bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 296: Targeting Macrophage Subsets Attenuates Cardiac Remodeling and Dysfunction Following Experimental Myocarditis in Rat
Authors: Rinkevich-Shop Shunit , Ben-Mordechai Tamar , Kain David , Feinberg Micha S. , Landa-Rouben Natalie , Leor Jonathan
Published: July 2021

Introduction:
The mechanism of myocarditis is complex and poorly understood. Monocytes and macrophages comprise the majority of infiltrates in myocarditis.

Hypothesis:
We aimed to test the hypothesis that macrophage subsets have a key role in the pathogenesis and progression of myocarditis, and that targeting macrophages would modulate disease progression and outcome.

Methods and Results:
Experimental autoimmune myocarditis (EAM) was induced in 67 male Lewis rats. Of them, 27 were sacrificed at different time points to assess M1 and M2- macrophage subsets by flow cytometry. The remaining rats were randomized either to early macrophage depletion (from day 8-14 after EAM induction), by intravenous injections of clodronate-liposomes (CL), or late depletion (from day 15-35 after induction of EAM), or PBS-liposome injections (control). Left ventricula (LV) remodeling and function were assessed by echocardiography; before induction of EAM, before CL treatment, and 35 days after induction of EAM. Macrophages in the heart peaked at 21 days after induction of EAM (25±7%,p=0.0008).The number of M1 macrophages in the heart was highest at 14 days after EAM induction (16.3±2%), while M2 macrophages peaked at day 21 (15.5±2.7%,p=0.003). Early macrophage depletion (8-14d) decreased LV diastolic dimension and volume by 5.3±3.9% and 10.4±8% (p=0.04). Late macrophage depletion (15-35d) increased LV wall thickness and decreased LV systolic dimension and volume by 10.2±4% and 21±8% (p=0.03). Furthermore, late macrophage depletion increased LV ejection fraction and fractional shortening by 14.6±6% (p=0.01) and 22±10% (p=0.02) 35 days after EAM.

Conclusions:
Our study describes, for the first time, the kinetics of M1 and M2 macrophages in ocarditis and shows that macrophages contribute to the progression of adverse remodeling and dysfunction. Thus, targeting macrophages could be a new therapeutic strategy to improve cardiac remodeling and function in myocarditis.
Abstract 304: Estrogen Plays a Protective Role in Advanced Heart Failure by Suppressing Cardiac Fibrosis Associated Genes via miR129 Induction
Authors: Sharma Salil , Iorga Andrea , Singh Harnek , Li Jingyaun , Eghbali Mansoureh
Published: July 2021

We have previously shown that short term treatment of estrogen(E2) can rescue advance heart failure(HF) and decreases associated fibrosis. We hypothesized that E2 can reduce fibrosis by regulating the levels of specific microRNAs including miR129-5p(miR129) through ERβ mediated mechanism.

We used transaortic constriction to induce HF in male mice, and once the ejection fraction (EF) reached ~30%, one group of animals was sacrificed (HF), and the other group received 17b-estradiol via a subcutaneous pellet implant (0.012mg/pellet, n=16) (E2) for 10 days. Sham-operated mice served as CTRL. Serial echocardiography was performed to monitor cardiac structure and function. Short-term E2 treatment rescued pressure overload-induced decompensated HF in mice by restoring the EF from 33.17±1.12% to 53.05±1.29 (p <0.001, n=16). E2 decreased both interstitial and perivascular fibrosis in HF. Microarray analysis comparing HF with E2 revealed ~70 microRNAs including miR129 regulated by E2. qPCR validation revealed that E2 treatment upregulates miR129 by 2 folds compared to HF restoring it to CTRL levels. Treatment of HF with ERβ agonist (DPN), but not ERα agonist (PPT) resulted in the upregulation of miR129 indicating the E2 mediated induction of miR129 is mediated through ERβ. In vitro, angiotensin II treatment significantly downregulated miR129 expression in neonatal rat fibroblasts (NRVF) which was restored by E2 and DPN but not by E2+ERβ antagonist (PHPT) further confirming the role of ERβ in regulating miR129. In vitro, OE of miR129 in both neonatal and adult rat cardiac fibroblasts (ARVF) resulted in significant downregulation of transcripts of many in-silico predicted pro-fibrotic target genes including EGFR, RUNX, GREM1, COL2A, PDGFA, PDGFRA and the transcription factor SOX4. OE of miR129 in fibroblasts also resulted in downregulation of EGFR protein. Gain of miR129 prevented the transition of fibroblasts to myofibroblasts in both NRVF and ARVF and inhibited fibroblast proliferation in vitro.

In conclusion, E2 treatment during HF induces miR129 likely through ERβ. MiR129 represses fibrosis by targeting key genes associated with cardiac fibrosis, inhibits fibroblast proliferation and fibroblast to myofibroblast transition.
Abstract 305: E2F1 Suppresses Cardiac Fibrosis
Authors: Biyashev Dauren , Boriboun Chan , Qin Gangjian
Published: July 2021

