bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P092: Simplified Monolayer Differentiation of Human-Induced Pluripotent Stem Cells to Functional Cardiac Myocytes
Authors: Lang Jennifer K , Fernandez Stanley , Cimato Thomas
Published: July 2021

Background:
Human induced pluripotent stem cells (hiPSCs) are an important model for cardiovascular research, drug discovery, and translational research applications. Commonly used methods to direct iPSCs to cardiac myocytes can be technically demanding. Prior studies have shown that both VEGF and endothelial cells promote differentiation of stem cells to cardiac myocytes. Furthermore, DMEM/F12 with 10% fetal calf serum (DMEM-FCS) has been shown to induce cardiac myocytes in an embryoid body (EB) system. The objective of this study was to determine if differentiation of hiPSCs using conditions that support endothelial cell differentiation would promote cardiac myocyte colony formation.

Methods:
Two hiPSC lines derived using non-genome integrating methods were maintained on Matrigel-coated surfaces under serum free conditions in mTeSR1 medium. We performed a comparison of monolayer myocyte differentiation efficiency using DMEM-FCS and endothelial cell medium (EC). Cells were maintained in iPSC medium (mTeSR1) as a negative control. The number of beating colonies derived under each growth condition was determined using phase microscopy at 4 weeks. Cardiac myocyte commitment was characterized using an α-MHC-GFP reporter vector and electrophysiologic action potentials on isolated beating colonies.

Results:
Differentiation of human iPSCs in EC medium induced substantial numbers of beating colonies 4 weeks after differentiation (2.29 ± 0.3 beating colonies/cm2 culture area, n=42). Unlike EB models of myocyte differentiation, no beating clusters were observed in our monolayer system with DMEM-FCS medium (n=14) (p<0.01). As expected, mTESR1 (n=12) did not induce any cardiac myocytes. All beating cell colonies expressed GFP driven by the cardiac specific α-MHC promoter. Electrophysiological studies confirmed the presence of action potentials with ventricular phenotypes.

Conclusions:
Differentiation of human iPSCs under monolayer conditions that support endothelial cells facilitates efficient induction of functional human cardiac myocytes. Our findings simplify the differentiation of iPSCs to cardiac myocytes, making research with human iPSCs more accessible to a broad range of cardiovascular investigators.
Abstract P095: An Expression-Based in Vivo Protein Trap Screen Enriches Cardiac Mutants and Reveals Cardiac Functions of Methionine Adenosyltransferase 2A in Zebrafish
Authors: Ding Yonghe , Huang Wei , Deng Yun , Jomok Beninio , Caron Alissa , Yang Jingchun , Lin Xueying , Ekker Stephen , Xu Xiaolei
Published: July 2021

The present forward mutagenesis screen strategies in vertebrates are difficult to identify genes and mutants affecting a specific organ of interest. Here, we report a strategy to enrich cardiac mutants by leveraging the transparency of zebrafish embryos and the high efficiency of pGBT-RP2 (RP2), a gene-breaking transposon-based
in vivo
protein trap cassette. By selectively raising up founder fish with Red Fluorescence Protein (RFP) reporter expression in the heart, we successfully generated 15 cardiac mutant lines. All tagged loci exhibit cardiac expression and the overall knockdown efficiency at transcript level is greater than 95%. One of the cardiac lines,
RP2_ #135,
is a homozygous embryonic lethal line that is caused by a RP2 insertion in
methionine adenosyltransferase 2a (mat2a)
. Taking advantage of the LoxP sites inside the RP2 vector, we further demonstrated that the cardiac phenotypes in
RP2_ #135
is due to a disrupted expression of
mat2a
in cardiomyocytes, as indicated by a conditional rescue assay using
Tg(cmlc2:Cre-YFP)
, a myocardium specific Cre transgenic line. Finally, we show that the reduced cardiomyocyte number, but not size, in the
mat2a
mutant is conveyed by the p53-mediated apoptosis pathway. Together, our data prove the feasibility of a facile approach to generate a vertebrate cardiac mutant collection with the following two unique features. First, the expression dynamics of each tagged gene is reported by RFP. Second, the causality between the tissue-specific gene expression and the resulted cardiac phenotypes can be attested genetically.
Abstract P104: Reactivation of Embryonic Gene Program due to CUG Repeat RNA Expression in Myotonic Dystrophy
Authors: Kalsotra Auinash , Singh Ravi , Creighton Chad , Cooper Thomas
Published: July 2021

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disease that affects multiple organ systems. Cardiac involvement, which is characterized by conduction defects and arrhythmias, is the second leading cause of death in DM1 patients. The causative mutation is a CTG expansion in the 3' untranslated region of DMPK gene resulting in aberrant expression of CUG repeat RNA that accumulates into nuclear foci and causes misregulation in alternative splicing. Here we show that heart-specific and inducible expression of CUG repeat RNA in a DM1 mouse model results in global reactivation of embryonic gene expression program in adult heart that is distinct from a general hypertrophic stress response. Using q-PCR TaqMan arrays, we identified 54 miRNAs that were differentially expressed in DM1 mouse hearts one week following induction of CUG repeat RNA. Interestingly, 83% (45/54) of them exhibited a developmental shift in expression towards the embryonic pattern. Because over 90% (41/45) of them were down regulated within 72 hr after induction of repeat RNA and only 2/22 examined decreased in two unrelated mouse models of heart disease, we conclude their reduced expression is specific to DM1 and not simply a general response to cardiac injury. Microarray studies revealed a developmental switch not only in the miRNA expression patterns but also a pervasive shift in mRNA steady state levels of a number of genes to embryonic stage. Intriguingly, we found that loss of MBNL1 or gain of CELF1 activity, two major RNA binding proteins disrupted in DM1, are not driving the miRNA misregulation since their expression is indistinguishable between wild type, MBNL1 knock out and CELF1 over expressing mice. Moreover, comparable decrease in ten out of ten primary miRNA transcripts examined suggests loss of expression is not due to a processing defect. Instead, we discovered that adult-to-embryonic shift in expression of select micro- and messenger RNAs in DM1 heart occurs due to specific inactivation of a Mef2 transcriptional program. We are currently determining causal contributions of this Mef2-miRNA circuitry in the developmental reprogramming of gene expression in DM1 as well as its direct role in cardiac manifestations of this disease.
Abstract P110: EBP50 Regulates Vascular Smooth Muscle Cell Growth by Skp2-Mediated Degradation of p21/cip1
Authors: Song Gyun Jee , Barrick Stacey , Leslie Kristen L , Fiaschi-Taesch Nathalie M , Bisello Alessandro
Published: July 2021

