bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 75: TNF Receptor 1 Signaling: a Mechanistic Link between Cardiac Inflammation and Fibrosis
Authors: Duerrschmid Clemens , Wang Yanlin , Entman Mark L , Haudek Sandra B
Published: July 2021

Background:
We previously showed in several models of cardiac hypertrophy and failure that, in the absence of cell death, cardiac interstitial fibrosis was mediated by the uptake of monocyte-derived fibroblast precursor cells; fibrosis and concurrent cardiac remodeling were blocked by deletion of monocyte chemoattractant protein-1 (MCP-1), as well as of tumor necrosis factor receptor-1 (TNFR1). We now investigated the cellular origin, kinetics of uptake, and subpopulation of fibroblast precursors in the angiotensin-II (Ang-II)-challenged heart.

Methods:
Mice with genetic deletion of TNFR1 (TNFR1-KO mice) were irradiated and rescued with bone marrow from wild-type (WT) mice, and were subjected to continuous infusion of Ang-II for 7 days. Flow cytometry was performed on isolated cells, immunostaining on perfusion-fixed tissue, quantitative PCR on whole heart mRNA isolations.

Results:
In WT mice, Ang-II induced the early uptake of monocytes which became M1 macrophages (CD86+CD45+) in an M1/Th1 environment. Monocytes entering the heart after 2-3 days polarized to M2 macrophages (CD301+CD45+) in an M2/Th2 milieu with concurrent appearance of collagen-producing CD301+ fibroblasts. M1 cells produced TNF, whereas M2 cells did not; both expressed TNFR1. Ang-II-exposed TNFR1-KO hearts showed similar cardiac infiltration of M1 cells, but the amount of M2 cells was significantly lower. They also had reduced expression of M1 and M2 cytokines, but not of Th1 and Th2 interleukins. Transplantation of WT bone marrow to TNFR1-KO mice before Ang-II exposure restored cardiac fibrosis, uptake of M2 cells, and expression of cytokines to levels observed in Ang-II-exposed WT animals. Many TNFR1+ cells in chimeric TNFR1-KO/WT were committed to the fibroblast lineage.

Conclusion:
Our data show that monocytic fibroblast precursor cells originated in the bone marrow, and that signaling through TNFR1 was required for their uptake and maturation into collagen-producing M2/fibroblasts that mediated the development of cardiac fibrosis in response to Ang-II. They also suggest a mechanistic link between inflammation and fibrosis, i.e. pro-inflammatory, M1-produced TNF initiates pro-fibrotic M2/fibroblast maturation via TNFR1 signaling.
Abstract 89: Mitochondrial Protein Acetylation in Aged Hearts in the Context of Ischemia-Reperfusion Injury
Authors: Nadtochiy Sergiy M , Urciuoli William R , Brookes Paul S , Porter George A
Published: July 2021

Introduction:
SIRT3 deficiency leads to increased mitochondrial acetylation, and enhances sensitivity of the heart to stress. Recently we demonstrated that a worse outcome of ischemia-reperfusion (IR) injury in aged hearts was associated with increased protein acetylation. The aim of this study was to investigate the mechanism of acetylation and the identity of acetylated targets in aged hearts in the context of IR injury.

Methods:
Mitochondria were isolated from WT adult (7 mo.), SIRT3
+/-
adult (7 mo.) and WT aged (18 mo.) hearts. SIRT3 protein expression and identification of acetylated proteins from SIRT3
+/-
adult and WT aged samples were investigated using proteomics (2D gel, western blot, peptide mass fingerprinting).

Results:
Mitochondrial acetylome analysis revealed that many hyperacetylated patterns were identical between adult SIRT3
+/-
and aged WT hearts, suggesting that increased protein acetylation in the aged WT heart might be due to SIRT3 inhibition. Several targets were indentified, including complex I NADH dehydrogenase flavoprotein 1, which is known to interact with SIRT3. In this regard, we demonstrated that respiratory complex I (CxI) activity was significantly inhibited in both SIRT3
+/-
adult and WT aged hearts. Analysis of SIRT3 protein revealed that negatively charged species of SIRT3 (relative to the bulk SIRT3 population), were lost in aged hearts. These alterations suggest post-translational modification of SIRT3 which may lead to loss of its deacetylase activity. Alternatively, we found that upregulation of the mitochondrial acetyltransferase GCN5L1 in aged hearts (examined by western blot) may also contribute to enhanced protein acetylation.

Conclusions:
These data support a connection between SIRT3 downregulation, mitochondrial protein acetylation, and exacerbation of IR injury in aging hearts. Furthermore, it is known that functional CxI is required for several modes of cardioprotection suggesting that downregulation of CxI in SIRT3
+/-
or WT aged hearts may underlie their poor recovery from IR injury.
Abstract 9: Development And Testing Of A Human Induced Pluripotetent Stem Cell Derived Cardiac Scaffold
Authors: Lancaster Jordan J , Sanchez Pablo , Juneman Elizabeth , Weigand Kyle , Bahl Josehp J , Goldman Steven
Published: July 2021

Introduction:
Cell-based regenerative therapies hold promise in providing treatment strategies for heart failure. Previous work from our laboratory has shown that tissue engineered cardiac constructs enhance improvements in cardiac function by providing structural and nutrient support, potentially aiding in transplanted cell survival, integration and re-population of injured tissues. Our current studies focus on the development and testing of second-generation cardiac constructs utilizing high purity human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).

