bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 210: Transcriptomic Changes During Induced Pluripotent Stem Cell-derived Neural Crest Cell Differentiation Highlight Genes Involved in Endocardial Cushion and Cardiac Outflow Tract Development
Authors: Jang Min Young , Pereira Alexandre C , McKean David M , Gorham Joshua M , DeLaughter Daniel M , Agarwal Radhika , Sharma Arun , Ward Tarsha L , Reichart Daniel T , Seidman Christine E , Seidman J. G
Published: October 2019

Rationale:
Neural crest cells (NCCs) play a critical role in normal cardiac development, and defects in NCCs likely cause congenital heart disease (CHD). NCCs are transient and multipotent migratory stem cells that give rise to diverse tissues, including cardiac structures such as the smooth muscles of the great arteries and semilunar valves. Induced pluripotent stem cells (iPSC) can be differentiated into NCCs, as demonstrated by expression of several marker proteins including NGFR and HNK1. To better define iPSC-NCCs and to better understand the progression of iPSCs to NCCs, we have compared the transcriptomes of iPSC and iPSC-NCCs. We have also begun to investigate the consequences of loss-of-function mutations in genes implicated in CHD on the differentiation of iPSC-NCCs.

Methods and Results:
PGP1 iPSCs were differentiated to NCCs and RNA was collected at 0, 5, 10, and 15 days of differentiation. RNAseq analysis showed that by day 15, 6483 genes were upregulated in NCC vs iPSCs, 6406 downregulated, and 6715 unchanged (FDR 5%). Enrichment analysis for the top 500 upregulated genes showed 4 cardiac gene ontology (GO) terms in the top 10, including ‘endocardial cushion morphogenesis’ (fold enrichment 11.85, p = 0.012). Notably, of 45 genes under GO term ‘endocardial cushion development’, 38 (84%) differentially expressed in NCCs by day 15 (FDR 5%). Next, we compared this RNAseq data with that of iPSC-derived cardiomyocyte (CM) differentiation in a subset of 253 genes previously implicated in CHD. Of these, 143 genes were differentially expressed in both iPSC-CMs and iPSC-NCCs. However, 27 genes (10.6%) including
MYH6, PITX2,
and
TBX5
were uniquely upregulated during CM differentiation, while 65 genes (25.7%) including
CHD4
and
NOTCH1
were uniquely upregulated during NCC differentiation.

Conclusion:
Transcriptomic changes during iPSC-NCC differentiation, assessed by both bulk and single cell RNAseq, reveal an upregulation of genes involved in cardiac development, particularly endocardial cushions and the outflow tract. Importantly, a subset of genes implicated in CHD are altered during iPSC-NCC differentiation but not in iPSC-CM differentiation. Thus, iPSC-NCCs offer a new model with which to investigate the pathogenesis and mechanisms of CHD.
Abstract 211: Substrate Elasticity Impacts Duchenne Muscular Dystrophy Cardiomyopathy Progression
Authors: Pardon Gaspard , Chang Alex C , Pruitt Beth L , Blau Helen M
Published: October 2019

Duchenne Muscular Dystrophy (DMD) is a X-link disease affecting ~1:3500 boys per year and culminating with heart failure in early adulthood. DMD results from >200 possible genetic mutations on dystrophin. The lack of dystrophin disrupts the anchoring of the cell sarcomere to the extracellular matrix (ECM), affecting the cardiac contraction. With disease progression, in an attempt to mitigate the subcellular defects, the tissue stiffness and ECM composition remodel in association with a dilated cardiomyopathy phenotype and fibrosis. Our hypothesis is that disease progression is accelerating because of this remodelling, through a positive feedback loop involving multiple mechanosensing pathways. Here, we use a single-cell assay platform to model the effect of fibrotic remodelling in DMD. This platform allows measuring the force production of single human-derived pluripotent stem cell (hiPSC) cardiomyocytes (CMs). The substrate stiffness can be controlled to match that of healthy (~10kPa) or fibrotic (~35kPa) tissue. In addition, single iPSC-CMs are patterned in an elongated 1:7 aspect ratio using microcontact printing of ECM protein. This enhances their intracellular structural maturity towards a more mature adult phenotype. This renders our in-vitro model more representative of the human pathology and greatly improves our measurements standardization. Our results show that single iPSC-CMs with DMD mutations have a dramatically reduced ability to produce force on stiffer substrates compared to their isogenic control. This loss of contractile function correlates with an increase in reactive oxygen species (ROS) and mitochondria dysfunction, as well as with other markers of stress response and cellular senescence.We are asking how the remodeling of the ECM stiffness and composition is signaled from the outside-in and affects the progression of the disease phenotype. The further development of our platform and approach will allow for more accurate in-vitro modeling of cardiac diseases and greatly increase our understanding of the underlying biophysics of mechanosensing.
Abstract 220: Gut Microbiota Alterations Associate With T Cell Activation and Adverse Cardiac Remodeling in Response to Cardiac Pressure Overload
Authors: Carrillo-salinas Francisco J , Ngwenyama Njabulo , Anastasiou Marina , Kaur Kuljeet , Aronovitz Mark , Alcaide Pilar
Published: October 2019

Background:
The complex syndrome of heart failure (HF), a leading cause of morbidity and mortality affecting more than 24 million people worldwide, is recently becoming associated with alterations in gut microbiota, also known as gut dysbiosis, as well as with T cell mediated systemic inflammation and T cell infiltration in the heart. Specifically, certain bacteria promote T cell activation, and T cell activation contributes to adverse cardiac remodeling and cardiac dysfunction in HF. We
hypothesize
that gut dysbiosis modulates cardiac remodeling and function in a T cell-dependent manner.

