bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 270: mTORC1 is Essential for Cardiomyocyte Homeostasis and Female Sex Related Cardioprotection
Authors: Guergen Dennis B , Beiner Nelli , Catar Rusan , Mielke Thorsten , Busch Hauke , Boerries Melanie , Dengjel Joern , Brink Marijke , Grahammer Florian , Huber Tobias B , Kusch Angelika , Dragun Duska
Published: June 2021

Mechanistic target of rapamycin (mTOR) kinase operates in two functionally and structurally distinct protein complexes mTORC1 and mTORC2. Raptor is a defining mTORC1 protein critical for cardiac growth in response to stress. Our previous studies showed sexual dimorphism in LV remodeling of normotensive DOCA-salt mice, where females retain normal LV architecture and males developed maladaptive LV hypertrophy. We hypothesized involvement of mTORC1 in regulation of this sexual dimorphism and induced cardiomyocyte (CM) specific raptor deletion (ra-cKO) in our model of normotensive mineralocorticoid challenge. Deletion of raptor in CM lead to decreased mTORC1 downstream target activation of 4E-BP1 and strong compensatory hyperactivation of mTORC2 target AKT in both sexes.

In contrast to healthy wild type females, female DOCA-salt ra-cKO mice developed severe dilative phenotype with tremendous interstitial fibrosis (15fold increase vs. WT) of similar extent as males. Functionally, ra-cKO females had stronger decrease in EF (51 vs. 26%) than ra-cKO males (50 vs. 32%). Greater loss of sarcomeric ultrastructure and mitochondrial morphology in transmission electron microscopy was observed in male ra-cKOs. Analysis of label-free proteomics and transcriptomics approaches revealed genotype and sex-specific clustering and identified significantly regulated single proteins or gene sets. Concordant to observed ultrastructural changes, down-regulation of genes for Z-disc, t-tubule and sarcomere organization was prominent in male hearts, whereas females displayed up-regulation of gene sets for inflammation, angiogenesis and mitochondrial function.

Intact mTORC1 is indispensible for CM homeostasis of both sexes as reflected by development of severe phenotype even by a mild stressor in our model. More specific, female sex-specific cardioprotection is completely lost in absence of mTORC1. Despite similar phenotypic changes in both sexes, sex-specific differences in regulation of gene expression points toward sex-specific functional selectivity of mTORC1 in regulation of CM homeostasis. Given the broad indications for mTOR inhibitors and especially used high concentration in oncology, one could anticipate adverse cardiac side effects.
Abstract 28: Automated Characterization and Quantification of Cardiomyocyte Proliferation and Apoptosis in Post Myocardial Infarction Tissue Images
Authors: Gross Polina , Honnorat Nicolas , Varol Erdem , Wallner Markus , Trappanese Danielle , Sharp Thomas , Starosta Timothy , Duran Jason , Koller Sarah , Davatzikos Christos , Houser Steven
Published: June 2021

Detection of cardiac cell death and cardiomyocyte (CM) proliferation after ischemic injury often hinges on quantitative image analysis of cardiac histopathology. Currently, the analysis of cardiac images has greatly relied on manual measurements of the number or percentages of apoptotic CM nuclei, as well as newly forming CM nuclei. The current manual methodology for image analysis is a tedious, subjective and inconsistent approach. Nuquantus (Nuclei quantification utility software) is a novel machine learning-based analytical method, which classifies and quantifies CM nuclei in post myocardial infarction (MI) fluorescent images. Nuquantus accelerated the image analysis by 6 fold from 12.6 ± 5.6 minutes per image by manual approach to 2.2 ± 0.9 minutes (mean ± standard deviation). The analysis variability reflected by the inter-observer agreement Fleiss Kappa was improved from 0.511 ± 0.006 to 0.695 ± 0.006 (Fleiss Kappa ± standard error (***P<0.0001). The percentage of proliferating CMs out of all proliferating cell types in the heart was compared between Nuquantus and manual approaches in 1 week post MI mouse heart. CM nuclei positive to 5-ethynyl-2′-deoxyuridine (EdU) and DAPI stainings were determined to undergo DNA synthesis in the following regions: Infarct Area 0.95 ± 0.26% vs. 0.24 ± 0.07%, Border Zone 0.67±0.40% vs. 0.21±0.10%, Remote Zone 1.39±0.80% vs. 1.40±0.60% and sham 3.85±2.56% vs. 1.32±1.32% (mean ± standard error) Nuquantus vs. manual count, respectively (N=4). Nuquantus identified and quantified CM nuclear DNA fragmentation in wild-type mouse hearts stained by TUNEL assay and DAPI. In the TUNEL negative controls, we detected 0% vs. 0.1±0.1% apoptotic CM nuclei, and in the positive TUNEL control 84.77±3.49% vs. 95.64±0.69% apoptotic CM nuclei (mean ± standard error, Nuquantus vs. manual). No statistical significance was detected between the different methods. In summary, Nuquantus provides innovative computerized methodology to reliably analyze cardiac tissue images, significantly facilitates image analysis and minimizes human bias. Moreover, validation of Nuquantus indicates that CM proliferation in the remodeling heart is a very rare event.
Abstract 281: Sigma-1 Receptor Dependent Pathway for a Protective Endoplasmic Reticulum Stress Response in Cardiomyocytes
Authors: Alam Shafiul , Orr A. Wayne , Pattillo Christopher B. , Bhuiyan Md. Shenuarin
Published: June 2021

Rationale:
We recently reported that Sigma 1 receptor (Sigmar1) is a molecular chaperone protein highly expressed in the heart. Studies involving different cancer and neuronal cell lines indicated Sigmar1 resides in the mitochondrion-associated ER membrane (MAM). However, the subcellular localization of Sigmar1 and the molecular function in ER-stress remains unknown in cardiomyocytes. Here we describe a function for Sigmar1 as an effector of an adaptive ER stress response.

