bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 031: β-Adrenergic Receptor-Mediated Transactivation Of Epidermal Growth Factor Receptor Decreases Cardiomyocyte Apoptosis Through Differential Activation Of ERK1/2 and Akt
Authors: Grisanti Laurel A , Carter Rhonda L , Yu Justine E , Repas Ashley R , Tilley Douglas G
Published: July 2021

β-adrenergic receptors (βAR) are critical regulators of cardiac function whose dysregulation during heart failure are associated with diminished function. However, βAR-mediated EGFR transactivation has been shown to relay cardioprotection in a mouse model of heart failure via unknown mechanisms. We hypothesized that transactivation of EGFR promotes survival via distinct cardiomyocyte signaling responses leading to decreases in apoptosis. To test this hypothesis, C57BL/6 mice were injected with isoproterenol (Iso) in the presence or absence of the EGFR antagonist AG1478 and ERK1/2 and Akt phosphorylation and subcellular distribution were assessed. Following 10 min Iso stimulation, increases in ERK1/2 and Akt phosphorylation were observed in cytosolic, plasma membrane and nuclear fractions. Phosphorylation of ERK1/2 were AG1478 sensitive in all three fractions while Akt phosphorylation occurred through EGFR-transactivation only in plasma membrane and nuclear fractions, which was confirmed in rat neonatal cardiomyocytes (RNCM). Additionally, EGFR-transactivation by βAR decreased apoptosis, as measured via caspase 3 activation/activity and TUNEL assay, which was sensitive to inhibition of both ERK1/2 and Akt signaling pathways. Increased phosphorylation of ERK1/2 and Akt in the nucleus and the ability to inhibit Iso-mediated changes in apoptosis with the transcriptional inhibitor Actinomycin D suggested that the cardioprotective effects of Iso-mediated EGFR transactivation may be influenced by changes in gene transcription. An Apoptotsis RT2 PCR Array was used to identify changes in transcript levels of 84 apoptotic genes. Of these, 12 were found to be altered in response to EGFR inhibition in the presence of Iso. These results demonstrate that βAR-mediated EGFR transactivation in the heart induces differential subcellular activation of ERK1/2 and Akt and leads to the promotion of cell survival, in part through the modulation of apoptotic gene expression in cardiomyocytes. Further understanding the downstream consequences of these effects in response to βAR-mediated EGFR transactivation could lead to improved therapies for the treatment of heart failure.
Abstract 042: Radiation-Associated Cardiovascular Risks for Future Deep-Space Missions
Authors: Yan Xinhua , Sasi Sharath P , Gee Hannah , Lee Juyong , Yang Yongyao , Song Jin , Carrozza Joseph , Goukassian David A
Published: July 2021

During the future Moon and Mars missions astronauts will be exposed to space radiation (IR) for extended time. The effect of cosmic IR during and after space flights on cardiovascular (CV) system is unknown. Nine-month old C57BL/6N male mice were IR once with proton 50 cGy or 56Fe 15 cGy, both at 1 GeV/nucleon. We evaluated IR-induced biological responses - underlying molecular mechanisms, calcium handling, signal transduction and gene expression. Cardiac function was assessed by echocardiography and hemodynamic measurements.

Left ventricular end diastolic pressure (LVEDP) was increased in 56Fe mice 1 and 3 months post-IR (p<0.001). One month post-IR, compared to control, proton- and 56Fe-IR sarcolemmal Na+-Ca2+ exchanger (NCX) (p<0.007) and sarco(endo)plasmic reticulum calcium-ATPase (SERCA2a, p<0.02) were both increased more than 200% and p-p38 was decreased 400% (p<0.05), suggesting activation of compensatory mechanisms in [Ca2+]i handling in these hearts.

By 3 months, compared to control, proton- and 56Fe-IR hearts SERCA2a and p-Creb1 was decreased 200-500% (p<0.02), suggesting reduced capacity in intracellular [Ca2+]i handling, suggesting that [Ca2+]i handling dysfunction combined with LVEDP increase in 56Fe-IR may be due to prolonged activation of compensatory mechanisms that lead to changes in SERCA2a and p-Creb1 levels.

By 10 months, compared to control, LVESP was decreased in proton- and 56Fe-IR (p<0.03), suggesting IR-associated decrease in contractile function. However, compared to age-matched controls (18 months), the LVEDP was increased (p<0.05) and dP/dt Min was decreased (p<0.02) in proton-IR but not 56Fe-IR mice. This data suggests that after 10 months proton- but not 56Fe-IR affects considerably contractile and relaxation functions during aging.

Our longitudinal 1, 3 and 10 months studies reveal that a single full body low dose proton- and 56Fe-IR have long-lasting negative effect on heart homeostasis during aging. The divergent effects of low dose proton vs. 56Fe-IR on heart function during aging suggest significantly different biological mechanisms responsible for this ion-dependent dichotomy over 10 months post-IR and necessitate further studies into underlying molecular mechanisms.
Abstract 056: The Impact of Periostin as A Regulator of Cardiac Remodeling
Authors: Azuma Junya , Taniyama Yoshiaki , Okayama Keita , Katsuragi Naruto , Masaki Iwabayashi , Otsu Rei , Iekushi Kazuma , Sanada Hirohumi , Rakugi Hiromi , Morishita Ryuichi
Published: July 2021

Background:
Periostin (PN) is a matricellular protein that is induced by mechanostress and regulates organ remodeling. We previously documented that over-expression of full-length periostin (periostin1:Pn1) resulted in ventricular dilation with enhanced interstitial collagen deposition. Controversially, supplement of PN splicing variant lacking exon 17 (periostin2:Pn2) was reported to accelerate cardiac repair with the induction of cardiomyocyte proliferation and angiogenesis. The aim of this study was to investigate the different roles of PN splicing isoforms in cardiac remodeling.

