bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1137: Combination Of Zebularine Treated Rat Bone Marrow Mesenchymal Stem Cells With 3d Collagen Scaffold Preserves Ventricular Remodeling In Rat Myocardial Infarction Model
Authors: Qazi Rida-e-Maria , Khan Irfan , Haneef Kanwal , Malick Tuba S , Naeem Nadia , Ahmad Waqas , Salim Asmat , Mohsin Sadia
Published: November 2022

Cardiovascular diseases (CVDs) are the leading cause of death globally due to immensely high morbidity and mortality ratio. The incidence of CVD, particularly myocardial infarction (MI), is expected to increase rapidly in the coming decades. MI causes the death of myocytes, which ultimately leads to heart failure. Advancements in medical and surgical treatments can only limit the disease progress but cannot improve the function of infarcted myocardium. Therefore, a promising treatment strategy for ischemic heart diseases is highly needed. Stem cell therapy holds great potential to regenerate the injured myocardium due to the self-renewal and differentiation potential of these cells. Among various types, mesenchymal stem cells (MSCs) are potential candidates for regenerating the damaged cardiac tissue due to their multi-lineage differentiation potential and ease of isolation. However, certain issues such as poor survival and engraftment can prevent them from fully restoring normal cardiac structure and function. The use of biomaterials provides suitable microenvironment to the cells to perform various biological functions, and can solve the poor cellular engraftment problem associated with cell transplantation alone. In this study, we used natural three dimensional (3D) collagen scaffold along with demethylating agent, zebularine, for
in vivo
transplantation in rat MI model. Treatment with zebularine is shown to enhance cardiac differentiation of MSCs. MSCs were isolated from rat bone marrow, characterized, seeded in collagen scaffold and treated with zebularine. Cytotoxicity analysis was performed to confirm the non-cytotoxic effect of the treatments, and gene and protein expression levels were analyzed using cardiac specific primers and antibodies, respectively.
In vitro
analysis revealed that treatment in the 3D environment significantly enhanced cardiac differentiation of MSCs both at gene and protein levels. For
in vivo
analysis, rat MI model was developed and zebularine treated MSC-seeded scaffold was transplanted and compared with the MI group, along with other groups in which only collagen scaffold and untreated MSC-seeded scaffold were transplanted. Echocardiographic analysis revealed enhanced functional improvement in zebularine treated MSC-seeded group as compared to the other groups. Histological analysis revealed tissue preservation, cardiac regeneration beneath the scaffold, reduction in the fibrotic scar and improvement in the ventricular wall thickness in the treated MSC group. This strategy is expected to overcome the problems associated with cell transplantation alone and to provide an effective therapeutic strategy for improved cardiac function.
Abstract P1138: Combination Of Pluripotency And Cardiac Transcription Factors Reprogram Skin Fibroblasts Into Cardiomyocytes
Authors: Ahmad Waqas , Qazi Rida-e-Maria , Muneer Rabbia , Khan Irfan , Salim Asmat , Khan Mohsin
Published: November 2022

Direct reprogramming of autologous somatic cells into cardiomyocytes is a novel approach which is being employed for cardiac regeneration. Multiple studies have combined different cardiac-specific factors that could directly reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, this approach needs heart biopsy for the isolation of autologous fibroblasts which involves extensive surgical procedures that require precision. In this study, we aimed to develop an approach which can directly reprogram somatic cells into cardiomyocyte-like cells using a combination of pluripotency and cardiac transcription factors. Skin fibroblasts were isolated from rat neonatal pups and were induced into cardiomyocyte-like cells using non-viral integration of cardiac transcription factors (GATA4, Mef2c and Nkx2.5) and OKSIM plasmid carrying the iPSC factors, Sox2, Oct4, Klf4 and cMyc. After 72 h, cells were analyzed for the expression of cardiac markers by qPCR and immunocytochemistry. Gene expression analysis showed significantly higher expression of cardiac markers GATA4, cMHC, Mef2c, cTnT, cTnI, and Nkx2.5 Immunostaining confirmed the expression of cardiac proteins GATA4, cMHC, cTnT, cTnI, and Nkx2.5. These results imply that the transfected cells started differentiating towards cardiac lineage. Transfected cells were also transplanted in rat myocardial infarction (MI) model immediately after ligation of left ventricle descending (LAD) artery. After 2 and 4 weeks of cell transplantation, the animals were assessed for cardiac function via echocardiography. They showed significant improvement in the cardiac function as compared to MI and non-transfected groups. After 4 weeks, the hearts were harvested, and histological analysis was performed for the assessment of fibrosis and left ventricular wall thickness. Rats transplanted with transfected cells showed significantly reduced fibrosis and increased wall thickness as compared to MI and non-transfected groups. In conclusion, transient and combined expression of cardiac transcription and iPSC factors in neonatal somatic cells leads to the transdifferentiation of skin cells into myogenic lineage. This approach can be useful for future therapeutic application for cardiovascular diseases.
Abstract P1140: Scaling Up Engineered Cardiac Tissue For Clinical Translation As A Regenerative Therapy
Authors: Dwyer Kiera , Kant Rajeev , Coulombe Kareen L
Published: November 2022

