bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 340: Short Telomeres Induce Autophagy and Modulate Cardiac Progenitor Cell Fate
Authors: Hariharan Nirmala , Matsumoto Collin , Emathinger Jacqueline , Daneshpooy Saba , Shin Minyoung , Levinson Aimee B , Quijada Pearl , Nguyen Nathalie , Sussman Mark
Published: July 2021

Aging severely limits myocardial regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesize that short telomeres induce autophagy and elicit the age-associated change in cardiac progenitor cell fate. We compared mouse strains with different telomere lengths (TL) for phenotypic characteristics of aging and also isolated CPCs from them. Naturally occurring wild mouse strain
Mus musculus castaneus
(CAST) possessing short telomeres (TL:18Kb) exhibits early cardiac aging with diastolic dysfunction, hypertrophy, fibrosis and increase in senescence markers p53 and p16, as compared to common lab strains FVB (TL:75Kb) and C57 (TL:50Kb). CAST CPCs with short TLs have altered cell fate as characterized by slower proliferation (p<0.01); increased senescence identified by beta-galactosidase activity (p<0.05); increased basal commitment as determined by expression of lineage markers smooth muscle actin, Tie2, and sarcomeric actinin (16.6, 1.7 and 1.75, p<0.05); as well as loss of quiescence marker expression. Consistent findings of altered cell fate are also evident in old CPCs isolated from aged mice with significantly shorter TLs. Cell fate changes occurring downstream from short TL are at least partially p53 dependent, as p53 inhibition rescues the irreversible cell cycle arrest observed in CAST CPCs. Mechanistically, short TLs induce autophagy, a catabolic protein degradation process. Autophagy flux is increased in CAST CPCs as evidenced by increased LC3 (p<0.05), reduced p62 expression (-52%, p<0.05) and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation, but not autolysosome formation reverses the cell fate to a more youthful phenotype. Overall the data suggests that short TLs activate autophagy to accommodate cell fate changes that tip the equilibrium away from quiescence and proliferation into differentiation and senescence, leading to age-associated exhaustion of CPCs. The study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to enhance the therapeutic effects of aged CPCs.
Abstract 345: Maturation of Cardiomyocytes via Rbfox1
Authors: Lee Josh Z , Gao Chen , Pushkarsky Ivan , Hu Chaowei , Xu Haodong , DiCarlo Dino , Wang Yibin
Published: July 2021

Background:
Cardiovascular disease is the leading cause of death in the world with a dearth of effective therapies. Heart undergoes a complex differentiation and maturation process throughout embryonic and post-natal stages. Intense efforts have been made in the study of cardiomyocyte differentiation, maturation and pathological remodeling. With the aid of stem cells, investigators are able to recapitulate events in early cardiac development and gain valuable insight in transcriptional regulatory networks directing early cardiomyocyte differentiation. However, the molecular network that determine myocyte maturation in postnatal development, which are especially important for understanding diseases and developing cell based clinical applications, are far less well characterized. Human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs) are lineage committed but remain immature and fetal-like in molecular, morphological and functional characteristics. Their application in cell based therapy or serving as a disease model for heart failure is limited, in part due to the lack of sufficient insight to promote their maturation into adult myocytes.

Results:
We find from previous studies that postnatal cardiomyocyte development is marked by global alternative splicing (AS) programming. In mouse heart, we find RNA splicing regulator RBFox1 is markedly induced in heart only in post-natal period and functions as a key regulator to post-natal global RNA alternative splicing reprogramming during cardiomyocyte maturation. Transcriptome analysis revealed that exogenous expression of RBFox1 promotes gene expression in neonatal ventricular myocytes reminiscent of mature adult heart, and results in cellular maturation based on sarcomere organization and calcium cycling characteristics. Furthermore, ectopic expression of rodent RBFox1 in human iPSC derived CMs resulted in similar maturation effects, implicating a conserved mechanism in human and rodent myocytes.

Conclusion:
RBFox1 mediated RNA splicing is an important contributor to post-natal myocyte maturation and it can be manipulated to promote cardiomyocyte maturation for cell-based therapy or disease modeling.
Abstract 352: Using Extracellular Matrix Biomolecules to Modify Poly(vinyl alcohol) Vascular Graft Surface Properties
Authors: Truong Katie P , Anderson Deirdre E , Yim Evelyn K , Hinds Monica T
Published: July 2021

The demand for biosynthetic vascular grafts is increasing due to the growing prevalence of occlusive arterial disease in the population. Autologous vessels are often used for treatment but are limited due to a patient’s pre-existing condition or previous surgeries. There is a clinical need for engineered small diameter blood vessel grafts with mechanical and physiologic properties that mimic native arteries. Poly(vinyl alcohol) hydrogel (PVA) has been previously shown to be a suitable biomaterial for grafting. However, its hydrophilic and bioinert properties prevent
in vivo
endothelialization. Previously, our work has shown that modification of the PVA surface with different biomolecules can alter endothelial growth and function. Coating with gelatin exhibited a pro-endothelial environment for tissue growth but displayed pro-thrombotic properties. When cyclicRGD (cRGD) peptide was incorporated in the PVA graft, we observed increased endothelial cell viability without an increase in platelet adhesion. We hypothesize that short peptide sequences of the basement membrane extracellular matrix will produce peak endothelial cell growth without compromising the hemocompatibility of the graft. Planar PVA underwent surface modification with different basement membrane proteins and peptides including collagen, laminin, cRGD, and YIGSR. To characterize the hydrophobicity of the material, we measured the static water contact angle on the surface of the PVA. Cell attachment and proliferation of primary endothelial cells were quantified with immunohistochemistry by staining for nuclei, actin, and VE-cadherin. Initial results showed increased hydrophobicity and cell attachment with the surface modifications. Currently, we are assessing thrombotic potential by obtaining plasma clotting time, and platelet attachment and activation during incubation with platelet rich plasma. In the future, we will measure cell migration and retention under flow and characterize the cell phenotype under pro-thrombotic and anti-thrombotic flow conditions using real time PCR. This work is a significant step toward advancing the fabrication of biosynthetic vascular grafts for the treatment of cardiovascular disease
Abstract 353: Bone Marrow Fibroblast Progenitor Cell-derived Exosomes Activate Resident Fibroblast and Augment Pressure Overload Induced Cardiac Fibrosis in IL10KO Mice
Authors: Verma Suresh K , Girikipathi Venkata N , Cimini Maria , Cheng Zhongjian , Khan Moshin , Truongcao May , Benedict Cindy , Goukassian David , Kishore Raj
Published: July 2021

