bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P551: Platelet Activation is Associated with Pulmonary Edema and Renal Injury in a Rat Model of Polymicrobial Sepsis
Authors: Ishimwe Jeanne , McCalmon Maggie L , Shields Corbin A , Gnam Ashley , Williams Jan M , Cornelius Denise C
Published: July 2021

Sepsis, life-threatening organ dysfunction due to a dysregulated host response to infection, is positively correlated with platelet activation. Furthermore, clinical studies have also shown that platelet activation is associated with sepsis severity, suggesting a role for platelets in sepsis pathophysiology. Despite this correlation, the underlying mechanisms by which activated platelets contribute to sepsis are under investigated. In preliminary studies, we set out to determine if platelet activation is associated with multi-organ dysfunction and injury in a rat model of chronic polymicrobial abdominal sepsis. Sepsis was induced via cecal ligation and puncture (CLP) followed by cecum removal 24 hours post-CLP. At 72 hours post-CLP, blood, urine, and tissues were collected for analysis. Platelet activation was measured via flow cytometry. Lung wet/dry ratio and plasma creatinine were measured to assess lung edema and renal injury, respectively. Platelet activation doubled in CLP rats versus Sham rats. Activated platelets increased from 3.8±1.7% of the gated population in Sham animals (n=5) to 9.2±1.9% of the gated population in CLP animals (n=5; p=0.07). Lung wet/dry ratio significantly increased from 3.9±0.2 in Sham (n=8) to 6.7±1 in CLP rats (n=8; p<0.05). Furthermore, plasma creatinine increased by 33% from 0.55±0.3 mg/dL in Sham animals (n=6) to 0.73±0.06 mg/dL in CLP rats (n=8; p<0.05), indicating a decrease in renal function. These data demonstrate, for the first time, an increase in platelet activation in response to CLP, and identifies an association of activated platelets with pulmonary edema and reduced renal function in the cecal ligation and puncture rat model of abdominal polymicrobial sepsis. Future studies will investigate the underlying mechanisms by which activated platelets contribute to multi-organ dysfunction and injury in sepsis.
Cellular Mechanisms Underlying the Low Cardiotoxicity of Istaroxime
Authors: Racioppi María Florencia , Burgos Juan Ignacio , Morell Malena , Gonano Luis Alberto , Vila Petroff Martín
Published: July 2021

Background

Istaroxime is an inhibitor of Na
+
/K
+
ATPase with proven efficacy to increase cardiac contractility and to accelerate relaxation attributable to a relief in phospholamban‐dependent inhibition of the sarcoplasmic reticulum Ca
2+
ATPase. We have previously shown that pharmacologic Na
+
/K
+
ATPase inhibition promotes calcium/calmodulin‐dependent kinase II activation, which mediates both cardiomyocyte death and arrhythmias. Here, we aim to compare the cardiotoxic effects promoted by classic pharmacologic Na
+
/K
+
ATPase inhibition versus istaroxime.

Methods and Results

Ventricular cardiomyocytes were treated with ouabain or istaroxime at previously tested equi‐inotropic concentrations to compare their impact on cell viability, apoptosis, and calcium/calmodulin‐dependent kinase II activation. In contrast to ouabain, istaroxime neither promoted calcium/calmodulin‐dependent kinase II activation nor cardiomyocyte death. In addition, we explored the differential behavior promoted by ouabain and istaroxime on spontaneous diastolic Ca
2+
release. In rat cardiomyocytes, istaroxime did not significantly increase Ca
2+
spark and wave frequency but increased the proportion of aborted Ca
2+
waves. Further insight was provided by studying cardiomyocytes from mice that do not express phospholamban. In this model, the lower Ca
2+
wave incidence observed with istaroxime remains present, suggesting that istaroxime‐dependent relief on phospholamban‐dependent sarcoplasmic reticulum Ca
2+
ATPase 2A inhibition is not the unique mechanism underlying the low arrhythmogenic profile of this drug.

Conclusions

Our results indicate that, different from ouabain, istaroxime can reach a significant inotropic effect without leading to calcium/calmodulin‐dependent kinase II–dependent cardiomyocyte death. Additionally, we provide novel insights regarding the low arrhythmogenic impact of istaroxime on cardiac Ca
2+
handling.
Cardiovascular Biomarkers of Obesity and Overlap With Cardiometabolic Dysfunction
Authors: Lau Emily S. , Paniagua Samantha M. , Zarbafian Shahrooz , Hoffman Udo , Long Michelle T. , Hwang Shih‐Jen , Courchesne Paul , Yao Chen , Ma Jiantao , Larson Martin G. , Levy Daniel , Shah Ravi V. , Ho Jennifer E.
Published: July 2021

Background

Obesity may be associated with a range of cardiometabolic manifestations. We hypothesized that proteomic profiling may provide insights into the biological pathways that contribute to various obesity‐associated cardiometabolic traits. We sought to identify proteomic signatures of obesity and examine overlap with related cardiometabolic traits, including abdominal adiposity, insulin resistance, and adipose depots.

Methods and Results

We measured 71 circulating cardiovascular disease protein biomarkers in 6981 participants (54% women; mean age, 49 years). We examined the associations of obesity, computed tomography measures of adiposity, cardiometabolic traits, and incident metabolic syndrome with biomarkers using multivariable regression models. Of the 71 biomarkers examined, 45 were significantly associated with obesity, of which 32 were positively associated and 13 were negatively associated with obesity (false discovery rate
q
<0.05 for all). There was significant overlap of biomarker profiles of obesity and cardiometabolic traits, but 23 biomarkers, including melanoma cell adhesion molecule (MCAM), growth differentiation factor‐15 (GDF15), and lipoprotein(a) (LPA) were unique to metabolic traits only. Using hierarchical clustering, we found that the protein biomarkers clustered along 3 main trait axes: adipose, metabolic, and lipid traits. In longitudinal analyses, 6 biomarkers were significantly associated with incident metabolic syndrome: apolipoprotein B (apoB), insulin‐like growth factor‐binding protein 2 (IGFBP2), plasma kallikrein (KLKB1), complement C2 (C2), fibrinogen (FBN), and N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP); false discovery rate
q
<0.05 for all.