Methods:
Coronary ligation was employed to induce cardiac remodeling in rat. A neutralizing antibody against exon17 (Pn1Ab) was injected intravenously every 6 days until day 21. Mice were followed ultrasonographically and then euthanized for histological analysis at day 56. In vitro study, the effect of each PN splicing isoform was assessed in rat cardiac fibroblast and HUVEC.

Results:
Administration of Pn1Ab resulted in 24.1 % decrease in infarct size and 38.2 % decrease in fibrotic area at day 56 post-operation (n=21, P<0.05). Consistently, echocardiography demonstrated 26.3 % better ejection fraction (P<0.05). Inhibition of fibrosis was associated with suppressed fibrosis-related gene expression such as collagen I, collagen III and TGF-beta1. Importantly, the number of myofibroblasts was significantly reduced in the Pn1Ab group, while neither cardiomyocyte proliferation nor angiogenesis was detected. In vitro study, Pn2 administration for HUVEC induced proliferation and tube formation however Pn1 did not. Pn1 administration accelerated maturation of myofibroblast more than Pn2.

Conclusions:
Pn1Ab preserved cardiac function through anti-fibrotic effect without cardiomyocyte proliferation and angiogenesis. Specific blockade of Pn1 without Pn2 will potentially lead to new therapeutic targets on a molecular basis to limit cardiac remodeling.
Abstract 062: Mitoubiquinone Decreases Reactive Oxygen Species Production and Improves Cardiac Efficiency in Chronic Volume Overload
Authors: Yancey Danielle M , Gladden James D , Guichard Jason L , Darley-Usmar Victor M , Dell’Italia Louis J , Zhou LuFang
Published: July 2021

Background:
The hemodynamic stress of left ventricular (LV) volume overload (VO) produces LV dysfunction accompanied by mitochondrial and cytoskeletal disruption in cardiomyocytes. Because mitochondria are both a source and target of reactive oxygen species (ROS), we hypothesize myocyte damage and LV dysfunction are mediated by mitochondrially produced ROS and can be attenuated by the mitochondrially targeted antioxidant, mitoubiquinone (MitoQ).

Methods:
Aortocaval fistula (ACF) was induced for 8 weeks in adult rats ± MitoQ. Echocardiography and high-fidelity LV pressure catheter recordings were used to study the LV end-systolic pressure-volume relationship and cardiac efficiency. Isolated cardiomyocytes were loaded with Carboxy-H2DFFDA (CM-DCF) and tetramethylrhodamine (TMRM) to measure mitochondrial ROS production and membrane potential.

Results:
Isolated cardiomyocyte studies demonstrated increased ROS production and decreased mitochondrial membrane potential in VO animals, both of which were attenuated with MitoQ. Treatment with MitoQ demonstrated a strong trend toward improvement in LV contractility, as cardiac efficiency improved significantly in MitoQ-treated VO animals. Untreated VO animals exhibited mitochondrial swelling and myofibrillar disruption that was prevented by MitoQ.

Conclusion:
These studies suggest an early interplay between mitochondrial-derived ROS production and cardiac ultrastructure and function.
Abstract 067: Long-Term Ivabradine Treatment Does Not Preserve Myocardial Perfusion and Coronary Reserve in Middle-Aged Rats with Large Myocardial Infarction
Authors: Dedkov Eduard I , Bogatyryov Yevgen , McCooey Daniela , Christensen Lance P , Tomanek Robert J
Published: July 2021

Background:
We have previously shown that 1-month treatment with ivabradine (IVA), the selective cardiac pacemaker I

f

current inhibitor, preserved myocardial perfusion and coronary perfusion reserve in post-MI middle-aged rats. However, the persistence of this cardioprotective effect after a prolonged period of IVA treatment remains to be determined.

Methods:
Acute MI was induced in 12-month-old male Sprague-Dawley rats by left coronary artery ligation. Twenty four hours later, the rats with a confirmed large transmural MI (>50% of the left ventricular (LV) free wall) were randomly assigned in two experimental groups. In a first group, rats were treated with IVA
i.p.
via osmotic pumps in a dose of 10.5 mg/kg/day for 3 months (MI+IVA). In a second group, rats received placebo treatment (5% dextrose) during the same time period (MI). Sham-operated rats served as an age-matched control. At the end of experimental period, myocardial perfusion (baseline and maximal coronary conductance per 100g of tissue) and coronary perfusion reserve (fold increase between baseline and maximal coronary conductance) were determined in non-infarcted LV free wall and interventricular septum by using the neutron-activated stable isotope-labeled microsphere technique.

Results:
During 3 months of IVA treatment, heart rate in MI+IVA rats was consistently reduced compared to untreated MI rats by mean of 30.6%. Nevertheless, we found that the infarct size and the extent of LV remodeling were relatively comparable between MI and MI+IVA rats three months after surgery. Moreover, the levels of baseline and maximal coronary conductance were similar in LV free wall and septum between two experimental groups. Consequently, IVA-treated rats revealed no difference in coronary perfusion reserve as compared to untreated post-MI animals (2.22±0.46 vs. 2.59±0.41 in LV free wall and 2.30±0.59 vs. 2.68±0.44 in septum, respectively). However, the rats of both post-MI groups had markedly reduced levels of maximal coronary blood flow as compared to non-infarcted controls (p≤0.01).