Results:
C57/BL6 mice were orally treated with a well-stablished wide spectrum antibiotic cocktail (ABX) and subjected to transverse aortic constriction (TAC), a model of non-ischemic HF. ABX was present during the duration of TAC (4 weeks), thus sterilizing the gut completely, or during 3 weeks before TAC followed by removal during the 4 weeks of TAC duration for spontaneous recovery of the gut microbiota (TAC-REC). The lack of gut microbiota showed a reduced T cell activation in the mediastinal lymph nodes and heart T cell infiltration, as well as cardiac fibrosis and cardiac hypertrophy were prevented in response to TAC. Nevertheless, gut dysbiosis induced by bacterial recolonization only partially recovered this phenotype. In addition, we sequenced 16S RNA to study the changes in bacterial populations in Sham, TAC, and TAC-REC. TAC induced changes in the gut microbiota compared to Sham surgery, which resulted in a higher relative abundance of several genera, such as Bacteroides, and lower relative abundance of Coprococcus, Lactobacillus and Bifidobacterium, these last two normally associated to an anti-inflammatory response. At the end of the experiment, bacterial recolonization in TAC-REC mice showed different relative bacterial abundance between TAC and TAC-REC, although there were no significant changes in diversity at 4 weeks post-TAC.

Conclusion:
Collectively, our findings suggest that changes in gut microbiota constitution modify T cell activation and migration to the heart in the TAC model. Exploring the role of gut microbiota in modulating adverse cardiac remodeling and cardiac function can become an optimal therapeutic target to treat HF patients.
Abstract 221: IKKe Deficiency Aggravates Cardiac Inflammation with Dysregulation of p52 and p38 in Myocardial Infarction
Authors: Cho Hyang Hee , Kim Yong Sook , Jun Ju Hee , Cho Dong Im , Cho Meeyoung , Ahn Youngkeun
Published: October 2019

Inhibitor of NF-κB kinase (IKK), an upstream of nuclear factor-kappa B (NF-κB), is a critical modulator for pathophysiological inflammation. IKKε is a non-classical IKK and has been studied in infectious diseases and cancers. However, the role of IKKε in a myocardial infarction (MI) has not been addressed. In this study, we used IKKε knockout mice to induce MI by coronary artery ligation. Cardiac function was analyzed by echocardiograph after 2 weeks, and fractional shortening (FS) was 16.36±4.46% in the wild type group and 13.47±1.21% in the knockout group. Cardiac fibrosis and macrophage infiltration deteriorated in the knockout group. Next, we investigated the inflammatory responses and found that the expression of inducible nitric oxide synthase (iNOS), an inflammatory marker, was much higher in both infarcted heart tissues and bone marrow-derived macrophages (BMDM) isolated from knockout mice than from wild type mice. Besides, cardiac macrophages displayed more inflammatory phenotype in the IKKε knockout group than in the wild type group. To explore the responsible mediator, we performed phosphorylated protein array and found phosphorylated p38 was significantly downregulated in the IKKε knockout BMDM. Conversely, both knockdown of p38 by siRNA and inhibition of p38 by SB203580 treatment in RAW264.7 cells upregulated iNOS induction. In the infarcted heart tissue, non-canonical NF-κB2 (p52) protein was dramatically upregulated in the IKKε knockout group than in the wild type group, while mRNA level was not different in the both groups. Immunohistochemical analysis showed nuclear accumulation of p52 in cardiomyocytes and fibroblasts in the peri-infarct lesion. Our data showed excessive inflammation in IKKε knockout mice was associated with inactivation of p38 in macrophages and upregulated p52 in the infarcted myocardium. Collectively, IKKε is involved in the control of inflammation resolution through modulating p38 activity and p52 post-translational modification in the infarcted myocardium.
Abstract 222: Single Cell Analysis of the Emergency Hematopoietic Response to Myocardial Infarction
Authors: King Kevin R , Calcagno David M , Ng Richard P , Toomu Avi , Huang Kenneth , Taghdiri Nika , Aguirre Aaron D , Fu Zhenxing
Published: October 2019

Myocardial infarction (MI) is the initiating event in ischemic heart disease, the most common cause of death in the world. Although MI-induced injury is sterile, it nevertheless elicits a vigorous emergency hematopoietic response in the bone marrow that supplies abundant myeloid cells to the heart, which remove dying cell debris and orchestrate healing, repair, and fibrosis. Ensemble methods such as flow sorting of immunostained cells and qPCR have provided some insights into infarct leukocyte phenotypes using candidate markers/genes, but the full diversity remains unknown. Here, we used single cell RNA-Seq to perform genome-wide transcriptomic profiling of >50,000 single cells from the hearts, blood, and bone marrow of infarcted and non-infarcted mice on days 1-4 after MI. We defined the diversification of myeloid cells, from their granulocytic and monocytic origins in the bone marrow, through the blood, and into the infarcted heart. This allowed construction of an atlas of MI-induced myeloid diversification and trafficking. Among the many observations enabled by this data were the origins of the type I interferon response. We recently discovered that MI induces a type I interferon response that can be targeted genetically or pharmacologically for therapeutic benefit. Our single cell data now reveal that interferon induced cells (IFNICs) derive from both neutrophilic and monocytic origins as early as 24 hours after MI. These cells are not only found within the heart, but can also be identified in the post-MI blood and bone marrow. Ongoing studies are investigating whether IFNICs can be detected in human blood after MI. Our results have clinical importance for understanding and modulating the immune response to myocardial injury. We show, using single cell transcriptomics, that post-MI treatment with anti-interferon alpha receptor antibody (IFNAR Ab) (the murine equivalent of a therapeutic antibody currently in Phase 3 clinical trials for lupus), completely abolishes MI-induced interferon signaling and shifts the intracardiac macrophage program towards a reparative posture. Our comprehensive atlas of the emergency hematopoietic response to MI can serve as a resource for others studying the inflammation in ischemic heart disease.
Abstract 227: Intravital Multiphoton Microscopy Reveals Increased Capillary Patrolling by Leukocytes and Cardiomyocyte Dysfunction in High Fat Diet Induced Hypertrophy
Authors: Small David M , Allan-Rahill Nathaniel H , Lamont Michael R , Djakpa Salomon , Jhala Marvarakumari G , Zhu Yvette , Nishimura Nozomi
Published: October 2019

