bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 454: Protein Phosphatase Pp2a Regulates Hypertrophic Response Through Hdac2 Dephosphorylation in the Heart
Authors: Min Hyun-Ki , Yoon Somy , Kwon Duk-Hwa , Kook Hyun , Eom Gwang Hyeon
Published: June 2021

Rationale:
Cardiac hypertrophy is an adaptive process to meet the hemodynamic demands from exogenous stresses, and histone deacetylase (HDAC) 2 plays central role in cardiac remodeling. Recently, we have suggested the importance of acetylation of HDAC2; however, specific phosphatase of HDAC2 remains unclear.

Objective:
We aimed to delineate the phosphatase of HDAC2 in the development of cardiac hypertrophy and to suggest therapeutic implications of those phosphatase in cardiac remodeling.

Methods and Results:
We performed complex-isolation assay in the heart and found that Hdac2 physically interacted with the Ppp2ca, and Hsp70. Ppp2ca kept Hdac2 unphosphorylated in the absence of hypertrophic stresses. Hypertrophic stresses-induced Hdac2 K75 acetylation, which then allowed Ppp2ca to dissociate from Hdac2, which led to phosphorylate Hdac2. The agonist-induced hypertrophy was significantly attenuated in transgenic mice heart expressing Ppp2ca. Forced expression of phosphorylation mimicking mutant of Hdac2, Hdac2 S394E, successfully overcame to antihypertrophic effects of Ppp2ca, whereas wild type of Hdac2 failed to do so. On the other hand, hypertrophic stresses induced Hsp70, one of the binding partners of Hdac2, which then preferentially bound to phosphorylated Hdac2 rather than to unphosphorylated one. The increase in expression of Hsp70 led to dissociate Ppp2ca from Hdac2. Hsp70 significantly increased phosphorylation of Hdac2 by protection from Ppp2ca. Cardiac hypertrophy was observed in the TgHsp70 mice and hyper-phosphorylation of Hdac2 was also detected. Double transgenic mice expressing both Ppp2ca and Hsp70 showed cardiac hypertrophy, which implicated that Hsp70 functioned as an endogenous regulator of Ppp2ca in the heart. TgHsp70-induced cardiac hypertrophy was significantly inhibited by adeno-Ppp2ca in a dose response fashion.

Conclusion:
Taken together, HDAC2 forms a complex with PP2A in the absence of hypertrophic stresses and remains inactivated. HDAC2 acetylation results in dissociation of PP2A and thereby phosphorylation, which is maintained by the association with HSP70 during development of cardiac hypertrophy. Hyun-Ki Min and Somy Yoon contributed equally to this work.
Abstract 465: Medical Device Materials and Blood Pressure Alterations in Mice
Authors: Jaimes Rafael , Sherman Meredith , Swiercz Adam , Muselimyan Narine , Marvar Paul , Posnack Nikki
Published: June 2021

Di-2-ethylhexyl phthalate (DEHP) is a plasticizer that is used to impart flexibility to polyvinyl chloride products. Patients have an increased exposure to phthalates through contact with DEHP-containing medical devices, including: storage bags containing blood, plasma, intravenous fluids, total parenteral nutrition, tubing associated with their administration, nasogastric tubes, enteral feeding tubes, catheters, extracorporeal membrane oxygenation (ECMO) circuits, hemodialysis tubing, respiratory masks and endotracheal tubes. Human health concerns pertaining to DEHP exposure are linked to its endocrine-disrupting properties. Accordingly, increased exposure has been associated with cancer, metabolic disturbances, reproductive and neurological disorders, and cardiovascular disease. As an example, epidemiological studies have shown a link between DEHP exposure and elevated systolic blood pressure in adolescents. Despite bans and restrictions on the use of DEHP-containing medical devices in other countries, there is currently no mandate from the Food & Drug Administration for the use of DEHP-free devices and storage containers. The objective of this study was to quantify the impact of in vivo DEHP exposure on cardiovascular function; thereby, providing additional information for regulatory decisions by the scientific, medical and regulatory community. Healthy C57BL/6 male mice were implanted with radiotelemetry transmitters; briefly, the transmitter catheter was placed in the carotid artery and biopotential leads were routed subcutaneously to collect electrocardiogram (ECG) signals. After surgical recovery, pre-exposure data was collected, and thereafter, animals were exposed to 0.2 mg/g DEHP or control diet. We observed a significant increase in systolic pressure in DEHP-treated (145
+
3 mmHg) vs control animals (136
+
1 mmHg). We also detected an increase in diastolic and mean arterial pressure in DEHP-treated (119
+
5 and 132
+
3 mmHg, respectively) vs control animals (107
+
2 and 121
+
2 mmHg). Our previous reports have shown that DEHP diminishes cardiac contractility, which suggests that these effects on blood pressure are likely attributed to alterations in sympathetic tone and/or an increase in vascular resistance.
Abstract 469: Gene Expression Profiling Reveals Genes and Pathways Involved in the Cardiac Protection of Valosin-containing Protein in the Heart
Authors: Stoll Shaunrick , Chen Xin , Wang Charles , Qiu Hongyu
Published: June 2021

Aims:
Valosin-containing protein (VCP) participates in signaling pathways essential for cell homeostasis in multiple tissues, yet its role in mammalian heart remains largely unknown. Our recent studies showed that overexpression of VCP protects cardiomyocytes from stress- induced cell death in both
in vitro
and
in vivo
, however, it is unknown which target genes and pathways are regulated by VCP through which the heart may be protected from stress-induced injury. Here we provided the first
in vivo
evidence of the VCP-regulated target genes by microarray gene expression profiling in a transgenic mouse model.

