bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 87: Loss of Mir-155 Attenuates Sepsis Induced Cardiac Dysfunction
Authors: Wang Hui , Bei Yihua , Shi Jing , Sun Wei , Huang Peipei , Liu Hui , Li Xinli , Xiao Junjie , Kong Xiangqing
Published: July 2021

Objective:
Sepsis induced cardiac dysfunction is featured by inflammation and metabolic repression. miR-155 is a typical multifunctional miRNA and loss of miR-155 has been shown to protect the heart from pathological cardiac hypertrophy while increased miR-155 could promote the formation of foam cell in atherogenesis. However, the role of miR-155 in sepsis induced cardiac dysfunction is unclear.

Methods:
E.coli lipopolysaccharide (LPS) (5mg/kg) was administered to C57BL/6 mice to create a sepsis-induced cardiac dysfunction model. Cardiac function was assessed by echocardiography 5-6 h post-LPS administration. Heart tissues were collected within 7-9 h after LPS treatment for the analysis of gene expressions. Tail vein injection of miR-155 antagomir (80mg/kg/d) or miR-155 agomirs (30mg/kg/d) for 3 consecutive days were used to decrease or increase miR-155 expressions in heart.

Results:
LPS induced a reduction of 15% in fractional shortening (%FS) and 25% in ejection fraction (%EF). Expression of miR-155 was increased by 2 fold in sepsis-induced cardiac dysfunction mouse model. Over-expression of miR-155 agomirs led to a decrease of 5% in FS and 10% in EF as compared to scramble controls. Aggravation of LPS induced cardiac dysfunction by miR-155 agomir was not associated with alteration in inflammation or cardiac metabolism. However, miR-155 agomir increased LPS- induced myocardium apoptosis and also elevated the ratio of Bax/Bcl-2 at the protein level. Intravenous injection of cholesterol-modified antisense oligonucleorides antagomirs of miR-155 markedly rescued the LPS induced heart failure and apoptosis. Western bloting indicated that miR-155 overexpression in
vivo
led to a significant inhibition of Pea15a while miR-155 knock-down caused a significant upregulation of Pea15a, indicating that Pea15a was a potential target gene of miR-155. Interestingly, plasma miR-155 levels were also found to be significantly increased in critically ill patients with sepsis compared to healthy controls.

Conclusion:
This study demonstrates that miR-155 regulates sepsis induced cardiac dysfunction and Pea15a is a potential targer gene of miR-155. Loss of miR-155 represents a novel therapeutic method for sepsis induced cardiac dysfunction
Abstract 88: Microrna-21* Controls Sepsis-induced Cardiac Dysfunction
Authors: Wang Hui , Bei Yihua , Shi Jing , Sun Wei , Liu Hui , Li Xinli , Xiao Junjie , Kong Xiangqing
Published: July 2021

Background:
Sepsis-induced cardiac dysfunction is charactered by cardiac contractility dysfunction, myocardial inflammation and cardiac metabolism abnormal. Dysfunction of microRNAs (miRNAs, miRs) contributes to a variety of human diseases. However, their roles in sepsis-induced cardiac dysfunction are unclear.

Methods and Results:
Cardiac dysfunction was induced by E.coli lipopolysaccharide (LPS) administration in mice and 8 dysregulated miRNAs were identified by miRNA arrays. Among them, miR-21* was found to be increased most obviously as determined by quantitative reverse transcription polymerase chain reactions. Inhibition of miR-21* in vivo by antagomir attenuated the reduction of factional shortening (FS) and ejection fraction (EF) induced by LPS administration while forced over-expression of miR-21* in vivo by agomir accelerated LPS-induced cardiac dysfunction. Besides that, S100A8 and S100A9, two genes related to cardiac contractility were also found to be regulated in vivo by injection of miR-21* agomirs and antagomirs. Interestingly, cardiac inflammation indictors such as TNF-α and IL-6 and cardiac metabolism regulators including PPAR family, CD36, FATP, GLUT1, GLUT4, PDK4 were not changed by miR-21* in vivo. These data indicate that miR-21* controls sepsis-induced cardiac dysfunction by direct affecting cardiac contractility instead of cardiac inflammation and metabolism. SORBS2 was identified as a target gene of miR-21* and it was decreased by miR-21* agomir and increased by miR-21* antagomir in vivo. In consist with this, circulating levels of miR-21* were also increased in patients with sepsis compared with healthy controls.