Background:
The study of functional cardiomyocyte adaptation and inflammatory cell behavior at the micro-scale in vivo has been challenging due to limited imaging tools. We recently developed intravital multiphoton microscopy (MPM) methods that enable visualization and quantification of cardiac dynamics at a cell-scale throughout the cardiac cycle. We aimed to determine the dynamic cellular changes that occur due to high fat diet (HFD) induced hypertrophy using intravital cardiac MPM.

Methods:
ApoE
-/-
C57Bl6 mice started a HFD at 6 weeks of age (ApoE
-/-
-HFD, n=11), while age-matched wild-type mice (WT-ND, n=10) were fed a normal chow diet. At 26-weeks, mice were assessed by cardiac echocardiography and intravital MPM in the intact beating heart. Intravenous injections of rhodamine-6G (R6g) labeled cardiomyocytes and leukocytes, and Texas-Red dextran labeled vasculature. 3D volumes were reconstructed throughout the cardiac cycle to quantify cell motion using automated algorithms.

Results:
ApoE
-/-
-HFD hearts underwent hypertrophy compared to WT-ND with increased heart weight-to-tibial length ratio (10±0.8 vs 13±1.1) and left ventricle wall thickness (1.07±0.03 mm vs 1.13±0.06 mm, respectively, p<0.05 for both) while ejection fraction remained similar (66±3 % vs 59±3 %). In vivo MPM demonstrated that cells move a greater total distance in each cardiac cycle in ApoE
-/-
-HFD vs WT-ND. Maximum displacement in the apex-base and anterior-posterior directions increased by 46 % in ApoE
-/-
-HFD compared to WT-ND (30 μm vs 14 μm). R6g+ leukocytes were visible moving in capillaries. The incidence of patrolling behavior (defined as slowing moving cells, visible for longer than one heart beat) increased in capillaries of ApoE
-/-
-HFD compared to WT-ND (3.4±0.5/min vs 0.12±0.1/min, p<0.01).

Conclusion:
These results suggest that hypertrophied cardiomyocytes increase myocardial displacement, and increased leukocyte patrolling behavior is associated with HFD induced cardiac hypertrophy. Intravital cardiac MPM provides a novel perspective to study HFD induced cardiac hypertrophy by capturing the simultaneous contributions of inflammatory cells and myocyte function in the beating heart.
Abstract 230: Disturbed Blood Flow-induced Platelet Transmigration Via C-type Lectin-like Receptor-2 Modulating Vascular Inflammation
Authors: Tang Chaojun , Sheng Yulan , You Tao , Wang Lei , Xie Zhanli , McDaniel Michael , Xia Lijun , Zhu Li
Published: October 2019

Atherosclerosis preferentially develops at sites under disturbed blood flow (d-flow) that leads to steep multidirectional gradients of wall shear stress at vascular predilection sites, promoting an oxidative, proapoptotic, and proinflammatory gene expression profile in endothelial cells. Sustained d-flow causes endothelial injury characterized by leukocyte infiltration, endothelial barrier dysfunction, and eventually the formation of atherosclerotic plaques. While previous studies suggest a proinflammatory role of platelets via promoting leukocyte activation in atherogenesis, whether and how platelets are regulated by d-flow remains largely unclear. In this study, we showed that d-flow induced by partial carotid ligation (PCL) causes platelet adhesion, endothelial damage and leukocyte recruitment in mice. Unexpectedly, PCL induces platelet transmigration into the subendothelial space. Transmigrated platelets at 2 days of PCL appeared inside the intercellular crevices beneath the damaged endothelium. After sustained d-flow (5-7 days after PCL), more adherent leukocytes and platelets with pseudopodia were observed in the seriously damaged endothelium. In investigating the mechanism of this unexpected observation, we found that mice with platelet-specific deficiency of C-type lectin-like receptor-2 (CLEC-2) showed ~80% reduction in platelet endothelial accumulation and no subendothelial localization of platelets under d-flow, indicating an essential role of CLEC-2 in mediating platelet transmigration. Enhanced platelet transmigration exacerbated monocyte accumulation, while leukocyte depletion or PSGL-1 deletion did not affect platelet transmigration under d-flow. Our results revealed unprecedented platelet transmigration mediated by CLEC-2 in the rheological regulation of vascular homeostasis, controlling vascular inflammation.
Abstract 235: A Micropeptide Regulator of Voltage-gated Potassium Channels Controls Cardiac Rhythm
Authors: Anderson Kelly M , Parks Xiaorong , Poosala Pornthida , Lindley Sean , Lopes Coeli , Anderson Douglas M
Published: October 2019

