bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 125: Role for β-Arrestins in Cardiac Inflammation and Regenerative Repair
Authors: Gumpert Anna M , Chen Mai , Brinks Henriette , Peppel Karsten , Gao Erhe , Koch Walter J
Published: July 2021

Myocardial ischemic injury is characterized by an extensive loss of myocytes due to apoptosis/necrosis followed by an immune/inflammatory response and reestablishment of tissue integrity by a fibrotic scar. Prevention of rupture due to scar formation however, compromises contractility and reduces cardiac function. As bone marrow derived stem/precursor cells (BMSCs) have the ability to trans-differentiate and aid in the regeneration of injured myocardium, we studied BMSCs and their response to inflammation. BM cell mobilization, egress from the BM and recruitment to the site of injury can be regulated by signals through G protein-coupled receptors (GPCRs). β-arrestins are known for their GPCR signaling termination and scaffolding functions. Here, we explored the potential role for β-arrestins expressed in total BM in mediation of the initiation and progression of inflammation. We investigated the role β-arrestin1 (βArr1) and β-arrestin2 (βArr2) with respect to modulation of regenerative competence of BMSCs and inflammatory cells and their contribution to cardiac repair following ischemic injury. We carried out BM transplants to determine whether βArrs may be involved in cardiac repair. WT mice were irradiated and received BM transplants from WT, βArr1 or βArr2 KO donor mice. Subsequent to BM reconstitution, mice underwent MI and their recovery and progress were followed. Interestingly, βArr1 and βArr2 KO chimeras had inferior outcomes than mice receiving WT BM. This included significantly decreased post-MI survival with βArr2 KO BM and both βArr chimeras had significantly lower cardiac function post-MI compared to WT BM recipients. Histology shows that both chimeras had larger infarcts, accelerated rate of hypertrophy, quicker induction of fibrosis, exacerbated cell infiltration and increased number of apoptotic cells. At 3d post-MI immune cell intrusion of βArr2KO chimeric hearts was highest and the blood plasma content of TNFα and IL-6 was increased. We conclude that βArrs play a novel role downstream of GPCR desensitization in cardiac progenitor/inflammatory cells in the BM and are critical in the heart’s response to ischemic injury via cardiac repair and regeneration.
Abstract 133: Novel Transgenic Mouse Lines for Identification of Cardiomyocyte Nuclei and Visualization of Their Cell Cycle Status In-vitro and In-vivo
Authors: Raulf Alexandra , Horder Hannes , Geisen Caroline , Grünberg Sabine , Freitag Patricia , Fleischmann Bernd K , Hesse Michael
Published: July 2021

The typical remodelling process after cardiac injury is scarring and compensatory hypertrophy. The limited regeneration potential of the adult heart is due to the post-mitotic status of cardiomyocytes (CMs), which are mostly binucleated. Nevertheless, there is evidence for CM turnover in the adult heart, but its extent is still under debate. One technical limitation of quantitations is the unequivocal identification of CMs and of CM cell division.

In order to enable a clear identification of CM nuclei in-vivo, we have developed a transgenic mouse line in which a fusion protein of the human histone 2B and the red fluorescence protein mCherry is specifically and persistently expressed in CM nuclei (αMHC-H2B-mCh). The fluorescence label allowed the investigation of CM percentages in native tissue slices, which properly reflect the cellular composition of the heart. We focused on regional (atrium versus left and right ventricle, apical versus basal slices) and developmental-stage dependent changes in the percentage of CM nuclei and binuclearity. We therefore analyzed time-points before/during and after terminal differentiation of CMs (postnatal day 3 (P3), P7 and 9 weeks). In addition, we also investigated endoreduplication and acytokinetic mitosis using time lapse microscopy in postnatal CMs to better understand cell biological mechanisms leading to terminal differentiation.

As currently huge efforts are invested for the search of substances that increase the regeneration potential of the heart, we established a novel screening assay for cell-cycle modifying substances in isolated, postnatal CMs. We crossed the αMHC-H2B-mCh with the CAG-eGFPanillin mouse line, which marks cell-cycle activity with a high resolution of M-phase. Analysis of binuclearity and of different eGFPanillin subcellular localizations will be helpful to understand, whether CMs complete cytokinesis. As a proof of principle we investigated the effects of cell cycle activating micro RNAs199 and 590.

Thus, our double transgenic mouse line will be useful to examine the plasticity of mono- and binuclear CMs and to unravel cell biological mechanisms leading to terminal differentiation of CMs.
Abstract 136: Gel Stiffness-tuned, Nanostructure-templated Alignment and Maturation of Cardiomyocytes For Cardiac Tissue Engineering
Authors: Sleep Eduard , Mantei Jason R , McClendon Mark T , Stupp Samuel I
Published: July 2021

Objective:
To investigate how the supramolecular structure and mechanical properties of hydrogels made of chemically-defined peptide amphiphiles (PAs) could influence i) the alignment of cardiomyocytes seeded in them and ii) the functional maturation of the cell construct.

Methods:
We generated a series of PAs with different peptide sequences that allowed us to make PA gels with stiffness values ranging three orders of magnitude. The nature of the gelation process provides these gels with same-direction oriented nanofibers along an elongated PA gel. We seeded these PA gels with either HL-1 cardiomyocytes or iPSC-derived cardiomyocytes and cultured them for up to two weeks. We then measured cell survival, proliferation and alignment over time and also the electrophysiological properties of the cell construct as a whole.

Results:
We found that cardiomyocytes responded to the alignment of the nanofibers in the gel by aligning to them and that this process can be tuned by changing the stiffness of the PA gel. This in turn influenced the investigated electrophysiological parameters of the cell construct suggesting a functional maturation induced by the PA gel properties.

