bhalla Ajay , O’Neill Mark , Williams Steven E.
Published: July 2021

Approximately one‐third of ischemic strokes are classified as cryptogenic strokes. The risk of stroke recurrence in these patients is significantly elevated with up to one‐third of patients with cryptogenic stroke experiencing a further stroke within 10 years. While anticoagulation is the mainstay of treatment for secondary stroke prevention in the context of documented atrial fibrillation (AF), it is estimated that up to 25% of patients with cryptogenic stroke have undiagnosed AF. Furthermore, the historical acceptance of a causal relationship between AF and stroke has recently come under scrutiny, with evidence to suggest that embolic stroke risk may be elevated even in the absence of documented atrial fibrillation attributable to the presence of electrical and structural changes constituting an atrial cardiomyopathy. More recently, the term
embolic stroke of unknown source
has garnered increasing interest as a subset of patients with cryptogenic stroke in whom a minimum set of diagnostic investigations has been performed, and a nonlacunar infarct highly suspicious of embolic etiology is suspected but in the absence of an identifiable secondary cause of stroke. The ongoing ARCADIA (Atrial Cardiopathy and Antithrombotic Drugs in Prevention After Cryptogenic Stroke) randomized trial and ATTICUS (Apixiban for Treatment of Embolic Stroke of Undetermined Source) study seek to further define this novel term. This review summarizes the relationship between AF, embolic stroke, and atrial cardiomyopathy and provides an overview of the clinical relevance of cardiac imaging, electrocardiographic, and serum biomarkers in the assessment of AF and secondary stroke risk. The implications of these findings on therapeutic considerations is considered and gaps in the literature identified as areas for future study in risk stratifying this cohort of patients.
Abstract 2: IL-10 Modulates Mobilization of Bone Marrow Endothelial Progenitor Cells and Enhances Their Survival and Angiogenic Properties in Ischemic Myocardium
Authors: Krishnamurthy Prasanna , Thal Melissa , Verma Suresh , Hoxha Eneda , Lambers Erin , Ramirez Veronica , Qin Gangjian , Losordo Douglas , Kishore Raj
Published: July 2021

Endothelial progenitor cells (EPC) transplantation has been shown to enhance neovascularization and improve myocardial infarction (MI)-induced ventricular dysfunctions. However, persistent inflammation in the ischemic myocardium, adversely affect EPC survival and function, thereby compromising full benefits of EPC-mediated vascular repair. We hypothesized that modulation of IL-10 signaling in EPCs enhances their mobilization, survival and function in ischemic myocardium after MI. GFP-labeled EPC were transplanted intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. EPC survival and EPC-mediated neovascularization and myocardial repair were evaluated. IL-10-treated mice showed increased number of GFP+EPCs retention that was associated with reduced EPC apoptosis in the myocardium (P<0.05). The engraftment of EPC into the vascular structures and the associated capillaries density was significantly higher in IL-10-treated mice (P<0.05). The above findings were corroborated with reduced infarct size, fibrosis and enhanced LV function (echocardiography) in IL-10+EPC group as compared to EPC+saline group. Invitro, IL-10-deficient EPCs showed higher LPS-induced apoptosis compared to WT-EPCs (P<0.05). IL-10 treatment induced VEGF expression in WT-EPCs which was abrogated by STAT3 inhibition (using curcurbitacin I). Furthermore, microRNA (miR) profile experiments identified significant increases in a number of pro-apoptotic and anti-angiogenic-related miRs in EPCs from IL-10 deficient mice. Interestingly, IL-10-deficient mice showed impaired MI-induced mobilization of bone marrow EPCs (Sca1+Flk1+ cells) into the circulation and the associated SDF-1 mRNA expression in the myocardium. Bone marrow transplantation studies involving replacement of IL-10-deficient marrow with WT marrow attenuated these effects. Invitro, LPS-induced CXCR4 expression was lower in IL-10-deficient EPCs as compared to WT-EPC. Taken together, our studies suggest that IL-10 enhances EPC mobilization, possibly in an SDF1-CXCR4 dependent manner and increased their survival and neovascularization and the associated myocardial repair, in part via activation of STAT3 signaling cascades.
Abstract P007: Cardiomyocyte Damage-Released Extracellular S100A1 Protein Promotes Regeneration of Infarcted Myocardium by Modulating Cardiac Fibroblast Function
Authors: Rohde David , Qiu Gang , Herzog Nicole , Katus Hugo A , Bierhaus Angelika , Peppel Karsten , Most Patrick
Published: July 2021

Background:
Similar to heart muscle-specific creatinkinase (CK-MB), S100A1 protein is released from damaged human cardiomyocytes in response to myocardial infarction (MI). Since S100A1-knock out (SKO) mice display rapid post-MI onset of adverse myocardial remodeling and accelerated transition to heart failure, we assessed the hypothesis that ischemia-related release of S100A1 protein modulates myocardial regeneration.