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-binding phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout (KO) mice. The molecular mechanisms underlying EBP50 actions in VSMC are unknown.

Genetic ablation of EBP50 reduced VSMC proliferation and was associated with increased (5-fold) expression of the cell cycle inhibitor p21cip1 both in vessels and in primary cells. No differences in mRNA levels of p21cip1 were observed in WT and KO cells. However, the half-life of p21cip1 in KO VSMC was significantly longer than in WT VSMC (80 min vs. 45 min) and p21cip1 levels were similar in WT and KO VSMC treated with the proteasome inhibitor MG132. These observations suggest that EBP50 regulates post-translational degradation of p21cip1. The S-phase kinase-associated protein 2 (skp2) is a component of the E3 ligase complex that degrades p21cip1. The C-terminal four amino acids of skp2 (ProSerCysLeu) are a canonical PDZ-binding sequence. Indeed, co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and skp2. Mutation of the C-terminal Leu to Ala (L424A-skp2) abrogated the interaction with EBP50. Skp2 expression was significantly lower in KO than in WT cells and inhibition of EBP50 expression by an shRNA lentivirus decreased skp2 expression in WT cells. Moreover, expression of skp2, but not of the mutant L424A-skp2, in WT cells reduced p21cip1 levels. Therefore, EBP50 regulates both expression and activity of skp2 with attendant effects on p21cip1 and VSMC proliferation.

Collectively, these experiments show that EBP50, by regulating skp2 and p21cip1 expression, controls VSMC proliferation and the progression of neointima formation. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide a mechanistic basis for the remarkable effect of this scaffolding protein on vascular remodeling.
Abstract P151: Differential Effects of Concurrent and Sequential Combined Lapatinib and Doxorubicin Treatment on Cardiac Function in Mice
Authors: Goukassian David , Sasi Sharath , Lee Juyong , Stone James R , Budiu Daniela , Lawson Christopher , Maysky Michael , Hlatky Lynn , Carrozza Joseph , Morgan James P , Klement Giannoula , Yan Xinhua
Published: July 2021

Background:

Clinical studies showed that Trastuzumab, a monoclonal antibody that blocks the HER2 receptor, exacerbated chemotherapy drug doxorubicin (DOX)-induced heart failure. Lapatinib, a small molecule inhibitor of HER2 and EGF receptor (EGFR), is currently studied in clinical trials for cancer therapy, in combination with DOX, concurrently and sequentially. Therefore, it is important to assess the cardiac effect of the combination treatment using lapatinib and DOX.

Methods:

Two mouse models were used. In the first model, mice were treated with a single dose of DOX (20 mg/kg, i.p.), concurrently with lapatinib (100 mg/kg, oral gavage, daily). In the second model, mice were treated with DOX (2 mg/kg, i.p. twice a week) for seven weeks, followed by lapatinib (100 mg/kg, oral gavage, daily) for three weeks. Survival was analyzed by the Kaplan-Meier method. Cardiac function was monitored by hemodynamic measurements.

Results:

When mice were treated concurrently with lapatinib and DOX, ten-day survival was significantly lower in DOX+lapatinib vs. DOX mice (13 vs. 0 %, n=7–8/group,
P
<0.001). Five to seven days after the treatment, cardiac contractile function was significantly lower in DOX+lapatinib vs. control mice [left ventricular systolic pressure (LVSP): 83 ± 2 vs. 93 ± 2 mmHg,
P
<0.05; dP/dtmin: 4020 ± 360 vs. 7112 ± 580 mmHg/sec,
P
<0.05; cardiac output (CO): 1236 ± 266 vs. 2734 ± 209 unit/min,
P
<0.05, n=5/group]; however, these indices were maintained in DOX mice (LVSP: 99 ±4 mmHg; dP/dtmin: 7787 ± 965 mmHg/sec; CO: 2315 ± 366 unit/min,
P
=NS vs. control, n=5–6/ group). On the other hand, when mice were treated sequentially with DOX and lapatinib, seventy-day survival was higher in DOX+lapatinib vs. DOX mice (63 vs. 43 %, n=7–8/ group). Cardiac output was decreased in DOX vs. control mice (1733±135 vs. 3438±268 unit/min,
P
<0.05, n=4–5/group). However, cardiac output was less depressed in DOX+lapatinib mice (2817±559 unit/min,
P
=NS vs. control, n=4/group).

Conclusions:

These results showed that concurrent and sequential combined lapatinib and DOX treatment produced opposite effects on survival and cardiac function in mice. This study suggests that the cardiac safety of lapatinib and DOX combination therapy depends on the specific treatment regimen.
Abstract P168: Activated Protein C Inhibits the Development of Myocardial Fibrosis
Authors: Sopel Mryanda J , Myers Tanya L , Lee Timothy D , Liwski Robert S , Legare Jean-Francois
Published: July 2021

Background:
Activated Protein C (aPC) is a naturally produced circulating anticoagulant with anti-inflammatory and cytoprotective properties proposed to have beneficial effects on the myocardium. Myocardial fibrosis is a pathophysiologic process evident in many cardiovascular diseases and is believed to directly contribute to eventual organ failure. Using a well-described model of myocardial fibrosis after Angiotensin II (AngII) infusion our aim was to investigate the novel therapeutic function of aPC in the development of fibrosis.