Conclusions:
We believe this study shows for the first time that the nanostructural and mechanical properties of hydrogels can be exploited to influence the maturation of cardiomyocytes/cell construct. This can have important implications for strategies that aim to use ESC- or iPSC-derived cardiomyocytes in tissue engineering, as there is a need for their proper maturation and function.
Abstract 139: Alterations to the Extracellular Matrix Composition Following Myocardial Infarction Impact Cardiac Progenitor Cell Fate In Vitro
Authors: Sullivan Kelly E , Sant Sharada , Burns Laura , Black Lauren D
Published: July 2021

Limitations associated with cardiac progenitor cell (CPC) therapy of myocardial infarction (MI) including poor engraftment, cell death and incomplete cardiac differentiation have hindered the efficacy of treatment in pre-clinical trials. Given that the extracellular environment plays an important role in regulating cell function and that it is significantly remodeled following MI, it is critical to understand how these changes impact the therapeutic potential of CPCs. In this study, we investigated how the alterations to the extracellular matrix (ECM) following MI impacted the regenerative potential of CPCs in vitro. Hearts were decellularized with 1% SDS prior to MI and 1 and 4 weeks post-MI (Fig A) and the composition of the left ventricle or scar was characterized through LC-MS/MS. While Periostin and Collagen I increased post-MI, Laminin decreased (Fig B). c-kit+ CPCs isolated from rat hearts 1 week post-MI were cultured on tissue culture plastic (TCP) coated with pepsin-solubilized ECM. Our results demonstrated that the healthy matrix promoted the expression of pro-angiogenic growth factors, while maintaining the cells in an undifferentiated state (Fig D,E). Alternatively, 1 week ECM promoted cell adherence (Fig C) and the expression of pro-survival growth factors (Fig D) and GATA-4 (Fig E). Cells cultured on 4 week ECM demonstrated significant differentiation towards vascular lineages through their expression of smooth muscle (TAGLN) and endothelial (VWF) markers. By characterizing how the changing ECM composition following MI impacts CPC fate, we may be able to develop therapeutic strategies that modulate cell fate/ function in vivo following implantation.
Abstract 145: Mitophagy Protects Against Statin-Induced Cell Death in Muscle Cells
Authors: Andres Allen , Ramesh Mridula , Hernandez Genaro , Gurney Michael , Linton Phyllis-Jean , Islam Moutusee , Gottlieb Roberta
Published: July 2021

Statins are a family of cholesterol lowering drugs associated with undisputable cardiovascular benefits. However, their use is associated with the risk of developing skeletal muscle myopathy which remains a significant barrier to maximizing the efficacy of statin therapy. Many recognize that the instances of statin-associated myopathy reported grossly underestimate the actual frequency of these events. Statin myopathy is a major reason for why many patients are unable to comply with their prescribed statin treatments. Identifying and understanding the molecular events involved in the development of statin-induced myopathy will pave the way to mitigating the unwanted effect of statin-myopathy.

We have identified that statins induce mitochondrial loss in both C2C12 myotubes, and in gastrocnemius muscle of mice evidenced by loss of mitochondrial markers Tom70 and Cox4 by western blot analysis. C2C12 myotubes were treated with 2μM simvastatin for 24 hours, and wild-type C57BL/6 mice were given a daily i.p. dose of 20mg/kg simvastatin for 2 weeks. Pretreatment of C2C12 cells with 100nM bafilomycin for 2 hours before statin treatment attenuated autophagic flux, and prevents the observed loss of mitochondria through Tom70 and Cox4 mitochondrial markers. Thus, the loss of mitochondria is attributable to autophagy (mitophagy). Statin treatment increases the expression of Bnip3 and p62/SQSTM1 (sequestosome 1) which have been identified as important facilitators of mitophagy. We found increased Bnip3 dimerization via western blot under non-reducing conditions which implies increased activity. Furthermore, we observed increased mitochondrial translocation of p62/SQSTM1. Based on our findings, we hypothesized that statin-induced mitophagy may play a key role in the development of muscle myopathy.

To investigate the role of mitophagy in statin myopathy, we silenced p62/SQSTM1 via RNAi prior to statin treatment. Surprisingly, reducing mitophagic flux via p62/SQSTM1 silencing resulted in increased destruction of myotubes. This was associated with increased levels of cytosolic cytochrome c. Our data combined suggests that mitophagy protects against statin-mediated cell death by sequestering and eliminating decrepit mitochondria via autophagy.
Abstract 150: S-nitrosylation Of Thioredoxin1 At Cysteine 73 Promotes Trans-nitrosylation, Autophagy And Cell Survival During Glucose Deprivation.
Authors: Nagarajan Narayani , Sciarretta Sebastiano , Sadoshima Junichi
Published: July 2021