Background:
Activated fibroblasts (myoFBs) play critical role in cardiac fibrosis, however, their origin in diseased heart remains uncertain. Previous studies suggest the contribution of bone marrow fibroblasts progenitor cells (FPC) in pressure overload (PO)-induced cardiac fibrosis and inflammation acts as catalyst in this process. Recently others and we have shown that paracrine mediators packaged in exosomes play important role in cardiac pathophysiology. Thus, we hypothesized that exosome-derived from IL10KO-FPC augments PO-induced resident cardiac fibroblast activation and therefore, aggravate cardiac fibrosis.

Methods and Results:
Cardiac fibrosis was induced in Wild-type (WT) and IL10-knockout (IL10KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in IL10KO mice. PO-enhanced FPC (Prominin1
+
cells) mobilization and homing in IL10KO mice compared to WT mice. To establish the IL10KO-FPC paracrine signaling, exosomes were isolated from WT and IL10KO BM-FPC culture media and characterized for proteins/miRNA. IL10 KO FPC-exosomes showed altered packaging of signature fibrotic miR and proteins. To explore whether FPC-exosomes modulate resident fibroblast activation, adult cardiac fibroblasts were treated with WT and IL10KO FPC-derived exosomes. IL10KO-FPC-derived exosomes exaggerate TGFβ
2
-induced activation of adult fibroblasts. These data suggest that fibrotic remodeling factors (miRs and/or proteins) packaged in IL10KO-FPC exosomes are sufficient to enhance the resident cardiac fibroblast activation and mediate cardiac fibrotic remodeling IL10 treatment significantly inhibits TGFβ
2
-induced FPC to myoFBs transition.

Conclusion:
Taken together, our findings suggest that paracrine factors secreted by BM-FPC augment resident cardiac fibroblast activation and fibrosis in pressure overloaded myocardium and IL10 negatively regulates this process. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 on PO-induced cardiac fibrosis and heart failure.
Abstract 36: Cardiac Progenitor Cell Lineage Tracing During Embryonic Cardiomyogenesis
Authors: Wang Bingyan J , Muliono Alvin , Alvarez Roberto , Sussman Mark A
Published: July 2021

Introduction:
Stem cell therapy represents great promises to myocardium regeneration. Multipotent c-Kit
pos
cardiac progenitor cells (CPCs) are able to differentiate into endothelial cells, smooth muscle cells, and cardiomyocytes. However, fundamental knowledge of CPC biology remains incomplete. Studies in rodent myocardial infarction model revealed that CPCs have poor long-term survival and engraftment after adoptive transfer, perhaps due to the severely damaged host environment. Therefore, it is critical to understand how CPCs interface with the recipient environment following transfer in order to enhance their true regenerative potentials.

Hypothesis:
Adoptively transferred stem cells are thought to survive and engraft best in an environment closely resembling their original habitat. Thus, we hypothesized that the embryonic environment provides the optimal spatiotemporal conditions to promote CPCs engraftment and commitment to cardiac fate.

Methods:
CPCs isolated from adult mouse hearts were expanded, fluorescence-tagged, and injected into blastocysts at E3.75 and
in utero
at E15.5. Embryos were analyzed following cardiogenesis by immunofluorescence for presence of CPC-derived tissues. Additionally, CPCs were injected intramyocardially at various stages from P0 to P7, to follow long-term adoptive transfer and assess CPCs lineage commitment.

Results and Conclusions:
At 48 hours post injection, donor CPCs were found anchoring in blastocoel and trophoblasts at E5.5, and were detected within the host myocardium at E17.5 predominantly at perivascular regions (n=4). Interestingly, CPCs also integrated into aminochorionic sac, indicating a novel non-cardiogenic fate of CPCs (n=5). CPCs injected at P3 stably engrafted into left ventricular myocardium by 14 days post injection (n=4), sharing gap junction proteins (ZO-1, Connexin-43) with neighboring cells. In conclusion, this study provides vivid evidence for the first time of CPC engraftment and survival
in vivo
under homeostasis during cardiogenesis. Future studies will assess the permissive environmental conditions, which may optimize their use in therapeutic applications, and the cardiogenic potential of CPCs in order to provide fundamental insights on CPCs biology.
Abstract 372: CD16 Monocytes Stimulate Arteriogenesis to Salvage the Ischemic Limb
Authors: Patel Ashish S , Ludwinski Francesca E , Kapoor Puja , Jones Paul R , Kerr Alexander , El-Sayed Tamer , Saha Prakash , Lyons Oliver T , Smith Alberto , Modarai Bijan
Published: July 2021

Introduction:
Cell therapy using unselected mononuclear cell populations has had modest benefits in patients with critical limb ischemia (CLI). We hypothesized that tissue-remodeling monocytes, identified by their expression of CD16 (CD16+ Mo), may be a novel cellular therapy for CLI.