Advances in cardiovascular interventions have significantly decreased mortality due to acute myocardial infarction (MI). However, heart failure (HF) as a main sequela remains a major and devastating health burden. Globally, ~13 million people suffer from HF with systolic dysfunction and have no clinically available treatment to remuscularize their failing heart. Regenerative therapies using cardiomyocytes derived from embryonic or human-induced pluripotent stem cells (hiPSC-CMs) have emerged in large animal models and clinical trials to stabilize cardiac function post MI. Delivery of CMs through epicardially-implanted engineered cardiac tissues (ECTs) is advantageous as increased CM maturity, improved engraftment and increased mechanical support of the cardiac wall have been reported as compared to intramuscular CM injection. To date, however, the lack of rigorous studies exploring ECTs scaled to clinically relevant size (>3x3cm) poses an immense hurdle to their use in clinical trials. To this end, we are focused on understanding how such ECT scale up impacts the electromechanical function of the tissue. Our work details the differentiation and proliferative expansion of hiPSC-CMs to achieve the quantity and quality of hiPSC-CMs required for clinically scaled ECTs. We fabricate ECTs by mixing hiPSC-CMs with 5% human cardiac fibroblasts in a collagen-1 hydrogel with electromechanical function assessed through optical mapping of action potential and tensile mechanical testing. We report compromised electromechanical function and structural organization of ECTs with scale up, which is ameliorated by maturation of hiPSC-CMs using drug and metabolic interventions. Because larger animals will require higher CM density in implants, we evaluated the impact of CM density on ECT formation and function. Increasing CM density (30x10
6
vs 5x10
6
hiPSC-CMs/mL) decreased ECT compaction by 70% while increasing active force generation by 5.5-fold and contractile upstroke velocity by 4.3-fold. In conclusion, we have identified the design space for fabricating ECT of clinically relevant size. By understanding the space and its functional impact, this work supports the translational feasibility of ECTs as a regenerative treatment for the failing heart.
Abstract P1142: Engineering Vascular Therapies With Angiogenic Factors For Cardiac Regeneration And Tissue Remodeling
Authors: Minor Alicia , Roser Stephanie , Zahiri Keyana , Song Elena , Kobayashi Momoka , Polucha Collin , Coulombe Kareen L
Published: November 2022

The limited regenerative capacity of the adult human heart presents a critical need to stabilize and support injured heart muscle to mitigate the progression of heart failure. The effectiveness of remuscularization therapies using human induced pluripotent stem cell derived cardiomyocytes depends host mediated angiogenesis to direct the formation of stable, perfused vascular networks within the engineered cardiac implant. We hypothesize that potent combinations of growth factors will stimulate angiogenesis in the myocardium and increase vascular development in engineered cardiac tissue. In a fractional factorial experiment, we assessed the effect of ten growth factors, cytokines and enzymes: VEGF, bFGF, Shh, PDGF, IGF-1, EGF, TGF-β1, Ang-1, MCP-1 and MMP-9 on
in vivo
angiogenesis in a subcutaneous Matrigel plug assay in the rat. Five factors were highly potent by metrics of vessel density and vascular cell recruitment in explants and warranted further investigation. We then designed a two-level, fractional factorial experiment using a defined collagen-alginate gel to deliver high/low doses of these 5 factors subcutaneously to elucidate main effects and two-way interactions that regulate vessel development
in vivo
. After 7 days,
Griffonia Simplicfolia lectin I
was administered to label perfused vasculature then explants were removed and processed for immunohistochemistry. Our data show that 3 unique cocktails yielded a 10-fold increase in lumen density, a 3.3-fold increase in lumen size, and a 3.7-fold increase in endothelial cell recruitment relative to unloaded controls. Ongoing analyses will determine which angiogenic cocktail to deliver in a customizable biomaterial-based vascular therapy for applications in the heart. This novel therapy will be delivered with an engineered cardiac tissue onto the epicardium in a rat I/R MI model to quantify vascular remodeling in the implant and underlying host tissue and whole heart function over 4 weeks. In conclusion, we have identified five potent angiogenic factors for modulating revascularization responses and our ongoing work aims to understand the potential synergy between revascularization and remuscularization therapies for heart regeneration.
Abstract P2008: Hypertrophic Cardiomyopathy Treatment Using Ultrasound And Microbubbles
Authors: Singh Davindra , He Stephanie , Helfield Brandon
Published: November 2022

Introduction:
Hypertrophic cardiomyopathy is a disease characterized by the abnormal growth of cardiomyocytes (CMs), a marker of which is the early downregulation of miR-1. The delivery of miR-1 to hypertrophic CMs presents as a promising treatment strategy, however there are still limitations in the safe and efficient delivery of gene therapeutics.Ultrasound (US) and microbubbles is an emerging approach to site-specific gene delivery. Microbubbles have long been employed in clinical cardiology as a diagnostic contrast agent. Recent work has demonstrated that under specific conditions, they are capable of forming temporary pores on neighboring cell membranes, allowing the delivery of otherwise impermeable macromolecules. This presents an exciting approach to the treatment of cardiovascular disease (CVD) as a targeted and inherently image-guided therapeutic delivery platform.

Objective:
The goal of this study is to determine the feasibility of viable US and microbubble mediated delivery of miR-1 to hypertrophic CMs with a view towards reversing the disease. Secondly, we aim to show a correlation between bubble echoes and resulting gene delivery to introduce clinical metrics for the translation of this technique.