Methods and results:
We generated a transgenic (TG) mouse that over-expressed 3.5 fold VCP specifically in the heart. Total RNA extracted from the heart tissues from both adult VCP TG and their litter-matched wild type (WT) mice (N=4/group) was assayed by using Affymetrix Mouse Genome 430 2.0 Array, which interrogates over 39,000 transcripts. Differentially expressed genes (DEGs) were defined by p-value ≤ 0.05 and FC (fold change) ≥ 1.5, and totally 581 DEGs were identified between VCP TG and WT. Applying a more stringent criteria (FDR ≤ 0.05 and FC ≥ 1.5), we identified 21 upregulated and 28 downregulated genes in VCP TG mouse hearts compared to WT. Among the top DEGs, there is a significant up-regulation in the genes regulating the stress response and oxidation in VCP TG versus WT, such as a kinase interacting protein by 2.6 folds which is a key regulator of cardiac stress; glutathione peroxidase 1 by 2.2 folds which is an antioxidant enzyme and anti-apoptotic mediators: protein kinase C delta by 2.3 folds and B-cell lymphoma 2 by 2.7 folds. VCP TG mice also exhibits an upregulation in the genes involved in cellular structure and contractility, e.g., alpha 1 actin by 3.3 folds, myosin light chain 9 by 8.2 folds and Tropomyosin 2 by 4.5 folds which is a gene regulating the binding of myosin and actin. Other DEGs include the upregulated-genes regulating cellular amino acid metabolism such as pterin-4 alpha-carbinolamine dehydratase by 9.7folds, phosphatidic acid phosphatase 2c by 3.4 folds and sortilin 1 by 2.4 folds.

Conclusion:
Significant difference of gene regulation exists between VCP TG and WT in the heart, which may be the mechanism of VCP-mediated cardiac protection.
Abstract 470: Computational Identification of NKX2-5 Binding Sites and Downstream Gene Targets Using Transcription Factor Motif and Human Heart-specific Experimental Data
Authors: Tan Nicholas , Chung Mina K , Smith Jonathan D , Van Wagoner David R , Barnard John
Published: June 2021

Background:
While NKX2-5 plays pivotal roles in human cardiac development and disease, no ChIP-Seq studies of NKX2-5 in human cardiac tissues currently exist, resulting in an incomplete understanding of its direct gene targets. Modern computational methods which identify binding sites using transcription factor motif and tissue-specific experimental data can help to fill this knowledge gap.

Objective:
To use computational methods to identify likely NKX2-5 binding sites and downstream gene targets using human heart-specific experimental data.

Methods:
Human cardiomyocyte DNAse hypersensitivity data (2 replicates) were downloaded from the Encyclopedia of DNA Elements (ENCODE) database. The position weight matrix (PWM) representing the transcription factor motif of NKX2-5 was obtained from JASPAR. We applied the Protein Interaction Quantification (PIQ) algorithm to detect NKX2-5 binding sites using the PWM and DNAse hypersensitivity data as inputs. RNA-Seq data from 108 human heart-specific samples (atrial appendages and left ventricles) were downloaded from the Genotype-Tissue Expression (GTEx) database. Protein-coding genes significantly expressed in the heart (RPKM <= 1 based on the GTEx RNA-Seq data) that were within 100kb of the predicted binding sites were then identified. Pathway analysis of these genes was performed using Ingenuity Pathway Analysis (IPA).

Results:
1283 binding sites for NKX2-5 were discovered by PIQ. Of 12698 protein-coding, heart-expressed genes, 625 were within 100kb of these binding sites. The identified genes were highly enriched in physiologic categories like “Vasculogenesis” and “Development of Cardiovascular Tissue” (p = 2.36x10-9 and 2.31x10-8 respectively). Notable genes included: cardiac transcription factors (MEF2A, TBX20); growth factors (TGFB2, BMP2); muscle and ion channel function (ACTA2, BIN1), and; calcium signaling (CALM2, CAMK2D).

Conclusion:
By using computational analyses of transcription factor motif and human heart-specific experimental data, we have identified candidate downstream targets of NKX2-5. Future work will include validation studies in an external cohort and analysis of associations between these candidate genes and cardiac disease.
Abstract 61: Ablation of BK
Ca
Channels Results in Cardiac Hypertrophy
Authors: Singh Harpreet , Shah Kajol , Ponnalagu Devsena , Chandrasekhar Sanjay , Kohut Andrew R , Meredith Andrea L , Gururaja Rao Shubha
Published: June 2021

Expression and activation of the large conductance calcium and voltage-gated potassium (BK
Ca
) channels encoded by
Kcnma1
gene is shown to be vital in cardioprotection from ischemia-reperfusion injury. BK
Ca
channels present in SA node cells regulate the heart rate, and in blood vessels play an active role in vascular relaxation. However, the role of BK
Ca
in regulation of structure and function of the heart is not fully-established. Using
Kcnma1
-/-
mice, we have observed structural changes in cardiomyocytes and compromised cardiac function as compared to wild type mice. Absence of BK
Ca
resulted in significant increase in size of adult cardiomyocytes (from 7.95
+
0.1 um
2
to 9.68
+
0.1 um
2
, p
<
0.01, n=480 cells each) and also increased cardiac fibrosis. Further to determine underlying signaling mechanisms in cardiac hypertrophy, we performed microarray analysis of RNAs isolated from wild type and

Kcnma1
-/-

mice (n=3) hearts. We found up regulation of a class of cardiac hypertrophy markers (myosin variants) and changes in the expression of several mitochondrial genes (such as ND4) directly associated with heart diseases in

Kcnma1
-/-

mice. To evaluate the functional consequence of absence of BK
Ca
, we performed high-resolution echocardiography on wild type and