E2F1 transcription factor is best known for regulation of cell cycle; its role in the cardiovascular system is not well understood. In a transcriptome analysis, we detected a significantly elevated level in the expression of collagen I and alpha-smooth muscle actin in the E2F1-null (E2F1-/-) mouse embryonic fibroblasts (MEFs) as compared to wild-type (WT) MEFs. Levels of Smad 2 and Smad 3 were also significantly higher in E2F1-/- MEFs. In addition, treatment with TGF-beta (10 ng/ml) induced a greater degree of Smad 2 and Smad 3 phosphorylation in E2F1-/- MEFs than in WT MEFs. Interestingly, these in vitro observations were corroborated with our results obtained from mouse heart samples: the basal levels of both total and phosphorylated Smad 2 were significantly higher in the E2F1-/- heart than in the WT heart (n=3). To understand the significance of these findings in the pathogenesis of cardiac fibrosis, we administered Angiotensin II (3 mg/kg/day) to animals for 7 or 14 days with a subcutaneous osmotic minipump. The total area of cardiac fibrosis was significantly greater in the E2F1-/- mice than in WT littermates (E2F1-/- vs. WT: 17+/-3.8% vs. 6+/-2.6%, p<0.05). Thus, we disclose a novel role of E2F1 in the control of Smad signaling that may limit the development of fibrosis in the stressed heart.
Abstract 308: Controlling Methylglyoxal To Prevent The Development Of Diabetic Cardiomyopathy
Authors: Vulesevic Branka , Ahmadi Ali , Milne Ross , Suuronen Erik
Published: July 2021

Hyperglycemia caused by diabetes leads to increased glucose oxidation and mitochondrial generation of superoxide. One of the most abundant by-products of glucose oxidation is methylglyoxal (MG). MG has been implicated in the development of diabetic complications. Diabetic cardiomyopathy, characterized by systolic and diastolic dysfunction, has a complex etiology, including the accumulation of MG-formed advanced glycation end products (AGEs). Methylglyoxal is detoxified by the activities of the enzymes glyoxalase 1 (GLO1) and glyoxalase 2. We have developed a mouse line that carries a transgene encoding human GLO1 (hGlo1) under the control of the preproendothelin promoter. The hGlo1 transgene has been shown to reduce MG-derived AGEs in tissues of streptozotocin (STZ)-induced hyperglycemic mice. We have also shown that over-expression of hGlo1, solely in the bone marrow, can rescue defective post-ischemic revascularization in STZ-treated mice. In this present study, the hGlo1 mice have been used to test the hypothesis that reduced MG levels can offset the development of diabetic cardiomyopathy. Diabetes was induced by intraperitoneal injection of STZ; 4 and 8 weeks post-STZ treatment, echocardiography of the wild type (WT) diabetic group showed systolic dysfunction. At 4 weeks of hyperglycemia, the ejection fraction (EF) was 54% and fraction shortening (FS) 37%. In contrast, cardiac function in the GLO1 diabetic mice as well as non-diabetic controls remained normal over time. Both the EF and FS were significantly lower in WT diabetic mice at 4 weeks post STZ-treatment compared to the control mice (p=0.02 and p=0.04, respectively). Western blots of heart tissue collected after 8 weeks of diabetes showed a significant increase in the expression of RAGE (receptor for AGE) in WT diabetic mice compared to other groups (p=0.038). Heart tissue collected from GLO1 diabetic and non-diabetic mice had increased GLO1 activity by 1.7-fold compared to the non transgenic mice. These results suggest that MG contributes to the development of diabetic cardiomyopathy and that reducing methylglyoxal levels could be a potential therapeutic target for prevention.
Abstract 309: Two SoxD Family Transcription Factors Influence Heart Function at Baseline and After Cardiac Overload
Authors: Beetz Nadine , Quiat Daniel , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

Background:
Sox6, a member of the SoxD family of transcription factors, has recently been established as a mediator of the actions of MyomiRs miR-208b and 499 in skeletal muscle. Sox6 suppresses the expression of slow skeletal muscle genes and its deletion promotes skeletal muscle fiber switching in mice. However, the role of Sox6 in the heart remains elusive. Given the importance of MyomiRs in regulating cardiac stress responses and the regulation of Sox6 by MyomiRs in the heart, we postulate that Sox6 is a central regulator of cardiac hypertrophy and heart failure.

Results:
Because of functional redundancy of Sox6 and Sox5 in multiple tissues,
Sox6
fl/fl
mice were crossbred with
Sox5
fl/fl
mice and mice expressing CRE recombinase under the control of the alphaMHC gene promoter to obtain
Sox5
fl/fl
/
Sox6
fl/fl
/CRE+ (
Sox5/6
cKO) animals or CRE- controls.
Sox5/6
cKO animals were viable and born at expected Mendelian ratios. At baseline,
Sox5/6
cKO mice did not show any signs of hypertrophy or cardiac failure as assessed by echocardiography at the age of 9 weeks. While cardiac size and heart function measured by echocardiography was unaltered in mice aged 4-5 months, cardiac catheterization revealed significant decreases in contractility in
Sox5/6
cKO animals. In addition, qPCR analysis of hearts showed increased expression of slow myosin
Myh7
and
Nppa
, a marker of cardiac stress, in
Sox5/6
cKO animals compared to controls. Following cardiac pressure overload for 3 weeks by transverse aortic constriction (TAC), significant decreases in cardiac function, as measured by echocardiography, were apparent in
Sox5/6
cKO compared to control mice.
Sox5/6
cKO mice subjected to TAC also showed signs of cardiac failure such as increased wet lung weight.

Conclusion:
Cardiac-specific deletion of
Sox6
and
Sox5
lead to reduction of cardiac function at baseline and exacerbated susceptibility to the development of cardiac failure after cardiac pressure overload. The contribution of each individual transcription factor to the observed phenotypes is currently being investigated.
Abstract 311: Measurements of cAMP Dynamics in the SERCA2 Microdomain in Adult Mouse Cardiomyocytes using a Targeted FRET Biosensor
Authors: Sprenger Julia U , Nikolaev Viacheslav O
Published: July 2021

PURPOSE:
cAMP is a central regulator of cardiac function and disease. This global second messenger acts in a compartmentalized fashion, and changes in cAMP dynamics are linked to cardiac diseases. In this project, we visualized cAMP signals directly in such microdomains to gain insights into the molecular mechanisms involved in cAMP compartmentation and its alterations in hypertrophy.