Methods:
High purity hiPSC-CMs were seeded and co-cultured onto a vicryl matrix embedded with biologically active human dermal fibroblasts. The hiPSC-CM constructs were maintained at 37°C and 5% CO2. Patches were constructed maintained as described for both in vitro and in vivo evaluation. Patches for in vivo evaluation were seeded, cultured and implanted onto the rat heart 3 weeks after left coronary artery ligation to assess improvements in LV function. Patches prepared for in vitro evaluation were seeded and cultured 1-10 days.

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. In addition hiPSC-CM constructs respond to electrical stimulation with increased rate of contraction while maintaining their synchrony. Post pacing, the hiPSC-CM constructs return to their intrinsic rhythm.

Conclusion:
These findings show that high purity hiPSC-CMs can be seeded and co-cultured onto a vicryl and fibroblast construct in manner permitting adhesion and electromechanical coupling of the hiPSC-CMs to form a fully contractile construct. This is supported by the observation that the hiPSC-CMs contract spontaneously and in a synchronized manner in a directional fashion.
Abstract 91: Alteration of Myocardial GRK2 Produces a Global Metabolic Phenotype.
Authors: Woodall Benjamin P , Castellini Meryl A , Cannavo Alessandro , Koch Walter J
Published: July 2021

A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. GRK2 levels increase in the heart post injury in an attempt to normalize sympathetic overdrive. While initially beneficial, this response becomes maladaptive. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. Recently, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Amongst these, our lab has identified GRK2 as a negative regulator of cardiac insulin signaling and this is through direct interaction and phosphorylation of the insulin receptor substrate-1 (IRS1). While continuing to study cardiac metabolic aspects of GRK2 manipulation we have uncovered the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. We found that transgenic mice with cardiac-specific overexpression of GRK2 (Tg-GRK2) show resistance to high fat diet (HFD) induced obesity. In contrast, Tg-βARKct mice with cardiac-specific expression of a peptide inhibitor of GRK2, known as the βARKct display an enhanced obesogenic phenotype when fed a HFD. We placed transgenic mice (and their non-transgenic littermate controls) on a high fat diet (60% kcal% fat) or a control diet (10% kcal% fat) for up to 16 weeks. Mice were housed in grouped cages at room temperature (18-22°C). HFD feeding increased white adipose mass in Tg-BARKct mice vs. NLC, whereas adipose mass in Tg-GRK2 mice is lower than NLC animals. Moreover, HFD Tg-βARKct mice exhibit elevated serum leptin levels relative to NLC, indicating development of leptin resistance. The novelty of this finding could bear great clinical significance, not least because these cardiac specific transgenes elicit a global metabolic phenotype implicating the heart as an endocrine organ and GRK2 activity is essential for this new and unexpected finding.
Abstract 99: Hypotonic Swelling Promotes Nitric Oxide Release in Cardiac Ventricular Myocytes
Authors: Gonano Luis , Morell Malena , Burgos Juan I , Vila Petroff Martin
Published: July 2021

Cardiac myocyte swelling occurs in multiple pathological situations and in particular contributes to the deleterious effects of ischemia and reperfusion by promoting contractile dysfunction. We investigated whether hypotonic swelling promotes nitric oxide (NO) release in cardiac myocytes and if so, whether it impacts on swelling induced contractile dysfunction. Perfusing rat cardiac myocytes, loaded with the NO sensor DAF-FM, with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient amplitude and significantly increased DAF-FM fluorescence. When cells were exposed to the HS supplemented with 2.5 mM of the NO synthase inhibitor L-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the NOS1 inhibitor, Nitroguanidine. In addition, Colchicine (an inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either L-NAME, Nitroguandine or the guanylate cyclase inhibitor, ODQ, suggesting that NOS1-derived NO provides contractile support via a GMP-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and the HS-induced increment in ryanodine receptor (RyR2) phosphorylation at site Ser2808 suggesting that in the context of hypotonic swelling, cGMP may contribute to preserve contractile function by enhancing SR Ca2+ release. Our findings suggest a novel mechanism for NO release in cardiac myocytes with putative pathophysiological relevance in the context of ischemia and reperfusion, where it may be cardioprotective by reducing the extent of contractile dysfunction associated with hypotonic swelling.
Abstract O-1: Klf5 Regulates Cardiac Pparα and Med13 and affects Cardiac Fatty Acid Metabolism And Obesity
Authors: Drosatos Konstantinos , Pollak Nina , Ntziachristos Panagiotis , Trent Chad M , Hu Yunying , Homma Shunichi , Aifantis Iannis , Goldberg Ira J
Published: July 2021