Conclusion:
Our data demonstrate that long-term IVA treatment does not provide sustainable improvement in LV myocardial perfusion and coronary perfusion reserve in middle-aged rats following large MI.
Abstract 072: Protein Kinase A prevents cardiomyocyte hypertrophy through abhydrolase domain containing 5 (ABHD5)-mediated proteolysis of Histone deacetylase 4(HDAC4)
Authors: Jebessa Zegeye H , Worst Barbara , Katus Hugo A , Backs Johannes
Published: July 2021

Histone deacetylase 4 (HDAC4) regulates numerous gene expression programs through its signal-dependent repression of the transcription factor myocyte enhancer factor 2 (MEF2). Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling promotes pathological cardiac remodeling by phosphorylating HDAC4, with consequent stimulation of MEF2 activity. Recently, we described a novel mechanism whereby protein kinase A (PKA) overcomes CaMKII-mediated activation of MEF2 by regulated proteolysis of HDAC4. PKA induces the generation of an N-terminal HDAC4 cleavage product (HDAC4-NT). HDAC4-NT inhibits MEF2 activity, thereby antagonizing the pro-hypertrophic actions of CaMKII signaling. However, the individual protease mediating HDAC4 proteolysis remains unknown. Using a number of group specific chemical protease inhibitors, we found that the PKA-dependent HDAC4 protease belongs to the family of serine proteases. To further identify the individual serine protease, we screened a human serine protease siRNA library and found abhydrolase domain containing 5 (ABHD5) to be required for PKA-induced HDAC4 proteolysis. The screening result was validated using three different siRNAs. Remarkably, forced adenoviral expression of ABHD5 led to robust production of HDAC4-NT in isolated neonatal rat ventricular myocytes (NRVMs) even in the absence of PKA. Furthermore, adenoviral expression of ABHD5 in NRVMs inhibited MEF2 transcriptional activity and attenuated cardiomyocyte hypertrophy. Interestingly, ABHD5 was initially described to be involved in lipid metabolism. Taken together, we show that ABHD5 is required for PKA-dependent HDAC4 proteolysis and sufficient for HDAC4-NT production as well as inhibition of cardiomyocyte hypertrophy. These data imply a so far unknown link between lipid metabolism and epigenetic regulation of cardioprotection.
Abstract 073: Late Onset of Wolff-Parkinson-White Syndrome in a 48 year old Post-Menopausal Woman
Authors: Bush Shane D
Published: July 2021

Introduction—
First described in 1930, Wolff-Parkinson-White syndrome, or WPW syndrome, is an abnormal extra electrical pathway in the heart that can lead to tachycardia, palpitations and rarely sudden cardiac death. The extra electrical pathway of Wolff-Parkinson-White syndrome is present at birth and is a congenital heart abnormality occurring in about 1 per 1,000. Young men have a higher incidence of WPW than women. Episodes of tachycardia often first occur when they are in their teens or early 20s. The definitive treatment of WPW is a destruction of the abnormal electrical pathway by radiofrequency catheter ablation with success rates of as high as 95%. If radiofrequency catheter ablation is successfully performed, the condition is generally considered cured. Recurrence rates are typically less than 5% after a successful ablation.

Case description—
A 48 year old post-menopausal woman with intermitted palpitations and syncope over approximately one month which were affecting her daily activities presented to our family practice center complaining of worsening palpitations and new onset tachycardia. An EKG revealed a positive delta wave. Unique features were late onset diagnosis at age of 48 year old post-menopausal female without any previous documentation of WPW syndrome on an EKG. WPW is usually diagnosed in men in their early 20’s. Additional cardiac workup included a 24hr Holter monitor; exercise EKG treadmill test and transthoracic echocardiography which helped confirm the initial diagnosis. The patient had catheter ablation approximately two months after the first symptoms appeared. The procedure involved a Thermocool ® catheter and 3D electroanatomic mapping using the Ensite ® system which led to a successful ablation of the accessory pathway.

Discussion—
This case involves a very rare condition with an atypical late onset. WPW is normally diagnosed in men at a young age. The unique and rare features of this case were the recent onset of palpitations, syncope and tachycardia, evolving at a fairly advanced age of 48 years old, without any previous documentation of WPW syndrome in an ECG. The patient also had a full recovery and had no further symptoms of palpations, syncope and tachycardia since undergoing the catheter ablation surgery.
Abstract 076: Lapatinib Reduced Cardiac Hypertrophy Induced By Combined Use Of A Pi3k-mtor Dual Inhibitor And Doxorubicin
Authors: Yan Xinhua , Yang Yongyao , Lee Juyong , Onufrak Jillian , Fuseler John , Price Robert , Borg Thomas K , Morgan James , Goukassian David , Carrozza Joseph
Published: July 2021

The HER2-PI3K-mTOR pathway is pivotal for regulating the physiological function of the heart. Currently, drugs targeting the HER2-PI3K-mTOR pathway are either approved or being tested in clinical trials for cancer therapy. It is, therefore, important to evaluate the cardiac effects of using these drugs. In addition, it is necessary to develop countermeasures to prevent the cardiac side effects if any.

Methods:
Three-month old female FVB/n mice were treated with lapatinib (an HER2 inhibitor) or BEZ235 (BEZ, a PI3K-mTOR dual inhibitor), alone or with doxorubicin (DOX). Cardiac function was monitored by echocardiography and hemodynamic measurements. Cardiac morphology was assessed by confocal microscopy and transmission electron microscopy. The activation of signaling molecules were measured by Western blot analysis.

Results:
BEZ alone induced cardiac hypertrophy and subsequent heart failure and lapatinib alone induced cardiac failure, in a course of 17 months, respectively. Combination of BEZ with DOX, either concurrently or sequentially, induced cardiac hypertrophy which was associated with higher mortality rate compared to DOX alone. Neuregulin1, a HER receptor ligand worsened, while Lapatinib alleviated, cardiac hypertrophy in these mice. Lapatinib increased the activation of AMPK in DOX+BEZ treated hearts. The cardiac effect of lapatinib was blocked by Compound C, an AMPK inhibitor. Metformin, an AMPK activator, alleviated DOX+BEZ induced cardiac hypertrophy.