Methods and Results:
After LAD ligation in C57/B6 mice, S100A1 serum levels peaked at 10 µg/ml 8 hours post-MI, precisely mirroring the time course previously observed in MI patients. RT-PCR analyses in post-MI whole heart samples revealed significantly lower I-CAM (−50%) and IL-10 (−75%) mRNA abundance as well as heightened Collagen-1 (+40%) and VEGF (+80%) expression in SKO vs. WT mice (p<0.05, n=6 in each group). Interestingly, injection of an S100A1-neutralizing antibody prior to MI in WT mice mimicked the abnormalities observed in post-ischemic SKO animals. To further elucidate extracellular S100A1 biological activity, cardiomyocytes, cardiac fibroblasts (CF), endothelial and smooth muscle cells were exposed to S100A1
in vitro
. A rapid internalization of S100A1 was exclusively found in CF, resulting in a phosphorylation of ERK1/2, JNK, and p38 with subsequent activation of NF-kappaB as assessed by Western Blot (WB) and EMSA. RT-PCR and WB analyses revealed significant alterations in CF gene expression in response to S100A1, including an increase in I-CAM (3,5-fold) and IL-10 (20-fold) mRNA levels and diminished Col-1 (−80%) expression. Similar effects were observed after direct injection of S100A1 protein into the left ventricular apical region of WT mice
in vivo
(S100A1- vs. PBS-injection, n=6). In SKO mice, intraperitoneal application of S100A1 prior to MI largely normalized the adverse gene expression pattern towards WT animals.

Conclusions:
Our study provides first evidence for cardiomyocyte damage-released S100A1 to act as an endogenous mediator of post-MI inflammation and tissue repair. Considering today's unability to manipulate these molecular mechanisms, extracellular S100A1 might represent a promising target for future therapies of MI.
Abstract P021: Adipose-Derived Stromal Cell Therapy Stabilizes Cardiac Function and Improves Border Zone Remodeling After Coronary Occlusion in Rats
Authors: Campos de Carvalho Antonio C , Bagno Luiza , Werneck de Castro João Pedro , Oliveira Patricia , Abreu Marcia , Rocha Nazareth , Kasai Brunswick Tais , Miranda Vivian , Goldenberg Regina
Published: July 2021

Recent studies have identified adipose tissue as a new source of mesenchymal stem cells for therapy. The purpose of this study was to investigate the therapy with rat adipose derived stromal cells (ASC) in a rat model of healed myocardial infarction (MI). ASC from inguinal subcutaneous adipose tissue of male Wistar rats were isolated by enzymatic digestion and filtration. Cells were then cultured until passage 3. Four weeks after ligation of the left coronary artery of female rats, a suspension of either 100µl with PBS + Matrigel + 2 x 106 ASC labeled with Hoechst (n=11) or 100µ;l of PBS + Matrigel (n=10) was injected along the borders of the ventricular wall scar tissue. A sham operated group (n=5) was submitted to the same surgical procedure except permanent ligation of left coronary artery. Cardiac performance was assessed by electro and echocardiogram. Echo was performed prior to injections (baseline-BL) and six weeks after injections (follow-up - FU), and values after treatment were normalized by values obtained before treatment. Hemodynamic measurements were performed 6 weeks after injections. All data are expressed as mean ± SEM. Student's paired or unpaired T test was used to compare the same group in two different times or two distinct groups, while two way ANOVA was used to compare more than two groups along different times and p was set at <0.05. All infarcted animals exhibited cardiac function impairment. Ejection fraction (EF), shortening fractional area (SFA) and left ventricular akynesia (LVA) were similar between infarcted groups before treatment. Six weeks after therapy, ASC group showed significant improvement in all three Echo indexes in comparison to vehicle group. In non-anesthetized animals dp/dt+ was also significantly higher in ASC when compared to vehicle. In agreement with functional improvement scar area was diminished in the ASC group. We conclude that ASC stabilize cardiac function in infarcted rats when administered directly to the myocardium.
Abstract P043: The COP9 Signalosome Regulates Autophagy
Authors: Su Huabo , Li Faqian , Ranek Mark J , Wei Ning , Wang Xuejun (XJ)
Published: July 2021

By indiscriminately degrading portions of cytoplasm for self-supply of nutrients, non-selective autophagy helps the cell to survive starvation. Selective autophagy, however, removes defective/surplus organelles and protein aggregates, thereby playing an important role in quality control in the cell.
A
utophagy is involved in the pathophysiology of a variety of disease, including common forms of heart disease. Mechanisms regulating autophagy, especially selective autophagy, remain poorly understood. The COP9 signalosome (CSN) is an evolutionarily conserved protein complex consisting of 8 subunits (CSN1 through CSN8). CSN was purported to regulate ubiquitin-proteasome system (UPS) mediated proteolysis. We recently reported UPS malfunction and the accumulation of ubiquitin positive aggregates in the cardiomyocytes of mice with perinatal cardiomyocyte-restricted Csn8 knockout (CR-Csn8KO), which displayed massive cardiomyocyte necrosis, congestive heart failure, and premature death. Here we report that Csn8/CSN is required for the removal of autophagosomes in cardiomyocytes, an exciting discovery that has not been reported in any types of cells. CR-Csn8KO mouse hearts show marked increases in LC3-II, indicative of increased autophagosomes. The increase in LC-II is accompanied by a significant increase in p62 protein levels, which is evident as early as 1 week of age, long before the accumulation of a surrogate UPS substrate becomes discernible. The increase in autophagosomes is confirmed by probing with a transgenic GFP-LC3 and by electron microscopy. Autophagic flux assessments reveal that the removal of autophagosomes in cardiomyocytes is impaired by Csn8 deficiency and the defective fusion between autophagosomes and lysosomes may be responsible. Rab7 transcript and protein levels in the heart are significantly decreased by Csn8 deficiency. Confocal microscopy reveals a striking reciprocal relationship between increases in GFP-LC3 puncta and the decreased Rab7 expression. Rab7 knockdown impairs the removal of autophagosomes in cultured cardiomyocytes. Hence, Csn8/CSN is a central regulator in not only the UPS but also autophagy. Csn8/CSN supports autophagosome-lysosome fusion likely by stimulating Rab7 expression.
Abstract P059: Cardiomyocyte Unfolded Protein Response Stimulates Autophagy
Authors: Wang Zhao V , Gillette Thomas G , Rothermel Beverly A , Hill Joseph A
Published: July 2021