Methods:
CMs were harvested from neonatal rat pups (1-3 days old) and hypertrophied using phenylephrine (100μM). Suspension of healthy or diseased CMs, microbubbles and free miR-1 were placed in a chamber within a 37°C degassed water bath. These samples were insonicated at a mechanical index (MI) ranging from 0.063-0.25 for 2 minutes (20 cycles; 5 kHz PRF) and bubble echoes were recorded via a co-aligned transducer to be processed offline. miR-1 delivery was confirmed with RT-qPCR, cell viablity assesed with MTT, protein expression was measured by immunoblotting and cell size via microscopy. All experiments preformed are at least n=2 independent samples.

Result and Conclusion:
Treated healthy and diseased CMs demonstrate an upregulation in miR-1 with increasing MI, from 0.063 to 0.188. At MI=0.12, a 2.22 and 1.95-fold increase in miR-1 was shown in diseased and healthy CMs respectively (
p=
0.07,
p=
0.02) while maintaining viability (86% and 96% respectively). These miR-1 levels begin to show the reversal of hypertrophy as quantified by increases in target protein expression (1.93-fold in TWF1; 1.38-fold in MEF2A; 1.36-fold in CX43) and cell size decrease (16%,
p
=0.035) back towards heathy levels. Echo data highlights that relative miR-1 expression positively correlates with bubble disruption (
r
=0.76,
p
=0.04), suggesting a means by which treatment efficiency can be monitored in a clinical setting.In conclusion, we demonstrated that US and microbubbles can be used to successfully delivery miR-1 to CMs, with the potential to reverse the disease phenotype. These findings demonstrate the feasibility of US as an image-guided delivery method for molecular therapeutics in CVD.
Abstract P2009: MPTM1: A Cardiac-Enriched Microprotein That Is Upregulated During Heart Failure
Authors: Kamradt Michael L , Makarewich Catherine A
Published: November 2022

Although they were originally misclassified as lncRNAs due to oversights in genome annotation methods, microproteins—proteins with open reading frames (ORFs) of 150 amino acids or less—execute critical roles in heart and skeletal muscle. We have identified a novel microprotein, MPTM1, that is enriched in cardiac and skeletal muscle tissues. The MPTM1 amino acid sequence is strongly conserved across multiple species, which indicates that it is a functional microprotein that has been maintained in response to evolutionary pressure. Genome-wide association studies (GWAS) of patients have demonstrated that MPTM1 is upregulated during heart failure. Additionally, MPTM1 expression strongly increases during skeletal muscle differentiation and maturation. In the present study, we assessed the hypothesis that MPTM1 promotes heart and skeletal muscle homeostasis using both
in vitro
and
in vivo
gain- and loss-of-function approaches. Furthermore, we generated C2C12 myoblast and NIH3T3 fibroblast cell lines null for MPTM1 using CRISPR/Cas9 gene editing methods. MPTM1 depletion in knockout cell lines resulted in striking membrane and nuclear abnormalities. Additionally, MPTM1 null C2C12 cells exhibited defects in myoblast differentiation characterized by premature myosin expression, altered cellular architecture, abnormal nuclear distribution, and the accumulation of intracellular vacuoles. We further evaluated MPTM1 cellular function using CRISPR/Cas9 homology-directed repair (HDR) to insert tandem affinity tags (3XHA-V5 or 3X FLAG) into the MPTM1 locus to assess binding partners
in vitro
using immunoprecipitation and mass spectrometry. Our results provide strong evidence that MPTM1 is a multi-localizing protein that is differentially recruited to the cytosol, nucleolus, and mitochondria, indicating it may act as a key intracellular signaling effector in response to stress. Additional studies in MPTM1 null mice are ongoing and will provide critical insight into MPTM1 function in the heart at under normal physiologic conditions and in response to pathological stress.
Abstract P2010: Cardiomyocyte Knockout Of Sorbs2 Rewires Cytoskeleton And Causes Dilated Cardiomyopathy
Authors: McLendon Jared M , zhang xiaoming , Matasic Daniel , Kumar Mohit , Koval Olha , Grumbach Isabella M , Sadayappan Sakthivel , London Barry , Boudreau Ryan L
Published: November 2022

Sorbs2 is a cardiomyocyte-enriched, cytoskeletal adaptor protein and there is growing interest in understanding its roles in cardiac biology and disease. While Sorbs2 global knockout mice display lethal cardiomyopathy with severe arrhythmias, the underlying mechanisms remain unclear, and if this results from intrinsic loss of Sorbs2 in cardiomyocytes (CM) is unknown, as Sorbs2 is also well-expressed in the nervous system and vasculature. Thus, the potential relevance of Sorbs2 in human cardiomyopathy is underexplored. We aim to characterize the effects and potential mechanisms of CM-specific deletion of Sorbs2 on cardiac structure and function in mice, and to further examine Sorbs2 dysregulation in failing hearts and explore potential links between Sorbs2 genetic variations and human cardiovascular disease phenotypes. We report that heart Sorbs2 expression is consistently upregulated in humans with ischemic and idiopathic cardiomyopathies, and in animal models of heart failure (HF). We generated mice with CM-specific loss of Sorbs2 (Sorbs2-cKO) and found early atrial and ventricular conduction anomalies, despite unaltered expression of primary action potential ion channels and gap junction proteins. At mid-life, Sorbs2-cKO mice exhibit impaired cardiac contractility with cardiomyofibers failing to maintain adequate mechanical tension. These mice develop progressive diastolic and systolic dysfunction, enlarged cardiac chambers, and die with congestive HF at ~1 year of age. Comprehensive survey of potential underlying mechanisms show that Sorbs2-cKO hearts have defective microtubule polymerization and compensatory upregulation of desmin, vinculin, and tubulins. Finally, consistent with our mouse observations, we identified suggestive links between Sorbs2 genetic variants and human cardiac phenotypes, including conduction abnormalities, atrial enlargement, and dilated cardiomyopathy. In conclusion, our studies show that Sorbs2 is essential for maintaining cytoskeletal structural integrity in CM, likely through strengthening the interactions between microtubules and other structural proteins at crosslink sites, and highlights its potential clinical relevance to cardiomyopathy.
Abstract P2019: Do Changes In Metabolism Or Lactation Mediate The Reversal Of Pregnancy-induced Cardiac Growth?
Authors: Schulman Emily B , Hill Bradford G , Collins Helen E
Published: November 2022