Objective:
The objective of this study was to elucidate functional roles of Simgar1 in ER-stress in cardiomyocytes.

Methods and Results:
Subcellular fractionation of the mouse heart showed extensive localization of Sigmar1 in the MAM and mitochondrial fraction. To define the function in an ER-stress response, we used small interfering RNA-mediated Sigmar1 knockdown and adenovirus-mediated overexpression in cultured neonatal rat ventricular cardiomyocytes. We treated with tunicamycin to induce ER stress. In cardiomyocytes, tunicamycin increased C/EBP-homologous protein (CHOP) expression; Sigmar1 overexpression significantly decreased the CHOP expression. We found that Sigmar1 overexpression was sufficient to activate the nuclear transport of spliced X-box binding protein 1 (Xbp1s) with minimal effects in other adaptive ER stress proteins. Sigmar1 knockdown decreased the nuclear transport of Xbp1s, increased the expression of nuclear CHOP and significantly increased LDH release. We also observed significant Sigmar1 expression dependent increases in mitochondrial respiration in cardiomyocytes under ER stress. Hence, Sigmar1 can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Xbp1s.

Conclusions:
Sigmar1 is an essential component of the adaptive ER stress response in cardiomyocytes. Sigmar1 can regulate ER-stress induced CHOP expression by activating XBP1s signaling in cardiomyocytes. Therefore, Sigmar1 residing at the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-response signaling between the ER and mitochondria.
Abstract 283: SGK1-Dependent Sirt3 Phosphorylation Regulates Mitochondrial Dynamics
Authors: Ellis Dallas , Scott Takara , Zhong Wei , Babayeva Oguljahan , Francis Sharon C
Published: June 2021

Mitochondrial dynamics (i.e. fusion and fission) is impaired in models of obesity and can result in target organ dysfunction. However, the mechanisms that regulate mitochondrial dynamics in the setting of obesity are not completely understood. The objectives of this study are to examine a role for and determine the molecular mechanisms of serum and glucocorticoid-inducible kinase 1 (SGK1) in obesity-related mitochondrial dynamics in the vasculature. We recently reported that aortic expression of SGK1 is elevated in a model of diet-induced obesity (DIO)
in vivo
and by resistin; a fat-derived adipokine, in human aortic smooth muscle cells (SMC)
in vitro
. To directly examine the effects of SMC-derived SGK1 on mitochondrial dynamics, wildtype and SMC-specific SGK1 knockout mice were subjected to DIO for eight weeks. Our results indicate that SMC-specific deletion of SGK1 induced a fused, elongated mitochondrial phenotype in aortic SMC
in vivo
and attenuated obesity-mediated arterial mitochondrial fragmentation suggesting a role for SGK1 in stimulation of mitochondrial fission. To determine the molecular mechanism for this effect, we performed a proteomic screen for novel SGK1 substrates and identified the mitochondrial deacetylase SIRT3 as a novel SGK1 target. Mass spectrometry indicates SGK1 phosphorylates SIRT3 on serine103. Increasing doses of resistin augmented SIRT3-S103 phosphorylation and caused a concomitant decrease in total SIRT3 in rat aortic SMC
in vitro
. To examine whether SGK1-dependent SIRT3 phosphorylation regulates the mitochondrial fission protein machinery; we evaluated total and activated levels of Drp1, the mitochondrial fission regulator, in response to ectopic expression of SIRT3 wildtype, phospho-memetic (S103D) and phospho-deficient (S103A) mutants. SIRT3-S103D increased total Drp1 and activated Drp1 protein levels an effect inhibited by SIRT3-S103A. These findings implicate elevated resistin observed during obesity in stimulation of SGK1 and subsequent phosphorylation of SIRT3 leading to activation of Drp1 and stimulation of arterial mitochondrial fragmentation.
Abstract 286: Role of ATP-sensitive Potassium Channel Openers in Prevention of Atrial Fibrillation
Authors: Ozcan Cevher , Li Zhenping
Published: June 2021

Introduction:
The ATP-sensitive potassium (K
ATP
) channel dysfunction causes susceptibility to adrenergic atrial fibrillation (AF). However, the therapeutic efficacy of K
ATP
activation in primary prevention of AF is not known. We hypothesized that K
ATP
channel opening drugs prevent AF with effective metabolic and mechanosensitive gating of K
ATP
channels and preserving electro-anatomical stability in atria by regulating oxidative and metabolic states.

Methods:
This hypothesis was tested in a novel mouse model of AF, cardiac specific LKB1 knockout (KO) mice. Male and female LKB1 KO mice were treated with K
ATP
channel openers, nicorandil (15 mg/kg/day) and diazoxide (10 mg/kg/day). Control group included untreated littermates of the study group. Incidence of AF, structural and electrophysiological property of atria and sex-based differences were analyzed at the 3 months of age in all groups.

Results:
The incidence of spontaneous AF was significant reduced in LKB1 KO male mice with nicorandil (0%, n=5) and diazoxide (33%, n=6) treatment compare to untreated mice (84.6%, n=13) (p<0.05). The treatment was also effective in female LKB1 KO mice as AF occurred in 37.5% of nicorandil (n=8) and 40% of diazoxide (n=5) (p<0.05) treated mice as oppose to 71% of untreated mice (n=24). The K
ATP
openers, particularly nicorandil, were more effective in male mice preventing AF. There was 100% risk reduction with nicorandil in male mice compare to 39% with diazoxide. Nicorandil demonstrated 52% risk reduction in female mice while it was 56% with diazoxide treatment. Prevention of AF was associated with preservation of atrial structural and electrical property including size, heart rate and conduction intervals. Biatrial enlargement was significantly prevented by K
ATP
openers compare to untreated mice. Incidence of AF was significantly higher in male mice than female with or without treatment.