Krüppel-like factors (KLF) have been associated with metabolic phenotypes. Our study focused on the metabolic role of cardiac KLF5, as it showed the highest increase among all KLFs that were detected by whole genome microarrays of energy-starved hearts obtained from lipopolysaccharide (LPS)-treated mice. Analysis of ppara promoter indicated two potential binding sites for c-Jun (AP-1 sites), the transcriptional factor that is activated by LPS and reduces cardiac PPARα expression: −792/-772 bp and −719/-698 bp prior to the transcription initiation site. This analysis showed that both AP-1 sites overlap with potential KLF-binding sites. Adenovirus-mediated expression of constitutively active c-Jun in a mouse cardiomyocyte cell line (HL-1) reduced PPARα gene expression, while treatment with Ad-KLF5 had the opposite effect. Chromatin immunoprecipitation analysis (ChIP) showed that c-Jun binds both −792/-772 bp and −719/-698 sites of ppara promoter while KLF5 binds on −792/-772 bp. ChIP analysis also showed that LPS promotes c-Jun binding on −792/-772 bp, which prohibits occupation of this region by KLF5. A cardiomyocyte-specific KLF5 knockout mouse (αMHC-KLF5-/-) had normal cardiac function but reduced cardiac expression of PPARα (50%) and other fatty acid metabolism-associated genes such as CD36 (40%), LpL (20%), PGC1α (45%), AOX (28%) and Cpt1 (45%). High fat diet (HFD)-fed αMHC-KLF5-/- mice had a more profound body weight increase (35%) compared to HFD-fed WT mice (15%), as well as larger white adipocytes and brown adipocytes (H&E) and increased hepatic neutral lipid accumulation (Oil-Red-O). The obesogenic effect of cardiomyocyte-specific deletion of KLF5 resembles the phenotype of the αMHC-MED13-/- mice. We showed that KLF5 ablation reduced cardiac MED13 levels despite lack of changes in the expression levels of miR-208, a known regulator of MED13. Infection of HL-1 cells with Ad-KLF5 increased MED13 gene expression. ChIP identified a KLF5 binding site on med13 gene promoter region (-730/-714 bp). Thus, KLF5 regulates cardiac PPARα and MED13 and affects cardiac and systemic fatty acid metabolism and obesity, thus indicating KLF5 as a potential target for cardiac dysfunction associated with energetic complications, as well as for obesity
Abstract 1: Chemical Biology of Cardiac Regeleration: an Instructive Hydrogel-based Platform for Heart Repair
Authors: Schneider Jay W , Goetsch Sean C , Kyrychenko Serge , Vegas Arturo , Anderson Daniel G , Hong Yi
Published: July 2021

Regeleration
is the myocardium’s natural adaptive remodeling response to implanted biopolymer hydrogels, a new heart failure treatment modality with promising success in early clinical trials. Classified as a medical device capable of long-term myocardial engraftment, implanted hydrogels of varying molecular composition provide mechanical bulking and scaffolding support that can stabilize or reverse adverse ventricular remodeling. Additionally, natural or synthetically designed hydrogels encoding specific bioactivities or signaling functions can directly regulate myocardial biology to mediate heart repair. To gain mechanistic insight into the molecular and cellular biology and biochemistry of the biopolymer-myocardial interface, we studied two clinically relevant hydrogels - seaweed-derived alginate (Alg) and myomatrix (MMx), extracellular matrix molecules prepared from decellularized pig heart - in a mouse model. Alg and MMx differentially activated signal transduction cascades, recruited different cell types and produced distinctive gene expression signatures and patterns of cardiomyocyte hypertrophy, including muscle enhancer factor-2 (MEF2) and fetal gene program (re)activation. Chemically modifying Alg’s backbone structure correspondingly altered myocardium’s biological response, demonstrating the synthetic tunability of this repair process. These observations demonstrate that implanted biopolymer hydrogels drive unexpectedly robust and versatile regelerative responses in myocardium, transducing physical and biochemical signals to the cardiac genome that contribute to hydrogel function, providing a potential therapeutic target for enhancing hydrogel-mediated heart repair without stem cells.
Abstract 110: Activation of the Homeostatic Intracellular Response Leads to Increased Myocardial Mitochondrial Turnover in Patients Undergoing Cardiac Surgery
Authors: Andres Allen M , Sengstock David , Jahania Salik , Dabir Reza , Gottlieb Roberta A , Mentzer Robert M
Published: July 2021

Background:
Previously we showed that the homeostatic intracellular repair response (HIR2) is activated in the hearts of patients undergoing cardiac surgery. Autophagy is a principal component of this beneficial response that clears fragile mitochondria and protein aggregates. Moreover, we have previously shown that mitochondrial elimination through autophagy (mitophagy) is a key element in ischemic preconditioning. Thus, an important mechanism of cardioprotection appears to involve the upregulation of autophagy which facilitates the clearance of vulnerable mitochondria to limit I/R injury. We hypothesized that this protective action leads to turnover of the existing mitochondrial population in the heart during the resolution of I/R injury. The purpose of this study was to examine if the mechanism of HIR2 extends to remodeling the existing mitochondrial population of the heart.

Study Design:
Autophagy and mitochondrial turnover were assessed in 10 patients undergoing coronary artery bypass or valve surgery requiring cardiopulmonary bypass. Biopsies of the right atrial appendage obtained before initiation and after weaning from cardiopulmonary bypass were processed to yield whole tissue lysates and mitochondria-enriched heavy membrane fractions. Samples were analyzed for autophagy by immunoblotting for LC3, Beclin-1, ATG5-12, and p62. Mitochondrial turnover was assessed by monitoring Tom70, Cox4, Drp1, p62 and Parkin in tissue lysates and heavy membrane fractions.

Results:
Heart surgery was associated with a robust increase in autophagy indicated by depletion of LC3, Beclin-1, ATG5-12 and p62, as well as the mitophagy and fission regulator Drp1. Parkin increased in the mitochondrial fraction after bypass. Surprisingly, post-bypass tissue lysates showed a marked increase in mitochondrial markers Tom70 and Cox4, suggesting mitochondrial biogenesis.

Conclusions:
These findings provide evidence for the first time in humans that coordinated mitophagy and biogenesis are part of the homeostatic response to I/R, pointing to the importance of studying this aspect of HIR2. Strategies designed to amplify HIR2 during cardiac stress may represent an entirely new approach to myocardial protection in patients undergoing heart surgery.
Abstract 13: Hippo Signaling Deletion During Heart Failure Reverses Functional Decline
Authors: Leach John , Heallen Todd , Zhang Min , Morikawa Yuka , Martin James
Published: July 2021

The heart has long been thought of as a static organ incapable of repair. Recent findings have challenged this view of the heart, and have demonstrated that mature cardiomyocytes are capable of re-entering the cell-cycle. However, there is still paucity in understanding endogenous mechanisms preventing cardiomyocyte self-renewal. Our approach is to apply developmental mechanisms of cardiomyocyte cell-cycle control to the damaged heart by altering the Hippo signaling pathway.