During pregnancy, the maternal heart undergoes physiologic growth, which reverses following parturition and is associated with the restoration of hemodynamic parameters. However, the molecular processes underlying the reversal of pregnancy-induced cardiac growth and the time frame in which these changes occur remain unclear. Because changes in cardiac metabolism have been shown to influence the remodeling in the maternal heart, changes in metabolism may also contribute to the reversal of pregnancy-induced remodeling. Therefore, the goal of this study was to examine the underlying processes that contribute to the structural and functional changes occurring in the maternal heart following parturition. Timed pregnancy studies in 12-week-old female FVB/NJ mice showed that heart weight to tibia length (HW:TL) and cardiomyocyte size were highest in lactating 1-week post-birth (PB) mice versus mid-pregnant (8 d), late-pregnant (LP; 16 d), and non-pregnant (NP) mice. Interestingly, we also found that HW:TL was significantly reduced in non-lactating 1-week post-birth mice (PBNL) compared with LP mice. To determine the time-course of the reversal of pregnancy-induced remodeling and to examine the impact of lactation, we examined HW:TL in PB and PBNL mice 1, 3, and 6 weeks following parturition. We find that HW:TL is significantly higher in lactating PB mice versus non-lactating PBNL mice at 1 (8.71 ± 0.05 v 7.09 ± 0.26 mg/mm) and 3 weeks following parturition (8.90 ± 0.40 v 6.92 ± 0.56 mg/mm); however, 6 weeks after parturition, HW:TL was comparable between PB (7.35 ± 0.29 mg/mm) and PBNL groups (7.05 ± 0.18 mg/mm). Nevertheless, HW:TL remained elevated for 6 weeks after parturition in PB and PBNL mice compared with NP mice. RNA-Seq showed reductions in metabolic transcripts in PB hearts, including
Pdk4
. Immunoblot analysis of key metabolic proteins showed that Pdk4 was reduced in hearts from 1 and 3 PB versus 1 and 3 weeks PBNL and NP. Together, these findings suggest that lactation influences the reversal of pregnancy-induced cardiac growth, which may be due in part to changes in cardiac metabolism.
Abstract P2023: Non-cardiomyocytes From Chronic Unloaded Post LVAD Heart Form Organized Complex Neovascular Structures When Compared To Those From Heart Failure.
Authors: Ranka Rajul , Cooke John P , Bhimaraj Arvind , Youker Keith A
Published: November 2022

Introduction:
Chronic unloading of the left ventricle (LV) has shown increased endothelial density suggestive of a vascular recovery. Mechanisms of such neovascularization have not been fully explored.

Hypothesis:
Non-myocyte transitions may play a role in neovascularization in cardiac recovery.

Methods:
We utilized fresh cardiac tissue obtained from patients at the time of LVAD (pre-LVAD core - representing HF) and from a LVAD patient at the time of transplant (Post-LVAD-representing chronic unloaded heart). Using disaggregation, cardiac non-myocytes were isolated, and the resulting pellet of cardiac non-myocytes was divided and plated in both fibroblast (FGM) and endothelial (EGM) growth media.

Results:
Cells from post-LVAD patient in EGM (grown on glass coverslip) showed a complex organizational network with the presence of CD31+ cells in vessel like structures surrounded by vimentin positive cells (See Figure). These networks were not observed in any of the other culture conditions. We also observed that cells from post-LVAD grew much faster compared to pre-LVAD cells.

Conclusions:
To our knowledge, this is the first-time disaggregated cells from human heart samples have been studied in culture. These preliminary observations support the ability of non-cardiomyocytes in a chronic unloaded and rested heart to form neovascular structures not seen in cells from the diseased state of heart failure. This platform can serve to further understand the behavior and role of complex non-myocyte interactions in cardiac recovery.
Abstract P2028: A Role For Pannexin 1 In Heart Failure Induced By Acute And Chronic Isoproterenol Administration
Authors: Pavelec Caitlin M , Wolf Matthew J , Yeudall Scott , Upchurch Clint , Leitinger Norbert
Published: November 2022