Conclusions:
Activation of sarcolemmal and mitochondrial K
ATP
channels prevents AF by preserving electro-anatomical property of atria and decreasing susceptibility to AF. Metabolic regulation of K
ATP
is essential in AF. Thus, the K
ATP
openers are novel upstream therapy for primary prevention of AF in vulnerable population.
Abstract 298: Pluripotent Stem Cell Microrna-294 as a Mediator of Cardiac Proliferative Response After Myocardial Injury
Authors: Khan Mohsin , Booth Brandon , Troupes Constantine D , Mohsin Sadia , Nickoloff Emily , Benedict Cynthia , Houser Steven R , Koch Walter J , Kishore Raj
Published: June 2021

Rationale:
Embryonic heart is characteristic of rapidly dividing cardiomyocytes required to build a working myocardium. In contrast, cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs regulating the unique cell cycle of ESCs and their characteristic pluripotency. Nevertheless, expression of miR-294, a member of the ESCC miRs is lost during developmental transitions from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied.

Objective:
To determine whether miR-294 drives cardiac proliferative response leading to augmentation of cardiac function after myocardial infarction.

Methods and Results:
miR expression analysis in the heart during development revealed elevated levels of miR-294 in the prenatal stages while the expression was lost in the neonates and adults as confirmed by qRT-PCR. Neonatal ventricular cardiomyocytes (NRVMs) treated with miR-294 mimic to showed elevated mRNA levels of cell cycle markers (
E2F family and cyclins
) concurrent with increased expression of p-histone 3, Ki67 and Aurora B kinase (G2/M) as confirmed by immunocytochemistry compared to control cells. Cardiac progenitor cells (CPCs) engineered with miR-294 lentivirus led to increased proliferation and metabolic activity. An inducible cardiac specific AAV-9 carrying miR-294 was administered in mice subjected to myocardial infarction. Increased cardiac function in mice receiving AAV-miR-294 was observed 8 weeks after injury. Increase myocyte proliferation was observed in the heart after miR-294 treatment as analyzed by BrdU uptake, p-Histone 3 and Aurora B expression by immunostaining. Concurrently, a decrease in infarct size along with decreased apoptosis was observed in the miR-294 hearts compared to the control. Furthermore, increased c-kit+ CPCs activation and proliferation was observed in the miR-294 receiving hearts.

Conclusion:
Ectopic expression of miR-294 recapitulates embryonic signaling and enhances cardiomyocyte ability to proliferate together with CPC activation and expansion leading to augmented cardiac function in mice after myocardial infarction.
Abstract 3: The Histone Methyltransferase Smyd1 is a Novel Transcriptional Regulator of Cardiac Fatty Acid Metabolism in Mice
Authors: Warren Junko S , Barton Dane W , Miller Mickey , Wang Li , Cox James , Yuzyuk Tatiana N , Zaitsev Alexey V , Franklin Sarah
Published: June 2021

Epigenetic control of metabolism in the healthy and diseased heart remains poorly understood. We recently demonstrated that chromatin-bound Smyd1, a muscle-specific histone methyltransferase, is significantly upregulated in a mouse model of pressure overload-induced heart failure (HF) and that inducible, cardiac-specific Smyd1 knock-out (Smyd1-KO) mice develop cellular hypertrophy and fulminate HF. Bioinformatic analysis of transcripts differentially regulated in these mice revealed that cardiac metabolism was the most perturbed biological function in the heart. However, it was not clear whether alterations in cardiac metabolism were a direct consequence of Smyd1 deletion or were secondary to developed HF. Here we hypothesized that Smyd1 directly regulates cardiac metabolism; the effects of which should be detectable in Smyd1-KO mice before overt cardiac dysfunction. To test this hypothesis we performed unbiased metabolomic analysis of Smyd1-KO mice using GC/MS and MS/MS (n=9 control, n=10 KO) combined with targeted gene expression analysis. Our results showed significant changes in the metabolic profile of Smyd1-KO mice at the earliest time point (3 weeks after tamoxifen treatment) in which Smyd1 protein expression was significantly reduced while cardiac function remained normal. The most profound difference, in energetics-associated pathways in these mice, was found in fatty acid β-oxidation, manifested by the decreased myocardial content of carnitine and free fatty acids and downregulation of their transporters, OCTN2 and CD36. In addition, mRNA levels of the PPAR-α complex (PPAR-α;RXR-α;PGC-1α), an established regulator of fatty acid β-oxidation, and its target genes (CPT1b;CD36;Acox1;MCAD) were significantly reduced in Smyd1-KO mice prior to the onset of cardiac dysfunction (all p<0.05). To identify whether Smyd1 directly controls gene expression of PPAR-α, we examined the PPAR-α loci using chromatin-immunoprecipitation followed by qPCR and observed significant binding of Smyd1 upstream of the PPAR-α transcriptional start site. Overall, this study identifies Smyd1 as a novel regulator of fatty acid metabolism and suggests that Smyd1 controls cardiac energetics directly by regulating gene expression of PPAR-α.
Abstract 300: Alda-1 Attenuates Acute Ischemia-reperfusion Mediated Cardiac Damage in Aldehyde Dehydrogenase 2 Mutant Diabetic Mice
Authors: Pan Guodong
Published: June 2021

Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme in heart, can remove 4-hydroxy-2-nonenal (4-HNE), a toxic by-products of oxidative stress induced by diabetes and ischemia-reperfusion (I/R) injury. A common inactivating mutation of ALDH2 (termed ALDH2*2) was found in 8% of the world’s population, which causes lower ALDH2 activity in mutation carriers. We hypothesized that Alda-1, the only known activator of both ALDH2 and ALDH2*2 mutation, is able to protect heart from I/R injury in diabetic mice with/without ALDH2*2 mutation. Adult male ALDH2*2 mutant and C57B6 wild-type (WT) mice at 3-4 months of age were made hyperglycemic with streptozotocin injection (150 mg/kg. i.p.). Three weeks after injection, Alzet osmotic pumps were implanted subcutaneously to deliver Alda-1 (10 mg/kg) or vehicle. Mice were sacrificed after one day of pump implantation. Hearts were isolated and subjected to 30-minute ischemic followed by 90-minute reperfusion in a Langendorff apparatus. The basal myocardial ALDH2 activity in diabetic ALDH2*2 mutant was significantly lower than in diabetic WT mice (0.50±0.23 vs 0.83±0.08 mmol/min/μg, -39.8%, p<0.05). Alda-1 significantly increased myocardial ALDH2 activity in both ALDH2*2 (1.17±0.38 mmol/min/μg, +134.0%, p<0.05) and WT (1.46±0.40 mmol/min/μg, +75.9%, p<0.05) diabetic mice. Compared with vehicle, Alda-1 significantly improved left ventricular pressure (LVP), and decreased infarcted areas (IA) both in ALDH2*2 (LVP: 4.30±2.03 vs 15.77±8.99 mmHg, +266.7%, p<0.05; IA: 75.17%±9.49 vs 40.46%±7.20, -46.2%, p<0.05) and WT (LVP: 14.22±7.92 vs 21.96±4.32 mmHg, +54.4%, p<0.05; IA: 42.44%±8.60 vs 28.61%±8.55, -32.6%, p<0.05) subjected to I/R injury. Western-blots showed that Alda-1 decreased levels of 4-HNE protein adducts, and increased levels of mitochondrial complex V in both ALDH2*2 and WT mice. Our data suggest that one-day Alda-1 treatment can confer cardio-protective effects against I/R injury in ALDH2*2 diabetic mice possibly accelerating the detoxification of toxic 4-HNE and thereby protecting mitochondria.
Abstract 305: Carvacrol Attenuates Diabetic Cardiomyopathy by Enhancing PI3K/AKT Pathway in STZ-induced Type 1 Diabetic C57BL/6J Mice
Authors: Hou Ning , Mai Yunpei , Chen Wenliang , Li Faqian , Luo Jiandong
Published: June 2021

Background:
Carvacrol (CAR), a monoterpenic phenol that occurs in many essential oils of the family Labiatae including
Origanum
,
Satureja
,
Thymbra
,
Thymus
, and
Corydothymus
species, possess a wide variety of pharmacological properties including antioxidant and anti-inflammatory potential. This present study was designed to investigate the cardiac protective effect of CAR on diabetic cardiomyopathy in STZ-induced type 1 diabetic mice and explore its potential molecular mechanism.

Methods:
Type 1 diabetes was induced by the intraperitoneal injection of streptozocin (STZ) to male mice at dose of 45 mg/kg body weight (BW). The diabetic animals were divided into three groups containing eight in each: Group I diabetes, Group II and II injected with CAR at 10 and 20 mg/kg BW respectively once daily for 6 weeks. Age matched male C57 mice were used as normal controls. The plasma concentrations of glucose, total cholesterol (TC) and triglycerides (TG) levels were enzymatically determined using commercial kits. The cardiac function was measured by echocardiography. Protein levels of p-PDK1/t-PDK1, p-AKT/t-AKT, p-GSKα/β/t-GSKα/β were detected by Western blotting.

Results:
STZ-induced C57BL/6J diabetic mice showed an elevation in serum glucose, TG and TC level. Compared to diabetic mice, administration of CAR resulted in significant decrease (
P
<0.05) in plasma glucose level in a dose dependent manner, but did not attenuate elevation in TG and TC levels. The abnormal diastolic function in type 1 diabetic mice was significantly reversed by CAR administration. Furthermore, western blotting showed that the expression of p-PDK1, p-AKT and p-GSKα/β were lower in diabetic hearts than C57 hearts while total PDK1, AKT and GSK α/β protein levels were no difference among groups. CAR administration attenuated these decreases in protein phosphorylation. These findings indicate that the impaired PI3K/AKT pathway induced by STZ in diabetic heart can be restored by CAR.

Conclusion:
Carvacrol has antidiabetic property and can be potentially used to prevent hyperglycemia and diabetic cardiomyopathy.
Abstract 307: Apoa-1 Binding Protein Regulates Retinal Angiogenesis via Control of Notch Signaling
Authors: Mao Renfang , Miller Yury , Fang Longhou
Published: June 2021