During development Hippo signaling regulates intrinsic organ size. The core mammalian Hippo pathway includes the Ste20-like serine/threonine kinases Mst1 and Mst2, homologous to the Drosophila Hippo kinase. A subsequent kinase cascade leads to the phosphorylation of the transcription factor Yap. Phosphorylated Yap is sequestered in the cytoplasm, thus preventing transcriptional activity. We previously demonstrated Hippo signaling controls cardiomyocyte proliferation during development to restrain heart size. Additionally, using both the Apex resection (AR) and LAD-ligation (MI) models of cardiac damage, in a Hippo signaling deletion mouse, we demonstrated cardiac regeneration. Indicated by preserved cardiac function and reduced fibrotic scar formation. Additionally, these hearts display cardiomyocyte proliferation as marked by EDU incorporation, pHH3, AurkB, and Ki67 staining.

We are taking a new approach to determine the effect of Hippo signaling deletion on the failing heart, by inducing Hippo deletion after fibrotic scar formation has already occurred. To Thus far our results indicate functional recovery of the failing heart only after inducible deletion of Hippo signaling. Consistent with our previous data, preliminary results indicate adult cardiomyocytes after Hippo deletion re-enter the cell cycle. By altering Hippo signaling during heart failure and subsequently inducing cardiomyocyte proliferation we have established recovery of cardiac function. These results will greatly advance strategies to induce cardiac repair.
Abstract 132: The First Mouse Mutants of a Novel Epigenetic Modifier, Rearranged L-Myc Fusion, Display Defects in Heart Development
Authors: Bourke Lauren M , Harten Sarah , Bharti Vandhana , Oey Harry , Whitelaw Nadia , Whitelaw Emma
Published: July 2021

Rearranged L-Myc Fusion, Rlf, was recently identified as a novel epigenetic modifier from a mouse N-ethyl-N-nitrosourea mutagenesis screen. The mice used in this study carry a multi-copy green fluorescent protein (GFP) transgene linked to an erythroid specific α-globin promoter that is sensitive to epigenetic silencing. Three independent mouse lines with mutations in Rlf were each found to have a decrease in GFP expression, suggesting Rlf acts an epigenetic modifier. Our study is the first to reveal a role for Rlf in epigenetics.

Preliminary phenotyping has found loss of Rlf results in perinatal lethality. Late gestation homozygous null Rlf mutants were found to weigh significantly less than their heterozygous or wild type littermates. Histological analysis of mid gestation embryos has identified a potential heart defect in homozygous mutants. Rlf mutants display a thin compact layer, an overabundance of trabeculae and a fenestrated interventricular septum. The Rlf mutant phenotype is reminiscent of ventricular noncompaction defects observed in humans, such as left ventricular noncompaction cardiomyopathy.

RNA-seq analysis of mid gestation Rlf wild type and null hearts, prior to the observation of a cardiac defect, was undertaken to determine which pathways may be regulated by Rlf in the heart. More genes were observed to be significantly down-regulated in Rlf mutant hearts compared to wild types. Pathway analysis of differentially expressed genes showed genes involved in cell-cell adhesion, cell signalling, glycosylation and the Notch pathway were dysregulated. These findings indicate that Rlf may play a critical role in cardiac development.

Whole genome bisulphite sequencing of different embryonic tissue/stages has shown loss of Rlf results in an increase in methylation at a large number of distinct loci across the genome. Many of which were found to overlap sites reported to be putative regulatory elements, and histone marks associated with active or poised regulatory elements in the heart.

Our data suggest Rlf plays a key role in regulating gene expression pathways during heart development. These are the first mouse mutants available to study how Rlf functions as an epigenetic modifier and the phenotypic consequences of Rlf inactivation.
Abstract 150: Association Between Serum Ghrelin Levels and Coronary Artery Disease: a Meta-analysis
Authors: Agasthi Pradyumna , Aloor Sivakanth , Chenna Avantika , Onwuanyi Anekwe
Published: July 2021

Background:
Ghrelin (GH) is a gastrointestinal endocrine peptide regulating multiple biological processes including adipogenesis, glucose metabolism, cell differentiation and proliferation. Recent studies demonstrated that GH inhibits pro-atherogenic changes in vessel wall via inhibition of nuclear factor - B activity, a transcriptional factor mediating production pro-inflammatory cytokines and adhesion molecule expression in the endothelium. The aim of the current study is to conduct a meta-analysis to evaluate the relationship between serum GH levels and coronary artery disease (CAD).

Methods:
We searched MEDLINE, CINHAL and COCHRANE databases for studies reporting serum GH levels in the CAD and non CAD study population. We included case controls, cohort and cross-sectional studies. We calculated the weighted standardized mean difference (SMD) in serum GH levels between the CAD and control groups.

Results:
Our search strategy yielded 285 articles and we included 10 studies enrolling 1855 participants. The median age of the CAD group was 62 yrs. (IQR 60 - 63) compared to 61 yrs. (IQR 58 - 65) in the control group. The median body mass index in the CAD group was 28 kg/m2 (IQR 27.9 - 28) compared to 27 kg/m2 (IQR 26 - 27) in the control group. The unweighted median serum GH levels in the CAD group were 0.66 ng/ml (IQR 0.3 - 1.6) compared to 0.76 ng/ml (IQR 0.38 - 4.9) in the control group. The SMD of GH level was -0.44 (95% CI -0.56,-0.31) p<0.001 comparing those in the CAD group and control group.