Methods:
C57Bl/6 mice were continuously infused with AngII (2.0 μ g/kg/min), AngII and aPC (0.4 μ g/kg/min), or saline for a 3d period. Hearts were harvested and processed for analysis. Cellular infiltration and collagen deposition were analyzed using histologic staining. Cellular apoptosis was assessed using a TUNEL assay. Quantitative RT-PCR was used to assess transcript levels of molecular mediators.

Results:
Infusion of AngII for 3d resulted in multifocal areas of myocardial cellular infiltration associated with significant collagen deposition compared to saline control animals (p≤0.01). The addition of aPC with AngII infusion inhibited this fibrotic response. Co-administration with aPC also inhibited the upregulation of the pro-fibrotic cytokine CTGF expression seen in AngII infused animals (p≤0.01). Apoptosis was also significantly inhibited when aPC was co-administered based on a decrease of in TUNEL positive cells and an increase expression ration of BCL/BAX suggesting an anti-apoptotic effect (p≤0.01), Furthermore, aPC also inhibited the up-regulation of the cell adhesion molecule p-selectin seen in AngII infused animals (p≤0.01).

Conclusion:
The co-administration of aPC in a model of myocardial fibrosis was able to abrogate completely the fibrotic response. The mechanism of action of aPC appears to be a decreased cellular adhesion molecule expression in the myocardium leading to decreased cellular infiltration and enhanced cellular survival. This data suggests that aPC has the potential as a therapeutic agent in cardiovascular diseases.
Abstract P195: Nuclear Translocation of GRK5 Following Hypertrophic Stimuli Is a Calmodulin-Dependent Process
Authors: Gold Jessica I , Martini Jeffrey S , Chuprun J Kurt , Koch Walter J
Published: July 2021

Maladaptive ventricular hypertrophy often precedes the pathological endpoint of Heart Failure (HF). G protein Coupled Receptors (GPCRs), such as the β-adrenergic receptor, play a crucial role in the development of cardiac hypertrophy. GPCR Kinase 5 (GRK5) is a primary GRK expressed in cardiomyocytes. Traditionally, it phosphorylates and desensitizes agonist-bound GPCRs. Recent, exciting data from our lab shows that GRK5 translocates into the nucleus and phosphorylates Histone Deacetylase 5 (HDAC5) in response to hypertrophic stimuli. This novel kinase activity of GRK5 causes nuclear export of HDAC5, enhancing transcription of hypertrophic genes regulated by myocyte elongation factor 2 (MEF2). We hypothesize that that nuclear GRK5 activity plays a critical role in cardiomyocytes, facilitating pathological hypertrophy and HF, and that hypertrophic GPCR signaling induces GRK5 nuclear translocation. To test this hypothesis, we utilized neonatal rat ventricular cardiomyocytes (NRVMs) to determine GRK5 localization following the hypertrophic stimulus of phenylephrine (PE) treatment. To determine the mechanism causing nuclear translocation, we analyzed the effects targeted inhibition of key post-GPCR hypertrophic signaling pathways on GRK5 cellular localization. Treatment of NRVMs with PE for 1 hour increases nuclear GRK5 accumulation by 100%. Chronic infusion of PE also lead to a 50% increase in nuclear GRK5 in the hearts of mice overexpressing cardiac-specific GRK5. Calmodulin inhibition (CaM) in NRVMs via calmidizolium chloride decreases nuclear GRK5 accumulation, while inhibition of PKC and PKD shows no significant effect. Using a luciferase assay, we have shown that overexpression of CaM increases MEF2 activity 3-fold and causes increased nuclear accumulation of GRK5. Further, overexpression of a GRK5 mutant that lacks CaM binding activity, results in a lack of PE- and CaM-induced nuclear translocation. We have now found that hypertrophic stimuli increases myocyte GRK5 nuclear translocation via a CaM-dependent process. Defining the role of CaM signaling in pathological GRK5 nuclear localization may offer a new therapeutic target for maladaptive hypertrophy and HF.
Abstract P197: Reversible Acetylation of GSK3β Regulates the Development of Cardiac Hypertrophy
Authors: Nagalingam Ravi Sundaresan , Pillai Vinodkumar B , Samant Sadhana , Wolfgeher Don , Gupta Madhu , Gupta Mahesh P
Published: July 2021