Conclusions:
BEZ or lapatinib treatment alone induced irreversible cardiac dysfunction in mice. Combined use of BEZ and DOX induced cardiac hypertrophy and early mortality, which were prevented by lapatinib. The cardioprotective effects of lapatinib may rely on activating AMPK in the heart.
Abstract 081: Liganded Cannabinoid Receptors Suppress Cardiac Myocyte Hypertrophy by AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) signaling
Authors: Lu Yan , Anderson Hope
Published: July 2021

Endocannabinoids are bioactive lipids that signal through CB1 and CB2 cannabinoid receptors. Despite an effort to avoid the psychoactive side effects mediated by central CB1 receptors, we previously found that selective activation of CB2 receptors is not sufficient to prevent cardiac myocyte hypertrophy. Thus, the objective of this study is to determine the effects of CB13, a peripherally-restricted agonist of CB1/CB2 receptors, on hypertrophy. The effects of CB-13 on endothelin-1 (ET1)-induced hypertrophy were determined using isolated neonatal rat myocytes. Hypertrophic indicators included myocyte enlargement, protein synthesis by [3H]-leucine incorporation, and fetal gene expression as brain natriuretic peptide (BNP) mRNA levels. AMPK and eNOS signaling were assessed by immunoblotting. ET1 increased myocyte size (122±4% vs. control; p<0.01), BNP expression (426±88% vs. control; p<0.01) and [3H]-leucine incorporation (147±13% vs. control; p<0.05). CB13 attenuated all three hypertrophic indicators (respectively, 104±3%, 210±32%, 90±8% vs. control; not significant). We next queried whether the anti-hypertrophic actions of CB13 were mediated by AMPK. CB13 increased total expression (222±45% vs. control; p<0.05) and phosphorylation (354±58% vs. control; p<0.01) of AMPK. Also, pretreatment with a chemical inhibitor of AMPK, compound C, attenuated the anti-hypertrophic actions of CB-13. AMPK is a regulator of cellular energy, so we determined that ET-1-induced mitochondrial depolarization, as assessed using the potential-sensitive dye, JC-1, was prevented by CB-13. In addition, CB-13 increased eNOS phosphorylation (237±51%; p<0.01), which suggests that AMPK-eNOS crosstalk, through anti-growth nitric oxide signaling, plays a role. These findings support the notion that agonism of CB1 and CB2 receptors by CB13, a synthetic endocannabinoid with poor brain-blood barrier penetration, prevents hypertrophy and mitochondrial dysfunction. These CB13 actions rely on AMPK signaling, and are associated with eNOS activation. In conclusion, cannabinoid-based treatment of heart disease remains a viable goal with therapeutic potential and warrants further study.
Abstract 091: Mitochondrial Calcium Currents Are Conducted By A Pore Encoded By The MCU Gene.
Authors: Chaudhuri Dipayan , Sancak Yasemin , Mootha Vamsi K , Clapham David E
Published: July 2021

Mitochondrial uptake of calcium (Ca
2+
) in cardiomyocytes is thought to regulate cell survival and match ATP production to energetic demands. Of multiple pathways allowing such entry, the mitochondrial Ca
2+
uniporter is a highly Ca
2+
-selective channel encoded by several recently-discovered genes. However, which of these several genes encodes the pore-forming subunit of the channel remains an open question. Using whole-mitoplast voltage-clamping, we show that RNAi-mediated knockdown of the
mitochondrial calcium uniporter
(
MCU
) gene reduces the uniporter current (

I
MiCa

), whereas overexpression increases it. Mutations in the putative pore domain alter key features of

I
MiCa

, such as its strong affinity for ruthenium red, a classic uniporter blocker. These analyses demonstrate that
MCU
encodes the pore-forming subunit of the uniporter channel.
Abstract 104: Effects of Pinacidil and Ca
2
+ on Sodium-loaded Rat Heart Mitochondria
Authors: Korotkov Sergey M , Nesterov Vladimir P , Brailovskaya Irina V , Emelyanova Larisa V , Konovalova Svetlana A , Furaev Victor V
Published: July 2021

Deterioration of the contractile parameters of the heart muscle caused by ischemia and followed reperfusion is known as the main postoperative complication which is related to Ca
2+
and Na
+
overload in cardiomyocytes and mitochondria. Pinacidil reduced the overload in ischemia/reperfusion experiments. The mechanism of this phenomenon is still not clear. We hypothesized that increased ion permeability of the inner mitochondrial membrane (IMM) followed drop of electrochemical potential (ΔΨ
mito
) can reduce the calcium. The aim of the study was to elucidate the effect of pinacidil (100 μM) and Ca
2+
(100 μM ) on swelling, oxygen consumption and ΔΨ
mito
of isolated sodium-loaded rat heart mitochondria (RHM(Na)) energized glutamate and malate. Pinacidil significantly enchanced the permeability of IMM to protons in ammonium nitrate medium. Also increased swelling of RHM(Na) energized with substrates in potassium acetate medium revealed that pinacidil increased potassium transport into matrix. Pinacidil stimulated oxygen consumption of RHM(Na) in State 4 and detained Ca
2+
-induced dissipation of ΔΨ
mito
. Under condition of Ca
2+
and Na
+
overload simulating ischemia/reperfusion, RHM(Na) oxygen consumption was not affected with pinacidil in State 3 and in the presence of 2,4-dinitrophenol. Cyclosporin A and ADP, the inhibitors of mitochondrial permeability transition pore (MPTP), markedly decreased Ca
2+
- induced swelling of RHM(Na) in nitrate ammonium or potassium acetate medium in the presence of pinacidil. Carboxyatractyloside, an inhibitor of cytosolic side-specific adenine nucleotide translocase, eliminated a pinacidil-stimulated oxygen consumption of succinate-energized RHMNa in State 4 regardless of the presence of Ca
2+
. Pinacidil was also concluded to accelerat potassium flux into energized RHM(Na) and promot MPTP opening in the low conduction state. Based on our data we suggested that the effect of pharmacological preconditioning induced by pinacidil could be due to it’s direct effect on mitochondria which is connected with above stimulation of the potassium permeability of the inner mitochondrial membrane and following reduce of the ΔΨ
mito
that thus prevent calcium overload of cardiomyocytes after ischemia/reperfusion in turn.
Abstract 107: Apocynin Attenuates Isoproterenol-induced Myocardial Injury: Implications For Targeting Nadph Oxidase In Myocardial Fibrogenesis
Authors: Chen Yu , Cui Jingang , Yang Qinbo , Jia Chenglin , Xiong Minqi , Wang Peiwei , Zhang Teng
Published: July 2021