Conclusions

We found that the proteomic architecture of obesity overlaps considerably with associated cardiometabolic traits, implying shared pathways. Despite overlap, hierarchical clustering of proteomic profiles identified 3 distinct clusters of cardiometabolic traits: adipose, metabolic, and lipid. Further exploration of these novel protein targets and associated pathways may provide insight into the mechanisms responsible for the progression from obesity to cardiometabolic disease.
Abstract P006: A Potential Role for β-Arrestins in Cardiac Regeneration
Authors: Gumpert Anna M , Chen Mai , Brinks Henriette , Peppel Karsten , Gao Erhe , Koch Walter J
Published: July 2021

Development of chronic heart failure (HF) syndrome following myocardial injury (MI) is characterized by an extensive loss of myocytes due to substantial apoptosis and necrosis. As bone marrow derived stem cells (BMSCs) are capable of transdifferentiating, they also show potential for regenerating the myocardium after infarction. Stem cell mobilization, egress from the bone marrow and homing to the site of injury can be regulated by signals through G protein-coupled receptors (GPCRs). β-arrestins are known for their signalling and scaffolding functions and as downstream regulators of GPCR desensitization and endocytosis in particular. We have begun to investigate whether β-arrestins play any role in cardiac precursor cell function concentrating on the properties of BMSCs. Using knockout (KO) mice, we investigated the role β-arrestin1 (βArr1) and β-arrestin2 (βArr2) with respect to modulation of regenerative competence of BMSCs and their contribution to cardiac repair following ischemic injury. First, BM-derived cells devoid of either βArr1 or βArr2 were found to proliferate and colonize in a significantly deficient manner compared to BM cells isolated from wild-type (WT) mice. In addition, the number of c-kit positive cardiac stem cells (as a % of total BM) were significantly lower in the βArr KO mice compared to WT. We carried out BM transplant studies to begin to determine whether the βArrs may play a role in cardiac repair. In our study, WT mice were irradiated and then received BM transplants from either WT donors as a control or BM from βArr1 or βArr2 KO mice. Subsequent to BM reconstitution, mice underwent a myocardial infarction (MI) and their condition was followed. Interestingly, chimeric mice with βArr1 and βArr2 KO BM had significantly poorer outcomes than mice receiving WT BM. This included significantly decreased post-MI survival with βArr2 KO BM and both βArr chimeras had significantly lower cardiac function post-MI compared to mice receiving WT BM. Additionally, our analyses of the BM cells and very small embryonic-like stem cells (VSELs) circulating in peripheral blood (PB), indicate that βArr-KO BM and PB contain fewer and less viable cardiac stem/precursor cells compared to WT transplanted controls.
Abstract P009: Notch Signaling Regulates Pluripotent Gene Expressions in Cardiac c-Kit+ Cells
Authors: Zakharova Liudmila , Gaballa Mohamed A
Published: July 2021

We and others have shown that transplantation of explant-derived cells (EDCs) obtained from cardiac biopsies improved cardiac function after myocardial infarction. The current study was designed to examine the molecular mechanisms regulating expression of pluripotent genes in these cells. Toward this end, EDCs were separated based on expression of c-Kit antigen after 21 days in culture. We found that Notch signaling was activated mainly in c-Kit+ cells, but to a lesser degree in c-Kit- cells. In addition, we found that the pluripotency markers Sca-1, Nanog and Sox2 were mainly expressed in c-Kit+ cells. Also, in c-Kit+ cells, forced activation of Notch signaling via over-expression of Notch intracellular domain (NICD) induced cellular and molecular changes typical of epithelial-mesenchymal transition (EMT), evident by a decrease in VE cadherin and increases in N-cadherin, MMP, and ICAM. . Suppression of Notch signaling was associated with c-Kit+ cells exhibition of an epithelial/endothelial morphology. This observation is further supported by the increase in Nanog, Sca1, Sox2, VEGFR2 gene expressions after Notch suppression. In contrast, overexpression of NICD resulted in down-regulation of pluripotency gene expressions. Furthermore, suppression of Notch signaling was coincided with stabilization of β-catenin and accumulation of phosphorylated glycogen synthase kinase 3 beta (pGSK3β) suggestive of a crosstalk between Notch and canonical Wnt pathway. Thus, Notch regulates expression of pluripotency genes and mesenchymal transition of c-Kit+ cardiac EDCs.
Abstract P011: Endothelial Differentiation and Lineage Tracing in Adipocyte-Derived Multipotent Cells
Authors: Jumabay Medet , Abdmaulen Raushan , Yao Yucheng , Bostrom Kristina
Published: July 2021