ApoA-1 binding protein (AIBP), a mediator of cholesterol efflux from endothelia cells to HDL, plays a vital role in zebrafish angiogenesis. Although it is evolutionary conserved from Drosophila to human, its role of angiogenesis in higher vertebrate has not been studied. Thus, we propose that the role of AIBP in angiogenesis is also conserved from zebrafish to mice to humans. Mouse models display tremendous power in the study of human cardiovascular disease as the murine and human vasculatures are highly similar. We therefore generated
Aibp
-/-
mice, which are viable and fertile. We then compared retinal vasculature development in
Aibp
-/-
mice and wild-type control mice by whole mount immunostaining. Our data show that AIBP deficiency in mice results in significantly enhanced angiogenesis, including an increase in the radial extension of vascular plexus, the development of a denser upper capillary layer, and more tip cells as well as filopodia in the retinas. Notch signaling pathway plays an essential role for proper angiogenesis. However, the role of cholesterol metabolism in the Notch pathway is poorly understood. We found the expressions of the Notch downstream targets Hes1, Hey1 and Hey2 were upregulated in
Aibp
-/-
retinas compared to controls. Furthermore, the protein level of Notch intracellular domain (NICD) was decreased in
Aibp
-/-
retinas. These results suggest that the activity of Notch signaling is decreased in retinas from
Aibp
-/-
mice. Mechanistically, we found AIBP positively regulates Notch signaling by affect on γ-secretase activity. Moreover, we performed Matrigel plug assay. Comparing that of wild-type, Matrigel plugs from
Aibp
-/-
mice has more VE-cadherin staining and higher VE-cadherin mRNA expression level; and shows lower Hey1 expression as well as decreased NICD protein levels. It indicates that Matrigel plugs from
Aibp
-/-
mice expresses more blood vessels and show lower Notch signaling activity. Our study demonstrated that AIBP inhibits angiogenesis by activating the Notch pathway and are the first to connect AIBP, a cholesterol metabolism regulator, to Notch signaling.
Abstract 31: First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVCs) Show Superior Angiogenic Potential in Angiogenic Potency Assays When Compared to Term HUCPVCs and Bone Marrow Stromal Cells
Authors: Iqbal Farwah , Szaraz Peter , Wu Jun , Gauthier-Fisher Andree , Li Ren-Ke , Librach Clifford
Published: June 2021

Introduction:
Cell therapy employing pro-angiogenic cell types is a promising option for promoting revascularization of ischemic tissues. First trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a young source of mesenchymal stromal cells (MSCs) that support blood vessels in the umbilical cord.

Objective:
We aimed to determine the angiogenic potential of FTM HUCPVCs using angiogenic potency assays and compare to older sources of MSCs: term HUCPVCs and bone marrow stromal cells (BMSCs).

Methods:
For the aortic ring assay, aortas were sectioned and embedded into Matrigel™. Fluorophore-labeled MSCs for testing were added to developing endothelial networks (Day0). MSC integration and network development were monitored by microscopy and quantification of endothelial networks was performed using ImageJ™ software (Day4) n=3. Using the Matrigel™ plug assay, 5.0 x10
5
MSCs were suspended with equal volumes of Matrigel™ and injected subcutaneously in 11-week-old nude mice and isolated after two weeks. Plug associated microvasculature was imaged and quantified n=3.

Results:
In the aortic ring assay, FTM HUCPVCs homed to endothelial networks and demonstrated greater endothelial cell coverage, when compared to term HUCPVCs and BMSCs. FTM HUCPVCs showed significantly greater network growth when compared to term HUCPVCs (
p
≤0.001), BMSCs (
p
≤0.001) and untreated endothelial networks (
p
≤0.001). FTM HUCPVC contributed to a significantly greater number of closed loops when compared to term HUCPVCs (
p
≤0.01), BMSCs (
p
≤0.001) and untreated networks (
p
≤0.05). At two weeks following injection of Matrigel plugs, FTM HUCPVC resulted in significantly greater blood vessel recruitment when compared to term HUCPVCs (
p
≤0.05), BMSCs (
p
≤0.01) and control media plugs (
p
≤0.01). Small tortuous blood vessels were found in significantly higher quantity in FTM HUCPVC injected plugs when compared to term HUCPVCs (
p
≤0.05), BMSCs
(p
≤0.01) and media plugs (
p
≤0.001).

Conclusions:
These studies demonstrate that FTM HUCPVCs have superior potential to augment, both the initiation of capillary formation and the development of functional, perfusable blood vessels, highlighting their potential for tissue regeneration following ischemic injury.
Abstract 311: Opposing Roles TCF7/LEF1 and TCF7L2 in Wnt/β-Catenin and BMP Signaling During Late Heart Development
Authors: Ye Bo , Xu Haodong , Li Faqian
Published: June 2021

T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors are important nuclear mediators of Wnt/β-catenin signaling. Their roles in cardiac development and diseases remain to be determined. We found that these TCF/LEF family members, including LEF1, TCF7, TCF7L1, and TCF7L2 had unique expression pattern during mouse heart development. At embryonic day (E) 13.5, LEF1 was one of the most abundant TCF/LEF members at mRNA level in the murine heart. By immunohistochemical staining, mesenchymal cells in valvular regions had much stronger intensity of LEF1 than cardiomyocytes in ventricles. Cardiomyocytes with weak to moderate nuclear staining for LEF1 were scattered in compact and trabecular layers. At E17.5, LEF1 mRNA levels dropped dramatically and its protein was no longer detected in cardiomyocytes. TCF7L1 mRNA levels also decreased from E13.5 to E17.5. Weak cytoplasmic, but not nuclear TCF7L1 signal was detected in cardiomyocytes at E13.5 by immunochemistry. TCF7 mRNA was the lowest among all four TCF/LEF members at E13.5 and it further decreased at E17.5. TCF7 protein was only detected by immunohistochemistry in mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. TCF7L2 mRNA became the most abundant TCF/LEFs at E17.5. Immunolocalization revealed that TCF7L2 formed an intensity gradient with the highest levels at the inner trabecular layer, opposite to the distribution of β-catenin. Wnt signaling activation by cardiac deletion of adenomatosis polyposis coli (
Apc
), a negative Wnt regulator, led to a dramatic increase in cyclin D2, Bmp4, LEF1, and TCF7. BMP mediators, SMAD1/5/8, were increasingly phosphorylated. Chromatin immunoprecipitation revealed that TCF7L2 was removed from and replaced by LEF1 and TCF7 in the promoter of
cyclin D2
and
Bmp4
to promote β-catenin recruitment. Furthermore, there was co-occupancy of TCF/LEF and SMAD in the adjacent TCF/LEF and SMAD binding sites of Wnt target genes. Finally, we proved that TCF7L2 was a transcriptional suppressor of
cyclin D2
and
Bmp 4
in a cardiac cell line by overexpression and knockdown experiments. Our results indicate that TCF7L2 is transcriptional repressor of Wnt/β-Catenin pathway in the heart.
Abstract 358: First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVCs) Maintain High Expression Levels of Angiogenic Factors and Have Superior Pro-angiogenic Effects on Endothelial Networks When Compared to Term HUCPVCs and BMSCs in an ex-vivo Rat Aortic Ring Assay
Authors: Iqbal Farwah , Szaraz Peter , Kenigsberg Shlomit , Gauthier-Fisher Andree , Librach Clifford
Published: June 2021