Methods:
We generated transgenic mice expressing a new Förster resonance energy transfer (FRET)-based cAMP sensor Epac1-camps-PLN to measure cAMP dynamics in the microdomain around the sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2). This sensor is targeted to SERCA2 via phospholamban (PLN).

Results:
Colocalization and cell fractionation analysis confirmed proper localization of the sensor in transgenic mouse hearts. qPCR analysis revealed a two-fold overexpression of PLN. However, no adverse cardiac phenotype could be detected by histological analysis and heart weight to body weight ratios. Local cAMP dynamics were measured using freshly isolated adult ventricular myocytes and compared to cAMP signals in the bulk cytosol using cardiomyocytes from Epac1-camps mice. We detected the predominant role of phosphodiesterases (PDEs) 4 and 3 in the SERCA2 compartment under basal conditions. These PDEs were responsible for shaping the microdomain and its segregation from the cytosolic compartment. Interestingly, beta1-adrenergic stimulation led to a stronger increase of local cAMP in the SERCA2 compartment compared to the bulk cytosol. 8 weeks after transverse aortic constriction (TAC), PDE4 activity was downregulated in the SERCA2 microdomain compared to sham cardiomyocytes.

Conclusion:
We successfully generated transgenic mice expressing the targeted Epac1-camps-PLN biosensor to visualize cAMP dynamics in the SERCA2 compartment. We could show distinct cAMP dynamics around the SERCA2 compartment compared to the bulk cytosol and uncovered its alterations in hypertrophied cardiomyocytes
Abstract 321: Deletion Of The Translational Repressors Eukaryotic Translation Initiation Factor 4e Binding Proteins 1 And 2 Protects Against Pressure Overload Induced Heart Failure
Authors: lu zhongbing , Hu Xinli , Huang Yimin , Xu Xin , zhang Ping , Peterson Mark , Polunovsky Vitaly A , Sonenberg Nahum , Bache Robert. J , Bitterman Peter B , Chen Yingjie
Published: July 2021

Assembly of the translation initiation machinery is negatively regulated by the eukaryotic translation initiation factor 4E binding proteins, which sequester the mRNA cap-binding protein eIF4E, thus preventing assembly of an intact initiation complex. However, the role of translational control on the development of congestive heart failure (CHF) has not been systematically examined. Here we perturbed translational control in mice by knockout of both 4E binding protein 1 (Eif4ebp1) and 2 (Eif4ebp2) (designated as Eif4ebp1/2 double knockout) to study its impact on left ventricular hypertrophy and CHF resulting from transverse aortic constriction. Eif4ebp1/2 double knockout caused a modest increase in left ventricular mass under basal conditions. However, following transverse aortic constriction, Eif4ebp1/2 double knockout profoundly attenuated the development of CHF and its attendant mortality. Examination of candidate genes involved in the mechanism revealed increased expression of transcription factors for genes governing energy metabolism and mitochondrial biogenesis with corresponding increases in the expression of their target genes. Our data indicate that removing physiological restraints on translation initiation exerts a profound cardiac protective effect against pressure overload induced CHF, suggesting that method(s) to disrupt the function of the 4E binding proteins may be a novel therapeutic approach for preventing or treating CHF.
Abstract 327: Cardiac Regulation of Metabolic Homeostasis by Med13
Authors: Grueter Chad E , Baskin Kedryn K , Kusminski Christine M , Holland William , Scherer Philipp E , Olson Eric N
Published: July 2021

Alterations in metabolism are a major component of cardiovascular disease associated with obesity and type 2 diabetes. The complex interplay between these three diseases poses a challenge for successful treatment and warrants further studies directed at understanding the intertissue communication between major metabolic organs. We previously identified a signaling pathway within the heart that modulates systemic energy homeostasis by regulation of Med13, a component of the kinase submodule of the Mediator Complex, in the heart. The Mediator Complex is a large, multiprotein complex that functions to integrate signal specific events with transcriptional activation and elongation in a context dependent manner. Our current work further delineates a mechanism by which Med13 in the heart functions to regulate whole body energy homeostasis. The increase in energy expenditure in Med13 transgenic (TG) mice is due in part to increased triglyceride uptake and beta-oxidation in white adipose tissue and liver. Additionally, the expression of Krebs Cycle and fatty acid oxidation genes are increased in adipose tissue and liver as measured by RNA seq and in metabolite production in Med13 Tg mice. Together, these results demonstrate the Mediator Complex regulates cardiac gene expression and metabolite production which communicates with energy depots within the body to modulate whole body energy homeostasis.
Abstract 329: HSF-1 Knockout Enhances Dietary Cholesterol Metabolism by Inducing CYP7A1 Gene Expression and Attenuates Atherosclerosis
Authors: Ilangovan Govindasamy , Karthikeyan Krishnamurthy
Published: July 2021

Objective:
Coronary heart disease and diabetes are highly prevalent among obese populations due to aberrant dietary cholesterol metabolism. Here we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis and dietary cholesterol metabolism.

Methods and Results:
Atherogenic western diet-induced weight gain was reduced in HSF-1 and LDLr double knock out mice (HSF-1
-/-
/LDLr
-/-
), compared to LDLr
-/-
mice. Atherosclerotic lesion growth in aortic arch and carotid regions was retarded. Also, repression of PPAR-γ2 and AMPKα expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. Furthermore, reduced heat shock proteins and their mRNA levels in atherosclerotic lesions correlated with reduction in lesion burden. In HSF-1
-/-
/LDLr
-/-
liver, higher cholesterol 7α hydroxylase (CYP7A1, the rate limiting enzyme in the synthesis of bile acid from cholesterol) and MDR1/p-glycoprotein (bile salt transporter across the hepatocyte canalicular membrane) gene expressions were observed, consistent with higher bile acid sequestration and larger hepatic bile ducts. HSF-1 deletion, however, upregulated both CYP7A1 enzyme and MDR1/p-glycoportein expression and activities, due to removal of its repressive binding in the CYP7A1 and MDR1 gene promoters. This increased the conversion of cholesterol into 7-α-hydroxycholesterol and bile acid, and dietary cholesterol metabolism.