Myocardial fibrosis results from cardiac injuries caused by various pathophysiological mechanisms including myocardial infarction, leading to destruction of myocardial architecture and progressive cardiac dysfunction. Oxidative stress is likely involved in myocardial ischemic injury and the subsequent tissue remodeling mediated by myocardial fibrogenesis. Our current study aimed to evaluate the implication of NADPH oxidase in overproduction of reactive oxygen species and its contribution to the pathogenesis of myocardial fibrogenesis after ischemic injuries. The effects of Apocynin, a selective NADPH oxidase inhibitor, were evaluated in the mouse model of isoproterenol-induced myocardial injury by histopathological approaches and whole-genome gene expression profiling. The results demonstrated that Apocynin was able to inhibit the development of ISO-induced myocardial necrotic lesions and fibrogenesis in a dose-dependent manner. Moreover, the preventive effects of Apocynin on myocardial injuries were associated with suppressed expression of genes implicated in inflammation responses and extracellular matrix, which were remarkably upregulated by isoproterenol administration.
In summary, o
ur study provides proof-of-concept for the involvement of NADPH oxidase-mediated ROS generation in myocardial ischemic injuries and fibrogenesis, which will benefit the mechanism-based therapeutic development targeting NADPH oxidase and oxidative stress in treating myocardial fibrosis and related disorders.
Abstract 109: Adiponectin Inhibits TNF-a-Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation
Authors: Ma Xin , Wang Yajing , Lau Wayne , Gao Erhe , Koch Walter
Published: July 2021

Anti-inflammatory and vascular protective actions of adiponectin (APN) are well-recognized. However, many fundamental questions remain unanswered. The current study attempted to identify the APN receptor subtype responsible for APN’s vascular protective action, and investigate the role of ceramidase activation in APN anti-inflammatory signaling. Wild type (WT) or gene manipulated HUVEC were treated with TNFα in the presence and absence of APN. The effect of APN on TNFα-induced inflammatory and oxidative/nitrative stress was determined. In WT HUVEC, APN significantly reduced TNFα-induced ICAM-1 expression and attenuated TNFα-induced superoxide and peroxynitrite formation (P<0.01). These anti-inflammatory actions were virtually abolished by AdipoR1-, but not AdipoR2-, knockdown (KD). Treatment with APN significantly increased neutral ceramidase (nCDase) activity (3.7-fold, P<0.01). AdipoR1-KD markedly (P0.05), reduced APN-induced nCDase activation. More importantly, siRNA mediated nCDase-KD markedly blocked the effect of APN upon TNFα-induced ICAM-1 expression (P0.05), and modestly inhibited APN anti-inflammatory effect (P87% of APN-induced nCDase activation was lost. Whereas APN treatment failed to inhibit TNFα-induced ICAM-1 expression, treatment with S1P or SEW (S1P receptor agonist) remained effective in Cav1-KD cells in reducing TNFα-induced ICAM-1 expression (P<0.01). AdipoR1 and Cav1 co-localized and co-precipitated in HUVECs. APN treatment did not affect this interaction. Moreover, re-expression of WT Cav1 in Cav1-KD cells restored nCDase activation in response to APN (P<0.01 vs. vehicle), whereas re-expression of a mutated Cav1 blocking AdipoR1/Cav1 interaction failed to restore APN-mediated nCDase activation. Finally, there is weak basal Cav1/nCDase interaction, which significantly increased following APN treatment. These results demonstrate for the first time that APN inhibits TNFα-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1- dependent fashion.
Abstract 117: MicroRNA-138 regulates S100A1 in Endothelial Cells
Authors: Sen Anagha , Ren Shumei , Sun Jianxin , Most Patrick , Peppel Karsten
Published: July 2021

Rationale:
The EF-hand Ca2+ sensor S100A1 is essential for proper endothelial nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in endothelial cells (ECs) subjected to hypoxia, rendering them dysfunctional.

Objective:
To determine if the 3’UTR mediates the rapid hypoxia-induced downregulation of S100A1 in ECs.

Methods and Results:
ECs transfected with a S100A1 - 3’ untranslated region (UTR) luciferase reporter construct displayed significantly reduced gene expression when subjected to gas or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (miR-138) could target the 3’UTR of S100A1. Hypoxia greatly increased miR-138 levels in ECs, but not in skeletal muscle C2C12 myotubes. Consistent with this finding, patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased miR-138 levels. Transfection of a miR-138 mimic into ECs reduced S100A1 - 3 ‘UTR reporter gene expression, while transfection of an anti miR-138 (antagomir) prevented the hypoxia-induced downregulation of the reporter gene. The increased levels of miR-138 are dependent on Hif1-α activation as treatment with siRNA against Hif1-α prevented S100A1 reporter gene downregulation after hypoxia. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression. Finally, ECs transfected with miR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus.

Conclusions:
Our study shows that miR-138 is an essential mediator of EC dysfunction via its ability to target the 3’UTR of S100A1 in a hypoxia-induced manner. MiR-138 might thus be an attractive target for the treatment of pathologies that are linked to endothelial dysfunction.
Abstract 128: Patches Of Atrial Appendage Tissue Improve Ventricular Function After Myocardial Infarction
Authors: Luo Jun , Weaver Matthew S , Bernardi Kristina G , Dennis James E , Allen Margaret D
Published: July 2021

Introduction:
Atrial appendage (AA) is an expendable autologous tissue source containing cardiomyocytes, inherent cardiac progenitors, and endothelial cells. We hypothesize that AA tissue can be employed as a unique hormonally-active external patch for infarct repair and that inducing the cytoprotective factor, heme oxygenase-1 (HO-1), would enhance survival in transplanted patch cardiomyocytes.

