bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 625: Mixed Lineage Kinase 3 Regulates Blood Pressure Through Kinase Independent Effects in the Vasculature
Authors: Calamaras Timothy D , Baumgartner Robert A , Aronovitz Mark , McCarthy Joseph , Tam Kelly , Kim Seung Kyum , Martin Gregory , Richards Daniel A , Baca Paulina , Jaffe Iris Z , BLANTON Robert M
Published: October 2019

Background:
Mixed lineage kinase 3 (MLK3) opposes pathologic cardiac remodeling, but its role in blood pressure (BP) has not been studied. MLK3 activates JNK signaling through kinase-dependent effects, and opposes RhoA activation through kinase-independent mechanisms, but the relevance of these mechanisms to BP is unknown. We investigated the effect of genetic deletion of MLK3 on BP.

Methods and Results:
Using ambulatory telemetric monitoring in 3 month old male mice, MLK3 -/- mice had significant hypertension compared to wild type (WT) littermates (WT systolic BP 121 ± 2 mmHg, MLK3-/- 162 ± 5 mmHg; n=3; p<0.05). The MLK3 kinase inhibitor URMC-099 (10 mg/kg IP) did not affect BP in WT mice. By contrast, inhibition of downstream RhoA dependent Kinase (ROCK) with Y-27632 (15 mg/kg) fully normalized BP in MLK3 -/- mice (SBP WT baseline 126 ± 2 mmHg; WT ROCK inhibitor 94 ± 2 mmHg; MLK-/- baseline 163 ±6 mmHg; MLK3 -/- ROCK inhibitor 94 ± 12 mmHg; n=5 WT, 9 MLK3-/-). Aortic pulse wave velocity was elevated in MLK-/- mice (2.7 ± 0.1 mm/ms WT vs 3.6 ± 0.2 mm/ms MLK3-/-; p<0.05) indicating increased aortic stiffness. Pressure myography in mesenteric resistance arterioles of MLK3 -/- mice revealed reduced distensibility compared to WT. Both pressure myography and direct histological measurement in resistance arterioles demonstrated reduced passive luminal diameter but preserved wall cross sectional area in MLK3-/- arterioles, indicating eutrophic remodeling. Compared with dispersed aortic smooth muscle cells from WT littermates, MLK3 -/- cells had increased actin stress fiber accumulation and cell area.

Summary and Conclusions:
These data demonstrate that MLK3 deletion leads to hypertension with increased arterial stiffness, reduced distensibility and eutrophic remodeling of resistance vessels, but retained BP responsiveness to downstream ROCK inhibition. Together with previous work, these findings support that MLK3 modulates cardiac remodeling through a kinase dependent mechanism while modulating blood pressure through kinase-independent effects. Because hypotension limits many heart failure therapies, delineating vascular versus cardiac mechanisms of MLK3 signaling has the potential to suggest novel approaches to heart failure treatment.
Abstract 627: Primary Cilia of the Cardiac Neural Crest Orchestrate Critical Aspects of Ventricular Maturation and Postnatal Cardiac Function: A Role for Hedgehog Signaling
Authors: Fitzsimons Lindsey A , Moran Adrian M , Tucker Kerry L
Published: October 2019

Primary cilia are tiny, plasma-membrane bound organelles that function to modulate intra- and extracellular signaling, in particular Hedgehog signaling, implicated in both normal development as well as in various disease pathologies, including congenital heart disease (CHD). Cardiac neural crest cells (CNCC) display primary cilia and function as a major progenitor cell population contributing to the developing heart. We hypothesized that damage to or loss of primary cilia in CNCC would impair normal heart development, leading to CHD. We present 1) a precise description of CHD’s resulting from loss of primary cilia of CNCC
in vivo
; 2) characterization of the functional, electrophysiological aspects of the perinatal phenotype; and 3) a preliminary investigation of the molecular mechanisms leading to this phenotype. Using a Wnt1:Cre-2, Ift88-targeted conditional elimination of primary cilia, and a Td-tomato reporter to track CNCC, we observed loss of cilia in CNCC at embryonic day E9.5, with notable CNCC contribution to the ventricular myocardium and pronounced disorganization of the endocardium by E10.5.The phenotype resulting from cilia loss in CNCC was characterized by a variety predicted CHDs in addition to disorganization of the ventricular endocardium, pronounced noncompaction of the ventricular myocardium, and perinatal lethality. To investigate potential changes in Hedgehog signaling resulting from cilia loss, we implemented an
in vivo
conditional knockout in which Wnt1:Cre-2 drives expression of a mutant, constitutively-active Hedgehog receptor. Noncompaction observed in all Wnt1:Cre-2/Ift88 mutants became exacerbated in a primary-cilia dependent fashion with the introduction of forced Hedgehog signaling in CNCC. These results support a critical role for both primary cilia and Hedgehog signaling in the development of the ventricular myocardium. Further electrophysiological assessments of Wnt1:Cre-2/Ift88 newborn mutant mice revealed bradycardia, conduction defects, and ECG tracings consistent with ventricular hypertrophy. These data collectively support a role for Hedgehog signaling via primary cilia of CNCC in the pathogenesis of outflow tract defects, VSD, and most notably, ventricular maturation CHDs.
Abstract 640: Indigo Naturalis, a Promising Herbal Medicine for Ulcerative Colitis, Can Induce Experimental Pulmonary Arterial Hypertension via Aryl Hydrocarbon Pathway
Authors: Hiraide Takahiro , Teratani Toshiaki , Kataoka Masaharu , Endo Jin , Sano Motoaki , Hakamata Yoji , Fukuda Keiichi , Kanai Takanori
Published: October 2019

Background:
Indigo naturalis is a Chinese herbal medicine, whose dramatical effects for treatment of ulcerative colitis have been reported. However, some patients who took indigo naturalis developed pulmonary arterial hypertension (PAH), which is a poor prognostic disease with right ventricular (RV) failure and progressive remodeling of pulmonary vessels.

Aims:
We examined the reproducibility in experimental animals and elucidate the mechanisms for indigo naturalis to induce PAH.