Introduction:
Features of an ideal cell type, that would be conducive to vascular regeneration include (1) expression of pro-angiogenic genes (2) secretion of factors that promote developing or regenerating vasculature, and (3) maintenance of pro-angiogenic properties in the microvascular niche.

Hypothesis:
FTM HUCPVCs, a young rich source of mesenchymal stromal-like cells (MSCs) are ideal candidates for vascular regeneration due to their high expression of pro-angiogenic factors.

Methods:
The paracrine angiogenic potential of 3 types of MSCs was evaluated and compared using
ex vivo
tissue culture of rat aortic rings. Aorta sections were embedded into Matrigel™, cells were added to transwell membranes (pore=0.1μm, EBM 2% FBS) and combined with aortic rings (Day 0). Radial network growth and total loop formation were monitored by microscopy. Endothelial networks were quantified by ImageJ
TM
software. p values were calculated using ANOVA. (N=3 experiments, n=3 replicates). At day 7, the MSCs were isolated from transwells, human cytokine gene expression levels were measured using human growth factor profiler qPCR array (Qiagen, normalizers: GAPDH, βACT). Ct>35 considered negligible.

Results:
In the transwell aortic ring assay, FTM HUCPVCs induced significantly greater network growths when compared to term HUCPVCs (p≤0.0001), BMSCs (p≤0.0001) and untreated rings (p≤0.05). Quantification of network loop formation showed that FTM HUCPVCs induced greater numbers of closed loops when compared to term HUCPVCs (p≤0.0001), BMSCs (p≤0.0001) and untreated networks (p≤0.0001). Human growth factor qPCR array showed a high expression of angiogenic factors (Ct<25 cycle) both at day0 and day7 of co-culture, including BMP1, GDNF, MDK, NRG1, PDGFc, VEGFa, and VEGFc. Most cytokine expression levels were maintained up to 7 days in co-culture with 1 gene upregulated (BMP6 ΔCt>3), and 3 downregulated genes (FGF19, FGF9, NRTN ΔCt>3) at day 7 when compared to day 1.

Conclusion:
Compared to older sources of MSCs, FTM HUCPVCs promote developing endothelial networks
in vitro
via paracrine mechanisms and maintain the high expression of pro-angiogenic factors when cultures with endothelial cells.
Abstract 362: Deletion of Gata4 and Gata6 in the c-Kit Lineage Augments Cellular Fusion in vivo
Authors: Maliken Bryan D , Kanisicak Onur , Molkentin Jeffery D
Published: June 2021

Cell therapy for treatment of ischemic heart disease has been under rigorous evaluation in recent years. Heart-derived c-Kit cells showed promising results in clinical trials with scar reduction and improved ejection fraction following coronary infusion. We previously developed a c-Kit lineage tracing model showing that these cells very rarely convert into
de novo
cardiomyocytes. To potentially reduce this rate to zero, and unequivocally evaluate cellular fusion processes in vivo, we deleted the cardiogenic transcription factors Gata4 and Gata6 in c-Kit cells using a Cre-loxP approach (Kit-Gata4/6 KO). We used the tamoxifen inducible Kit-MerCreMer allele crossed into Gata4/6 homozygous LoxP targeted background and the Rosa26-eGFP reporter, which were given tamoxifen at weaning to delete Gata4 and Gata6 in all c-Kit expressing cells and show them and their progeny as eGFP positive. Unexpectedly, we observed a greater than 10-fold increase in Kit lineage-traced cardiomyocytes in some Kit-Gata4/6 KO mice compared to Kit only controls with up to 4 months of treatment. Exploration of other tissues revealed a dramatic increase in presumed Kit-lineage traced skeletal muscle fibers in Kit-Gata4/6 KO mice as well. However, investigation of this effect suggested that this increase in presumed Kit-lineage traced cardiomyocytes and skeletal muscle fibers was due to an alteration in the immune cell compartment of these mice that generated greater rates of fusion. Indeed, analysis of Kit-derived hematopoietic lineages present in the heart showed a 6-fold increase in leukocyte infiltration (CD45
+
) and a 60-fold increase in dendritic cells (CD11c
+
) from Kit-Gata4/6 KO mice. Kit-eGFP
+
bone marrow was also transplanted into mice expressing a membrane tomato reporter. Kit-Gata4/6 KO bone marrow showed a 3-fold increase in Kit-eGFP cells, although all these myocytes had the membrane tomato tag indicting that these events were all due to fusion. Hence, alterations in c-Kit cell activity likely impacts the activity of the immune system and the fusigenic activity of derived cells.
Abstract 371: The Role of Survivin in Cardiac Regeneration
Authors: Tsang Tien-Jui
Published: June 2021