Kcnma1
-/-

mice. Under anesthesia (1.5% isoflurane), left ventricle of

Kcnma1
-/-

mice showed significant reduction (p
<
0.05) in ejection fraction (56
+
2 %, n=7) as compared to wild type (74
+
3 %, n=6) as well as fractional shortening (23
+
3 %, n=7, and 39
+
3 %, n=6, respectively). Similarly, right ventricle had a lower ejection fraction (35.7
+
4% vs 56.9
+
5 %, n
>
5) in

Kcnma1
-/-

as compared to wild type mice. In agreement with our histopathology and microarray data,

Kcnma1
-/-

mice showed increased posterior wall thickness (0.75
+
0.3 mm
vs
0.62
+
0.1 mm) and interventricular septum thickness (0.83
+
0.4 mm, n=7
vs
0.68
+
0.3 mm, n=6) . Together, these data imply that BK
Ca
plays a direct role in cardiac hypertrophy and cardiac function.
Abstract 74: Cardioprotective Effect of Growth Hormone Releasing Hormone in Mouse Model of Heart Failure with Preserved Ejection Fraction
Authors: Kanashiro-Takeuchi Rosemeire M , Takeuchi Lauro M , Dulce Raul A , Hardin Darrell , Zuttion Marilia , Balkan Wayne , Cai Ren Z , Schally Andrew V , Hare Joshua M
Published: June 2021

Heart failure with preserved ejection fraction (HFpEF) is characterized by impaired relaxation, ventricular stiffening and fibrosis. We previously showed that activation of GHRH receptor markedly reduces fibrosis in rat and swine models of ischemic myocardial injury. Therefore, we hypothesized that activation of GHRH receptor signaling can improve diastolic dysfunction in a mouse model of HFpEF.

C57BL6N mice (n=4-5) were implanted with a mini-osmotic pump to deliver angiotensin-II (Ang-II: 0.8 mg/kg/day) for 4 weeks and randomly assigned to receive daily injections of GHRH-Agonist (GHRH-A [MR-409]: 100 μg/kg) or vehicle (DMSO+propylene-glycol). Cardiac performance was assessed by serial echocardiography and hemodynamic analysis.

Chronic administration of Ang-II resulted in increased end-diastolic pressure (EDP, p=0.0186) with no changes in EF (p=ns) or end-systolic pressure (ESP, p=ns) in comparison to control mice. Isovolumetric relaxation time (IVRT, p<0.05) and end-diastolic pressure-volume relationship (EDPVR, p=0.0229) were markedly increased in the Ang-II group consistent with increased ventricular stiffness and poor myocardial relaxation. MR-409 treatment reset these parameters to normal levels (table 1).

Our findings demonstrate that chronic administration of Ang-II mediates structural and functional changes that mimic HFpEF. Importantly, MR-409 treatment reduces Ang-II-induced elevation of EDP, EDPVR and IVRT; thus preventing HFpEF–like effects, suggesting that activation of the GHRH receptor signaling pathways represents a potential new therapeutic approach for HFpEF.
Abstract 80: PI3Kα Regulates Biomechanical Stress-induced Cytoskeletal Remodeling: A Critical Role of Gelsolin
Authors: Patel Vaibhav B , Zhabyeyev Pavel , McLean Brent A , Fan Dong , Basu Ratnadeep , DesAulniers Jessica , Kassiri Zamaneh , Vanhaesebroeck Bart , McCulloch Christopher A , Oudit Gavin Y
Published: June 2021

Background:
Biomechanical stress and cytoskeletal remodeling are key determinants in pressure overload-induced heart failure. Class Ia phosphoinositide 3-kinases (PI3Ks) mediate a variety of cellular activities, in response to agonist binding to cell-surface receptors, by generating the phosphatidylinositol (3,4,5)-trisphosphate (PIP
3
) phosphoinositide lipid. Gelsolin is a Ca
2+
- and phosphoinositide-regulated actin filament severing and capping protein that is upregulated in failing human hearts and animal models of heart failure.

Hypothesis:
We hypothesize that PI3Kα regulates cytoskeletal remodeling through PIP
3
-mediated regulation of gelsolin. In addition, loss of gelsolin could attenuate the adverse cytoskeletal remodeling and result in increased resistance to the development of heart failure in response to pressure-overload.

Methods and Results:
Loss of p110α kinase activity, in two different transgenic models (PI3Kα dominant-negative (PI3KαDN) and cardiomyocyte-specific PI3Kα-null), resulted in dilated cardiomyopathy and markedly worsened cardiac dysfunction in response to transverse aortic constriction-induced pressure overload. Increased levels of mechanosensor proteins along with decreased F/G-actin ratio exhibited an uncoupling between cardiac mechanotransduction and cytoskeletal remodeling in p110α-null mice. Gelsolin activity was markedly increased in the p110α-null hearts in response to pressure-overload, whereas loss of gelsolin in PI3KαDN/gelsolin-null double mutant mice prevented the adverse cytoskeletal remodeling and preserved the cardiac function. In a murine model of chronic heart failure, loss of gelsolin prevented the pressure overload-induced cardiac dysfunction, fibrosis, and impaired cardiomyocyte contractility resulting in increased survival. Loss of gelsolin also mitigated the biomechanical stress-induced adverse cytoskeletal remodeling, via the attenuation of actin severing activity.

