bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 286: Smyd1 is Required for Cell Survival During Glucose Deprivation
Authors: Sabry Amira , Cawley Keiko , Sciuto Katie , Horiuchi Amanda , Nix David , Stubborn Chris , Saijoh Yukio , Zaitsev Alexey V , Warren Junco S
Published: October 2019

Our recent study demonstrated that the histone methyltransferase Smyd1 positively regulates cardiac metabolism through transcriptional control of PGC-1α, a key regulator of mitochondrial energetics. However, it is largely unknown whether Smyd1 plays a role in adaptive responses to metabolic stress in cardiomyocytes. Here, we hypothesized that Smyd1 is required for cell survival during metabolic stress through maintaining energetic balance. To address this hypothesis, neonatal rat ventricular myocytes (NRVMs) were cultured in glucose-free media for 24 hours and real-time quantitative PCR was performed. We found that Smyd1 and its downstream target PGC-1α were upregulated during glucose starvation, concurrent with the significant increase of mRNA levels of
PPARα
and
CPT1b
, the key regulators of fatty acid β-oxidation (all p<0.05), indicating enhanced energetics. Control NRVMs were able to withstand 24-hr glucose starvation, maintaining mitochondrial inner membrane potential (MIMP) and the cell membrane integrity. However, siRNA-mediated
Smyd1
knockdown (siSmyd1) prior to glucose starvation led to massive cell death, evidenced by the dissipation of MIMP and the uptake of the normally cell-impermeable indicator YO-PRO. To better understand the mechanism of siSmyd-NRVMs vulnerability to glucose starvation, we examined the expression of genes involved in metabolism and apoptosis at the early stage of glucose starvation (3 hr), when autophagy was activated as evidenced by upregulation of LC3 and FoxO1. We found that the upregulation of PGC-1α, PPARα, CPT1b, as well as LC3 and FoxO1, adaptively induced by glucose starvation in control NRVMs, were all abolished by
Smyd1
knockdown, concomitant with a significant increase in gene expression of tumor necrosis factor receptor type 1-associated DEATH domain protein (Tradd). Moreover, RNA-seq analysis revealed that
Smyd1
-knockdown
per se
led to upregulation of the genes involved in regulation of apoptosis and cell death (i.e. Tradd, Casp8, Ripk3, Smad1). These data suggest that upregulation of Smyd1 in response to metabolic stress is a critical adaptive mechanism for cell survival, operating via enhancement of fatty acid metabolism, activation of autophagy, and suppression of cell death signaling.
Abstract 297: Therapeutic Effects of SK Channel Activator on Cardioprotection in the Setting of Cardioplegic Ischemia-Reperfusion and Cardiopulmonary Bypass
Authors: Zhang Zhiqi , Sodha Neel R , Pavlov Vasile , Aboulgheit Ahmad , Clements Richard , Kolodziejczak Martin A , Sellke Frank W , Feng Jun
Published: October 2019

Background:
Inactivation of small-conductance calcium-activated potassium (SK) channels contributes to coronary microvascular dysfunction in patients after cardioplegic arrest, cardiopulmonary bypass (CP/CPB) and cardiac surgery. SK-channel activation is cardioprotective in rodent isolated heart model of myocardial infarction and ischemia/reperfusion. In the current study, we investigated the impact of SK activation on myocardial protection in a clinically relevant large animal model of pigs subjected to cardioplegic ischemia/reperfusion and CPB.

Methods and Results:
Ten Yorkshire pigs were subjected to 1 hour of CP/CPB followed by 1 hour of reperfusion. Pigs received either cold blood hyperkalemic CP alone (CP control) or CP containing the selective SK channel activator NS309 (10
-6
M, n = 5/group). Left ventricular (LV) function was assessed by using pressure-volume loop analysis. Coronary arteriolar relaxation response (in-vitro) to the endothelium-dependent vasodilators adenosine diphosphate (ADP, 10
-9
-10
-4
M) and substance P (10
-12
-10
-7
M) and independent vasodilator sodium nitroprusside (SNP) was evaluated with video-microscopy. Inclusion of NS309 in cold blood CP tends to improve the recovery of LV function compared with the CP controls. CP/CPB and reperfusion significantly reduced endothelium-dependent relaxation response to ADP, 10
-5
M) and substance P (10
-8
M) as compared to control (p<0.05). In contrast, CP-containing NS309 significant improved the recovery of endothelium-dependent relaxation response to ADP and substance P as compared with the control group (p<0.05, respectively).

Conclusion:
In pig model of CP/CPB, inclusion of the selective SK channel activator in the cold blood cardioplegia improves recovery of coronary arteriolar endothelial function and microvascular relaxation, which may contribute to increased myocardial perfusion and improved LV function.
Abstract 301: Bone Marrow Niche-Mediates Cardiac Repair is Associated With Adiponectin in Retnla Deficiency
Authors: Kim Yong Sook , Jeong Hye-yun , Kang Hye-jin , Kim Mi Ra , Ahn Youngkeun
Published: October 2019

Background:
Resistin like α (Retnla) is known to be upregulated in anti-inflammatory macrophages and to modulate lipid profile in atherosclerosis. However, the role of Retnla is unknown in the cardiac pathology.

Methods:
Myocardial infarction (MI) was induced in wild type, Retnla knockout (KO), and Retnla transgenic (TG) mice. Cardiomyocytes, cardiac fibroblasts, macrophages and bone marrow cells were treated with Retnla protein for mechanistic studies. For an adoptive transfer, bone marrow cells were isolated from KO mice to transplant to the infarcted heart.