Background:
Autophagy is an evolutionarily conserved process of protein and organelle recycling. Under basal conditions, autophagy is critical for protein and organelle quality control. This cannibalization mechanism, however, can be detrimental under certain conditions, and dysregulation of autophagy has been implicated in numerous diseases. Recently, activation of autophagic flux has been reported in cardiac hypertrophy, heart failure, myocardial infarction, and ischemia/reperfusion injury. The unfolded protein response (UPR) is a cellular mechanism triggered by folding stress in the ER. When protein folding capacity, governed by ER resident chaperones, is overwhelmed by misfolded proteins, ER stress ensues, stimulating chaperone protein expression, ER associated degradation, and ultimately cell death if the stress is not remediated. Recent studies in yeast suggest the UPR can directly activate autophagy by phosphorylating ATG1, a critical upstream kinase required during autophagy initiation. However, whether and how ER stress, which is active in cardiac disease, regulates autophagy in heart is unknown.

Methods and Results:
Using neonatal rat ventricular cardiomyocytes in culture, we found the classical ER stress inducer, tunicamycin, triggers profound UPR signaling and autophagy up-regulation. The processing of LC3-II, an indication of autophagy activity, is dramatically increased. As multiple pathways are involved in ER stress, we focused on the IRE1/XBP1 branch. With cardiomyocyte-specific over-expression by lentivirus in vitro, we observed robust activation of autophagy. Further, we found that in vivo over-expression of XBP1s in cardiomyocytes triggered autophagy, as evidenced by real-time PCR and immunoblotting assays. As autophagy markers can accumulate due to blockage of lysosomal degradation, we quantified the lysosomal proteins, cathepsin D and LAMP1, finding each to be increased, suggesting that autophagic activity and flux per se are enhanced.

Conclusions:
Taken together, our data suggest that the XBP1s arm of the UPR pathway activates autophagic gene expression, autophagosome assembly, and autophagic flux.
Abstract P063: BMP-2 Upregulates PTEN Expression and Induces Apoptosis of Pulmonary Artery Smooth Muscle Cells Under Hypoxia
Authors: Pi Weifeng , Su LiPing , Zhu Jian , Xu Weiguo
Published: July 2021

Background:
Primary pulmonary hypertension is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs).

Aim:
To investigate the role of BMP-2 in regulation of PTEN and apoptosis of PASMCs under hypoxia.

Methods:
Normal human PASMCs were cultured in basal medium (BM) or growth medium (GM) and treated with BMP-2 from 5–80 ng/ml under hypoxia (5% CO
2
+ 94% N
2
+1% O
2
) for 72 hours. Gene expression of PTEN, caspase−3, −8, and −9, AKT-1 and -2 were determined by quantitative RT-PCR (QRT-PCR). Protein expression levels of PTEN, AKT and phosph-AKT (pAKT) were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4 was used to determine whether Smad signaling pathway was involved in the regulation of PTEN expression by BMP-2. bpV(HOpic) (PTEN inhibitor) and GW9662 (PPAR-r antagonist) were also used.

Results:
Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferative rate was achieved at 40–60ng/ml under hypoxia (BM=77.2±4%, GM=80±2.8%). Increased gene expression levels of PTEN, caspases−3, −8 and −9 were found, while AKT-1, and -2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. Though AKT was unchanged in all treated samples, reduced pAKT was found in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2 in both mediums. PTEN expression was unchanged when Smad-4 expression was inhibited. However pre-treat PASMCs with bpV(HOpic) and GW9662 (PPAR-r inhibitor) inhibited PTEN protein expression and recovered PASMCs proliferation rate.