Conclusions:
We have identified a novel role of gelsolin as a mediator of adverse cytoskeletal remodeling leading to heart failure, where PI3Kα is a key regulator of gelsolin activity.
Abstract 82: Reduction of SHP2’s Phosphatase Activity Improves Cardiac Adaptation to Pressure and Volume Overload
Authors: Roy Rajika , Crombie Kathryn , Douglas Diana , Krenz Maike
Published: June 2021

Since the inability of the heart to adapt to pathological stress results in heart failure, supporting cardiac stress adaptation may improve clinical outcome. We hypothesized that the protein tyrosine phosphatase SHP2 controls adaptation by modulating several signaling pathways involved. To test whether SHP2 plays an active role in stress adaptation, we generated transgenic (TG) mice with cardiomyocyte-specific overexpression of Q510E-SHP2, which is a dominant-negative mutant (dnSHP2). Under unstressed conditions, TG mice do not exhibit a cardiac phenotype. TG and nonTG (NTG) mice were subjected to pressure overload by transverse aortic constriction (TAC) or to volume overload by inducing large myocardial infarction (MI) via coronary artery ligation. Expression of dnSHP2 significantly improved stress adaptation after TAC (n=17 TG, n=13 NTG). Twelve weeks after banding, TG mice exhibited well preserved contractile function with significantly higher fractional shortening (FS) [TG 33.2±1.7 vs. NTG 22.2±1.5 %, p<0.001] and ejection fraction (EF) [TG 67.7±2.7 vs. NTG 50.3±3.0 %, p<0.001]. dnSHP2 TGs also had smaller left ventricular (LV) chamber dimensions [LV inner diameter at end-diastole (LVIDd) TG 4.4±0.2 vs. NTG 5.1±0.2 mm, p=0.02]. There were no significant differences in ventricular wall thickness or heart weights, indicating that the degree of hypertrophy in response to TAC was not affected by dnSHP2 expression. In response to volume overload induced by large MI (n=22 TG, n=24 NTG), TG hearts displayed only a trend towards improved contractile function [FS TG 21.3±1.5 vs. NTG 18.9±1.1%, EF TG 48.4±2.7 vs. NTG 44.2±2.1%]. However, chamber dimensions were better preserved in TGs [LVIDd TG 5.1±0.1 vs. NTG 5.7±0.2 mm, p=0.03]. In summary, dnSHP2 expression ameliorated LV chamber dilation and increased contractile function under pressure overload. In TGs subjected to volume overload, reduced LV dimensions were observed but with only a trend towards improved contractile function. Taken together, it is evident that reducing SHP2’s phosphatase activity indeed improves cardiac stress adapation, making SHP2 a potential novel therapeutic target.
Abstract 83: Autophagy Protects the Heart Against Pressure Overload Induced Heart Failure
Authors: Shirakabe Akihiro , Ikeda Yoshiyuki , Saito Toshiro , Zhai Peiyong , Sadoshima Junichi
Published: June 2021

Autophagy is an important mechanism for the degradation of cytosolic proteins and organelles. We investigated how autophagy is regulated in the heart in response to pressure overload (PO). Mice were subjected to transverse aortic constriction (TAC) at multiple time points between 1 hours and 30 days. Left ventricular (LV) weight/tibial length (TL) was significantly elevated at Day 5 (6.21 ± 0.10 vs 4.59 ± 0.10, p<0.05) and thereafter. Ejection fraction (EF) was maintained at Day 7 (82.1±3.4 vs 78.4±3.2%), but gradually decreased thereafter (at Day 30; 51.0±4.5, p<0.05). The level of LC3II was rapidly increased and peaked at 3 hour (2.4 fold , p<0.05), returned to normal by 24 hours, and then significantly decreased at Day 5 (-40.0%, p<0.05) and thereafter. Autophagic flux was evaluated with tandem fluorescent LC3. At 6 hours, both GFP/RFP double positive (yellow) dots and RFP dots were significantly increased in the TAC group compared to the sham group with or without chloroquine (CQ) (yellow 12±2 vs 4±0 CQ(-), 23±3 vs 9±1 CQ(+); RFP 13±2 vs 5±1 CQ(-), 19±1 vs 13±1 CQ(+)). On the other hand, both yellow and RFP dots were significantly decreased at Day7 and thereafter in the TAC group compared to the sham group with or without CQ. These data suggest that autophagic flux is activated transiently after TAC, but is inactivated after Day 5. To examine the functional significance of autophagy during PO,
beclin1
heterozygous KO (
beclin1
-hetKO) mice,
atg7
cardiac specific KO (Atg7-CKO) mice, and cardiac-specific U6-shRNA
beclin1
(U6shRNA
beclin1
) mice were subjected to TAC. At Day 7 or 14, decreases in EF (60.7 ± 4.8%, 53.8 ± 2.1% and 46.7 ± 5.9%, p<0.05) and increases in lung weight/TL (8.43 ± 0.87, 11.04 ± 4.16 and 18.76 ± 3.77, p<0.05) were exacerbated in
beclin1
-hetKO,
atg7
-CKO and U6shRNA
beclin1
mice compared to in control mice. These results suggest that PO inhibits autophagy after Day5, which coincides with the development of cardiac dysfunction. Since heart failure is exacerbated by further suppression of autophagy, autophagy during PO protects the heart from cardiac dysfunction.
Abstract 88: Cardiac-specific Deletion of Protein Tyrosine Phosphatase 1B Exacerbates Pressure Overload-induced Hypertrophy and Heart Failure in Mice
Authors: Coulis Gerald , Bergeron Alexandre , Shi Yanfen , Labbe David , Tremblay Michel , Karsenty Gerard , Boivin Benoit
Published: June 2021

