bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 013: Role for β-Arrestins in Stem/Precursor Cell Function and Cardiac Regeneration
Authors: Gumpert Anna M , Chen Mai , Brinks Henriette , Bae Jang-Whan , Peppel Karsten , Gao Erhe , Koch Walter J
Published: July 2021

Chronic heart failure after myocardial injury (MI) is characterized by an extensive loss of myocytes due to considerable cell death. Bone marrow derived stem cells (BMSCs) can transdifferentiate and show potential for regenerating the myocardium after MI. Stem cell mobilization, egress from the bone marrow and recruitment to the site of injury can be regulated by signals through G protein coupled receptors (GPCRs). βArrestins have signalling and scaffolding functions and act as downstream regulators of GPCR desensitization and endocytosis. We explored the potential role for βArrestins in cardiac precursor cell function, concentrating on BMSCs. Using knockout (KO) mice, we investigated the role βArrestin1 (βArr1) and βArrestin2 (βArr2), their modulation of regenerative competence of BMSCs and their contribution to cardiac repair after ischemic injury. in vitro, we observed that BM derived cells devoid of either βArr1 or βArr2 are slower to proliferate, colonize and migrate, compared to wild type (WT) BM cells. We also observed elevated cell death in βArr2 deficient cells following oxidative stress. Additionally, the number of cKit+ stem cells, thought to be potential cardiac precursor cells, was significantly lower in the BM and blood of βArr KO vs WT. Similarly, BM and blood of the chimeras contained fewer and less viable cardiac stem/precursor cells pre and post MI, compared to WT transplanted controls. In our in vivo study, we carried out BM transplants to determine whether the βArrs may be involved in cardiac repair. WT mice were irradiated and received BM transplants from WT, βArr1 KO or βArr2 KO mice. Following BM reconstitution, mice underwent MI and their recovery was monitored. Interestingly, chimeric mice with βArr1 and βArr2 KO BM had significantly inferior outcomes, including significantly decreased post MI survival with βArr2 KO BM and both βArr chimeras had significantly lower cardiac function post MI than mice receiving WT BM. Histology revealed that both chimeras developed larger infarcts and hypertrophy at an faster rate. We conclude that βArrs play a novel role downstream of GPCR desensitization in cardiac progenitor cells in BM and appear to be critically involved in the heart’s response to ischemic injury via cardiac repair and regeneration.
Abstract 017: Hyperglycemia-induced Posttranscriptional Modification Of Proinflammatory Cytokine Mrna Stability Is Mediated Via Hur/PKC-delta Signaling Pathway
Authors: Verma Suresh K , Mackie Alexander R , Vaughan Erin E , Abramova Tatiana V , kishore Raj , Krishnamurthy Prasanna
Published: July 2021

Patients with diabetes are predisposed to increased risk of cardiovascular diseases. Persistent interaction of infiltrating macrophages and resident fibroblasts play a critical role in cardiac fibrosis. However, the signaling mechanism is not clear. We hypothesized that macrophage ELAV1 (mRNA stabilizing protein) modulates profibrotic mediators and extracellular matrix turnover by binding to 3′UTR and regulating the mRNA stability of TGF-beta and MMP-9 in hyperglycemic conditions. Mice receiving intramyocardial injection of HuR-specific shRNA showed significant reduction in infarct size and fibrosis area. Reduced fibrosis was associated with decrease in TGF-beta and MMP-9 expression in the myocardium. Conditioned media (CM) from high glucose (HG) treated macrophages significantly increased profibrogenic response (increased mRNA expression of Col1a1, Col3a1 and fibronectin) in fibroblast cell line as compared to fibroblasts incubated with CM from low glucose (LG)-treated macrophages. Knockdown of ELAV1 in HG-treated macrophages abrogated the profibrotic effects in fibroblasts. Indirect immunofluroscence of bone marrow-derived macrophages (BMM) demonstrated that HG increases nuclear ELAV1 export to the cytoplasm. Pharmacological inhibition of Protein kinase C-delta (PKCd) blocked HG-induced ELAV1 nuclear to cytoplasmic translocation. In vitro, stable knockdown of ELAV1 in mouse macrophage cell line RAW 264.7 reduced mRNA expression of TGF-beta and MMP-9 following LPS challenge, accompanied by a marked reduction in the mRNA stability of these genes. Our study here establishes an ELAV1/TGF-beta/MMP-9/PKC-delta signaling axis in the macrophages controlling the profibrogenic responses in fibroblasts, the major contributor in the pathogenesis of fibrosis. Therefore, targeting this signaling pathway might be of therapy value for cardiac fibrosis in diabetic patients.
Abstract 018: Rat Ckit+ Cardiac Progenitor Cells Release Pro-survival Micrornas In Exosomes Following Hypoxic Stimulation.
Authors: Gray Warren D , Finn Nnenna , Searles Charles , Davis Michael E
Published: July 2021

Introduction:
Cardiovascular disease is the leading cause of morbidity and mortality among developed nations, and acute myocardial infarction is the major subgroup. The need exists for cardiac therapeutic systems that mitigate tissue damage and induce regeneration within and around the infarcted region. Cardiac progenitor cells (CPCs) and other stem cell types have been attractive candidates for therapies. However, results suggest that regenerative or protective effects may occur through paracrine mechanisms. One such way may be through cell-cell transfer of microRNA (miR) via cell-secreted exosomes, which contain protein, mRNA, and miR. Cell-based therapies face substantial limitations, and therapies avoid these limitations and mimic paracrine efforts—such as delivery of harvested exosomes—have yet to be developed due in part to lack of characterization.

Hypothesis:
We hypothesized that in response to hypoxic conditions, CPCs secrete a pro-regenerative miRnome within exosomes.

Methods:
We used an Affymetrix MicroRNA GeneChip microarray to identify the miR populations that were present in CPC-conditioned media after hypoxic and normoxic treatments. Exosomes were isolated via ultracentrifugation (100,000xG) and validated by anti-CD9 immunohistochemistry and dynamic light scattering. We used RT-qPCR to quantify levels of miR upregulation in secreted exosomes.