Voltage-gated potassium (K
+
) channels (VGKCs) are essential regulators of the cardiac action potential and are required to maintain normal heart rhythm and contractility. Voltage-dependent activation, conductance and membrane trafficking of K
+
channels are tightly controlled by a family of single transmembrane regulatory cofactors (KCNE1-5), which co-assemble with pore forming K
+
channels in cell membranes. Human mutations in voltage-gated K
+
channels and their regulatory subunits are associated with Long QT syndrome (LQTS), a condition in which the repolarization of the heart is delayed and can lead to arrhythmias, syncope and sudden death. Here we identify a cardiac-enriched transmembrane micropeptide encoded by a small open reading frame, which we named KCNEmini, due to its sequence and structural homology with members of the KCNE family of VGKC regulators. We show that KCNEmini directly binds to and co-localizes with VGKCs in the plasma membrane and functions as a novel regulator of K
+
channel kinetics. Disruption of KCNEmini in mice resulted in QT interval prolongation and cardiac dysfunction. These studies shed light on a previously unrecognized regulator of K
+
handling in the heart, which may be an important future therapeutic target for the early diagnosis and treatment of human lethal cardiac arrhythmias.
Abstract 237: Identifying the Transcriptome Signature of Calcium Channel Blocker in Human iPSC-Derived Cardiomyocytes
Authors: Lam Chi Keung , Tian Lei , Belbachir Nadjet , Wnorowski Alexa , Shrestha Rajani , Ma Ning , Kitani Tomoya , Rhee June W , Wu Joseph C
Published: October 2019

Calcium channel blockers (CCBs) are important in treating cardiovascular diseases and the acute pharmacological actions of CCBs in the hearts have been extensively studied. However, we lack the knowledge of the drug-specific effect on human cardiomyocyte transcriptome and potential physiological change after long-term exposure as patients are usually prescribed with these medications for their lifetime after diagnosis. Thus, we aimed to simulate chronic CCB treatment in human cardiomyocytes and subsequently examine both the functional and transcriptomic alterations. We differentiated cardiomyocytes from three human induced pluripotent stem cell (iPSC) lines and exposed them to four different CCBs—nifedipine, amlodipine, diltiazem, and verapamil—at their physiological serum concentrations for two weeks. Without inducing cell death and damage to myofilament structure, CCBs elicited line specific inhibition on calcium kinetics and contractility. While all four CCBs exerted comparable inhibition on calcium kinetics, verapamil applied the strongest inhibition on cardiomyocyte contractile function. By examining cardiomyocyte transcriptome after treatment, we identified little overlap in their transcriptome signatures. Verapamil is the only inhibitor that reduced the expression of contraction related genes, such as myosin heavy chain and troponin I, consistent with its depressive effects on contractile function. Moreover, the alterations in these gene may help explain how HCM patients respond to verapamil in relieving outflow tract obstruction. In conclusion, we identified the distinct transcriptome signatures of different CCBs in human cardiomyocytes, suggesting that although the four inhibitors act on the same target, they may have distinct effects on normal cardiac cell physiology. The application of iPSC platform and transcriptomic findings may allow us to identify responders to verapamil treatment.
Abstract 239: Selective Right-sided Electrical Remodeling In A Pure Model Of Pulmonary Hypertension Promotes Micro-reentrant Arrhythmias
Authors: Strauss Benjamin , Obus Emerson , Bedoya Nestor , Sassi Yassine , Akar Fadi G
Published: October 2019

Background:
Pulmonary arterial hypertension (PAH) is characterized by elevated mean PA pressures that increase right ventricular (RV) afterload, leading to RV failure. Whereas structural and mechanical decompensation in PAH are well established, the extent of electrophysiological (EP) remodeling and arrhythmia propensity are far less clear. This is, in large part, because PAH-related EP properties have been exclusively studied in the monocrotaline (MCT) model, which is confounded by major extra-pulmonary toxicities, including liver failure and direct myocardial damage. To address this knowledge gap, we performed a comprehensive evaluation of the EP substrate, including arrhythmia dynamics, in a new pure model of PAH.

Methods:
Sprague Dawley rats underwent surgical left pneumonectomy followed by injection of the VEGF inhibitor Sugen 5416 (S/P), or underwent no intervention (CTRL). 5 wks later, rats were euthanized for high-resolution optical action potential (AP) mapping studies in ex vivo perfused hearts.

Results:
6/6 S/P vs 0/5 CTRL hearts were prone to pacing-induced VT (P<.05). Underlying this differential susceptibility was disproportionate RV sided prolongation of AP duration in S/P. This, in turn, promoted the formation of right-sided AP alternans at physiological rates in S/P but not CTRL. Correspondingly, KCND2 mRNA transcripts were downregulated by 80X in the RV of S/P relative to CTRL. While propagation was impaired at all rates in S/P, the most profound decrease in conduction velocity (53%) was always observed immediately prior to VT initiation. Nav1.5 and Cx43 mRNA expression were not altered in S/P. In contrast Col1a1, Col3a1, and fibronectin transcripts were upregulated implicating fibrosis in impaired conduction. The average wavelength was significantly decreased in S/P relative to CTRL (P<.05). Once generated, VT was sustained by multiple microreentrant circuits likely facilitated by the short wavelengths.