We previously showed that so-called de-differentiated fat (DFAT) cells, which are derived from mature white adipocytes, spontaneously differentiate into beating cardiomyocytes. Our aim in this study was to investigate if DFAT cells also differentiate into endothelial cells (ECs) in vitro, and to further examine the cellular origin of DFAT cells as well as adipose stromal cells (ASCs) using lineage tracing. First, we examined DFAT and ASCs prepared from aP2-Cre+/+;LacZ ROSA(R26R)+/+ double transgenic mice, which express LacZ under the aP2 promoter. The results revealed that 99.9% of DFAT cells and 45% of the ASCs stained positive for LacZ, supporting that the DFAT cells and part of the ASCs are of adipocytic origin. Second, we allowed newly isolated DFAT cells to spontaneously undergo EC differentiation, which was monitored by expression of EC lineage markers as determined by real-time PCR, immunofluorescence, and FACS. Expression of the EC markers CD31 and VE-cadherin increased progressively during 2 weeks in culture, the percentage of CD31(+) cells increased from 0.0% to 8.3%, and the cells formed multi-cellular tube structures when placed in Matrigel™/Collagen gels. The data supported that a fraction of the DFAT cells differentiate into ECs. Furthermore, the EC differentiation could be enhanced in DFAT cells by treatment with bone morphogenetic protein (BMP)-4 and BMP-9. In addition to EC differentiation, the DFAT cells also expressed markers of other cardiovascular lineages including smooth muscle cells and pericytes. The multipotency of DFAT cells suggests that cellular de-differentiation might be a way for differentiated cells to regain stem cell-like properties. Thus, white mature adipocytes maybe a new stem cell source for cardiovascular regeneration.
Abstract P014: Oxidative Stress Mediated Regulation of Antioxidants in Cardiac Progenitor Cells
Authors: Iyer Gokulakrishnan , Davis Michael E
Published: July 2021

Cardiac diseases are the leading causes of death throughout the world and transplantation of endogenous myocardial progenitor population with robust cardiovascular lineage differentiation potential is a promising therapeutic strategy. Therefore, in vitro expansion and transplantation of cardiac progenitor cells (CPCs) is currently in early clinical testing as a potential treatment for severe cardiac dysfunction. However, poor survival and engraftment of cells is one of the major limitations of cell transplantation therapy. Oxidative stress is increased in the ischemic myocardium and indirect inferences suggest the vulnerability of CPCs to oxidative stress. In this study, we show that in vitro, resident c-kit positive CPCs isolated from rat myocardium are significantly (p<0.05) resistant to superoxide-induced apoptosis compared to cardiomyocytes as analyzed by the number of sub-G1 population following xanthine/xanthine oxidase treatment. Interestingly, CPCs have two to four fold higher basal SOD1 and SOD2 activities (p<0.01) compared to cardiomyocytes and endothelial cells. Superoxide treatment increased expression of SOD1 (p<0.01), SOD2 (p<0.01), and glutathione peroxidase (p<0.05) mRNAs within 6 h of treatment compared to control cells. Recent studies suggest the involvement of AKT in controlling cell death, survival and also expression of SOD enzymes. Therefore, we investigated the involvement of AKT in CPCs subjected to oxidative stress. Western blot analysis revealed that the amount of phosphorylated AKT increased significantly within 10 minutes of xanthine/xanthine oxidase treatment. In addition, treatment with LY294002 - a PI3 kinase/AKT inhibitor, increased apoptosis in CPCs treated with superoxide. Our studies demonstrate a novel finding in which resident progenitor cells are protected from oxidative injury by containing higher basal levels of antioxidants as compared to myocytes. Moreover, under oxidant challenge antioxidant levels are regulated, possibly in an AKT-dependent manner. Further elucidation of this pathway may lead to novel therapeutic opportunities.
Abstract P023: HDAC Inhibition Promotes Cardiogenesis and the Survival of Embryonic Stem Cells Through Proteasome-Dependent Pathway
Authors: Zhao Ting C , Chen Hongping , DeNicola Megan , Zhao Yu , Qin Xin , Zhang Ling , Zhuang Shugang , Liu Paul , Ma Julio
Published: July 2021

Objectives:
Histone deacetylase (HDAC) inhibition plays a crucial role in mediating cardiogenesis and myocardial protection, whereas HDAC degradation has recently attracted attention in mediating the biological function of HDACs. However, it remains unknown whether HDAC inhibition modulates cardiogenesis and embryonic stem cell (ESC) survival through the proteasome pathway.

Methods and Results:
Using the well-established mouse CGR8 mouse ESC culture, we evaluated the impact of HDAC inhibition and proteasome pathway on the cell death, viability and apoptosis in ESCs in response to oxidant stress (100μ mol/L hydrogen peroxides). We demonstrated that HDAC inhibitors, both TSA (50 nmol/L) and sodium butyrate (200 μ mol/L) that causes the pronounced reduction of HDAC4 activity, decreased cell death and increased viability of ESCs. HDAC inhibition reduced the cleaved caspase 3, 6, 9, PARP and TUNE-positive ESCs, which were abrogated with MG132 (0.5 μ mol/L), a specific proteasome inhibitor. Furthermore, we employed
in vitro
“hanging drop” methods to carry out two weeks of embryoid bodies (EB) culture to assess the effect of HDAC inhibition and proteasome pathway on cardiogenesis. HDAC inhibition stimulates the growth of EB, which is associated with faster spontaneous rhythmic contraction. HDAC inhibition increased the up-regulation of GATA4, MEF2, NKX2.5, cardiac actin, and α-SMA mRNA and protein levels that were abrogated by MG132. Immunostaining analysis demonstrates that trichostatin A and sodium butyrate resulted in a significant increase in cardiac lineage commitments that were blocked by the proteasome inhibition. Notably, HDAC inhibitors led to noticeable HDAC4 degradation, which was effectively prevented by MG132. Luciferase assay demonstrates an activation of MEF2 cardiac transcriptional factor by HDAC inhibition, which was repressed by MG132, revealing that the degradation of HDAC4 allows the activation of MEF2.