Methods:
AA patches (5x7mm) were transplanted from donor F344 rats onto hearts in isogenic recipients immediately after acute myocardial infarction (MI). Recipient rats were randomized to receive acute MI only, or external AA patches from donors pretreated with PBS or with the HO-1 inducer, cobalt protoporphyrin (CoPP), 5mg/kg, 24 hrs prior to AA harvest (n=9/group). Echocardiography was performed at baseline and at 4 wks, at which point hearts were excised and fixed for image analysis.

Results:
At 4 wks post-MI, % fractional shortening was 41% higher (P < 0.01) in HO-1-induced and 32% higher (P = 0.03) in PBS-treated AA patch implant groups vs. MI-only controls. In atrial patches, HO-1 induction significantly reduced collagen deposition compared to PBS-pretreated patches (10 ± 3% vs. 16 ± 5% of ventricular area, P < 0.05), increased atrial cardiomyocyte-containing graft area by 35% (Fig. 1), and preserved connexin 43 expression in atrial grafts.

Conclusions:
External implantation of cardiomyocyte patches derived from AA tissue increased ventricular function after acute MI. Preemptive induction of pro-survival factor HO-1 showed additional advantages in preserving cardiomyocyte mass and gap junctions, and reducing fibrosis in transplanted cardiomyocyte patches.
Abstract 143: A Novel TAK1 Signaling Network Regulating Programmed Necrosis and Myocardial Remodeling
Authors: Liu Qinghang , Li Lei , Chen Yi , Doan Jessica , Molkentin Jeffery
Published: July 2021

We recently identified a novel signaling molecule, TAK1 (TGFβ-activated kinase 1, also known as MAP3K7), as a key regulator of the hypertrophic signaling network. Importantly, TAK1 is activated in mouse models of heart failure as well as in diseased human myocardium. Here, we defined a previously unidentified, novel role for TAK1 in promoting cardiac cell survival and homeostasis using cardiac-specific gene-targeted mice. Indeed, cardiac-specific ablation of TAK1 in mice using a Cre-LoxP system showed enhanced pathological cardiac remodeling and massive cell death, and these mice gradually developed heart failure and spontaneous death. Remarkably, ablation of TNF receptor 1 (TNFR1) largely rescued the pathological phenotype of TAK1-deficient mice, preventing early lethality and cardiac fibrosis, suggesting that TNFR1 signaling is critical in mediating adverse remodeling and heart failure associated with TAK1 deficiency. Genetic or pharmacological inactivation of TAK1 in cardiomyocytes markedly induced programmed necrosis and apoptosis in response to TNFα. Conversely, overexpression of the constitutively active TAK1 mutant, or TAK1 plus its activator TAB1, protected cardiomyocytes from TNFα-induced cell death. Mechanistically, inactivation of TAK1 promoted formation of the necroptotic cell death complex consisting of RIP1, RIP3, caspase 8, and FADD. Genetic ablation of RIP1, RIP3, caspase 8, or FADD largely blocked TNFα-induced cell death in TAK1-deficient cells, whereas deletion of Bax/Bak or cyclophilin D showed no effects. Further, IKK/NFκB-mediated cell survival signaling was greatly impaired in TAK1-deficient cardiomyocytes. Taken together, our data indicate that TAK1 functions as a critical “molecular switch” in TNFα-induced programmed necrosis in cardiomyocytes, by interacting with the RIP1/3-caspase 8-FADD cell death pathway as well as the IKK-NFκB cell survival pathway. These findings thus define an important TAK1-mediated cardio-protective signaling network in the heart, which may suggest new therapeutic strategies in the treatment of heart disease.
Abstract 153: Inhibition of the 5-a-reductase Reduces Cardiac Hypertrophy and Improves Cardiac Function
Authors: Zwadlo Carolin , Froese Natali , Bauersachs Johann , Heineke Joerg
Published: July 2021

Objectives:
Left ventricular hypertrophy (LVH) is an independent risk factor for increased cardiovascular mortality and a precursor of heart failure. Gender-specific differences point to a pivotal role of androgens in the development of pathological LVH. Dihydrotestosterone (DHT) is metabolized from testosterone via the enzyme 5-α-reductase. The 5-α-reductase is upregulated in the hypertrophied myocardium, leading to our assumption that DHT rather than testosterone is the crucial component in the development of LVH and might therefore constitute a potential therapeutic target.

Methods:
One week after transverse aortic constriction (TAC) or sham surgery male wild-type mice were treated for 2 weeks via an oralgastric tube with the 5-α-reductase inhibitor finasteride (daily dose 25mg/kg BW) or were left untreated (controls). Male and female transgenic Gαq (TG, a model of dilative cardiomyopathy) or non-transgenic mice were treated with finasteride for 6 weeks.

Results:
Cardiac hypertrophy after TAC was dramatically reduced by finasteride in male mice (heart weight/ body weight ratio, HW/BW in mg/g: control 6.65±0.35 versus finasteride treated 5.23±0.3; p<0.01). The reduced hypertrophy in these mice was accompanied by a reduction in cardiomyocyte diameter, ANP expression and fibrosis, but increased capillary density and Serca2a expression. Accordingly, finasteride also markedly reduced hypertrophy in isolated primary rat cardiomyocytes
in vitro
. Amelioration of hypertrophy by finasteride was associated with blunted activation of the prohypertrophic kinase mTOR
in vitro
and
in vivo
. Left ventricular dilation in male Gαq TG mice was markedly reduced by treatment with finasteride, which also led to an improvement in left ventricular function (determined as fractional area change in % by echocardiography: finasteride 44.72±1.71 vs. control 32.8±3.84, p<0.05) and a similar trend was observed in female mice. Interestingly, finasteride reduced pulmonary congestion in male and female mice alike.