Results:
TUNEL staining showed the number of apoptotic cardiomyocytes was higher in the Retnla KO mice than in the TG mice 1 day after MI. Cardiac function and angiogenesis were significantly better in the Retnla KO group, while were worsen in the TG group. Retnla is an anti-inflammatory marker in macrophages, but the expressions of inflammation-related genes were not different between both groups. Interestingly, however, the numbers of macrophages isolated from bone marrow, peritoneum, and cardiac tissue of Retnla KO mice were higher than in the wild type mice. Moreover, phospho-histone H3-positive proliferating cells in the bone marrow were much more abundant in the Retnla KO mice than in the wild type mice. Adiponectin was highly expressed in bone marrow of KO mice, and we transplanted bone marrow cells from KO mice to recipient wild type mice. Two weeks after MI, recipient group transplanted with bone marrow cells from KO mice showed better angiogenesis, reserved cardiac function, and more anti-inflammatory macrophages. In Retnla-treated cardiomyocytes, bax, phopho-p38, and p21 were notably upregulated, while bcl2 and adiponectin were significantly reduced. To examine whether Retnla acted negatively on adiponectin, we administrated a synthetic adiponectin receptor agonist AdipoRon to Retnla-treated cardiomyocytes. AdipoRon blunted the effect of Retnla on cardiomyocytes. These data showed better cardiac recovery was associated with bone marrow-derived adiponectin in the Retnla KO group.

Conclusion:
Our study suggested that substantial cardiac recovery was strongly associated with adiponectin derived from highly proliferating bone marrow cells in Retnla KO mice.
Abstract 302: Role of Acidic pH in Linking SIRT1 and Cardioprotective Metabolism
Authors: Kulkarni Chaitanya A , Milliken Alexander S , Brookes Paul S
Published: October 2019

Metabolic acidosis is the hallmark of the ischemic period of ischemia-reperfusion injury and acidic pH is cardioprotective via unclear mechanisms. We previously reported induction of neomorphic activities in several dehydrogenase enzymes under acidic pH, leading to accumulation of non-canonical metabolite L-2-hydroxyglutarate (2-HG). Lysine deacetylase SIRT1 is required for cardioprotection via ischemic preconditioning (IPC) and 2-HG levels are elevated in IPC in a SIRT1 dependent manner. We hypothesized that acidic pH may be an independent variable that partly drives the metabolic character of not only ischemia, but also IPC. This hypothesis was tested using LC-MS/MS based metabolomics analysis of perfused hearts. Exposure of hearts to ischemia, hypoxia, IPC or intracellular acidosis resulted in partially overlapping metabolomes, with a number of metabolic adaptations previously attributed as either ischemic or IPC-driven, being induced by acidic pH alone. Furthermore, we observed that SIRT1 activator β-nicotinamide adenine mononucleotide induced cellular acidification in isolated cardiomyocytes. In the same cells it was observed that SIRT1 activation resulted in inhibition of sodium proton exchanger NHE1. These results suggest (i) a novel signaling mechanism in which SIRT1 is a cellular pH regulator via NHE1, and (ii) a role for acidic pH as a link between SIRT1 and cardioprotective metabolic adaptations.
Abstract 303: Exercise-induced Autophagy and Nadph Oxidase 2 Downregulation Prevents Cardiotoxicity Induced by Doxorubicin
Authors: Lee Youngil , Kwon Insu , Jang Yongchul , Cosio-Lima Ludmila
Published: October 2019

Endurance exercise (EXE) preconditioning prior to DOX treatment confers cardioprotection; however, whether EXE postconditioning (i.e., EXE intervention after the completion of DOX treatment) is cardioprotective remains unknown. Thus, the aim of the present study was to investigate if EXE postconditioning provide cardioprotection by testing the hypothesis that EXE-autophagy upregulation and NADPH oxidase 2 (NOX2) downregulation would be linked to cardioprotection against DOX-induced cardiotoxicity. C57BL/6 male mice were assigned into three groups: control (CON, n=10), doxorubicin (DOX, n=10), and doxorubicin + endurance exercise (DOX+EXE, n=10). Animals assigned to DOX and DOX+EXE groups were intraperitoneally injected with DOX (5 mg/kg each week for four weeks). 48 hours after the last DOX treatment, the mice assigned to DOX+EXE performed EXE on a motorized treadmill at a speed of 13-15 m/min for 60 min per day for four weeks. EXE prevented DOX-induced apoptosis and mitigated tissue damages. While DOX did not modulate auto/mitophagy, EXE significantly enhanced its flux (increased LC3-II levels, reduced p62 levels, and increased autophagosomes with mitochondria) along with increased mitochondrial fission (DRP1) and reduced fusion markers (OPA1 and MFN2). Interestingly, EXE-induced autophagy against DOX occurred in the absence of alterations of autophagy inducer AMPK or autophagy inhibitor mTOR signaling. EXE prohibited DOX-induced oxidative damages by suppressing NOX2 levels but without modulating other key antioxidant enzymes including MnSOD, CuZnSOD, CATALSE, and GPX1/2. Our data provide novel findings that EXE-induced auto/mitophagy promotion and NOX2 downregulation are linked to cardioprotection against DOX-induced cardiotoxicity. Importantly, our study shows that EXE postconditioning intervention is effective and efficacious to prevent DOX-induced cardiac injuries.
Abstract 311: Signal Transducer and Activator of Transcription 3 Inhibits the Opening of Mitochondrial Calcium Uniporter Against Cardiac Ischemia/reperfusion in Hydrogen Peroxide Postconditioning
Authors: Wu Lan , Tan Ji-liang , Chen Zhong-yan , Huang Gang
Published: October 2019