Results:
We found seven miRs upregulated (1.3- to 7.9-fold) due to hypoxia stimulation. Within that miRnome, two miRs ([[Unable to Display Character: ‐]]20a, [[Unable to Display Character: ‐]]210) are known to exert cardioprotection in infarct models, providing evidence of a potential beneficial paracrine effect from CPCs. Four of the remaining miRs (-15b, -17, -103, -199a) have been shown to regulate angiogenesis, proliferation, apoptosis, and fibrosis in various cell and tissue types. One miR ([[Unable to Display Character: ‐]]292) has been largely unexplored, but predicted mRNA targets include CTGF, which is involved in cell adhesion and fibrosis.

Conclusions:
The strong evidence of anti-apoptotic and anti-fibrotic potential of this miRnome indicates that the cocktail may serve as a powerful modulator of cardiac remodeling. Future work will investigate applying miR-containing exosomes from hypoxia-stimulated CPCs as a therapy to rescue the infarcted heart.
Abstract 028: CCN1 Enables Fas Ligand-Induced Apoptosis by Elevating Fas Expression in Primary Cardiomyocytes or by Increasing Cytoplasmic Smac in Cardiomyoblast H9c2 Cells
Authors: Su Bor-Chyuan , Mo Fan-E
Published: July 2021

Fas/Fas ligand (FasL) is implicated in cardiac ischemia/reperfusion injury. However, cardiomyocytes in culture are resistant to FasL-induced apoptosis, suggesting that additional factor(s) are required for FasL-induced apoptosis. Matricellular protein CCN1 has been demonstrated to promote cytotoxicity of FasL in human skin fibroblasts. CCN1 is induced in a variety of cardiac pathologies. We assessed the hypothesis that CCN1 may be involved in the regulation of FasL-induced apoptosis in cardiomyocytes. We found that either FasL or CCN1 did not induce cell death in neonatal rat ventricular cardiomyocytes (NRVM). Interestingly, the combination of FasL+CCN1 generated 2-fold induction of apoptosis (vs. control p<0.001). An integrin-α
6
β
1
-binding defective mutant CCN1, CCN1-DM failed to exert synergy with FasL to induce apoptosis, indicating a critical role of α
6
β
1
. The engagement between CCN1 and α
6
β
1
instigated the elevation of cellular reactive oxygen species (ROS), the activation of mitogen activated protein kinase p38, and followed by the induction of cell surface display of Fas, thereby sensitizing NRVM to FasL-induced apoptosis. Pretreatment of the p38 inhibitor SB202190 abolished the CCN1-induced cell-surface Fas expression and the apoptosis induced by FasL+CCN1. In addition, we tested the interaction between CCN1 and FasL on the cardiomyoblast H9c2 cells. We found that FasL or CCN1 alone did not cause apoptosis in H9c2, and required the combination of FasL+CCN1 to induced apoptosis (vs. control p<0.001) in H9c2 cells, reminiscent of the observation in NRVM. Mechanistically, CCN1 acted through binding to integrin α
6
β
1
, ROS generation, and p38 activation, however, did not increase the expression of cell surface Fas for its synergy with FasL in H9c2 cells. Instead, CCN1 induced Bax translocation to mitochondria, which in turn led to the release of Smac from mitochondria to cytosol. The cytosolic Smac functions to neutralize XIAP. Smac is critical for CCN1 action, because the knockdown of Smac blunted the apoptotic activities of CCN1. In conclusion, CCN1 may play a detrimental role in a stressed heart to both the differentiated cardiomyocytes and the proliferative cardioblasts through distinct signaling mechanisms.
Abstract 034: The Effect Of Simvastatin On P-wave Dispersion And Echocardiographic Parameters Among Hypertensive Patients
Authors: Pomer Pamela M , Elcano Janet W
Published: July 2021

Introduction:
Rising evidence suggests that statins have a positive effect on the prevention of occurrence of atrial fibrillation (AF) in hypertensive patients. P-wave dispersion (PWD) has been known to be an electrocardiographic predictor for the development of AF. We therefore aimed to investigate on the possible effects of statins on p-wave dispersion in hypertensive patients.

Method:
A total of 27 patients (22 females; mean age, 45±11 years) with hypertension and without history of AF were included in this prospective study. P wave durations and PWD, left ventricular mass (LVMI), left atrial volume index (LAVI), and ratio of early diastolic transmitral flow velocity (E) to early diastolic mitral annular velocity (e) (E/e’) were measured and compared before and after statin therapy (simvastatin 40mg/day for 3 months).

Results:
After 3 months of statin therapy, there was no significant change in PWD (pre-statin 39.7 ±10.7, post-statin 33 ± 8.8, p-value 0.30). Baseline and follow up blood pressure, heart rate, LVMI, LAVI and E/e’ ratio were not significantly different before and after simvastatin therapy. No patient developed AF during follow up.

Conclusion:
Simvastatin (40mg/day) therapy for 3 months did not show significant change on PWD, LVMI, LAVI and E/e’ ratio in hypertensive patients.
Abstract 045: Highly-reactive Lipid Aldehydes Are Downstream Mediators Of Oxidant-induced Spontaneous Ca Waves
Authors: Hwang Hyun Seok , Davies Sean , Knollmann Bjorn C
Published: July 2021

Background:
Reactive oxygen species (ROS) can activate ryanodine receptor (RyR2) Ca release channel and generate arrhythmogenic Ca waves (SCWs) in myocytes. ROS also cause lipid peroxidation in cell membranes that produce highly-reactive g-ketoaldehydes (IsoK), which can react immediately with proteins (RyR2) and accumulate in ischemic human heart. We previously found that specific IsoK scavengers reduce infarct size and prevent ventricular arrhythmia in an MI mouse model, suggesting a biological role of IsoK in oxidant heart injury. We hypothesize that IsoK mediate in part the ROS-induced SCW in myocytes.