Cardiac hypertrophy involves the re-expression of a foetal gene program that occurs when cardiomyocytes are continuously exposed to stresses. The enlargement initially improves cardiac function, however, this compensatory hypertrophy predisposes individuals to arrhythmias, pathological hypertrophy and heart failure. Given the importance of reactive oxygen species (ROS) in the transition from cardiac hypertrophy to heart failure and the documented inhibition of PTPs by ROS, we hypothesized and explored whether specific PTPs could act as checkpoints in this process. We have identified PTP1B as a target of ROS in hearts undergoing hypertrophy. To better understand the role of PTP1B inhibition in cardiac hypertrophy, we generated cardiomyocyte-specific PTP1B knockout (PTP1B cKO) mice. Subjecting PTP1B cKO mice to pressure overload (PO) caused a dramatic left ventricular dilation and several distinctive features of heart failure when compared to control mice subjected to PO for the same period. Characterization of the mRNAs expressed in the hypertrophy-associated foetal gene program revealed that although PO led to increased mRNA levels of ANF and BNP, the increased expression of β-MHC observed in control mice subjected to PO was compromised in PTP1B cKO-PO mice. Since PTP1B inactivation can lead to the inactivation of AGO2 and compromise miRNA-mediated mRNAs repression, we investigated whether PTP1B regulated AGO2 phosphorylation and association with mRNAs in this context. We observed that AGO2 phosphotyrosine-393 levels were elevated and that AGO2 was a substrate of PTP1B in myocytes and in hearts undergoing hypertrophy. We also found changes in AGO2-mRNA associations between control- and PTP1B cKO-PO hearts and identified MED13 as a regulator of β-MHC expression that was differentially regulated by AGO2 in PTP1B cKO-PO hearts. Since increased expression of β-MHC contributes to the compensatory response that initially improves cardiac function, we will propose a model in which PTP1B inhibition regulates AGO2 activity and contributes to heart failure.
Abstract 92: Platelet Derived Growth Factor Receptor Alpha Signaling is Required for Cardiac Fibroblast Survival and Proliferation
Authors: Ivey Malina J , Tallquist Michelle D
Published: June 2021

Cardiac fibrosis contributes significantly to heart disease and is a hallmark of decreased cardiac function. Currently, there are no treatments that attenuate fibrosis, but identification of signaling pathways required for fibroblast function would provide some potential targets. PDGFRα is a receptor tyrosine kinase that is required for fibroblast formation in the developing heart, and preliminary data indicates that it is also required for maintenance of resident fibroblasts and expansion of activated fibroblasts after injury. Preliminary experiments demonstrate that loss of PDGFRα expression in adult cardiac fibroblasts results in 50% reduction in the number of the resident fibroblasts by 4 days after gene deletion. This was further validated using an independent fibroblast marker, collagen1a1GFP. Based on the low basal level of fibroblast proliferation, we hypothesize that PDGFRα signaling is essential for fibroblast survival and that fibroblasts undergo rapid turnover in the absence of PDGFRα signaling. Future studies will determine the exact mechanism of this loss. We have also begun to elucidate which PDGFRα downstream signals promote fibroblast maintenance. Using a PDGFRα-dependent-PI3K-deficient mouse model, preliminary data indicates that PDGFRα-dependent PI3K signaling is essential for cell survival. We are also investigating the role of PDGFRα signaling after myocardial infarction. Using recently described genetic tools to follow fibroblasts after injury, we have determined that fibroblasts reach their peak of proliferation within a week after permanent left anterior descending artery ligation. This injury-induced proliferation is reduced by 50% after deletion of PDGFRα. Therefore, we have demonstrated that PDGFRα has a role in fibroblast maintenance in the healthy heart, as well as a role in fibroblast proliferation after injury. Our studies will continue to illuminate additional roles for PDGFRα in the fibroblast, as well as the implications of fibroblast loss on other cell types and overall heart function.
Abstract 93: The Role of the Stretch-responsive Genes Mlf1 and Rnd1 for Cardiac Physiology and Pathophysiology
Authors: Kluge Annika , Pott Jost , Rangrez Ashraf Y , Bernt Alexander , Stiebeling Katharina , Frey Norbert , Frank Derk
Published: June 2021

Cardiac remodeling is a typical change in function and morphology of the heart in response to pathological biomechanical stress. Despite substantial research in this area several underlying mechanisms remain unclear. We thus devised a screening experiment of dynamic stretch of neonatal cardiomyocytes (NRVCM) which revealed several genes which have not yet been implicated in cardiac remodeling before. In further analyses we focused on
Mlf1
and
Rnd1
, both showing significant expression in heart as well as in skeletal and liver. Conformational qRT-PCR revealed upregulation of Rnd1 mRNA (5.94x) after 24 h of stretch. Mlf1 mRNA was significantly downregulated (qRT-PCR 0.34x). Stimulation with phenylephrine (PE) induced Rnd1 mRNA (3.98x), while Mlf1 mRNA levels remained unchanged. Furthermore, in line with the
in vitro
results we found upregulation of Rnd1 (2.1x, p<0.001) and downregulation of the cardiac Mlf1 expression (var. 1 0.45x, var. 2 0.36x, both p<0.001) in mice after transverse aortic constriction.

Adenoviral overexpression of Mlf1 in NRVCM induced the fetal gene program (e.g.,
nppa
,
nppb
). However, a significant decrease in cell size was found. We noticed the consistent findings after stretch or PE-stimulation. Rnd1 overexpression also led to induction of
nppa
,
nppb
and Rcan1.4, but caused an increase in cell size.

To analyze whether Mlf1 and Rnd1 also influenced cell proliferation, we analyzed Mlf1/Rnd1-infected cells for the expression of proliferation markers Ki67 and phosphohistone H3 (PHH3). Mlf1 overexpression led to an increase in Ki67+ cells of about 5.92 fold, and in PHH3+ cells of 2.48 fold (p<0.001), Rnd1 overexpression showed an increase in Ki67+ cells of about 6.9 fold and in PHH3+ cells of 5,25 fold (p<0.001).