Conclusion:
BMP-2 can increase PTEN expression under hypoxia in a dose dependent pattern. BMP-2 can increase apoptosis of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPAR-r signalling pathway, instead of BMP/Smad signalling pathway.
Abstract P087: Hydralazine and Nitroglycerin Combination Improves Excitation-Contraction Coupling in Failing Cardiomyocytes
Authors: Dulce Raul A , Gonzalez Daniel R , Hare Joshua M
Published: July 2021

Although hydralazine and organic nitrates have proven clinical benefits for heart failure, the underlying mechanism of action of this combination remains obscure. Spontaneously Hypertensive Heart Failure (SHHF) rats are a representative heart failure model which shares common phenotypic features with human failing hearts. We have recently shown that SHHF cardiomyocytes exhibit depressed contractility and calcium transient amplitude in response to increasing rate and increased sarcoplasmic reticulum (SR) calcium leak, typical characteristics observed in heart failure. The purpose of this study was to test the hypothesis that hydralazine, alone or in combination with nitroglycerin, restores calcium cycling and contractile performance in cardiomyocytes from SHHF rats. We measured sarcomere length (SL) shortening and calcium transient amplitude (Δ[Ca
2+
]
i
) in isolated cardiac myocytes from male SHHF rats (22–24 months old, n=4) and their normotensive controls Wistar-Kyoto rats (18–22 months old, n=4) during pacing (0.5–4 Hz). Both SL shortening and Δ[Ca
2+
]
i
responses to increasing pacing in SHHF cardiomyocytes were augmented by 10 μM hydralazine. The same concentration of nitroglycerin alone did not significantly affect these parameters. In response to the combination of these two drugs, there was a strong trend toward improved contractility and normal Δ[Ca
2+
]
i
. We also assessed SR Ca
2+
leak by a brief incubation with tetracaine following by a caffeine challenge, a regimen designed to test SR Ca
2+
load. At matched SR Ca
2+
content, Ca
2+
leak was not significantly changed by nitroglycerin or hydralazine alone. However, in combination they restored the leak in SHHF myocytes to the normal levels seen in WKY myocytes. Hydralazine improves cardiac excitation-contraction coupling that is impaired in this model, whereas nitroglycerin does not exert a significant effect on this response. However, when administered together, nitroglycerin appears to modulate the enhancing effect induced by hydralazine and helps to restore normal SR calcium leakage. These data offer new insights into the mechanisms underlying the actions of hydralazine and organic nitrates on heart failure.
Abstract P090: The Contractile Effect of Angiotensin II Type 1 Receptor Autoantibodies on Human Placental Vessels
Authors: Song Li , Zheng Ronghua , Zhang Suli , Bai Kehua , Yang Lihong , Liu Huirong
Published: July 2021

Background:
Decreased placental perfusion induced by abnormal placental vascular contraction is one of the pathological basis of preeclampsia. It has been reported that the sera titers of the autoantibody against the second extracellular loop of angiotensin II type 1 receptor (AT1-AA) were negatively correlated with placental blood flow in preeclampsia. Our previous study has found that AT1-AA could induce contraction of rat thoracic aorta and coronary rings by activing angiotensin II type 1 receptor (AT1R). However, there is no direct evidence for explaining whether AT1-AA might cause vasoconstriction on human placental blood vessels.

Methods:
The SD rats were immunized with the synthetic peptide corresponding to the sequence of the second extracellular loop of the human AT1 receptor (AT1R-EC
II
), and anti-AT1R antibody (AT1R-Ab) was extracted. The expression of AT1R on human placental vessels was determined by immunohistochemistry. The effects of AT1R-Ab on placental vessels were measured with isolated vascular ring technique.

Results:
(1) AT1R was highly expressed in the human placental artery, vein and vascular endothelial cells. (2) AT1R-Ab (10 mmol/L) respectively enhanced the contraction of the placental arteries and veins (29.21% ± 3.7%
vs
. 21.35% ± 2.8%,
P
>0.05), which could be completely reversed when AT1R was blocked by AT1R inhibitor. (3) AT1R-Ab (0.1, 1 and 10 mmol/L) induced placental vasoconstriction of normal human. The percentage of maximal contraction was 2.73% ± 1.11%,

4.00% ± 3.2% and 33.30% ± 5.6%, respectively. There was significantly difference between the three groups for contraction amplitude induced by different concentrations of AT1R-Ab (
P
<0.01). (4) AT1R-Ab (0.1, 1 and 10 mmol/L) induced placental vasoconstriction of preeclampsia. The percentage of maximal contraction was 1.74% ± 0.3%, 5.58% ± 1.41%

and 3.73% ± 2.5%, respectively. There was significantly difference between the three groups for contraction amplitude induced by different concentrations of AT1R-Ab (
P
<0.01).

Conclusion:
AT1R-Ab could induce human placental vasoconstriction in a concentration-dependent manner, which suggested that AT1-AA might be involved in the pathogenesis of preeclampsia by directly contracted placental blood vessles.
Abstract P112: Estradiol Induces Physiological Hypertrophic Growth in the Healthy Mouse Heart
Authors: Kararigas Georgios , Nguyen Ba Tiep , Jarry Hubertus , Regitz-Zagrosek Vera
Published: July 2021

