bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 416: B-Raf Loss Suppresses Extracellular-Regulated Kinase Activation and Cardiomyocyte Proliferation
Authors: Chattergoon Natasha N , McCrohan Sara , Thornburg Kent L , Stork Philip
Published: July 2021

Objectives:
The postnatal heart does not retain the proliferative capacity it had during fetal life. Unlike large mammals, murine cardiomyocytes (CM) continue to divide into the first week of life before terminal differentiation and binucleation. We hypothesized that B-Raf regulates ERK activation in newborn CM and that loss of B-Raf suppresses cyclin levels and reduced proliferation. To test this, we determined whether loss of B-Raf disrupts the
ERK
(extracellular-regulated kinase) cascade and impairs CM growth.

Methods:
CM specific knockout (KO) of B-Raf was generated using CRE/lox (floxed B-Raf x αMHC CRE) resulting in a truncated, unstable B-Raf and a null phenotype. KO mice (α-MHC-CRE / B-Raf
lox/lox
) were compared to CRE negative / B-Raf
lox/lox
mice (wild type; WT). Hearts from 3d and 8d old pups were harvested for molecular analysis of B-Raf signaling and cell cycle markers. Hearts from 3d old pups were harvested and CMs isolated for culture using a trypsin/DNAse digestion. The cells were treated with Isoproterenol (Iso;10uM), forskolin (20uM), and IGF-1 (1ng/ml) for 15 min to determine if the loss of B-Raf results in reduced activation of ERK.

Results:
Heart weight to body weight (HW/BW) ratio was less in 3d KO versus 3d WT (n=50, p<0.05). HW/BW ratio became greater in 8d KO; there was no difference in 3d and 8d HW/BW in WT animals. Baseline B-Raf and phosphorylated ERK levels were reduced in KO hearts (*p<0.05). Cell cycle inhibitors p21 and p53 were increased in 3d KO hearts with decreased levels of all cyclins (p<0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with increased cyclin levels (p<0.05). In vitro ERK activation was blunted in KO CMs by forskolin and Iso compared to IGF-1.

Conclusions:
ERK activation was suppressed in KO hearts resulting in smaller newborn hearts but which exceeded normal HW/BW by 8d. This is may represent premature hypertrophy as the proliferative period of CM development had ended. Cell cycle analysis supports reduced CM mitotis among 8d CM. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.
Abstract 417: Adenosine Diphosphate Ribosyl Cyclase via PI3K-Akt and FOXO3a Up-regulating MMP-9 to Enhance Vascular Smooth Muscle Cell Proliferation and Migration in Mice
Authors: Li Yuming , Li Haitao , Wang Xinfang , Wang Junya , Li Zhongqiu
Published: July 2021

The current study was designed to explore the mechanisms of vascular smooth muscle cell (VSMC) proliferation and migration induced by adenosine diphosphate ribosyl cyclase(ADPRC). In this study, 32 Male ApoE-/- mice(6 weeks old, 18-22g)on a C57BL/6J background were divided into four groups, which received normal chow (n=8, NC group), high-fat Western-type diet (n=8, 0.25% cholesterol, 21% fat,HFD group), high-fat Western-type diet,infusion of 2,2′-dihydroxyazobenzene(DHAB, a ADPRC inhibitor, 2mg/kg/day, n=8, HFD-DHAB group) intraperitoneally or high-fat Western-type diet,infusion of LY294002(a Inhibitor of Akt, 5mg/kg/d, n=8, HFD-LY group) intraperitoneally, for 10 weeks. 8 male C57BL/6J mice served as control. After 10 weeks, mice were anesthetized with chloral hydrate, aorta was removed and immediately frozen in liquid nitrogen. Aortic atherosclerotic lesions, VSMC proliferation and migration were assessed by histomorphological observation, smooth muscle actin-α(α-SMA)and proliferating cell nuclear antigen (PCNA) examination. ADPRC expression and alterations of Akt, FOXO3a, phospho-FOXO3a and MMP-9 were determined by RT-PCR, Western Blot, Immunohistochemistry or Immunofluorescence. The results showed that, in aortic atherosclerotic lesions derived from atherosclerotic mice of HFD group, an increased VSMC proliferation and migration, reflected by the up-regulation of α-SMA and PCNA expression, were observed followed by increased expression of ADPRC, Akt, FOXO3a, phospho-FOXO3a and MMP-9. The enhanced expression of ADPRC and followed alterations of FOXO3a, phospho-FOXO3a, MMP-9 as well as α-SMA, PCNA, VSMC proliferation and migration were absent in NC group and C57BL/6J control mice. Treatment with DHAB or LY294002 reversed VSMC proliferation, migration and expression of Akt, FOXO3a, phospho-FOXO3a and MMP-9 in HFD-DHAB and HFD-LY group. These data shows that high-fat Western-type diet induced ADPRC may via PI3K-Akt to phosphorylate FOXO3a up-regulating MMP-9 to enhance vascular smooth muscle cell proliferation and migration in mice.
Abstract 420: Delineating the Caspase-dependent Targets and Signal Pathways That Promote Pathological Cardiac Hypertrophy
Authors: Putinski Charis , Abdul-Ghani Mohammad , Stiles Rebecca , Brunette Steve , Dick Sarah A , Fernando Pasan , Megeney Lynn A
Published: July 2021