Taken together, we could identify two stretch-sensitive genes which influence the process of cardiac hypertrophy and cell proliferation. Current work focuses on a knockdown of both genes in vivo and on the identification of potential protein-protein interactions to complete the characterization of Mlf1 and Rnd1.
Abstract 94: Menopausal Female Mice are Hypersensitive to Cardiovascular Disease
Authors: Danilo Christiane A , Constantopoulos Eleni , Sanchez Jessica N , Pollow Dennis P , Brooks Heddwen L , Lipovka Yulia , Konhilas John P
Published: June 2021

Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. The mechanisms responsible for loss of protection and rise in CVD onset are unknown. A novel model of menopause utilizing 4-vinylcyclohexene diepoxide (VCD) induces gradual ovarian failure, preserving the “perimenopause” transitional period and androgen secreting capacity of residual ovarian tissue. Our central hypothesis states that menopausal females are hypersensitive to CVD-induced pathological cardiac remodeling. To address this hypothesis, we instigated menopause in 2 month-old females by daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days); control mice received sesame oil as vehicle. Vaginal cytology was used to monitor estrous cycles and determine when cycling ceased. Mice were considered menopausal after 15 consecutive days in persistent diestrus, non-cycling. Female mice from cycling, perimenopausal and menopausal groups were subjected to 30 minutes of ischemia followed by 2 days of reperfusion (I/R). Following reperfusion, hearts were rapidly excised, sectioned and double-stained with Evans Blue and triphenyltetrazolium chloride to define the area at risk (AAR) and infarct size as area of necrosis (AON). On average, menopausal females displayed a significant (p<0.01) increase in AON relative to the AAR when compared to perimenopausal and cycling mice. Next, female mice (cycling, perimenopausal and menopausal) received the hypertensive agent angiotensin II (Ang II, 800 ng/kg/min via alzet s.q. mini-pump, 14 days). Ang II infusion induced a significant exacerbation of hypertension (measured via tail cuff) in menopausal females. In addition, menopausal females demonstrated worsened pathological cardiac remodeling measured by functional (echocardiography), cellular (myocardial fibrosis) and molecular (fetal gene program) assessments. Using a novel model of menopause (VCD) combined with CVD by I/R or infusion of the hypertensive agent Ang II, we demonstrated that menopausal mice are more susceptible, or hypersensitive to pathological cardiac remodeling compared to cycling and perimenopausal mice.
Abstract 95: The Role of TGFβ Signaling in a Fibrotic cMyBP-C HCM/HF Model
Authors: Meng Qinghang , Bhandary Bidur , Bhuiyan Md. Shenuarin , Osinska Hanna , Robbins Jeffrey
Published: June 2021

Purpose:
Hypertrophic cardiomyopathy (HCM) is considered one of the most common genetic heart disorders with a prevalence of about 1 in 500 people, with 35% of those affected being attributed to mutations within the gene encoding cardiac myosin-binding protein C (cMyBP-C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40kDa fragment in mouse cardiomyocytes (

Mybp3
40kDa

) leads to HCM, fibrosis and heart failure, mimicking human disease progression. The transforming growth factor-β (TGFβ) signaling pathway has been implicated in a variety of fibrotic processes. The goal of this study is to define the role of TGFβ signaling in distinct cell populations, the cardiomyocyte and fibroblast, in the cMyBP-C HCM/HF model.

Methods and results:
Masson’s Trichrome staining, PCR arrays, immunohistochemistry and western blots were performed to characterize the fibrotic progression in

Mybp3
40kDa

transgenic mice. Cardiac fibrosis was initially detected 4 weeks after transgene expression. Extensive interstitial fibrosis and severe atrial fibrosis were detected at 16 weeks. Both canonical and non-canonical TGFβ pathways were active during fibrotic progression. To specifically block TGFβ signaling in

Mybp3
40kDa

transgenic mice, compound mutant mice were generated, in which the
tgfbr1
or
tgfbr2
alleles were ablated, either in cardiomyocytes or in activated fibroblasts (myofibroblasts) by αMHC-Cre or Periostin-MerCreMer-Cre respectively. Blockage of TGFβ signaling in either cardiomyocytes or myofibroblasts alleviated cardiac fibrosis. Furthermore, treatment with the non-canonical TGFβ signaling inhibitor MMI-0100 also alleviated cardiac fibrosis and increased the life span of the

Mybp3
40kDa

transgenic mice.

Conclusions:
TGFβ signaling is activated in the

Mybp3
40kD

HCM/HF model. Genetic or pharmaceutical inhibition of TGFβ signaling inhibited fibrosis and increased the life span in this model.
Abstract 1: Dual Antiplatelet Therapy is More Effective for Multiple Acute Infarcts: Subgroup Analysis of Chance
Authors: Wang Yilong , Jing Jing , Wang Yongjun
Published: June 2021

Background and Purpose:
TIAregistry.org project showed that TIA and minor stroke patients with multiple acute infarctions (MAIs), which represent mechanisms of embolism, are at higher risk of recurrent stroke as compared to those with none or single acute infarction (SAI). We hypothesis that patient with MAIs may be more susceptible to dual antiplatelet therapy.

Methods:
We compared clopidogrel plus aspirin with aspirin alone with regard to their effectiveness and safety for prevention of stroke recurrence among non-cardiac TIA and minor ischemic stroke patients with different infarction patterns (no acute infarction, SAI, or MAIs) in the imaging subgroup from the Clopidogrel in High-Risk Patients with Acute Non-disabling Cerebrovascular Events (CHANCE) trial. The efficacy and safety outcome were stroke recurrence and moderate-to-severe bleeding event at 90 days respectively.

Results:
Overall, 1089 patients at 45 centers in CHANCE trial were included. The rate of recurrent stroke was 14.2%, 8.7%, and 2.0% in patients with MAIs, SAI and no infarction respectively at 90 days. Increased risk of stroke recurrence was observed in patients with MAIs (HR, 5.9; 95% CI, 2.3-15.2) and SAI (HR, 4.1; 95% CI, 1.6-10.7) (Figure A) as compared to those without infarction. The dual antiplatelet treatment was superior to aspirin alone for reducing the risk of recurrent stroke in patients with MAIs (HR 0.48; 95% CI, 0.25- 0.92;
P
= 0.03) but not in patients with SAI and without acute infarction with P=0.046 for interaction (Figure B-D). The effect of treatment assignment on bleeding did not vary among groups with different infarction patterns (
P
>0.05 for all).