Conclusion:
miR-21* controls sepsis-induced cardiac dysfunction by regulating SORBS2. Inhibition of miR-21* represents a novel therapeutic strategy for sepsis-induced cardiac dysfunction.
Abstract 89: MitoTimer Mouse: a Novel Tool for the Study of Mitochondrial Turnover in vivo
Authors: Stotland Aleksandr B , Ramil Jennifer , Gottlieb Roberta A
Published: July 2021

In order to study mitochondrial turnover at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein developed by Terskikh et al. The Timer protein transitions from green fluorescence to a more stable red conformation as it matures over a span of 48 h. Furthermore, the protein is very stable under physiological conditions, insensitive to variations in ionic strength, and changes in pH between 7.0 and 8.0. Notably, Timer maturation from green to red is significantly slowed in deoxygenated buffer, suggesting that molecular oxygen plays a part in fluorophore maturation. We fused the Timer protein with the mitochondrial signal sequence from the cytochrome c oxidase subunit VIII (COX8) to target the protein to the inner membrane of the mitochondria, and further cloned the protein into a construct with a cardiac-restricted α-myosin heavy chain promoter. This construct was used to create the α-MHC MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice reveals a remarkable degree of heterogeneity in the ratio of red-to- green fluorescence of MitoTimer in cardiac tissue. Furthermore, individual mitochondria within cardiomyocytes display a higher red-to-green fluorescence, implying a block in import of newly synthesized MitoTimer that would be caused by the lack of a high membrane potential, indicative of older, dysfunctional mitochondria. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart.
Abstract 9: Temperature-responsive Cell Delivery Biopolymers for Cardiac Tissue Engineering
Authors: Pena Brisa , Martinelli Valentina , Bosi Susanna , Sucharov Carmen , Jeong Mark , Taylor Matthew R , Prato Maurizio , Long Carlin S , Shandas Robin , Park Daewon , Mestroni Luisa
Published: July 2021

Background:
Advances in cell therapy and material science have made tissue engineering a promising strategy for heart regeneration. We developed an injectable biomimetic reverse thermal gel (RTG) that is liquid at room temperature but gel-like at body temperature, with the ultimate goal of being able to serve as a vehicle for cell-based delivery (liquid) to targeted tissue areas (gel-phase at 37°C). In this study we tested the suitability of this biomimetic RTG on cell viability.

Methods and results:
We tested different biomimetic RTG systems with and without the chemical incorporation of lysine. In vitro 3D culture experiments were performed with neonatal rat ventricular myocytes (NRVM) by mixing 3x104 cells with 50 μl of polymeric solution and allowing gel formation at 37°C. The cultured cells were incubated for 21 days. For controls we used NRVMs plated on 2D traditional gelatin coated dishes. We found that the 3D polymeric matrix induces rapid coordinated contraction with improved functionality when compared with standard 2D-cultured NRVM. By immunostaining for the morphology of the sarcomere (alpha-actinin) and DAPI, we also observed that the 3D polymeric matrix stimulates cells to spread and form 3D syncytia.

Conclusion:
These proof-of-concept results demonstrate long-term cell viability in this unique biomimetic system and therefore provide feasibility of a polymeric cell delivery system that permits reversible liquid-to-gel transition at body temperature. These results offer potential for a tissue engineering approach to cardiac regeneration.
Abstract 96: A Unique Jervell Lange-Nielsen Syndrome Mutation Modeled in Induced Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Bersell Kevin , Yang Tao , Roden Dan
Published: July 2021

Introduction:
Current screening for mutations in human disease is turning increasingly to next-generation methods that map short reads to a reference sequence. We report here an unusual variant that was undetected by next generation sequencing in a patient diagnosed with Jervell Lange-Nielsen syndrome (JLNS) and initial results in an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model.

Methods and Results:
A diagnosis of JLNS was made in a middle-aged woman with congenital deafness and QT intervals as long as 800 msec. However, next-generation sequencing found only a heterozygous
KCNQ1
mutation, R518X. Convinced by the clinical phenotype that a second causative variant was highly likely, we used Sanger sequencing of PCR KCNQ1 amplicons to identify a 36-basepair poly-adenine tract, encoding 12 lysines, inserted within the coding sequence at the 5’ end of exon 15. Electrophysiological studies in patient-specific IPSC-CMs revealed marked prolongation of ventricular-like action potentials (Figure).