Reactive oxygen species (ROS) generated during early reperfusion activated signal transducer and activator of transcription 3 (STAT3) contribute to intermittent hypobaric hypoxia (IHH)-afforded cardioprotection against ischemia/reperfusion (I/R)-induced mitochondrial calcium overload, but its mechanisms are not fully understood. This study investigated whether ROS signaling activated STAT3 is involved in moderate hydrogen peroxide postconditioning (H
2
O
2
PoC) mimicked IHH against cardiac I/R injury and the possible downstream target of STAT3 in mitochondria that mediates H
2
O
2
PoC-afforded cardioprotection. Moderate H
2
O
2
PoC not only improved the post-ischemic recovery of myocardial contractile performance and reduce the infarct size in isolated rat I/R hearts, but also alleviated cytosolic Ca
2+
and mitochondrial Ca
2+
overload and improved Ca
2+
transients and cell contraction in rat I/R cardiomyocytes. However, the cardioprotective effects of moderate H
2
O
2
PoC was abrogated by janus kinase 2 (JAK2)/STAT3 inhibitor AG490 in rat hearts as well as adenovirus-delivered shRNAs specific for STAT3 and the opener of mitochondrial calcium uniporter (MCU) spermine, respectively, in rat adult cardiomyoctes with simulated I/R. Besides, the moderate H
2
O
2
PoC-afforded cardioprotection abrogated by spermine could be rescued by STAT3 overexpression in rat cardiomyoctes with simulated I/R. Moreover, H
2
O
2
PoC enhanced the expression of serine 727 phosphorylation of STAT3 and STAT3 in mitochondria of rat cardiomyoctes with simulated I/R. Furthermore, immunofluorescence and co-immunoprecipitation assay revealed a co-localization/interaction of STAT3 and MCU in rat cardiomyoctes with moderate H
2
O
2
PoC at reperfusion 5 min and reperfusion 30 min but not in I/R cells. Co-immunoprecipitation further confirmed that STAT3 interacted with the N-terminal domain (NTD) of MCU in rat cardiomyoctes with moderate H
2
O
2
PoC. These findings indicate that STAT3 mediates the cardioprotection of moderate H
2
O
2
PoC against I/R injury by alleviating mitochondrial calcium overload through inhibiting MCU opening via the interaction with the NTD of MCU. Our finds provide new insight into the mechanisms of STAT3 in the cardioprotection.
Abstract 313: Inhibition of Resistin Protects against Heart Failure Injury by Targeting Gadd45α
Authors: Zhao Baoyin , Lebeche Djamel
Published: October 2019

Background:
Resistin is an adipocyte-secreted hormone, increased Resistin levels are associated with the development of insulin resistance, diabetes, and cardiovascular disease. However, the role of Resistin in heart failure is still unclear. The aim of the present study is to investigate the molecular mechanisms underlying Resistin’s action in heart failure.

Methods:
C57BL/6 (WT) and Resistin knockout (RKO) mice were used. A mice transverse aortic constriction (TAC) induced heart failure model was established, and cardiac function and hemodynamic changes were measured by echocardiography and left ventricular pressure volume loops. A recombinant adeno-associated virus 9 (AAV9) vector was used to deliver resistin gene into the myocardium. RNA sequence was used to determine differentially expressed mRNA in heart from RKO and WT mice subjected to TAC or sham surgeries. Quantitative real-time PCR (qRT-PCR) and Western blot analyses were used to verify the expression levels of various fibrosis-related factors (collagen I, Fibronectin, and CTGF) and hypertrophy-related markers (ANF, β-MHC and Acta1).

Results:
Resistin inhibition attenuated while resistin overexpression (via AAV9-Resistin) exacerbated TAC-induced hypertrophy and cardiac dysfunction in WT and Resistin KO mice. Improvement in cardiac function was associated with decreased myocardial fibrosis as evidenced by histology and decreased mRNA levels of collagen I, fibronectin and CTGF in RKO-TAC hearts compared to WT or RKO-AAV9. In addition, myocardial tissue expression levels of ANF, β-MHC and Acta1 were also reduced in the RKO-TAC hearts. RNA sequencing analysis reveals that Gadd45a was significantly increased in RKO-TAC mice. The expression level of Gadd45a was verified by qRT-PCR and Western blot. Gadd45a expression was significantly downregulated in the RKO-TAC hearts following AAV9-Resistin delivery, suggesting that Resistin may control the regulation of Gadd45a.

Conclusions:
Resistin gene knockout increased Gadd45a expression in mouse model of heart failure, significantly reduced myocardial fibrosis and hypertrophy and improved heart function. The results of this study indicate that inhibition of resistin is a potential therapeutic method for treating heart failure.
Abstract 327: MicroRNA-574-FAM210A Axis Maintains Mitochondrial Translational Homeostasis and Influences Pathological Cardiac Remodeling
Authors: Yao Peng , Wu Jiangbin , Subbaiah Kadiam C. Venkata , Jiang Feng , Hedaya Omar , Tang Wai Hong Wilson , Small Eric , Yan Chen
Published: October 2019

Rationale:
Translational control is a critical regulatory step in altering protein synthesis under disease conditions. Aberrant synthesis of mitochondrial proteins impairs cardiac function and causes heart disease. However, the mechanism of translational control in mitochondria and during cardiac disease remains underexplored.

Objective:
We have found that multiple pathogenic cardiac stressors induce the expression of miR-574 guide and passenger strands (miR-574-5p/3p) in humans and mice. Here, we aim to define a new miR-574-FAM210A axis that regulates cardiac mitochondrial translational homeostasis and prevents adverse cardiac remodeling.

Methods and Results:
Echocardiography, histology and biochemical analyses were used to evaluate the cardiac function of miR-574 knockout mice, which exhibit severe cardiac hypertrophy, fibrosis, and cardiac dysfunction under heart failure-triggering stresses. miR-574-5p/3p mimics that were delivered systematically using nanoparticles reduced cardiac pathogenesis with disease insults. Transcriptome analysis of miR-574-null hearts has uncovered
FAM210A
as a common target mRNA for miR-574-5p/3p. Interactome and translational state analyses identify a FAM210A-bearing trimeric complex that promotes the translation of mitochondrial encoded electron transport chain genes and modulates mitochondrial activities. Moreover, the phenotypic characterization of tamoxifen-inducible CM-specific Fam210a knockout mice suggests that FAM210A plays a critical role in maintaining normal cardiac mitochondrial morphology and function as well as cardiac health and organismal viability.