Methods and Results:
Rats with 12-week intake of low-dose indigo naturalis (60 mg/kg body weight; corresponding to therapeutic dose for patients) did not induce PAH. However, RV and pulmonary vessel remodeling were identified in rats fed with high-dose indigo naturalis (600 mg/kg body weight) for 12 weeks. Significantly elevated RV systolic pressure was identified in rats administered with vascular endothelial growth factor-2 receptor blocker (Sugen 5416) and fed with indigo naturalis for 8 weeks compared to that of rats fed with normal diet (38.6 [38.2, 40.0] mmHg) vs. 31.9 [31.4, 34.6] mmHg, p < 0.001). Indigo, a major compound of indigo naturalis, also induced mild PAH. Indigo works as a ligand of aryl hydrocarbon receptor (AhR), and the mRNA levels of CYP1A1, downstream of AhR signal, were elevated in lungs. Rats with indigo naturalis and AhR antagonist gavage developed less pulmonary vessel remodeling than rats without AhR antagonist.

Conclusions:
The combination of Sugen 5416 and indigo naturalis can be a novel unique approach to generate rat PAH model without a hypoxic chamber. The effects of indigo naturalis to PAH might be mediated at least in part through activation of AhR-CYP1A1 pathway.
Abstract 646: Myocardial Ischemia/Reperfusion Impairs Adipocyte Endocrine Function via Exosome-Mediated Endoplasmic Reticulum Dysfunction
Authors: Gan Lu , Xie Dina , Lau Wayne , Christopher Theodore , Lopez Bernard , Zhang Ling , Liu Jing , Gao Erhe , Koch Walter , Wang Yajing , Ma Xinliang
Published: October 2019

By incompletely understood mechanisms, MI patients sustain systemic metabolic disorder. Adipocytes are an important cellular type regulating energy homeostasis. The impact of MI upon adipocyte function remains unknown. Exosomes (Exo) are critical vehicles mediating organ-organ communication. However, whether and how Exo may mediate post-MI cardiomyocyte/adipocyte communication have not been previously investigated. Adult male mice were subjected to MI/R. Serum Exo were isolated 3 hours after R and incubated with 3T3L cells for 24 hours. Compared to control, MI/R Exo significantly altered the expression of 17 genes known to be important in adipocyte function. GO analysis revealed that genes associated with endoplasmic reticulum (ER) function and adipocyte endocrine function are the primary two pathways altered by MI/R Exo. Venn analysis identified 11 mi-RNAs as cardiac-enriched, adipocyte-poor, and ER function-related miRNAs. RT-qPCR confirmed the miR-23a/27a/24-2 family members are the most markedly increased mi-RNAs in MI/R Exo. Incubation of 3T3L cells with mi-R27a mimic significantly downregulated EDEM3, DsBA-L, and PPARγ, and upregulated PERK and CHOP. Conversely, mi-R27a inhibitor significantly decreased the impact of MI/R Exo upon ER function genes. Additional studies demonstrated EDEM3 and PPARγ (two critical molecules maintaining ER function and adipocyte endocrine function) to be direct targets of mi-R27a. One of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by PPARγ at the transcriptional level and by DsBA-L at the post-translational level. We next determined whether MI/R Exo may affect adiponectin expression/assembly. Incubation of 3T3L cells with MI/R Exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by miR27a mimic. Finally, in vivo administration of GW4869 (Exo biogenesis inhibitor) or miR27a inhibitor attenuated adipocyte ER dysfunction and restored plasma adiponectin level in MI/R animals. We demonstrate for the first time that MI/R causes significant adipocyte ER and endocrine dysfunction by Exo mediated cardiomyocyte/adipocyte communication via miR-23a/27a/24-2.
Abstract 653: The Long Noncoding RNA Landscape of the High Altitude Induced Thromboembolic Disorder: Role of Endogenous miRNA Sponge
Authors: Jha Prabhash K , Vijay Aatira , Prabhakar Amit , Chatterjee Tathagat , Bajaj Nitin , Nair Velu , Kumar Bhuvnesh , Ashraf Mohammad Z , Sharma Manish
Published: October 2019

Background:
The pathophysiology of Deep vein thrombosis (DVT) is considered as multifactorial, where thrombus formation is interplay of genetic and acquired risk factors. Lately, different pipelines have been developed to identify novel lncRNAs in different diseases, however a little is known about the expression profile and roles of lncRNAs in human subjects developing DVT at high altitude.

Methods:
In the present study, using RNA sequencing, we compared peripheral blood lncRNA expression profile in human High Altitude deep Vein Thrombosis (HA-DVT) patients with high altitude control and sea level control human subjects. We used DESeq to identify differentially expressed (DE) genes. We identified the noncoding RNAs by annotation database that includes lncRNAs from the NONCODE 3.0 human noncoding RNA database. This was followed by the functional characterization of the identified DE lncRNAs by Co-expressed protein-coding genes analysis. We further performed
in silico
putative lncRNA-miRNA association study to unravel the endogenous miRNA sponge associated with our candidate lncRNAs. Finally, to validate the
in silico
findings, siRNA knockdown assay of the candidate LncRNAs was conducted in primary endothelial cells

Results:
We identified 973 differentially expressed lncRNAs. Co-expressed protein-coding genes analysis resulted in a list of 722 coexpressed protein-coding genes with a Pearson correlation coefficients >0.7. The functional annotation of co-expressed genes and putative proteins revealed their involvement in the hypoxia, immune response and coagulation cascade. Putative lncRNA-miRNA association analysis revealed several miRNAs associated with cardiovascular disorders. Through its miRNA response elements (MREs) to compete for miR-143 and miR-15, lncRNA-LINC00659 and UXT-AS1 regulates the expression of prothrombotic genes. Furthermore, in vitro RNA interference (siRNA) simultaneously suppressed lncRNAs and target gene mRNA level.