Heart failure continues to be the major cause of mortality in many countries. How to reduce the loss of cardiac function after injury continues to be a major challenge in cardioprotective treatment. Survivin (SVV) is the smallest member in IAP (Inhibitor of apoptosis) family and has multiple functions such as inhibiting cell apoptosis and enhancing cell proliferation. SVV expresses in nucleus of proliferating cardiomyocyte during heart development but disappear in adult stage. However after injury, SVV is induced in the cytoplasm of cardiomyocyte in peri-infarct region. We established inducible conditional SVV knockout transgenic mouse to investigate the importance of SVV after myocardial infarction. Cardiac-specific SVV knockout mice exhibit poor cardiac function and more apoptotic cardiomyocytes compared to wild-type mice after injury. In order to confirm localization is related to the function of SVV, we have established adenovirus carrying SVV or SVV fused with NLS (nuclear-localization signal). SVV locates in cytoplasm after virus infection but NLS-SVV could be detected in both cytoplasm and nucleus. Both SVV and NLS-SVV inhibited cell apoptosis
in vitro
but only NLS-SVV enhanced neonatal mouse cardiomyocyte proliferation. Mice received intramyocardial injection of virus shows better ejection fraction after myocardial infarction and less apoptotic cardiomyocytes in peri-infarct region, and more H3P-positive cardiomyocytes are observed in border zone overexpressing NLS-SVV group. We believe this dual functional SVV can be applied in future cardioprotective therapy.
Abstract 373: Identification of Novel Anti-necrotic Factor TGN91
Authors: Karch Jason , Brody Matthew J , Kanisicak Onur , Sargent Michelle A , Molkentin Jeffery D
Published: June 2021

Loss of cardiomyocytes by necrotic cell death is the direct mechanistic basis for mortality due to myocardial infarction injury, as well as ensuing heart failure associated with an array of cardiomyopathies. Further definition of the regulated necrotic pathway and identification of novel effectors will be essential in designing new therapies. To identify novel regulators of necrotic cell death we performed an unbiased lentiviral-based overexpression screen comprising of over 13,000 human cDNAs. In order to mimic an ischemic insult, the calcium ionophore ionomycin was used to induce necrotic cell death in conjunction with each individually encoded cDNA in the lentiviruses. After 24 hours of calcium overload cell viability was assessed to determine if a cDNA had a protective or maladaptive effect on necrotic cell death. One completely novel cDNA that we named Trans-Golgi Network protein 91 (TGN91) was found to be significantly protective against regulated necrosis. TGN91 is a multipass transmembrane protein that is conserved from humans to drosophila, although its function is unknown. TGN91 is most abundant in the heart compared to other tissues in the mouse and the protein resides within the Golgi. The expression of TGN91 increases in human ischemic failing hearts compared to non-ischemic failing and normal hearts. Lastly, we generated transgenic mice overexpressing TGN91 specifically in cardiomyocytes and our preliminary results suggest that these mice are protected against ischemic injury. The identification of TGN91 not only provides a novel putative regulator of the necrotic pathway, but also advocates for the Golgi as a regulatory site in necrotic cell death.
Abstract 387: Cardiac Myosin-binding Protein C Structural Dynamics - a Time-resolved FRET Biosensor for Enhanced Contractility in Heart Failure Therapeutic Discovery
Authors: Bunch Thomas A , Lepak Victoria C , Colson Brett A
Published: June 2021

We have created a high-resolution time-resolved spectroscopy molecular biosensor for heart failure therapeutic discovery based on the structural dynamics of cardiac myosin-binding protein C (cMyBP-C). Drugs of interest mimic phosphorylation states of cMyBP-C, which influences myocardial contractility. β-adrenergic stimulation enhances contractility in myocardium, in part due to Protein Kinase A (PKA)-mediated phosphorylation of cMyBP-C. Recent structural and functional results have provided an understanding for how cMyBP-C physiologically regulates inotropy and lusiotropy in the heart by modulating actin-myosin interactions. Phosphorylation introduces a structural change within the regulatory M domain of cMyBP-C, leading to altered actin-myosin binding, and regulation of myocardial contraction. Previous molecular dynamics simulations suggested a specific rotation within the M domain upon phosphorylation that facilitates bending and exposes a putative binding site that is only accessible in the phosphorylated molecule. We hypothesized that these structural rearrangements in nanometer spatial orientation could be measured spectroscopically in mechanistic studies via introduction of site-directed probes within the M domain of cMyBP-C using time-resolved fluorescence resonance energy transfer (TR-FRET). Moreover, we hypothesized that this fluorescently-labeled recombinant muscle protein could be used as a TR-FRET biosensor for detecting potential drugs that alter the M domain structure in specific ways. These drugs will be particularly relevant to enhancement of myocardial contractility and represent novel therapeutics for heart failure and cardiomyopathy. Here, we engineered pairs of cysteine residues within the M domain for FRET labeling and tested the effects of phosphorylation to influence the tertiary structure and dynamics of cMyBP-C. We have characterized cMyBP-C’s molecular basis for fine-tuning contraction and our biosensor is well aligned for high-throughput spectroscopic screens for drugs that perturb structure specific to phosphorylation state. Compound discovery using this assay is applicable to development of novel cardiac disease therapies by enhancing contractility to alleviate dysfunction.
Abstract 390: Dysregulation of ATF2 in Dilated Cardiomyopathy Caused by FBXO32 Mutation
Authors: Alyacoub Nadya , Awad Salma , Kunhi Mohmad , Al-Habeeb Walid , Poizat Coralie
Published: June 2021

Background:
Dilated cardiomyopathy (DCM) is a common form of cardiomyopathy causing systolic dysfunction and heart failure. Rare variants in more than 30 genes mostly encoding sarcomeric proteins and proteins of the extracellular matrix have been implicated in familial DCM to date. We recently identified a novel mutation (Gly243Arg) in
FBXO32
causing familial DCM through abnormal SKP1/CUL/F-BOX (SCF) complex formation and defects in proteins regulating the autophagy/lysosome machinery (Al-Yacoub, Genome Biology, 2016).