Glycogen synthase kinase 3β (GSK3β) is a critical regulator of diverse cellular functions involved in the maintenance of cellular structure, function and survival. In the heart, GSK3β has been shown to play a key role in antagonizing the development of cardiac hypertrophy. It has been shown that GSK3β activity is inhibited by phosphorylation at Ser9 during the development of cardiac hypertrophy. However, recent studies indicated that blocking the Ser9 phosphorylation alone is not sufficient to protect GSK3β activity, suggesting that alternative mechanisms might be involved in regulation of GSK3β activity. In the present study, we tested the hypothesis whether GSK3β is acetylated in established hypertrophy, which leads to inhibition of GSK3β activity, and thereby promoting progression and maintenance of cardiac hypertrophy. We examined acetylation, phosphorylation and catalytic activity of GSK3β in pressure overload model of cardiac hypertrophy at different stages of hypertrophy development. The results showed that GSK3β catalytic activity was significantly down-regulated throughout the course of hypertrophy development. However, the inhibitory phosphorylation of GSK3β was observed only at initial stages, but not at later stages of pressure overload (4 weeks). Interestingly, we observed a significantly increased acetylation of GSK3β, which corresponded to the reduced activity at this later stage of hypertrophy. We then determined the impact of GSK3β acetylation on its catalytic activity, by measuring the ability of GSK3β to phosphorylate the substrate glycogen synthase. The results showed that acetylation decreased the catalytic activity of the GSK3β by almost 3 fold. Next, we performed mass spectrometry to find out the target lysine residues of GSK3β. Proteomic analysis revealed that multiple lysine residues in different regions of GSK3β were acetylated. By using site directed mutagenesis, we found that acetylation inhibits the catalytic activity of GSK3β by suppressing its substrate binding ability, which is independent of inhibitory-Ser 9 phosphorylation. These studies for the first time show a phosphorylation independent mechanism regulating GSK3β activity during progression and maintenance of cardiac hypertrophy.
Abstract P212: Haploinsufficiency of Target of Rapamycin Attenuates Different Forms of Cardiomyopathies in Adult Zebrafish
Authors: Sun Xiaojing , Ding Yonghe , Huang Wei , Hoage Tiffany , Redfield Margaret , Kushwaha Sudhir , Sivasubbu Sridhar , Lin Xueying , Ekker Stephen , Xu Xiaolei
Published: July 2021

In contrast to the wide application of zebrafish embryos in developmental genetic studies, the value of an adult zebrafish to dissect signaling pathways in human diseases such as cardiomyopathy remains largely elusive. Previously, we have established anemic
tr265/tr265
fish as the first adult zebrafish model of cardiomyopathy. Here, we generate the second adult zebrafish cardiomyopathy model induced by injection of a single bolus of doxorubicin (DOX). Despite their different pathogenesis, cardiac enlargement induced by either anemia or DOX can be effectively attenuated by inhibition of target of rapamycin (TOR) signaling via rapamycin treatment. However, along the progression of both models, we have also detected dynamic TOR activity and distinct effects of TOR signaling inhibition at different stages of pathogenesis. To assess the long term effects of TOR haploinsufficiency, we utilized a
zebrafish target of rapamycin (ztor)
mutant that was identified from a mutagenesis screen. We show that sustained TOR inhibition in
ztor/+
improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the anti-hypertrophy, anti-apoptosis, and pro-autophagy function of TOR signaling inhibition. Together, our results prove adult zebrafish as a novel vertebrate model for human cardiomyopathies and provide genetic evidences for a cardioprotective function of TOR signaling inhibition in different forms of cardiomyopathies.
Abstract P214: Reduction of Hexokinase II Exaggerated Cardiac Hypertrophy via Increased ROS Production and Mitochondrial Permeability Transition
Authors: Wyatt Eugene J , Wu Rongxue , Chawla Kusum , Ghanefar Mohsen , Ardehali Hossein
Published: July 2021

Rationale/Objective:
Cardiac hypertrophy is associated with a metabolic switch in substrate utilization from fatty acids to glucose. Levels of Hexokinase II (HKII), a key regulator of glucose metabolism, increase in response to hypertrophic stimulation and we hypothesized that a reduction in HKII could attenuate the hypertrophic response.

Methods/Results
: Contrary to our hypothesis, heterozygous knockout of HKII (HKII+/-) resulted in exaggerated cardiac hypertrophy after the induction of pressure overload by trans-aortic constriction (TAC). HKII+/- mice demonstrated increased heart size, interstitial fibrosis, and pulmonary edema compared to wild-type (WT) 4 weeks after TAC. This was associated with reduced cardiac function and increased mortality in the HKII+/- mice by the 8-week time point, indicating an accelerated transition to heart failure. In primary cultures of neonatal rat cardiomyocytes (NRCM), siRNA knockdown of HKII exacerbated the hypertrophic response to Angiotensin II (AngII) compared to control siRNA. Mechanistically, reduction of HKII resulted in increased ROS levels after hypertrophic stimulation relative to controls. Treatment of NRCM with the antioxidant
N
-acetylcysteine (NAC) attenuated the hypertrophic response to AngII, and abrogated the increased hypertrophy observed with HKII knockdown. In addition to its enzymatic activity, HKII can bind the outer membrane of the mitochondria, and this interaction increased with hypertrophic agonists. Dissociation of HKII from the mitochondria using a synthetic cell permeable peptide resulted in
de novo
hypertrophy compared with scrambled peptide control, and this was also attenuated with NAC treatment. Further investigation into the source of ROS revealed that HKII knockdown (+AngII) or mitochondrial dissociation resulted in increased mitochondrial permeability pore (mPTP) opening in the absence of cell death.

Conclusions:
Overall, the data suggest that HKII and its binding to the mitochondria serve as a negative regulator of hypertrophy by decreasing ROS production, possibly through regulation of the mPTP.
Abstract P215: β3-Adrenergic Receptor Mediated NOS Signaling in Cardiomyocytes
Authors: Watts Vabren L , Niu Xiaolin , Miller Karen L , Barouch Lili A
Published: July 2021