Conclusions:
This transcriptome profile describes several potential mechanisms of interaction between lncRNAs, the coding genes, miRNAs and regulatory transcription factors that define the thrombotic signature and may be used in establishing lncRNAs as biomarker in HA-DVT.
Abstract 657: Long Noncoding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation
Authors: Kang Xuedong , Zhao Yan , Wu Joseph , Nelson Stanley F , Touma Marlin
Published: October 2019

Introduction:
Emerged as critical epigenetic regulators of the transcriptome, the long noncoding RNAs (lncRNAs) play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction in congenital heart disease. In our work, we identified a novel lncRNA that regulates myogenic differentiation.

Methods:
Antisense oligonucleotides (GapmeR) were used to suppress Ppp1r1b-lncRNA. Chromatin immunoprecipitation (CHIP) and chromatin isolation by RNA purification (CHIRP) were used to analyze gene specific histone modification level and lncRNA:chromatin interaction, respectively. RNA pull-down and RNA immunoprecipitation (RIP) were used to identify the protein molecules that interact with Ppp1r1b-lncRNA.

Results:
By silencing Ppp1r1b-lncRNA, C2C12, a skeletal myoblast cell line, and human-induced pluripotent stem cell derived cardiomyocytes failed to develop fully differentiated myotubes. Analysis of GapmeR injected neonatal mouse heart (
in vivo
) and siRNA silenced human myoblasts (
in vitro
) further confirmed the role of Ppp1r1b-lncRNA in normal myogenesis. Members of the MyoD family of muscle-specific transcription factors were not induced during myogenic differentiation in C2C12 cells treated with Ppp1r1b-lncRNA specific GapmeR. Proteins essential for striated muscle development were also suppressed in Ppp1r1b-lncRNA- deficient cells. Histone modification analysis revealed enrichment of histone tri-methylation at Myogenin and MyoD1 promoters in GapmeR treated C2C12 cells. Subsequently, lncRNA- protein complex isolation and lncRNA-DNA interaction assays confirmed Ppp1r1b-lncRNA function.

Conclusions:
Our findings support important role of Ppp1r1b-lncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of key myogenic genes. Particularly, the identification of EZH2 in pulled Pp1r1b-lncRNA: protein complex supports that Polycomb repressive complex 2 is involved in Ppp1r1b-lncRNA modulated myoblast differentiation. The recovery of MyoD1 promoter DNA in isolated lncRNA: chromatin DNA interactome implies that Ppp1r1b-lncRNA regulates transcription by recruiting PRC2 complex to its target genes.
Abstract 700: Recellularization of Acellular Xenogenic Scaffold With Autologous Human Mesenchymal Stem Cells Rescues the Xenoreactive Immune Response
Authors: Bozso Sabin J , Kang Jimmy , Adam Benjamin , Moon Michael C , Freed Darren H , Nagendran Jayan , Nagendran Jeevan
Published: October 2019

Background:
Pre-clinical evidence suggests that structural valve deterioration (SVD) of bioprosthetic xenogenic tissue heart valves (XTHV) is a result of chronic immune rejection. Recellularization of XTHVs with human autologous cells represents one potential strategy to prevent this xenoreactive immune response and mitigate SVD.

Methods:
Bone marrow, human pericardium and heparinized whole blood was collected from adult patients undergoing elective cardiac surgery. Decellularized bovine pericardium underwent recellularization with hMSCs, which were isolated and cultured from the bone marrow, via co-incubation to allow for cell seeding. To examine the cell mediated immune response, we performed immunofluorescent staining for CD68+ macrophages and CD3+ T-cells and performed a proliferation assay to quantify T-cell proliferation. To investigate the humoral immune response, we performed an enzyme linked immunosorbent assay for pro-inflammatory cytokines TNF-α and INF-γ. Serum and PBMCs were collected for subsequent biochemical and flow cytometric analysis.

Results:
We show that decellularized bovine pericardium, exposed to human serum, had significantly decreased T-cell activation, represented by TNF-αand INF-γexpression, as well as a significant decrease in T-cell proliferation, when compared to wild-type bovine pericardium (p<0.01). Moreover, when decellularized bovine pericardium was recellularized with autologous hMSCs and exposed to human serum, there was an additional decrease in TNF-α, INF-γexpression along with a further significant decrease in T-cell proliferation, when compared to both wild-type and decellularized bovine pericardium (p<0.01). Importantly, recellularized XTHVs exposed to human serum had an equivalent expression of TNF-α, INF-γ and T-cell proliferation when compared to native human pericardium exposed to autologous human serum.

Conclusions:
Taken together, our data suggest that autologous human MSC recellularization of decellularized bovine pericardium abrogates the xenoreactive immune response. As such, autologous hMSC recellularization of an acellular xenogenic scaffolds may be an effective approach to decrease the progression of bioprosthetic SVD.
Abstract 730: Celltype-Specific Functions in Dilated Cardiomyopathy Caused by the
LMNA
Gene Mutation
Authors: Sikder Kunal , Phillips Elizabeth , Zhong Zhijiu , Wang Nadan , Margulies Kenneth B , Choi Jason C
Published: October 2019