Conclusions:
HSF-1 ablation not only eliminates heat shock response to retard atherosclerosis, but it also transcriptionally upregulates CYP7A1 and MDR1/P-gp axis to increase cholesterol metabolism. Therefore, HSF-1 is a metabolic regulator of dietary cholesterol and a major contributor to heart disease among obese population.
Abstract 330: Glycogen Synthase Kinase-3a Negatively Regulates Fatty Acid Metabolism During Lipid-induced Insulin Resistance
Authors: Nakamura Michinari , Sadoshima Junichi
Published: July 2021

Obesity and insulin resistance lead to ectopic lipid accumulation and impaired cardiac metabolism, resulting in cardiovascular diseases. Peroxisome proliferator-activated receptor α (PPARα) is highly expressed in the heart and serves as a key regulator of fatty acid metabolism. However, the underlying mechanisms responsible for the development of cardiac dysfunction in these pathologies are still poorly understood. GSK-3α was activated, as evidenced by a decrease in S21 phosphorylation, during insulin resistance with normoglycemia in the hearts of obese mice fed a high-fat diet and
ob/ob
mice. To evaluate the functional significance of GSK-3α upregulation with regard to metabolism, we applied 50 μM of BSA-conjugated palmitic acid to cardiomyocytes
in vitro
for three days. This intervention elicited ectopic lipid accumulation, as evaluated with Oil Red O staining, and a 2.0-fold activation of GSK-3α, similar to lipid-induced insulin resistance and dyslipidemia in the heart
in vivo
. In this condition, downregulation of GSK-3α with shRNA-GSK-3α in cardiomyocytes increased cell viability, ATP synthesis, and fatty acid oxidation, but not glycolysis. Downregulation of GSK-3α also increased the activity of PPRE-luciferase (1.5 fold, p<0.05) and mRNA expression of genes involved in fatty acid metabolism in response to palmitic acid, including
Acox1
and
Cpt1b
. Overexpression of GSK-3α induced a rightward shift of the dose response curve where the activity of the PPARα reporter was plotted against the dose of WY14643, a PPARα agonist. GSK-3α, but not GSK-3β, directly interacted with and phosphorylated PPARα
in vitro
. Collectively, these results suggest that GSK-3α negatively regulates ligand-dependent activity of PPARα through phosphorylation of PPARα, thereby inhibiting fatty acid metabolism during lipid-induced insulin resistance. GSK-3α may be a novel therapeutic target for metabolic disorders.
Abstract 333: Deficiency of DJ-1 Leads to Increased Injury following Myocardial Ischemia by Enhancing Apoptosis and Oxidative Stress
Authors: Lambert Jonathan P , Nicholson Chad K , Calvert John W
Published: July 2021

Background:
DJ-1 is a ubiquitously expressed protein that has typically been associated with the development of early onset Parkinson’s disease. Recent data suggests that it also plays a role in the cellular response to stress. Although much is known about DJ-1 in the brain, very little has been investigated in the heart. Here, we tested the hypothesis that a deficiency in DJ-1 would enhance myocardial ischemia-reperfusion (MI/R) injury.

Methods and Results:
Wild-type (WT) control and DJ-1 knockout (DJ-1 KO) mice were subjected to 45 min of left coronary artery ischemia followed by 24 hrs of reperfusion. The deficiency of DJ-1 significantly increased myocardial infarct size relative to both the area-at-risk and entire left ventricle, as well as increased circulating troponin-I levels (Panels A-B). Echocardiography and hemodynamic analysis at 1 week of reperfusion revealed that DJ-1 KO mice experienced greater left ventricular dilatation and hypertrophy, displayed exacerbated left ventricular dysfunction, and displayed worse contractility and relaxation when compared to WT controls. In an effort to evaluate the signaling mechanism responsible for the increased injury in DJ-1 KO mice, additional WT and KO animals were subjected to 45 min of ischemia followed by 4 hrs of reperfusion. DJ-1 KO hearts were found to display higher levels of oxidative stress, greater caspase-3 activity (Panel C), enhanced phosphorylation of Jnk, and enhanced activation of the mitochondrial fission protein, dynamin-related protein 1 (Drp1).

Conclusions:
These findings provide important information that DJ-1 plays a protective role in heart against acute MI/R injury.
Abstract 335: Activation of BKCa Channels With Rottlerin Greatly Improves Cardiac Functional Improvement Following Surgical Ischemia Associated With Cardioplegic Arrest.
Authors: Clements Richard T , Cordeiro Brenda
Published: July 2021

Introduction:
BKCa channels are thought to provide protection during ischemic insults in the heart. Rottlerin, has been implicated as a potent BKCa activator, however this has not been firmly established. The purpose of this study was two fold: 1) assess the specificity of rottlerin for BKCa channels and 2) investigate the efficacy of BKCa channel activation as a cardioprotective agent during cardioplegia/reperfusion (CP/R).

Methods:
Wild type and BKCa knock-out mice with and without rottlerin (only administered in CP) were subjected to an isolated heart model of CP/R. Hearts were perfused for 30 min at 37C (baseline), followed by intermittent cold crystalloid cardioplegia (intermittent St Thomas II, 10C), and normothermic, normoxic, reperfusion for 30 min. In additional studies, mechanism of rottlerin-induced cardioprotection was investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential (TMRE) and ROS (DCFDA) production.