Beta3 -adrenergic receptors play a pivotal role in modulating cardiac function, though their precise role in the heart remains controversial. We have recently demonstrated alterations in Ca
2+
-dependent NOS isoforms and decreased NOS activity in left ventricular tissue of beta3
-/-
mice after pressure overload. However, the exact manner in which beta3-AR signaling regulates these isoforms to stimulate NOS activity at the cardiomyocyte level is not well understood. In this study we used a specific beta3-AR agonist,
BRL37344
(BRL), to assess the role of beta3-AR in eNOS and nNOS regulation in hypertrophied isolated neonatal rat ventricular cardiomyocytes (NRVM). To induce hypertrophy we pretreated cells with norepinephrine for 72 hours, which resulted in a 70% increase in cell size and a 25% increase in beta3-AR mRNA expression as compared with non-hypertrophied cells, analyzed by immunocytochemistry and real-time PCR. In hypertrophied cardiomyocytes, BRL administration lead to a time-dependent 5-fold increase in NOS activity, measured by the arginine-to-citrulline conversion assay. beta3-activation also caused a 1.5-fold increase in nNOS phosphorylation at positive regulatory site Ser1416, and dephosphorylation of negative regulatory site Ser847 as compared with unstimulated control. NOS activity and nNOS phosphorylation overlapped in time. In addition BRL induced phosphorylation eNOS-Ser114, which indicates eNOS deactivation. Pretreatment with pertussis toxin (PTX) suppressed BRL-induced nNOS-Ser1416 phosphorylation, nNOS-Ser847 dephosphorylation, and NOS activity, suggesting G
i/o
dependency. Taken together, our data suggest that BRL regulates NOS signaling in ventricular cardiomyocytes via phosphorylation regulation of nNOS. To our knowledge this is first study to demonstrate a role for nNOS phosphorylation as a key factor in beta3-AR signaling. These results contribute significantly to our understanding the negative inotropic properties of myocardial beta3-AR at cellular levels during cardiac sympathetic overstimulation, and will ultimately aid in drug discoveries that target the molecular mechanisms associated heart failure.
Abstract P234: S-nitrosylation of Cyclophilin D Attenuates Mitochondrial Permeability Transition Pore Opening: A Critical Role for Cysteine 203 Residue
Authors: Nguyen Tiffany T , Stevens Mark V , Kohr Mark J , Steenbergen Charles , Sack Michael N , Murphy Elizabeth
Published: July 2021

S-nitrosylation (SNO), a reversible, redox-dependent post-translational modification, has emerged as an important mechanism for dynamic regulation of many proteins. Our previous studies have shown that protein S-nitrosylation (SNO) plays a protective role in myocardial ischemia/reperfusion (IR) injury. The primary mediator of cell death in I/R injury is activation of the mitochondrial permeability transition pore (mPTP). Using a proteomic approach, we have previously found that cyclophilin D (CypD), a critical mPTP regulator, can be SNO on cysteine 203 (C203). To investigate whether SNO of CypD might attenuate mPTP activation, we mutated cysteine 203 of CypD, to a serine residue (C203S) and determined its effects on mPTP opening by assessing H
2
O
2
-induced mPTP opening using the calcein AM-cobalt chloride quenching method. Treatment of CypD
-/-
mouse embryonic fibroblasts (MEFs) with H
2
O
2
resulted loss in an ≈50 % loss of mPTP opening as compared to WT MEFs (n=5, p<0.05), consistent with the protective role of CypD in mPTP activation. Addition of a nitric oxide donor, GSNO, to CypD
-/-
MEFs did not further reduce mPTP opening; however, WT MEFs treated GSNO attenuated mPTP opening by half. To elucidate the role of SNO of C203 on CypD, we infected CypD
-/-
MEFs with a C203S-CypD vector. C203S-CypD re-constituted MEFs were also resistant to mPTP opening in the presence or absence of GSNO. This suggests that C203 is required for mPTP activation. To determine whether
in vivo
expression of C203S-CypD would alter mPTP opening, we generated adenovirus vectors encoding WT CypD or mutated C203S-CypD and injected these viral particles into CypD
-/-
mice
via
tail-vein. Mitochondria isolated from livers of CypD
-/-
mice or mice expressing C203S-CypD were resistant to Ca
2+
-induced swelling as compared to WT CypD reconstituted mice. In summary, our results indicate that C203 of CypD is required for mPTP opening and for the first time shows that SNO of C203 on CypD acts to attenuate mPTP activation.
Abstract P257: Isoform-Specific Effects of the Transcription Factor Sister-of-Mammalian Grainyhead on Endothelial Cells and in Vivo
Authors: Mlynek Arne , Lukosz Margarete , Graf Martin , Winkler Christoph , Haendeler Judith , Altschmied Joachim
Published: July 2021

Apoptosis and reduced migratory capacity of human endothelial cells (EC) are hallmarks for the development of atherosclerosis. TNFalpha has been described as one apoptotic stimulus, which is increased during cardiovascular disease. However, recent findings support the hypothesis that TNFalpha can induce survival genes before committing cells to apoptosis. In a screen for anti-apoptotic genes regulated by TNFalpha we have identified the transcription factor Sister-of-Mammalian Grainyhead/Grainyhead-like 3 (SOM/GRHL3). In humans two RNAs are transcribed from the gene, one of which is alternatively spliced, yielding the protein isoforms SOM1 and SOM3, the latter being an N-terminally truncated version. We have found that both isoforms are expressed in EC. Since nothing is known about the function of these proteins in EC, we investigated their functional properties and role in migration and apoptosis. To analyze their transcription factor activity we established a SOM-dependent reporter system by inserting tandem SOM binding sites and corresponding mutants upstream of a minimal promoter driving luciferase expression. To assess transcriptional activation by SOM1 and SOM3 we cotransfected these reporters with expression vectors for both proteins. In contrast to previously published work, in which isolated SOM domains fused to a Gal4 DNA binding domain were used, we found that both full length proteins are active transcription factors. We next investigated the influence of SOM1 and SOM3 on EC functions. Surprisingly, overexpression of isoform 1 induced migration and inhibited apoptosis, whereas isoform 3 had opposite effects. Along the same lines, SOM1, but not SOM3 activated endothelial nitric oxide synthase and Akt. To investigate whether these isoforms have different functions also in vivo, we overexpressed them in zebrafish embryos. SOM3 but not SOM1 overexpression led to increased lethality, a strong reduction in normal phenotype and a 10 fold higher frequency in heavy deformations. The effects observed on EC migration and apoptosis as well as on zebrafish development suggest that these isoforms activate different sets of target genes, which we are currently identifying by microarray analysis.
Abstract P274: Cardiac-Specific Knockout of
Capn4
Attenuates Myocardial Remodeling and Improves Function After Myocardial Infarction in Mice
Authors: Ma Jian , Wei Meng , Wang Hao , Liu Weihua , Zhu Wei , Li Ying , Lacefield James , Greer Peter , Karmazyn Morris , Peng Tianqing
Published: July 2021