Conclusion:
Long inserts of the type we identified here have not been previously reported in the long QT syndromes. We speculate that next generation-based short reads containing this variant could not be mapped to a reference sequence and thus this type of variant will be missed by next-generation analysis unless bioinformatics filters are specifically modified to include this possibility. Validation of this long QT syndrome iPSC-CM model provides a human cell based platform for drug discovery and mechanistic studies to further our understanding of disease pathogenesis.
Abstract 110: Statin Therapy Alters the Transcriptome of Ventricular Fibroblasts From Human Failing Heart
Authors: Ross Gracious R , Edwards Stacie , Rizvi Farhan , Werner Paul , Tajik A Jamil , Jahangir Arshad
Published: July 2021

Introduction:
The mechanical and electrical dysfunction in heart failure (HF) is associated with excessive cardiac fibrosis (CF). Activation of human ventricular fibroblasts (hVF) and transdifferentiation to myofibroblasts underlies the increased CF. We recently reported that statin therapy reduced differentiation of hVF in HF patients. However, the underlying mechanism is not known. Therefore, we studied the effect of statin therapy on the transcriptome of hVF from HF patients.

Hypothesis:
We tested the hypothesis that statin therapy alters the expression of differentiation-associated transcription factors (TF) in hVFs from HF patients.

Methods:
Primary cultures of hVF obtained from HF patients undergoing left ventricular assist device implantation either under statin therapy for at least 1 year (n=3) or not (n=3). The extent of transcriptomic changes induced by statin therapy in hVFs was studied from total RNA using RT
2
Profiler
TM
PCR array - human transcription factors (Qiagen, Catalog No: PAHS-075Z ) run on Roche LightCycler 96-well block. Fold change was calculated by 2
-ΔΔCt
method. Data were analyzed by Student’s t test, and
P
value <0.05 was considered significant.

Results:
Out of the 84 related genes profiled, statin therapy upregulated significantly (P<0.05) at least two-fold the following genes: CREB1 (Cyclic AMP-responsive element-binding protein 1), SMAD1, TCF7L2 (transcription factor 7-like 2 ), MEF2A(myocyte enhancer factor-2), ATF1(activating transcription factor 1), and SP3. CREB1, SMAD1, TCFL2, and MEF2A are mainly involved in signaling pathways of G-protein coupled receptors, bone morphogenetic proteins, Wnt, and mitogen-activated protein kinases/extracellular signal-regulated kinases, respectively, while ATF1 and SP3 are involved in various signaling pathways. TFAP2A (transcription factor AP-2 alpha) tends to be downregulated by two-fold, however, did not reach statistical significance.

Conclusion:
Statin therapy mitigates differentiation of hVFs from human failing heart patients by associated changes in the transcriptome. Selective targeting of hVF transcription factor may be a potential therapeutic strategy to de-differentiate myofibroblasts and mitigate the progression of CF and HF.
Abstract 112: Cardiac-specific Constitutive Activation of Nrf2 Induces Reductive Stress and Pathological Cardiac Remodeling
Authors: Shanmugam Gobinath , Narasimhan Madhusudhanan , Fernandes Jolyn , Whitehead Kevin , Litovsky Silvio H , Hoidal John R , Palaniappan Sethu , Kensler Thomas W , E Abel Dale , Jones Dean P , Rajasekaran Namakkal-Soorapppan
Published: July 2021

Background:
Heart failure is a growing cause of human morbidity and mortality. Supplementations of free radical scavenging antioxidants have largely failed to protect the myocardium from oxidative stress diseases. While endogenous transcriptional activation of antioxidants appears to be promising, their chronic effects are unknown. Here, we tested a hypothesis that chronic activation of antioxidant system will result in reductive stress (RS) and lead to pathological cardiac hypertrophy.

Methods:
Novel transgenic (TG) mice expressing constitutively active Nrf2 in the heart (α-MHC-caNrf2-TG) and their littermates were used to study the effects on structure and function of the myocardium. Myocardial glutathione redox state (GSH/GSSG), transcript levels (qPCR), and protein (immunoblotting) for Nrf2-related antioxidants and structure and function analysis (echocardiography - Vevo2100 Imager) in Non-transgenic (NTg), TG-low and TG-high mice (n=6-12/gp.) were performed at 6-8 months of age. Further, changes in cardiomyocytes and rate of survival in TG mice were analyzed.