Methods and Results:
Flow cytometry showed that the proportion of circulating CD16+ Mo was greater in CLI patients (n=25) compared with matched controls (n=15, P<0.0001). Removal of ischemia following revascularization or amputation resulted in a fall in CD16+ Mo to control levels (P<0.05). CLI CD16+ Mo expressed higher levels of the adhesive proteins VLA, ICAM-1 and CD11c compared with controls (P<0.05). Conditioned media from these cells contained higher levels of HB-EGF, PlGF, endoglin, VEGF-C and VEGF-D (P<0.05) and induced greater endothelial cell tubule formation (P<0.05) compared with CD16- Mo from the same patients (n=9). CD16+ Mo preferentially migrated towards ischemic muscle supernatants isolated from CLI patients (n=7, P<0.02). CD16+ and CD16- Mo were isolated from 12 CLI patients and 1x10
6
cells injected into the adductor muscles of nude athymic mice following femoral artery excision. More ischemic hindlimbs were salvaged when treated with CD16+ compared with CD16- Mo (83% [10/12] vs 17% [2/12] limbs, P<0.05) and this was associated with enhanced arteriogenesis (αSMA-stained vessels, P<0.05).

Conclusion:
Circulating CD16+ Mo from CLI patients have increased expression of adhesion markers, are preferentially retained within ischemic muscle and promote robust arteriogenesis and limb salvage in experimental HLI. This monocyte subset may be an effective cellular therapy for CLI.
Abstract 385: Cardiac Myosin Binding Protein-C Mutants Disrupt Ubiquitin Proteasome System and Hsc70 Functions
Authors: Glazier Amelia A , Helms Adam , Hafeez Neha , Kotlo Srisha , Yob Jaime , Tang Vi , Day Sharlene
Published: July 2021

The most commonly mutated gene in hypertrophic cardiomyopathy (HCM) is cardiac myosin binding protein C (MYBPC3). Over 90% of MYBPC3 mutations are nonsense, but whether these mutations manifest in loss- or gain-of-function is unresolved. Evidence suggests MYBPC3 mutants impact protein quality control mechanisms. The objective of this study was to evaluate interactions of MYBPC3 with proteostatic systems and test the hypothesis that these interactions affect protein homeostasis in cardiomyocytes.

WT and mutant MYBPC3 constructs were expressed in neonatal rat ventricular myocytes (NRVMs) via adenovirus. Mutant MYBPC3 induced ubiquitin proteasome system reporter GFPu accumulation (fold increase in GFPu-positive cells vs control: WT 138±14.0%, mutant 198±27.2%, mean±SEM, p<0.05 vs control and WT), indicating proteasome dysfunction. Affinity purification/mass spectrometry identified molecular chaperones Hsp70 and Hsc70 as prominent interactors with MYBPC3. We observed MYBPC3 degradation by cycloheximide chase in response to Hsc70 siRNA knockdown or pharmacological treatment with Hsp70 activator YM-1. Hsc70 knockdown slowed degradation of WT and mutant MYBPC3 (WT control t
½
=5.47±0.70 hr, WT Hsc70 knockdown t
½
=13.5±1.62 hr; mutant control t
½
=3.42±0.61 hr, mutant Hsc70 knockdown t
½
=9.87±0.95 hr), while YM-1 treatment accelerated degradation (WT DMSO t
½
=10.2±3.28 hr, WT YM-1 t
½
=3.16±0.61 hr; mutant DMSO t
½
=11.7±2.67 hr, mutant YM-1 t
½
=1.37±0.16 hr). We then evaluated whether transferrin uptake via clathrin mediated endocytosis, a critical Hsc70-dependent activity, was affected by mutant MYBPC3. Transferrin uptake was significantly decreased in NRVMs expressing mutant MYBPC3 compared to WT and untreated controls (transferrin-positive cells: control 22.93±3.34%, WT 17.47±0.70%, mutant 9.30±1.63%, mean±SEM, p<0.05 vs control and WT).

In conclusion, we have demonstrated that Hsp70 chaperones interact with MYBPC3 in cardiomyocytes and affect MYBPC3 degradation, suggesting MYBPC3 is a client of Hsp70 and Hsc70. Additionally, expression of mutant MYBPC3 causes ubiquitin proteasome impairment and interferes with normal Hsc70 function. These results support our hypothesis that mutant MYBPC3 affects protein homeostasis in HCM.
Abstract 386: Identification of Genetic Regulatory Networks for Insulin Resistance in Multiple Populations of Diverse Ethnicities
Authors: Shu Le , Zhao Yuqi ,   The GENESIS Consortium, Lusis Aldons J , Hao Ke , Quertermous Thomas , Knowles Joshua W , Yang Xia
Published: July 2021