Background
- Calpain has been implicated in myocardial injury after myocardial infarction (MI). However, no direct evidence is available on the role of calpain in post-MI myocardial remodelling and dysfunction. The present study investigated the effects of cardiomyocyte-specific deletion of
Capn4
, essential for calpain-1 and calpain-2 activities on myocardial remodelling and dysfunction following MI.

Methods and Results
- A novel mouse model with cardiomyocyte-specific deletion of
Capn4
(
Capn4-ko
) was generated. MI was induced by left coronary artery ligation. Deficiency of
Capn4
significantly reduced the protein levels and activities of calpain-1 and calpain-2 in the
Capn4-ko
heart.
In vivo
cardiac function was relatively improved in
Capn4-ko
mice at 7 and 30 days after MI compared with their wild-type littermates. Deletion of
Capn4
reduced cardiac apoptosis, limited infarct expansion and infarct zone thinning, and prevented left ventricle dilation in
Capn4-ko
mice. Furthermore, myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly attenuated in
Capn4-ko
mice, which were accompanied by down-regulation of pro-fibrotic genes and hypertrophic genes. These effects of
Capn4
knockout correlated with down-regulation of inflammatory mediators and normalization of matrix metalloproteinase (MMP)-9 activity in the non-infarct area of
Capn4-ko
mice after MI.
In vivo
mouse model of endotoxemia confirmed that calpain activation resulted in inflammatory gene expression and MMP-9 activity in the heart.

Conclusions
- Cardiomyocyte-specific knockout of calpain attenuates myocardial adverse remodelling and improves myocardial function after MI. These beneficial effects of calpain disruption may result from inhibition of cardiac apoptosis, inflammation and MMP-9 activity.
Abstract P318: Vasoprotective Effects of an Apo A-I Mimetic Peptide
Authors: White C , Datta Geeta , Parks Dale , Anantharamaiah G
Published: July 2021

Hepatic ischemia/reperfusion (I/R) injury is associated with several pathologic states and contributes to the development of systemic vascular complications. High density lipoprotein (HDL) and apolipoprotein (apo) A-1 possess antioxidant and anti-inflammatory properties and reduce tissue injury in several models of I/R. In the current study, we tested the hypothesis that the synthetic apoA-I mimetic peptide 4F, similar to HDL and apoA-I, exerts cytoprotective effects in a murine model of hepatic I/R injury. C57BL/6 mice were randomized to receive 4F (5 mg/kg, IP) or an equivalent volume of saline vehicle. Mice were then subjected to 45 min of segmental hepatic ischemia, followed by 24 hr reperfusion. Sham-operated mice served as a control. Hepatic I/R injury was associated with an increase in alanine transaminase (ALT: 980±95U/mL) and xanthine oxidase (XO: 700±100U/mL) release compared to sham-operated controls (ALT=27±8U/mL; XO=100±10U/mL). Endothelium-dependent relaxation was impaired in aortae of I/R mice (R
max
=20%) compared to controls (R
max
=72%). This response was associated with a decrease in eNOS protein and an increase in iNOS and plasma levels of nitric oxide metabolites (NOx). Further, immunohistochemical studies revealed an increase in the nitration (3-NT) and chlorination (3-CT) of protein tyrosine residues in aortic sections from I/R mice that was associated with an increase in tissue myeloperoxidase (MPO) content. 4F treatment significantly improved the relaxation response in the aorta of I/R mice (R
max
=60%), prevented the upregulation of iNOS and NOx and reduced tissue protein nitration/chlorination. 4F treatment was further associated with a reduction in hepatocellular injury in I/R mice, as revealed by a decrease in ALT (110±15U/mL) and XO (270±50U/mL) release. Collectively, these data suggest that 4F reduces systemic vascular injury via cytoprotective effects at the level of the liver. ApoA-I mimetic peptide-based therapies may be effective in reducing vascular complications associated with clinical procedures such as transplantation.
Abstract P339: The Mechanistic Relationship Between GRK2 and eNOS During Cardiac Ischemia/Reperfusion Injury
Authors: Huang Zheng M , Gao Erhe , Shang Xiying , Tian Xufan , Qiu Gang , Chuprun J Kurt , Lefer David J , Koch Walter J
Published: July 2021

Background:
Previous studies have shown that both cardiac-specific GRK2 transgenic (TG) (αMHC-GRK2) mice and eNOS knockout (KO) mice have larger infarcts after I/R compared to control mice. In contrast, cardiac-targeted eNOS TG mice and cardiac specific GRK2 KO mice show cardioprotection after I/R, suggesting a dynamic interaction between the two proteins. Mechanistically, GRK2 can be inhibited by cellular NO through S-nitrosylation with Cys340 being the major site; meanwhile GRK2 has been shown to directly bind to and inhibit Akt, which is a strong activator of eNOS. Aim: to investigate the potential novel interaction between GRK2 and eNOS, and the consequent functional impact on the protein activity and cardiac phenotype after I/R injury.

Methods:
αMHC-GRK2 mice were crossed with either eNOS TG or eNOS KO mice. All mice were subjected to sham or 30min myocardial ischemia via coronary artery ligation followed by 24hrs of reperfusion. Infarct size, cardiac function, and tissue apoptosis were examined. Co-IP was used to test the interaction between GRK2 and eNOS, and phosphorylation of eNOS was studied in neonatal myocytes.