Estradiol-17beta (E2) has been shown to exert anti-hypertrophic actions by either attenuating or blunting the development of left ventricular hypertrophy. However, the vast majority of these studies have been performed in stressed or diseased hearts. Consequently, very little is known about the actions of E2 in the stress- and disease-free heart. The aim of our study was to identify and characterize structurally and molecularly the role of E2 in the healthy heart. Female C57Bl/6J mice were ovariectomized at the age of two months. Mice were randomly assigned into groups feeding on either an E2-containing (n = 19) or soy-free (Ctrl; n = 19) diet for three months. Following this, all mice were sacrificed and hearts were collected for weight measurement. Left ventricles were analyzed structurally by immunohistochemistry and molecularly by genome-wide expression profiling. E2 led to an increase in the heart weight (11%; P < 0.001) and the heart-to-body weight ratio (32%; P < 0.001) compared to Ctrl mice. Cardiomyocyte cross-sectional area revealed cardiomyocyte hypertrophy in E2 (n = 6) compared to Ctrl (n = 5) mice (32%; P = 0.004). Analysis of the left ventricular transcriptome identified 1059 probe sets (adjusted P ≤ 0.05) differentially expressed between E2 (n = 5) and Ctrl (n = 5). Hypergeometric testing for Gene Ontology showed most genes to be associated with cell cycle, regulation of growth, cell and tissue development. Pathway analysis revealed 140 pathways (adjusted P = 0.05) modulated between the two groups, such as the DNA replication and Wnt signaling pathways. Next, we tested the hypothesis that this hypertrophic effect of E2 is of the physiological type. To this extent, we identified that angiogenesis was increased with cardiac growth as determined by the microarray analysis and VEGF-A protein levels assessed by Western blotting. Furthermore, the embryonic gene program was not activated and no fibrosis was observed in the E2-treated group. In conclusion, our study is the first to demonstrate pro-hypertrophic actions of E2 in the healthy heart through the modulation of growth-related genes and pathways. Due to that we have characterized the hypertrophic effect of E2 as physiological, we expect this effect to be beneficial for the heart.
Abstract P114: Evidence of Transplacental Antibody Transfer in Proposed Animal Model for Hypoplastic Left Heart Syndrome
Authors: Cole Charles R , Basu Mitali , Baker R Scott , Lam Chris , Blanco Adita , Cunningham Madeleine , Eghtesady Pirooz
Published: July 2021

Background:

The pathogenesis of Hypoplastic Left Heart Syndrome (HLHS), a congenital heart disease with significant morbidity and mortality, remains unknown. We previously proposed a hypothesis wherein HLHS represents a type of rheumatic heart disease in the fetus; trans-placental passage of maternal anti-strep or anti-cardiac myosin (CM) antibodies are postulated to play a key role in the pathogenesis of disease. This is a first report of an animal model that we have developed to assess our hypothesis.

Methods:

Female Lewis rats (∼ 8 weeks old) were immunized with either streptococcal antigen M type 5 S. pyogenes (PepM5; n=6), rat CM (n=8) or saline (controls; n=5) with three booster injections administered at 2-week intervals. Serum titers of acquired PepM5 or CM antibodies were determined by ELISA assays every 7–14 days. No boosters were administered during gestation. Trans-uterine echocardiography was performed near term (E19-21) to determine fetal number and viability then cesarean section was performed under anesthesia to deliver the progeny. Maternal and fetal serum and hearts were harvested for analysis.

Results:

All rats immunized with PepM5 had elevated serum anti-PepM5 antibody titers (>1:12800) and two of these animals also had elevated anti-CM titers (1:800). The offspring of these PepM5 immunized animals had elevated anti-PepM5 antibody titers (≥1:6400), but no CM elevation. Rats immunized with CM had a variable response ranging from anti-CM titers of 1:1600 to >1:12800; there were two non-responders. Their fetuses had anti-CM titers that ranged from 1:100 to 1:800. None of the controls had detectable serum titers. Fetal CM titers of ≥1:200 correlate with maternal peak CM titers of ≥1:6400 and/or maternal harvest titers of ≥1:800. Thus far, 6 of the CM treated fetuses have evidence of left-sided morphologic abnormalities along a variable spectrum; all of these fetuses had CM titers of ≥1:200.

Conclusion:

We have documented maternal antibody response and trans-placental antibody transfer from maternal rats immunized with CM or PepM5 prior to pregnancy. Anti-CM antibody does not cross the placental as readily as PepM5. Preliminary histologic findings demonstrate probable HLHS phenotype, which appears to correlate with fetal antibody titer.
Abstract P119: S100A6 Regulates Endothelial Cell Physiology
Authors: Ren Shumei , Peppel Karsten , Most Patrick
Published: July 2021

Endothelial cell dysfunction is central to the development of cardiovascular pathologies. While Calcium cycling is of recognized importance to myocyte physiology and cardiac perfomance, the role of Ca2+ signaling in endothelial cell (EC) physiology remains relatively unexplored. In this study we investigated the role of S100A6 (calcyclin), an EF-hand type Ca2+ -binding protein that modulates target protein function dependent upon intracellular Ca2+ signaling. S100A6 displays cytoplasmic distribution in quiescent, confluent HUVEC, but translocates to the nucleus upon serum stimulation. Oxidative stress, induced by incubation of HUVEC with H¬2O2 (150 uM) leads to redistribution of nuclear S100A6 to the cytosol and cell cycle arrest. Knockdown of S100A6 by siRNA transfection reduced S100A6 levels by over 70% and diminished DNA synthesis by more than 75% (±12%, n=9, p<0.01), as well as expression of the proliferation markers PCNA and KI-67 (by more than 70% (±15%, n=9, p<0.01). Reduced S100A6 levels in HUVEC lead to an increase in cellular senescence, as measured by the expression of senescence associated β-galactosidase expression (by more than 6-fold, n=4, p<0.01) and a reduction of EC tube formation on matrigel matrix (by more than 90%, n=3, p<0.01). Reduction of S100A6 increased the expression of the cell cycle control and DNA repair-associated gene BRCA2, but did not change in the expression of BRCA1, cyclins or p53 (by RT-PCR array). We conclude that Ca2+ regulation by S100A6 is essential for multiple aspects of EC physiology.
Abstract P120: Increased [ca
2+
]
e
Enhances Adipocyte Development In Bone Marrow Stroma
Authors: Hashimoto Ryota , Katoh Youichi , Itoh Seigo , Iesaki Takafumi , Daida Hiroyuki , Nakazato Yuji , Okada Takao
Published: July 2021