Although cardiac hypertrophy is initially an adaptive response, chronic stress on the heart is a maladaptive process that inevitably leads to end-stage heart failure. Interestingly, this pathological process is also characterized by cell behaviors associated with apoptosis. We previously demonstrated the essential role of the intrinsic cell death pathway during cardiac hypertrophy; however, the caspase-dependent pathways and cleavage targets remain elusive. To this aim, we evaluated a myocyte enhancer factor 2 (MEF2) transcription factor inhibitor, histone deacetylase 3 (HDAC3), and gelsolin as potential caspase cleavage substrates involved in the induction and/or maintenance of cardiac hypertrophy. In vitro cleavage assays were completed with effector caspase and recombinant substrate protein which demonstrated caspase-dependent cleavage. HDAC3 cleavage was observed during early stages of hypertrophy and reduced in the presence of a caspase inhibitor. Luciferase assays demonstrated that the transcriptional activity of MEF2 is dependent on intact caspase function suggesting caspase-directed HDAC3 cleavage may serve as a novel regulatory mechanism to alleviate MEF2 suppression to engage the hypertrophy gene expression program. Unlike HDAC3, caspase mediated gelsolin cleavage occurs at latter stages and is coincident with the cytoskeletal alterations that occur during this process. As gelsolin is a potent actin capping/severing enzyme, we hypothesize that caspase-mediated gelsolin activation acts as a key regulatory step in the structural rearrangements that allow for hypertrophy to occur. We have generated adenoviral vectors containing caspase cleavage mutants and cleaved forms of HDAC3 and gelsolin and will discuss the impact of these modified substrates on the hypertrophy process in vitro and in vivo. Collectively, this work suggests that caspase signalling acts to engage both the transcriptional program and cytoskeletal accommodations that characterize cardiac hypertrophy. Importantly, these observations suggest that identification of inhibitors that suppress caspase activity and/or activity of its cognate substrates may offer novel therapeutic targets to limit the development of pathological hypertrophy.
Abstract 421: Nck Promotes Podosome Formation and Function in Vascular Endothelial Cells
Authors: Chaki Sankar P , Barhoumi Rola , Rivera Gonzalo M
Published: July 2021

Podosomes are actin rich adhesion structures capable of extracellular matrix remodeling (ECM) and facilitate invasive cell migration. ECM remodeling and migration of endothelial cells (ECs) are prerequisites for angiogenesis, an important process in development and cardiovascular disease. In this study using a combination of molecular genetics and high resolution microscopy, we have demonstrated that adaptor proteins Nck1 and Nck2, that links signaling by tyrosine phosphorylation with actin dynamics, promote podosome formation and function. Expression of human Nck1-YFP or human Nck2-mCherry induces podosome formation in human umbilical vein endothelial cells. Silencing of Nck by retroviral expression of short hairpin RNA disrupted podosome formation in Src transformed endothelial cells. Both number of cells with podosomes and number of podosomes per cell decreased significantly (P<0.05) compared with control and rescued cells. Functionally, Nck silenced cells were deficient in fluorescent gelatin matrix degradation, an effect that could be almost completely reversed by expression of siRNA-resistant Nck2. Further, overexpression of Nck in Src transformed endothelial cells induced an even higher (p<0.05) index of matrix degradation when compared with Src transformed control cells. Overall, a high degree of colocalization between F-actin and areas of degradation was observed. However, areas of degradation without cellular/F-actin colocalization [[Unable to Display Character: –]] presumably due to the highly motile nature of HUVEC [[Unable to Display Character: –]] were also observed. Mechanistically we found that Nck promote podosome biogenesis through interaction with p62Dok. Collectively, these results provide strong support for a critical role of Nck adaptors in the invasive program of endothelial cells which is important in health and diseases.
Abstract 424: Activation of the S1P3 Receptors is Responsible for S1P-mediated RhoA Activation and Cardioprotection
Authors: Yung Bryan S , Xiang Sunny Y , Purcell Nicole , Rosen Hugh , Chun Jerold , Heller Brown Joan , Miyamoto Shigeki
Published: July 2021

Sphingosine-1-phoshpate (S1P) is a bioactive lysophospholipid, generated and released at sites of tissue injury. S1P signals through a variety of G-protein coupled receptor subtypes and there are three major sub-types, S1P
1
, S1P
2
, and S1P
3
, to mediate cardiovascular responses. S1P
2
and S1P
3
receptors couple to Gα
i
, Gα
12
, Gα
13
and Gα
q
and we first examined the contribution of S1P
2
and S1P
3
to cardiac hypertrophy using S1P
2
and S1P
3
knockout (KO) mice and found that there is no difference in hypertrophy induced by pressure-overload. We previously showed that S1P provides cardioprotection against oxidative stress such as ischemia/reperfusion in which RhoA activation and its downstream effector PKD1 play an important role. It has not, however, been determined which S1P receptor subtype is responsible for S1P mediated cardioprotection. We knocked down the three major S1P receptors using siRNA in neonatal rat ventricular myocytes (NRVMs) and assessed RhoA and PKD1 activation induced by S1P. Knockdown of S1P
3
abolished RhoA activation and largely attenuated phosphorylation of PKD1 while knockdown of S1P
1
and S1P
2
did not. Using siRNA or pertussis toxin to inhibit different G-proteins, we further established that S1P