Results:
CP and 30 min reperfusion decreased LVDP (58.7 +/- 5 % baseline, n=8 ), +/- dP/dt ( 71.1+/- 6.4, 59.2 +/-6.6, % baseline respectively) and coronary flow (CF) (66.8+/-7.4, %baseline) in wild type mice. Rottlerin, (100 (n=3), 500 nM (n=5)) delivered in the CP solution dose dependently increased the recovery of LV function and CF to near baseline levels (500 nM rottlerin, LVDP: 94.1+/- 2.3, +/-dPdt: 99.6 +/- 7.2, 111.0+/- 15.0, % baseline, and CF: 128.9 +/-30.0 %baseline ). BKCa KO hearts treated with (n=4)or without (n=3) 500nM rottlerin, were similar to wt CP hearts, showing no improved cardioprotection (BKCa KO + 500 nM rott: LVDP: 57.7+/- 4.0, +/-dP/dt 69.5 +/-12.0, 59.8+/- 4.9, and CF 73 +/- 6.7). H9c2 cells subjected to in vitro CP/R, displayed reduced mitochondrial membrane potential (39% +/-.09 % decrease relative to control, p<.01) and increased ROS generation (50%+/-.04 increase, p<.01), both of which were dose dependently normalized by rottlerin (100 nM-1 uM) (minimum n=8).

Conclusions:
Activation of BKCa channels profoundly rescues ischemic damage associated with CP, likely via mitochondrial effects on improved mitochondrial membrane potential and reduced ROS generation.
Abstract 336: A 14-3-3 Mode-1 Binding Motif Promotes Gap Junction Internalization During Acute Cardiac Ischemia
Authors: Smyth James W , Sanchez Jose M , Lamouille Samy , Hong Ting-Ting , Vogan Jacob M , Shaw Robin M
Published: July 2021

During each heartbeat, robust cell-cell electrical coupling via connexin 43 (Cx43) gap junctions allows billions of individual cardiomyocytes to contract in synchrony. Cx43 turns over rapidly, and altered Cx43 trafficking during disease contributes to the arrhythmias of sudden cardiac death. The overall phosphorylation status of the Cx43 protein is known to regulate gap junction coupling, but the role of many residue specific phosphorylation events remains unknown. One such residue, Ser373, forms a mode-1 14-3-3 binding motif upon phosphorylation. Given that 14-3-3 proteins are known to regulate protein trafficking, we hypothesized a role for Cx43
Ser373
phosphorylation in regulation of Cx43 gap junction coupling. Using Langendorff-perfused mouse hearts we find robust phosphorylation of Cx43 at Ser373 and Ser368 after 30 min of no-flow ischemia. In human cell lines, a S373A mutation ablated Cx43/14-3-3 complexing and
35
S pulse-chase revealed Cx43
S373A
also experiences a longer half-life than wild-type Cx43. Previous reports have implicated phosphorylation of Cx43
Ser368
in PKC mediated Cx43 internalization. We find that upon activation of PKC, the Cx43
S373A
mutant undergoes lower and more transient levels of phosphorylation at Ser368 than wild-type Cx43. Consistent with these data, siRNA-mediated ablation of 14-3-3 expression results in enlargement of gap junction plaque formation at cell-cell borders. In conclusion, we propose that phosphorylation of Cx43
Ser373
results in 14-3-3 binding which promotes and maintains phosphorylation of Cx43
Ser368
and the subsequent internalization of gap junction channels. These results identify for the first time a specific role for 14-3-3 proteins in regulation of Cx43 internalization during acute ischemia and contribute to the development of therapies aimed at preserving or enhancing gap junction coupling in the heart.
Abstract 338: Thioredoxin-Interacting Protein Contributes to Aggravated Myocardial Ischemia/Reperfusion Injury by Hyperglycemia
Authors: Zhang Haifeng , Xing Wenjuan , Ji Lele , Su Hui , Sun Xin , Tie Ru , Liang Xiangyan , Tian Fei , Tao Ling , Gao Feng
Published: July 2021

Hyperglycemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin-interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether hyperglycemia enhances Txnip expression and consequently exacerbates myocardial ischemia/reperfusion (MI/R) injury. Adult male SD rats were subjected to MI/R (30 min/4 h) and treated with saline or high glucose (HG, 500 g/L, 4 ml/kg/h, intravenously throughout the whole MI/R).
In vitro
study was performed on cultured neonatal rat cardiomyocytes subjected to simulated ischemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium.
In vivo
HG infusion significantly reduced the ± LV d
P
/dt
max
by 13.2 and 14.1% respectively (n=8,
P
<0.05), increased infarct size and myocardial apoptosis (
P
<0.05) and increased superoxide accumulation (
P
<0.01) compared with those in the saline group. Meanwhile, Txnip expression was enhanced (Ratio of Txnip/β-actin: 1.22 ± 0.16 vs. 0.92 ± 0.05 of MI/R+saline, n=6-8,
P
<0.05) whereas thioredoxin activity was inhibited (0.50 ± 0.05 vs. 1.41 ± 0.06 μmol/min/mg protein,
P
<0.01) following HG treatment in MI/R hearts. Additionally, HG significantly activated p38 MAPK and inhibited Akt in I/R hearts (
P
<0.05). In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity (n=6,
P
<0.05). Overexpression of Txnip enhanced HG-induced superoxide generation and aggravated cell apoptosis (n=6,
P
<0.05), while Txnip RNAi significantly blunted the deleterious effects of HG (
P
<0.05). Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG-induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury as evidenced by reduced infarction size (23.7 ± 1.6% vs. 43.1 ± 4.8%, n=6,
P
<0.05) and caspase 3 activity (
P
<0.05) in HG-treated rats. Hyperglycemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequent amplification of cardiac injury following MI/R.
Abstract 342: Increased Myocardial Ischemic/Reperfusion Injury in Renal Failure Involves Cardiac Adiponectin Signal Deficiency
Authors: Tao Ling , Song Yanbin , Zhang Junyi , Huang Weidong , Liu Yi , Sun Lu , Zhang Haifeng , Wang Haichang , Qu Yan
Published: July 2021