Conclusions:
miR-574-5p and miR-574-3p protect against pathological cardiac remodeling through regulation of FAM210A, a key translational control factor of mitochondrial encoded genes. Thus, we discovered a novel miR-574-FAM210A pathway that modulates cardiac mitochondrial translational homeostasis and influences cardiac remodeling in heart failure.
Abstract 330: A HaloTag-TEV Genetic Cassette for Mechanically Probing Native Titin
Authors: Rivas-Pardo Jaime Andrés , Li Yong , Mártonfalvi Zsolt , Tapia-Rojo Rafael , Unger Andreas , Fernández-Trasancos Ángel , Herrero-Galán Elías , Velázquez-Carreras Diana , Linke Wolfgang A , Fernández Julio M , Alegre-Cebollada Jorge
Published: October 2019

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces interact with titin in the sarcomere, enabling muscle contraction. However, testing these mechanical hypotheses on native titin in its natural environment has remained inaccessible to conventional genetics, biophysics and molecular biology tools. To overcome these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in titin. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of native titin under physiological forces using recently developed magnetic tweezers. At physiological pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors.
Abstract 333: Cardiomyocyte Hypercontractility is an Adaptational Response to Stiffness in High-Fat Diet Mice
Authors: Janssens Johannes V , Raaijmakers Antonia J , Koutsifeli Parisa , Mellor Kimberley M , Curl Claire L , Delbridge Lea M
Published: October 2019

Diabetic heart disease typically features a restrictive/ hypertrophic cardiac phenotype characterised by diastolic dysfunction with preserved systolic performance. Little is known about the early adaptational mechanisms that underpin maintenance of systolic performance in diabetic diastolic dysfunction and how this may contribute to disease progression. This study aimed to evaluate cardiomyocyte contractility in a high-fat diet (HFD) mouse model of diastolic dysfunction. Echocardiography (GE Vivid 9) was performed in 33 wk old male C57Bl/6 mice fed a high-fat diet (HFD, 43% kcal fat, 24 wks duration). Isolated cardiomyocytes (paced 2Hz, 2.0mM Ca
2+
, 37C) were subjected to progressive axial stretch. Sarcomere length/ shortening, tension and intracellular calcium transients (Fura-2AM, 5μM) were simultaneously measured (Myostretcher, Ionoptix). HFD hearts displayed
in vivo
(22.2±1.4 vs 16.7±1.0 E/E’; p<0.05) and
in vitro
diastolic dysfunction (0.244±0.039 vs 0.075±0.017 nN/pl/ %cell stretch; p<0.05). HFD cardiomyocytes exhibited length-dependent hypercontractility with augmented developed tension amplitude at maximal stretch (4.891±0.942 vs 2.484±0.677 nN/pl; p<0.05), but not basal stretch. Hypercontractility could not be attributed to a difference in Ca
2+
transient amplitude but was correlated with length dependence of Ca
2+
sensitivity (n=17 cells, R
2
=0.4039 p<0.05). During progressive cardiomyocyte stretch, systolic tension slopes were strongly correlated with diastolic tension slopes (cardiomyocyte stiffness) (n=30 cells; R
2
=0.8442, p<0.05) while there was no difference in the ratio of systolic/diastolic tension slopes indicating maintained length-dependent activation. These findings provide the first evidence that cardiomyocyte hypercontractility and stiffness are strongly linked. High-fat diet cardiomyocytes may operate near maximal contractile capacity to maintain basal cardiac output, potentially limiting capacity for cardiac output increase in response to physiological stressors. An aberrant myofilament post-translational modification profile represents a potential mechanism through which cardiac dysfunction arises during the development of diabetes.
Abstract 334: Cardiac Myosin Binding Protein C Phosphorylation Regulates Calcium Homeostasis
Authors: Kumar Mohit , Haghighi Kobra , Kranias Evangelia , Sadayappan Sakthivel
Published: October 2019

Rationale:
Cardiac myosin binding protein-C (cMyBP-C) is heavily phosphorylated to regulate normal cardiac function under basal conditions. However, its phosphorylation level is significantly decreased in patients with heart failure and atrial fibrillation. Furthermore, decreased cMyBP-C phosphorylation causes reduced myofilament contractility and decreased calcium sensitivity. The impact of such decreases in cMyBP-C phosphorylation and myofilament calcium sensitivity on the overall calcium handling and potential induction of arrhythmogenesis is not known.

Objective:
To determine the necessity and sufficiency of cMyBP-C phosphorylation to regulate calcium cycling and contractility at the isolated cardiomyocyte level.

Methods and Results:
Contractile properties and calcium kinetics were measured in intact cardiomyocytes isolated from 3-month-old, mixed sex, mice expressing phospho-ablated (S273A/S282A/S302A) cMyBP-C (AAA) or phospho-mimetic (S273D/S282D/S302D) cMyBP-C (DDD) and nontransgenic (NTG) control mice. AAA cells displayed a significant decrease in fractional shortening compared to NTG and DDD cells (9.4%
vs
12.8% in NTG, and 12.3% in DDD, p<0.0001). Similarly, AAA myocytes demonstrated abnormal calcium kinetics with increased diastolic calcium levels (20%, p<0.001
vs
. NTG and DDD) and prolonged decay time of the calcium transient (26%, p<0.01
vs
NTG and DDD) when compared with NTG and DDD myocytes. Caffeine-induced calcium release in AAA myocytes indicated no change in SR calcium content, while sodium-calcium exchanger function, assessed as the time constant (τau) of calcium decline, was increased (89%, p<0.01
vs
NTG). However, these depressive effects in AAA myocytes were relieved by isoproterenol (100 nmol/L) stimulation. Furthermore, stress conditions (2 Hz + ISO) increased after-contractions in AAA cardiomyocytes (60% in AAA
vs
13% in NTG, p<0.001 and 15% in DDD, p<0.001). In addition, preliminary data indicate increased susceptibility of the AAA mice to arrhythmias under stress conditions in vivo.