Results:
1).αMHC-GRK2/eNOS TG hybrid mice showed a significantly reduced infarct size after I/R compared to αMHC-GRK2 mice, accompanied by improved cardiac function measured by echocardiography and hemodynamics, and significantly less apoptosis tested by TUNEL assay, implying a rescue effect by eNOS. 2). αMHC-GRK2/eNOS KO mice exhibited a bigger infarct size compared to either αMHC-GRK2 mice or eNOS KO mice. 3). CO-IP confirmed the interaction between GRK2 and eNOS in cardiac tissue, which was increased upon β-AR agonist treatment. 4). In neonatal ventricular myocytes, GRK2 overexpression significantly decreased eNOS phosphorylation at Ser1177 after exposure to H2O2, while GRK2 knockdown by siRNA led to slight increase in pSer1177.

Conclusions:
eNOS interacts with and may be a downstream target of GRK2 in the heart. Decreased activation of eNOS may mediate the deleterious effect of GRK2 overexpression during cardiac I/R injury.
Abstract P345: The Neuregulin Glial Growth Factor 2 (GGF2) Produces Sustained Improvement in Left Ventricular Function in Rats with Both Early and Established Heart Failure
Authors: Ganguly Anindita , Troy Erika , Srinivas Maya , Vecchione Andrea , Sarmiere Patrick , Hu Tao , Iaci Jennifer , Hackett Craig , Button Donald , Caggiano Anthony , Parry Tom J
Published: July 2021

Neuregulin-1β
is essential for fetal cardiac development and adult cardiac function. Previous reports indicate that neuregulins improve left ventricular function in heart failure models, however the duration of the functional improvements with early or late initiation of neuregulin treatment has not been characterized. The present studies examine the effects of early and delayed initiation of intravenous GGF2 treatment on left ventricular (LV) function in rats with myocardial infarction (MI). Rats underwent surgically-induced MI by left anterior coronary artery ligation. Treatment with vehicle or GGF2 (2.6 mg/kg) was initiated at 2 or 16 w post-MI and continued once or twice weekly or once every two weeks for the in-life duration of the study (approximately 40 weeks). LV function was assessed echocardiographically up to once weekly for the duration of the study. Early and delayed initiation of GGF2 treatment caused sustained and significant improvement (p < 0.05) in both ejection fraction (EF) and fractional shortening (FS) in all regimens tested. The greatest improvements were seen with the once weekly dosing paradigm after early initiation (average EF (%) at 40 weeks post initiation of dosing: vehicle = 44.4 ± 6.0, n = 8 rats, vs. GGF2 = 64.7±6.1. n = 9 rats) and twice weekly dosing paradigm after delayed initiation (average EF (%) at 4 weeks post initiation of dosing: vehicle = 34.18±1.6, n = 7 rats, vs. GGF2 = 50.69±4.68, n = 7 rats). In addition, LV function improved when rats were re-challenged with GGF2 following an extended wash out period. This observation indicates potential efficacy for treatment paradigms that utilize intermittent dosing. These findings suggest that GGF2 produces sustained improvement in LV function after early or delayed initiation of treatment following MI in rats.
Abstract 10: Antifibrotic Effect of CCN5 in a Murine Model of Heart Failure
Authors: Jeong Dongtak , Kho Changwon , Lee Ahyoung , Park Woo Jin , Hajjar Roger
Published: July 2021

CCN family members are matricellular proteins with diverse roles in cell function. Recently, we showed that the differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. Since it is reported that an underlying morphological correlate of diastolic dysfunction is cardiac fibrosis, which leads to increased stiffness of the heart, we aimed to evaluate the role of CCN5 on cardiac fibrosis and function by the gene delivery using the cardiotropic AAV9 vector. We generated pressure-overload heart failure models in mouse by TAC operation. After 8-10 weeks of TAC on mice, HF was confirmed by Echocardiography. In those HF mice, AAV9-GFP (control) and AAV9-CCN5 were addressed by IV. Two more months later, cardiac function was evaluated by echocardiography and invasive hemodynamics. Protein and RNA expression levels of CCN5, several types of collagen and conventional TGF-beta signaling related genes were evaluated by western blot and quantitative real time PCR analysis. First, we were able to achieve about 4-5 fold increase of CCN5 expression by AAV9-CCN5 injection without any change in heart function. Second, CCN5 expression level in blood was not significantly altered after AAV9-CCN5 gene transfer because it may be the result of the cardiac tropism of the vector used. The HF model by TAC surgery was confirmed with echocardiography (FS (%)). Overall average FS (%) in HF was 41.87+/− 5.27 (n=16) and in non-surgery control mice was 58.39 +/− 2.06(n=4). After AAV9 injection, cardiac function of CCN5 injected mice was sustained but AAV9-GFP injected mice showed severe cardiac dysfunction and dilation (AAV-GFP (24.29+/− 9.11) vs AAV-CCN5 (42.66 +/− 4.73)). Third, western blot analysis showed that the downstream effectors, namely TGF-beta signaling pathways were significantly down-regulated in CCN5 injected mice. In addition, fibrotic area of the heart was tremendously reduced. Finally, CCN5 expression is significantly decreased in human heart failure patients compared to those in nonfailing donors. Taken together our data would indicate that CCN5 may be a promising therapeutic target to reduce cardiac fibrosis.
Abstract 103: Cardiac Grk2 Blockade Augments β2-Adrenergic Receptor-Dependent Contractility and Survival Post Myocardial Infarction
Authors: Lymperopoulos Anastasios , Rengo Giuseppe , Gao Erhe , Koch Walter J
Published: July 2021