Chronic kidney disease (CKD) is associated with increased incidence as well as adverse outcomes of acute myocardial infarction. Adiponectin (APN), known as a cardioprotective factor, has been observed decreased in cardiovascular disorders including myocardial infarction. However, plasma APN levels are significantly increased and are inversely related to the risk of cardiovascular morbidity and mortality in patients with CKD. The mechanism underlying the paradoxical relationship between high APN and poor cardiac outcome remains unclear. In the present study, subtotal nephrectomized (SN) and sham-operated mice were randomly subjected to a 30 min myocardial ischemia followed by 24 hrs reperfusion with or without human recombinant gAd treatment (2μg/g/d, 7days). Compared with sham WT mice, SN mice displayed significantly depressed cardiac function and enlarged infarct size following MI/R. TUNEL staining and caspase-3 activity assay demonstrated markedly increased cardiomyocyte apoptosis in SN mice following MI/R. What’s more, increased plasma total APN levels was observed in SN mice 4 weeks after MI/R. Importantly, SN caused further decreased cardiac function and larger infarct size in APN knockout (KO) mice compared with those in SN WT mice subjected to MI/R. However, gAd treatment significantly enhanced cardiac function and reduced infarct size and apoptosis in both WT and KO mice. Further, we found that SN KO mice showed more reduced eNOS phosphorylation, upregulated iNOS expression and ·O
2
-
production and consequently increased peroxynitrite production in cardiac tissue than SN WT animals, which can be partly reversed by administration of gAd. AdipoR1 expression was reduced 4-wk after SN whereas AdipoR2 showed no significant change. More importantly, AMPK activation was also inhibited after SN and exogenous gAd supplementation reversed this change. In conclusion, the present study demonstrates that renal dysfunction increases cardiac susceptibility to ischemic/reperfusion injury, which is associated with downregulated APN/AdipoR1/AMPK signaling and increased oxidative/nitrative stress in local myocardium, and provides the first evidence for the benefits of exogenous supplement of APN on cardiac outcomes in renal failure.
Abstract 343: Upregulated S1P Lyase Exerts Cardioprotective Effects through Inhibiting S1P/HIF-1a-Mediated Oxidative/Nitrative Stress during Myocardial Ischemia/Reperfusion
Authors: Tao Ling , Yan Wenjun , Zhou Fen , Zhang Fuyang , Sun Lu , Wang Haichang , Qu Yan
Published: July 2021

Sphingosine-1-phosphate (S1P) signaling directs a diverse array of biological processes, such as anti-apoptosis, promoting in[[Unable to Display Character: ﬂ]]ammatory, and inducing fibrosis. Therefore, S1P levels need to be tightly controlled through the delicate interplay of its generating enzymes and its degrading enzymes. S1P lyase (SPL1) is a stress-activated enzyme responsible for irreversible S1P catabolism. This study was designed to determine the role of SPL1 in ischemia/reperfusion (I/R) induced cardiac dysfunction and the underlying mechanisms. Mice were subjected to 40 minutes left coronary artery occlusion followed by reperfusion for 24 hours, 1 week, or 4 weeks. First, we observed that SPL1 expression in cardiac tissue was upregulated by about 4-fold in response to I/R (n=6,
P
<0.01 versus sham). Further, oral administration of THI (an SPL1 inhibitor, 25 mg/L, daily) increased plasma S1P level and deteriorated I/R-induced apoptosis and fibrosis determined 1 week after I/R (all
P
<0.05 versus I/R + saline group), and cardiac dysfunction (ejection fraction: 39.1 ± 3.1% in I/R + THI group versus 50.7 ± 2.8% in I/R + saline group determined 4 weeks after I/R,
P
<0.05). After 1 week of I/R, hearts from THI-fed mice exhibited increased hypoxia-inducible factor-1α (HIF-1α) expression, along with significant oxidative/nitrative damage as evidenced by gp91
phox
, inducible nitric oxide synthase (iNOS), and 3-nitrotyrosine abundance (all
P
<0.05 versus vehicle). To define the underlying mechanisms of SPL1 in I/R-induced heart injury, neonatal rat ventricular myocytes were cultured and submitted to hypoxia/reoxygenation (H/R, 9 hours/3 hours) with or without THI treatment. Importantly, THI significantly increased gp91
phox
, iNOS, and 3-nitrotyrosine level, as well as apoptosis (n=8, all
P
<0.05 versus H/R), all of which were inhibited by pretreatment with HIF-1α siRNA (all
P
<0.05 versus scramble RNA). The present study demonstrated for the first time that SPL1 is remarkably upregulated during I/R and protects heart from I/R-induced S1P/HIF-1α-mediated oxidative/nitrative stress, suggesting SPL1 as a potential targets of therapy for ischemic heart disease.
Abstract 344: FNDC5, a PGC1-a-Dependent Myokine, Increases Glucose Utilization and Fatty-Acid Oxidation by AMPK Signaling Pathway
Authors: Tao Ling , Liu Yi , Xin Chao , Huang Weidong , Zhang Lijian , Zhu Di , Sun Lu , Wang Haichang
Published: July 2021