Background:
Bone marrow stroma contains adipocytes, osteoblasts, and lymphohematopoietic donor cells. With age, fatty marrow gradually predominates in bone marrow stroma and is a factor underlying age-related fracture and anemia. Thus, it is important to understand the mechanism of adipocyte development in bone marrow stroma. Bone marrow Ca
2+
levels can reach high concentrations of 8 to 40 mM, while circulating plasma Ca
2+
levels normally range from 2.3 to 2.6 mM. However, the effects of a high extracellular calcium concentration ([Ca
2+
]
e
) on adipocyte development in bone marrow stroma remain largely unknown.

Methods and Results:
We studied the effects of high [Ca
2+
]
e
on adipocyte development in bone marrow stroma. First, we used the fura-2 method to examine whether a change in [Ca
2+
]
e
alters [Ca
2+
]
i
levels in bone marrow stromal cells. Changes of [Ca
2+
]
e
from 1.8 mM to 5.4 mM and 10.8 mM significantly increased [Ca
2+
]
i
by 1.1 and 1.3 times, respectively. Next, bone marrow stromal cells were cultured for 14 days in high [Ca
2+
]
e
(5.4 mM and 10.8 mM) and normal [Ca
2+
]
e
(1.8 mM) conditions. Adipocyte development was monitored by Oil Red O staining of cytoplasmic lipids and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). In 5.4 mM and 10.8 mM [Ca
2+
]
e
, Oil Red O-stained cells increased significantly by 1.4 and 2.3 times, respectively, and GPDH activity increased significantly by 1.7 and 2.3 times, respectively, compared with the respective values in 1.8 mM [Ca
2+
]
e
.

Conclusions:
These results indicate that high [Ca
2+
]
e
induces an increase of [Ca
2+
]
i
, which enhances adipocyte development in bone marrow stroma. Further studies are required to determine the influx pathway of Ca
2+
, since prevention of Ca
2+
influx into bone marrow stromal cells might suppress development of fatty marrow and reduce age-related fracture and anemia.
Abstract P122: Characterization of Ventricular Assist Device Mediated Sensitization in the Bridge to Heart Transplant Patient
Authors: Kwon Murray H , Zhang Jennifer Q , Abrahamyan Ani , Zhang Zilu K , Gjertson David W , Ardehali Abbas , Kobashigawa Jon , Shemin Richard J , Reed Elaine F
Published: July 2021

Purpose:
To clarify patterns of anti-HLA antibody expression (sensitization) occurring in patients bridged to transplantation (BTT) with ventricular assist devices (VADs).

Methods:
The study is a retrospective review of 68 patients undergoing BTT with either Heartmate II (HMII) axial flow LVAD or paracorporal BIVAD from January 2007 to July 2010 at UCLA Medical Center.

Results:
Five of 15 (33.3%) HMII pts became sensitized during treatment compared to 29 of 53 (54.7%) BIVAD patients, p=0.24.
Table 1
shows common etiologies for patient sensitization of which only PRBC transfusion was statistically significant. [
table 1
] Multiple variable analysis comparing BIVAD vs. HMII while controlling for previous cardiac surgery, pregnancy, and PRBC transfusion demonstrated an Odds Ratio of 5.20, p=0.029 (robust variance estimator). Of sensitized patients, all 5 (100%) of the HMII patients had pre-existing antibodies prior to VAD placement compared to 11 of 29 (62.1%) BIVAD patients, p=0.016. Maximum cumulative MFIs for BIVAD were 46,259 ± 66,349 vs. 42540 ± 12840 for HMII, p=0.90. Time to maximum antibody expression was shorter for the HMII group (34 ± 28 days vs. 5.8 ± 9 days, p=0.04).

Table 1.

Sensitization Risk Factors

Antibodies +
Antibodies −
p-value

Previous Cardiac Surgery
23.5%
8.8%
0.19

Pregnancy
20.6%
2.9%
0.05

PRBC
52.1±34.7
35.9±22.3
0.04

FFP
29.4±14.4
23.6±15.2
0.15

Platelets
9.8±9.7
6.8±6.7
0.20

Cryoprecipitate
3.2±2.6
3.1±2.6
0.74

Blood products expressed as mean units ± std dev

Conclusion:
BIVADs were associated with a five fold increased risk for sensitization when accounting for other risk factors. HMII patients required pre-sensitization to express antibodies during their treatment interval whereas BIVAD patients developed
de novo
antibodies. Although the peak cumulative MFIs were similar for both VAD types, the days to reach this peak were significantly less in the HMII group. These data suggest that sensitization in HMII patients may not be due to antigenic stimulation from the device itself.
Abstract P123: β- and γ-Catenin Cooperate to Maintain Mechanoelectrical Coupling in the Heart
Authors: Swope David , Cheng Lan , Li Jifen , Radice Glenn
Published: July 2021