Conclusion:
Serum GH levels are significantly and inversely associated with CAD. Current findings warrant the need to further investigate the role of GH in the pathogenesis of CAD.
Abstract 163: Regulation of Adult Cardiomyocyte Proliferation and Repair by Tbx20
Authors: Yutzey Katherine E , Guo Minzhe , Xiang Fu-Li
Published: July 2021

Background:
In the adult heart, there is increasing evidence that cardiomyocytes can proliferate, but these rates are exceedingly low and are not sufficient for myocardial repair after injury. In the embryonic heart, the T-box transcription factor Tbx20 is required for cardiomyocyte proliferation, and Tbx20 overexpression (Tbx20OE) promotes fetal characteristics in adult cardiomyocytes when initiated before birth in mice. We hypothesize that Tbx20OE, when induced in adult cardiomyocytes, promotes proliferation and improves cardiac repair after injury.

Methods and Results:
Mice were generated with tamoxifen-inducible Tbx20OE specifically in differentiated cardiomyocytes driven by αMHCMerCreMer. Tbx20OE initiated in adult cardiomyocytes leads to increased numbers of mononucleated proliferating cardiomyocytes, increased expression of cyclins, and increased total numbers of cardiomyocytes. No changes in cardiac function or heart weight/body weight ratios were observed, although the average size of cardiomyocytes with Tbx20OE is smaller than controls. In addition cardiac proliferative pathways, including AKT, Yap1, and IGF1, are activated, while cell cycle inhibitors, including Meis1 and p21, are decreased. In order to determine if Tbx20 promotes cardiac repair after injury, Tbx20OE was induced 3 days post-myocardial infarction (MI). In Tbx20OE mice subjected to MI, cardiac function was significantly improved, the infarct scar size was smaller, and increased pHH3+ cardiomyocytes were observed. Likewise, capillary density and expression of VegfA and bFGF are increased in the injured myocardium with Tbx20OE. Ongoing studies will determine direct regulatory interactions of Tbx20 with genes and regulatory pathways that promote cardiomyocyte proliferation and repair in adults.

Conclusions:
Tbx20OE in adult CM activates cell proliferation also improves cardiac function and repair in mice when induced post-MI.
Abstract 167: Mir-195-3p/-5p Decrease Cardiac Fibroblast Proliferation and the Transdifferentiation into Myofibroblasts
Authors: Shen Shutong , Wang Xiuzhi , Xu Dongjie , Tao Lichan , Wu Xiaoting , Xiao Junjie , Li Xinli
Published: July 2021

Aims:
MicroRNAs (miRNAs, miRs) contribute to many essential physiological and pathological processes including fibrosis. This study aims at investigating the role of miR-195-3p/-5p in cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts.

Methods and results:
In isolated primary neonatal cardiac fibrobasts (NRCFs), forced expression of miR-195-3p/-5p with agomiRs could attenuate fibrobast proliferation as determined by EdU and Ki67 staining while inhibition of miR-195-3p/-5p with antagomiRs could increase fibrobast proliferation. By quantitative reverse transcription polymerase chain reactions (RT-PCRs) and western blotting (WB), α-SMA (a marker of myofibroblast transdifferentiation) was found to be suppressed in the miR-195-3p/-5p agomiR-treated NRCFs at both mRNA and protein levels, while was increased in the miR-195-3p/-5p antagomiR-treated NRCFs. Moreover, Chek-1 was identified as a target gene of miR-195-3p/-5p responsible for cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts by RT-PCR and WB and immunofluorescent staining. Silencing of Chek-1 attenuates cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts as detected by α-SMA/EDU staining. In addition, Chek-1 mediated the effects of miR-195-3p/-5p in cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts.

Conclusion:
Therefore, miR-195-3p/-5p might be promising therapeutic targets for cardiac fibrosis.
Abstract 172: Interleukin-10-mediated Activation of AKT and Bcl2 Inhibits Chronic Angiotensin II-induced Pathological Autophagy
Authors: Verma Suresh K , Krishnamurthy Prasanna , Girikipathi Venkata N , Abramova Tatiana , Khan Moshin , Nickoloff Emily , Johnson Jennifer , Benedict Cynthia , Kishore Raj
Published: July 2021

Rationale:
Although, autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological (stress-induced exaggerated/defective) autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation of autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly inhibited pressure overload-induced hypertrophy and heart failure, but role of IL-10 in regulation of pathological autophagy is not known.

Hypothesis:
We tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and this process, in part, led to improved cardiac function.

Methods and Results:
Pathological autophagy was induced in wild type (WT) and IL10-knockout (IL-10 KO) mice by angiotensin II (Ang II for 28 days) infusion. Ang II-induced left ventricular (LV) dysfunction and hypertrophic remodeling were accentuated in IL-10 KO mice compared to WT mice. IL-10 KO mice showed exaggerated autophagy as observed by Electron Microscopy and Western blotting (beclin 1, LC3 II/I and CHOP) with reduced AKT phosphorylation at serine-473. In neonatal rat ventricular cardiomyocytes (NRCM), Ang II treatment enhanced beclin1, LC3 and CHOP protein levels and inhibited AKT and 4EBP1 phosphorylation and Bcl2 levels. Interestingly, IL-10 inhibited Ang II-induced autophagic marker proteins. Additionally, IL-10 restored Ang II-induced suppression of AKT and 4EBP1 phosphrylation and restoration of Bcl2 protein level. Pharmacological inhibition of AKT via PI3K inhibitor (LY290002), reversed IL-10 responses on the Ang II-induced pathological autophagy, confirming that IL-10 mediated inhibition of autophagy is AKT dependent. Finally, as physical interaction of Bcl2 with beclin 1 is important to inhibit autophagy, we performed immunoprecipitation pull-down experiments, which showed Ang II disrupts the physical interaction of beclin 1 with Bcl2 and IL-10 reestablished this physical interaction to reduce autophagy.