Mutations in the
LMNA
gene, encoding lamins A and C (lamin A/C) cause a diverse group of human diseases termed laminopathies. The most prevalent laminopathy is dilated cardiomyopathy (herein referred to as
LMNA
cardiomyopathy) characterized by variable onset of fibrosis/pathological remodeling that always progresses to heart failure. Despite recent progress, how cell type-specific effects of
LMNA
mutations are integrated at the tissue level to engender complex pathologies in a heterocellular organ such as the heart are not well understood. Using a conditional deletion model that permits cardiomyocyte (CM)-specific
Lmna
deletion coupled with translating ribosome affinity purification, we found that CM-specific lamin A/C depletion caused rapid fibrosis and severe cardiac dysfunction accompanied by endoplasmic reticulum (ER) stress. At the molecular level, CM-specific translating mRNA profiling identified increased expression of MED25, a member of the Mediator complex implicated in regulating ER stress responses, prior to the onset of cardiac dysfunction. In contrast, lamin A/C depletion selectively in cardiac fibroblasts (CFs) displayed no immediate cardiac pathology whereas a concurrent depletion in both CMs and CFs resulted in lesser fibrosis and pathological remodeling. These results suggest that lamin A/C play an important role in CF biology and that reactive CFs underlie the rapid onset and the exaggerated
LMNA
cardiomyopathy symptoms observed in mice with CM-only
Lmna
deletion. Mechanistically, we found that increased matrix stiffness elevated the expression of MED25 and ER stress markers in lamin A/C-depleted CMs
in vitro
, indicating that the physical component of fibrosis may underlie the observed molecular changes. Taken together, our results suggest lamin A/C-depleted CFs mediate a brake on cardiomyopathy development and interactions between CFs and CMs are important determinants of the rate of progression and the severity of
LMNA
cardiomyopathy. Therefore, strategies targeting lamin A/C function in CFs may hold therapeutic potential for patients with
LMNA
cardiomyopathy as well as for other forms of cardiomyopathy in which fibrosis is integral to disease pathogenesis.
Abstract 731: Sorafenib Induces Cardiotoxicity via Damage to Cardiac Endothelial Cells
Authors: Elmadani Manar , Szabo Zoltan , Raatikainen Sami , Ruizhu Lin , Alakoski Tarja , Piuhola Jarkko , Yrjolä Raisa , Miinalainen Ilkka , Åström Pirjo , Alitalo Kari , Tehunen Olli , Magga Johanna , Kerkelä Risto
Published: October 2019

Aim:
Sorafenib (Nexavar) is a multitargeted tyrosine kinase inhibitor that is used for treatment of hepatocellular carcinoma and renal cell carcinoma. Cancer treatment with sorafenib has been reported to be associated with cardiotoxic side effects such as hypertension, myocardial ischemia, thrombosis and pulmonary embolism, whereas incidence of LV dysfunction is rare. Aim of the current study was to identify the mechanism of sorafenib cardiotoxicity.

Results:
Healthy mice were treated with sorafenib (50 mg/kg/d) for 4 weeks. Echocardiography analysis revealed no difference in LV systolic function, but LV posterior wall thickness and relative wall thickness were significantly increased, and LV volume decreased in sorafenib treated mice. Hearts of sorafenib treated mice showed a 10-fold increase in expression of heart failure marker gene ANP and a 3-fold increase in the number of apoptotic cells. Electron microscopy analysis indicated damage to cardiac endothelial cells with no evidence of cardiomyocyte loss and co-staining of cardiac sections with endothelial marker CD31 revealed that ~80% of apoptotic cells in sorafenib treated hearts were endothelial cells (ECs). To assess for the time course for EC damage, we treated mice with sorafenib for 8h, 24h, 48h and 72h. The increase in EC death was already detected after 8h treatment and peaking at 24h-48h. RNA profiling analysis showed that expression of angiopoietin-1 (Angpt1) was reduced, whereas expression of Angpt2 was induced in hearts of mice treated with sorafenib for 4 weeks and in EC fractions from hearts of mice treated with sorafenib for 72h. Angpt-1 overexpression induced protective signaling pathways in hearts of mice treated with sorafenib for 48h, but did not rescue from acute EC injury. Treatment of Angpt1 overexpressing mice with sorafenib for 4 weeks resulted in capillary rarefaction and heart failure, evidenced by a decrease in LV systolic function and a 40-fold increase in ANP expression.

Conclusion:
our data shows that sorafenib cardiotoxicity is due to endothelial cell damage in the heart and results in heart failure with preserved ejection fraction. Furthermore, antagonizing the function of Angpt2 by overexpression of Angpt1 is detrimental during sorafenib treatment.
Abstract 754: The Relationship Between Obstructive Sleep Apnea and Coronary Plaque Instability: An Optical Frequency Domain Imaging Study
Authors: Konishi Takao
Published: October 2019

Background and purpose:
Obstructive sleep apnea (OSA) is associated with coronary artery disease (CAD) and with an increased risk of myocardial infarction, stroke or death due to cardiovascular disease. Optical frequency-domain imaging (OFDI) is a useful modality for evaluating the characteristics of atherosclerotic plaque. The purpose of the study was to use OFDI to investigate the association of OSA with coronary plaque characteristics in patients undergoing percutaneous coronary intervention (PCI).

Methods:
We retrospectively analyzed OFDI data for coronary artery plaques from 15 patients with OSA and 35 non-OSA patients treated between October 2015 and October 2018. Plaque morphology was evaluated for 70 lesions, including 21 from patients with OSA and 49 from non-OSA patients.

Results:
Compared with the non-OSA group, patients with OSA had significantly higher prevalences of thinned cap fibroatheroma (TCFA) (67% vs. 35%, P=0.014) and microchannels (86% vs. 55%, P=0.014); a significantly higher mean lipid index (1,392±982 vs. 817±699, P=0.021), macrophage grade (8.4±6.4 vs. 4.8±4.5, P=0.030), and maximum number of microchannels (1.5±1.0 vs. 0.7±0.7, P=0.001); and a significantly lower mean minimum fibrous cap thickness (69.4±28.7 vs. 96.1±51.8 μm, P=0.008).