Objective:
To explore in more details the mechanisms by which the defective SCF
FBXO32
complex leads to the development of DCM.

Methodology:
Using a PCR-based microarray, we screened for mRNAs significantly dysregulated in the heart of the patient carrying the
FBXO32
mutation compared to control and idiopathic human hearts. Subsequently, we validated dysregulation of a candidate gene using immunoblot analysis and tested the effect of the mutant or wild-type FBXO32 on the novel candidate identified in primary neonatal rat cardiomyocytes.

Results:
We found a robust up-regulation in mRNA expression of the Activating transcription Factor 2 (ATF2), a member of the leucine zipper family of DNA binding proteins, which plays a critical role in cardiac development. ATF2 protein level was also strongly increased in the heart with the
FBXO32
mutation compared to control hearts and to hearts of idiopathic origin. Expression of the mutant FBXO32 protein in primary cardiomyocytes enhanced ATF2 protein expression compared to cells expressing the wild-type FBXO32 protein. Since FBXO32 is member of the SCF complex and has ubiquitin ligase activity, experiments are now investigating whether FBXO32 directly regulates ATF2 protein stability and the role of ATF2 in autophagy flux regulation in dilated cardiomyopathy.

Conclusion:
Our results indicate that abnormal SCF activity due to the
FBXO32
mutation stabilizes the AFT2 transcription factor and suggest a new mechanism by aberrant SCF activity causes DCM in human.
Abstract 393: The Intercalated Disc Protein Myozap: A Novel Player in Cardiac Proteinopathy
Authors: Rangrez Ashraf Y , Frank Derk , Cassidy Liam , Christen Lynn , Geurink Inka , Bernt Alexander , Böttner Martina , Milting Hendrik , Tholey Andreas , Robbins Jeffrey , Frey Norbert
Published: June 2021

Background:
A growing number of cardiac muscle diseases are characterized by depositions of misfolded proteins, including cardiac amyloidosis and desmin-releated cardiomyopathy (DRM). The continued presence and chronic accumulation of misfolded or unfolded proteins can lead to aggregation and/or the formation of soluble peptides that are proteotoxic. This in turn leads to compromised protein quality control and precipitates a downward spiral of the cell’s ability to maintain homeostasis and may eventually result in cell death. We recently identified massive protein aggregates in the hearts of transgenic mice overexpressing the intercalated disc (ID) protein myozap (Myozap-tg). We now sought to investigate the precise composition of these aggregates and the role of Myozap in other proteinopathies such as DRM.

Methods and Results:
We employed multi-dimensional proteomics, transcriptomics, confocal microscopy, and molecular biology approaches to decipher the underlying causes and consequences of protein aggregate formation in Myozap-tg mice. Transcriptome profiling of these mice revealed striking upregulation of autophagy, protein synthesis, and pro-inflammatory pathways, whereas protein degradation pathways were down-regulated. Surprisingly, proteomics analyses revealed Desmin and α-crystallin B (CryAB) as the major constituents of the aggregates, which was further validated by confocal microscopy. Moreover, we identified the presence of toxic preamyloid oligomers in Myozap-tg mouse hearts, a hallmark in many protein aggregation-based diseases including DRM. Most interestingly, we also observed co-localization of Myozap with protein aggregates observed in both transgenic mouse hearts overexpressing mutant Desmin (D7) and mutant CryAB (R120G), as well as in human DRM patients.

Conclusion:
The present study implies a new role for Myozap, which was previously reported to affect cardiac SRF signaling: (1) Myozap accumulates in various forms of experimental and human protein aggregation cardiomyopathy, suggesting involvement in protein homoestasis. (2) The fact that Myozap is now the third ID protein (after desmin and CryAB) to cause cardiac proteinopathy points to a general role of the ID in its molecular pathogenesis.
Abstract 396: Regulation of Cardiomyocyte Proliferation by the Hippo Pathway and Dystrophin Complex
Authors: Morikawa Yuka , Leach John , Heallen Todd , Tao Ge , Martin James F
Published: June 2021

Regeneration in mammalian hearts is limited due to the extremely low renewal rate of cardiomyocytes and their inability to reenter the cell cycle. In rodent hearts, endogenous regenerative capacity exists during development but is rapidly repressed after birth, at which time growth is by hypertrophy. During the developmental and neonatal periods, heart regeneration occurs through proliferation of pre-existing cardiomyocytes. Our approach of activating heart regeneration is to uncover the mechanisms responsible for repression of cardiomyocyte proliferation. The Hippo pathway controls heart size by repressing cardiomyocyte proliferation during development. By deleting
Salv
, a modulator of the Hippo pathway, we found that myocardial damage in postnatal and adult hearts was repaired both anatomically and functionally. This heart repair occurred primary through proliferation of preexisting cardiomyocytes. During repair, cardiomyocytes reenter the cell cycle;
de novo
DNA synthesis, karyokinesis, and cytokinesis all take place. The dystrophin glycoprotein complex (DGC) is essential for muscle maintenance by anchoring the cytoskeleton and extracellular matrix. Disruption of the DGC results in muscular dystrophies, including Duchenne muscular dystrophy, resulting in both skeletal and cardiac myopathies. Recently the DGC was shown to regulate cardiomyocyte proliferation and we found that the DGC and the Hippo pathway components directly interact. To address if the DGC and the Hippo pathway coordinately regulate cardiomyocyte proliferation, we conditionally deleted
Salv
in the mouse model of muscular dystrophy, the mdx line. We found that simultaneous disruption of both the DGC and Hippo pathway leads an increased
de novo
DNA synthesis and cytokinesis in cardiomyocytes after heart damage. Our findings provide new insights into the mechanisms leading to heart repair through proliferation of endogenous cardiomyocytes.