Conclusion:
Dephosphorylation of cMyBP-C is sufficient to reduce sarcomere contractility and impair calcium cycling resulting in spontaneous after-contractions and arrhythmias under stress conditions.
Abstract 346: Tuberin S1365 Phosphorylation Regulates Mechanistic Target of Rapamycin Complex 1 (mTORC1) Pathological Signaling While Sustaining Metabolic Sensor Function
Authors: Dunkerly-Eyring Brittany L , Vera Miguel P , Oeing Christian U , Ranek Mark J , Kass David A
Published: October 2019

Rationale:
The Mechanistic Target of Rapamycin complex 1 (mTORC1) integrates signaling and sensory inputs to maintain cardiomyocyte homeostasis, and itself is negatively regulated by the signaling nexus tuberin (TSC2). We identified a novel TSC2 phosphorylation site S1365 (pS1365) targeted by protein kinase G (PKG), which suppressed hormonal growth factor (PE or ET1)-stimulated mTORC1 activity to attenuate pathological cardiomyocyte hypertrophy. This was recapitulated during growth factor stimulation with expression of a phospho-null (S1365A) or a phospho-mimetic (S1365E) TSC2 that exacerbated or blunted mTORC1 activation, respectively. The nature of TSC2 pS1365 as a potential metabolic sensor is unknown and will provide mechanistic insight into the TSC2 kinase input hierarchy that regulates the homeostatic function of mTORC1.

Objective:
To determine how pS1365 affects the ability of TSC2 to integrate metabolic dependent signals to regulate mTORC1.

Methods/Results:
TSC2 KO MEFs were infected with TSC2 WT or S1365A adenovirus, and then stimulated with ET1 (hormonal stress that also activates mTORC1), and 2-DG (AMPK stimulation). Both groups responded with similar decreases in mTORC1 activation regardless of pS1365. Phosphorylation of the AMPK site on TSC2 (S1387) was increased in all groups despite the presence of a phospho-null S1365. Both MEFs and neonatal rat cardiomyocytes (NRCMs) infected with TSC2 WT, S1365A, or S1365E adenovirus similarly increased mTORC1 activation with insulin (PI3K-Akt-TSC2 pathway) treatment. Serum withdrawal from NRCMs reduced mTORC1 activation in all groups regardless of whether a WT, S1365A, or S1365E TSC2 was expressed. In NRCMs subject to hypoxia (a combination of Erk, Akt, AMPK signaling), there was a similar observation with only nominal changes between WT, S1365A, and S1365E TSC2.

Conclusions:
The energy and nutrient sensing role of the TSC2-mTORC1 pathway remains intact regardless of the phospho-status of TSC2 S1365. These findings provide important mechanistic insight into the function of TSC2 pS1365 as a potent suppressor of pathological mTORC1 activation while not affecting the ability of TSC2 to respond to the metabolic dependent signals – AMPK (energy), PI3K-Akt (insulin), and serum starvation.
Abstract 348: Homeobox A4 Suppresses Vascular Smooth Muscle Cell Phenotypic Switching as a Novel Regulator of YAP/TEAD Transcriptional Activity
Authors: Kimura Masahiro , Horie Takahiro , Kimura Takeshi , Ono Koh
Published: October 2019

The Hippo signaling pathway is involved in the pathophysiology of various cardiovascular diseases. Yes-associated protein (YAP) and transcriptional enhancer activator domain (TEAD) transcriptional factors, the main transcriptional complex of the Hippo pathway, were recently identified as modulators of phenotypic switching of vascular smooth muscle cells (VSMCs). However, the intrinsic regulator of YAP/TEAD-mediated gene expressions in VSMCs remains to be elucidated, then we sought to investigate a novel regulator of YAP/TEAD transactivation involved in vascular diseases.

We first investigated novel YAP/TEAD regulators using lentiviral shRNA library in HEK 293T-based TEAD-responsive reporter cell line and detected Homeobox A4 (HOXA4) as a potent repressor of YAP/TEAD transcriptional activity. HOXA4 attenuated YAP/TEAD-mediated gene expression independently of YAP phosphorylation, and co-immunoprecipitation assays revealed that HOXA4 interacts with TEADs but not YAP. Mechanistically, HOXA4 attenuated YAP/TEAD-mediated transcription by competing with YAP for TEAD binding. We also clarified that the expression of HOXA4 is relatively abundant in the vasculature, especially in VSMCs. In vitro experiments in human VSMCs showed HOXA4 maintains the differentiation state of VSMCs via inhibition of YAP/TEAD-induced phenotypic switching characterized by cell morphology, cell proliferation, and gene expression patterns. We generated Hoxa4-decifient mice and confirmed the downregulation of smooth muscle-specific contractile genes and the exacerbation of vascular remodeling after carotid artery ligation in vivo.