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart-muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome including plakoglobin (also known as γ-catenin). Plakoglobin (PG) is a unique cell adhesion molecule as it is located in both desmosomes and adherens junctions located at the intercalated disc (ID) where it functions to link cadherins to the cytoskeleton. Redistribution of PG away from ID is found in virtually all cases of ARVC, regardless of the specific mutation, suggesting that PG plays a fundamental role in the pathogenesis of ARVC. To investigate the role of PG in ARVC, we generated an inducible cardiac-restricted knockout (CKO) of the
PG
gene in mice. Despite gap junction remodelling, PG CKO mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin, showed increased association with the gap junction protein, connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin protects PG mutant animals from sudden arrhythmic death, we generated mice lacking both PG and β-catenin specifically in the heart (i.e. double knockout, DKO). The PG/β-catenin DKO mice exhibited acute cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD 3 – 5 months (124 day median survival) after deleting the genes. The PG/β-catenin DKO hearts were susceptible to induced arrhythmias (8/11 DKO versus 0/9 WT,
p
< 0.01) consistent with the SCD phenotype. Furthermore, spontaneous lethal arrhythmias were captured in PG/β-catenin DKO mice with telemetric monitoring. In contrast to the PG and β-catenin single mutants, N-cadherin was significantly reduced at the ID in the PG/β-catenin DKO mice. Consistent with decreased desmoglein-2 and plakophilin-2 at the ID, quantitative analysis of TEM images demonstrated a significant reduction in the number of desmosomes (7.36 ± 3.68 DKO versus 36.37 ± 4.44 WT per 100-µm ID; n = 10 fields per heart;
p
< 0.001). In conclusion, these studies suggest that increasing β-catenin interaction with Cx43 may enhance gap junction function in ARVC patients, thus improving electrical conduction in the heart.
Abstract P155: HSF-1 Deletion Induces MDR1 Gene in the Heart and Protects from Doxorubicin-Induced Cardiotoxicity
Authors: Krishnamurthy Karthikeyan , Druhan Lawrence , Ilangovan Govindasamy
Published: July 2021

Heat shock factor 1 (HSF-1) has been found to be a frontline responder of different stresses in eukaryotic cells. Deletion of HSF-1 has been reported to develop defects in mice. In the present work we report that deletion of HSF-1 induced multidrug resistance 1 (MDR1) gene and P-glycoprotein (P-gp) expression. Both RT-PCR and western blotting, confirmed the higher level of MDR1 gene expression and P-gp protein in HSF-1 knock out mouse hearts. P-gp is well known ATP binding cassette, which extrudes intracellular drugs upon binding ATP. Hence the phenotype of the P-gp pump in HSF-1
−/−
mice was studied in the form of Doxorubicin (Dox) extrusion in the heart. Cardiomyocytes isolated from HSF-1
−/−
and HSF-1
+/−
mice retained very less intracellular Dox, compared to wild type counterparts. Similarly, both HSF-1
+/−
and HSF-1
−/−
mice were less susceptible for Dox-induced cardiotoxicity, as seen from reduced cardiac dysfunction in these group against Dox (as confirmed by cardiac MRI and echocardiography). Further confirmatory studies revealed that deletion of HSF-1 enhances NF-B, which subsequently induces MDR1 gene. These results reveal that inactivation of HSF-1 in the heart will be a potential approach to prevent Dox-induced cardiotoxicity.
Abstract P204: Sex Differences and the Role of Estrogen Receptor β in Exercise-Induced Myocardial Hypertrophy
Authors: Dworatzek Elke , Mahmoodzadeh Shokoufeh , Brozová Eva , Nawrath Karina , Schubert Carola , Gustafsson Jan-Ake , Regitz-Zagrosek Vera
Published: July 2021