Methods:
We compared the effect of H
2
O
2
, synthetic IsoK, the specific IsoK scavenger (SA, salicylamine), its inactive homolog (4-SA, 4-OH-salicylamine), and the ROS scavenger (MPG, 2-mercaptopropionyl glycine) in field-stimulated mouse ventricular myocytes loaded with Fura-2AM.

Results:
Thirty min exposure to H
2
O
2
(10μM) or IsoK (1μM) both generated SCWs (H
2
O
2
37%, IsoK 37%, p<0.05 vs.vehicle, VEH). MPG, (100μM, 60 min) prevented H
2
O
2
-induced SCWs, but had no effect on IsoK-induced SCWs (
Fig.A
). Pre-treatment with SA (8 μM, 60 min) significantly reduced H
2
O
2
-induced SCWs, whereas 4-SA had no effect on H
2
O
2
-induced SCWs (%SCWs, H
2
O
2
; 53%, SA+H
2
O
2
; 27%*, 4-SA+H
2
O
2
; 56%. *p<0.05 vs. VEH,
Fig. B
). In the absence of H
2
O
2
, MPG, SA, or 4-SA had no significant effect on Ca handling in myocytes.

Conclusion:
IsoK directly activate SCWs independent of ROS production in myocytes. Specific IsoK scavengers inhibit 50% of ROS-induced SCWs. Hence, our data suggested that IsoK are biologically-relevant downstream mediators of oxidant-induced RyR2 dysfunction.
Abstract 058: Novel Agonist of Adenosine Receptor Ameliorates Ventricular and Vascular Dysfunction in Monocrotaline-induced Pulmonary Arterial Hypertension in Rats
Authors: Zapata-Sudo Gisele , Alencar Allan K , Pereira Sharlene L , Ferraz Emanelle , Nascimento José H , Tesch Roberta , Maia Rodolfo , Sant’Anna Carlos M , Fraga Carlos A , Barreiro Eliezer J , Sudo Roberto T
Published: July 2021

Aims:
Pulmonary arterial hypertension (PAH) consists of increased pulmonary vascular resistance and remodeling and right ventricular hypertrophy. This work investigated the effects of a new
N
-acylhydrazone derivative, (E)-N’-(3,4-dimethoxybenzylidene)-4-methoxybenzohydrazide (LASSBio-1386), in rats with monocrotaline (MCT)-induced PAH.

Methods and Results:
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Male Wistar rats received a single i.p. injection of MCT (60 mg/kg) for PAH induction. Experimental groups were: control, MCT + vehicle (DMSO), MCT + LASSBio-1386 (50 mg/kg p.o.). The animals were treated with vehicle or LASSBio-1386 for 14 days after the onset of disease (n = 6). Right ventricular systolic pressure (RVSP) and relation between RV weight to body weight (RV/BW) were analyzed. Transthoracic echocardiography was performed to determine pulmonary acceleration time (PAT), pulmonary artery diameter and RV wall thickness. Pulmonary vascular morphometry was analyzed using images of terminal arterioles and wall thickness was measured. The parameters evaluated are shown in table 1.

In addition, LASSBio-1386-induced vasorelaxation was mediated partially by the activation of A2A adenosine receptors, with an IC50 of 6.2 ± 1.2 µM. Docking analysis in the A
2A
crystal structure was performed using the program GOLD 5.1 and showed the interaction of the compound with A
2A
receptor.

Conclusions:
LASSBio-1386 effectively reversed right ventricular hypertrophy and pulmonary vascular remodeling in rats with MCT-induced PAH through activation of adenosine receptor.
Abstract 059: N-acylhydrazone Derivative Improves Cardiac Hypertrophy and Dysfunction in Spontaneously Hypertensive Rats Submitted to Myocardial Infarction
Authors: Zapata-Sudo Gisele , Silva Jaqueline S , Pereira Sharlene L , Maia Rodolfo C , Fraga Carlos A , Barreiro Eliezer J , Sudo Roberto T
Published: July 2021

Aims:
Ventricular dysfunction is observed in arterial hypertension and myocardial infarction (MI). The
N
-acylhydrazone derivative, 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294), which has been described as a potent cardiac inotropic agent with vasodilator properties, was orally administrated in spontaneously hypertensive rats (SHR) submitted to experimental MI.

Methods and Results:
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Male SHR (12 - 14 week-old) were randomly divided in sham-operated (SHAM) and infarcted groups (MI) and were treated or not with LASSBio-294 (20 mg/kg p.o.) during 8 weeks. Under sevoflurane anesthesia, the animals were submitted to a ligature of the anterior descending coronary artery to produce the MI. After 8 weeks, rats were submitted to treadmill test and also, the following hemodynamic parameters were analyzed: left ventricular end diastolic pressure (LVEDP), left ventricular end-systolic pressure (LVESP) and LV contractility (dp/dt
max
). Hypertrophy was measured using the relation between heart weight to body weight (heart/BW). The volume fraction of collagen (%) was determined by measuring the area of stained tissue within a given field by picrosirius red staining. The results are presented in the following table.

Conclusions:
Treatment of infarcted SHR with LASSBio-294 reduced intolerance to exercise, ventricular dysfunction, cardiac hypertrophy and fibrosis.
Abstract 060: CCN1/a
6
β
1
Mediates Myocardial Injuries Induced by Work Overload or by Doxorubicin in Mice
Authors: Hsu Pei-Ling I , Mo Fan-E
Published: July 2021

Introduction:
Matricellular protein CCN1 is expressed in myocardial infarction, pressure overload, and ischemia in mice, and in patients with a failing heart. Despite its well-documented angiogenic activities, CCN1 promotes fibroblast apoptosis in some contexts. The role of CCN1 in an injured heart was not clear. We assessed the hypothesis that CCN1 plays a detrimental role and mediates cardiac injury through its proapoptotic activities.