Insulin resistance (IR) is a critical pathogenic factor for highly prevalent modern cardiometabolic diseases, including coronary artery disease (CAD) and type 2 diabetes (T2D). However, the molecular circuitries underlying IR remain to be elucidated. The GENEticS of Insulin Sensitivity Consortium (GENESIS) conducted genome-wide association studies (GWAS) for direct measures of IR using euglycemic clamp or insulin suppression test. We sought to identify gene networks and their key intervening drivers for IR by performing a comprehensive integrative analysis leveraging GWAS data from seven GENESIS cohorts representing three ethnic groups - Europeans, Asians and Hispanics, along with expression quantitative trait loci, ENCODE, and tissue-specific gene network models (both co-expression and graphical models) from IR relevant tissues. Integration of the multi-ethnic GWAS with diverse functional genomics information captured shared IR pathways and networks across ethnicities that are independent of body mass index, including GLUT4 translocation regulation, insulin signaling, MAPK signaling, interleukin signaling, extracellular matrix, branched-chain amino acids metabolisms, cell cycle, and oxidative phosphorylation. Further integration of these GWAS-informed IR processes with graphical gene networks uncovered potential key regulators including
HADH, COX5A, VCAN
and
TOP2A
, whose network neighbors are consistently enriched for the genetic association signals of IR across ethnicities, and show significant correlation with IR, fasting glucose and insulin levels in the transcriptomic-wide association data from a Hybrid Mouse Diversity Panel comprised of >100 strains fed with high-fat diet. Findings from this in-depth assessment of genetic and functional data from multiple human cohorts provide new understanding of the pathways, gene networks and potential regulators contributing to IR. These results will also facilitate future functional investigations to unveil how DNA variations translate into IR.
Abstract 398: Growth Hormone Releasing Hormone Agonist (GHRH-A) Restores Cardiac Function in a Rodent Model of Heart FailureWith Preserved Ejection Fraction (HFpEF)
Authors: Kanashiro-Takeuchi Rosemeire M , Takeuchi Lauro M , Yuan Chen C , Katz Diana , Hardin Darrell , Zuttion Marilia , Balkan Wayne , Cai Renzhi , Schally Andrew V , Hare Joshua M
Published: July 2021

Background:
Roughly half of patients with heart failure (HF) have preserved EF (HFpEF) and this rate is increasing. The pathophysiology of HFpEF is unclear and treatment of HFpEF remains a critical unmet need.

Hypothesis:
Growth hormone releasing hormone agonist (GHRH-A) restores cardiac function in a rodent model of HFpEF.

Methods:
C57BL/6N mice (n=4-5) received angiotensin-II (Ang-II: 0.8 mg/kg/day) via mini-osmotic pump for 4 weeks with concurrent daily administration of GHRH-A (MR-356: 200 μg/kg) or vehicle (DMSO+propylene-glycol). Echocardiography was assessed at baseline and 4 weeks after Alzet pump placement. Hemodynamic studies were performed and the titin N2BA/N2B ratio measured.

Results:
Ang-II administration increased end-diastolic pressure (EDP, p=0.0186) with no changes in EF (p=ns) or end-systolic pressure (ESP, p=ns) in comparison to control mice. Isovolumetric relaxation time (IVRT, p<0.05) and end-diastolic pressure-volume relationship (EDPVR, p=0.0229) were significantly increased in the Ang-II/vehicle group, consistent with increased ventricular stiffness and impaired relaxation. Importantly, GHRH-A treatment reset these parameters to normal conditions (table). HFpEF mice exhibited higher HW/BW ratios and lung weight. The titin N2BA/N2B ratio, which was increased (p<0.05) in the Ang-II group, was restored by GHRH-A treatment.

Conclusion:
Chronic administration of Ang-II mediates structural and functional changes that mimic HFpEF. GHRH-A treatment improves diastolic dysfunction and impaired relaxation. Therefore, GHRH-A therapy may be beneficial in the treatment of HFpEF.
Abstract 399: The Roles of NAD
+
-sensitive Protein Acetylation in Diastolic Dysfunction
Authors: Lee Chi Fung , Chavez Juan , Chiao Ying Ann , Bruce James , Regnier Michael , Tian Rong
Published: July 2021

Half of the heart failure (HF) patients in US have preserved ejection fraction but diastolic dysfunction. Without a specific medication, better understanding on the mechanisms of diastolic dysfunction is urgently needed. NAD
+
-sensitive lysine acetylation emerges as regulators of HF pathogenesis. We generated mouse models to manipulate NAD
+
redox states in the heart. We used cardiac Ndufs4-KO mice (cKO) as a model of reduced NAD
+
/NADH ratio and cardiac NAMPT over expression (cNAMPT), as a model of elevated NAD
+
levels. Using labelled-free quantitative proteomics, we identified NAD
+
-sensitive changes of acetylation landscapes in the heart. Muscle contraction is one of the top categories of acetylated proteins identified by gene ontology analysis. Analysis of non-mitochondrial acetylome showed that many proteins regulating SR Ca
2+
homeostasis and myofilament movement were hyperacetylated in cKO hearts. Therefore, we hypothesized that increased acetylation of components of SR Ca
2+
homeostasis and myofilament movement impairs diastolic function. Using Doppler echocardiography, we observed a decline in E’/A’ ratio and increased myocardial performance index in cKO hearts. Systolic function and cardiac geometry were normal in cKO hearts. In addition, elevation NAD
+
levels by cNAMPT expression (cKO:cNAMPT) normalized protein hyperacetylation and reversed diastolic dysfunction in cKO. These data support that increased acetylation contributes to the decline in diastolic function in cKO hearts. Future experiments using isolated myofibril and cardiomyocytes will determine the roles of protein acetylation to regulate myofilament, SR Ca
2+
homeostasis and thus diastolic dysfunction.
Abstract 411: The Activated Fibroblast Secretome Drives Cardiomyocyte Dedifferentiation in Pulmonary Hypertension-induced Right Ventricular Dysfunction: a Role for Interleukin-1β?
Authors: Bruns Danielle R , Tatman Philip D , Stenmark Kurt R , Buttrick Peter M , Walker Lori A
Published: July 2021