Conclusions:
This OFDI analysis suggests that OSA is associated with unstable plaque characteristics in patients with CAD. More intensive medical management for stabilization of coronary atherosclerotic plaque is required in patients with OSA.
Abstract 757: β-arrestin-Biased β2-Adrenergic Receptor Signaling Mediates Novel Mechanisms of Cardiomyocyte Contractility
Authors: Okyere Ama Dedo , de Lucia Claudio , Song Jianliang , Carter Rhonda L , Benovic Jeffrey L , Koch Walter J , Cheung Joseph Y , Tilley Douglas G
Published: October 2019

During heart failure, chronically decreased cardiac output can be treated with positive inotropes, but classic inotropes such as β-adrenergic receptor (βAR) agonists that increase cAMP-dependent Ca
2+
mobilization and contractility ultimately enhance patient mortality. Thus, an alternate approach would be to enhance cardiomyocyte contractility without alterations in cAMP and Ca
2+
levels, such as regulation of sarcomeric proteins. Recently, we demonstrated that a small lipidated pepducin designed from the 1
st
intracellular loop of β2AR (ICL1-9) enhanced cardiomyocyte contractility in a Ca
2+
-independent, β-arrestin-dependent manner, yet the complete mechanisms remained unclear. We also showed that β2AR stimulation in hearts in vivo or neonatal rat ventricular myocytes (NRVM) in vitro activated RhoA in a βarr-dependent manner. Therefore, we sought to determine both the proximal and distal mechanisms by which ICL1-9 enhances cardiomyocyte contractility. Using adult murine cardiomyocytes isolated from wild-type C57Bl/6J mice, we measured basal, ICL1-9- and isoproterenol (ISO, as a positive control)-promoted contractility either alone or in the presence of inhibitors of Gα
i
activity (Pertussis toxin), ROCK1 (Y-27632), myosin light chain kinase (ML7), and protein kinase D (CID755673). Inhibition of Gα
i
activity prior to ICL1-9 stimulation led to a decreased contractile response. Consistent with RhoA activation by ICL1-9, ROCK1 inhibition was able to attenuate ICL1-9-mediated contractility, as was inhibition of MLCK. Interestingly, we observed that inhibition of PKD also attenuated ICL1-9-mediated contractility. These data suggest that ICL1-9 acts proximally to engage a β2AR/Gα
i
/βarr signaling axis, which may distally increase the activation of kinases including PKD, MLCK, and ROCK to alter the regulation of sarcomeric proteins.
Abstract 759: Circulating Preoperative microRNA-29, Biomarkers of Collagen Synthesis, and Age Predictive of Postoperative Atrial Fibrillation After Coronary Artery Bypass Grafting
Authors: Rizvi Farhan , Mirza Mahek , Olet Susan , Albrecht Melissa , Edwards Stacie , Emelyanova Larisa , Kress David , Ross Gracious R , Holmuhamedov Ekhson , Tajik A. Jamil , Khanderia Bijoy K , Jahangir Arshad
Published: October 2019

Background:
Postoperative atrial fibrillation (PoAF) is a common complication occurring in 35-50% patients within 2-3 days after cardiac surgery. Identification of patients, especially those with no prior history of atrial fibrillation before surgery, is a challenge. A pre-existing atrial substrate appears to be an important factor in the development of PoAF. The aim of this study was to assess the role of biomarkers in identifying patients at risk of PoAF in a pathophysiology-based risk predictive model by combining clinical and biochemical risk factors.

Methods:
Preoperative blood from patients undergoing cardiac surgery with no previous history of AF was assessed for circulating levels of biomarkers reflecting collagen synthesis/degradation and extracellular matrix remodeling using ELISA. Reverse transcriptase polymerase chain reaction assessed microRNA29 in the serum and correlated to extent of atrial fibrosis. Echocardiographic evaluation of LA mechanics was performed preoperatively using M-mode, 2D Doppler, and 3D Speckle tracking.

Results:
Out of 55 patients, 31 patients (56.4%) who developed PoAF after surgery during their hospital stay were older in age (70.0 ± 4.0 years vs.63.4±9.9; p<0.01) with abnormal global longitudinal stain (6.9±0.69 vs.10.9±0.93, p=0.007), higher amino-terminal peptide procollagen III (PIIINP) levels (101.1±42.7 vs.36.6±20.0; p=0.043) with increased collagen to myocardial ratio (0.20±0.09 vs. 0.09±0.01, p= 0.026), and reduced preoperative circulating microRNA-29a, -29b and -29c levels. By combining the clinical risk factors, circulating biochemical and molecular biomarkers, we developed a model that identified PoAF patients (AUC=0.7987; 95% CI, 0.6174—0.98) with reduced preoperative atrial ejection fraction (32±2% vs. 42±2 %; p=0.01) as an independent risk factor for PoAF.

Conclusion:
Our study developed a noninvasive tool to identify those who are at risk for new-onset PoAF in patients with no previous history of AF when combining age, biomarkers of collagen synthesis and microRNA-29a.
Abstract 773: Neonatal and Adult Cardiac Transcriptome Analysis Reveals Distinct Mechanisms in Noncompaction Dilated Cardiomyopathy Development Induced by p.K69R-MLP (Muscle Lim Protein) Mutation i
n vivo
Authors: Purevjav Enkhsaikhan , Xu Fuyi , Munkhsaikhan Undral , Alberson Neely , Sabau Ramona , Lu Lu , Towbin Jeffrey A
Published: October 2019

Introduction:
Pediatric and adult dilated cardiomyopathy (DCM) represent distinct pathological entities, however, the basis of this phenomena remains obscure. The pathogenic variant p.K69R in MLP/
CSRP3
induced DCM in adult Mlp
K69R
knock-in mice. A noncompacted ventricular wall and dilated ventricles were seen in embryonic and neonatal Mlp
K69R
hearts, reminiscent of that seen in infant variant carriers.

Hypothesis:
Mlp-
K69R associated genetic modifiers govern DCM phenotypes in neonatal and adult heart. The study was to find genetic modifiers associated with divergent neonatal and adult phenotypes induced by
Mlp-
K69R.

Methods:
RNA extracted from neonatal and adult Mlp
K69R
and Mlp
WT
mouse myocardium was applied to Affymetrix microarray and QPCR analysis. Data were normalized, subjected to differentially expression genes and gene set enrichment analysis.