Conclusion:
In this pure model of PAH, we document highly selective RV-sided EP remodeling that facilitates the initiation of multi-wavelet micro-reentry underlying VT. The S/P model represents a severe form of PAH that allows the study of EP properties without the confounding influence of extra-pulmonary toxicity.
Abstract 246: Applying a Quantitative, Cell Surface Glycoproteomic Approach to Understanding the Role of Human Cardiac Fibroblasts in Advanced Heart Failure
Authors: Berg Luecke Linda , Buchberger Amanda R , Waas Matthew , Mahr Claudius , Gundry Rebekah L
Published: October 2019

Heart Failure (HF) is a complex clinical syndrome defined by the heart’s inability to adequately perfuse the human body. The clinical progression of HF has been linked to the pathological activation of CF, which contributes to adverse remodeling and cardiac fibrosis. Previous studies have shown that unloading the failing heart
via
mechanical circulatory support devices contributes to reverse remodeling, improving both cardiac structure and function. However, despite the importance of CF in biology and disease, little is known regarding CF-specific mechanisms and functions during cardiac remodeling and reverse remodeling. Moreover, most human studies have relied on
in vitro
culture systems to investigate CF function, despite the fact that extended culturing leads to phenotypic changes in CF. Here, we apply state-of-the-art mass spectrometry approaches to discover and quantify cell surface and extracellular matrix (ECM) proteins to 1) develop an optimized CF culture system that allows for the preservation of their
in situ
phenotype and 2) study CF isolated from failing hearts, including failing hearts treated with mechanical circulatory support to identify novel targets for tracking and studying disease. To date, we have identified >600 surface
N-
glycoproteins on primary human CF (n=12), including those with known relevance to CF (
e.g.
BGN, BSG, FBN1) and those not previously described within CF (
e.g.
HHIP, NFACS, GPNMB). In CF samples that were passaged
ex vivo
, cell surface and ECM
N-
glycoproteins that are associated with a phenotype switch (
e.g.
COL5A1, ECM1, FBLN5, LAMB2) were detected and changes in their abundance correlated with increased expression of α-SMA. Our studies also identified cell surface and ECM proteins that have not been previously associated with the heart and those with known relevance to HF, cardiac remodeling and fibrosis (
e.g.
BGN, GLUT8, ITGA11). Current efforts are underway to quantify surface proteins of interest to determine how their abundance changes during reverse remodeling in the context of HF. Overall, this work allowed for the development of an optimized CF culture system and revealed new cell-type specific molecular targets to study and track reverse remodeling in the context of advanced HF.
Abstract 248: Microfibrillar-Associated Protein 4 Regulates Maladaptive Cardiac Remodeling
Authors: Dorn Lisa E , Lawrence William , Petrosino Jennifer M , Xu Xianyao , Hund Thomas J , Schlosser Anders , Sorensen Grith L , Accornero Federica
Published: October 2019

Cardiac hypertrophy is a well-known risk factor for heart failure. Cardiomyocytes are often considered the ultimate culprit of cardiac remodeling following stress, despite comprising less than half of the heart’s total cells. Recent work has begun to recognize the importance of non-myocyte populations in the heart, yet how signaling between these and cardiomyocytes can dictate cardiac remodeling is still largely unknown. We have found that Microfibrillar-associated protein 4 (MFAP4) is a novel mediator of intercellular communication in the heart, and is specifically required for cardiac adaptation to stress. MFAP4 is an extracellular matrix protein that has been previously shown as a necessary component of arterial remodeling following injury, but its role in cardiac hypertrophy has not been explored. Our data revealed that, in the heart, MFAP4 is selectively secreted by vascular smooth muscle cells and endothelial cells in response to transforming growth factor β (TGFβ) activation. To determine the role of MFAP4 in cardiac hypertrophy, we subjected MFAP4-deficient mice to two forms of stress: chronic pressure overload or 1 week administration of the pro-fibrotic agent Angiotensin II (AngII). MFAP4-deficient mice developed accelerated cardiac hypertrophy, fibrotic remodeling, and marked cardiac functional defects with pressure overload when compared to wild-type animals. Also, AngII administration was sufficient to exacerbate cardiac hypertrophy prior to marked cardiac functional decline in MFAP4-deficient mice, suggesting a primary role for MFAP4 in regulating cardiomyocyte growth. Furthermore, aging-induced cardiac stress led to left ventricular dilation and spontaneous development of functional defects in otherwise unstressed MFAP4-deficient mice. Mechanistically, MFAP4 possess an integrin-binding domain and can affect cardiac homeostasis downstream of cardiomyocyte integrins via altered signaling through the FAK-ERK cascade, well-known to dictate cardiomyocyte geometric remodeling. Overall, our study demonstrates a critical role for MFAP4 on cardiac hypertrophy and function post-injury, underscoring the importance of non-myocyte-derived factors on cardiomyocyte pathophysiology.
Abstract 256: Cardiac Fibroblast-derived Exosomes Mediate Endothelial Dysfunction and Heart Failure
Authors: Ranjan Prabhat , Kumari Rajesh , Krishnamurthy Prasanna , Kishore Raj , Verma Suresh K
Published: October 2019

Objective:
Endothelial cells (ECs) play critical role to maintain the normal heart function. Others and we have previously shown that stress-induced chronic inflammation mediates ECs dysfunction and restoration of IL10 (an anti-inflammatory cytokine) preserve ECs function, but, its mechanism is not clearly known. Here we hypothesized that activated cardiac fibroblasts-derived exosomes (FB-Exo) mediates cardiac ECs dysfunction and leads for cardiac pathology and IL10 treatment negatively regulates the FB-Exo contents and thus improve ECs function and biology.

Methods:
Human aortic endothelial cells (HAECs), human umbilical endothelial cells (HUVEC) and cardiac endothelial cells were cultured in EC growth media. Cells were treated with TGFβ/IL10/FB-exo. Exosomes were isolated from fibroblast condition media by ultracentrifugation and characterized by nanosight & electron microscopy.