Chronic exposure to hypobaric hypoxia (HH) causes pulmonary hypertension (PH) and right ventricular (RV) dysfunction, a strong predictor of survival in a variety of clinical contexts. Inflammatory-fibrotic remodeling underlies PH-induced RV dysfunction, a process regulated in part by cardiac fibroblasts (Cfib). In response to insult, Cfib undergo phenotypic changes including hypersecretion of bioactive molecules which may contribute to ventricular dysfunction. We hypothesized that persistent changes in the activated Cfib phenotype would be mediated through DNA methylation and that secretion of signals from the activated Cfib would contribute to cardiomyocyte dysfunction. We tested this hypothesis using RV Cfib derived from the neonatal calf exposed to HH- a novel large animal model with significant resonance with human disease. HH exposure resulted in an epigenetically modified pro-inflammatory Cfib characterized by global DNA hypomethylation, elevated IL-1β signaling, and activation of a myofibroblast phenotype. IL-1β treatment of control Cfib also elicited DNA hypomethylation and phenotype conversion, albeit less robustly than Cfib derived from the HH-exposed RV. Treatment of adult rat ventricular myocytes (ARVM) with conditioned media from HH-derived Cfib caused dedifferentiation of ARVM to a neonatal-like phenotype characterized by activation of the fetal gene program. Immunodepletion of IL-1β from HH conditioned media attenuated expression of fetal genes and myocyte dedifferentiation. However, IL-1β treatment alone was not sufficient to cause dedifferentiation and fetal gene expression, suggesting other secreted (inflammatory) factors contribute to myocyte disarray and dysfunction. Together, these data suggest a key role of the epigenetically modified activated Cfib in the pathogenesis of PH-induced RV dysfunction, mediated in part by IL-1β signaling. Ongoing investigations will identify other pathways of Cfib-mediated ventricular dysfunction to facilitate manipulation of the fibroblast for therapeutic benefit in right heart disease.
Abstract 416: NF-kB-Mir-23a-27a: a Critical Modulator in Post-myocardial Infarction Remodeling
Authors: Gupta Sudhiranjan , Li Li
Published: July 2021

Myocardial infarction (MI) is one of the leading causes of death around the world. Myocardial ischemia contributes to the development of MI and initiates cardiomyocyte (CM) apoptosis, myofibroblasts differentiation, fibrosis and left ventricle (LV) remodeling leading to cardiac dysfunction. Previously, we have demonstrated the direct involvement of miRNAs (a new class of post-translational regulator) and NF-κB in LV remodeling. However, NF-κB-mediated miRNA modulation and miRNA/gene networks in post-MI remodeling remain elusive. Furthermore, with an emerging interest to combine cell transplantation with gene therapy, mesenchymal stem cells (MSC) are considered most beneficial of the stem cells in regenerative medicine. We have a paucity of data in genetically modified MSC on their mobilization within infarcted myocardium. We identified a panel of novel dysregulated miRNAs in the wild type (WT) mice subjected to MI using conventional miRNA array. These miRNAs were restored in cardiac-specific IκBα triple-mutant mice (3M, resistant to NF-κB activation) mice indicated NF-κB-dependent regulation. Among them, miR-23a and miR-27a were significantly upregulated in MI. Our study has identified PPARγ-GSK3β-Bcl
2
-axis, a
bona-fide
target for miR-23a/-27a after unbiased
in vitro
screening. Our hypothesis is that NF-κB-dependent miR-23a/-27a is a pathogenic niche, exacerbates post-MI remodeling through several target pathways; and inhibition of NF-κB, miR-23a/-27a and transplantation of 3M-MSC mitigates the cardiac damage and abrogates the cardiac damage by restoring PPARγ-GSK3β-Bcl
2
-axis. Inhibition of NF-κB normalized miR-23a/-27a expression and reduced CM death in hypoxia/ reoxygenation (H/R) model. Our data also indicated that miR-23a is a key regulator in simulated H/R-induced injury. Inhibition of miR-23a showed reduction of cell death and improved mitochondrial function in H/R model. Interestingly, 3M-MSC showed significant reduction in apoptosis, inflammation and oxidative stress in H/R-induced CM injury, compared to WT-MSC. Our findings conclude that miR-23a/-27a could be a triggering factor and 3M-MSC provides a better protection in post-MI remodelling indicate new mechanistic information for therapeutic benefit.
Abstract 420: MMI-0100 Inhibits Cardiac Fibrosis in a Model of Cardiac Myosin Binding Protein C Hypertrophic Cardiomyopathy
Authors: Meng Qinghang , Bhandary Bidur , Xu Na , Osinska Hanna , Shay-Winkler Kritton , Willis Monte , Robbins Jeffrey
Published: July 2021

Background:
Hypertrophic cardiomyopathy is considered one of the most common genetic heart disorders, occurring with a frequency of about 1 in 200, with approximately 30% of those affected showing mutations within the cardiac myosin-binding protein C (cMyBP-C) gene (
MYBPC3
). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40kDa fragment in mouse cardiomyocytes (MyBP-C
40k
) leads to hypertrophic cardiomyopathy), fibrosis and heart failure. Fibrosis can occur in many cardiovascular diseases and the p38-MK2 signaling pathway has been implicated in a variety of fibrotic processes. Recent studies demonstrated that MK2 inhibition using the cell-permeant peptide inhibitor MMI-0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI-0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP-C
40k
cardiomyocyte expression.

Methods and Results:
Non-transgenic and MyBP-C
40k
transgenic (Dtg) mice were given MMI-0100 or PBS daily for 30 weeks. In control groups, long term MMI-0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy or probability of survival. In the Dtg group, MMI-0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy and prolonged survival.

Conclusions:
Pharmaceutical inhibition of p38-MK2 signaling via MMI-0100 treatment is beneficial in the context of fibrotic MyBPC
40k
disease.
Abstract 43: A20 Plays as a Regulator in Tnfα-induced Injury of Human Umbilical Vein Endothelial Cells Through Tak1-dependent Pathway
Authors: Li Lei , Lin Jiafeng , Chu Maoping , Ji Kangting , Wu Lianpin
Published: July 2021

A20, a negative regulator of nuclear factor κB signaling, has been shown to attenuate atherosclerotic events. Transforming growth factor beta-activated kinase 1 (TAK1) plays a critical role in TNFα-induced atherosclerosis via endothelial nitric oxide (NO) synthase (eNOS) uncoupling and NO reduction.