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of cardiac dysfunction and fibrosis, we use an acute (14 day) and a chronic (28 day) model of adrenergic stimulation to induce heart failure in two novel mouse models: 1) We have generated mice with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP-flanked alleles of
Panx1
to mice expressing Cre recombinase under the control of the cardiomyocyte specific
MyHC6
promoter (Panx1
MyHC6-Cre
). 2) Using mice expressing the tamoxifen-inducible MerCreMer system under the control of the
Tnnt2
promoter we generated mice with tamoxifen-inducible deletion of Panx1 in cardiomyocytes (Panx1
Tnnt2-MerCreMer
). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline as measured by echocardiography. However, after acute isoproterenol treatment (15 mg/kg/day, i.p.), Panx1
MyHC6-Cre
mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy. Furthermore, after chronic administration of isoproterenol (15 mg/kg/day, osmotic pump) Panx1
MyHC6-Cre
mice were still protected from cardiac hypertrophy compared to Panx1
fl/fl
littermates.
In vitro
, treatment of H9c2 cardiomyocytes with isoproterenol led to Panx1-dependent release of ATP in a dose-dependent manner. This was demonstrated with both pharmacological blockade by spironolactone and siRNA-mediated knock-down of Panx1. Taken together, these data demonstrates that Panx1 deficiency in cardiomyocytes protects against isoproterenol-induced cardiac dysfunction and that ATP is released through Panx1 in response to isoproterenol. Further elucidating the role of Panx1 in cardiomyocytes in regulating cardiac function and fibrosis will identify Panx1 as a novel therapeutic target to prevent heart failure.
Abstract P2029: The Role Of Runx1 In Cardiomyocyte Cell Cycle Activity And Its Impact On Cardiac Regeneration
Authors: Akins Kaelin A , Swift Samantha , Kolell Mary , Flinn Michael , Omeara Caitlin , Patterson Michaela
Published: November 2022

Factors responsible for cardiomyocyte proliferation may serve as a potential therapeutic to stimulate endogenous myocardial regeneration following insult, such as ischemic injury. A previously published forward genetics approach assessing the frequency of a rare and presumed proliferation-competent subpopulation of cardiomyocytes, mononuclear diploid cardiomyocytes (MNDCMs), led us to the transcription factor RUNX1. It is established that RUNX1 induction in cardiomyocytes increases after injury. Here, we examine the effect of RUNX1 on cardiomyocyte cell cycle and establishment of the MNDCM population during postnatal development and cardiac regeneration using both cardiomyocyte-specific gain-and loss of function mouse models. We hypothesize that RUNX1 overexpression (OE) increases cardiomyocyte cell cycle activity with expansion of the MNDCM population, thereby extending the neonatal regenerative window and positively impacting adult cardiac remodeling post injury. During postnatal development, RUNX1 KO decreased postnatal cardiomyocyte cell cycle activity, while RUNX1 OE extended the period of cell cycle activation. This extension observed in RUNX1 OE mice is complete with cytokinesis resulting in an expansion of the MNDCM population and total cardiomyocyte endowment. To determine whether RUNX1 could similarly regulate cardiomyocyte cell cycle in a regenerative and non-regenerative model, we induce P6 and 8-week MIs to measure cell cycle activity and cardiac function post injury. RUNX1 OE neonatal mice with a P6 MI displayed no difference in cardiomyocyte cell cycle activity 7 post injury compared to control littermates. However, RUNX1 OE in adult mice with an 8-week MI showed increased cardiomyocyte cell cycle activity with completion of cytokinesis 2 weeks post injury and limited improvement in cardiac function 28 days post injury. RUNX1 influences cardiomyocyte cell cycle activation in the context of normal postnatal development and adult MI. Conversely, this phenomenon does not appear to translate to the neonatal injury context. We are examining this possible discrepancy with further experiments to fully understand .
Abstract P2035: Palmitoylation Of Rac1 Is Required For Cardiomyocyte Hypertrophy And Oxidative Stress
Authors: Teuber James P , Subramani Arasakumar , Farhan Yasin , Brody Matthew
Published: November 2022

Cardiac hypertrophy, the enlargement of cardiomyocytes in response to an increased workload, is a precursor to maladaptive cardiac remodeling and heart failure. Palmitoylation is a post-translational lipid modification which controls the subcellular localization, trafficking, and activity of proteins but has not been extensively studied in the setting of cardiac disease. Previous work from our group shows that transgenic mice with cardiomyocyte-specific overexpression of zDHHC3, a S-acyltransferase which catalyzes palmitoylation, develop severe cardiac hypertrophy which progresses to lethal dilated cardiomyopathy. Palmitoylated levels, as well as total expression, of the small GTPase Rac1 were significantly elevated in the hearts of these transgenic mice compared to control mice suggesting that palmitoylation of Rac1 at Cys-178 may drive this phenotype. Rac1 is required for the development of cardiac hypertrophy and relatedly plays an important role in mediating myocardial oxidative stress as a regulatory subunit of the NADPH oxidase 2 (Nox2) complex. Interestingly, transgenic mice overexpressing zDHHC3 in cardiomyocytes also display increased myocardial oxidative stress. Because of the known importance of Rac1 in regulating cardiac remodeling, we sought to define the role of palmitoylation in controlling Rac1 signaling in cardiomyocytes. To do this, we overexpressed a constitutively active Rac1 mutant, Rac1-G12V, or a constitutively active, palmitoylation-deficient Rac1 mutant, Rac1-G12V/C178S, in neonatal rat cardiac myocytes (NRCMs). Our data suggest that inhibition of Rac1 palmitoylation at Cys-178 protects NRCMs from hypertrophy and oxidative stress induced by hyperactive Rac1 signaling. Furthermore, we found that overexpression of palmitoylation-deficient Rac1 protects against oxidative stress induced by angiotensin II in NRCMs. Future work will interrogate if inhibition of Rac1 palmitoylation at Cys-178 in cardiomyocytes protects against the development of cardiac hypertrophy and myocardial oxidative stress
in vivo
.
Abstract P2037: Activation Of A2A Adenosine Receptor Reduces Cardiac Dysfunction Resulting From Pulmonary Hypertension In Rats
Authors: Da Silva Jaqueline S , Rocha Bruna , Gomide Gabriel F , Nascimento-Carlos Bianca , Montagnoli Tadeu , Dematté Bruno E , Maia Rodolfo , Barreiro Eliezer J , Zapata-Sudo Gisele
Published: November 2022