Results:
Kaplan-Meier survival plots demonstrated 10 and 40% mortality in TG-low and TG-high, respectively, compared to NTG by 60 weeks of age. The myocardial glutathione and its redox ratio (GSH/GSSG) were significantly increased (p<0.05) in the TG-low and TG-high compared with NTg mice indicates development of RS. A significant increase in Nrf2-ARE (promoter) binding with increased expression of antioxidant genes and proteins (p<0.05) were noted in TG vs. NTg mice. Increased heart-to-body weight and heart weight to tibia length ratios were prominent in TG-high relative to NTg or TG-low mice. Histological analyses (WGA, H&E staining) showed increased cardiomyocyte size, ventricular wall thickening and decreased chamber volume in TG mice. Echocardiography analyses revealed significant hypertrophic cardiomyopathy with abnormally increased ejection fraction (HCM
i
EF) due to chronic reductive stress.

Conclusion:
Thus, basal attenuation of the obligatory oxidative signaling with chronic activation of Nrf2-antioxidants could shift the redox equilibrium to “reductive” side and thereby causing pathological cardiac remodeling.
Abstract 122: Adenylate Kinase Isoform Network and AMP Metabolic Signaling in Heart Regeneration
Authors: Zhang Song , Dzeja Petras
Published: July 2021

Metabolic signaling mechanisms of tissue regeneration is still an enigma. Energy state and metabolite signals regulate cell commitment to self-renewal, lineage specification, differentiation and regeneration. In a favorable metabolic environment, cells can grow and proliferate, however when energy is low - metabolic monitoring system sends signals to cell cycle checkpoint to halt cell division and preserve fuel resources. We demonstrate that loss of heart regenerative capacity after birth in mice is associated with marked changes in metabolome, AK-catalyzed phosphotransfer flux (β-ATP[
18
O]), ATP turnover (γ-ATP[
18
O]) and AMP-AMPK signaling along with changes in expression levels of p21, cyclins A and E, pGSK3β and thymidine kinase. Marked reduction of thymidine phosphorylation capacity prevents DNA synthesis and cell proliferation. It emerges, that in adult heart augmented ATP turnover and AMP signal dynamics is misread by AMPK-sensor as "low energy" state inducing blockade of cell cycle metabolic checkpoint and cardiomyocyte proliferation and regeneration after injury. This occurs through augmented adenylate kinase (AK)-mediated AMP signaling which turns on AMPK consequently silencing p53/p21/cyclin cell cycle checkpoint. Changes in expression levels AK1, AK1β, AK2 and AK5 isoforms occur with arrest of heart regeneration. Protein knockdown using siRNA and CRISPR/Cas9 approach indicates that AK2 is critical for cardiomyocyte mitochondrial biogenesis and network formation. Furthermore, we have discovered that deficiency of the AK2 isoform, which is localized in mitochondria intermembrane-intra-cristae space, arrests developmental programming and is embryonically lethal in mice. The uncovered shift in metabolic signaling mechanisms opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.
Abstract 128: Mitochondrial CaMKII Plays Important Physiological and Pathological Roles
Authors: Wang Qinchuan , Luczak Elizabeth , Viswanathan Meera , Wu Yuejin , Wei An-Chi , Gupta Ashish , Corradini Eleonora , Heck Albert , Cammarato Anthony , Weiss Robert , Anderson Mark
Published: July 2021

Calcium/calmodulin-dependent protein kinase II (CaMKII) is known for its roles in fight-or-flight responses, where it mobilizes ATP-consuming processes to maximize power output by regulating cellular Ca
2+
. We hypothesized that CaMKII may also influence ATP-generating processes in a feedforward mechanism to match ATP consumption with demand. Most cellular ATP is produced by oxidative phosphorylation (OXPHOS) in mitochondria; therefore, we generated transgenic animals whose mitochondrial CaMKII activity is boosted or inhibited by overexpressing mitochondrial targeted CaMKII (mtCaMKII), or a specific CaMKII inhibitor (mtCaMKII
N
). We found that mitochondria from mtCaMKII mice have increased activity of pyruvate dehydrogenase and various TCA cycle enzymes. However, these mitochondria failed to provide sufficient ATP for cardiac function, likely due to detrimental remodeling of electron transport chain complex I. As a result, mtCaMKII mice develop a unique dilated cardiomyopathy soon after birth, which could be rescued by overexpressing the mitochondrial but not the myofibrillar form of creatine kinase. mtCaMKII
N
mice are protected against lipopolysaccharides (LPS)-induced mortality, ischemia/reperfusion injury, adverse cardiac remodeling after myocardial infarction and asthma. Furthermore,
Drosophila melanogaster
overexpressing mtCaMKII
N
have longer lifespan under heat stress (29
o
C) and are resistant to paraquat, an ROS-inducing agent. However, in the mice, inhibiting mitochondrial CaMKII reduces metabolic fitness required to sustain the isoproterenol-induced fight-or-flight response, leading to elevated utilization of the cardiac glycogen store and increased lactate production. mtCaMKII
N
mice have reduced spontaneous activity and a higher tendency to gain fat mass when fed a high-fat diet; mtCaMKII
N
flies have a shorter lifespan in the absence of thermal stress (25
o
C). We are using phosphoproteomics, metabolomics and
in silico
modeling approaches to determine the mechanisms by which mtCaMKII regulates energy metabolism and stress responses. Our results have provided important insights into the physiological and pathological roles of mitochondrial CaMKII.
Abstract 13: Suppressing Microtubule Detyrosination Reduces Stiffness and Improves Contractility in Human Cardiomyocytes
Authors: Prosser Benjamin L , Bedi Kenneth , Caporizzo Matthew , Chen Christina , Robison Patrick , Margulies Kenneth
Published: July 2021