Conclusions:
Taken together, our study is the first to demonstrate that HDAC inhibition through proteasome pathway forms a novel signaling to determine the cardiac lineage commitment and elicit the survival pathway, which is dependent on specific HDAC4 degradation and subsequent MEF2 activation of ESCs.
Abstract P028: The Effect of Cotransplantation of Human Embryonic Stem Cell--Derived Cardiomyocytes and Mesenchymal Stem Cells on Ventricular Function and Remodeling After Myocardial Infarction in Nude Rats
Authors: Dai Wangde , Kearns-Jonker Mary , Gerczuk Paul , Gunthart Mirja , Girish Nandini , Pera Martin , Mummery Christine , Kloner Robert A
Published: July 2021

We determined whether co-transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and mesenchymal stem cells (MSCs) had additive effects on left ventricular (LV) function and remodeling compared with hESC-CMs treatment alone in a rat myocardial infarction model. One week after myocardial infarction induced by left coronary ligation, nude rats received hESC-CMs (n=15), hESC-CMs + MSCs (n=16), hESC-CMs + MSCs transduced to over-express hemeoxygenase 1(HO-1) (n=14), or saline (n=19). At 4 weeks after treatment, LV function was assessed by left ventriculography, echocardiography and Millar catheter. Some hearts were processed for histology. The LV ejection fraction (LVEF) in sham noninfarcted hearts was 78.1±1.8% (n=5) in the nude rat model. LVEF in the 3 cell treated groups (hESC-CMs: 67.6±1.4%; hESC-CMs + MSCs: 67.2±1.6%; and hESC-CMs + MSCs with HO-1: 66.3±1.7%) were comparable, and significantly higher than in the saline group (60.6±1.2%, n=19; p=0.0022). There was a trend for less left ventricular akinesis and dyskinesis (expressed as % of LV circumference) assessed by left ventriculography at 8.96±1.9% in hESC-CMs group, 8.37±1.67% in hESC-CMs + MSCs group and 4.57±1% in hESC-CMs + MSCs with HO-1 group compared to 10.73±1.76% in the control group (p=0.056). There was a nonsignificant trend for LV fractional shortening assessed by echocardiography to be greater in the 3 cell groups (32.1±3.9% in hESC-CMs; 30.2±2% in hESC-CMs + MSCs; 31.0±1.9% in hESC-CMs + MSCs with HO-1) compared to 24.8±2.2% in the saline group (p=0.18). Expansion index reflecting thinning and dilatation of the infarct was significantly worse in the control group at 0.71±0.05 versus the other 3 groups at 0.32±0.05 (p=0.0039). Thus, cell therapy by hESC-CMs alone or combination transplantation of hESC-CMs and MSCs (with or without HO-1) significantly improved LV function assessed by left ventriculography and reduced expansion index. However, co-transplantation of hESC-CMs and MSCs did not provide better functional improvement compared with hESC-CMs treatment alone after left coronary artery occlusion in nude rats over a period of 4 weeks, suggesting that there may be a ceiling effect above which LV function can not further improve after cell therapy.
Abstract P041: A Novel Preclinical Strategy for Identifying Cardiotoxic Kinase Inhibitors and Mechanisms of Cardiotoxicity
Authors: Cheng Hui , Kari Gabor , Rodeck Ulrich , Dicker Adam , Force Thomas
Published: July 2021

Rationale:
1) Despite intense interest in strategies to predict which tyrosine kinase inhibitor (TKI) cancer therapeutics may be associated with cardiotoxicity, current approaches are inadequate. 2) Sorafenib is a TKI of concern since it inhibits growth factor receptors and Raf-1/B-Raf, kinases that are upstream of ERKs and signal cardiomyocyte survival in the setting of stress.

Objectives:
1) Explore the potential use of zebrafish as a pre-clinical model to predict cardiotoxicity. 2) Determine whether sorafenib has associated cardiotoxicity and, if so, define the mechanisms.

Methods and Results:
We find that the zebrafish model is readily able to discriminate a TKI with little or no cardiotoxicity (gefitinib) from one with demonstrated cardiotoxicity (sunitinib). Sorafenib, like sunitinib, leads to cardiomyocyte apoptosis, contractile dysfunction and ventricular dilatation in zebrafish. In cultured rat cardiomyocytes, sorafenib induces cell death. This can be rescued by adenovirus-mediated gene transfer of constitutively active MEK1 which restores ERK activity even in the presence of sorafenib. While growth factor-induced activation of ERKs requires Raf, α-adrenergic-induced activation of ERKs does not. Consequently, activation of α-adrenergic signaling virtually abrogates sorafenib-induced cell death. Consistently, inhibition of α-adrenergic signaling with prazosin augments sorafenib-induced contractile dysfunction in zebrafish.

Conclusions:
1) Zebrafish may be a valuable pre-clinical tool to predict cardiotoxicity. 2) We identify a here-to-fore unknown pathway that bypasses Raf to activate ERKs, thereby limiting sorafenib cardiotoxicity. Importantly, given that the majority of men over the age of 60 have prostatic hypertrophy, for which α-adrenergic antagonists are the primary therapy, our findings suggest the consequences of the concomitant use of these agents with sorafenib should be explored.
Abstract P045: Two Forms of Autophagy in the Heart: Constitutive Autophagy via an LC3-Dependent Pathway and Starvation-Induced Autophagy via a Rab9-Dependent Pathway
Authors: Monden Yoshiya , Maejima Yasuhiro , Komatsu Masaaki , Kundu Mondira , Sadoshima Junichi
Published: July 2021