Conclusion:
Our data provides a novel role of IL-10 in regulation of pathological autophagy and thus can act as a potential therapeutic molecule in treatment of chronic heart disease.
Abstract 175: Pro-survival Function of Mef2 in Cardiomyocytes is Enhanced by β-blockers
Authors: Hashemi Sara , Salma Jahan , Wales Stephanie , McDermott John C
Published: July 2021

β1-adrenergic receptor (β1-AR) stimulation increases apoptosis in cardiomyocytes via activation of cAMP/ protein kinase A (PKA) signaling. β-adrenergic receptor antagonists, or β-blockers, oppose the action of PKA signaling by blocking the β1-receptor and effectively inhibit apoptosis and heart failure. The Myocyte Enhancer Factor 2 (MEF2) proteins have been implicated as nuclear targets for signaling cascades involved in muscle-gene expression and have important roles in proliferation, apoptosis and survival in multiple cell types. We previously reported that PKA signaling represses MEF2 activity. Here, we assessed whether β-blockers can inhibit neonatal cardiac myocyte apoptosis by interfering with PKA dependent MEF2 repression. We show that siRNA mediated MEF2 loss of function induced cardiomyocyte apoptosis. β1AR activation by isoproterenol treatment represses MEF2 transcriptional activity and promotes apoptosis in neonatal cardiomyocytes and, importantly, this effect was reversed in cells expressing a PKA resistant form of MEF2D (S121/190A), as indicated by FACS analysis. We also report that a β-blocker, Atenolol, antagonizes isoproterenol induced apoptosis and also acutely enhanced MEF2 transcriptional activity. We observed that β-adrenergic stimulation modulated MEF2 cellular localization in neonatal cardiomyocytes and this was reversed by atenolol treatment. In addition, we also document that Kru[[Unable to Display Character: ̈]]ppel-like factor 6 (KLF6) is an important MEF2 target gene and loss of function analysis using siRNA-mediated knockdown of KLF6 expression resulted in cardiomyocyte apoptosis. Collectively, these observations, establish that MEF2 plays an important pro-survival role in cardiomyocytes which can be modulated by β-adrenergic signaling. These observations have important clinical implications and may contribute to novel strategies for preventing cardiomyocyte apoptosis associated with heart pathology.
Abstract 176: Interleukin 17 A, Hepatosplenic Mansonic Schistosomiasis and Atherosclerosis
Authors: Oliveira Dinaldo C , Santos Diego , Alcantara Thamires , Coutinho Ana L , Brandt Carlos
Published: July 2021

Introduction:
The immune system plays an important role in the pathophysiology of schistosomiasis and atherosclerosis due to its activation of various immune cells and release of different inflammatory mediators. However, the pathologies of these diseases are not fully understood. The main aim of this study was to detect atherosclerotic disease in patients with mansonic schistosomiasis, while the secondary objectives were evaluate the serum concentrations of interleukin 17A (IL 17 A) and 22 (IL 22).

Hypothesis:
Atherosclerosis is not common in patients with mansonic schistosomiasis.

Methods:
This study included 30 patients (14 men, 16 women; mean age = 59.87 ± 7.50 years) with hepatosplenic mansonic schistosomiasis that agreed to participate in the study by signing the Free and Informed Consent Form. Ten healthy volunteers (four men, six women) were selected as controls for interleukin concentrations. Atherosclerosis was evaluated by measuring the carotid artery intima-media thickness (IMT ≥1.5 mm are indicative of atherosclerotic plaques). Shapiro-Wilk test was necessary to determine whether the variables followed a normal distribution. The continuous variables were evaluated using student’s t test. P value ≤ 0.05 was considered statistically significant.

Results:
The main clinical characteristics of the patients were as follows: benign arrhythmias, 19 patients (63%); hypertension, 17 (56%); smoking, 12 (40%); hepatitis B or C, nine (30%); peripheral vascular insufficiency, nine (30%); family history of CAD, nine (30%); diabetes mellitus, one (3.3%); leukemia, one (3.3%); and non-cardiac surgery, 24 (80%). There were no differences in the IL 17 A concentrations (15.63 ± 0.00 pg/ml vs 15.63 ± 0.00 pg/ml, p = 1) and in the IL 22 concentrations (7.81 ± 0.00 pg/ml vs 7. 81 ± 0.00 pg/ml, p = 1) between patients and controls. The overall mean of the intimal medial thickness was 0.7 ± 0.2 mm

Conclusions:
None of patients had atheroma. The serum concentrations of IL 17A and IL 22 were equal between patients and controls (concentrations undetectable or low). It is possible that the immune response of patients with hepatosplenic mansonic schistosomiasis may attenuate the development of atherosclerosis.
Abstract 182: Characterization of Electrophysiological and Conduction Parameters of the Heart in Trpm4-deficient Mice
Authors: Jacobs Griet , Kecskes Miklos , Vennekens Rudi
Published: July 2021