Aims:
In the study, we investigated the hypothesis that A20 protected endothelial cells induced by TNFα through modulating eNOS activity and TAK1 signalling. Human umbilical vein endothelial cells (HUVECs) were stimulated by TNFα. The impact of A20 on cell apoptosis, eNOS expression and NO production and related TAK1 pathway were detected. Both eNOS and NO production were remarkably reduced, TAK1and p38 MAPK phosphorylation, HUVECs apoptosis increased after TNFα stimulation for 2 hrs, all of which were effectively attenuated by A20 over-expression. Inhibition of A20 significantly activated TAK1, p38 MAPK phosphorylation, and cell apoptosis, but eNOS expression, NO production decreased. Furthermore, p38 MAPK expression was suppressed by A20 over-expression, but re-enhanced by inhibiting A20 or activation of TAK1. Furtherly, TNFα-induced suppression of eNOS and NO production were largely prevented by silencing p38 MAPK. Collectively, our results suggested that A20-mediated TAK1 inactivation suppresses p38 MAPK and regulated MAPK/eNOS pathway, which contributes to endothelial cell survival and function preservation.
Abstract 440: A Lamin A/C Haploinsufficiency Model of Cardiomyopathy Using Patient-Specific iPSC Derived Cardiomyocytes Demonstrates Impaired Contractility
Authors: Parikh Shan , Wang Lili , Bersell Kevin , Blair Marcia , Murad Andrea , Wells Quinn S , Knollmann Bjorn C
Published: July 2021

Background:
Lamin A/C (LMNA) mutations are the second most common cause of familial dilated cardiomyopathy (DCM). LMNA encodes nuclear lamins which are intermediate filaments of the inner nuclear membrane. Patients with LMNA DCM present with early signs of arrhythmias and often display a rapid deterioration in cardiac function. Previous induced pluripotent derived stem cell (iPSC) modeling of cardiac laminopathy did not demonstrate defects in cardiomyocyte (CM) contractility nor provide evidence of arrhythmogenesis. Developing a robust human model that recapitulates disease manifestations of LMNA cardiomyopathy is a critical step for understanding CM-intrinsic defects contributing to disease pathogenesis.

Purpose:
As such, our lab hypothesized that iPSC-CMs derived from a laminopathy patient would recapitulate the contractile dysfunction of the disease if studied in monolayer using flexible matrigel substrate.

Methods:
Patients with symptomology indicative of LMNA DCM were recruited for clinical testing. Peripheral blood mononuclear cells were isolated and reprogrammed into iPSCs. CMs were generated using a chemical differentiation method and then subjected to molecular and functional assessment. Cardioexcyte96 based detection of impedance was utilized for analysis of contractility of iPSC-CMs in a monolayer.

Results/Conclusions:
We identified a large pedigree with DCM in which affected individuals had a putatively pathogenic mutation within LMNA. Western blot analyses demonstrated that lamin A/C can be detected in iPSC-CMs with reduced expression in mutant cells. Mutant iPSC-CMs in a monolayer demonstrate both increased time for contraction (+55.1 msec P<.0001) and relaxation (+44.8 msec P<.0001) compared to control. Our results are the first to provide evidence for contractile dysfunction in an iPSC-CM model of cardiac laminopathy. In addition, this work highlights the utility of impedance based measurement of contractility as a robust outcome that can be applied to high throughput discovery of therapeutic interventions in this disease model.
Abstract 442: Modeling Endothelial Dysfunction in LMNA-Related Dilated Cardiomyopathy
Authors: Sayed Nazish , Liu Chun , Himmati Farhan , Zhang Joe , Termglinchan Vittavat , Moonen Jan-Ranier , Stack Jon , Chen Haodong , Matsa Elena , Sallam Karim , Rabinovitch Marlene , Wu Joseph C
Published: July 2021

Background:
Mutations in the gene that encodes the nuclear envelope proteins lamin A and C (LMNA) accounts for 6% of all cases of DCM. However, the molecular mechanisms that underlie “cardiolaminopathy” remain elusive, and it is unknown why mutations in this ubiquitously expressed gene have such a disproportionate effect on the heart.

Hypothesis:
Despite the fact that LMNA is abundantly expressed in endothelial cells (ECs) and mutations in LMNA are known to induce EC dysfunction, little is known about the EC-specific phenotype of LMNA-related DCM. As EC dysfunction has been known to contribute to DCM, we hypothesize that EC dysfunction due to LMNA mutation has a significant impact on the pathogenesis and disease progression of DCM.

Results:
Intriguingly, our preliminary data showed that iPSC-ECs derived from patients (n=5) harboring the LMNA-mutation exhibit decrease functionality as seen by impaired angiogenesis and decreased NO production (control iPSC-ECs vs LMNA iPSC-ECs; p<0.05). Similarly, genome editing of isogenic iPSC lines enabled us to recapitulate the EC disease phenotype further allowing us to dissect the effects of LMNA mutations on EC function. LMNA corrected iPSC-ECs (via use of CRISPR/Cas9 genome editing tool to correct the single mutated copy in heterozygous patient’s iPSCs) showed restoration of EC function. Whole genome RNA-sequencing identified Krüppel-like Factor 2 (KLF2) as a potential transcript responsible for EC dysfunction in LMNA-mutated patients. Importantly, treatment of LMNA-mutated ECs with KLF2 agonists showed rescue of EC dysfunction. Furthermore, iPSC-derived cardiomyocytes (iPSC-CMs) from LMNA-mutated patients that exhibited DCM phenotype, showed improvement in CM physiology when co-cultured with iPSC-ECs treated with KLF2 agonists.