Results:
TGFβ treatment significantly induced ECs dysfunction as shown by Matrigel assay, real-time Q-PCR and immunostaining data. IL10 treatment significantly restored TGFβ-induced endothelial dysfunction as evident by immunostaining and western blot (CD31, Smad2/3 and αSMA) data. Furthermore, exosomes were isolated from saline/AngII treated neonatal heart fibroblast conditioned media by ultracentrifugation and characterized. We found that exosomes-derived from Ang II/TGF treated fibroblast were enriched with TGFβ and collagen-1α. Matrigel tube formation and quantitative PCR results suggest that activated fibroblasts secrete factors which triggered endothelial dysfunction (Matrigel/qPCR). Intriguingly, Ang II treated fibroblast-derived exosomes strongly promote endothelial cell differentiation to fibroblasts like cells.

Conclusions:
Taken together, this study demonstrates that Ang II/TGFb treated fibroblasts-derived exosomes are enriched in pro-fibrotic factors and can lead to endothelial dysfunction and promotes cardiac fibrosis in PO myocardium. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 and modification of fibroblast-derived exosomal content on endothelial function and cardiac fibrosis failing heart.
Abstract 258: Elucidating Sex Differences in Stress-related Atrial Remodeling and AF Risk in a Mouse Model of Atrial Dysfunction
Authors: Bledzka Kamila M , Manaserh Iyad H , Koch Walter J , Grondolsky Jessica , Schumacher Sarah M
Published: October 2019

Atrial fibrillation (AF) is the most common arrhythmia and a major risk factor for cardiovascular mortality and stroke. Despite significant advances in AF diagnosis and management, major gender discrepancies in treatment and clinical outcomes remain. It is unclear if these are related to treatment disparities or to underlying differences in the structure and biochemical makeup of the female versus male heart. How lifestyle, diet, comorbidities and risk factors contribute to this clinical dimorphism are not well understood. My laboratory has recently developed a double-transgenic mouse line that recapitulates this gender dimorphism. These mice were generated by crossing lines with cardiac expression of an amino and carboxyl terminal fragment, βARKnt (residues 50-145) and βARKct (residues 495-689), of the prototypical G protein-coupled receptor (GPCR) kinase GRK2 (originally βARK1). This line was created to investigate the therapeutic potential of dual regulation of β-adrenergic receptor signaling independent of canonical GRK2 activity. Since preliminary data demonstrate atrial dysfunction in both genders, but structural remodeling in females only, this model presents a unique tool to investigate sex differences in the molecular and cellular mechanisms of atrial remodeling. Masson’s trichrome staining of 4-chamber paraffin sections demonstrated an increase in wall thickness in the βARKnt and βARKnt/βARKct mice in agreement with the increased heart weight. Interestingly, these data revealed a significant increase in left and right atrial size in the double transgenic female hearts only, with no apparent alteration in the male atria. Preliminary echo recordings demonstrated significant alterations in E/A ratio (ventricular relaxation/atrial contraction) in the βARKnt and even more pronounced in the βARKnt/βARKct male and female mice. These data suggest that although no remodeling is evident at baseline, the male βARKnt/βARKct mice have underlying atrial dysfunction that may be more susceptible to additional cardiovascular stress. A better understanding of gender differences in the underlying mechanisms of atrial susceptibility may facilitate the development of safer and more effective approaches for AF management and prevention.
Abstract 270: Loss of S100A1 Protein Negatively Impacts Glucose Metabolism and Energy Homeostasis in Cardiomyocytes
Authors: Abdalla Fadwa , Schneider Andrea , A.Katus Hugo , Busch Martin , Most Patrick
Published: October 2019

Introduction:
As a true metabolic omnivore, the heart is able to utilize different substrates for energy production. In the hypertrophied and early failing state, cardiac muscle leans towards glucose as a major carbon substrate to meet its energetic demand. Given that S100A1 gene therapy reverses the compromised energetic balance of the failing heart, we were interested in determining the specific role of the EF-hand calcium sensor S1001A1 in cardiomyocyte energy metabolism.

Methods and results:
To study metabolic changes in response to different carbon substrates, we used neonatal rat ventricular cardiomyocytes (NRVMs), which were subjected to electrical stimulation (2Hz) in culture and adenoviral-mediated anti-S100A1 shRNA silencing or scr-shRNA, and either fed with glucose, oleate or lactate only for 24hrs. Of note, lack of S100A1 expression only impaired energy metabolism of cardiomyocytes in response to glucose as reflected by a significantly decreased ATP content and subsequently enhanced AMP/ATP and p-AMPK/AMPK ratios with consecutive elevated p-ACC/ACC and Glut4 protein levels. In contrast, energy metabolism appeared to remain normal in response to oleate in S100A1-deprived cells. Targeted metabolomics yielded a tremendous decrease in glucose-6-phosphate levels as the first key step in glucose metabolism despite enhanced AMPK activation in glucose-fed S100A1-defiecient cardiomyocytes.

Conclusion:
Our novel in vitro data indicate that S100A1 protein may be crucial for cardiomyocytes to meet their energetic demand when glucose is the major substrate. Since the latter becomes the preferred substrate for functionally compromised heart over fatty acids, lack of S100A1 expression - as a result of fetal gene reprogramming - most likely impairs the ability of the heart to sufficiently utilize glucose to meet its energetic demand. These data shed new mechanistic light on the sustained therapeutic efficacy of S100A1 gene therapy of heart failure where S100A1 may be a key factor enabling the heart to sufficiently use glucose to reverse its compromised energetic state. Further studies answering these pressing questions are underway for an informed design of a best-case patient enrollment scenario for a first-in-men clinical study.
Abstract 272: Cardioprotective Effects of Plant-derived Fat in Low Carbohydrate Diet on the Progression of Heart Failure
Authors: Bujo Satoshi , Toko Haruhiro , Harada Mutsuo , Guo Jiaxi , Ishizuka Masato , Yanagisawa-Murakami Haruka , Komuro Issei
Published: October 2019

Background:
The association between low carbohydrate diet (LCD) and cardiovascular disease and mortality remains unknown. Results from meta-analyses have reported that cardiovascular mortality increased when the carbohydrate in LCD was replaced for animal-derived fat, whereas mortality decreased when the substitution was plant-derived fat. However, the molecular mechanism is yet to be elucidated. We aimed to assess the effects of the LCD under two conditions, LCD with animal-derived fat (LCD-A) and LCD with plant-derived fat (LCD-P), on mouse cardiac function.