TRPM4 is a Ca2+-activated non-selective cation channel that belongs to the family of the Transient Receptor Potential (TRP) ion channels. Importantly, TRPM4 is impermeable for Ca2+ and is involved in different Ca2+-dependent cell functions, such as exocytosis, contraction and cell death. Trpm4 is known to be expressed in atrial and ventricular cardiomyocytes. The interest in the functional role of TRPM4 in the heart has risen further by the discovery of Trpm4 mutations that are linked to cardiac conduction disorders, including Progressive Familial Heart Block type I (PFHBI) and Brugada Syndrome. Both gain-of-function and loss-of-function mutation were described in patients with cardiac conduction diseases. Recently, our group showed that TRPM4 plays a role during the late repolarization phase of the action potential in murine ventricular cardiomyocytes and that deletion of the Trpm4 gene leads to shorter ventricular action potentials. To characterize if deletion of Trpm4 has an effect on the conduction properties of the heart, an in depth electrophysiological study was performed in living mice. An octapolar catheter was inserted into the right atrium and ventricle of the heart to measure intracardial electrograms. The atrial-His (AH) and His-ventricular (HV) intervals were calculated and no differences were found between WT and Trpm4-deficient mice. Additionally, more detailed conduction parameters of the heart were determined by use of programmed electrical stimulation (PES) protocols. Sinus node recovery time (SNRT) was not different between WT and Trpm4-deficient mice. Effective refractory period of atrium (AERP), AV node (AVNERP) and ventricle (VERP) were in the same range in WT and Trpm4-deficient mice. Wenckebach periodicity, the parameter for AV nodal conduction, was also not different between WT and Trpm4-deficient mice. These results suggest that deletion of Trpm4 has no effects on the conduction properties of the murine heart.
Abstract 191: Deletion or Knockdown of Myotonic Dystrophy Protein Kinase Does Not Affect Cardiac Conduction or Ejection Fraction in Mice
Authors: Carrell Samuel , Auerbach David , Pandey Sanjay , Bennett Frank , Dirksen Robert , Thornton Charles
Published: July 2021

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, causes dominantly-inherited muscle weakness, defects of cardiac conduction, variable LV dysfunction, and risk of sudden death. The genetic basis is an expanded CTG repeat in the 3’ untranslated region of DMPK. DM1 patients are functionally hemizygous for DMPK protein, due to nuclear retention of mRNA having expanded repeats. The cardiac aspects are attributed to DMPK loss, toxicity of RNA with expanded repeats, or both. Dmpk heterozygous (+/-) and homozygous knockout (-/-) mice were reported to show AV conduction abnormalities resembling DM1 (Berul et al, JCI, 1999). In an effort to reduce RNA toxicity, antisense oligonucleotides (ASOs) targeting DMPK mRNA have recently entered clinical trials. DM1 phenotypes in skeletal muscle were corrected by ASO knockdown of toxic RNA in mice (Wheeler et al, Nature, 2012). While ASOs may have similar potential to mitigate RNA toxicity in the heart, there is risk of aggravated DMPK deficiency. To reexamine the role of DMPK in the conduction system we studied mice with Dmpk gene deletion or ASO knockdown. We obtained ECGs and echocardiograms on Dmpk -/- and +/- mice, compared to WT littermates. The +/- mice were treated with Dmpk-targeting ASOs or saline. Subcutaneous injection of 50 mg/kg/wk ASO was started at age 2 months, then shifted to biweekly injections after 6 weeks. Dmpk expression in hearts of +/- mice was ~50% of WT, and was further reduced by ASOs (84 ± 3% decrease of mRNA, 93 ± 2% decrease of protein, relative to WT). Surface ECGs and echocardiography at 6 and 10 months showed no differences of heart rate, cardiac conduction, or ejection fraction in WT, saline-treated +/-, ASO-treated +/-, or -/- mice. Conscious, unrestrained ECGs obtained at 11-12 months by radiotelemetry showed no differences among WT, saline-treated +/-, ASO-treated +/-, or -/- mice. We conclude that ASOs can induce posttranscriptional silencing of Dmpk in murine hearts. Constitutive absence of DMPK did not impact cardiac conduction or contractility, and the same was true for ASO knockdown to levels <15% of WT. Our data support the idea that cardiac dysfunction in DM1 results mainly from RNA toxicity, which potentially could be prevented or alleviated by ASOs.
Abstract 2: Mesenchymal Stem Cell Therapy Prevented Doxorubicin Induced Cardiac Dysfunction in Diabetics Rats
Authors: Dhingra Sanjiv , Ammar Hania Ibrahim , Nashed Mira Barsoum , Ammar Rasha Ibrahim , Gabr Hala , Elsayed Hany Elsebaee , Sequiera Glen Lester , Abu-El Rub Ejlal , Sareen Niketa
Published: July 2021

Background:
It is now established that having diabetes not only increases the chances of cancer also it complicates cancer treatment therapy. Doxorubicin (DOX) is a well known anticancer drug, however the clinical use of DOX was limited due to its cardiotoxic effects. One of the major concerns with DOX therapy has been its toxicity in patients who are less robust and more prone to toxic side effects, particularly patients with comorbid diseases such as diabetes mellitus. Several studies have demonstrated that mesenchymal stem cell (MSC) therapy has the potential to restore cardiac function following DOX induced cardiac injury. However, there is no study available on the effects of MSC therapy on DOX induced cardiac dysfunction in diabetics.