Thioredoxin-1 (Trx1) is a key antioxidant protein that is known to play a protective role in the heart during oxidative stress mainly through its oxidoreductase activity. Trx1 can be S-nitrosylated and, in turn, can trans-nitrosylate other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is not known. Here, we investigated the role of Trx1-mediated protein S-nitrosylation in the regulation of CM survival in response to stress. Using the biotin-switch assay, we found that wild-type Trx1 (Trx1WT) was S-nitrosylated, whereas the extent of S-nitrosylation was attenuated in Trx1C73S, suggesting that Trx1 is S-nitrosylated at Cys73. Also, we observed that the redox activity of Trx1 was intact in the Trx1C73S mutant. Overall protein S-nitrosylation in rat neonatal CMs was increased in response to 4 hrs of glucose deprivation (GD). Using biotin-switch assay and immunocytochemistry (fluorescent staining of s-nitrosylated cysteines), we observed that overexpression of Trx1WT increased, whereas short-hairpin RNA-mediated knockdown of Trx1 (shTrx1) or overexpression of Trx1C73S decreased, total protein S-nitrosylation in response to GD. These results suggest that Trx1Cys73 plays a key role in the regulation of protein S-nitrosylation in CMs during GD. Overexpression of Trx1 increased CM survival after 24 hrs of GD (Trx1WT vs. LacZ: propidium iodide assay, 0.5 ± 0.08-fold, p<0.01). Conversely, shTrx1 or overexpression of Trx1C73S increased cell death during GD (Trx1C73S vs. LacZ: 1.7 ± 0.034-fold, p<0.05). Autophagy is a pro-survival mechanism during GD. Therefore, we tested the effect of Trx1 on autophagy. After 4 hrs of GD, knockdown of Trx1 or overexpression of Trx1C73S decreased autophagy compared to control cells (LC3-II/LC3-I, 0.7-fold; autophagosomes, 0.83 ± 0.16-fold; autolysosomes, 0.62 ± 0.13-fold, p<0.005). Taken altogether, our results suggest that Trx1 promotes autophagy during GD through a trans-nitrosylation dependent mechanism. S-nitrosylation of Trx1 at Cys73 is associated with an overall increase in protein S-nitrosylation in CMs and promotes autophagy and thus, cell survival during GD.
Abstract 151: MCL-1 Promotes Mitochondrial Fusion and Survival in Cardiac Myocytes
Authors: Thomas Robert L , Kuo Jennifer , Gustafsson Åsa B
Published: July 2021

Myeloid cell leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein that is highly expressed in myocardium, but little is known about its function in myocytes. Recently, we reported that MCL-1 is essential for myocardial homeostasis and autophagy. Cardiac-specific deletion of MCL-1 in mice led to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Despite extensive mitochondrial damage, MCL-1 deficient hearts failed to activate mitochondrial autophagy. Parkin, an E3 ubiquitin ligase, normally translocates to damaged mitochondria to promote mitochondrial autophagy, but loss of MCL-1 resulted in cytosolic accumulation of Parkin. However, we found no evidence that MCL-1 functions as a mitochondrial Parkin receptor or substrate. Instead, loss of MCL-1 reduced mitochondrial accumulation of PINK1, which is involved in Parkin recruitment. Additionally, we identified mitochondrial outer membrane (OM) and matrix isoforms of MCL-1 in mouse hearts and found that the two forms respond differently to ischemic injury. Four hours after myocardial infarction, MCL-1
OM
levels were reduced by 40% in border zone tissue. After 24 hours, MCL-1
OM
levels returned to baseline. Meanwhile, MCL-1
Matrix
levels were preserved at four hours, and increased significantly compared to control 24 hours after infarction. These changes correlated with increased expression of HSP70, a chaperone protein that stabilizes MCL-1 and participates in import of mitochondrial proteins. Overexpression of MCL-1
Matrix
promoted mitochondrial fusion in fibroblasts under baseline conditions and protected cells against FCCP-mediated mitochondrial fission. While 94.1% (1269 of 1349) of control cells exhibited fragmented mitochondria after two hours of FCCP treatment, mitochondria were only fragmented in 41.3% (391 of 947) of cells overexpressing MCL-1
Matrix
. These data suggest MCL-1 isoforms play different roles in the cellular stress response. MCL-1
OM
protects against apoptosis, whereas MCL-1
Matrix
protects mitochondria by promoting fusion. In addition, upregulation of HSP70 may preserve mitochondrial function and cell viability in damaged cardiac myocytes by increasing the stability and mitochondrial import of MCL-1.
Abstract 152: IL-10 Inhibits Angiotensin II-induced Pathological Autophagy in Myocardium.
Authors: Verma Suresh K , Krishnamurthy Prasanna , Abramova Tatiana , Srikanth Garikipati , Khan Mohsin , Mackie Alexander R , Klingler Aiko , Ramirez Veronica , Misener Sol , Qin Gangjian , Kishore Raj
Published: July 2021

Background:
In heart, persistent pressure overload causes pathological autophagy leading to cardiac cell death and heart failure. The role IL-10, a pleiotropic anti-inflammatory cytokine, on pathological autophagy is largely unknown. Here we hypothesized that IL-10 inhibits stress-induced pathological autophagy and therefore attenuates cardiac cells death and improve heart function.

Method and Results:
Cardiac stress was induced in C57 BL/6 mice by Angiotensin II treatment (Ang II-1.2mg.kg b.wt/day for 28 days) using mini osmotic pumps. Ang II treatment markedly induced autophagy in mice as measured by electron microscopy (autophagosome numbers) and Western blotting (Becline1 and LC3II proteins expression). Interestingly, systemic recombinant mouse IL-10 administration markedly inhibited Ang II-induced autophagy. To further understand the mechanism of IL-10 protection, neonatal rat ventricular myocytes (NRCM) were transfected with monomeric Red Fluorescent Protein-Enhanced Green Fluorescent Protein (mRFP-EGFP) tandem fluorescent-tagged LC3 (tfLC3) adenovirus (to measure autophagic flux) and then treated with AngII (1μM) and/or IL-10 (20ng/mL), in vitro. Ang II treatment significantly increased the numbers of both yellow (merged EGFP and mRFP signals) and red puncta, indicating active formation of both autophagosomes and autolysosomes, however, this flux was strongly inhibited by IL-10. Furthermore, Ang II significantly increased the Beclin1 and LC3II proteins expression, which was markedly reduced by IL-10 as measured by Western blot analysis. In addition, Ang II-inhibited AKT signaling (anti-autophagic signaling component) was strongly enhanced by IL-10. Ang II-induced autophagic signaling was mimicked by AKT inhibitor, suggesting AKT as the downstream target of IL-10 effects.