FNDC5 is a hormone secreted by myocytes that could reduce obesity and insulin resistance, However, the exact effect of FNDC5 on glucose and lipid metabolism remain poorly identified; More importantly, the signaling pathways that mediate the metabolic effects of FNDC5 is completely unknown. Here we showed that FNDC5 stimulates β-oxidation and glucose uptake in C2C12 cells in a dose- and time-dependent fashion in vitro (n=8, all P<0.01). In vivo study revealed that FNDC5 also enhanced glucose tolerance in diabetic mice and increased the glucose uptake evidenced by increased [18F] FDG accumulation in hearts by PET scan (n=6, all P<0.05). FNDC5 decreased the expression of gluconeogenesis related molecules (PEPCK and G6Pase) and increased the phosphorylation of ACC, a key modulator of fatty-acid oxidation, both in hepatocytes and C2C12 cells (n=3, all P<0.05). In parallel with its stimulation of β-oxidation and glucose uptake, FNDC5 increased the phosphorylation of AMPK both in hepatocytes and C2C12 cells in a dose- and time-dependent fashion in vitro and in vivo. More importantly, the β-oxidation and glucose uptake, the expression of PEPCK and G6Pase and the phosphorylation of ACC induced by FNDC5 were attenuated by AMPK inhibitor in hepatocytes and C2C12 cells (P<0.05). Most importantly, the FNDC5 induced glucose uptake and phosphorylation of ACC were attenuated in AMPK-DN mice (n=6, all P<0.05). The glucose-lowering effect of FNDC5 in diabetic mice was also attenuated by AMPK inhibitor. Our data presents the direct evidence that FNDC5 stimulates glucose utilization and fatty-acid oxidation by AMPK signaling pathway, suggesting that FNDC5 be a novel pharmacological approach for type 2 diabetes.
Abstract 346: Genome-wide Bisulfite Sequencing After Isoproterenol Identifies DNA Methylation Modules Influencing Heart Failure Susceptibility
Authors: Chen Haodong , Orozco Luz D , Rubbi Liudmilla , Wang Jessica , Rau Christoph D , Chapski Douglas , Ren Shuxun , Wang Yibin , Pellegrini Matteo , Lusis Aldons J , Vondriska Thomas M
Published: July 2021

DNA methylation is an epigenetic mechanism that controls gene expression. Recent studies of human samples linked dysregulation of DNA methylation to cardiovascular diseases. However, whether DNA methylation is mechanistically involved in disease pathogenesis is unknown. To determine how DNA methylation participates in the development of heart failure, we measured the DNA methylome in healthy and hypertrophic mouse hearts (and in isolated cardiac myocytes in parallel experiments) using reduced representational bisulfite sequencing. Isoproterenol (ISO) minipumps were implanted in two mouse strains with opposite phenotypes: BUB/BnJ, which is resistant to ISO-induced hypertrophy/failure, and BALB/cJ, which is susceptible. DNA and mRNA were isolated from hearts and myocytes and analyzed by bisulfite sequencing and microarrays, respectively. The results reveal three levels of information about the methylome: basal differences between the resistant and susceptible strains, loci affected in a disease-associated manner, and strain-specific changes in methylation following ISO treatment_which serve as novel targets to understand the genetic basis of differential incidence of heart failure. We observed 1,122 loci undergoing a >20% change in methylation after ISO in either strain (p<0.05). Our data also showed significant ISO-induced methylation differences in genes related to myofibril assembly (GO:0031032, p=6e-7) and increased heart weight (MP:0002833, p=2e-6). We selected the top 15,000 promoter-associated CpGs with high variation among samples and carried out an unbiased weighted correlation network analysis (WGCNA) of these methylation data. We identified a module of promoter-associated CpGs whose demethylation is highly correlated with ISO treatment in both strains (75 CpGs, Pearson correlation=0.898, p=0.001). We also identified modules with strain-specific responses to ISO treatment. Interestingly, we found a module of 109 CpGs whose methylation is increased in BALB/cJ strain after ISO treatment, but decreased in BUB/BnJ strain. Our data demonstrate global changes in DNA methylation in the adult heart during the development of disease and reveal networks of modified loci that influence heart failure susceptibility.
Abstract 347: Zebrafish Insertional Cardiac (ZIC) Mutants: a Living Genomic Resource for Cardiac Biology
Authors: Liu Weibin , Ding Yonghe , Gore Beninio , Ekker Stephen C , Xu Xiaolei
Published: July 2021

The human genome encodes about 20,000 genes and only a fraction of them are expressed in the heart. We hypothesized that annotation of this category of genes may facilitate our understanding of cardiac development and function. Recently, we reported a pilot insertional mutagenesis screening in zebrafish to annotate cardiac gene functions using a gene-break transposon (GBT). It demonstrated the feasibility of generation of a Zebrafish Insertional Cardiac (ZIC) mutant collection by screening gene expression in the embryonic heart. Here, we report an alternative strategy to identify cardiac mutants based on adult heart expression, and explore the application of expanded ZIC lines for understanding cardiac biology. From 203 GBT lines, we identified 34 lines with detectable expression in the dissected adult hearts. Various expression levels of the tagged genes were detected in embryonic hearts. Based on their tissue-specific expression, the candidate ZIC lines can be grouped into myocardium lines, endocardium lines, or both. Based on their subcellular expression, myocardium ZIC lines can be further categorized into sarcomeric lines, mitochondrial lines, and/or nucleic lines. Phenotypic analysis is being conducted to identify recessive mutants including embryonic lethal lines. We will present the current status of our ZIC collection, and discuss its application as a genomic resource platform for elucidating cardiac biology. Our data suggest that ~15% genes in the whole genome might have detectable cardiac expression. The sensitivity of screening for cardiac genes at adult stage is higher than that at embryonic stage (34/203 versus 18/322). We plan to expand the ZIC collection by screening 1,000 GBT lines that are being generated at Mayo Clinic. It is expected that this living mutant collection of cardiac genes will open doors to systematic annotation of novel cardiac genes and forward cardiac mutagenesis screening.
Abstract 348: A Novel Self Expanding Stent Technology for treatment of Aortic Aneurysms - Finite Element Analysis
Authors: Arokiaraj Mark
Published: July 2021

Background:
To investigate a novel self-expanding Nitinol stent model in treatment of aortic aneurysms.