Pulmonary hypertension (PH) is characterized by extensive pulmonary vascular remodeling, leading to right ventricle (RV) hypertrophy and dysfunction. This work evaluates the hypothesis that the activation of adenosine A2A receptor (AR-A2) by LASBio-1900 could interfere with the cardiac and vascular dysfunction on monocrotaline (MCT)-induced PH in rats. After 14 days of PH induction using a single injection of MCT (60 mg/kg i.p.), twelve male Wistar rats were randomly divided in groups and treated orally either with vehicle or LASSBio-1900 (180 μmol/kg/day). Hemodynamic parameters were obtained using the echocardiography and summarized in table 1.

LASSBio-1900 reduced RV hypertrophy observed in PH because the Fulton index altered from 55.4 ± 2.3 to 35.3 ± 5.9% (p<0.05). HP increased RV systolic and diastolic pressure from 19.2 ± 2.0 and 4.7 ± 1.0 to 51.5 ± 5.2 and 11.9 ± 1.3 mmHg, respectively. Ventricular dysfunction was recovered with LASSBio-1900 treatment reducing pressures to 28.4 ± 4.0 and 6.5 ± 0.8 mmHg, indicating improvement of RV dysfunction. Pulmonary arteriole (PA) muscularization was evaluated by alpha- smooth muscle actin stained and vessel medial wall area was expressed as the percentage of the portion positively stained relative to total transversal area. The increase of the wall thickness of distal PA from 61.7 ± 1.1 to 83.24 ± 1.82 % was reduced by LASSBio-1900 (75.2 ± 3.4 %). Perivascular collagen in PA was evaluated through picrosirius red staining and interstitial fibrosis was measured by obtaining the total collagen area per arteriole area. PH increased the perivascular collagen deposition in 15.1 ± 1.3% however, treatment with LASSBio-1900 reduced to 8.7 ± 1.4% (p<0.05). Intense inflammation process on PH was confirmed with the increase of iNOS expression from 4.5 ± 0.3 (control) to 19.0 ± 0.9% (p<0.05) with reduction to 8.7 ± 1.4% when treated with LASSBio-1900.

In conclusion, the agonist of AR-A2, named LASSBio-1900 improved RV function and pulmonary structural alteration in PH, indicating a new alternative for treatment of PH-induced RV dysfunction.
Abstract P2044: The Cardioprotective Egr-1 And Maladaptive Sp-1 Zinc Finger Transcription Factor Reciprocally Regulates Expression Of The Cardiomyocyte Performance Enhancing Protein S100a1
Authors: Busch Martin , Bui Thien-Kim , Adrian Lukas , Guenther Fabian , Rettel Mandy , Stein Frank , Haas Jan , Klett Hagen , Boerries Melanie , Meder Benjamin , Dieterich Christoph , Katus Hugo A , Most Patrick
Published: November 2022

Background and Hypothesis:
Expression of the cardiomyocyte (CM) protein S100A1, which improves contractile performance of the heart, sharply increases during postnatal myocardial maturation but declines rapidly in failing hearts. We therefore hypothesized that CMs are wired with transcriptional factors (TFs) that positively and negatively regulate S100A1’s gene locus activity. Understanding these reciprocal circuits may be relevant for advanced therapeutic modulation of S100A1’s abundance in diseased hearts.

Methods and Results:
H9C2 rat cardiomyoblasts, an animal-free
in vitro
tool, displayed a strong concordant rise in S100A1 mRNA and protein levels (8.1+/-1.1 vs. cont.; n=9, p<0.05) amongst other CM markers (i.e., SERCA2a) over a 5-day CM differentiation protocol. Overall TF activity during this process was computationally inferred from TF binding site (TFBS) assessments in promoters of all actively regulated transcripts provided by a time-resolved (undifferentiated, day 0, 2 and 5) transcriptome analysis. From these TFs, EGR1 and SP1 amongst eight others were chosen due to their abundance both in differentiated H9C2-CMs and adult rat hearts and TFBS in the -1000 to +500 bp rat S100A1 gene promoter region due to a 5’ RACE-PCR based transcription start site identification in our model. To capture the relevant TFs from this group, we next co-incubated nuclear extracts from H9C2-CMs with biotinylated fragments of the aforementioned S100A1 promoter region coupled to streptavidin beads. S100A1 promoter-bound TFs were identified by mass spectrometry and only factors with a >2-fold enrichment over control were selected delivering EGR1 and SP1 as top hits. Subsequent siRNA-mediated silencing of both TFs yielded an EGR1 and SP1 knock-down dose-dependent inhibition (80%; p<0.05 vs. contr., n=9) and amplification (126%; p<0.05 vs. contr., n=9) of the rise both in S100A1 mRNA and protein levels in the H9C2-CM model that validated our comp./exp. pipeline and biological relevance of our TF hits, respectively.