Autophagy is an intracellular bulk degradation process in which cytosolic proteins/organelles are sequestered into double-membrane vesicles termed autophagosomes to be fused with lysosomes for degradation. Recent evidence suggests that an alternative form of autophagy, which utilizes distinct sources of membrane for autophagosome formation, may exist. Whether alternative autophagy exists in the heart is unknown. Cardiac-specific
Atg7
-KO (
Atg7
-CKO) and systemic
Ulk1
-KO mice were subjected to starvation for 48 hours. LC3-II expression and p62 degradation were increased in wild-type (WT) mice after starvation (ST), indicating increased autophagy. In
Atg7
-CKO mice, LC3-II expression was reduced and p62 was markedly increased at baseline and in response to ST, indicating inhibition of LC3-dependent autophagy. In contrast, in
Ulk1
-KO mice, neither LC3-II nor p62 was affected at baseline or in response to ST. However, autolysosome formation evaluated by Lamp2/Rab9 staining, indicators of unconventional autophagy, was abolished. Furthermore, the level of polyubiquitinated protein was increased after ST. Echocardiographic analyses showed that fractional shortening (FS) was maintained after ST in WT mice (38%). However, in
Atg7
-CKO mice, FS deteriorated at baseline (22%) but was not further affected by ST (20%). In contrast, in
Ulk1
-KO mice, FS was relatively preserved at baseline (32%) but remarkably decreased during ST (19%). After glucose deprivation (GD) GFP-LC3 was co-localized with calnexin (ER marker) in Ad-sh-Control- and Ad-sh-Ulk1-transduced, but not in Ad-sh-Atg7-transduced, cultured cardiomyocytes (CMs) in vitro. In contrast, Lamp2 was co-localized with TGN46 (Golgi marker) in Ad-sh-Control and Ad-sh-Atg7-transduced, but not in Ad-sh-Ulk1-transduced, CMs after GD. These results suggest that the autophagosomes are derived from ER in LC3-dependent autophagy and Golgi in Rab9-dependent autophagy in CMs. Atg7 is required for maintaining cardiac function at baseline in an LC3-dependent manner, whereas Ulk1 is required for preserving cardiac function during ST, possibly through stimulation of Rab9-dependent autophagy. These results support the presence of alternative autophagy in the heart.
Abstract P070: NEU3 Sialidase Overexpression Activates Cell Survival in Murine Skeletal Myoblasts C2C12 and Protects Them from Hypoxia
Authors: Anastasia Luigi , Scaringi Raffaella , Papini Nadia , Garatti Andrea , Menicanti Lorenzo , Allevi Pietro , Venerando Bruno , Tettamanti Guido , Gelfi Cecilia , Leri Annarosa
Published: July 2021

Membrane-bound sialidase NEU3 increase during skeletal muscle differentiation has been shown to protect myoblasts from apoptosis and drive the differentiation process [1]. Thus, the objective of this study was to assess whether up-regulation of NEU3 would enhance the ability of murine skeletal muscle cells to resist to hypoxia, ultimately opposing cell death. We found that C2C12 myoblasts overexpressing NEU3 (L-NEU3) became highly resistant to 1% oxygen or 200 mM deferoxamine induced hypoxia. Moreover, L-NEU3 myoblasts survived a seven-day treatment of combined hypoxia and low serum (2% horse serum used to induce myoblast differentiation), without any significant cell loss. On the contrary, wild type C2C12 could not resist to these culturing conditions and all died within 48h. Real Time PCR showed NEU3 expression increase during all hypoxic treatments both in C2C12 and L-NEU3 cells, suggesting an endogenous NEU3 activation under these conditions. Moreover, we found that NEU3 over-expression activated pro-survival signalling pathways through up-regulation and activation of EGF receptor. Overall, our data support the hypothesis that NEU3 may play a critical role in the response of skeletal myoblasts to hypoxia and the preservation of cell viability by activating pro-survival signalling pathways. [1] Anastasia L. et al. J.Biol.Chem. 2008, 283 (52): 36265–36271.
Abstract P076: Essential Role of Neuronal Nitric Oxide Synthase in Ca
2+
/Calmodulin-Dependent Protein Kinase II Activation in Cardiac Myocytes During β-Adrenergic Stimulation
Authors: Tang Lifei , Roof Steve , Ziolo Mark
Published: July 2021

RATIONALE: Stimulation of the beta-adrenergic (beta-AR) pathway leads to positive inotropy, and is the major regulator of heart function. In addition to the traditional PKA pathway, activation of Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) and neuronal nitric oxide synthase (NOS1) signaling also play important roles in the positive inotropy by modulating ryanodine receptor (RyR) activity.

OBJECTIVE: The upstream activators of CaMKII during beta-AR stimulation are not well defined. The purpose of this study is to investigate if there is any cross-talk between the CaMKII and NOS1 signaling pathways.

METHODS AND RESULTS: Myocytes were isolated from wildtype (WT, C57Bl/6) and NOS1
−/−
mice. Ca
2+
transients (Fluo-4) and cell shortening (edge detection) were simultaneously measured. RyR activity was measured using the SR Ca
2+
leak/load relationship. CaMKII was acutely inhibited by KN93. In WT myocytes, KN93 decreased beta-AR stimulated contraction (Ca2+ transients (Fluo-4) and cell shortening). In NOS1
−/−
myocytes, beta-AR stimulated contraction was blunted compared to WT, and KN93 had no further effect on contraction. Furthermore, beta-AR stimulated RyR activity was blunted in NOS1
−/−
compared to WT myocytes. As with contraction, KN93 decreased beta-AR stimulated RyR activity in WT myocytes, but had no effect in NOS1
−/−
myocytes.

CONCLUSION: These data suggest that NOS1 is required for CaMKII-mediated RyR activation which contributes to positive inotropy during beta-AR stimulation. Further study of this pathway is warranted since CAMKII expression and activity are increased in cardiac hypertrophy and heart failure. A better understanding of the NOS1/CaMKII pathway during beta-AR stimulation has beneficial therapeutic potential for heart diseases.
Abstract P078: Dysregulated Calcium Handling in a Conditional Cardiac Myosin Binding Protein C Knockout Model of Hypertrophic Cardiomyopathy
Authors: Capes E M , Chen Peter P , Fitzsimons Daniel P , Valdivia Hector H , Moss Richard L
Published: July 2021