Methods and Results:
To test the role of CCN1 in cardiac injury, we employed two different myocardial injury models in mice, including a work-overload-induced injury created by isoproterenol treatment (ISO; 100 mg/kg/day; s.c. for 5 days; n= 6 for each group) and an injury induced by the cardiotoxicity of doxorubicin (DOX, single dose of 15 mg/kg; i.p. sacrificed after 14 days).
Ccn1
expression was induced in the damaged myocardium in both injury models. A line of knock-in mice carrying an apoptosis-defective
Ccn1
mutant allele,
Ccn1-dm
, which has disrupted integrin α
6
β
1
binding sites, were tested in the ISO- or DOX -induced cardiac injury. Myocardial damage was seen in tissues from wile-type (WT) hearts after receiving ISO.

Ccn1
dm/dm

(DM) mice possessed remarkable resistance against ISO or DOX treatments and exhibited no tissue damage or fibrosis compared to WT mice after H&E or Masson’s trichrome stainings. DM mice were resistant to both ISO- and DOX-induced cardiac cell apoptosis, indicating that CCN1 is critically mediating cardiomyocyte apoptotic death in cardiac injury. Moreover, we found that death factor Fas ligand (FasL) and its receptor Fas were upregulated in WT mice receiving ISO or DOX treatments by immunohistochemical staining, compared with the PBS-control. 8-OHdG-positive, a marker for oxidative stress, cardiomyocytes were increased by ISO or DOX treatments as well. In contrast, the expression of Fas/FasL, and the 8-OHdG-positive cardiomyocytes in the myocardium of DM mice were not changed by ISO or DOX.

Conclusions:
We identify CCN1 as a novel pathophysiological regulator of cardiomyocyte apoptosis in cardiac injury. Blocking apoptotic function of CCN1 effectively prevents myocardial injury in mice. CCN1 and its receptor α
6
β
1
represent promising future therapeutic targets in cardiac injury.
Abstract 061: Interstitial Matrix Flow-Regulation:Myocardial Fibroblasts Respond to Colloidosmotic Pressure
Authors: McGee Maria P , Morykwas Michael , Wang Rui , Jordan James , Argenta Louis
Published: July 2021

Ischemia-reperfusion injury induces large differences in hydration potential between ischemic and nonischemic areas of the myocardium. They influence the rate and volume of fluid transfer in myocardial explants; temperature-dependent hydration-potential differences of approximately
100 mmHg
in at-risk areas, as opposed to contiguous areas that are not at risk, suggest prompt interstitial fluid-transfer control mechanisms
(Circ. Res
.
2012;11:A235)
.

Aim:
We adapted osmotic-stress techniques to determine whether myocardial fibroblasts, which are known to respond to mechanical and flow signals, also respond to hydration-potential changes.

Methods:
Fibroblasts were isolated from midwall regions of the left ventricles of healthy pigs using standard procedures. They were incorporated into 3-dimensional collagen gels of

500 mm
3

volume at

150 cells/mm
3

and equilibrated overnight in cultures using the nontoxic, inert polymer polyethylene glycol 8000 (molecular radius ~26.5 Å; concentration range 0-10% w/w) to adjust colloidosmotic pressure from approximately
5 to 205 mmHg
. After the gels were detached from the dish, fluid flux was derived from any time-dependent changes in their dimensions.

Results:
The volume of gels without fibroblasts did not change significantly at any coloidosmotic pressure. In those with fibroblasts, the volume decreased, more slowly in those subjected to higher-than-plasma pressure levels. Progression curves conformed well to a two-exponential term model

(R
2
>0.9)

suggesting that two parallel processes contribute to matrix efflux, one relatively fast, with decay constant
0.274 ± 0.014 (n = 3)
, and another ~50-fold slower. Initial rates were calculated from the fitted curves, and linear regression analysis used to examine their dependence on colloidosmotic pressure. Initial efflux rates decreased with pressure, mean slope, -

0.29 ± 0.08 µl/h/mmHg (R
2
= 0.7; P-value = 0.006).

Conclusion:
In vitro
, fibroblasts in collagen matrices regulate fluid efflux in response to colloidosmotic stresses within the range of hydration-potential differences measured in myocardial explants after ischemia-reperfusion injury.
Abstract 064: The Lipoxigenase Inhibitor Nordihydroguaiaretic Acid Prevents the Development of Hypoxia-Induced Pulmonary Hypertension in Mice
Authors: Iorga Andrea , Wong Gabriel , Mai Denise , Li Jingyuan , Sharma Salil , Eghbali Mansoureh
Published: July 2021

Pulmonary hypertension (PH) is a chronic lung disease characterized by progressively elevated pulmonary arterial pressures and severe pulmonary vascular remodeling resulting from interactions between oxidized lipoprotein deposition and increased endothelial proliferation. Previously we have shown increased plasma levels of biological oxidation products such as hydroxyoctadecadienoic acids (HODEs) and hydroxyeicosatetraenoic acids (HETEs) in the rat monocrotaline model of PH. Here we investigated the role of HETEs and HODEs in the development of PH and whether their inhibition with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) attenuates the progression of PH. Mice were placed in a hypoxic chamber with O2 concentrations of ≤10% for 21 days and either left untreated to develop PH (n=7) or treated with NDGA daily (10mg/kg/day, i.p., n=4) from day 1. Direct RV catheterization was terminally performed to record RV pressure (RVP). Pulmonary arteriolar thickening and oxidized lipid deposition were assessed by staining lung sections with Masson’s Trichrome or with α-smooth muscle actin and E-06 (marker for oxidized low-density lipoproteins). In vitro, human pulmonary artery smooth muscle cell (hPASMC) proliferation was assessed by MTT assays in the absence or presence of 12-HETE (100ng/ml), 9-HODE (1µg/ml) and 13-HODE (1µg/ml) alone or together with NDGA (10, 25 and 50µM). In-vitro, HETE/HODE treatment increased hPASMC proliferation ~ 2-fold when compared to untreated cells and NDGA significantly inhibited the proliferative effects of all three oxidized lipids. In-vivo, NDGA treatment prevented the development of PH. RVP was lower in the NDGA-treated group vs. the PH group (24.01±1.39mmHg vs. 36.91±5.74mmHg, p<0.05) and was comparable to control normoxic mice (20.93±2.52mmHg). RV hypertrophy index was significantly elevated in the PH mice versus control mice (0.38±0.03 vs. 0.28±0.02 (p<0.001), while NDGA treatment completely prevented the development of RV hypertrophy (0.28±0.04). Lung sections demonstrated arteriolar thickening and E-06 positive deposits in the PH group, which was prevented by NDGA therapy. We conclude that oxidized fatty acid deposition and accumulation might play a role in the development of PH.
Abstract 065: Cardiac Fibrosis and Heart Failure Caused by DsRed Tetramers Involves the Induction of Tissue Inhibitor of Metalloproteinase-1
Authors: Chen Tsung-Hsien , Liu Shan-Wen , Chen Mei-Ru , Lin Kurt M
Published: July 2021