Conclusion:
Inhibition of pathological autophagy is a novel mechanism for cardio-protective effects of IL-10.
Abstract 16: Cardiosphere Derived Cells from Pediatric End-Stage Heart Failure Patients Have Enhanced Functional Activity Due to the Heat Shock Response
Authors: Sharma Sudhish , MIshra Rachana , Simpson David , Chen Ling , Colletti Evan , Deshmukh Savitha , Kaushal Sunjay
Published: July 2021

Background:
The potential of novel cell based therapies using cardiosphere derived cells (CDCs) to replace or repair damaged cardiac tissue is an attractive therapy and Phase I clinical trials in adults report encouraging results. We have previously demonstrated that age plays an important role in controlling the functional activity of CDCs and now we will determine how the heart’s physiology may change the functional activity of pediatric end-stage heart failure (ESHF) derived CDCs when compared to age-match control derived CDCs.

Methods and Results:
Our results shows that ESFH derived CDCs have significantly increased numbers of CSCs, including c-kit+, ISL-1+ and Sca-1+ cells. They are functionally more potent in an infarcted rodent model as compared to hCDCs derived from congenital heart disease (CHD) age-matched controls which have normal myocardium. The functional recovery was mediated in part by increased secretion of cytokines, SDF-1α and VEGF-A, which stimulated more angiogenesis, recruitment of endogenous stem cells, and proliferation of cardiomyocytes. The possible mechanism for this increased cytokine secretion was due to the activation of heat shock response (HSR), supported by three lines of evidence. First, gain of function studies demonstrated that the HSR induced the low functioning CHD-derived CDCs to significantly recover the injured myocardium, even more than the ESHF-derived CDCs by increasing cytokine secretion. Secondly, loss-of function studies targeting the HSR down regulating the ability of the ESHF-derived CDCs to secrete cytokines and thus functionally recover the injured myocardium. Finally, the HSR alone increased the number of the endogenous cardiac stem cell population and recovered the injured myocardium. We will present the results showing that activation of HSR expands cardiac stem cells (CSCs) without losing their phenotypic characteristics.

Conclusion:
Collectively, the present work demonstrates a novel biological activity of the HSR in its ability to increase the number of functional CSCs while maintaining the ‘stemness’ of these cells that have direct implications for future clinical trials.
Abstract 170: Hippo Signaling Regulates Epicardial Derived Cell Fate through Controlling Mechanical Property
Authors: Xiao Yang , Zhang Min , Morikawa Yuka , Martin James F
Published: July 2021

Epicardium is the outmost layer of heart and regulates heart development by contributing to the heart tissue and secreting signaling molecules. Epicardial-derived cells, EPDC, is a group of multi-potent cells mainly derive into cardiac fibroblasts and smooth muscle cells. Hippo signaling pathway controls organ growth through regulating cell proliferation. Yap, the effector of Hippo pathway has been shown to regulate mechanosensing and cell fate. However, the function of Hippo signaling in EPDC development has not been studied yet.

To study the role of Hippo signaling in development of epicardium and EPDC, we used epicardial specific line, Wt1
CreERT2
, to conditionally delete the components of the pathway, Lats1/2. Cre activity was induced at embryonic day (E)11.5 and Lats1/2 CKO hearts exhibited disorganized coronary vasculature. We used genetic lineage tracing approach to track EPDC and revealed increased endothelial cells deriving from EPDC, suggesting that EPDC changed cell fate. We observed that shape of the epicardial cells became rounded with decreased F-actin in Lats1/2 CKO, which suggested that cell tension was reduced. Since mechanical properties of cellular microenvironment are known to regulate cell shape and influence cell fate determination, we hypothesize that Hippo pathway regulates mechanical property to control cell fate in EPDC. Transcription analysis showed that signals regulating cytoskeleton organization were disrupted, which suggests Hippo regulates cytoskeleton gene expression and affects mechanical force that cell sensed autonomously. Proliferation assay using EdU incorporation showed increased proliferation in EPDC of Lats1/2 CKO was observed, which suggests the changes of external tension cell sensed. To recapitulate the changes of external tension, we plated EPDC onto different stiffness substrate. EPDC of Lats1/2 CKO differentiated into endothelial cells on the soft substrate. On the other hand, Lats1/2 CKO did not derive endothelial cells on the hard substrate and wild type EPDC did not differentiate into endothelial cells on neither soft nor hard substrate.

Taken together, our results suggested that Hippo pathway affected EPDC fate determination through controlling internal and external mechanical cues.
Abstract 181: Myozap Deficiency Promotes Adverse Remodeling And Cardiomyopathy In Response To Biomechanical Stress
Authors: Rangrez Ashraf Y , Frank Derk , Poyanmehr Reza , Bernt Alexander , Frey Norbert
Published: July 2021

Background:
Myozap is a new addition to the list of intercalated disc (ID) proteins, which we previously identified using a bioinformatics screen. In vitro characterization of Myozap revealed that it activates Rho-dependent SRF signaling. Moreover, cardiac restricted overexpression of Myozap in mice resulted in protein aggregate-associated cardiomyopathy, whereas, knockdown of its ortholog in Zebrafish led to severe contractile dysfunction and cardiomyopathy. The objective of the current study was to elucidate the cardiac consequences of targeted deletion of Myozap in mice.