Results:
We found 1024 genes in adult and 252 genes in neonatal heart that were significantly altered in Mlp
K69R
vs
WT littermates. 23 of these genes were mutually altered in adult and neonatal mutants. In adult mutants, 15 sarcomeric genes were significantly over-expressed compared to WT littermates. The mutation altered expression of genes involved in sarcolemmal and desmosomes unity. Calcium handling, integrin-associated networks, and heart failure-associated genes were upregulated in mutants
vs
WT littermates. In neonates, genes involved in cardiac and vascular development (
Rhox2c, Tbx3, Mir23a, Tbc1d7
), cell adhesion and migration (
Tff2
), and apoptosis (
Mir297a-2
) were significantly altered. Two cardio-protective miRNAs,
Mir98
and
Mir451a
, were highly reciprocally altered in both, adult and neonatal, mutant
vs
WT hearts. Their expression was increased in mutant adult hearts, but decreased in neonatal mutant hearts, suggesting novel candidates associated with K69R-MLP-induced DCM and noncompaction phenotypes.

Conclusions:
Differential transcriptomes in neonatal and adult hearts suggest that pathogenic K69R-MLP variant induces DCM and noncompaction phenotypes
via
distinct underlying genetic modifiers. Contrary to mutant adult mice, mutant newborn hearts show significant decrease in
Mir98
and
Mir451a,
which may predispose them to developing myocardial noncompaction.
Abstract 776:
MYBPC3
Mutations in Indian Patients With Hypertrophic Cardiomyopathy
Authors: Wankhade Varsha W
Published: October 2019

Introduction:
Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is characterized by thickening of the ventricular wall, myocyte disarray, and fibrosis, resulting in impaired cardiac function and leading to adverse ventricular remodeling, heart failure and increased risk for sudden cardiac death. Importantly, mutations in the gene encoding cardiac myosin binding protein-C (
MYBPC3
), a muscle contractile protein,account for approximately 50% of the reported HCM cases. However, the prevalence of penetrance of
MYBPC3
mutations in Indian patients with HCM is yet to be systematically studied.

Methods:
The Institutional Human Ethics Committee at the University of Puneapproved the present study. HCM patients undergoing treatment in theCardiology Unit of Bharti Hospital, Pune, were identified. With the approval of written informed consent, blood samples were obtained from all patients and their relatives and screened by single-strand conformation polymorphism (SSCP) and direct DNA sequencing, followed by correlation with clinical outcome.

Results:
Of the 23 probands screened in this study, one, #16, showed a mutation in exon 07 (A/G) at chromosomal position 47370041, causing a protein change at position NP_000247.2:p.S236G, and reported as SNP status ID rs386584787. The mutation was localizedto the immunoglobulin domain of cardiac myosin binding protein-C, causing an amino acid change from serine to glycine with a score of 0.00, as shown by thePolyPhen-2 (Polymorphism Phenotyping v2) tool.G>C transversion of intron variant in exon 21 of
MYBPC3
results in a new exon splicing enhancer (ESE) site which may alter splicing. G>A transition at g. 20167 is an intron variant of a previously reported status (rs373904644). This polymorphism results in the creation of an intronic splicingenhancer (ISE) site. The proband had left ventricular wall thickness of 24 mm, ejection fraction of 60 (%), and left ventricular internal diameter of 30 mm, suggesting early onset of HCM.

Conclusion:
The
MYBPC3
mutations might play a key role in thedevelopment of HCM in the Indian population, indicating the soundness of establishing a systematic screening of all HCM patients.
Abstract 781: Doxorubicin-Induced Cardiotoxicity and Novel GATA4-Targeted Compounds
Authors: Karhu Tuuli , Kinnunen Sini , Talman Virpi , Ruskoaho Heikki
Published: October 2019

Doxorubicin (DOX) is a widely used anticancer drug which, unfortunately, induces dose-related cardiotoxicity. A deep understanding of DOX’s toxicity is still unclear, partly because most
in vitro
studies have evaluated the effects of short-term high-dose DOX treatments. Thus, a more precise model of DOX cardiotoxicity is needed to more accurately simulate clinical scenario.

The first aim of this study was to establish an
in vitro
model of long-term low-dose administration of DOX utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The second aim was to investigate if novel GATA4-targeted compounds can protect CMs from DOX toxicity.

Acute toxicity was studied in both primary neonatal rat ventricular cardiomyocytes (NRVCs) and hiPSC-CMs. The cells were exposed to 0.1-3 μM DOX for 48 h, after which the lactate dehydrogenase and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out. To study long-term toxicity, hiPSC-CMs were exposed to 100 nM DOX for up to 14 days. MTT assay and high-content analysis were carried out after 4, 7 and 14-day exposures. The effect of the test compound was studied by exposing the cells simultaneously to DOX and the compound.

A 48-hour exposure to DOX at 1 and 3 μM concentrations induced more than 62% reductions in both NRVC and hiPSC-CM viability, whereas a 14-day exposure to 100 nM DOX induced a 26% reduction in hiPSC-CM viability. A 4-day exposure to 100 nM DOX induced a 3.1-fold increase in the percentage of cells positive for pro-B-type natriuretic peptide (proBNP) compared to control. When the cells were exposed simultaneously to 100 nM DOX and the test compound (10 μM), the percentage of proBNP+ cells increased only 1.4-fold compared to control. However, over 14-day exposure the compound at 10 μM concentration reduced hiPSC-CM viability 50% compared to control.

A long-term exposure of hiPSC-CMs can be utilized as an
in vitro
model to investigate the mechanisms of delayed DOX-induced cardiotoxicity. The GATA4-targeted test compound exhibited cardioprotective potential against subacute DOX toxicity. Over chronic exposure the compound was, however, toxic to CMs, indicating that further structural optimization is required to develop non-toxic derivatives.
Abstract 783: Epicardial Contribution to Arrhythmogenic Cardiomyopathy
Authors: Kohela Arwa , van Kampen Sebastiaan , Moens Tara , Monshouwer-Kloots Jantine , Molenaar Bas , Wehrens Martijn , Vink Arjan , Chen Huei-Sheng Vincent , van Rooij Eva
Published: October 2019