Whereas aggregation of intracellular proteins was linked to the initiation of cardiac myopathy, the sequence of participating events, including myocyte apoptosis, autophagy, necrosis and fibrosis as the underlying mechanisms leading to heart failure, was not clear. Green fluorescent protein (GFP) and its derivatives induced cardiac dysfunction in mice when expressed in high quantity; however, the mechanism underlying the aggregation of fluorescent protein leading to heart failure remains unexplored.We created a transgenic mouse with switchable expression of the GFP monomer or the expression of DsRed, a red fluorescent protein (RFP) tetramer that tends to aggregate into a large protein complex. GFP mice were free of cardiac symptoms; in contrast, RFP mice with homozygous DsRed alleles developed myocyte necrosis, carditis, ventricular hypertrophy and fibrosis, left atrium thrombosis, dilated heart failure and death at the age of approximately five months. The hemizygote mice displayed similar symptoms at a later age. The expression of the microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) and transglutaminase 2, and the expression of many myopathy- and fibrosis-related genes were significantly induced in the hearts of two-month-old RFP mice. Together with the findings of increased autophagosomes, lysosomes and dysfunctional mitochondria, these results suggest a marked induction of myocyte autophagy and fibrosis as the main underlying mechanism of heart failure in RFP mice. Interestingly, apoptosis was not elevated in RFP hearts. One of the most up-regulated genes in the early stage RFP heart was the tissue inhibitor of matrix metalloproteinases type 1 (TIMP-1), corroborating the role of TIMP-1 in cardiac remodeling and anti-apoptotic activity. The heart-origin of the morbidity in RFP mice was confirmed by expressing DsRed tetramers specifically in cardiac tissues, and the same phenotypes as in RFP mice were observed.

In summary, in cardiac myocytes under the stress of protein aggregation, strong induction of TIMP-1 and down-regulation of MMP activity may play a significant role in enhancing the synthesis of extracellular matrix, resulting in fibrosis and heart failure.
Abstract 066: MEF2-induced Loss of Sarcomeres is Mediated by an Alternative Splice Variant of DMPK
Authors: Elzenaar Ies , van der Made Inge , Wijnen Wino J , Ehler Elisabeth , De Windt Leon J , Pinto Yigal M , van Oort Ralph J
Published: July 2021

The pathology of heart failure is characterized by poorly contracting and dilated ventricles. Although this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres, the mechanism underlying these changes in cardiomyocyte structure remains to be elucidated. We have previously identified the transcription factor myocyte enhancer factor-2 (MEF2) as important trigger for adverse cardiomyocyte remodeling. Here, we use microarray analysis and gain- and loss- of function approaches to identify MEF2 target genes involved in structural remodeling of the cardiomyocyte.

Isolated neonatal rat cardiomyocytes infected with adenoviruses expressing MEF2 underwent cellular elongation associated with loss of sarcomeric structure. Microarray analysis revealed myotonic dystrophy protein kinase (DMPK) as MEF2 target gene, which we verified by chromatin immunoprecipitation experiments. siRNA mediated knockdown of DMPK prevented MEF2-induced cardiomyocyte elongation and loss of sarcomeres. Interestingly, RT-PCR analysis of known DMPK splice variants demonstrated a relative increase of the DMPK E isoform in failing mouse hearts. To test the role of this specific splice isoform, we generated adenoviruses expressing DMPK E or a kinase dead mutant DMPK E. Overexpression of wildtype DMPK E, but not of the kinase dead mutant, in cardiomyocytes resulted in severe loss of sarcomeric structure. Moreover, quantitative PCR analysis showed a decrease in mRNA levels for several sarcomeric genes after overexpression of DMPK E. These genes are known targets of the transcription factor serum response factor (SRF) and DMPK is known to phosphorylate SRF. Therefore, we tested the effect of DMPK on SRF activity in luciferase experiments, which demonstrated that DMPK E is an inhibitor of SRF transcriptional activity.

Our data indicate that MEF2 induces loss of sarcomeres, which is mediated by at least one specific splice variant of DMPK. Moreover, increased expression of this DMPK splice variant results in a decrease in sarcomeric gene expression, which possibly involves inhibition of SRF transcriptional activity. Together, these results assign a novel function to MEF2 and DMPK in adverse cardiomyocyte remodeling during heart failure development.
Abstract 070: Interaction between Mena, Connexin43, and Rac1 at the Intercalated Disc
Authors: Ram Rashmi , Wescott Andrew P , Thomas Tamlyn , Dirksen Robert T , Blaxall Burns C
Published: July 2021

Background:
We previously reported that Mena, a member of Ena/VASP family of actin regulatory protein, is associated with heart failure (HF). Mena is localized to the intercalated disc (ICD) and interacts with ICD proteins implicated in HF. The ICD mediates force transmission and electrical coupling between cardiomyocytes. Slowed conduction in HF is associated with Cx43 remodeling, the predominant connexin isoform in the heart. Mena-/- mice develop cardiac dysfunction, conduction abnormalities, ICD disorganization, and Cx43 lateralization. We hypothesized that: 1) Mena’s interaction with the cytoskeleton is critical for ICD stabilization and maintenance of electrical function; 2) Mena may modulate signal(s) from the cardiac sarcolemma to the ICD for control of expression and localization of Cx43 through the regulation of the small GTPase Rac1.