The microtubule contribution to myocyte mechanics has been a controversial topic over the years. Utilizing high-speed, super-resolution imaging, we were recently able to directly observe microtubule behavior in working myocytes (Robison et al.,
Science
2016). Strikingly, we found that microtubules buckle like springs between sarcomeric attachment points, providing a mechanical resistance that limits sarcomere shortening and stretch. Further, we identified that post-translational “detyrosination” of microtubules regulates their attachment to the sarcomere, and thus the microtubule contribution to both passive and active mechanics.

Here we present new data identifying microtubule detyrosination as a compelling therapeutic target for the treatment of human heart failure. Using quantitative mass spectrometry, we have probed the cytoskeletal changes that occur during the progression of human heart failure in over 40 patient samples at different stages and etiologies of disease. We find that progressive upregulation and stabilization of the structural cytoskeleton, particularly microtubules and intermediate filaments, is a robust hallmark of human heart failure.

Next, we have performed detailed biophysical studies on isolated myocytes from explanted failing and non-failing human hearts. Using advanced imaging, single myocyte tensile tests and atomic force microscopy (e.g. Prosser et al.,
Science
2011; Robison et al.
Science
2016), we have interrogated the contribution of detyrosinated microtubules to the active and passive mechanics of human myocytes. We find that by reducing microtubule detyrosination, we can robustly improve contractile function. Suppressing detyrosination significantly lowers passive stiffness at physiologic rates, while robustly improving contraction velocity, fractional shortening, and relaxation speed. Of note, the improvement in mechanics correlates with the severity of disease, as myocytes from end-stage patients show greater benefits than those from non-failing or compensated hypertrophic hearts. In conclusion, our work demonstrates pre-clinical efficacy for suppressing detyrosinated microtubules to improve myocyte mechanics in human heart failure.
Abstract 136: Understanding Multi-allelic Heterozygous Variant Contributions to Dilated Cardiomyopathy
Authors: Happe Cassandra , Deacon Dekker , Erbe Rossin , Tenerelli Kevin , Tran Vivien , Alder Eric , Chi Neil , Engler Adam
Published: July 2021

Dilated cardiomyopathy (DCM) is a multivariate disease with poorly understood mechanisms, but recently 30+ different mutations have been suggested to contribute to disease pathology. We have identified the first incidence where a family with high DCM prevalance is caused by the co-segregation of two heterozygous mutations in dissimilar cytoskeletal proteins, i.e. α-tropomyosin (TPM1; +/c.G97A) and vinculin (VCL; +/c.659dupA). To better understand the disease mechanism, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from the family cohort carrying the variants and modeled these variants in human embryonic stem cell-derived CMs (hESC-CMs) via CRISPR to discern the functional consequences that could induce DCM. Affected CMs contracted with decreased energy compared to non-carrier control CMs. Decreased sodium and potassium channel expression accompanied slowed action potential kinetics in affected vs. non-carrier-derived CMs, which together suggests that VCL and/or TPM1 mutations may cause unique downstream transcriptome regulation that leads to the dysfunction we observed
in vitro
. To assess the combinatorial regulation by mutations in these dissimilar genes, hESC-CMs were engineered to mirror altered protein expression; these cells exhibited prolonged calcium transients and contractions with decreased energy and irregular timing, suggesting that reduced VCL creates CMs with dysfunctional mechanical properties, resulting in part from prolonged Ca
2+
handling, which would likely adversely affect TPM1 mutant phenotypes leading to DCM when the variants co-segregated. Given the lack of disease in single variant carriers, these data provide a unique set of analyses that result in identification of how dissimilar but co-segregating variants can result in disease.
Abstract 154: CCDC80 Functions as a Protein Kinase GI Substrate and is Secreted by Cardiac Myocytes
Authors: Blanton Robert M , Cooper Craig , Hergruetter Anja , Aronovitz Mark , Calamaras Timothy D
Published: July 2021