Conclusions:
Patients with MAIs had the highest risk for stroke recurrence among patients with different infarction patterns. Patients with MAIs may benefit the most clinically from dual antiplatelet therapy and dual antiplatelet therapy does not increase the risk of hemorrhage among any groups.
Abstract 108: Additional Value of Intracranial Vessel Wall Imaging to Determine the Etiology of Ischemic Stroke
Authors: Schaafsma Joanna D , Mikulis David J , Coutinho Jonathan , Jaigobin Cheryl S , Mandell Daniel M
Published: June 2021

Objective:
High-resolution MR-imaging of the intracranial arterial wall is a promising technique for diagnosis of intracranial arteriopathies in patients with ischemic stroke. We aimed to evaluate the additional value of vessel wall imaging (VWI) to the standard work-up of ischemic stroke patients.

Methods:
We selected all patients with ischemic stroke who had intracranial VWI at our institute to evaluate possible intracranial arteriopathy, such as atherosclerosis, dissection, vasculitis, or reversible cerebral vasoconstriction syndrome. Two observers, who were blinded to the VWI, first determined the most likely stroke etiology based on the standard work-up (clinical history, brain parenchyma imaging, vessel lumen imaging, laboratory results, and cardiac work-up). Then VWI was reviewed to assess whether this would change the suspected stroke etiology or whether the differential diagnosis could be narrowed down.

Results:
Between 2006 and 2014, 199 patients with ischemic stroke, mean age 55 (IQ-range: 44-67) had VWI. VWI provided additional information to the standard stroke work-up in 128 patients (64%). In 38/199 patients (19%) the conclusion on stroke etiology was altered based on VWI and in 90/199 patients (45%) the differential diagnosis was further narrowed after VWI. VWI did not have additional value when the most likely stroke etiology based on the standard work-up remained the same (50/199 patients; 25%), when the differential diagnosis could not be narrowed down (16/199; 8%), or in case of poor image quality (5/199 patients; 3%). Patients under the age of 46 benefited more often from VWI than older adults (Odds Ratio 3.5; 95%CI: 1.7-7.6).

Conclusion:
VWI provided additional information to the conventional stroke work-up in almost two-thirds of patients suspected to have intracranial arteriopathy. Next step is to determine how frequently this additional information resulted in altered therapy.
Abstract 111: Plasma Levels of Oxidative Stress Marker ADMA (Asymmetric Dimethylarginine) is Reduced by Successful PFO Closure
Authors: Deng Wenjun , Wickham Thomas , Fisher Lindsay , Oyer Maxwell , Chou I-Ying Richard , Song Bo , Silverman Scott , McMullin David , Inglessis-Azuaje Ignacio , Palacios Igor , Buonanno Ferdinando S , Lo Eng H , Ning MingMing
Published: June 2021

Background:
Patent foramen ovale (PFO) is an independent risk factor of ischemic stroke. It enables the mixing of venous and arterial blood and therefore serves as a conduit for venous clots and vasoactive factors to enter into arterial circulation and contribute to a prothrombotic status. Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, contributes to vascular disease and has been linked with increased levels of homocysteine, which creates additional oxidative stress by decreasing the production of dimethylarginine dimethlyaminohydrolase (DDAH) and further inhibiting the clearance of ADMA. We previously identified a significant reduction of homocysteine by PFO closure. Here we study the influence of PFO on ADMA levels, a marker of oxidative stress.

Method:
97 PFO-related stroke patients were prospectively recruited in accordance with IRB, of which 61 received PFO closure and 36 underwent medical therapy alone. Peripheral venous blood was collected at baseline (BL) and 1 year follow-up (FU) post treatments. Plasma ADMA was quantified by mass spectrometry.

Result:
Compared to baseline, plasma ADMA levels were statistically significantly reduced post PFO closure (p = 0.0026), while no changes were observed for the patients treated with medications alone (p = 0.5500) (Figure 1A). Moreover, among the patients receiving PFO closure, the reduction of ADMA was only pronounced for those without residual shunting (p = 0.0009) but not for those with residual shunting (p = 0.4557) (Figure 1B, C).

Conclusion:
PFO closure reduced oxidative stress marker ADMA in the circulation, but no changes were observed in patients on medical treatment only or those with residual shunting post PFO closure. Our results support the hypothesis that PFO-related right-to-left interatrial blood shunting may causally contribute to the high level of vasoactive factors in circulation. Further studies on an expanded cohort of PFO patients are ongoing.
Abstract 112: Recrudescence of Neurological Deficits After Stroke: Clinical-imaging Phenotype, Triggers and Risk Factors
Authors: Singhal Aneesh B , Saka Esen , Silverman Scott B , Schwamm Lee H , Topcuoglu Mehmet A
Published: June 2021

Background:
Re-emergence of prior stroke-related deficits or post-stroke recrudescence (
PSR
) has not been adequately studied.

Methods:
Using preliminary criteria for PSR (transient recurrence or worsening of residual post-stroke deficits; chronic brain infarct or hemorrhage but no acute vascular lesion on MRI including DWI; no severe ipsilateral cerebral artery stenosis; no evidence for seizure), we identified 153 patients (145 prior infarcts, 8 hypertensive ICH) with 164 admissions for PSR from 2000-2015. Clinical-imaging features of PSR were characterized. Triggers of PSR were identified by comparing PSR admissions to adjacent admissions without PSR (n=65). Risk factors for PSR were identified by comparing PSR cases to 1861 controls without PSR in our prospective institutional stroke registry.