Arrhythmogenic cardiomyopathy (ACM) is an inherited disease mainly caused by desmosomal gene mutations and characterized by myocardial loss, replacement with fibro-fatty tissue, arrhythmias and sudden cardiac death. To date, it is unclear which cell type and molecular mechanisms contribute to the fibro-fatty phenotype. The epicardium is the outer mesothelial layer of the heart which has the capacity to undergo epithelial-to-mesenchymal transition (EMT) and differentiate into various cardiac cell types. The aim of this study is to investigate whether epicardial cells contribute to the excess fibro-fatty infiltration seen in ACM patients. To this end, we differentiated induced pluripotent stem cells (iPSCs) from an ACM patient with a haploinsufficiency-causing mutation in the desmosomal gene plakophilin-2 (PKP2) (c.2013delC), and an isogenic control into epicardial cells. While Western blot, flow cytometry, qPCR and immunofluorescence imaging indicated comparable epicardial differentiation efficiencies, RNA-sequencing revealed a profound increased expression of fibroblast and lipid markers only in mutant epicardial cells. These data were corroborated by the spontaneous accumulation of lipid droplets and adipogenic markers in the PKP2 mutant epicardial cells. Single cell sequencing analysis revealed a significant induction of Activating Enhancer-Binding Protein 2 (AP2) family of transcription factors in a subset of PKP2 mutant epicardial cells, which are known to play roles in EMT and lipid biogenesis. siRNA-mediated knock down of AP2 members in mutant epicardial cells significantly reduced the expression of fat and fibroblast markers, suggesting an AP2-mediated fibro-fatty signalling in epicardial cells. The PKP2-dependence of these findings was further validated in healthy iPSC-epicardial cells treated with siRNAs targeting PKP2 which recapitulated the observations made in the mutant cells. Using this human
in vitro
model system, we were able to show the epicardial role during fibro-fatty tissue replacement upon PKP2 suppression. Ongoing experiments, including studies on explanted ACM hearts and a PKP2 c.2013delC knock-in mouse model, aim at further elucidating the molecular mechanisms of ACM pathogenesis.
Abstract 795: TRAF2-GRK2 Interaction Mediates Non-Canonical Desensitization of Beta-Adrenergic Receptors to TNF-α
Authors: Mukherjee Sromona , Maradumane Mohan , Prasad Sathyamangla V. N
Published: October 2019

β- Adrenergic receptor (βAR), a prototypical G-protein coupled receptor (GPCR) is a key regulator of cardiac function. Impaired G-protein coupling (desensitization) of βARs is a key hallmark of heart failure. Desensitization occurs by phosphorylation of βARs by GRK2 to sympathetic overdrive leading to β-arrestin binding and endocytosis. Our studies show that TNFα-induces βAR phosphorylation/desensitization via GRK2 recruitment that is independent of the classical Gβγ mechanism suggesting a non-canonical pathway of GRK2 recruitment. Comorbid conditions like obesity/ hypertension have a higher risk of heart failure and is associated with increase in TNFα, a known cardio-depressant. β-blockers are known to be contra-indicative in obesity which we believe is due to βAR desensitization through non-canonical GRK2 recruitment to the βAR in response to elevated TNFα. However the underlying mechanisms for βAR desensitization are not known and as TNFα recruits TRAF2 to mediate downstream signaling, we hypothesized that TRAF2 recruits GRK2 to the plasma membrane causing receptor phosphorylation and desensitization. Our study shows TNFα drives βAR dysfunction by recruiting GRK2 to βAR complex through TRAF2. Our studies indicate that TRAF2 and GRK2 form a cytosolic complex and is recruited to the receptor complex post-TNFα treatment. Pharmacologic inhibition of GRK2 results in decreased βAR phosphorylation after TNFα. Furthermore, siRNA knockdown of TRAF2 results in reduction of βAR phosphorylation indicating the key role for TRAF2 in TNFα-mediated desensitization. Given that nothing is known about TRAF2 and GRK2 interaction we performed molecular docking using Haddock software to identify that the N-terminal region of GRK2 may interact with TRAF2. Overexpression of GRK2 N-terminal domain as dominant negative strategy resulted in reduced β2AR phosphorylation in response to TNFα, suggesting the requirement of GRK2 N-terminal in non-canonical β2AR desensitization. We are currently using cardiac specific GRK2 and TRAF2 knockout mice to assess their role in cardiac function in response to TNFα showing the role of GRK2-TRAF2-TNFR axis in mediating agonist-independent βAR desensitization/dysfunction.
Abstract 796: Dysfunctional Immune Cell Clock Contributes to Heart Failure Progression
Authors: Rana Samir , Zhou Guihua , Hamid Tariq , Rokosh Gregg , Ismahil M. Ameen , Young Martin E. , Prabhu Sumanth D.
Published: October 2019

Objective:
Inappropriately sustained activation of monocytes/macrophages contributes to heart failure (HF) progression. Time-of-day greatly influences cardiovascular and immune systems, in part through cell autonomous circadian clocks. Here, we propose that disruption of monocyte/macrophage circadian clocks contributes to pathologic inflammation and HF progression following myocardial infarction (MI).

Methods:
MI-induced HF was achieved by permanent ligation of coronary artery in myeloid-specific
Bmal1
knockout (MBK) mice and wild-type (WT) mice. Left ventricular (LV) function was assessed echocardiography. Flow cytometry was utilized for immune cell profiling. RNAseq and qRT-PCR was utilized for gene-expression studies. Tissue fibrosis was quantified using Masson-trichrome staining.

Results:
WT (C57Bl/6) HF mice exhibited altered Ly6C
hi
monocyte circadian clock gene expression as compared with sham-operated mice, with significantly reduced expression of the key clock effector
Bmal1
. Genetic disruption of the monocyte clock in MBK mice had no effect on macrophage inflammatory markers during unstressed, baseline conditions. However, MBK-HF mice (4 w post-MI) exhibited increased infiltrating CCR2
+
heart macrophages (2642±803 vs. 937±190 per heart, p<0.05), augmented cardiac fibrosis (22±1 vs. 14±3 %, p<0.05), greater LV dysfunction [ejection fraction 19.7±2 vs. 28.4±4 %, p<0.05), increased LV end-diastolic and end-systolic volume (EDV 140.4±10 vs. 100±8 μl, p<0.01; ESV 115±10 vs. 74±9 μl, p<0.01) and cardiac hypertrophy (heart weight/tibia length 12.1±0.6 vs. 10.5±0.4 mg/mm, p<0.05) as compared with WT HF littermate control mice. Moreover, circulating monocyte levels in MBK-HF mice were significantly increased at zeitgeber time 6 versus WT HF littermate controls (296±58 vs. 131±17 cells/μl, p<0.05).