Background:
Protein kinase G I alpha (PKGIa) inhibits cardiac hypertrophy, remodeling, and dysfunction. Downstream PKGI substrates remain incompletely understood and represent potential novel therapeutic targets for myocardial disease. We previously identified through a molecular screen that PKGIa binds and phosphorylates the protein coiled-coiled domain containing 80 (Ccdc80; also termed SSG1 and URB) in vascular smooth muscle cells. Previous work also identified that Ccdc80 is secreted from adipocytes. However, the expression and secretion of Ccdc80 from the cardiac myocyte has not been investigated. The current study tested the hypothesis that Ccdc80 is expressed in and secreted from the cardiac myocyte.

Results:
In cultured rat cardiac myocytes (CM), we detected Ccdc80 by western blot. Western blot for Ccdc80 also detected a band of the predicted Ccdc80 molecular weight present in media from these cells, but not in uncultured media. Ccdc80 could be detected in the human left ventricle (LV), though expression did not differ between hearts of normal controls and patients with hypertrophic cardiomyopathy. In the setting of LV pressure overload induced by transaortic constriction (TAC), we observed an increase in Ccdc80 expression in 1 week TAC LVs, compared with sham LVs (5.0 +/- 0.3 arbitrary densitometric units in sham versus 9.6 +/- 0.9 in TAC; n=4 per group).

Conclusion:
Taken together, our findings identify that the PKGIa substrate Ccdc80 expresses in cardiac myocytes, becomes secreted from CMs, resides in the human heart, and increases in expression in the mouse LV in response to pressure overload. Given the anti-remodeling role of PKGIa, these findings support future studies to understand the in vivo role of Ccdc80 in the cardiovascular system. Future studies will also explore the significance of Ccdc80 secretion from the CM and its potential regulation by PKG.
Abstract 157: Alterations in Signaling Pathways During Regression of Pathological Cardiac Hypertrophy
Authors: Langager Deanna , Leinwand Leslie
Published: July 2021

Introduction:
Cardiac hypertrophy is initially, a compensatory mechanism to maintain cardiac output when there is an increased load on the heart. However, if cardiac hypertrophy persists for an extended time, there can be maladaptive changes to the myocardium. Even when the underlying cause of hypertrophy is treated, regression is often minimal or absent. Clinical cases of cardiac regression do exist, including patients receiving bariatric surgery or a left ventricular assist device. While many of the mechanisms leading to cardiac hypertrophy are well understood, little is known about the mechanisms of reversal of hypertrophy and why it is sometimes irreversible. We hypothesized that a reversal of isoproterenol (Iso) induced cardiac hypertrophy in the mouse will be observed within 7 days following the removal of the stimulus and we will be able to identify alterations in signaling pathways.

Methods:
We induced pathological cardiac hypertrophy with Iso for 7 days, at which peak hypertrophy is achieved. To identify if/when regression occurs, the Iso treatment was stopped and the mice were monitored for 7 days. Heart weights were measured at peak hypertrophy, post-drug days 1, 2, 3 & 7, along with vehicle treated mice (8/group). We used left ventricle tissue for protein analysis and protein degradation activity assays.

Results:
Regression from cardiac hypertrophy occurs by post-drug day 7 (p=0.016) in the Iso mouse model. p-Akt is increased with Iso treatment and returns to vehicle control levels by post-drug day 7. There is a decrease in p-mTOR and an increase in LC3-II levels at post-drug day 7, indicating a possible role of autophagy in cardiac regression. In addition, there was a decrease in cell size when neonatal rat ventricular myocytes were treated with the Akt inhibitor, Wortmannin, following phenylephrine induced hypertrophy.