Our results demonstrate HOXA4 is a novel repressor of VSMC phenotypic switching by inhibiting YAP/TEAD-mediated transcription. These findings give us a better understanding of the vascular pathophysiology and a novel therapeutic approach for vascular remodeling.
Abstract 351: Suppression of Store Operated Ca
2+
Entry Components, dStim and dOrai, Results in Dilated Cardiomyopathy
Authors: Petersen Courtney , Smyth Jeremy T , Wolf Matthew J
Published: October 2019

Cardiac disease is a leading cause of morbidity and mortality throughout the western world with current treatment options limited to palliative pharmacological or invasive therapy. The discovery of curative treatments will rely on thorough understanding of the molecular mechanisms that govern cardiac patho-physiology. Significantly, irregularities in calcium (Ca
2+
) homeostasis are a major contributing factor to the pathogenesis of cardiac diseases, and targeting Ca
2+
signaling mechanisms may therefore be an important approach to novel therapeutic development. Our lab has recently identified a requirement for the store operated Ca
2+
entry (SOCE) pathway, whereby Ca
2+
enters the cell in response to endo/sarcoplasmic reticulum (ER/SR) store depletion, in
Drosophila
cardiac physiology. Heart-specific suppression of key SOCE pathway components, Stim and Orai, induced dilated cardiomyopathy characterized by enlarged end-diastolic and end-systolic diameters and decreased fractional shortening. Animals with SOCE suppression had a significant delay in development and ultimately died considerably earlier than controls, suggesting pathological impairment of cardiac function. Additionally, Stim and Orai suppressed hearts exhibited highly disorganized or disrupted myofibrils, suggesting either defective heart development, tissue remodeling, or degeneration resulting from disrupted Ca
2+
homeostasis. We are currently testing these possibilities by restricting Stim and Orai suppression to specific lifecycle stages, and we are analyzing Ca
2+
transients in intact hearts to determine whether SOCE is required for contractile Ca
2+
cycling or may have a contraction-independent signaling role. Collectively, our results demonstrate an essential role for SOCE in normal cardiac physiology, and present how powerful genetic tools and
in vivo
analyses in
Drosophila
will allow us to define the mechanistic basis for this requirement.
Abstract 363: Systems Network Genomic Analysis Reveals the Role Of MURC/Cavin-4 in Cardiac Ischemia/reperfusion Injury
Authors: Nishi Masahiro , Ogata Takehiro , Nakanishi Naohiko , Higuchi Yusuke , Sakamoto Akira , Tsuji Yumika , Matoba Satoaki
Published: October 2019

Background:
Ischemia/reperfusion (I/R) injury is a critical issue in the development of treatment strategies for ischemic heart disease. Muscle-restricted coiled-coil protein (MURC)/Cavin-4, which is a component of caveolae, is involved in the pathophysiology of dilated cardiomyopathy and cardiac hypertrophy. However, the role of MURC in cardiac I/R injury remains unknown.

Objective:
To elucidate the role of MURC in cardiac I/R injury.

Methods and Results:
The systems network genomic analysis based on PC-corr network inference on microarray data between wild-type and MURC knockout (KO) mouse hearts described a network of discriminating genes associated with reactive oxygen species (ROS). To demonstrate the prediction, we investigated the effect of MURC deletion in cardiac I/R injury. MURC deletion in IR-injured mouse hearts decreased infarct size and preserved heart contraction with the inhibition of ROS production and ROS-related gene expressions such as EGR1 and DDIT4 as well as EGR1 protein level. PC-corr network inference integrated with a protein-protein interaction network prediction showed that MURC is also involved in the apoptotic pathway. We confirmed the upregulated activity of STAT3, which is a transcription factor of anti-apoptotic signaling, with the increase of BCL2 mRNA expression and protein level and the decrease of cleaved Caspase 3 protein level in MURC KO compared with WT mouse hearts after I/R. TUNEL assay showed that MURC modulates the apoptosis in cardiomyocytes exposed to hydrogen peroxide. STAT3 inhibitor cancelled the cardioprotective effect of MURC deletion in I/R-injured heart and the anti-apoptotic effect of MURC knockdown in cardiomyocytes.

Conclusions:
Our findings suggest that MURC plays a pivotal role in the regulation of ROS-induced cell death and STAT3-meditated anti-apoptotic signaling in cardiac I/R injury. MURC may be a therapeutic target for cardiac I/R injury.
Abstract 364: Klotho Gene Deficiency Causes Heart Failure via Disruption of Phosphate Metabolism
Authors: Chen Kai , Sun Zhongjie
Published: October 2019

Heart failure is the major cause of mortality for patients with chronic kidney damage (CKD). Although a decrease in plasma klotho levels has been linked to chronic kidney disease (CKD), the relationship between Klotho and heart failure is unclear. Here, using Klotho mutant homozygous (KL -/-) mice, we found that Klotho deficiency caused hyperphosphatemia and heart failure. Normalization of serum phosphorous by dietary phosphate restriction rescued Klotho deficiency-induced heart failure in male mice. However, low phosphate diet did not rescue hyperphosphatemia and heart failure in KL (-/-) female mice. Therefore, hyperphosphatemia may be an important mechanism of Klotho deficiency-induced heart failure. Dietary phosphate restriction did not prevent estrogen depletion in KL (-/-) female mice, suggesting that estrogen depletion may involve in Klotho deficiency induced hyperphosphatemia and heart failure. Normalization of serum estrogen level by
17β-estradiol
prevented cardiac remodeling and heart dysfunction in KL (-/-) female mice. Moreover, treatment with
17β-estradiol
normalized phosphate metabolism via regulating renal NaPi co-transporter expression. Treatment with
17β-estradiol
abolished mitochondrial dysfunction and cardiac apoptosis in Klotho deficient mice. This study demonstrates for the first time that hyperphosphatemia is an important mediator of heart failure which can be prevented by dietary phosphate restriction in male KL (-/-) mice. Estrogen deficiency mediates hyperphosphatemia leading to heart failure due to klotho deficiency in female mice.
Abstract 370: A Novel Algorithm for the Collective Integration of Single Cell Rna-seq During Embryogenesis
Authors: Gong Wuming , Singh Bhairab N , Das Satyabrata , Garry Mary G , Garry Daniel J
Published: October 2019

Background:
Single cell RNA-seq (scRNA-seq) over specified time periods has been widely used to dissect the cell populations during mammalian embryogenesis. Integrating such scRNA-seq data from different developmental stages and from different laboratories is critical to comprehensively define and understand the molecular dynamics and systematically reconstruct the lineage trajectories.