regulates RhoA activation through Gα
13
, but not Gα
12
, Gα
q
,

or Gα
i
. To investigate the role of S1P
3
receptors in the adult heart, hearts were isolated from wild-type or S1P
3
KO adult mice, perfused in the Langendorff mode and subjected to ex vivo ischemia/reperfusion. As previously reported, S1P perfusion significantly reduced infarct size induced by ischemia/reperfusion in WT hearts (by 50%), but this protection was abolished in the S1P
3
KO mouse heart. To further confirm the role of S1P
3
in cardioprotection we perfused WT mouse hearts with an S1P
3
-specific agonist CYM-51736. We observed that CYM-51736 attenuated the infarct size to a similar degree as that observed with S1P. Our findings reveal that activation of the S1P
3
receptor coupling to Gα
13
and subsequent RhoA activation is responsible for cardioprotection against ischemia/reperfusion. Accordingly specific drug targeting of S1P
3
receptors could provide therapeutic benefits in ischemic heart disease without the undesirable effects of global activation of other cardiac S1P receptors.
Abstract 425: Induced Pluripotent Stem Cell Derived Cardiomyocyte Tissue Engineered Scaffold Improves Left Ventricular Function and Electro-Mechanical Coupling in Rats with Heart Failure
Authors: Lancaster Jordan J , Juneman Elizabeth , Sanchez Pablo , Weigand Kyle , Moukabary Talal , Lahood Nicole , Pandey Amitabh , Bahl Joseph J , Goldman Steven
Published: July 2021

Background:
Chronic Heart Failure (CHF) is the leading cause of hospital readmissions in the United States. It may result from systolic or diastolic dysfunction, which often coexists. Here we report the effects of delivering human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) via a bioengineered patch on left ventricular function in rats with CHF.

Methods:
Adult male Sprague-Dawley rats underwent left coronary artery ligation and were randomized to Sham, CHF, and hiPSC-CM patch. High purity human hiPSC-CMs were obtained from Cellular Dynamics International, seeded and co-cultured onto a vicryl matrix embedded with human dermal fibroblasts. Echocardiography was performed at 3 and 6 weeks post-randomization. Hemodynamic pressure measurements were performed at 6 weeks post-ligation with Millar solid state micromanometer catheters. Open chest Electrophysiologic (EP) mapping was performed at 6 weeks post ligation.

Results:
Patches constructed with hiPSC-CMs displayed synchronized and spontaneous contractions within 48hrs of culture which developed in robustness over time. At maximal robustness, contractions were visualized across the full thickness of the construct. Contractions were recorded at 36+5 beats BPM. Three weeks after implantation, the hiPSC-CM patch decreased LV EDP (45%), Tau (29%), E/e’ (23%) and increased, EF (14%), e’ (20%), and e’/a’ (36%) versus CHF. EP studies show electro-mechanical coupling between the patch and the native myocardium with normal activation through the patch and increases (P<0.05) voltage amplitude in CHF versus hiPSC-CM patch treated rats (1±0.5 mV vs 6±1.5mV).

Conclusion:
Cardiac patch implantation with human iPSC derived cardiomyocytes is an effective and feasible method of treating CHF with improvements in systolic function, diastolic function, and electro-mechanical coupling in rats with CHF.
Abstract 44: Telomere and Mitochondrial Dysfunction in Duchenne Muscular Dystrophy
Authors: Chang Alex C , Ong Sang-Ging , Wu Joseph , Blau Helen M
Published: July 2021

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease that is result of mutations in the dystrophin gene and is the most common myopathic disease in humans with a prevalence of one in every 3500 males. Dystrophin is crucial for the formation of a dystrophin-glycoprotein complex (DGC), which connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix in both skeletal and cardiac muscles. In the heart, loss of dystrophin leads to increased fibrosis and death in the third decade of life due to dilated cardiomyopathy. A conundrum in studying and developing therapies for DMD has been the lack of a mouse model that fully recapitulates the clinical phenotype, as mice that lack dystrophin (mdx model), unlike patients, exhibit only mild skeletal muscle defects, essentially no cardiac defects and have a relatively normal lifespan. Our lab reasoned that the difference in the manifestation of the disease in mice and humans could be telomere length, as mice have substantially longer telomeres than humans. We created a novel mouse model with shortened telomere lengths (similar to humans) that fully recapitulates the skeletal muscle (Cell. 2010;143:1059-1071; the mdx/mTRKO model) and cardiac muscle phenotype of DMD (Nat Cell Biol. 2013; 15:895-904; dilated cardiomyopathy). Interestingly, we observed a relative 45% reduction in cardiomyocyte telomere length in our mdx/mTRKO animals (3 animals per group, N = 300-400) as well as patient samples (4 DMD patient samples, N = 40-95). Here we present new evidence of mitochondrial dysfunction and telomere dysfunction.
Abstract 50: GATA4-Dependent Control of Myocyte Protein Quality Mediated by HSPB7
Authors: Mercer Emily J , Evans Todd
Published: July 2021