Conclusion:
Regression of Iso-induced cardiac hypertrophy occurs in the mouse after 7 days following the removal of the stimulus. The Akt pathway is activated with Iso treatment and when this pathway is inactivated during regression, autophagy is activated, which may be an important mechanism to degrade proteins and lead to a decrease in cardiac hypertrophy. Finally, when the Akt pathway is inhibited
in vitro
, hypertrophic cells regress.
Abstract 162: MicroRNAs Mediate the Hemodynamic Regulation of Endothelium-to-Smooth Muscle Signaling
Authors: Zhou Jing , Zhu Juanjuan , Liu Yuefeng , Chien Shu
Published: July 2021

Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study has demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (LS) to ECs. In the current study we found that atherogenic oscillatory shear (OS), but not atheroprotective pulsatile shear (PS), promotes the regulated exocytosis, particularly the secretion of non-membrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs), VAMP3 and SNAP23. Quantitative PCR arrays identify 15 EC-secreted miRNAs whose levels in the vesicle-poor supernatant of flow perfusate are differentially regulated (P<0.05) by OS versus PS. The fold changes of 11 miRNAs are either greater than 2.0 or less than 0.5. Knockdown of VAMP3 and SNAP23 reduces the secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mTORC1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern-specificity of SNAREs activation and its contribution to the miRNA-mediated EC-SMC communication, uncovering potential targets for future diagnosis and therapeutic interventions for proliferative vascular diseases.
Abstract 172: Vascular Smooth Muscle Cell Activation is Regulated by miR-25 Induced miR-9 Suppression of Nox4 and Myocardin
Authors: Schickling Brandon M , Takapoo Maysam , Devor Eric J , Miller Francis J
Published: July 2021

Vascular smooth muscle cell (SMC) de-differentiation with subsequent migration and proliferation into the subendothelial space is central to the progression of cardiovascular diseases. The Nox4 NADPH oxidase (Nox4) is implicated in maintaining the differentiated phenotype of SMC in part through myocardin, a master regulator of SMC gene expression. However, this process is poorly understood. We hypothesized that microRNAs (miR)-mediate changes in Nox4 expression and regulate SMC differentiation. Treatment of human SMCs with a miR-9 or miR-25 mimic silenced Nox4 mRNA through binding to the Nox4 3’UTR. However, only miR-25 was sufficient to downregulate Nox4 protein levels. We found that miR-25 induced the expression of miR-9 through a novel mechanism involving demethylation of the miR-9 promoter by Tet methylcytosine dioxygenase 2 (TET2). Inhibition of miR-9 induction by miR-25 with a miR-9 inhibitor restored Nox4 protein expression to basal levels. Furthermore, the miR-25-mediated decrease in Nox4 protein was ameliorated by inhibiting the proteasome with MG132. These data suggest a novel mechanism wherein miR-9 and miR-25 regulate Nox4 through both translational suppression and proteosomal degradation. Overexpression of miR-9 or miR-25 in human SMCs (1) suppressed myocardin mRNA and protein expression; (2) decreased expression of multiple SMC differentiation genes; and (3) was sufficient to induce cell migration. Thrombin and tumor necrosis factor increased the expression of miR-9 and miR-25 in human SMCs and inhibition of miR-9 prevented thrombin-mediated decrease in myocardin and SMC migration. Mir-9 and miR-25 levels were increased in SMCs derived from balloon injured rat aorta as compared to medial SMCs and in murine carotid artery ten days post carotid injury. A miR-9 inhibitor decreased neointimal formation by more than 50% in following partial carotid ligation in mice. These findings identify miR-9/Nox4 as a novel regulatory pathway of SMC differentiation and a potential therapeutic target in vascular disease.
Abstract 189: HuR, RNA Stabilizing Protein, Regulation of Pluripotency and Differentiation in iPSCs
Authors: Suresh Babu Sahana , Rajasingh Johnson , Wong Wing Tak , Krishnamurthy Prasanna
Published: July 2021

Background:
The Hu family of RNA-binding proteins, HuR (also known as ELAVL1 or human embryonic lethal abnormal vision-like protein), binds to the 3’-untranslated region of mRNAs and regulates transcript stability and translation. Global deletion of HuR is embryonically lethal in mice and plays a critical role in progenitor cell survival and biology. Induced-pluripotent stem cells (iPSC) have distinct transcriptional machinery for the maintenance of pluripotency and achievement of differentiation. However, the exact role of HuR in pluripotency or differentiation of iPSC to cardiomyocytes (iCM) remains unclear.

Methods:
HuR knockdown in human dermal fibroblast-derived iPSCs was achieved by CRISPR/Cas9 or lentiviral shRNA transduction and subsequently differentiated into cardiomyocytes (iCM). Then, the expression of HuR, pluripotency and cardiomyocyte markers were evaluated on days 0, 1, 3, 6, 8 and 17 following the initiation of differentiation.