Heritable cardiomyopathy (HCM) is the leading cause of sudden cardiac arrest (SCA) in young people, affecting 1 in 500 individuals. HCM is chiefly caused by mutations in myofibrillar proteins of the cardiac sarcomere, and cardiac myosin binding protein-C (cMyBP-C, encoded by MYBPC3) is one of the most commonly affected. cMyBP-C, an accessory protein that binds tightly to myosin, has an important role in thick filament regulation. Mice with genetic ablation of MYBPC3 exhibit cardiac hypertrophy, reduced ejection fraction, and increased relaxation times
in vivo
. Experiments with explanted hearts from these mice exhibit greater susceptibility to arrhythmias compared to WT, suggesting derangement of Ca
2+
handling. The molecular mechanisms underlying the progression of HCM are poorly understood, and are difficult to tease apart in constitutive knock out models due to potential compensatory changes that can mask important aspects of the disease phenotype. We used a tamoxifen-induced conditional MYBPC3 knock out mouse to investigate the onset and progression of the disease before development of overt hypertrophy. We recorded Ca
2+
transients in enzymatically isolated cardiomyocytes at several pacing frequencies using a META LSM 510 confocal microscope. We found that cell shortening in conditional CMs (con-CMs) was reduced by as much as 54% (e.g., 5.46% ± 0.869 in con-CMs, vs. 11.91% ± 0.423 shortening in WT-CMs). Ca
2+
release was often dissynchronous, possibly indicating remodeling at the diads. Ca
2+
transient amplitude was significantly lower in con-CMs across all pacing frequencies (reduced by at least 22%; e.g., fluorescence intensity ratios of 7.983 ± 0.537 in con-CMs vs. 10.332 ± 0.502 in WT CMs). Further, the time constant of transient decay (tau) was dramatically increased in con-CMs (mean 217.27 ± 18.052 msec for con-CMs vs. 107.44 ± 4.045 msec in WT CMs). In some cells (∼5%), this prolongation of transient decay was so profound (e.g., up to 600 milliseconds) that a second Ca
2+
release occurred before [Ca
2+
]
i
could return to baseline. These data suggest that conditional knock out of cMyBP-C produces not only contractile dysfunction, but also pro-arrhythmic alterations in Ca
2+
handling.
Abstract P079: Cardiac Contractility in Response to Isoproterenol from Gravin Knockout (AKAP12) Mice
Authors: McConnell Bradley , Yin Xing , Li Zhitao , Wijaya Cori , Diaz Andrea , Siddiqui Muhammad , Rabaha'h Abeer , Guillory Ashley , Ga Wei Dong
Published: July 2021

Gravin (AKAP12), one of the A-Kinase-Binding-Proteins (AKAPs), serves as a scaffold protein linking β
2
-adrenergic receptor (β
2
-AR), phosphatase 2B and several kinases including Protein Kinase A (PKA) and Protein Kinase C (PKC). The presence of gravin facilitates signal transduction of β
2
-AR and thus affects cardiac excitation-contraction coupling. We previously showed decreased cardiac hypertrophy, increased contractility and decreased arrhythmias in gravin-KO mice, following chronic β-AR stimulation. This response was associated with enhanced [Ca
2+
]
i
homeostasis. Here, we test whether cardiac contraction is also affected in gravin knock-out (gravin-KO) mice. Trabeculae or small papillary muscles from the right ventricles were mounted between a force transducer and a motor arm, and superfused with K-H solution (pH 7.4) at room temperature. Developed force increased as external Ca
2+
([Ca
2+
]
o
) was raised from 1 to 10 mM in both gravin-KO and wide-type (WT) muscles. Developed force and intracellular Ca
2+
transient (Ca
2+
i
) increased in a dose dependent manner as [Ca
2+
]
o
was raised. Gravin-KO muscles had lower Ca
2+
i
at any given [Ca
2+
]
o
, but had similar force at [Ca
2+
]
o
>4.0 mM; at higher [Ca
2+
]
o
s, developed forces was significantly lower. Isoproterenol (ISO, 0.05-200nM) increased force and Ca
2+
i
in both groups of muscles. However, force and Ca
2+
i
started to blunt in gravin-KO muscle at higher ISO doses and became significantly lower at ISO does >50 nM. Both force and Ca
2+
i
were lower at a given rest interval in gravin-KO muscles with no changes in recirculation fraction (RF). These results show that gravin-KO muscles maintain their response to both Ca
2+
and ISO with reduced capacity at higher doses, and to rest potentiations with lower magnitudes, but with no changes in recirculation fraction of Ca
2+
through the sarcoplasmic reticulum (SR). These findings suggest: (1) gravin-KO muscles may likely have smaller Ca
2+
content in the SR; (2) gravin plays an important modulatory role in the argumentation of force by Ca
2+
and β
2
-AR stimulation; (3) at physiological [Ca
2+
]
o
, gravin-KO increases myofilament Ca
2+
responsiveness. Ongoing experiments are focused on changes myofilament Ca
2+
responsiveness in gravin-KO mice.
Abstract P107: Interactions of the BDNF Val66Met Polymorphism, Gender, and High-Carbohydrate Diets on Risk Factors for Cardiovascular Diseases
Authors: Hu Minshan , Li Ronghui , Zhang Zhen , Song Yongyan , Li Yuanhao , Fang Ding Zhi
Published: July 2021

Background_

Brain-derived neurotrophic factor (BDNF) is a member of the “neurotrophin” family of growth factors, it was lately found involved in cardiovascular diseases(CVDs), somehow whether BDNF has any effect on dyslipidemia has not been detected. We hypothesized that Val (Valine) 66Met (Methionine ) polymorphism of BDNF could change risk factors of CVDs including serum lipids profile difference, and interaction with a high-CHO diet could modified these factors.

Methods and Results_

Fifty-six young and healthy Chinese subjects were recruited. A stabilization diet of 54% carbohydrate was first given to exclude these subjects’ own various diet background interferences, and then a high-CHO diet of 70% carbohydrate was followed to study its intervention effect. The serum lipid profiles were analyzed at baseline, after the stabilization diet and after the high-CHO diet. After the stabilization diet, significant difference among BDNF Val66Met polymorphism was found for the females’ body mass index (BMI) with the means appeared at Val/Val>Val/Met>Met/Met. After the high-CHO diet, most of the cardiovascular risk factors were changed to a more favorable direction and the effect has gender difference. But interestingly, both TAG and food intake notably increased in female Met carriers.