Conclusion:
This study is a first step towards understanding the molecular mechanisms of cardiolaminopathy by modeling endothelial dysfunction using patient-specific iPSCs. Moreover, our results suggest that improving EC function in cardiolaminopathy patients could have a significant impact on the pathogenesis of DCM. Results from this work could potentially lead to new strategies that could improve the management of DCM patients.
Abstract 450: Brg1 Attenuates Cardiac Dysfunction by Activating Sonic Hedgehog Pathway in Acute Myocardial Infarction
Authors: Hou Ning , Li Sujuan , Liang Guanfeng , Qiu Xiaoxia , Luo Jiandong
Published: July 2021

Background:
Brahma-related gene 1 (Brg1), the core ATPase subunit of a large chromatin remodeling complex, plays critical role in the regulation of gene expression during cardiac growth, differentiation. In adults, Brg1 is turned off in cardiomyocytes and reactivated by cardiac stress. How Brg1 in myocardial infarction (MI) is poorly understood. The sonic hedgehog (Shh) pathway was activated to protect the injured cardiomyocytes during the development of MI. However, by which Shh activation is mediated in MI still remains to be determined.

Methods and results:
Adult male C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery for MI model. We investigated Brg1 activation and how it regulated the gene expressions of Shh pathway in peri-infarct zone in MI mice. Our data demonstrated that in peri-infarct zone, the protein of Brg1 was significantly increased 7 days after MI compared with the sham group in control mice, accompanied by upregulation of shh, patched-1 (Ptch1), and glioma-associated oncogene-1(Gli1). We further revealed that with adenoviral intramyocardial injection, the Brg1 overexpression reduced the percentage myocardial infarct, improved cardiac dysfunction, decreased the number of apoptotic cells and upregulated the ratio of Bcl-2/Bax in MI mice. Conversely, shRNA-mediated knockdown of Brg1 enlarged the percentage myocardial infarct, exacerbated cardiac dysfunction, increased the apoptotic cells and downregulated the ratio of Bcl-2/Bax. More importantly, the Brg1 overexpression increased Shh, Ptch1 and Gli1 expressions, whereas Brg1 knockdown suppressed Shh, Ptch1 and Gli1 expressions. Chromatin immunoprecipitation confirmed that Brg1 were recruited to the regulatory elements of Shh, Ptch1 and Gli1. This recruitment was associated with transcriptional upregulation of these three genes.

Conclusions:
This study indicated that in MI mice, the myocardial Brg1 is upregulated and that the upregulated Brg1 contributes to protection of cardiac function and activation of Shh pathway. Strategies that are aimed at augment the Brg1-Shh pathway may offer useful means for preventing and treating cardiac dysfunction in patients with MI.

Key words: Acute myocardial infarction, Brg1, Shh pathway
Abstract 464: Two Different Microdomains of β
1
-adrenoreceptor Signaling Revealed by Live Cell Imaging
Authors: Froese Alexander , Nikolaev Viacheslav O
Published: July 2021

Background:
3’,5’-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger and a crucial regulator of cardiac function and disease. In cardiomyocytes, it is produced predominantly after activation of β
1
-adrenergic receptors (β
1
-ARs) by catecholamines and acts intracellularly in discrete functionally relevant microdomains formed, for example, around calcium-handling proteins. Previously, we reported that β
1
-ARs are distributed across various cardiomyocyte membrane areas, including transverse (T)-tubules and cell crests. However, it is unknown whether these two β
1
-AR pools contribute differentially to the regulation of cardiac contractility and gene expression.

Methods and Results:
To directly visualize receptor-microdomain communication in cardiomyocytes, we established a combination of scanning ion conductance microscopy (SICM) with transgenically expressed targeted Förster resonance energy transfer (FRET)-based biosensors. Using this approach, we measured local cAMP responses in distinct microdomains of mouse ventricular cardiomyocytes (such as plasma membrane, cytosol and nucleus) after localized stimulation of β
1
-AR on different membrane structures of healthy and diseased cardiomyocytes. Using a plasma membrane targeted cAMP biosensor, we found that β
1
-AR stimulation at the crest induced stronger cAMP signals compared to β
1
-AR stimulated in the T-tubuli where cAMP was highly confined by PDE3. This difference was abolished in a pressure overload hypertrophy model due to submembrane redistribution of PDEs. Interestingly, crest β
1
-AR signals could propagate deeper inside the cell, inducing higher nuclear cAMP responses than recorded from receptors stimulated in the T-tubules.

Conclusions:
in the present study, we have demonstrated that β
1
-ARs located in T-tubuli and cell crests form two differentially regulated cAMP microdomains, each having its typical PDE repertoire and generating distinct second messenger signals. More detailed understanding of these two microdomains at different subsarcolemmal locations may contribute to new therapeutic strategies including more specific β-blockers.
Abstract 466: Cardiac Activation of β-catenin in Mice Leads to Prolongation of QRS and Susceptibility to Arrhythmia by Inhibiting Na
+
Channel Activity Through Suppression of Na
V
1.5 Expression
Authors: Huo Rong , Hu Chaowei , Lu Yan , Wang Ning , Ye Bo , Deb Arjun , Li Faqian , Xu Haodong
Published: July 2021

Introduction:
In vitro
studies showed that activation of β-catenin suppresses Na
V
1.5 expression by inhibiting
SCN5a
promoter activity, leading to a decrease of Na
+
channel activity. How β-catenin regulates cardiac electrophysiological phenotype is unknown.

Hypothesis:
We hypothesized that cardiac activation of β-catenin regulates electrophysiological phenotype by suppressing Na
V
1.5 expression.