Conclusion/Broader Impact:
Monocyte/macrophage circadian gene dysregulation resulting from Bmal1 loss-of-function exacerbates pathological LV remodeling, systemic and cardiac inflammation, and tissue fibrosis in chronic ischemic HF. Restoration of circadian clock function in innate immune cells may represent a potential therapeutic approach to immunomodulation in chronic HF.
Abstract 807: Rad Ablation as a Treatment to Target Cardiac Inotropy via L-type Calcium Channel Function
Authors: Ahern Brooke , Levitan Bryana , Veeranki Sudhakar , Shah Mihir , Ali Nemat , Sebastian Andrea , Li Jiayang , Stelzer Julian E , Andres Doug , Satin Jonathan
Published: October 2019

The L-type Ca
2+
channel current (I
Ca,L
) provides trigger Ca
2+
to contribute to cardiac contraction. Rad GTPase associates with the L-type Ca
2+
channel (LTCC) and serves as an endogenous inhibitor of LTCC activity. Overexpression of Rad blocks I
Ca,L
; absence of Rad increases I
Ca,L
. Rad attenuates β-adrenergic receptor (β-AR) signaling. Chronic β-AR stimulation associates with Ca
2+
mishandling and can promote signaling that progresses towards heart failure. Early studies of global, constitutive Rad-knockout mice (gRadKO) suggested that elevated Ca
2+
dynamics leads to pathological cardiac hypertrophy; however, Rad is also expressed in non-cardiac tissues.

Our objective is to test the hypothesis that increased myocardial I
Ca,L
via Rad deletion safely enhances cardiac function without driving pathological remodeling.

We created a cardiac-restricted inducible Rad knockout mouse (Rad
Δ/Δ
).
In vivo
function was measured with echocardiography. We examined I
Ca,L
through whole cell configuration of the patch clamp technique, assessed Ca
2+
handling, and sarcomere dynamics.

Unlike gRadKO, Rad
Δ/Δ
showed no elevation of fetal gene program, nor fibrosis, and no change to aortic pressure. Rad
Δ/Δ
had a sustained increase of inotropy without structural or functional remodeling (EF: Rad
Δ/Δ
=76 ± 2%, n=16; Rad
fl/fl
=59 ± 4%, n=7; p=0.001.) I
Ca,L
was significantly increased, with Rad loss mirroring a β-AR modulated phenotype on basal I
Ca,L
(max. conductance: Rad
Δ/Δ
=254 ± 19 pS/pF, n=15; Rad
fl/fl
=144 ± 12 pS/pF, n=18; p<10
-4
). Contrary to models of chronic β-AR stimulation, Rad
Δ/Δ
retained β-AR signaling shown
in vivo
using isoproterenol, and by preserved phosphorylation of protein regulators of Ca
2+
reuptake and contractility. Rad
Δ/Δ
cardiomyocytes show enhanced cytosolic Ca
2+
handling (Decay of Ca
2+
transient: Rad
Δ/Δ
= 0.07 ± 0.003 (F
340
/F
380
)/s, n=67, Rad
fl/fl
= 0.10 ± 0.005, n=69; p<10
-4
), increased contractile function, and elevated SERCA2a expression.

These new findings challenge the canonical assumption that increased myocardial Ca
2+
necessarily promotes pathology. We conclude that cardiac hypertrophy in gRadKO was caused by non-cardiac tissue effects, and myocardial Rad deletion is a promising cardiac inotropic therapeutic direction.
Abstract 809: Chronic Testosterone Deficiency Increases Late Inward Sodium Current in Ventricular Myocytes from Aging Male C57BL/6 Mice
Authors: Heinze-milne Stefan , Banga Shubham , Ayaz Omar , Howlett Susan
Published: October 2019

Clinical studies suggest that testosterone modulates the electrical activity of the heart and that low testosterone promotes cardiac arrhythmias. Here we investigated cellular mechanisms involved in the electrophysiological effects of chronic testosterone deficiency on the heart in aging male mice. Male C57BL/6 mice were subjected to either a gonadectomy (GDX) or a sham surgery at 1 month of age and then aged to 16-18 months. Ventricular myocytes were isolated and transmembrane voltage plus ionic currents were recorded with microelectrodes (20-25 MΩ; current clamp & discontinuous single electrode voltage clamp; 37
o
C). Cells were paced at 2 and 4 Hz. Action potential duration at 90% repolarization (APD
90
) was prolonged by GDX (57.9 ± 1.7 vs 108.2 ± 14.1 msec; p<0.05), while APD
50
and resting membrane potential were unchanged. We next determined whether an increase in late inward sodium current (I
Na-L
) contributed to the increase in APD
90
in GDX cells. Voltage clamp studies showed that I
Na-L
was significantly larger in GDX cells compared to sham cells (-0.70 ± 0.16 vs -1.53 ± 0.27 pA/pF; p<0.05). When myocytes were superfused with the I
Na-L
antagonist ranolazine (10 μM), the increase in APD
90
was blocked in cells from GDX mice, so that APD
90
no longer differed between the two groups. By contrast, ranolazine had no effect on either APD
50
or resting membrane potential. Ranolazine also blocked the increase in I
Na-L
in cells from GDX mice. Prolongation of the action potential in GDX myocytes was associated with a significant increase in the incidence of early afterdepolarizations (EADs) when compared to sham controls. EADs were also inhibited by ranolazine. These data demonstrate that long-term GDX prolongs APD
90
at least in part by increasing the magnitude of I
Na-L
. These findings also suggest that an increase in I
Na-L
may promote arrhythmias in older men with low circulating testosterone levels.