ß
1
- and ß
2
-adrenergic receptors (ßARs) are G-protein coupled receptors (GPCRs) that play clearly distinct roles in cardiac physiology/pathology: cardiomyocyte contraction is readily stimulated by ß
1
AR but not ß
2
AR signaling, and ß
1
AR signaling is largely pro-apoptotic in the heart, in contrast to the cardioprotective ß
2
AR signaling. These differences might be due to assembly of different macromolecular complexes containing phosphodiesterases (PDEs), which constrain pro-contractile 3'-5'-adenosine monophosphate (cAMP) signaling. Additionally, ß
2
AR is readily phosphorylated by GPCR kinase (GRK)-2, which then recruits ß-arrestin (ßarr), a universal GPCR adapter/scaffolding molecule, to the receptor. ßarrs reduce ß
2
AR pro-contractile signaling by desensitizing the receptor and can either increase or decrease apoptosis via interactions with several downstream effectors. Herein, we sought to investigate the effect of ßarr recruitment blockade on ß
2
AR-dependent contractility and survival in vivo. To this end, we crossed ß
1
AR knockout (B1KO) mice, lacking the ß
1
AR, with M27 mice, which overexpress, specifically in cardiac myocytes, the GRK2 inhibitor GRK2ct (or ßARKct). By blocking GRK2-mediated phosphorylation, ßarr binding to ß
2
AR is prevented. We studied the offspring both under normal conditions and after surgically induced myocardial infarction (MI). Contractility was significantly augmented in M27/B1KO mice compared to control B1KO's, both in healthy mice (ejection fraction (EF): 69+1.8% vs. 57+1.7%, respectively, p<0.05, n=8) and at 4 weeks post-MI (EF: 42.6+0.1% vs. 25+3.9%, respectively, p<0.05, n=8). In addition, M27/B1KO mice exhibited less cardiac dilatation, increased survival and decreased cardiac apoptosis and infarct size, compared to B1KO's, at 4 weeks post-MI. At the molecular level, M27/B1KO hearts displayed significantly less membrane recruitment of PDEs, upregulation of the anti-apoptotic Bcl-2 and a more favorable inflammatory cytokine profile vs. B1KO hearts. Thus, cardiac GRK2 inhibition by GRK2ct increases both ß
2
AR-dependent contractility and survival in post-MI heart failure and could be pursued in situations where ß
2
AR agonism is beneficial, e.g. in patients awaiting cardiac transplantation.
Abstract 104: Protein Kinase GIα Functions in the Cardiac Myocyte as a Tonic Inhibitor of Pathologic Cardiac Hypertrophy In Vivo
Authors: Blanton Robert M , Mendoza James P , Aronovitz Mark , Kass David A , Mendelsohn Michael E , Karas Richard H
Published: July 2021

Objectives:
We and others previously demonstrated that activation of the NO-cGMP-Protein Kinase G (PKG) pathway inhibits cardiac hypertrophy and remodeling in vivo. However, it remains untested whether PKG specifically in the cardiac myocyte (CM) mediates these effects. We therefore tested the hypothesis that PKGIα inhibits pathologic cardiac hypertrophy through a specific role in the CM.

Methods and Results:
We created and characterized mice with CM-restricted excision of PKGIα. Mice were generated in which the PKGI exon 1 (specific for the Iα isoform) was flanked by loxP sites. We crossed these PKGIα
fl/fl
mice with αMHC-Cre mice which constitutively express Cre recombinase selectively in the CM. The resultant PKGIα
fl/fl
/ αMHC-Cre+/- mice were compared with PKGIα
fl/fl
/ αMHC-Cre-/- littermate controls (termed PKG

CMKO

and Ctrl, respectively). By age 3 months (n=5 per genotype), male PKG

CMKO

mice developed atrial and LV hypertrophy compared with Ctrl littermates PKG

CMKO

atrial weight/tibia length 0.33 ± 0.03 mg/mm vs 0.22 ± .01 in Ctrl,
P
<0.05; PKG

CMKO

LV/TL 5.0 ± 0.2 mg/mm vs 4.1 ± 0.4 in Ctrl,
P
<0.05). LV CM cross sectional area also increased in the 3 month old PKG

CMKO

mice (9445 ± 282 pixels PKG

CMKO

vs 8273 ± 213 in Ctrl, n>400 cells/genotype, 5 hearts per genotype;
P
<0.001). The systolic index end systolic elastance was decreased in 3 month old PKG

CMKO

mice (PKG

CMKO

3.1 ± 0.4 mmHg/μ l vs 6.1 ± 1.0 in Ctrl-;
P
<0.05). Importantly, blood pressure did not differ between genotypes. By age 6 months, PKG

CMKO

mice developed early mortality (3 of 4 PKG

CMKO

males died at 6 months of age vs 0 of 4 Ctrl males). Total heart and atrial weights of male mice (n=3 PKG

CMKO

, 4 Ctrl) increased in PKG

CMKO

mice (heart weight/tibia length 10.8 ± 1.7 mg/mm in PKG

CMKO

vs 7.1 ± 0.6 in Ctrl;
P
<0.05; atrial weight/tibia length 2.3 ± 1.1 mg/mm in PKG

CMKO

vs 0.30 ± 0.1 Ctrl,
P
<0.05). LV fractional shortening percentage, recorded at 6 months age, trended lower in the PKG

CMKO

mice as well (35 ± 3% PKG

CMKO

vs 44 ± 4% Ctrl,
P
0.09).

Conclusions:
These data provide the first evidence that PKGIα functions in the CM as a tonic inhibitor of age-dependent pathologic hypertrophy, supporting further study of PKGIα as a therapeutic target in the prevention and treatment of LV remodeling and congestive heart failure.