Methods and Results:
We generated a Myozap null mutant (MZP-ko) by global deletion of Myozap in mice. Unchallenged MZP-ko mice did not exhibit a baseline cardiac phenotype. However, upon biomechanical stress due to TAC (transverse aortic constriction), deficiency for Myozap led to accelerated cardiac hypertrophy (significant increases in the heart to body weight, left ventricular to body weight ratios) and fibrosis, accompanied by “super”-induction of the hypertrophic gene program (ANF/BNP). Moreover, MZP-ko mice revealed a severe reduction of fractional shortening and signs heart failure (increased lung/body weights) as well as a markedly increased mortality in response to TAC). Additional molecular data exhibited a significant decrease in the levels of native and phosphorylated Connexin 43 after transverse aortic constriction (TAC) in MZP-ko mice compared to wildtype animals. Finally, we observed a downregulation of dysbindin, a novel interaction partner of Myozap and known inducer of ERK1/2 signaling in TAC operated MZP-ko mice. Consistently, activation of ERK1/2 in response to TAC was blunted compared to wild-type littermates.

Conclusions:
We here show that myozap deficiency in vivo leads to a maladaptative response to increased biomechanical stress associated with cardiomyopathy, heart failure and cardiac death. Mechanistically, this phenotype can at least in part be explained by the interruption of the interaction between myozap and dysbindin with subsequent loss of ERK1/2 activation. Taken into a larger perspective, our data imply an essential role of the ID and its associated proteins in cardiac remodeling.
Abstract 183: Branched-Chain Amino Acid Catabolic Reprogramming in Heart Failure
Authors: Sun Haipeng , Olson Kristine , Gao Chen , Prosdocimo Domenick A , Zhou Meiyi , Jeyaraj Darwin , Youn Ji-Youn , Ren Shuxun , Cai Hua , Ping Peipei , Schulze Paul C , Lynch Christopher , Jain Mukesh K , Wang Yibin
Published: July 2021

Abnormal branched-chain amino acids (BCAA) catabolism has been strongly linked with cardiovascular and metabolic diseases in recent studies. In heart, metabolic reprogramming associated with the onset of heart failure has been established based on fetal-like changes in fatty acid and glucose metabolism, while little is known about the changes in amino acid homeostasis. In this report, we demonstrated a signature of suppressed BCAA catabolism in failing rodent and human hearts. The branched-chain α-keto acids (BCKA), intermediate of BCAA catabolism, accumulates, closely coordinated with reduced expression of key enzymes of BCAA catabolism in pathologically stressed myocardium. The down-regulation of these genes mimics a similar expression pattern observed in neonatal heart, indicating a fetal-like genetic reprogramming. Using both
in vitro
and
in vivo
models, we identified KLF15 as a key transcriptional regulator of the BCAA catabolic circuitry in heart. Genetic inactivation of BCAA catabolic pathway resulted in elevated cardiac BCKA levels and promoted cardiac dysfunction in response to mechanical overload, associated with increased oxidative stress and impaired mitochondrial respiration. Taken together, our data established for the first time that BCAA catabolic reprogramming is an integral component of metabolic remodeling that contributes to heart failure progression.
Abstract 184: Exposure To Air Pollution In Utero Causes Persistent Cardiovascular Dysfunction
Authors: Wold Loren , Gorr Matthew , Eichenseer Clayton , Youtz Dane , Velten Markus
Published: July 2021

Exposure to air pollutants during pregnancy can have devastating effects on offspring, inducing intrauterine growth retardation and maturation deficits. These developmental insufficiencies can affect the formation and function of the adult heart. We hypothesized that exposure to air pollution
in utero
would induce abnormal cardiac function at adulthood. FVB mice were exposed (6h/day, 7d/wk) to environmentally relevant concentrations of ambient particulate matter (PM2.5) or filtered air (FA) beginning when animals were paired for breeding. After birth, both groups remained in FA. Cardiac echocardiography was performed at 10 weeks of age. Birth weight was reduced in pups exposed to PM2.5 during intrauterine development compared to FA exposed pups, and litter size did not differ significantly between groups. Echocardiography revealed reduced left ventricular fractional shortening with greater left ventricular end systolic diameter in PM2.5 exposed mice at 10 weeks of age. These results were similar to data from mice exposed perinatally (until weaning). This study supports the hypothesis that exposure to air pollution
in utero
can lead to heart dysfunction at adulthood.
Abstract 210: Progressive Mitochondrial Protein Acetylation and Microsteatosis is Associated with Diastolic Dysfunction in a Hypertrophic Cardiomyopathy Model
Authors: Stram Amanda R , Wagner Gregory R , Pride P M , Payne R M
Published: July 2021