Methods and Results:
Diabetes was induced in male Wistar rats by streptozotcin injection (STZ, 65mg/kg body weight, i.p.). After 4 weeks of STZ injection, blood glucose levels in STZ group (301.58±23.97mg/dl) were significantly greater than control group (83.51±7.91mg/dl). These diabetic rats were treated with adriamycin (2.5mg/kg body weight, i.p) 3 times/week for two weeks (AD group); or with adriamycin+bone marrow MSCs (BM-MSC; 2x106 cells, via tail vein) or with adriamycin+adipose tissue derived MSCs (AD-MSC; 2x106 cells, via tail vein). Echocardiographic measurements showed a significant decline in cardiac function (%EF) following adriamycin treatment. Both BM-MSC and AT-MSC treatment improved %EF at 4 weeks. After 4 weeks of MSC injection, hearts from all the groups were excised and subjected to retrograde Langendorff perfusion and baseline levels of left ventricular developed pressure (LVDP), maximum rate of pressure rise dp/dt max and rate pressure product (RPP) were recorded. AD treatment caused a significant decrease in LVDP, dp/dt max and RPP levels. Both BM-MSCs and AD-MSCs injection significantly improved all these parameters.

Conclusion:
Both BM-MSC and AT-MSC were equally effective in preventing deterioration of cardiac function following doxorubicin therapy in diabetic rats. Furthermore, these findings should act as a stimulus for further research on the benefits of MSC therapy for diabetic subjects suffering from cancer.
Abstract 205: Mlip is a New Integrator of Physiological Stress Required For Proper Cardiac Adaptation
Authors: Cattin Marie-Elodie , Wang Jessica , Roeske Cassandra , Mak Esther , Thorn Stephanie L , DaSilva Jean N , Wang Yibin , Lusis Aldon J , Burgon Patrick G
Published: July 2021

Aging and diseases are generally a result of the inability of tissues to properly adapt to stress. The heart is particularly vulnerable to disequilibrium in homeostasis as its regenerative capacities are limited. Many molecular players have been identified as cardiac gate-keepers and integrators of stress, all essential to preserve cardiac function. However, the molecular basis of the relationship between aging and the pathogenesis of cardiac dysfunction remains poorly understood. Muscle enriched A-type lamin interacting protein (MLIP) is a unique mammalian protein of unknown function that was recently identified through its interaction with A-type lamins in the heart and as a modulator of cardiac hypertrophy in vitro. Here we report that young Mlip deficient mice develop dilated cardiomyopathy, manifested by an increase in heart mass with reduced cardiac function. Through global gene expression profiling of the Mlip deficient hearts, we identified a deregulation in mTOR signalling, a key stress sensing canonical pathway. Analysis of mTOR regulatory proteins (AMPK and AKT) revealed hyperactivation of the mTOR pathway and a deregulated integration of these two stress/nutrient sensors. These data support the notion that Mlip deficient hearts have impaired cardiac adaptation due to deregulated mTOR activity resulting in maladaptive remodeling, and the development of dilated cardiomyopathy. These results are further supported by a genetic association between Mlip and early response to pro-hypertrophic stimulus. Collectively, these results demonstrate that Mlip is required for normal integration of physiological stress (postnatal cardiac growth, isoproterenol-induced hypertrophy) through the regulation of the AMPK/Akt/mTOR pathway to maintain cardiac homeostasis in the adult heart.
Abstract 214: MicroRNA-451 Aggravates Lipotoxic Cardiomyopathy Through Suppression of the LKB1/AMPK Signaling Pathway
Authors: Kuwabara Yasuhide , Horie Takahiro , Baba Osamu , Kita Toru , Kimura Takeshi , Ono Koh
Published: July 2021

Rationale:
In some type 2 diabetes mellitus (T2D) patients without hypertension, cardiac hypertrophy and attenuated cardiac function are observed, and this insult is termed “diabetic cardiomyopathy.” Tons of evidence suggests that microRNAs are involved in cardiac diseases. However, the functions of microRNAs in the diabetic cardiomyopathy induced by T2D and obesity are not fully understood.

Methods and Results:
C57BL/6 mice were fed a high-fat diet (HFD) for 20 weeks, which induced obesity and T2D. MicroRNA microarray and real-time PCR revealed that miR-451 levels were significantly increased in the T2D mouse hearts (n=4-5, p<0.05). Because excess supply of saturated fatty acids is a cause of diabetic cardiomyopathy, we stimulated neonatal rat cardiac myocytes (NRCMs) with palmitate in physiological albumin concentration and confirmed that miR-451 expression was increased in a dose-dependent manner (n=6-12, p<0.01). Loss of miR-451 function ameliorated palmitate-induced lipotoxicity in NRCMs (n=4, p<0.05). Calcium-binding protein 39 (Cab39) is a scaffold protein of liver kinase B1 (LKB1), an upstream kinase of AMP-activated protein kinase (AMPK). Cab39 was a direct target of miR-451 in NRCMs and Cab39 overexpression rescued the palmitate-induced lipotoxicity in NRCMs (n=4, p<0.01). To clarify miR-451 functions in vivo, we generated cardiomyocyte-specific miR-451 knockout (cKO) mice. HFD-induced cardiac hypertrophy and contractile reserves were ameliorated in cKO mice compared with HFD-fed control mice. Protein levels of Cab39 and phosphorylated AMPK were increased and phosphorylated mammalian target of rapamycin (mTOR) was reduced in HFD-fed cKO mouse hearts compared with HFD-fed control mouse hearts (n=10-12, p<0.05). We also measured the lipotoxic intermediates, triglyceride and ceramide, in these mouse hearts using HPLC-evaporative light scattering detector (ELSD). Although there was no difference in triglyceride levels (n=3-5), ceramide level was decreased in HFD-fed cKO mice compared with HFD-fed control mice (n=3-5, p<0.05).

Conclusions:
Our results indicate that miR-451 exacerbates diabetic cardiomyopathy. miR-451 is a potential therapeutic target for cardiac disease caused by T2D and obesity.