Results:
PSR occurred 3.9±0.6y after stroke, lasted 18.4±20.4 hrs, and 69% resolved on Day 1. Mean age 67±16y, 60% women. Neuro-worsening was usually abrupt, mild, with motor-sensory or language dysfunction. No patient had isolated gaze paresis, hemianopia or neglect. During PSR the NIHSS worsened by mean 2.5±1.9 points; deficits were limited to a single subscale NIHSS item in 38%. The underlying chronic strokes were variably-sized, predominantly affected white-matter tracts, and 73% involved the MCA territory. Infection, hypotension, hyponatremia, insomnia/stress and benzodiazepine use were higher (p<0.05) during PSR as compared to adjacent admissions without PSR. As compared to the ischemic stroke registry controls, the subgroup of 145 cases with PSR after ischemic stroke had more women, African-American and Other races, diabetes, dyslipidemia, smoking, and more severe NIHSS scores at the time of index stroke (all p<0.05). The PSR group had more small-vessel infarcts and more strokes from “other definite” causes. Six patients with PSR received iv tPA without hemorrhagic complications.

Conclusion:
This detailed characterization of PSR should enable prompt diagnosis and distinguish PSR from mimics such as TIA, migraine, Todd’s paralysis, Uhthoff’s phenomenon, and others. This is the first large comprehensive study of PSR. Prospective studies are required to validate our proposed diagnostic criteria and decipher underlying mechanisms.
Abstract 137: Neighborhood Income and 30-day Readmissions for Stroke and Transient Ischemic Attack
Authors: Lichtman Judith H , Leifheit-Limson Erica C , Wang Yun , Goldstein Larry B
Published: June 2021

Background:
Residence in a socioeconomically challenged neighborhood is associated with increased stroke incidence and shorter post-stroke survival. Little is known about the relationship between socioeconomic status and 30-day post-stroke readmissions. We used a nationally representative readmission database that includes both insured and uninsured patients to determine whether there is a relationship between community-level income and 30-day readmissions after stroke and transient ischemic attack (TIA).

Methods:
Hospitalizations were identified in the 2013 Nationwide Readmissions Database for patients aged ≥18y with subarachnoid hemorrhage (SAH; ICD-9 430), intracerebral hemorrhage (ICH; ICD-9 431), ischemic stroke (IS; ICD-9 433, 434, 436), and TIA (ICD-9 435). We used mixed logistic regression models with hospital-specific random effects to assess the relationship between community income level (measured according to the median household income quartile for a patient’s ZIP code) and 30-day readmissions. Models were stratified by age and adjusted for demographic and clinical characteristics.

Results:
There were 7,061 hospitalizations for SAH, 17,325 for ICH, 212,306 for IS, and 67,606 for TIA. In unadjusted analyses, 30-day readmission rates decreased with increasing income quartile for younger patients hospitalized with IS and SAH (figure). In adjusted analyses, this association persisted only among those with IS aged 18-44y for whom each quartile increase in income was associated with a 7% decrease in 30-day readmission (figure). There were no significant associations between income and 30-day readmission for SAH, ICH, and TIA.

Conclusions:
Overall, community income was not associated with readmission for hemorrhagic stroke and TIA, but higher income was associated with lower 30-day readmission for younger IS patients. Possible explanatory factors such as better access to post-stroke care warrant further research for this subgroup.
Abstract 138: Plasma Soluble ST2 Concentration Predicts Functional Outcome and Hemorrhagic Transformation After Acute Ischemic Stroke
Authors: Wolcott Zoe , Batra Ayush , Khoury Jane , Sperling Matthew , Meyer Brett C , Walsh Kyle B , Adeoye Opeolu , Broderick Joseph P , Kimberly W. T
Published: June 2021

Introduction:
Soluble ST2 (sST2) is a member of the interleukin-1 receptor family that predicts mortality and outcome in cardiovascular disease. We aimed to determine whether baseline sST2 predicts functional outcome after stroke and examined its potential role in hemorrhagic transformation (HT).

Methods:
We measured plasma sST2 and matrix metalloproteinase-9 (MMP-9) in 646 patients who presented with acute ischemic stroke and were enrolled in the Specialized Program of Translational Research in Acute Stroke (SPOTRIAS) network biomarker study. Soluble ST2 and total MMP-9 were measured by ELISA. Functional outcome was assessed at 3 months with the modified Rankin Scale (mRS), with good outcome defined as mRS 0-2, and poor outcome defined as mRS 3-6. HT was classified in 246 patients with available CT scans, using the ECASS III criteria. The relationships between sST2, outcome, and HT were evaluated using multivariate logistic regression.

Results:
Median admission sST2 level was 35.0 ng/mL [IQR 25.7—49.8] in 646 patients (mean age 69 years; 44% women). The plasma concentration of sST2 was independently associated with poor outcome (OR 3.06, 95% CI 1.15-8.31,
P
=0.02) and mortality (OR 8.94, 95% CI 2.13-39.55,
P
=0.003) after adjustment for age, sex, NIHSS, admission glucose, cardioembolic stroke subtype, and tPA treatment. Of the 246 patients with imaging data available, HT occurred in 37 (15%) patients: 18 (48%) with hemorrhagic infarction type 1 (HI1), 11 (30%) with hemorrhagic infarction type 2 (HI2), 4 (11%) with parenchymal hemorrhage type 1 (PH1) and 4 (11%) with parenchymal hemorrhage type 2 (PH2). Admission sST2 level was associated with the subsequent development of any HT (
P
=0.03) and with hemorrhagic infarction type 2 or greater (HI2, PH1, or PH2;
P
=0.008). Elevated sST2 independently predicted HI2 or greater after adjustment for age, MMP-9, blood glucose level, and tPA treatment (OR 9.27, 95% CI, 1.05-86.76,
P
=0.045).

Conclusion:
Plasma sST2 levels predicted mortality, functional outcome, and risk of HT after ischemic stroke. Further study of the potential link between sST2 and the brain injury response after stroke is warranted.