We tested the hypothesis that increased mitochondrial protein acetylation is associated with impaired fatty acid metabolism and diastolic dysfunction in Friedreich’s Ataxia (FRDA). FRDA results from deficiency of the mitochondrial protein, frataxin (FXN), and causes hypertrophic cardiomyopathy. FRDA hearts show decreased ATP production. We previously showed that FXN loss results in loss of activity of the NAD+-dependent mitochondrial deacetylase, sirtuin 3 (SIRT3), and cardiac mitochondrial protein hyperacetylation in a mouse model of FRDA. Long-chain and medium chain acyl CoA dehydrogenases (LCAD, MCAD) are targets of SIRT3, suggesting that abnormal acetylation may alter fatty acid metabolism. A cardiac specific mouse model with conditional deletion of FXN in heart and skeletal muscle (FXN MCK-Cre-/-) was compared to healthy controls (FXNfl/fl). Mice underwent echocardiogram and left heart catheterization in vivo at age 30 and 65 days. Heart lysate was examined for overall lysine acetylation at ages 30, 45 and 70 days, and acetylated LCAD and MCAD. Myocardial cells were stained with oil red to detect lipids. Hearts of FXN MCK-Cre-/- animals showed mitochondrial hyperacetylation, increased LCAD and MCAD acetylation, and microsteatosis compared to controls. Diastolic dysfunction was evident in FXN MCK-Cre-/- at day 65 by increased mitral valve E/A ratios and IVRT (p<0.04 and p<0.03, respectively, n=7), prolonged tau, and decreased -dP/dt (p<0.02 and p <0.03, respectively, n=6). Cardiac mechanics at day 65 showed systolic failure with reduced EF and FS (p<0.002 and p<0.003, respectively, n=7). Comparison of FXN deficient mice and controls at day 65 trended toward flattened ESPVR and left shifted EDPVR curves, but differences were not significant. Early diastolic dysfunction was evident at day 30 in FXN MCK-Cre-/- by high E/A ratio and EDP compared to day 65 controls (p <0.03, n=7, and p<0.03, n=6, respectively). We conclude that progressive mitochondrial protein acetylation and abnormal fatty acid metabolism is associated with early diastolic dysfunction and later systolic failure. This metabolic dysregulation and pathophysiology may share characteristics with other metabolic heart disorders, such as diabetes and metabolic syndrome.
Abstract 216: Traditional Chinese Medicine Tongxinluo Modulates The Secretion Of Cytokines In Vitro By Cardiac Microvascular Endothelial Cells In Ischemia/Reperfusion Injury.
Authors: Cui Hehe , Li Xiangdong , Wang Zhigang , Li Na , Qi Kang , Li Qing , Jin Chen , Yang Yuejin
Published: July 2021

Cardiac microvascular endothelial cells (CMECs) regulate the function of cardiomyocytes and blood cells in myocardial ischemia/ reperfusion injury (MIRI) via paracrine methods. Tongxinluo (TXL) is a Traditional Chinese Medicine compound to treat angina pectoris in China, which is constituted of ginseng and other 11 natural products. It is proved to improve the endothelial function and be protective against MIRI. Thus, we aimed to find alterations in paracrine function of CMECs under the hypoxia/reoxygenation (H/R) situation and its modulation by TXL.

CMECs were exposed to different concentrations of TXL for 30 min and then subjected to H/R for 12 h/2 h. Apoptotic rates were measured to determine the optimal concentration. Protein antibody arrays were used to find the alterations of cytokines in conditioned medium (CM) secreted by CMECs. Gene Ontology project was adopted to describe the functions of changed cytokines.

TXL inhibited apoptosis of CMECs dose-dependently under H/R and reached its peak effect at 800 μg/mL. Thirty-three types of cytokines were significantly changed by H/R (19 factors decreased and 14 increased), and TXL at 800μg/ml changed 121 types of cytokines compared to the H/R group (93 factors decreased and 28 increased). The cytokines with significant alterations were involved in cell differentiation and proliferation, positive chemotaxis, and endothelial cell migration. Among these cytokines, bFGF, PDGF and IL-7 were attenuated by H/R and further decreased by TXL, while IL-21, MCP-2 and GROa were increased by H/R but decreased by TXL.

The study elucidated the paracrine function of CMECs in MIRI and partly illuminated the protective mechanisms of TXL.
Abstract 222: Intralipid-induced Cardioprotection In Late Pregnancy Is Fully Abolished By Inhibition Of Stat3, But Not Pi3k Signaling Pathway
Authors: li Jingyuan , Eghbali Mansoureh
Published: July 2021

We have recently shown that the heart of late pregnant (LP) rodent is more prone to ischemia/reperfusion (I/R) injury compared to non-pregnant. Here we explored the therapeutic potential of Intralipid (ITLD) in the protection of LP hearts against I/R injury, and investigate the involvement of the signal transducer and activator of transcription-3 (STAT3) and phosphoinositide 3-kinase (PI3K) signaling pathways in ITLD-induced cardioprotetion. Isolated LP mouse hearts were subjected to 20 min ischemia followed by 40 min reperfusion with 1) Krebs Henseleit buffer (CTRL group), 2) 1% intralipid (ITLD group) or 3) ITLD+STAT3 inhibitor Stattic (20 μM, Stattic group), and 4) ITLD+PI3K inhibitor LY294002 (45 μM). The heart function and the infarct size were measured. The Intralipid-induced cardioprotection was fully abolished by Stattic, as RPP was significantly lower in the presence of Stattic (RPP=8881±1331 mmHg*beats/min vs. 1186±563 mmHg*beats/min in ITLD+Stattic, p<0.01) at the end of reperfusion. In fact, all of the hemodynamic indexes in ITLD+Stattic were not significantly different from CTRL. The infarct size was also significantly larger in ITLD+Stattic group when compared to Intralipid alone (47.9±2.5% in ITLD+Stattic vs. 21.7±2.6 % in ITLD, p<0.01). The Intralipid-induced cardioprotection was only partially abolished by LY294002, as at the end of 40 min reperfusion the RPP was significantly lower compared to the group treated with Intralipid alone, but still significantly higher than ITLD+Stattic ((RPP=8881±1331 mmHg*beats/min in ITLD vs. 5212±1955 mmHg*beats/min in ITLD+LY, p<0.05; RPP=5212±1955 mmHg*beats/min in ITLD+LY vs.1186±563 mmHg*beats/min in ITLD+Stattic, p<0.05). The infarct size was also larger when compared to Intralipid alone (32.8±3.1% in ITLD+LY vs. 21.7±2.6% in ITLD, p<0.05), but lower than ITLD+Stattic group (32.8±3.1% in ITLD+LY vs 47.9±2.5%, p<0.05). In conclusion, Intralipid protects the late pregnant heart against I/R injury via the STAT3 rather than the PI3K signaling pathway.
Abstract 223: The Role Of Angiogenesis In The Myocardial Ischemia/reperfusion Injury In Pregnancy
Authors: li Jingyuan , Arany Zoltan Pierre , Eghbali Mansoureh
Published: July 2021