The transcription factor GATA4 plays essential roles in heart development, including regulation of cardiomyocyte stress response. GATA4 depletion leads to defective cardiogenesis while GATA4 overexpression is protective in stress models including myocardial ischemia and cardiotoxin administration. However, the molecular basis for GATA4 function remains poorly understood. We discovered that expression of small heat shock protein beta 7 (HSPB7) is regulated by GATA4 and that HSPB7 depletion causes defects in cardiomorphogenesis. The goal of our study is to determine the cellular and molecular mechanism of action of HSPB7 in cardiomyogenesis. Through proteomic mass spectrometry and co-immunoprecipitation, we identified Filamin C (FLNC), a large sarcomeric protein, as an HSPB7 binding partner. FLNC mutations lead to pathological aggregate formation and progressive myopathy. Previous work suggests HSPB7 may prevent damaged protein aggregation through autophagic pathways, and our studies in zebrafish embryos suggest HSPB7 depletion alters the morphology and cytoskeletal architecture of ventricular cardiomyocytes, in particular the trabeculae. We hypothesize that HSPB7 facilitates myocyte function by processing damaged FLNC to prevent aggregation. Preliminary data suggest that BAG3 is also a part of this chaperone complex, linking HSPB7 to the process of Chaperone Assisted Selective Autophagy. Interestingly, disturbing autophagic pathways in the chick embryo precipitates dextrocardia, a phenotype observed in HPSB7 morphant embryos. We also generated a spectrum of defined HSPB7 mutant alleles using TALEN technology. Four such alleles have been bred to homozygosity including a suspected N-terminal truncation that deletes a putative regulatory polyserine stretch. Although homozygous mutants do not exhibit an overt cardiac phenotype, we hypothesize that loss of this polyserine domain may result in a hypomorphic HSPB7 protein. Exposure of mutant embryos to various stressors, including increased temperature (32°C) and the autophagy inducer rapamycin (1μM), led to an increase in phenotypes we have observed in HSPB7 morphants, including dextrocardia and the incidence of defects in cardiac morphology.
Abstract 51: Thymosin β4 Targets Wisp1 to Protect Cardiomyocytes in AngII- induced Cardiac Hypertrophy
Authors: Gupta Sudhiranjan , Li Li , Guleria Rakesh , Baker Kenneth M
Published: July 2021

Background:
Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tβ4 in cardiomyocytes hypertrophy is currently unknown. The purpose of this study is to dissect the cardio-protective mechanism of Tβ4 in Ang II induced cardiac hypertrophy.

Methods:
Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were stimulated with Ang II and expression of cell sizes, hypertrophy marker genes and Wnt signaling components was evaluated by quantitative real-time PCR, western blotting and fluorescent microscopy. Selected target gene Wisp-1 was either overexpressed or silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 in Ang II-induced cardiac hypertrophy was evaluated.

Results:
Pre-treatment of Tβ4 resulted in reduction of cell sizes, hypertrophy marker genes and WNT-associated gene expression and levels induced by Ang II in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl
2
ratio in the cardiomyocytes. Wisp-1 overexpression promotes cardiac hypertrophy and was reversed by pretreatment with Tβ4. Knocking down of Wisp1 partly rescue the cells from hypertrophic response after Tβ4 treatment.

Conclusion:
This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes hypertrophy and its capability to selectively target Wisp-1 in neonatal cardiomyocytes thus preventing cell death, thereby, protecting the myocardium. Wisp-1 promotes the cardiac hypertrophy which was prevented by Tβ4 treatment.
Abstract 52: PPAR-γ Targeted by MicroRNA-130a Regulates Angiotensin II-Induced Cardiac Fibrosis
Authors: Gupta Sudhiranjan , Li Li
Published: July 2021

Aims:
Cardiac fibrosis which occurs due to disruption of extracellular matrix network resulted in the accumulation of excess collagens and other matrix components leading to myocardial dysfunction.

Angiotensin II (Ang II), a critical effector of this system has been implicated in the development of hypertension-induced cardiac fibrosis. In recent years, miRNAs have identified as an attractive targets for therapeutic intervention in various disease pathologies including cardiac fibrosis. However, the exact effect and underlying mechanism of miRNAs in cardiac fibrosis remains unclear. Here, we sought to investigate and test our hypothesis that miR-130a plays a critical role in the development of myocardial fibrosis by restoring PPARγ level.

Methods and Results:
We have identified a panel of novel miRNAs
via
miRNA array in Ang II infused mice heart. Among them, we found that miR-130a was upregulated both in pressure overload and Ang II infused models targeting PPARγ. Overexpressing miR-130a in cardiac fibroblast promoted the pro-fibrotic gene expression (collagen I/III, fibronectin and CTGF) and myofibroblasts differentiation. Inhibition miR-130a reversed the process and weakened these activities. Using luciferase-linked constitutive and dominant negative constructs of PPARγ, we determined the underlying mechanism of cardiac fibrosis occurred
via
targeting PPARγ. The
in vivo
inhibition of miR-130a by subcutaneous injections of LNA-based anti-miR-130a in mice subjected to Ang II infusion significantly reduced the severity of cardiac fibrosis, hypertrophy. The protective mechanism is associated with restoration of PPARγ level, reduction of pro-fibrotic genes and apoptosis; reversion of myofibroblasts differentiation and improved cardiac function.

Conclusions:
Our findings provide evidence that miR-130a plays a critical role in the progression of cardiac fibrosis by directly targeting PPARγ, and that inhibition of miR-130a reversed the cardiac fibrosis. We conclude that miR-130a may be a new marker for cardiac fibrosis and inhibition of miR-130a would be a promising strategy in the treatment of cardiac fibrosis.
Abstract 53: PP1, a Derivative of Salubrinal, Ameliorate Ventricular Function After Myocardial Infarction in Rats
Authors: Liu Chunlei , He Yunyun , He Kunlun
Published: July 2021

Objective:
salubrinal, an inhibitor of ER-stress, has potent effect on cell injury. In this study, we investigated the effect of salubrinal and its derivative-PP1 on chronic heart failure induced by myocardial infarction ( MI).

Methods:
Male wistar rats were randomly divided into 5 groups:i) sham-operated; ii) vehicle (MI+ DMSO); iii) MI + salubrinal; iv) MI + PP1; v) MI + kaptopril. After 6 weeks treatment, transthoracic echocardiographic and hemodynamic parameters were evaluated. The cardiac tissue sections were subjected to TUNEL to assess the level of apoptosis. The expression of mRNA and protein involved in apoptosis, autophagy, ER stress was analyzed using real-time reverse transcription-polymerase chain reaction and western blotting, respectively.