Methods and Results:
Interaction of Mena with Cx43 and Rac1 was evaluated in neonatal rat ventricular myocytes (NRVM), HeLa cells, and in transgenic mice overexpressing constitutively active V12Rac1 (RacET). Pull down assay using GST-PAK1 demonstrated a direct association of Mena with active Rac1-GTP in vitro. In NRVMs, siRNA knockdown of Mena significantly increased activity of Rac1-GTPase by 2-fold (p=0.05, n=5). Western blot analysis revealed increased total Cx43 expression in Mena siRNA (1.11±0.2) compared to scramble (0.33±0.1, p<0.01, n=5). Enhanced Cx43 expression was associated with increased rate of recovery after photobleaching between cardiomyocytes by gap-FRAP analysis (Tau 153.0±4.8 vs. 73.0±1.8 sec, p<0.0001). Ongoing experiments with optical mapping of NRVM monolayers will determine whether conduction velocity may be enhanced after Mena knockdown. Confocal imaging revealed altered Cx43 trafficking and localization with Mena knockdown. Additionally in ventricles of RacET hearts, Mena expression was increased by 59% (p=0.01, n=4) compared to control, along with lateral redistribution of Cx43.

Conclusions:
Our results suggest that Mena is a critical regulator of the ICD at the intersection of Cx43 and Rac1. Increased Rac1 activation is associated with lateralized Cx43, and enhanced cellular coupling. Mena may modulate Cx43 localization through the regulation of Rac1 activity.
Abstract 075: Novel Role for GRK2 in Cardiac Hypertrophy and Heart Failure
Authors: Schumacher-Bass Sarah M , Gao Erhe , Chuprun Kurt , Gold Jessica I , Koch Walter J
Published: July 2021

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated, increasing phosphorylation, desensitization and down-regulation of β-adrenergic receptors (βARs) and other cardiac GPCRs. Increased GRK2 has been shown to participate in adverse remodeling and contractile dysfunction during HF, while GRK2 inhibition via a carboxy-terminal peptide, βARKct, enhances heart function and can prevent HF development. Mounting evidence supports the idea of a dynamic GRK2 “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several novel GRK2 interacting partners are important for adaptive and maladaptive myocyte growth including Gq, the signaling trigger for maladaptive cardiac hypertrophy, leading to HF. Importantly, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain (termed βARK-RGS). This domain directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. Therefore, this domain of GRK2 may alter hypertrophic responses in the heart and represent a novel role for GRK2 and also a potential therapeutic target to limit maladaptive cardiac hypertrophy. We have begun to address this by generation of novel transgenic mice with cardiac-specific expression of the RGS domain of GRK2. Data from mice with cardiac expression of βARK-RGS demonstrate anti-hypertrophic effects in a trans-aortic constriction (TAC) model of pressure overload hypertrophy. Echocardiographic analysis post-TAC revealed reduced left ventricular posterior wall thickness in βARK-RGS compared to non-transgenic littermate controls (1.34 vs 1.57 mm LVPWd at 4 weeks). RT-PCR analysis found decreased hypertrophic factor transcripts, such as ANF for which the nearly 18-fold increase post TAC was completely inhibited in βARK-RGS mice. Other hypertrophic phenotypic markers have been studied and mechanistic characterization is underway. These data support our hypothesis that the RGS domain of GRK2 may serve as a non-canonical inhibitor of Gq-mediated hypertrophic signaling in the heart and highlight how this research may pave the way for novel GRK2-based therapeutic approaches to prevent hypertrophy and HF.
Abstract 078: Differential Phosphorylation of Inositol 1,4,5-triphophate Receptor by CaMKIId During Cardiac Remodeling
Authors: Sankar Natesan
Published: July 2021

Natesan Sankar
1
, Sukriti Dewan
1
, Joshua Maxwell
1
, Donald Bers
2
, Joan Heller Brown
3
, Jeffery Molkentin
4
, Pieter deTombe
1
, Gregory Mignery
1
.
1
Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL.
2
Department of Physiology, Department of Pharmacology University of California at Davis, Davis, CA.
3
Department of Pharmacology, University of California, San Diego, La Jolla, CA.
4
Howard Hughes Medical Institute, Molecular Cardiovascular Biology, Cincinnati Children’s Hospital, Cincinnati, OH. In cardiac myocytes the type-2 isoform of the inositol 1,4,5-triphosphate receptor (InsP
3
R2) Ca
2+
release channel is expressed predominantly in the nuclear envelope. InsP
3
R2 releases intracellular Ca
2+
bidirectionally towards the cytoplasm and nucleoplasm in response to an array of pro-hypertrophic signaling. Thus, InsP
3
R2 mediated Ca
2+
release may contribute to both Excitation-Contraction Coupling (ECC) and Excitation-Transcription Coupling (ETC) during normal and pathophysiologic conditions such as cardiac remodeling. However, the regulation of InsP
3
R2 mediated Ca
2+
release and its role in ECC and ETC during cardiac remodeling is not fully understood. We have shown that CaMKIIδ and InsP
3
R2 forms a signaling complex in the heart and CaMKII mediated phosphorylation of InsP
3
R2 at S150 modulates its intrinsic Ca
2+
channel activity. Here we show that InsP
3
R2 is differentially phosphorylated by CaMKIIδ
B
and CaMKIIδ
C
, the predominant nucleoplasmic and cytoplasmic isoforms respectively, in cardiac myocytes. Using adult rat cardiac myocytes we show that the differential phosphorylation by CaMKII of InsP
3
R2 at S150 leads to elevated nuclear Ca
2+
signaling and diminished release towards the cytoplasm. Additionally we show that the InsP
3
R2 was phosphorylated in the hearts of Angiotensin II infused and pressure overload induced cardiac remodeling animal models. Finally, we show that there was an increase in InsP
3
R2 phosphorylation in human heart-failure samples compared to non-failing hearts. Collectively our studies demonstrate that, CaMKIIδ mediated regulation of InsP
3
R2 Ca
2+
channel activity contributes to ECC and ETC during all the phases of cardiac remodeling processes.
Abstract 082: Deletion of Mitochondrial Aldehyde Dehydrogenase 2 Restrains Cardiac Fatty Acid Oxidation during Pressure-Overload Induced Heart Failure
Authors: Fan Fan , Sun Aijun , Xia Guang , Zou Yunzeng , Hu Kai , Ge Junbo
Published: July 2021

Rationale:
Mitochondrial Aldehyde dehydrogenase 2 (ALDH2) is regarded as an inner protector of cardiac diseases, which downregulation has been reported to induce cardiac metabolic remodeling and augmented glucose uptake. However, it has not been investigated whether myocardial fatty acid oxidation (FAO) could be regulated by ALDH2.