Angiogenesis plays an important role in the pathogenesis of cardiovascular disease. Pro-angiogenic and anti-angiogenic treatments have provided new insights into the impact of angiogenesis-based approaches on coronary artery disease. We have recently reported that the hearts of late pregnant (LP) mice are more prone to ischemia/reperfusion (I/R) injury compared to non pregnant(NP) mice. Provided the significant change of angiogenesis status in pregnancy, here we explored whether stimulating the angiogenesis with VEGF is able to protect the heart against I/R injury in late pregnancy, and whether anti-antigenic treatment with soluble endoglin(sENG), an anti-angiogenic factor, aggravates cardiac I/R injury in NP. Pregnant mice at day 12 either received daily injection of VEGF (100 ug/kg daily subcutaneous injection) or PBS(LP CTRL) for 7 days, and at day 19 the LP mice hearts were subjected to 20 min ischemia followed by 40 min reperfusion in Langendorff. NP mice either received a single adenovirus sENG(2х10 8particles via tail vein injection) or vehicle(NP CTRL), and 10 days later NP mice were subjected to 20 min ischemia followed by 40 min reperfusion in Langendorff. The heart function was recorded throughout the experiments, and the infarct size was measured by TTC staining at the end of experiments. Exogenous VEGF treatment significantly improved the cardiac function of LP mice after ischemia. The rate pressure product (RPP) at the end of reperfusion was improved from 1617±287 mmHg*beats/min (n=6) in LP CTRL to 11287±1783 mmHg*beats/min (n=3) in the VEGF group(p<0.01). The infarct size was also significantly reduced by VEGF treatment to 25.0±4.3% (n=3) from 57.4±5.2%(n=6) in CTRL (p<0.01). While sENG aggravated the cardiac I/R injury in NP, as the RPP at the end of reperfusion in the sENG group (4523±1281 mmHg*beats/min, n=4) was significantly lower compared with NP CTRL group(12818±1213 mmHg*beats/min, n=6)(p<0.01). Furthermore, the infarct size in the sENG group was markedly higher compared with NP CTRL group (34.0±3.3% (n=4) vs. 16.3±1.4%(n=6) in NP CTRL, p<0.05). In conclusion, anti-angiogenic treatment aggravates the cardiac I/R injury in NP, while angiogenic therapy protects the heart against I/R injury in LP.
Abstract 241: Cardiac TIMP-4 Dictates cardiomyocyte contractility and differentiation of embryonic stem cells into cardiomyocytes: Road to Therapy
Authors: Chaturvedi Pankaj , Kalani Anuradha , Familtseva Anastasia , Kamat Pradip K , Metriveli Naira , Tyagi Suresh C
Published: July 2021

The remarkable nature of cardiomyocytes for contractility is attributed to the extracellular matrix which is maintained by the balance between MMPs (Matrix Metalloproteases) and TIMPs (Tissue Inhibitors of Matrix Metalloproteases). Any deviation from this delicate balance of MMP/TIMP is a hallmark of cardiovascular pathologies including myocardial infarction (MI). TIMP4, which is the least studied molecule, is deficient in failing hearts and mice lacking TIMP4 show poor regeneration capacity after MI. Therefore, we hypothesize that TIMP4 helps in cardiac regeneration by alleviating contractility and inducing the differentiation of cardiac progenitor cells into cardiomyocytes. To validate this hypothesis, we transfected cardiomyocytes with TIMP4 and TIMP4-siRNA and observed that there was increase in contractility in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. To explain this we looked into the calcium channel genes and found increase in the expression of serca2a (sarcoplasmic reticulum calcium ATPase2a) in the TIMP4 transformed myocytes. Serca2a is tightly regulated by mir122a and we found decrease in the expression of mir122a in the TIMP4 transfected cells as compared to the TIMP4-siRNA cells. To observe the effect of TIMP4 in differentiation of cardiac progenitor cells we treated mouse embryonic stem cells with cardiac extract and cardiac extract minus TIMP4 (using TIMP4 monoclonal antibody). The cells treated with cardiac extract showed cardiac phenotype in terms of Ckit+, GATA4+ and Nkx2.5 expression. This is a novel report on the influence of TIMP4 on contractility and inducing the differentiation of stem cells to cardiomyocytes. In view of the failure of MMP9 inhibitors for cardiac therapy in clinical trials, TIMP4 provides and alternative approach, being an indigenous molecule, a natural inhibitor of MMP9 and efficient ROS scavenger.
Abstract 243: Profilin-1 Phosphorylation Regulates Post-ischemic Cardiac Repair By Induction Of Vascular Stem Cell Homing
Authors: Fan Yi
Published: July 2021