Conclusion:
Finasteride treatment reduces hypertrophy and eccentric cardiac remodelling in mice, indicating a possible involvement of DHT in these processes as well as a potential benefit of 5-α-reductase inhibition in cardiac disease.
Abstract 157: A Highly Reproducible, Clinically Relevant Large Animal Model of Myocardial Ischemia and Heart Failure
Authors: Goodchild Traci , Sweet Michael , Hutchison Ross , Frohwein Stephen , Raines Andrew , Chang Carlos , Fallon Anna , Matheny Robert
Published: July 2021

Background:
Heart failure (HF) is the leading cause of mortality in United States. Animal models used to test novel HF therapeutics are inadequate as pathological features of human HF are not replicated. Available large-animal swine myocardial ischemia (MI) models mimic human HF; however, do not achieve ejection fractions (EF) less than 40%.

Objective:
We sought to develop a reproducible swine MI model with EF below 35%.

Methods:
Yucatan miniature pigs (n=18) were anesthetized, catheterized and collagen suspension (COL) of microfibrillar hemostat (INSTAT MCH, Ethicon) mixed in contrast and saline solution was injected into the left anterior descending coronary artery (LAD) under fluoroscopic guidance. COL injections started distally and filled side branches during catheter retraction. Complete transient LAD occlusion was confirmed at 2hrs by coronary angiography. Cardiac function was evaluated at 3mos using a 1.5T MRI System (Siemens Magnetom Avanto). LV volumes at end systole (LVESD) and diastole (LVEDD), EF, wall thickness (WT) in area at risk (AAR) and normal zones (NZ) were calculated. Uninjected Yucatan pigs (n=3) served as controls.

Results:
Twelve of 18 pigs survived COL infarction. At 3mos, EF was reduced in COL injected pigs compared to control (27.0±2.3 vs. 61.6±1.3%, respectively, P<0.001). LVESV and LVEDV were 4- and 2-fold higher and WT in AAR and NZ were reduced 6- and 2-fold, respectively in COL injected compared to controls.

Conclusions:
A reproducible large animal model of MI was produced by catheter based LAD collagen delivery resulting in EFs below 35% and progression to HF. By closely resembles human HF, it a highly useful for testing potential HF therapies.
Abstract 159: Cardiac Hypertrophy and Sudden Death in a Mouse Model of STIM1 Overexpression
Authors: Goonasekera Sanjeewa A , van Berlo Jop , Burr Adam R , Correll Robert N , York Allen J , Sargent Michelle A , Chen Xiongwen , Houser Steven R , Molkentin Jeffery D
Published: July 2021

Background:
STIM1, an ER/SR resident Ca
2+
sensing protein regulates Ca
2+
entry following internal Ca
2+
store depletion in a broad range of tissues and cell types. However their putative roles in excitable tissue such as cardiac myocytes is uncertain.

Results:
Here we generated a mouse model of STIM1 overexpression in cardiac and skeletal muscle. Western blot analysis suggested approximately 4-6 fold STIM1 overexpression in Tg mouse hearts compared to Ntg littermates. Immunocytochemistry carried out in ventricular myocytes revealed that STIM1 and the cardiac ryanodine receptor (RyR2) co-localize. Functionally, the amplitude of Ca
2+
entry following SR Ca
2+
depletion was 2-fold greater in myocytes isolated from STIM1 Tg mice compared to NTg littermates. Echocardiographic analysis in STIM1 Tg mice showed age dependent remodeling of the myocardium with a significant decrease in fractional shortening at 16 weeks of age (14.4.5±3.8 in STIM1 Tg vs. 36.9±1.5 in Ntg). These changes were accompanied by a significant increase in heart weight to tibia length (13.6 +/- 1.4 vs 6.5 +/- 0.24) and increased lung weight to tibia length ratio (11.6+/- 2.1 vs 8.1 +/- 0.38) in STIM1 Tg mice compared to Ntg littermates. Photometry experiments in isolated ventricular myocytes demonstrated significantly increased Ca
2+
transient amplitude with an unexpected decrease in the SR Ca
2+
load associated with STIM1 overexpression. In addition transgenic mice showed increased calcineurin-nuclear factor of activated T cells (NFAT) activation in vivo, increased CaMKII activity, interstitial fibrosis and exaggerated hypertrophy following two weeks of neuroendocrine agonist or pressure overload stimulation.

Conclusion:
Our observations suggest that STIM1 overexpression by itself can lead to cardiac hypertrophy and contribute to pathological cardiac remodeling and possibly sudden cardiac death. The molecular mechanisms underlying these phenomena are currently under investigation.
Abstract 163: Inhibition of p38a Regulates SERCA2a Function
Authors: Kaikkonen Leena , Magga Johanna , Ronkainen Veli-Pekka , Koivisto Elina , Perjes ábel , Chuprun Kurt , Vinge Leif E , Aro Jani , Ulvila Johanna , Alakoski Tarja , Bibb James , Szokodi Istvan , Koch Walter J , Ruskoaho Heikki , Kerkelä Risto
Published: July 2021

Rationale:
p38 MAPK is a negative regulator of cardiac contractile function, but the underlying molecular mechanism is not well understood.

Objective:
To determine the molecular mechanism mediating the positive inotropic effect of p38 inhibition.