Objective:
We hypothesized that global deletion of ALDH2 would deteriorate heart function and energy generation, inhibit cardiac FAO and promote the development of pressure-overload induced heart failure.

Methods and Results:
ALDH2 knockout (ALDH2-/-) did not led to considerable birth defects and cardiac dysfunction in base line. Pressure overload were induced by transverse aortic constriction (TAC) for 4weeks. In echocardiographic and hemodynamic test, left ventricular systolic functions were decreased in ALDH2-/- TAC mice compared with wild type (WT) TAC mice. Myocardial morphology and subcellular structure examinations shown ALDH2-/- TAC mice exhibited cardiac hypertrophy with a significant mitochondrial destroy relative to WT-TAC. Meanwhile, a decreased FAO and ATP production were detected (54±3.6% and 77±1.5%, p<0.05,respectively) in ALDH2-/- sham myocardium, which were aggravated by pressure overload. Therefore, ALDH2 deletion accelerated cardiac energy remolding. Furthermore, ALDH2-/- repressed AMP-activated protein kinase (AMPK) phosphorylation(49±4.6%, p<0.05) and subsequently inhibited the activation of peroxisome proliferator-activated receptor a (PPARa) (31±2.9%, p<0.05) - carnitine palmitoyl transferase 1 (CPT1) (81.5±5.2%, p<0.05) - fatty acid transportation pathway, which induced inefficient of FAO.

Conclusions:
These data of present study suggest that ALDH2 deficiency promotes heart failure and cardiac metabolic remodeling by inhibiting myocardial FAO through AMPK-PPARa-CPT1 pathway.
Abstract 085: Anti-Inflammatory Treatment Reverses The Age-Related Increase In Catecholamine Biosynthetic Enzyme Tyrosine Hydroxylase And NYP In The Adrenal Medulla
Authors: Tumer Nihal , Foster Thomas C , Ormerod Brandi , Scarpace Philip J
Published: July 2021

Aging is associated with both elevated blood pressure and decreased baroreceptor sensitivity. These have been attributed to increased sympathetic nerve activity with age. One marker of sympathoactivation is protein levels of tyrosine hydoxylase (TH) in the adrenal medulla (AM), and TH levels markedly increase with age. There is also an increase in inflammation with age. To test the hypothesis that increased inflammation with age contributes to the elevated TH levels in the AM, we examined treatment with the anti-inflammatory agents, indomethacin (10 mg/kg, BID) or rosiglitazone (1 mg/kg, BID) on TH synthesis in F344 rats of three ages, 5, 12, 18 months. In addition, NPY, a peptide that is synthesized and co-released with catecholamines in the AM, as well as angiotensin II receptor subtypes, AT1 and AT2 protein levels were assessed in the AM following the anti-inflammatory treatments. Indomethacin and rosiglitazone were administered orally as part of a frozen strawberry treat for 16 days. Although there were no changes in TH or NPY levels between the 5 mo and 12 mo old control animals, TH and NPY were elevated by 27% and 45% (P<0.05), respectively in the 18 compared with 5 mo rats. These age-related increases were completely reversed by treatment with indomethacin or rosiglitazone (P<0.05). There was no effect of either treatment on AT1 or AT2 receptor protein, although AT1 receptor protein levels diminished slightly with age. These data indicate that anti-inflammatory treatment reverses the elevated TH and NPY in AM with age. Increased inflammation with age indirectly through elevated catecholamine synthesis may be contributing to the increased prevalence of hypertension with age.
Abstract 087: Reversal of Aging-Induced Contrast Changes of Cardiac SERCA 2a and Inducible Nitric Oxide Synthase in Mice Deficient in Beta3-Adrenoreceptor: Insights into the Molecular Mechanism of Cardiac Aging
Authors: Cheng Che Ping , Cheng Heng-Jie , Zhou Peng , Li Tian-Kai , Kitzman Dalane W , Kritchevsky Stephen B , Little William C
Published: July 2021

Background:
We have shown previously that aging-induced cardiac dysfunction and β-adrenergic desensitization were prevented in β
3
-adrenergic receptor (AR) knockout (β
3
KO) aged mice. However, the molecular mechanism is unclear. We hypothesize that reversal of aging-induced alterations of cardiomyocyte SR Ca
2+
-ATPase (SERCA 2a) and inducible nitric oxide (NO) synthase by β
3
-AR deficiency may play a key role for the protective effect.

Methods:
We compared SERCA 2a, iNOS, β
1
- and β
3
-AR protein expression, myocyte contractile, and [Ca
2+
]
i
transient ([Ca
2+
]
iT
) responses to isoproterenol (ISO, 10
-8
M) in cardiomyocytes obtained from 2 young (Y) (~6 mo) and 2 aged (A) (~26-30 mo) groups (5/group) of wild-type (WT) and β
3
KO mice, respectively.