Methods:
Here, we describe a novel algorithm to integrate heterogenous temporal scRNA-seq datasets and to preserve the global developmental trajectories. scNCA not only successfully corrected the batch effects, but also preserved the global structure of gene expression. For both balanced and imbalanced synthetic scRNA-seq data, we found that scNCA had significantly better performance of batch correction than mnnCorrect and Seurat alignment, on mixing cells from different batches, revealing the global trajectories, and bringing together the cells from the same lineage.

Results:
We applied this algorithm and approach to integrate 3,387 single cells from seven heterogenous temporal scRNA-seq datasets, and reconstructed the cell atlas of early mouse cardiovascular development from E6.5 to E9.5. Using this integrated atlas, we identified an Etv2 downstream target,
Ebf1
, as an important transcription factor for mouse endothelial development.

Conclusions:
In summary, we presented scNCA as a novel tool to correct the batch effect of temporal scRNA-seq. We used scNCA to integrate 3,387 single cells from seven heterogenous temporal scRNA-seq datasets of mouse early cardiovascular development , and identified an Etv2 downstream target,
Ebf1
, as an important transcription factor for mouse endothelial development. We provide the R/TensorFlow implementation of scNCA at https://github.com/gongx030/scNCA. The integrated mouse early cardiovascular development data can be explored at https://heartmap.umn.edu/scNCA.
Abstract 374: Etv2 Transcriptionally Regulates
Yes1
and Promotes Cell Proliferation During Embryogenesis
Authors: Singh Bhairab , Gong Wuming , Das Satyabrata , Theisen Joshua , Sierra-Pagan Javier , Yannopoulos Demetris , Skie Erik , Shah Pruthvi , Garry Mary , Garry Daniel
Published: October 2019

Etv2, an Ets-transcription factor, governs the specification of the earliest hemato-endothelial progenitors during embryogenesis. While the transcriptional networks during hemato-endothelial development have been well described, the mechanistic details are incompletely defined. In the present study, we described a new role for Etv2 as a regulator of cellular proliferation via
Yes1
in mesodermal lineages. Analysis of an Etv2-ChIPseq dataset revealed significant enrichment of Etv2 peaks in the upstream regions of cell cycle regulatory genes relative to non-cell cycle genes. Our bulk-RNAseq analysis using the doxycycline-inducible Etv2 ES/EB system showed increased levels of cell cycle genes including
E2f4, Gadd45g
and
Ccne1
as early as 6h following Etv2 induction. Further, EdU-incorporation studies demonstrated that the induction of Etv2 resulted in a ~2.5-fold increase in cellular proliferation, supporting a proliferative role for Etv2 during differentiation. Next, we identified
Yes1
as the top-ranked candidate that was expressed in

Etv2-EYFP
+

cells at E7.75 and E8.25 using single cell RNA-seq analysis. Doxycycline-mediated induction of Etv2 led to an increase in
Yes1
transcripts in a dose-dependent fashion. In contrast, the level of
Yes1
was reduced in
Etv2 null
embryoid bodies. Using bioinformatics algorithms, biochemical, and molecular biology techniques, we show that Etv2 binds to the promoter region of
Yes1
and functions as a direct upstream regulator of
Yes1
during embryogenesis. These studies enhance our understanding of the mechanisms whereby Etv2 governs mesodermal fate decisions early during embryogenesis.
Abstract 404: Deterministic Paracrine Repair of Injured Myocardium using Microfluidic Cocooning of Heart Explant-Derived Cells
Authors: Kanda Pusphinder , Benavente-Babace Ainara , Godin Michel , Davis Darryl R
Published: October 2019

Previous work has shown that cocooning heart explant-derived cells (EDCs) within protective nanoporous gel capsules before intra-myocardial injection increases the retention of transplanted cells and the paracrine production of nanoparticles to improve post infarct cardiac function. In this study, we investigated the influence of cocoon size and intracapsular cell number on cell-treatment outcomes using a newly developed microfluidic-based (MF) cellular cocooning platform.

Methods/Results:
Traditional vortex-based encapsulation (Vx) inherently provides cocoons of varying diameters (30-100 μm; 68±5 μm). By altering the flow pressure ratios and the nozzle diameters within the MF chip, we encapsulated human EDCs within small (51±1 μm, MF50) and large (90±1 μm, MF90) diameter nanoporous gel cocoons for comparison with standard Vx-defined capsules (71±1 μm, MF70). MF cocooning mirrored the expected Poisson distribution with smaller cocoons having a greater proportion of single cells while larger diameter cocoons contained greater proportions of multicellular aggregates. Immunodeficient mice underwent left coronary artery ligation 1 week before randomization to echocardiographic guided intra-myocardial injection of EDCs (suspended or variable diameter cocoons) or vehicle. Increasing cocoon diameter stimulated progressive salutary effects on post-infarct function (ejection fraction), scar burden and newly generated peri-infarct blood vessels (isolectin B4+) and cardiomyocytes (BrdU+/TNT+) 4 weeks after treatment. Bioluminescent imaging of luciferase tagged cells revealed increasing cocoon diameter reduced the rate of cell clearance from injured tissues. Disrupting cell-cell contact within the capsules (using a custom antibody cocktail to block E/P-selectin and N-cadherin) reduced the amount and profile of pro-healing cytokines + nanoparticles delivered to injured myocardium.