Methods and Results:
The effect of p38 inhibition on calcium regulatory proteins and calcium cycling was investigated in isolated neonatal and adult rodent cardiomyocytes using chemical inhibitors, recombinant adenoviruses and RNAi. Chemical inhibition of p38 enhanced phospholamban (PLB) phosphorylation at serine 16 residue. This was recapitulated with dominant negative p38α adenovirus and by p38α RNAi, but not with dominant negative p38β adenovirus. Chemical inhibition of p38 and dominant negative p38α resulted in significant decrease in Ca
2+
-transient decay time in adult rat cardiomyocytes. Inhibition of p38α, but not p38β, induced an increase in phosphorylation of protein phosphatase inhibitor-1 at the protein kinase A site threonine 35. Analysis of underlying mechanisms revealed that p38 inhibition increased phosphorylation of G protein-coupled receptor kinase 2 (GRK2), but not GRK3 or GRK5. p38 inhibition- induced PLB phosphorylation was abolished in cardiomyocytes overexpressing wild type GRK2. In contrast, increased PLB phosphorylation upon p38 inhibition was not reduced in cardiomyocytes overexpressing GRK2 harboring a non-phosphorylatable mutation in serine 670 residue. Finally, p38 inhibition-induced shortening of Ca
2+
-transient decay time was absent in cardiomyocytes overexpressing wild type GRK2.

Conclusions:
Inhibition of p38α regulates protein phosphatase inhibitor-1 resulting in increased PLB phosphorylation and enhanced SERCA2a function.
Abstract 165: Chronic Ethanol Consumption Impacts Post-Myocardial Infarction Cardiac Function and Modulates Gene Expression in Cardiac Cell Types through Alteration of Histone 3 Lysine 79 Methylation
Authors: Mackie Alexander R , Vaughan Erin E , Verma Suresh K , Krishnamurthy Prasanna , Ramirez Veronica , Ito Aiko , Abramova Tatiana V , Misener Sol , Kishore Raj
Published: July 2021

The beliefs surrounding the cardiac effects of chronic ethanol (EtOH) consumption in humans are known to be dictated by the frequency of ingestion. Studies have established that moderate consumption (i.e. 1-2 drinks/day) imparts a cardiac benefit to patients by reducing the frequency of adverse cardiovascular events (ACE). EtOH consumption beyond moderate levels (i.e. >2 drinks per day) is associated with a significant increase in ACEs. Despite this knowledge, little is known regarding the functional impact of chronic EtOH consumption on post-myocardial infarct repair or the cellular mechanisms involved in this process. Therefore, we investigated the post-AMI functional consequences of chronic ethanol consumption in mice. Mice received chronic ethanol via the Lieber-DeCarli paradigm and each group of mice received either: 0%, 1% (moderate) or 5% (high) ethanol v/v in an isocaloric fashion for 8 weeks. After 8 weeks, mice then underwent a 60 minute ischemic/reperfusion injury and the subsequent assessment of their cardiac function at 1, 2 and 4 weeks after AMI. As early as two weeks post-AMI, mice fed the 1% EtOH displayed significant improvements in ejection fraction, systolic ventricular volumes and infarct size as compared to control mice. Conversely, mice that consumed the 5% EtOH diet displayed diminished ejection fraction and increased systolic chamber volume and infarct size. To investigate the mechanistic basis behind these changes, isolated murine cardiac fibroblasts (CFBs) were exposed to EtOH using a “chronic” 5 day treatment paradigm in which media ± EtOH was replaced daily. CFB lysates were then harvested and several epigenetic histone marks were assessed via western blotting. Specifically, methylation of histone 3 lysine 79 (H3K79) was significantly increased at the moderate dose (0.1% v/v) and severely diminished at the high dose (0.5% v/v). H3K79 is exclusively methylated by the methyl-transferase Dot1, suggesting that chronic ethanol may have biphasic dose effects on the regulation of genes influenced by H3K79 methylation. This data represents the first indication that chronic EtOH may affect cells of the post-ischemic heart by influencing their gene expression profiles through manipulation of their epigenetic fingerprint.
Abstract 166: Endothelial Overexpression of LOX-1 Increases Stroke Size in vivo
Authors: Akhmedov Alexander , Spescha Remo D , Paneni Francesco , Camici Giovani G , Luescher Thomas F
Published: July 2021

Background—
Stroke is one of the most common causes of death and long term disability worldwide primarily affecting the elderly population. Lectin-like oxidized LDL receptor 1 (LOX-1) is the receptor for oxidized LDL identified in endothelial cells. Binding of OxLDL to LOX-1 induces several cellular events in endothelial cells, such as activation of transcription factor NF-kB, upregulation of MCP-1, and reduction in intracellular NO. Accumulating evidence suggests that LOX-1 is involved in endothelial dysfunction, inflammation, atherogenesis, myocardial infarction, and intimal thickening after balloon catheter injury. Interestingly, a recent study demonstrated that acetylsalicylic acid (aspirin), which could prevent ischemic stroke, inhibited Ox-LDL-mediated LOX-1 expression in human coronary endothelial cells. The expression of LOX-1 was increased at a transient ischemic core site in the rat middle cerebral artery occlusion model. These data suggest that LOX-1 expression induces atherosclerosis in the brain and is the precipitating cause of ischemic stroke. Therefore, the goal of the present study was to investigate the role of endothelial LOX-1 in stroke using experimental mouse model.

Methods and Results—
12-week-old male
LOX-1TG
generated recently in our group and wild-type (WT) mice were applied for a transient middle cerebral artery occlusion (MCAO) model to induce ischemia/reperfusion (I/R) brain injury.
LOX-1TG
mice developed 24h post-MCAO significantly larger infarcts in the brain compared to WT (81.51±8.84 vs. 46.41±10.13, n=7,
p
< 0.05) as assessed morphologically using Triphenyltetrazolium chloride (TTC) staining. Moreover,
LOX-1TG
showed higher neurological deficit in RotaRod (35.57±8.92 vs. 66.14±10.63, n=7,
p
< 0.05) and Bederson tests (2.22±0.14 vs. 1.25±0.30, n=9-12,
p
< 0.05) - two experimental physiological tests for neurological function.

Conclusions—
Thus, our data suggest that LOX-1 plays a critical role in the ischemic stroke when expressed at unphysiological levels. Such
LOX-1
-associated phenotype could be due to the endothelial dysfunction. Therefore, LOX-1 may represent novel therapeutic targets for preventing ischemic stroke.