Results:
The VW/BW and LVW/BW were reduced by salubrial and PP1.The LVEDD, LVESD were significantly decreased while the EF and FS were greatest increased by salubrinal and PP1. The hemodynamic parameters including CO, SV, LVEDV, LVESV, LVEDP, LVESP, +dp/dtmax, -dp/dtmax, +dv/dtmax, -dv/dtmax, Ea, Tau were ameliorated. By calculating the apoptotic index, we found that salubrinal and PP1 have a great anti-apoptotic effect. The mechanism analysis demonsted that salubrinal and PP1 have a cell protection effect through downregualtion of PERK, GRP78, chop, caspase12 and upregulation of p62 , LC3II, ATF4 as detected by real-time PCR and western blotting.

Conclusions:
The current study suggested that salubrinal and PP1 have a cell protection effect involed in ER stress related autophay and apoptosis.This may implicate that salubrinal and PP1 may be a potent compound for the treatment of chronic heart failure.
Abstract 54: Effect of Salubrinal and PP1 on Cardiomyocytes Protection After Myocardial Infarction in Rats
Authors: Liu Chunlei , He Yunyun , He Kunlun
Published: July 2021

Objective:
salubrinal, an inhibitor of ER-stress, has potent effect on cell injury. In this study, we investigated the effect of salubrinal and its dervative-PP1 on cell protection aganinst myocardial infarction ( MI).

Methods:
Male wistar rats were randomly divided into 5 groups:i) sham-operated; ii) vehicle (MI+ DMSO); iii) MI + salubrinal; iv) MI + PP1; v) MI + metoprolol. After 24 hours treatment, heart tissue was havested and stained by 2,3,5-triphenyltetrazolium chloride (TTC). Serum was havested for the detection of CK-MB, cTNT, CRP. The changes in histomophology were observed using hematoxylin and eosin (HE) staining. The expression of mRNA and protein involved in apoptosis, autophagy, ER stress was analyzed using real-time reverse transcription-polymerase chain reaction and western blotting, respectively.

Results:
Salubrinal and PP1 decreased the infarction area and the level of CK-MB, cTNT, CRP. The mechanism analysis demonsted that salubrinal and PP1 have a cell protection through a ER-stress related pathway, showing upregualtion of PERK, eIF2α, ATF4, GRP78, GADD34, p62 , LC3IIand down regualtion of chop, caspase12 as detected by real-time PCR and western blotting.

Conclusions:
The current study suggested that salubrinal and PP1 have a cell protection effect involed in ER stress related autophay and apoptosis.This may implicated that salubrinal and PP1 may be a potent compound for the treatment of MI.
Abstract 60: Cardiac-specific Deletion of HuR Reduces Pathological Hypertrophy and Ventricular Remodeling Following Transverse Aortic Constriction
Authors: Tranter Michael , Kraynik Stephen , Anthony Sarah R , Slone Samuel , Nieman Michelle L , John Lorenz N , Rubinstein Jack
Published: July 2021

The RNA binding protein HuR (Human antigen R) interacts with specific AU-rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. However, the role of HuR in cardiac physiology is largely unknown. Our results show that HuR undergoes cytoplasmic translocation, indicative of its activation, in hypertrophic mouse cardiac myocytes at 8 weeks post-transverse aortic constriction (TAC). To determine the role of HuR in the development of cardiac hypertrophy, we have created a novel mouse model of cardiac myocyte-specific deletion of HuR. While cardiac-specific HuR deletion mice do not show an overt basal phenotype, they have preserved ejection fraction and reduced ventricular remodeling (hypertrophy and chamber dilation) compared to wild-type littermates at 8 weeks following TAC. Furthermore, HuR activation in the hypertrophic heart is strongly co-localized with regions of fibrosis. To this end, we show that HuR knockdown reduced hypertrophy and pro-fibrotic TGF-β gene expression in a phenylephrine treated neonatal rat ventricular myocyte (NRVM) model of hypertrophy. Thus, our results suggest that HuR activation in cardiac myocytes promotes pathogical hypertrophy and initiation of cardiac fibrosis via myocyte expression of TGF-β. These findings are significant as we have identified HuR as a novel mediator of pathological hypertrophy and suggest a pro-fibrotic role as a potential mechanism for this effect.
Abstract 63: Absence of Fibulin-2 Attenuates Cardiac Hypertrophy and Fibrosis in Transgenic Mice Over-expressing Circulating Transforming Growth Factor-beta
Authors: Tsuda Takeshi , Khan Shaukat
Published: July 2021

Background:
TGF-β is a potent growth factor that induces myocardial hypertrophy and fibrosis. However, the interaction between circulating TGF-β and local myocardial TGF-β in inducing cardiac hypertrophy is not well understood. Fibulin-2 is an ECM protein that mediates TGF-β activation during cardiac hypertrophy in response to chronic angiotensin (Ang) II infusion. We tested the hypothesis that fibulin-2 mediates systemic TGF-β-induced cardiac hypertrophy.