Conclusions_

The above results indicate that although Met mutation could be protective to CVDs in females, after interacting with high-CHO diet, due to the rising triacylglycerol (TAG) and food intake, the risk for carbohydrate-induced hypertriacylglycerolemia happens in these female Met carriers was
actually
increased.
Abstract P111: Rheb-mTOR Signaling Pathway Regulates Cardiomyocyte Mass During Post-Neonatal Period
Authors: Tamai Takahito , Hikoso Shungo , Murakawa Tomokazu , Oyabu Jota , Oka Takafumi , Taneike Manabu , Takeda Toshihiro , Yamaguchi Osamu , Nakayama Hiroyuki , Nishida Kazuhiko , Komuro Issei , Otsu Kinya
Published: July 2021

Rheb (Ras homologue enriched in brain) is a major activator of mTOR. Rheb-mTOR pathway is a critical mechanism for maintenance of homeostasis, cell growth and stress response by regulating both protein synthesis and degradation. In this study, we attempted to clarify the role of Rheb-mTOR pathway in the heart using cardiac-specific Rheb-deficient mice (Rheb
−/−
). We generated floxed Rheb mice and crossed them with transgenic mice expressing Cre recombinase in cardiac-specific mannner to generate Rheb
−/−
. Rheb
−/−
were born in Mendelian ratio, but they started to die 8 days after birth and all of them had died until 10 days after birth. Echocardiographic analysis revealed that chamber dimension and contractile function of Rheb
−/−
were indistinguishable from those of control mice (Rheb
+/+
) 5 days after birth. However, Rheb
−/−
exhibited cardiac dilatation and reduced contractility 8 days after birth (LV end diastolic dimension, Rheb
−/−
: 2.5±0.2 mm vs. Rheb
+/+
: 2.1±0.2 mm, p<0.01, fractional shortening, Rheb
−/−
: 19.7 ± 9.7 % vs. Rheb
+/+
: 48.6 ± 8.8 %, p<0.01). These suggest that Rheb
−/−
died of cardiac dysfunction and heart failure. Heart weight and cross-sectional area of cardiomyocytes were significantly lower in Rheb
−/−
8 days after birth. Electron microscopic analysis revealed that the area of sarcomere was significantly lower in Rheb
−/−
cardiomyocytes. Expressions of sarcomeric proteins, such as myosin heavy chain, actin or desmin, were decreased in Rheb
−/−
, while the mRNA expression of desmin was significantly increased in Rheb
−/−
. Thus impairment of cardiomyocyte growth observed in Rheb
−/−
could be due to either increased degradation or decreased translation. Although autophagic activity was enhanced in Rheb
−/−
heart, ablation of Atg5, an essential molecule for autophagy, could not prevent premature death of Rheb
−/−
. On the other hand, polysome analysis revealed that the mRNA translation activity had decreased in Rheb
−/−
heart compared with Rheb
+/+
. Thus, we concluded that Rheb-mTOR pathway in the heart is essential to regulate mRNA translation activity and protein synthesis, thereby to cardiomyocyte growth in neonatal period.
Abstract P117: Does Mammalian Target of Rapamycin Complex-2 Regulate Protein Degradation Pathways in the Heart?
Authors: Shende Pankaj S , Morandi Christian , Brink Marijke
Published: July 2021

Background:
Mammalian target of rapamycin (mTOR) occurs in the cell in two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2, which contain raptor and rictor, respectively. We have recently demonstrated that mTORC1 activity is required for the hypertrophic response to aortic constriction and for the normal cardiac homeostasis. Moreover, we showed that raptor deletion causes Akt hyperphosphorylation and lower gene expression of Atrogin-1 and MuRF1, two muscle specific E3 enzymes part of the proteasomal degradation pathway. These results suggested that, as a counter-regulatory response to mTORC1 inactivation, mTORC2 reduces protein degradation via phosphorylation of Akt at Ser473. It has previously been shown that the phosphorylation state of Akt regulates Atrogin-1 and MuRF1 gene expression at the transcriptional level via FoxO. In the present study, we have tested whether mTORC2 inactivation induces the ubiquitin-proteasomal degradation pathway.

Methods and results:
In 10 week-old male mice, transgenic for MerCreMer driven by the α-MHC promoter and homozygous for floxed rictor, deletion of the rictor gene was induced by tamoxifen. Protein and RNA extracts were analyzed at three weeks after tamoxifen by Western blotting and qPCR, respectively. The rictor gene was efficiently ablated from the heart as its protein levels were reduced. Phosphorylation of Akt and PKC-α, direct targets of mTORC2, was abolished, identifying these signaling molecules as downstream targets of mTORC2 in the heart. However, the reduced Akt phosphorylation was not associated with any changes in the mRNA levels of Atrogin-1, MuRF1, and MuRF3.