We and others found sex differences in physiological myocardial hypertrophy (MH) in mice subjected to voluntary cage-wheel running (VCR) and forced exercise training. Female mice showed significantly more MH, suggesting the involvement of estrogen (E2) and estrogen receptors (ER). We therefore investigated the underlying mechanisms leading to sex differences in physiological MH and the role of E2 and ER beta (ERß). Male and female C57/Bl6 wild-type (WT) and ERß-deficient mice (ERß-/-) at the age of 12 weeks performed 8 weeks of VCR or were kept sedentary (sed). Exercise performance was monitored daily and left ventricular mass (LVM) was examined by echocardiography. RNA and protein were analyzed by Real-Time PCR and western blot. Luciferase-reporter Assays were performed with PGC-1a-, MEF2A- and MEF2C-promoter deletion constructs in a human cardiac myocyte cell line (AC16 cells) with/without E2 treatment. Female WT-mice run more than their male counterparts (6.7km/day vs. 4.2km/day; p<0.001). Females showed significant greater increase in LVM and cardiomyocyte diameter in response to exercise compared to males. VCR led to a significant activation of AKT and p38-MAPK signalling in female running-mice, but not in males. Mitochondrial biogenesis associated genes MEF2A and ATP5K mRNA expression were significantly higher in female VCR mice. Female and male ERß-/- mice showed similar running performance compared with WT-mice (6.3km/day vs. 2.7km/day; respectively; p<0.001); however they showed no changes in LVM compared to sed-controls. In contrast to WT animals, female ERß-/- mice showed no increase in AKT and p38-MAPK phosphorylation or up-regulation of mitochondrial key enzymes. E2-treatment of AC16 cells up-regulated mitochondrial target genes (PGC-1a, MEF2A NRF1/2 and TFAM) and led to nuclear translocation of PGC-1a. E2 increased transcriptional activity of PGC-1a and MEF2A/C in an ER dependent manner in cardiomyocytes. Female hearts develop more physiological MH due to exercise, characterized by a stronger activation of pathways and genes involved in the regulation of mitochondrial function. ERß is necessary for the development of physiological hypertrophy and for the activation of p38- MAPK and AKT pathways in female mice.
Abstract P221: Retinoblastoma Protein--Associated Proteins 48 and 46 Are Novel p300/GATA4-Binding Partners Involved in Hypertrophic Responses in Cardiomyocytes
Authors: Sunagawa Yoichi , Katanasaka Yasufumi , Terada Taishi , Watanabe Yuichi , Wada Hiromichi , Shimatsu Akira , Fujita Masatoshi , Hasegawa Koji , Morimoto Tatsuya
Published: July 2021

Background:
A zinc finger protein GATA4 is one of hypertrophy-responsive transcription factors, and increases its DNA-binding and transcriptional activities in response to hypertrophic stimuli in cardiomyocytes. Activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. Here, we show that retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48, RbAp46), components of NuRD (nucleosome remodeling and deacetylase) complex that has been implicated in chromatin remodeling and transcriptional repression associated with histone deacetylation, are novel components of p300/GATA4 complex. However, the precise functional relationships among p300, GATA4, RbAp48, and RbAp46 remain unknown.

Methods and Results:
A series of GST pull-down assays revealed that the C-terminal domain of RbAp48/46 bound to the N-terminal transcriptional activation domain of GATA4 and C/H-3 domain of p300, respectively. Immunoprecipitation followed by western blotting demonstrated that RbAp48/46 repressed p300-induced acetylation of GATA4 and histones. While overexpressions of RbAp48/46 inhibited p300/GATA4-induced atrial natriuretic factors (ANF) and endotheline-1 (ET-1) promoter activities, knockdown of neither RbAp48 nor RbAp46 by RNAi enhanced these promoter activities in HEK293 cells. Stimulation of cardiomyocytes with phenylephrine (PE) decreased the binding of GATA4/p300 with RbAp48/46. RbAp48/46 repressed PE-induced hypertrophic responses such as myofibrillar organization, increase in cell size and promoter activation of the ANF and ET-1 in cardiomyocytes.

Conclusion:
These findings demonstrate that RbAp48 and RbAp46 form a functional protein complex with GATA4/p300 and regulated hypertrophic responses in cardiomyocytes.
Abstract P225: Possible Involvement of Homer-1b/c in Gq-Mediated Hypertrophy in Cardiomyocytes
Authors: Grubb David R , Luo Jieting , Iliades Peter , Woodcock Elizabeth A
Published: July 2021

Receptor activation of Gq causes hypertrophy in cardiomyocytes, via the activation of phospholipase Cβ 1b (PLCβ1b). PLCβ1b, localizes to the cardiac sarcolemma through an interaction with the multi-domain scaffolding molecule Shank-3 (SH3 and multiple ankyrin repeat domains protein 3; Grubb et al., 2011), which is required for PLC activation and for hypertrophic responses. In the CNS, Shank-3 forms higher order oligomeric complexes with three isoforms of Homer protein homolog 1 (Homer-1), Homer-1a, Homer-1b and Homer-1c. Homer-1b and Homer-1c link G-protein coupled receptors, ionotropic receptors, canonical transient receptor potential channel (TrpC) and intracellular calcium store regulators into a signaling complex. Homer-1a acts as a natural dominant negative, in dynamic competition with Homer-1b and Homer-1c. Neonatal rat ventricular myocytes (NRVM) infected with adenovirus expressing either Gαq(Q209L) (constitutively active Gαq), or its immediate down-stream effector, PLCβ1b, increased Homer-1b/c transcription. Incubation with phenylephrine/propranalol (α
1
-adrenergic agonist, PE/Pro) also increased Homer-1b/c, but not Homer-1a, mRNA. All treatments caused cardiomyocyte hypertrophy. There was no comparable increase in Homer-1b/c mRNA in NRVM expressing PLCβ1a (inactive splice variant) or incubated with fetal calf serum to induce hypertrophy by Gq-independent mechanisms. Homer-1b/c protein induced by PLCβ1b, Gαq or PE/Pro was primarily localized close to the sarcolemma along with Shank3, PLCβ1b and TrpC4. We conclude that Gαq/PLCβ1b-mediated signaling leads to the up-regulation of Homer-1b/c, that co-localizes with a signaling complex close to the sacrolemma. Induction of Homer-1b/c may be critical in facilitating localized Ca
2+
signaling and thereby promoting Gq dependent hypertrophy.