Methods:
A novel large self-expanding Nitinol stent method was designed with strut thickness of 70µM x 70µM width and it was deployed in a virtual aneurysm model of 6cm wide x 6cm long fusiform hyper-elastic anisotropic model. At cell width of 9mm, there was no buckling of the deployed stent. The peak wall stress and stress-strain properties of the aortic aneurysm wall; and the adjacent normal segments of aorta were studied at various pressures with or without stent. The radial force of the stents was tested after parametric variations. A prototype 300µM x 150µM stent with cell width of 9mm was evaluated similarly for maximal stress distribution after embedding in the aortic wall and with a tissue overgrowth of 1mm over the stent.

Results:
The 300µM x 150µM stent reduced the peak wall stress by 70% in the aneurysm and 50% reduction in compliance after a tissue overgrowth of 1mm over the stent struts.

Conclusions:
There is potential for a novel Nitinol based self-expanding stent in the therapy of aortic aneurysms.
Abstract 352: MicroRNAs Play A Crucial Role In The Induction Of Endothelial-to-Mesenchymal Transition-Mediated Cardiac Fibrosis.
Authors: Nagpal Varun , Eren Mesut , Michaels Marissa A , Vaughan Douglas E
Published: July 2021

Introduction:
Fibroblast-like cells derived from aberrant activation of endothelial-to-mesenchymal transition (EndMT) are an important contributor to cardiac fibrosis. TGF-beta signaling plays a pivotal role in the induction of EndMT and cardiac fibrosis. Our recent studies have shown that specific miRNAs are differentially regulated during TGF-beta-induced EndMT and blocking TGF-beta-receptor I (TbetaR1) kinase inhibits TGF-beta-induced EndMT.

Hypothesis:
We hypothesize that miRNAs that promote EndMT will potentially exacerbate cardiac fibrosis, and knockdown of these miRNAs will attenuate EndMT-mediated cardiac fibrosis.

Results and Methods:
We investigated the levels of miRNAs and profibrotic markers in the failing human myocardium compared to healthy human heart tissue. Our results indicate that miRNAs upregulated during EndMT were significantly elevated in the failing human myocardium. In addition, failing human myocardium exhibited significant upregulation of profibrotic markers including alpha-SMA, Col1 and PAI-1. Next, primary cultures of mouse cardiac endothelial cells treated with TGF-beta or SB431542 (TbetaR1 kinase inhibitor) were evaluated for EndMT markers using bright field microscopy, fluorescence microscopy, western blot analysis and qRT PCR. We observed that blocking TbetaR1 kinase by SB431542 inhibits specific miRNAs which were upregulated during TGF-beta-induced EndMT. In addition,
in silico
analysis revealed that these miRNAs target key TGF-beta effectors, which was further confirmed by western blot analysis. Furthermore, overexpression of specific miRNAs using mimics resulted in the induction of EndMT. Next, miRNA mimics in combination with TGF-beta substantially potentiated TGF-beta-induced EndMT. Finally, knockdown of these miRNAs using inhibitors or Cy3-tagged antagomiRs significantly attenuated TGF-beta-induced cardiac EndMT.

Conclusions:
Our results indicate that TbetaR1 kinase-induced expression of miRNAs is involved in cardiac EndMT. Thus, miRNAs may promote profibrotic signaling in EndMT-derived fibroblast-like cells, which may contribute to fibrogenesis in the human heart.
Abstract 355: In Vivo Targeting Of GTF2B Selectively Inhibits Transcription Of Cardiomyopathy-related Genes And Partially Blunts Development Of Cardiac Hypertrophy
Authors: Sayed Danish , Yang Zhi , He Minzhen , Abdellatif Maha
Published: July 2021

Transcriptional profiling of cardiac genome during hypertrophy identified two categories of genes with distinct modes of regulation. The first set of genes involved in the cells essential functions (e.g. RNA splicing) and whose transcription is expected to be incremental and contribute to the increasing cardiac mass is regulated by promoter clearance of RNA polymerase II (pol II). On the other hand, the second set that include genes with specialized function and show a robust increase in expression upon growth stimulus (cytoskeletal, extracellular matrix) are regulated by de novo pol II recruitment to promoters. Our goal was to identify the transcriptional mechanisms that distinguish these two sets of genes and then to selectively inhibit those that participate in contractile dysfunction, while preserving the expression of genes necessary for essential functions. General Transcription factor IIB (GTF2B), is one of the essential components of transcription machinery and is required for pol II recruitment. Thus, we hypothesized that inhibition of GTF2B would result in inhibition of only the specialized genes, sparing the essential genes. Our in vitro results with shRNA mediated inhibition of GTF2B in hypertrophying neonatal myocytes showed decreased expression of genes that required de novo pol II recruitment for transcription (eg. ACTA1), while no change was observed in the genes regulated by promoter clearance of pol II (Vdac1). Similarly, preliminary results with in vivo knockdown of GTF2B (~80% reduction in mRNA and ~36% in protein) via intravenous injection of modified antisense oligo in mice subjected to transaortic coarctation (TAC) showed inhibition of only cardiomyopathy-related genes that require pol II recruitment (ANF), while expression of essential genes (Vdac1) remained unchanged. Inhibition of GTF2B restricted increase in TAC-induced heart wt to 9%, compared to 29% in TAC hearts with control oligo. Echocardiography showed partial normalization of ejection fraction with GTF2B inhibitor during TAC from 61.5% to 66.4% compared to sham hearts with 71%. Thus, we conclude that by targeting GTF2B we can selectively restrict the expression of detrimental genes during hypertrophy, thereby delaying the onset of cardiac dysfunction and failure