Conclusion:
Our study suggests that mTORC2-induced phosphorylation of Akt is not required for the maintenance of low expression levels of these genes. Further studies are ongoing to identify the factors that modulate Atrogin-1 and MuRF1 gene transcription in the heart.
Abstract P129: G Protein--Coupled Receptor Kinase-2 (GRK2) Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts
Authors: D'Souza Karen M , Malhotra Ricky , Philip Jennifer L , Staron Michelle L , Theccanat Tiju , Akhter Shahab A
Published: July 2021

Cardiac fibroblasts (CF) make up 70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. The objective of this study was to investigate the potential role of GRK2 in modulating CF transformation to myofibroblasts and collagen synthesis in adult human CF isolated from normal and failing left ventricles. CF isolated from failing ventricles showed a significant increase in expression of collagen I, III and VI compared with controls. α-SMA was increased 2-fold over controls, consistent with CF transformation to myofibroblasts. Baseline collagen synthesis was elevated 2-fold in failing CF and was not inhibited by isoproterenol (ISO)-stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in failing CF as assessed by basal and ISO-stimulated cAMP production. The primary mechanism appears to be a 2.5-fold increase in GRK2 activity as GRK2 phosphorylates and uncouples agonist-occupied β-ARs. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following ISO stimulation. In contrast, siRNA-mediated knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity by adenoviral-mediated βARKct expression or GRK2 knockdown in failing CF led to a significant decline in collagen and α-SMA expression. GRK2 inhibition restored β-AR signaling and ISO-stimulated inhibition of collagen synthesis and also significantly decreased collagen synthesis in response to TGFβ stimulation. In conclusion, GRK2 appears to play a significant role in regulating CF transformation and collagen synthesis in adult human CF and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.
Abstract P130: Estrogen Receptor β Mediates the Rescue of Cardiac Function in Advanced Heart Failure by Promoting Neoangiogenesis and Reducing Fibrosis
Authors: Iorga Andrea , Partow-Navid Rod , Matori Humann , Li Jingyuan , Umar Soban , Eghbali Mansoureh
Published: July 2021

Estrogen can act via the estrogen receptor alpha (ERa) or estrogen receptor beta (ERb) to exert its biological effects, and both of these receptors are present in the heart. We have previously shown that short-term estrogen (E2) treatment can rescue pressure overload-induced decompensated heart failure (HF) in mice, and that this rescue is achieved mainly through the ERb. Furthermore, E2 has been shown to regulate angiogenesis in different tissues. Because HF has been associated with decreased angiogenesis and increased fibrosis, here we investigated whether the E2-induced rescue of HF by the selective ERb agonist DPN can regulate cardiac fibrosis and neoangiogenesis. We used transaortic constriction to induce HF, and once the ejection fraction (EF) reached ∼30%, one group of animals was sacrificed (HF group), and the other three groups received either 17b-estradiol via a subcutaneous pellet implant (0.012mg/pellet, n=16), selective ERa agonist (PPT, 0.625mg/kg/day), or selective ERb agonist (DPN, 0.625mg/kg/day) for 10 days. Serial echocardiography was performed to monitor cardiac structure and function. As expected, E2 rescued HF by restoring EF from 33.17±1.12% to 53.05±1.29%. Mice treated with DPN had a significant EF improvement from 33.17±1.12% to 45.25±2.1% (n=7), while the EF of PPT-treated mice did not improve (31.09±2.3%, n=6). Similarly, only the fractional shortening of DPN-treated mice improved from 15.7±0.58% in HF to 21.95±1.65% with DPN treatment vs. 14.72±1.24% with PPT. Next, we examined whether promotion of cardiac neoangiogenesis and suppression of fibrosis by the selective ERb agonist are possible mechanisms in the rescue action of HF by DPN. DPN treatment was able to reverse the interstitial and perivascular fibrosis observed in HF, while PPT had no effect. The selective ERb agonist also stimulated neoangiogenesis, as the capillary density was increased from 0.46±0.04 microvessels/cardiomyocyte in HF to 0.67±0.07 with DPN treatment, whereas PPT treatment had no effect (0.43±0.03). Our data strongly suggests that upregulation of cardiac neoangiogenesis and reversal of fibrosis are pivotal mechanisms in rescuing advanced HF by the estrogen receptor beta agonist DPN.
Abstract P135: Hyperpolarized Carbon-13 Magnetic Resonance Reveals Early- and Late-Onset Changes to Metabolism in the Failing Heart
Authors: Schroeder Marie , Lau Angus Z , Chen Albert P , Barry Jennifer , Tyler Damian J , Clarke Kieran , Connelly Kim A , Wright Graham , Cunningham Charles H
Published: July 2021

Disordered metabolic substrate utilisation has been implicated in the pathogenesis of heart failure (HF). Hyperpolarised (HYP) 13C magnetic resonance, a technique in which the fate of 13C-labelled metabolites can be followed using MR imaging or spectroscopy, has enabled non-invasive assessment of metabolism. The aim of this study was to monitor carbohydrate metabolism alongside cardiac structure, function, and energetics, throughout HF progression. HF was induced in pigs (n=5) by right ventricular pacing at 188 bpm for 5 weeks. Pigs were examined at weekly time points: cine MRI assessed cardiac structure and function, HYP 13C2-pyruvate was administered intravenously and 13C MRS was used to assess 13C-glutamate production via Krebs cycle, 31P MRS assessed myocardial energetics, and HYP 13C1-pyruvate was administered to enable MRI of H13CO3- production from pyruvate dehydrogenase (PDH). At baseline, pigs had a normal left ventricular (LV) cardiac index (CI) and end diastolic volume (EDVi). The PCr/ATP was 2.3 ± 0.2. The 13C-glutamate/13C2-pyruvate was 4.3 ± 0.9%, and the H13CO3-/13C1-pyruvate ratio was 1.6 ± 0.2%. After 1–2 weeks of pacing, CI decreased to 3.3 ± 0.5 l/min/m2, PCr/ATP decreased to 1.7 ± 0.1, and 13C-glutamate/13C2-pyruvate decreased to 2.1 ± 0.6%. With the onset of HF, EDVi increased to 140.3 ± 14.1 ml/m2 and H13CO3-/13C1-pyruvate decreased to 0.5 ± 0.2%. In conclusion, we observed an early defect in Krebs' cycle that occurred alongside impaired cardiac energetics and function. Carbohydrate oxidation via PDH was maintained until the onset of HF. These results encourage use of metabolic therapies to delay/prevent the onset of heart failure in patients.