Methods:
Using 10-week old male C57B/6J mice, we performed transverse aortic constriction surgery to generate a pressure overload model of heart failure and subjected them to either normal diet, LCD-A or LCD-P for 4 weeks. Cardiac function was measured by echocardiography at 4 weeks after surgery, and the extent of inflammation was assessed by immunohistological analysis. Expression levels of genes were assessed with RNA sequence.

Results:
LCD-A accelerated left ventricular dilatation and systolic dysfunction (P<0.01 vs. normal diet), whereas LCD-P ameliorated cardiac hypertrophy (P<0.01 vs. normal diet) as revealed by echocardiograpy. Immunohistological analysis demonstrated that LCD-A resulted in a significant increase in the infiltration of F4/80-positive macrophages, reflecting exacerbated inflammation (P<0.01 vs. LCD-P, P<0.01 vs. normal diet). Consistent with histological findings, inflammation-related and cell cycle-related gene expressions were upregulated only in LCD-A. On the other hand, mitochondrial fatty acid oxidation gene expression including PPARα targets such as
Cpt1a, Slc25a20, Acadvl, Hadha
and
Acaa2
were upregulated only in LCD-P (P<0.05, vs. normal diet), which may indicate the preserved energy metabolism during the pathogenic process of heart failure.

Conclusions:
These results suggest that the effects of the LCD on cardiac function vary between the sources of fat alternatives to carbohydrate intake. LCD-P induces PPARα activation, an important regulator of mitochondrial lipid metabolism, that may be therapeutically relevant for heart failure treatment. Further studies are required to unveil the precise molecular mechanism.
Abstract 273: The Role of Estrogen in Protection of Skeletal Muscle Function in Diastolic Dysfunction
Authors: Chatterjee Somik , Li Shumin , Zhang Aijun , Vedula Indira , AlRukby Judy A , Hamilton Dale J , Gupte Anisha A
Published: October 2019

Diastolic dysfunction (DD) is prevalent in elderly post-menopausal women. In addition to impaired cardiac relaxation and output, DD is associated with loss of skeletal muscle mass, muscle strength and function, and exercise capacity. Understanding the mechanisms of estrogen (E2)-mediated regulation of skeletal muscle structure and function in DD can have broader implications in understanding the progression of heart disease in post-menopausal women. In muscle, E2 regulates autophagy and mitophagy, processes that orchestrate degradation and recycling of damaged cellular components to maintain healthy tissue and conserve energy. However, this role of E2 under a physiological stress such as DD, is largely unknown. Our study aimed to understand the relationship between E2, autophagy/mitophagy and muscle function in DD. We hypothesized that estrogen receptor (ER) signaling plays a direct role in regulation of autophagy and mitophagy-mediated maintenance of muscle mass and muscle mitochondrial function in DD. Sham or ovariectomy (OVX) surgeries were performed to induce E2-deficiency, followed by induction of DD by administering hypertension agents L-NAME and angiotensin II. NMR analysis revealed significantly reduced total lean mass in OVX mice with DD, further confirmed by direct measurement of gastrocnemius muscle mass. Expression of ERα target-genes PDK4 and STAT3 were reduced in muscle of OVX mice with DD. DD significantly reduced LC3B II/I ratio in muscle, indicative of reduced autophagy. Consistent with this, expression of transcription factor FoxO3, a master-regulator of multiple autophagy and mitophagy genes, was reduced in OVX mice with DD compared to sham control mice. Expression of SDHA gene coding for mitochondrial respiratory chain subunit II, was reduced in sham and OVX mice with DD compared to sham control mice. Mitochondrial respiratory function measured from isolated muscle mitochondria, was reduced in sham and OVX mice with DD compared to sham control mice, and in OVX mice with DD compared to OVX control mice, indicating compromised skeletal muscle mitochondrial function. Our findings indicate that OVX-induced E2-deficiency and DD leads to muscle impairments, including defects in mitochondrial function.
Abstract 275: Modulation of Energy Metabolism by Metformin Prevents Diet Induced Cardiac Dysfunction in a Mouse Model of Adult Congenital Heart Disease
Authors: Pandey Raghav , Wilmanns Julia C , Hon Olivia , Rosenthal Nadia A , Furtado Milena B , Costa Mauro W
Published: October 2019

Objective:
Congenital heart disease (CHD) is the most frequent birth defect worldwide. Improved surgical and treatment interventions have led to a significant increase in the number of adult patients with CHD, now referred to as ACHD. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown.

Methods:
We used a newly characterized mouse genetic model of ACHD to investigate the consequences and the mechanisms associated with combined obesity and ACHD predisposition and metabolic intervention studies by metformin.

Results:
ACHD mice placed under metabolic stress (high fat diet) displayed decreased left ventricular ejection fraction. Comprehensive physiological, biochemical, and molecular analysis showed that ACHD hearts exhibited early changes in energy metabolism with increased glucose dependence. These changes preceded cardiac dysfunction mediated by exposure to high fat diet and were associated with increased disease severity. Restoration of metabolic balance by metformin lead to improved liver function in both control and ACHD mice and prevented the development of heart dysfunction in ACHD predisposed mice. Metabolomic analysis of these animals revealed that metformin leads to an ACHD specific increase in metabolites associated with fat acid oxidation, likely reflecting upregulation of FAO.