Neovascularization, the formation of new blood vessels, is fundamental to cardiac repair and regeneration in ischemic heart disease. After myocardial infarction (MI), vascular stem cells (VSC) are mobilized from bone marrow and recruited to the ischemic site, de novo generating new blood vessels to promote cardiac recovery. Our previous studies have revealed that phosphorylation of profilin-1 (Pfn-1) induces endothelial cell migration and sprouting angiogenesis. Here, we show that Pfn-1 phosphorylation regulates VSC homing to ischemic site and cardiac repair after MI through protease expression. Vascular lineage-specific knock-in of phosphorylation-dead Pfn-1(Y129F) mutant in mice show that Pfn-1 phosphorylation is critical for ischemia-induced neovascularization and cardiac function recovery after MI. Deficiency in Pfn-1 phosphorylation inhibits VSC homing to the ischemic hindlimb, suggesting a critical role of Pfn-1 phosphorylation in VSC recruitment. Mechanistic studies show that Pfn-1 phosphorylation is required for vascular endothelial growth factor (VEGF)-A-induced expression of metalloproteinase (MMP) −2 and −9 and cell migration in VSC. Therefore, these studies identify a critical role of Pfn-1 phosphorylation in VSC homing and neovascularization after MI, and suggest that Pfn-1 phosphorylation may represent as a therapeutic target for treating ischemic heart disease.
Abstract 245: Plasminogen Augments Cxcl12/cxcr4-regulated Stem Cell Homing And Contributes To Cardiac Repair After Myocardial Infarction.
Authors: Gong Yanqing , Zhao Yujing , Fan Yi , Hoover-Plow Jane
Published: July 2021

Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Bone marrow (BM)-derived stem cells promote tissue repair and regeneration after MI. Thrombolytic treatment with plasminogen (Plg) activators significantly improves the clinical outcome in MI by restoration of cardiac perfusion. In addition to its canonical function, Plg is critical for cardiac repair, wound healing and liver injury, however, the mechanism for Plg-regulated tissue repair remains unclear. Here, we show a novel role of Plg in stem cell-mediated neovascularization and cardiac repair after MI. Our data show that Granulocyte colony-stimulating factor (G-CSF), a stem cell mobilizer, significantly increased neovascularization and decreased infarct size in the infarct area, and improved ejection fraction and LV internal diameter by echocardiogram in wild-type mice. No improvement in tissue repair and heart function was observed in Plg deficient (Plg-/-) mice indicating that Plg is required for stem cell-regulated cardiac repair after MI. In vivo tracking of GFP-expressing BM cells after BM transplantation revealed that in Plg-/- mice, recruitment of BM-derived stem cells (GFP+c-kit+ cells) to the infarcted heart and stem cell-derived vessels and arteries are dramatically decreased (by 11 fold) suggesting that Plg may regulate stem cell homing to the lesion sites and subsequently contribute stem cell-mediated tissue regeneration. Mechanistic studies show that Plg up-regulated CXCR4 expression on stem cell in vivo and in vitro, suggesting Plg may promotes stem cell homing by induction of CXCR4 expression in stem cells. Stem cell migration was enhanced by endogenous Plg in vitro, however, AMD3100, a CXCR4 antagonist, significantly inhibited Plg-regulated stem cell migration. Furthermore, lentiviral reconstitution of CXCR4 expression in BM cells rescued stem cell homing to the infarcted heart in Plg-/- mice, indicating that Plg mediates stem cell homing through regulating CXCR4 expression. These findings identified a novel role of Plg in cardiac repair by promoting stem cell homing to the injured heart after MI. Thus, targeting Plg may offer a new therapeutic strategy for strengthening stem cell-mediated cardiac repair and regeneration after MI.
Abstract 248: Comparison Of Transplantation Of Bone Marrow-derived Stem Cells, Adipose-derived Stem Cells And Endometrium- Derived Stem Cells In The Infarcted Heart
Authors: Hu Xin Yang , Wang Kan , Wang Jian-an
Published: July 2021

Background:
A variety of adult stem cells have been transplanted into the infarcted heart to cure myocardial infarction(MI), however, comparative studies are lacking to show more suitable source of cells for transplantation. Mesenchymal stem cells hold promise for myocardial regeneration therapy. Derivation of these cells from the endometrium tissue might be easier compared to bone marrow and adipose tissue. However,the in vivo fate of endometrium stem cells (EnSCs) in the infarcted heart has never been compared directly to mesenchymal cells derived from bone marrow(BMMSCs) and adipose tissue(AdMSCs).

Methods:
EnSCs, AdMSCs and BMMSCs were isolated from healthy donors were characterized using flow cytometry for surface markers identification and microscopy for cell morphology. They were characterized with β-actin promoter driving firefly luciferase and green fluorescent protein (Fluc-GFP) double fusion reporter gene, and were characterized using flow cytometry, bioluminescence imaging (BLI) and luminometry. Cell proliferation was tested by CCK-8 kit, colony forming unit(CFU) was stained by crystal violet staining and apoptosis ratio were detected by TUNEL assay. Rat (n=8/group) underwent myocardial infarction followed by intramyocardial injection of 5х105 EnSCs, AdMSCs and BMMSCs, or saline (negative control). Cell survival was measured using BLI for 6 weeks and cardiac function was monitored by echocardiography and hemodynamics analysis. Ventricular morphology was assessed using histology.

Results:
EnSCs, AdMSCs and BMMSCs were CD29+, CD90+, CD105+, shared similar morphology, but EnSCs had best proliferation, colony-forming and anti-apoptosis activity of 3 types of MSCs. Cells expressed Fluc reporter genes in a number-dependent fashion, as confirmed by luminometry. After cardiac transplantation, transplantation of EnSCs was better capable of preserving ventricular function and dimensions than others, as confirmed by echo test, PV-loops and histology.

Conclusions:
This is the first study comparing the in in vitro and in vivo behavior of 3 types of MSCs in the infarcted heart. AdMSCs and BMMSCs do not tolerate well in the cardiac environment, resulting in more cell death andworse cardiac function than EnSCs groups.