Conclusions:
Increasing cocoon diameter and cell occupancy within protective nanoporous gel cocoons boosts paracrine-mediated repair of damaged myocardium by slowing clearance of cells from injured tissues and the number of cytokines + nanoparticles secreted by micro-encapsulated cells.
Abstract 410: A Novel Combination Therapy Using First Trimester Human Umbilical Cord-Derived Mesenchymal Stromal Cells and Endothelial Progenitor Cells Significantly Improves Angiogenesis and Cardiac Recovery Following Myocardial Infarction
Authors: Iqbal Farwah , Johnston Alexander , Mander Poonam , Wyse Brandon , Wu Jun , Li Ren-Ke , Szaraz Peter , Librach Clifford
Published: October 2019

Introduction:
Umbilical cord-derived MSC show pericyte-like characteristics and demonstrate significant angiogenic potential via paracrine and direct support to the vasculature. Endothelial progenitor cells (EPC) have the potential to initiate vascular structures while maintaining paracrine properties following ischemic injury.

Hypothesis:
A combination therapy of first trimester umbilical cord MSC (FTM HUCPVC) with rat EPC will promote significant angiogenesis, cardiomyocyte survival and lead to functional cardiac recovery in a rat model of MI.

Methods:
MI was induced by LAD ligation on 8-week-old Fox
rnu
rats. 1 week later, rats with notable decrease (<30%) in ejection fraction (EF) were randomly separated into 4 treatment groups (n=9) and received intramyocardial injection of:
G1:
Media;
G2:
FTM HUPVC (3х10
6
cells/rat);
G3:
EPC (2.2х10
6
cells/rat);
G4: FTM HUCPVC - EPC combination
(1:2, total cells 3х10
6
cells/rat). Endpoint analysis was at 5-days and 4-weeks after injection.

Results:
5-days after injection, exclusively
G4
animals showed significant cardiac recovery, improved EF and FS compared to
G1
-
G2
-
G3
(
p
<0.01). IHC analysis showed significantly less apoptosis (caspase-3), more protease activity and sarcomeric actin in combination groups
G4
compared to
G1-G2-G3
(
p
<0.001). More FTM HUCPVC and EPC colocalized adjunct to small capillaries in
G4
. Significant increase of human Angpt-2 was detected in
G4
compared to
G1-G2-G3
(p<0.0001). Analysis at 4-weeks post-injection showed significant improvements in end-systolic volume (128μl) and EF (43%) in
G4
compared to
G2
(160μl) (35%)
G3
(180μl) (30%) respectively
(B)
. LV contractility (dpdt, tau) significantly improved in
G4
compared to
G1
,
G2
,
G3
(p<0.001). Significant reduction in scar tissue and increased LV myocardial mass was found in
G4 and G2.
IHC analysis showed significantly more and larger (9um) capillaries localized in LAD injured myocardium in
G4
compared to
G1-G2-G3
(
p
<0.001). Significantly more sarcomeric actinin and connexin-43 was identified in
G4
compared to
G1-G2-G3
.

Conclusions:
Our results show the superior potential of FTM HUCPVC - EPC combination cell therapy for faster and greater cardiovascular repair and recovery compared to single-cell-type treatments for MI.
Abstract 417: A 3D iPSC-derived Scaffold-Assisted Microfluidic Model of Ventricular Ejection
Authors: Michas Christos , Nautiyal Pranjal , Agarwal Arvind , White Alice E , Chen Christopher S
Published: October 2019

Organ-on-a-chip technology fosters the potential of in vitro models of cardiac function. Nevertheless, modeling the ventricular ejection has been challenging due to the lack of 3D scaffolds of ventricular-like geometry that can accommodate the tissue while providing structural stability and contractile directionality. Using two-photon direct laser writing, we fabricate a concave, 1mm
3
scaffold that is then embedded in a microfluidic, organ-on-a-chip system. We seed the system with iPSC-derived cardiac tissue and demonstrate the ability of the scaffold to preserve the 3D concave tissue structure, promote cardiomyocyte alignment and generate significant ejection fractions. The presented system sets the foundation for novel in vitro studies on ventricular function and remodeling.
Abstract 419: Beneficial Effects of scaRNA Modulated Human iPSC-derived Cardiomyocytes Exposed Under Hypoxic Conditions
Authors: Rajasingh Sheeja , Sigamani Vinoth , Czirok Andras , Isai Dona G , Selvam Vijay , Rajasingh Johnson
Published: October 2019

Introduction:
Studies have shown that mRNA splicing is regulated by small cajal-body associated RNAs (scaRNAs) and plays a significant role in mammalian cardiac development and differentiation, but the potential contribution to human in vitro differentiation of iPSCs into CMCs remains unknown. In this study, the comparison of scaRNA20 modified and unmodified iPSCs derived-CMCs (iCMCs) will allow us to understand the role of scaRNAs in cardiac differentiation.

Hypothesis:
We hypothesize that the scaRNA20 modulated iCMCs are capable of withstanding better at low-oxygen environment than the normal iCMCs which will facilitate for cardiac therapy.

Methods and Results:
To show how ischemic cardiomyopathy effects on CMC contractility, skin fibroblast derived normal iCMCs (SF-N-iCMCs) and scaRNA20-OE-iCMCs were subjected to hypoxic condition (1% O
2
). Our particle image velocimetry (PIV) analysis shows that the contractility is reduced in normal iCMCs whereas the scaRNA20-OE-iCMCs maintained the contractility under hypoxic condition
(Fig. A-C)
. Moreover, the qRT-PCR data show that the SF-N-iCMCs exposed to hypoxic condition decreased the expression of scaRNA20
(Fig. D).
These data suggest that the scaRNA20-OE-iCMCs are capable of withstanding the low-oxygen environment at the infarcted heart.

Conclusions:
We report for the first time the beneficial effects of scaRNA20 modulated CMCs prior to transplantation will be an effective therapy for cardiac regeneration. 