Conclusion:
Our study identified the known cardioprotective EGR1 and maladaptive SP1 as novel positive and negative TF regulators of CM S100A1 expression and targets for therapeutic S100A1 gene locus modulation i.e., by SP1 TFBS gene-editing.
Abstract P2045: Transcriptome Versus Proteomic Discrepancy Is Associated With Structural-functional Dys-synchrony In Reductive Stress Myocardium
Authors: Sunny Sini , David Cynthia L , Parsawar Krishna , pogwizd Steven , Namakkal-Soorappan Rajasekaran N
Published: November 2022

Background:
A real-time action of Nrf2 in response to a stress is vital to preserve the redox homeostasis, but its activation under unstressed settings tilt the redox balance towards the reductive arm, leading to reductive stress (RS). In this study, we identified that >50% mismatch between mRNA and protein levels in RS myocardium undergoing structural/functional dys-synchrony.

Methods:
NGS-RNA Seq (for RNA), Tandem Mass Tagging (TMT) Mass spectrometry (for proteins) and speckle tracking strain echocardiography (STE) using Vevo2100 system was performed in the cardiac-specific constitutively active Nrf2 transgenic (CaNrf2-TG/TG) and non-transgenic (NTG) mice (n=4/group) at ~6-7 months of age.

Results:
Comparison of NGS and TMT data in TG vs. NTG hearts showed surprising results. Out of 104 commonly found gene products in NGS and proteins at a FC≥1.5, the levels of 50 proteins are not consistent with their RNA expression. The proteins that are steadily expressed according to their respective mRNA levels are classified as “transcription-sync proteins (T-SP)” and the ones that do not match are termed as “transcription-nonsync proteins. While changes in Nrf2-driven antioxidant genes (
Nqo1, Gsr, Gsta1-4, and Gstm1
) were grouped under “T-SP”, other transcripts including
Atp5pb, Ces1d, Cmbl, Cox7a1, Cpt1b, Fah and Gstk1
did not quantitatively match with their translated products in TG hearts. Further, STE demonstrated an impaired wall motion (diastolic and systolic velocity), increased fractional shortening (20% vs 50%; p<0.0001), decreased global longitudinal strain (-10% vs -25%, p<0.0002), increased IVRT (6.46 vs 13.8 ms; p<0.0001) and IVCT (8.06 vs 12.5 ms; p<0.0005) in TG vs. NTG mice. Changes in the systolic and diastolic functions with structural abnormalities demonstrate cardiac dys-synchronicity in TG hearts with chronic RS exhibiting impaired transcriptome and proteome profiles.

Conclusions:
Our comparative analysis of RNA and protein in RS hearts demonstrated that 50% of the proteins do not match with the quantitative changes of their transcripts. We highlight that the RS could be a novel mechanism for disproportionate transcription vs translation, which leads to structural and functional dys-synchrony in the TG hearts.
Abstract P2048: Matricellular Protein Cilp1 Promotes Myocardial Fibrosis In Response To Myocardial Infarction
Authors: Zhang Qingjun , He Yu , Li Yongnan , Liao Jun , Zang Qun S , Liu Zhiping
Published: November 2022

Rationale:
Cartilage intermediate layer protein 1 (Cilp1) is a secreted extracellular matrix (ECM) protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive.

Objective:
To establish the function and mechanism of action of Cilp1 in post-myocardial infarction (MI) cardiac remodeling.

Methods and Results:
We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of heart failure patients. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with N-terminal half of the protein (NCilp1) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared to wild-type (WT) littermates. Conversely, NCilp1-Tg mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 signaling were downregulated in KO hearts compared to WT hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and ECM gene expression analysis indicated that while MI caused significant stiffness in WT hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway.

Conclusions:
Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.
Abstract P2049: Myofibroblast Depletion Of Yap And Wwtr1 Improves Cardiac Function After Myocardial Infarction Via Ccn3 Suppression
Authors: Flinn Michael A , Alvarez Santiago , Knas Makenna C , Alencar Victor , Paddock Samantha , Zhou Xiaoxu , Buddell Tyler , Jamal Ayana , Liu Pengyuan , Drnevich Jenny , Patterson Michaela , Link Brian , Omeara Caitlin
Published: November 2022

The Hippo-Yap pathway is an area of interest in cardiac fibroblasts, as inhibition Lats1/2 promotes substantial fibrosis and Yap/Wwtr1 facilitate maturation of fibroblasts to myofibroblasts. While the Hippo-Yap pathway has been characterized in resting and activated fibroblasts, we take the novel approach of investigating its role in myofibroblasts. We assess depletion of Yap alone (

Yap
fl/fl
;Postn
MCM

) or Yap and Wwtr1 (

Yap
fl/fl
;Wwtr1
fl/+
;Postn
MCM

) in myofibroblasts immediately following myocardial infarction and focus on identifying and validating downstream factors mediating pathological remodeling in mice. Following injury, depletion of Yap alone had no effect on heart function, but depletion of Yap and Wwtr1 resulted in improved ejection fraction and fractional shortening at 60 days post injury.