Materials & Methods and Results:
We created double mutant mice by crossing TGF-β over-expressing transgenic mice (TG) and fibulin-2 knockout mice (KO). TG are known to produce excessive mature porcine TGF-β from the liver. TG/WT developed significant myocardial hypertrophy at 8 weeks compared with non-TG (NTG) groups. Hypertrophy in KO/TG was significantly attenuated compared with TG/WT. Myocardial TGF-β mRNA up-regulation was significantly higher in TG/WT than in TG/KO or NGT groups, so was Smad2 activation, but myocardial TGF-β bioactivity measured by PAI-1 promoter/luciferase bioassay was comparable among all four groups. Serum carrier-bound TGF-β (total TGF-β) in WT/TG was significantly higher than that in KO/TG and NTG groups, but free TGF-β level was equally elevated in TG groups compared with NTG groups. The difference in hypertrophy between WT/TG and KO/TG may be attributed to increased myocardial TGF-β[[Unable to Display Character: ]]mRNA and elevated serum total TGF-β in WT/TG, not directly to bioactive myocardial TGF-β[[Unable to Display Character: ]]or serum free TGF-β. Endogenous TGF-β mRNA levels in kidney and liver were equally increased in TG groups compared with NTG groups, and were same in all 4 groups in lung, suggesting fibulin-2 was not involved in TGF-β-induced TGF-β synthesis in these organs.

Conclusion:
Systemic TGF-β-induced-endogenous TGF-β up-regulation was mediated by fibulin-2 in the myocardium. After secretion, endogenous myocardial TGF-β may be either sequestrated into insoluble ECM or directly released into circulation as a soluble carrier-bound form. It is circulating endogenous TGF-β secreted from the heart that is primarily responsible for myocardial hypertrophy. Heart is a major source of circulating endogenous TGF-β in the TG/WT, which assumes an endocrine role in inducing myocardial hypertrophy.
Abstract 64: Conditional Knockout of Activin Like Kinase-1 (ALK-1) Leads to Heart Failure Without Maladaptive Remodeling
Authors: Morine Kevin , Paruchuri Vikram , Qiao Xiaoying , Mackey Emily , Levine Jonathan , Ughreja Keshan , Nepali Prerna , Karas Richard , Oh Paul , Kapur Navin K
Published: July 2021

Activin like kinase 1 (ALK1) mediates signaling via the TGFb family of ligands. ALK1 activity promotes endothelial proliferation and migration. Reduced ALK1 activity is associated with arteriovenous malformations. No studies have examined the effect of global ALK1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of ALK1 promote maladaptive cardiac remodeling. Methods: We employed an ALK1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre recombinase mediated excision of floxed ALK1 alleles. Tamoxifen treated wild-type (WT-Tam; n=5) and vehicle treated ALK1-cKO mice (cKO-Veh; n=5) served as controls for tamoxifen treated ALK1-cKO mice (cKO-Tam; n=15). Results: ALK1 cKO-Tam mice demonstrated reduced 14-day survival compared to cKO-Veh controls (33% vs 100%, respectively, p<0.01). Seven days after treatment, ALK1 cKO mice began to exhibit reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14 days total body mass was reduced, but LV and lung mass increased in cKO-Tam not cKO-Veh mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume increased in cKO-Tam, not cKO-Veh mice. LV ALK1 mRNA and protein levels were reduced in cKO-Tam, not cKO-Veh mice. LV levels of other TGFb-family ligands and receptors (ALK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFB1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-Tam mice, but LV levels of b-MHC, SerCA, and calcineurin were unchanged. No increase in cardiac fibrosis Type I collagen, CTGF, or PAI-1 levels were observed between groups. No differences were observed for any variable studied between cKO-Veh and WT-Tam mice. Conclusion: Global deletion of ALK1 is associated with the development of high output heart failure without maladaptive remodeling. Future studies exploring the functional role of ALK1 in cardiac remodeling are required.
Abstract 66: Discovery of a Novel Pharmaceutical Class as Potential Heart Failure Treatment
Authors: Feaster Tromondae K , Hempel Jonathan E , Williams Charles H , Frist Audrey Y , Hwang Hyun S , Knollmann Bjorn C , Hong Charles C
Published: July 2021

Utilizing an unbiased
in vivo
phenotypic chemical screening platform in zebrafish embryos, our laboratory has identified a number of novel compounds with high selectivity for a wide range of cellular targets, including kinases (CK2a, DRAK2, DYRK2 and bone morphogenetic protein receptors), GPCRs (lysophosphatidic acid receptor 1, and extracellular proton sensor), p300 histone acetyltransferase, and phosphodiesterase-4 (PDE4). While the compounds we discovered have therapeutic implications for a wide range of diseases, our translational work has focused on addressing the cardiovascular diseases. Heart failure (HF) is a leading cause of disability and mortality in US, affecting about 6 million Americans, and the incidence of heart failure is anticipated to increase substantially in the coming decades. Yet, current HF pharmaceuticals are palliative, and the outlook for HF drug pipeline is uncertain. Within this backdrop, we recently discovered eggmanone, an extraordinarily selective PDE4 inhibitor which has no known off-target. An usual feature of eggmanone is that it increases cAMP levels specifically in distinct cellular microdomains, without raising the total cellular cAMP content. In isolated mouse cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), eggmanone increases cardiac contractility by targeting a discrete myocyte microdomain without causing significant changes in myocyte calcium cycling. Importantly, eggmanone enhances systolic function in mice with failing hearts without increasing the heart rate. These results raise the exciting possibility that a microdomain-specific PDE4 inhibitor like eggmanone may be useful as an inotropic therapy for HF which avoids the pitfalls of traditional PDE inhibitors, whose utility has been limited by proarrhythmia, tachyphylaxis and cardiotoxicity.
Abstract 67: miR-19 Deficiency Impairs Cardiac Repolarization in Zebrafish
Authors: Benz Alexander , Katus Hugo A , Hassel David
Published: July 2021