Conclusions:
This study reveals that early metabolic impairment reinforces heart dysfunction in ACHD predisposed individuals and diet or pharmacological interventions can be used to modulate heart function and attenuate heart failure. Our current hypothesis is that metformin treatment leads to normalization of energy use by ACHD heart by enhancing FAO and we are currently performing CRISPR/Cas9 mediated deletion of key metabolic genes to characterize their role in ACHD. This data indicates that early manipulation of energy metabolism may be an important avenue for intervention in ACHD patients to prevent or delay onset of heart failure and secondary comorbidities.
Abstract 279: Increased Drp1 Acetylation Mediates Lipid Overload-induced Cardiomyocyte Death & Heart Dysfunction
Authors: Hu Qingxun , Zhang Huiliang , Cortés Nicolás G , Wu Dan , Wang Pei , Smith Eric , Wang Mingyi , Sheu Shey-Shing , Wang Wang
Published: October 2019

Rationale:
Lipid overload can cause diabetic cardiomyopathy (DCM), which is characterized by cardiomyocyte death, myocardial remodeling, and eventually contractile dysfunction. How excessive lipid supply impacts cardiac metabolism leading to DCM remains poorly understood.

Objective:
To investigate the role and mechanism of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), in lipid overload-induced cardiomyocyte death and DCM.

Methods and Results:
Mice fed with high fat diet (HFD) for 18 weeks exhibited DCM phenotype including myocardial insulin resistance, fibrosis, hypertrophy, cardiomyocyte death and mild contractile dysfunction. Cultured adult cardiomyocytes incubated with high levels of palmitate also exhibited increased cell death and openings of the mitochondrial permeability transition pore. Mitochondrial fission was stimulated by HFD in the heart or by palmitate incubation in cultured adult cardiomyocytes. Mechanistically, lipid overload increased acetyl-CoA level and decreased NAD
+
level in the heart and in adult cardiomyocytes, leading to upregulation of Drp1 acetylation at lysine 642 (K642). Elevated Drp1 acetylation increased Drp1 protein level, its oligomerization and its phosphorylation at the activating serine 616 (S616) site. The excessively activated Drp1 translocated to mitochondria, induced fission, and bound with VDAC1 to promote cardiomyocyte death. Preventing Drp1 acetylation (K642R mutation), inhibiting Drp1 activity, or supplementing NAD
+
ameliorated palmitate-induced fission and cardiomyocyte death. In the hearts of HFD-fed monkeys, we observed increased Drp1 acetylation, oligomerization, and phosphorylation, which were accompanied with markers of DCM.

Conclusions:
These findings uncover a novel mechanism mediating lipid overload-induced heart hypertrophy and dysfunction. Excessive lipid supply created an intracellular environment that facilitated Drp1 acetylation, which increased its activity and mitochondrial translocation causing cardiomyocyte death. Thus, Drp1 could be a critical mediator for metabolic stress-induced heart dysfunction as well as a potential target for therapy.
Abstract 285: Oxidized Low Density Lipoprotein and Angiotensin II Causes Vascular Senescence Via AT1R Signal-mediate Mitochondrial Fission
Authors: Uchikado Yoshihiro , Ikeda Yoshiyuki , Sasaki Yuichi , Akasaki Yuichi , Ohishi Mitsuru
Published: October 2019

Introduction:
Metabolic stress including oxidized low density lipoprotein (ox-LDL) and angiotensin II (AngII) cause mitochondrial dysfunction and vascular senescence. Mitochondria are highly dynamic organelles that constantly change their morphology to fusion or fission. This study aims to clarify how mitochondrial dynamics are involved in the etiology of vascular senescence.

Methods:
VSMC and HUVEC were stimulated by either ox-LDL or AngII. We also conducted in vivo experiment using C57BL6 (WT), apolipoprotein E (ApoE) deficient and the double knockout of ApoE and AT1R (DKO) mice.

Results:
Either ox-LDL or AngII induced excessive mitochondrial fission though phosphorylation of Drp1 at Ser616, mitochondrial dysfunction assessed by JC1, reactive oxygen species production measured with Mito-Sox Red and Amplex assay and cellular senescence analyzed by senescence associated beta galactosidase staining and immunoblot of p53 and p21 in cells. Administration of mdivi-1, a specific inhibitor of Drp1, restored imbalance of mitochondrial dynamics and these detrimental alterations. Treatment with angiotensin II type1 receptor (AT1R) inhibitor also inactivated Drp1 and improved imbalance of mitochondrial dynamics, mitochondrial dysfunction and cellular senescence. These results suggest that ox-LDL- or AngII-induced mitochondrial dysfunction and cellular senescence were derived from Drp1-dependent mitochondrial fission. Administration of AT1R inhibitor to HUVEC or SMC with ox-LDL prevented cells against excessive mitochondrial fission, its dysfunction and cellular senescence. The degree of vascular senescence was higher, the number of fused mitochondria and mitochondrial function were lower and mitochondrial oxidative stress were higher in either C57BL6 with AngII infusion or ApoE KO mice than those of control or WT mice, respectively. Treatment with mdivi-1 to these mice reduced mitochondrial fission, oxidative stress and attenuated vascular senescence. The number of fused mitochondria and its function were higher and the degree of vascular senescence were lower in DKO than those in ApoE KO mice.

Conclusion:
Either ox-LDL or AngIIcauses cellular and vascular senescence through AT1R signal-mediated mitochondrial fission.