Results:
Compared with YWT, AWT myocytes had significantly decreased protein levels of SERCA 2a (AWT: 0.22 vs YWT: 0.61) and β
1
-AR (0.34 vs 0.56), but increased iNOS (0.49 vs 0.24) and β
3
-AR (0.29 vs 0.14). These changes were associated with reduced basal cell contraction (dL/dt
max
) (84.3 vs 124.8 μm/s), relaxation (dR/dt
max
) (-66.1 vs -98.8 μm/s), and [Ca
2+
]
iT
(0.19 vs 0.23). This was accompanied by diminished ISO-stimulated inotropic response. In AWT myocytes, ISO caused significantly less increases in dL/dt
max
(34% vs 82%), dR/dt
max
(22% vs 60%), and [Ca
2+
]
iT
(15% vs 35%). Compared with YWT, Yβ
3
KO did not alter basal myocyte contraction and relaxation and response to ISO stimulation, but had significantly increased protein levels of SERCA 2a (Yβ
3
KO: 1.3 vs YWT: 0.61) and reduced iNOS (0.17 vs 0.24) with relatively unchanged β
1
-AR (0.63 vs 0.60). Aβ
3
KO mice had similar alterations. Importantly, in contrast to AWT, in Aβ
3
KO myocytes, the increased SERCA 2a (1.1) and reduced iNOS (0.19) correlated with normal basal cell contraction and relaxation with preserved ISO-stimulated inotropic response. ISO caused similar increases in dL/dt
max
(82% vs 84%) and [Ca
2+
]
iT
(31% vs 33%) compared to Yβ
3
KO mice.

Conclusions:
β
3
-AR deficiency prevents aging-caused downregulation of cardiac β
1
-ARs and reverses increased iNOS and decreased SERCA 2a, leading to the preservation of myocyte function, [Ca
2+
]
iT
, and β-adrenergic reserve in aged hearts. Thus, blocking β
3
-AR may provide a new strategy for myocardial aging.
Abstract 088: JNK2 Impairs Atrial Conduction And Enhances Arrhythmogenicity In Mouse Atria
Authors: Yan Jiajie , Zhao Weiwei , Zhang Qiang , Fast Vladimir G , Prabhu Sumanth D , Ai Xun
Published: July 2021

The c-Jun N-terminal kinase (JNK) is a stress-activated kinase that is important in the development of cardiovascular diseases. We have previously shown that enhanced JNK activation was associated with suppressed gap junction Cx43, slow conduction, and increased atrial fibrillation (AF) in aged rabbit left atrium (LA). In an atrial cellular model (HL-1), we further demonstrated that JNK activation downregulated Cx43 and enhanced arrhythmogenicity. However, direct evidence for JNK inducing atrial arrhythmia in vivo is lacking. In the present study, we found that JNK activator anisomycin (aniso) treatment in wild-type (WT) mouse in vivo resulted in a 30% decrease in Cx43 protein in LA vs that of vehicle-treated WT LA (p<0.001; n=4, 6; each sample contained mixed LAs from 5 mice). From optical mapping recordings in the intact LA, conduction velocity (CV) in aniso-treated WT mice slowed by 36% (30±2 vs. 47±1 cm/sec in vehicle-treated WT, pacing cycle length (CL) = 100ms, p<0.01; n=4, 3). Additional in vivo treatment with SP600125 (a specific JNK inhibitor) reversed aniso-induced Cx43 suppression (n=3) and CV slowing (50±2 cm/sec; n=6) as compared with WT LA treated with aniso alone (p<0.001). Moreover, we assessed atrial arrhythmia susceptibility in vivo using burst pacing delivered via an octapolar cardiac catheter. JNK activated hearts exhibited an increased incidence of pacing-induced AF (13%; n=7), while no AF was induced in WT-vehicle mice (n=4). To further confirm the specific role of JNK activation in atrial arrhythmogenicity, JNK2 (one of the cardiac JNK isoforms) knockout mice (JNK2KO) were treated with aniso in vivo. Similar to the effect of JNK inhibition by SP600125, JNK2 knockout prevented aniso-induced Cx43 suppression (n=3) and slowing of CV (47±1cm/sec, CL=100ms; n=4) compared with aniso-treated WT (p<0.05 & p<0.001, respectively). Furthermore, a dramatically decreased susceptibility to pacing-induced AF (1.7%; n=6) was found in aniso-treated JNK2KO vs aniso-treated WT mice. In conclusion, our results strongly suggest that JNK, especially JNK2, activation contributes to impaired cell-cell communication that in turn promotes AF development. Modulation of JNK2 could be a novel therapeutic approach to prevent and treat AF in the elderly.
Abstract 094: Antithrombin Exerts a Cardioprotective Effect against Myocardial Ischemic/reperfusion Injury through Induction of Prostacyclin and Inhibition of NF-kB Signaling
Authors: Li Ji , Wang Jingying , Wang Yanqing , Wang Jinli , Gao Junjie , Yawson Emmanuel , Manithody Chandrashekhara , Rezaie Alireza R
Published: July 2021

Antithrombin (AT) is a plasma serpin inhibitor that regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to its anticoagulant activity, AT also possesses potent antiinflammatory and antiangiogenic properties. In this study, we investigated the antiinflammatory activity of wild-type AT (AT-WT) and a reactive center loop mutant of AT (AT-RCL) not capable of inhibiting thrombin in a mouse model of ischemia/reperfusion injury in which the left anterior descending coronary artery (LAD) was occluded and released then. The results demonstrate that AT markedly reduces the myocardial infarct size (p<0.05 vs. vehicle) by a mechanism that is independent of its anticoagulant activity. Thus, the AT-RCL mutant, lacking any detectable reactivity with thrombin, attenuated myocardial infarct size to the same extent as the AT-WT in the acute injury model. Further studies revealed that AT binds to vascular heparan sulfate proteoglycans via its heparin-binding domain to exert its protective activity as evidenced by the therapeutic AT-binding pentasaccharide fragment of heparin (fondaparinux) abrogating the cardioprotective activity of AT. We further demonstrate that AT up-regulates the production of prostacyclin in myocardial tissues (p<0.05 vs. vehicle) and inhibits the production of proinflammatory cytokines TNF-α and IL-6 (p<0.05 vs. vehicle) in vivo by attenuating the ischemia/reperfusion-induced JNK and NF-κB signaling pathways. These results suggest that AT, through its antiinflammatory signaling effect, may have therapeutic potential for reducing cardiac ischemia/reperfusion injury and that a normal cardioprotective activity for the AT-RCL variant renders it a superior drug candidate if bleeding becomes a concern in the treatment.