The most common outcome of heart failure (HF) is sudden cardiac death which results mostly from prolonged action potential duration (APD) and arrhythmias. During the pathogenesis and progression of HF, a vast number of signaling pathways are altered. microRNAs are small noncoding RNAs that post-transcriptionally finetune gene expression. Interestingly, several microRNAs are dysregulated during HF, suggesting a potential involvement in the development and progression of the disease. Here, we identified miR-19 as an important regulator of heart function. Zebrafish lacking miR-19 developed severe bradycardia and reduced cardiac contractility. While the mammalian genome encodes for two isoforms of miR-19, zebrafish express four members (19a-d). We found that the reduction of miR-19b specifically is sufficient to cause bradycardia and reduced cardiac contractility. Imaging of ventricular APs from whole hearts revealed that APD is significantly prolonged and repolarization is impaired in miR-19b deficient zebrafish. By qRT-PCR experiments we showed that the expression of several cardiac ion channels is altered. Moreover, miR-19b deficiency results in increased sensitivity to an AV-Block, which is a characteristic feature of long QT-Syndrome in zebrafish. In conclusion, we identified miR-19b as a novel and essential modulator of the electrical activity of the heart and establish miR-19b as a potential candidate gene causative for human long QT syndrome.
Abstract 77: MiR-125b Regulates Myofibroblast Transition and Cardiac Fibrosis
Authors: Nagpal Varun , Rai Rahul , Place Aaron T , Ghosh Asish K , Vaughan Douglas E
Published: July 2021

Transforming growth factor-β (TGF-β)-induced fibroblast-to-myofibroblast transition (FMT) is a critical determinant of cardiac fibrosis. However, the contribution of microRNAs leading to TGF-β-induced FMT and cardiac fibrosis are not well-understood. Our results elucidate that blocking the canonical TGF-β pathway protects from FMT in primary cultures of human cardiac fibroblasts and that miR-125b is significantly upregulated during cardiac FMT. Furthermore, we observed significant upregulation of miR-125b in fibrotic human myocardium and two murine models of cardiac fibrosis. Importantly, we discovered that miR-125b is sufficient to induce cardiac FMT. In contrast, the knockdown of miR-125b using an antagomir approach attenuated TGF-β-induced FMT. In silico analysis and biochemical analysis revealed that miR-125b directly targets multiple anti-fibrotic mediators including p53 and apelin. In addition, miR-125b also plays a potent role in the regulation of fibroblast proliferation, an important cause of cardiac fibrosis. Finally, miR-125b was successfully inhibited in vivo by the systemic delivery of locked nucleic acid (LNA) targeted against miR-125b both in the presence and absence of Angiotensin II (Ang II). These results demonstrated that LNA-125b protected against Ang II-induced proliferation and fibrosis in the mouse heart in vivo. We conclude that TGF-β-induced miR-125b is an important regulator of both fibroblast proliferation and FMT, and miR-125b inhibits key anti-fibrotic mediators to promote cardiac fibrosis. We propose that miR-125b may serve as a novel therapeutic target for the preventative therapy for fibrosis.
Abstract 82: Mnsodtg Mice Exhibit Improved Heart and Mitochondrial Function During Chronic Chagas Disease
Authors: Wen Jianjun , Porter Craig , Herndon David , Garg Nisha J
Published: July 2021

Background:
We observed that mitochondrial reactive oxygen species (mtROS) plays very important roles in the pregression of chagesic disease (CD). In this study, we utilized genetically-modified mice to scavenge mtROS to investigate the impact of improved ROS scavenging capacity on heart function in CD.

Methods and Results:
C57BL/6 mice (wild-type, MnSODtg, MnSOD+/-) were infected with Trypanosoma cruzi(Tc). Chronically infected mice (≥120dpi) exhibited a substantial decrease in heart tissue MnSOD gene expression, protein level, enzyme activity and antioxidant level; decrease of heart dysfunction via lower of SV, CO, EF, FS and LVPW,s, and increase of ESV/EDS and LVID;s; enhancement of hypertrophy by increase of IVS, LV mass and areas duo to augmentation of collagen expressions. One of our novel observations was that sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) lost its role of maintenance of low cytoplasm free calcium and mediated calcium uptake to intracellular store in Tc-induced chronic chagasic disease. Studies of fresh heart slices using O2K confirmed that Tc diminished heart mitochondrial function like decrease of oxygen flux and respiratory control ratio (RCR), which were caused by enhancements of ROS. Myocardial mitochondrial damage was pronounced and associated with a >x% decline in mitochondrial oxygen flux in chronically infected wild-type and MnSOD transgenic mice. Imaging of intact heart for cardiomyocytes and collagen by the nonlinear optical microscopy techniques showed significant increase in collagen (>x0-fold) in chronically infected wild-type mice; while MnSODtg mice exhibited a basal increase in collagen that did not change during chronic phase. Chronically infected MnSODtg mice exhibited a marginal decline in Tc-induced heart function, heart hypertrophy, mitochondrial dysfunction

Conclusions:
Overexpression of MnSOD inhibited Tc-induced oxidative damage od heart tissue. , suggesting that enhancing the mitochondrial ROS scavenging capacity was beneficial in controlling the inflammatory and oxidative pathology, and cardiac remodeling responses that are hallmarks of chronic Chagas disease.
Abstract 83: Modulation of Macrophage Function via Metabolism by Trypanosoma cruzi
Authors: Koo Sue-Jie , Garg Nisha J
Published: July 2021