Yap
fl/fl

,

Wwtr1
fl/+
;Postn
MCM

hearts also displayed smaller scars, reduced interstitial fibrosis, and more denatured collagen. Single cell RNA sequencing of interstitial cells 7 days post injury showed suppression of pro-fibrotic and pro-inflammatory genes and suppression of a matrifibrocyte phenotype in fibroblasts derived from the

Yap
fl/fl

,

Wwtr1
fl/+
;Postn
MCM

heart. Analysis of
in vivo
and
in vitro
transcriptomics revealed depletion of Yap/Wwtr1 resulted in dramatically decreased fibroblast expression of the matricellular protein Ccn3. Administration of recombinant CCN3 to mice following injury aggravated cardiac function and scarring over the course of 28 days. Thus, we show Yap/Wwtr1 depletion in myofibroblasts attenuates fibrosis and significantly improves cardiac outcomes after injury and identify Ccn3 as a novel factor that contributes to adverse cardiac function. Given therapeutic strategies would likely be implemented after myocardial infarction when myofibroblasts are already activated, our work suggests targeting Yap and Wwtr1, or Ccn3 directly as an approach for suppressing pathological remodeling.
Abstract P2060: Comprehensive Analysis Of 3’ End Sequencing Reveals Novel Polyadenylation Signals In Human Left Ventricle Failure
Authors: Neupane Rahul , Yalamanchili Hari Krishna , Youker Keith , Wagner Eric J , Bhimaraj Arvind , Karmouty-Quintana Harry , Guha Ashrith , Amirthalingam Thandavarayan Rajarajan
Published: November 2022

Background:
Alternative polyadenylation (APA) regulates the genes post-transcriptionally and generates mRNA isoforms with different lengths of 3’ untranslated regions (3’UTR). APA dictates diverse cellular processes such as development, differentiation, metabolism, and protein localization. The dysregulation of APA leading to global 3’UTR changes has been well studied in several human diseases including cancer. However, the role of APA in the progression of cardiac fibrosis and eventually the left ventricle (LV) heart failure in patients remains elusive.

Objective:
To identify whether APA regulates the 3’UTR length in the failing LV hearts compared to the healthy hearts.

Methods and Results:
We used Poly(A)-Click-Seq to sequence the 3’ end of the transcripts and the PolyA-miner algorithm to comprehensively quantify the APA events in healthy and failing human LV specimens. We used the Poly-A index, a metric that determines the magnitude and position of the 3’UTR changes, to determine the shortening versus lengthening of 3′UTRs. Based on these scores, we identified more than 1000 genes with a significant shift in cleavage site usage in failing LV hearts compared to healthy. Moreover, the enrichment of these differentially regulated APA candidates revealed pathways involved in the progression of heart failure and molecular functions related to mRNA binding. So, by examining the differential polyadenylation events in these hearts, we identified disease-specific APA signatures. In addition, we also found the dysregulation in the core APA machinery proteins such as cleavage factor Im (CFIm) 25, 59, and 68 in the LV failing hearts compared to the healthy.

Conclusion:
Hence, we demonstrated that the regulation of the 3’UTR length through APA provides an extra layer of gene expression in human LV failure. Our results provide genome-wide polyadenylation maps of the human heart and show that APA of mRNA is dynamic in the progression of LV failure.
Abstract P2065: Ischemic Injury Aggravated Muscle-specific Mir-499-5p-impaired Angiogenic Property Of Endothelial Cell In Hindlimb Of Diabetic Db/db Mice: Role Of Small Extracellular Vesicles
Authors: Cheng Zhongjian A , Mallaredy Vandana , TRUONGCAO MAY , Wang Chunlin , Gonzalez Carolina , Cimini Maria , Huang Grace , Lucckese Anna Maria , Ibetti Jessica , Benedict Cindy , Rajan Sudarsan , Garikipati Venkata , Verma Suresh , Koch Walter J , Kishore Raj
Published: November 2022

Background:
Recently, skeletal muscle cells (SKMCs) have been reported to be critical for regulation of EC function in limbs. miR-499, a muscle specific microRNA (miR), was found to be modulated in diabetes and ischemic injury. Here, we studied the role of miR-499 in EC dysfunction in diabetic ischemic limb injury.

Methods:
ECs and SKMCs were isolated from ischemic or non-ischemic hindlimb (IHL) of male db/+ and db/db mice. Serum- and SKMC-derived small extracellular vesicles/exosomes (EV/Exo) were isolated by gradient ultracentrifugation. Ischemic hindlimb (IHL) surgery was conducted by unilateral ligation of formal artery.

Results:
miR-499-5p level was increased in SKMCs and ECs of db/db mice which was synergistically increased by ischemic injury. Overexpression of miR-499-5p impaired tube formation and migratory activity of ECs. Intramuscular injection of anti-miR-499-5p lentivirus improved blood prefusion and neovascularization in IHL of db/db mice. Mechanistically, miR-499-5p level was enhanced in serum- and SKMC-derived EV/Exo from db/db mice which was synergistically increased by ischemic injury. Diabetic SKMC-EV/Exo impaired blood perfusion in wildtype mice. Anti-miR-499-5p rescued diabetic SKMC-EV/Exo-impaired EC function. Co-culture of diabetic SKMCs with wildtype ECs increased miR-499-5p expression in ECs which was inhibited by EV/Exo inhibitor GW4869. Sex-determining region Y-box 6 (SOX6), the most attractive gene targeted by miR-499-5p, was decreased in ECs from db/db mice which was synergistically reduced by ischemic injury. SOX6 siRNA impaired pro-angiogenic factor secretion and function of ECs. Anti-miR-499-5p significantly enhanced SOX6 level in SKMs from IHL of db/db mice. Finally, overexpression of SOX6 by transduction of lentivirus improved EC function of db/db mice.

Conclusions:
Enhanced miR-499-5p expression in ECs of SKMs from hindlimb of db/db mice is synergistically increased by ischemic injury. EV/Exo transfer miR-499-5p from SKMCs to ECs. miR-499-5p impairs angiogenic property of EC via, at least partially, SOX6/proangiogenic factors axis. miR-499-5p may be a novel target for treatment of critical limb ischemia in diabetic patients.