Methods:
Adult mice with cardiac-specific, tamoxifen-induced deletion of β-catenin
exon3
, leading to cardiac activation of β-catenin (β-catenin
exon3
-/-
) were generated, and the effects of cardiac activation of β-catenin on the electrophysiological remodeling were assessed by electrocardiogram (ECG) recording. Class Ic antiarrhythmic reagent, flecainide, was administered to evaluate susceptibility to ventricular tachycardia (VT). Cardiac structure and function were evaluated by histologic and echocardiographic examinations, respectively. Western blot and qRT-PCR were performed to determine the levels of Na
V
1.5 and β-catenin expression in mouse hearts. Whole-cell recording technique was utilized to record Na
+
currents and action potentials (APs) from ventricular myocytes.

Results:
Histologic and echocardiographic examinations showed that β-catenin
exon3
-/-
mice had normal cardiac structure and function. Compared to wild type (WT) mice, the ratio of heart/body weight was not changed and the duration of QRS was significantly prolonged in β-catenin
exon3
-/-
mice. VT was induced by flecainide in 60% of β-catenin
exon3
-/-
mice but not in WT mice. Western blot and qRT-PCR showed that Na
V
1.5 protein and mRNA were significantly decreased in β-catenin
exon3
-/-
hearts, compared to WT hearts. Maximal upstroke velocity and amplitude of APs and Na
+
currents were significantly decreased in β-catenin
exon3
-/-
ventricular myocytes, compared to WT cells.

Conclusion:
Cardiac activation of β-catenin leads to prolongation of QRS and susceptibility to VT by decreasing Na
V
1.5 expression and Na
+
channel activity.
Abstract 483: Effect of Glucose on Endothelial Cell Expression of Inflammation-Related miRNAs
Authors: Hijmans Jamie , Levy Ma’ayan , Reiakvam Whitney , Bruster Lillian , Stockelman Kelly , Goldthwaite Zoë , Bammert Tyler , Greiner Jared , DeSouza Christopher
Published: July 2021

Hyperglycemia is an independent risk factor for cardiovascular disease (CVD). A key factor underlying hyperglycemia-related CVD risk is endovascular inflammation. The mechanisms underlying hyperglycemia induced endovascular inflammation are not clear. It is now recognized that microRNAs (miRs) play a pivotal role in regulating cellular inflammation pathways, vascular health and, in-turn, cardiovascular disease (CVD). It is currently unknown whether high glucose concentrations adversely affect the expression of inflammation-related miRs in endothelial cells; if so, miR dysregulation may provide novel insight into the proinflammatory endothelial phenotype associated with hyperglycemia. The aim of this study was to determine the effect of high glucose concentrations on the expression of inflammation-related miRs (miR-34a, -92a, -126, -146a, -150, -155 and -181b). Cultured human umbilical vein endothelial cells were harvested on the 3rd passage and plated in 6-well plates at a density of 5.0x10
5
cells/condition. Cells were incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a hyperglycemic state) or 5mM D-glucose (control condition) for 48 hours. Thereafter, RNA was extracted from 1.0x10
5
cells and miRs were reverse transcribed and expression was determined by RT-PCR. Cellular expression was normalized to RNU6 and calculated as fold change in ΔΔCt from control (N=6, experimental units). There was no effect of high glucose concentrations on the expression of proinflammatory miRs: miR-34a (0.93±0.18 fold), -92a (0.93±0.07 fold) and -155 (0.85±0.10 fold). However, cellular expression of anti-inflammatory miRs: miR-146a (0.64±0.06 fold), -150 (0.69±0.12 fold), and -181b (0.73±0.07 fold), were significantly reduced (~60%, ~45%, and ~40% respectively; P<0.05) in response to the high glucose condition. Lower cellular expression of miR-146a, miR-150 and miR-181b is consistent with a pro-inflammatory endothelial phenotype. These miRs directly target and suppress NF-kB activation and immune cell activation, primary mediators of cellular inflammation. The seminal and novel finding of this study is that high glucose concentration compromises endothelial cell expression of key anti-inflammatory miRs.
Abstract 50: Atrial Fibrillation is Associated with Higher Stroke Rates in Takotsubo Cardiomyopathy
Authors: Akinjero Akintunde M , Adegbala Oluwole , Akinjero Nike E , Edo-Osagie Eseosa , Akinyemiju Tomi
Published: July 2021

Background:
The prognosis of Takotsubo Cardiomyopathy (TTCM) is worse than in the general population. It is unclear how atrial fibrillation (AF) impacts this prognosis. We sought to evaluate the effect of concurrent AF on outcomes in patients with TTCM.

Methods:
We used the Healthcare Cost and Utilization Project (HCUP) Nationwide Inpatient Sample (NIS) to extract all hospitalizations between 2007 and 2011 with concurrent diagnosis of AF and TTCM. The ICD-9 CM codes for AF and TTCM were used. We compared patients admitted for TTCM who had coexisting AF to those without. We excluded patients below the age of 18 as well as those diagnosed with TTCM who later underwent percutaneous coronary intervention (PCI). Multivariate regression was used to assess the independent effect of coexisting AF on clinical outcomes (length of stay (LOS), stroke, and in-hospital mortality).

Results:
A total of 13,136 TTCM patients were studied. Of these, 2,083 (15.86%) had coexisting AF. Compared with those without, TTCM patients with coexisting AF had a greater multivariate-adjusted risk for increased stroke rate (aOR=1.66, 95% CI=1.27-2.18, Table 1). We found no significant association with in-hospital mortality (aOR=1.21, 95% CI=0.96-1.52) or LOS (aOR=1.21, 95% CI= 0.83-1.58).

Conclusions:
In this large, nationally representative study, we found higher stroke rates in patients with coexisting AF and TTCM. Our findings suggest the need for closer monitoring during hospitalization.