Chagas heart disease is an inflammatory cardiomyopathy which presents with mononuclear infiltrates in the interstitium and myocardial fibrosis in the chronic phase. Incomplete clearance by macrophages of the etiologic agent,
Trypanosoma cruzi
, is a significant cause of chronic disease development in approximately 30% of those serologically positive for the blood-borne parasite. The differential metabolic status, anaerobic glycolysis and mitochondria-dependent oxidative phosphorylation, are respectively associated with pro-inflammatory (M1) and anti-inflammatory (M2) functional activation of macrophages. Reactive oxygen species (ROS) have been shown to be an intracellular signal for glycolysis while peroxisome proliferator-activated receptors (PPARs) that enhance fatty acid oxidation provide transcription control of macrophage functional state. In our studies using diverse
T. cruzi
isolates, we showed that SylvioX10 (virulent), but not TCC (non-virulent), isolates are able to differentially control extracellular and intracellular ROS levels in macrophages. We found in macrophages infected with SylvioX10, the nuclear expression of PPAR-α was increased by 18 hours post-infection, and mitochondrial metabolic activity was similar to that of not-infected and M2 controls; which indicates anti-inflammatory function of macrophages, and therefore prohibiting
T. cruzi
clearance. In our ongoing studies, we are examining the impact of PPAR-α inhibitors in modulating the metabolic gene expression profile, functional phenotype and parasite survival in macrophages. Our data will provide the first indication that host macrophages have deficient pro-inflammatory capacity due to sub-optimal glucose oxidation, and enhancing the metabolism that supports
T. cruzi
clearance will provide a valuable basis for a strategy to arrest Chagas disease progression.
Abstract 84: Expression of the Transcription Factors PPAR Alpha and Gamma, and Their Target Genes in the Heart of Obese Mice
Authors: Ruiz-Velasco Andrea , Rios Amelia , Escalante Bruno
Published: July 2021

The Peroxisome-Proliferator Activated Receptors (PPARs) have been identified as key regulators of energy metabolism by sensing fatty acid availability and activating the transcription of enzymes involved in fatty acid and glucose metabolism. In the heart, PPARs have been proven to participate in the development of diseases, such as cardiac hypertrophy and diabetic cardiomyopathy, conditions that have been associated to obesity. Furthermore, the use of PPARs activator drugs is frequent for the treatment of obesity-dependent diseases, such as hypoglycemiant (fibrates) and anti-diabetic (thiazolidinediones) drugs. Therefore, the objective of this project was to characterize the expression of PPARs and some of their target genes involved in fatty acid and glucose metabolism in the heart of obese mice. Mice (C57BL/6) were fed with either a standard chow diet (control group) or a high fat diet (obese group) for three months. Hearts were weighed prior to quantifying protein expression of two PPARs (alpha and gamma), and both mRNA and protein expression of three enzymes (phosphofructokinase (PFK), glycerol-3-phosphate dehydrogenase (GPD1) and glycerol-3-phosphate acyltransferase (GPAT)). Protein levels were measured by western blot, while mRNA levels were quantified by real-time PCR. The obese group presented 41% increase in body weight compared with control mice. However, no change was observed in obese hearts weight compared with control group, meaning that hypertrophy had not been developed yet. The expression of PPAR-alpha was increased 42% in obese mice compared with the control group, suggesting a possible activation of the fatty acid oxidation pathway. PPAR-gamma together with GPD1 and GPAT showed no differences at any level; however, PFK presented 90% increase in mRNA level and 54% increase in protein level in obese mice. Interestingly, decrease in PFK has been reported in any altered PPAR-alpha condition, contrary to our results. These results suggest that there is a dysregulation of the heart’s energetic pathways mediated by PPARs in earlier stages of obesity.
Abstract 86: Exercise Prevents Cardiac Injury and Improves Mitochondrial Biogenesis in Advanced Diabetic Cardiomyopathy With Pgc-1α and Akt Activation
Authors: Wang Hui , Lu Yan , Sun Wei , Xiao Junjie , Kong Xiangqing
Published: July 2021

Background/Aims:
Diabetic cardiomyopathy (DCM) represents the major cause of morbidity and mortality among diabetics. Exercise has been reported to be effective to protect the heart from cardiac injury during the development of DCM. However, the potential cardioprotective effect of exercise in advanced DCM remains unclear.

Methods:
Seven-week old male C57BL/6 wild-type or db/db mice were either subjected to a running exercise program for 15 weeks or kept sedentary. Cardiac function, myocardial apoptosis and fibrosis, and mitochondrial biogenesis were examined for evaluation of cardiac injury.

Results:
A reduction in ejection fraction and fractional shortening in db/db mice was significantly reversed by exercise training. DCM induced remarkable cardiomyocyte apoptosis and increased ratio of Bax/Bcl-2 at the protein level. Meanwhile, DCM caused slightly myocardial fibrosis with elevated mRNA levels of collagen I and collagen III. Also, DCM resulted in a reduction of mitochondrial DNA (mtDNA) replication and transcription, together with reduced mtDNA content and impaired mitochondrial ultrastructure. All of these changes could be abolished by exercise training. Furthermore, DCM-associated inhibition of PGC-1α and Akt signaling was significantly activated by exercise, indicating that exercise-induced activation of PGC-1α and Akt signaling might be responsible for mediating cardioprotective effect of exercise in DCM.

Conclusion:
Exercise preserves cardiac function, prevents myocardial apoptosis and fibrosis, and improves mitochondrial biogenesis in the late stage of DCM. Exercise-induced activation of PGC-1α and Akt signaling might be promising therapeutic targets for advanced DCM.