Results:
At basal level, HuR expression was higher in the iPSCs compared to dermal fibroblasts. Upon differentiation of iPSCs into iCM, HuR mRNA expression gradually reduced with significantly lower levels on day 17. As expected, pluripotency markers gradually reduced upon differentiation with significantly lower levels from day 6 onwards. We observed a corresponding increase in ISL1, MESP1 (mesoderm/cardiac progenitor markers) from day 3 through day 8 with a steep fall from day 8 to day 17. This was associated with Myosin light chain-2V and GATA4 expression increases from day 8 through day 17. Interestingly, knockdown of HuR resulted in clumps of colonies with differentiated cells and a corresponding increase in cardiac-troponin positive cells. However, as a general observation, HuR knockdown reduced beating intensity compared to wild type cells.

Conclusions:
Based on these data, we could speculate that HuR might be necessary for maintenance of pluripotency and loss of which renders cells to differentiate in culture. HuR knockdown yields higher number of c-troponin positive cells but its effect on functional maturity of iCM needs to be further evaluated.
Abstract 196: Genetically Modified Human Mesenchymal Stromal Cells (MSCs) Help Improve Glucose Homeostasis by Reducing Inflammation and Promotes Browning of Visceral Fat
Authors: Sen Sabyasachi , Domingues Cleyton , Kundu Nabanita , Ahmadi Neeki
Published: July 2021

Background:
MSCs are multipotent cells that can home-in to a site of inflammation. Upregulation of specific antioxidants in MSCs reduces intracellular inflammation and ROS formation in a hyperglycemic condition.

Hypothesis:
Antioxidant over-expressed MSCs will reach fat depots, reduce local & systemic inflammation and improve glucose homeostasis in diet-induced obese (DIO, 60% and 45% fat diet) hyperglycemic mouse models.

Methods:
We used GFP-containing adenoviral constructs to upregulate intracellular (SOD1, SOD2, Catalase) and extracellular (SOD3) antioxidants in human adipose-derived MSCs. The modified MSCs were delivered in DIO mice.

Results:
In-vitro, SOD2 (mitochondrial anti-oxidant) upregulation showed reduced inflammatory markers IL6 and TNFa mRNA while PCG1A mRNA (a gene upstream of UCP1), upregulated. SOD2 upregulated MSC delivery in both DIO models demonstrated improved glucose tolerance test (GTT) at week 4 compared to SOD1-MSC and Null-MSC (control). Catalase-MSC delivery not only improved GTT but also improved insulin tolerance test (ITT) in 60% DIO mice. Interestingly, RT-PCR of pericardial fat showed significant increases in mRNA expression of both UCP1 (25-100,000-fold) and PRDM-16 (2-10-fold) in both DIO mice models that received antioxidants upregulated MSCs, compared to mice receiving Null MSCs. For omental fat, an increase in mRNA expression of UCP1 was observed in 60% fat DIO mice (1,000-6000 fold) for SODs 1-3 and catalase, while for 45% DIO mice only those receiving SOD1 & SOD2 upregulated MSCs presented UCP1 mRNA upregulation (1,000 to 11,000-fold). Omental Fat histology showed less hyperplastic fat with SOD2 and Catalase-MSCs. UCP1 staining of omental fat was also positive with SOD2-MSC. Inflammatory molecules such as IL-6 and TNF alpha levels by ELISA, were reduced with SOD2-MSC in 60% DIO mice model.

Conclusion:
We conclude that delivery of antioxidant upregulated MSCs to the inflamed adipocyte depots in diabetic DIO models appear to upregulate UCP1 and PRDM-16 in visceral fat while reducing systemic inflammatory markers, which may explain improvements noted in GTT and ITT. Delivery of modified MSC is a novel & robust therapeutic tool that improves glucose homeostasis in diet induced diabetes.
Abstract 198: Multipotent Placenta-derived Cdx2 Cells Possess in vitro and in vivo Cardiomyogenic Potential
Authors: Vadakke Madathil Sangeetha , Ranjan Amaresh , Yoon Jesse , Tripodi Joseph , Raedschelders Koen , Parker Sarah , Najfeld Vesna , Van Eyk Jennifer , Chaudhry Hina
Published: July 2021

Stem cell-based therapies for cardiac regeneration are of crucial importance and an ideal cell-type is yet to be established. We previously reported that fetal cells from placenta “home” to injured maternal heart and approximately 40% (40/100) of the migrating cells expressed homeodomain protein Cdx2. This interesting observation led us to hypothesize that placental Cdx2 could be a novel cell target for cardiac differentiation. To understand this phenomenon, we employed a cre-lox strategy that labeled Cdx2 cells in placenta with e-GFP and induced myocardial infarction (MI) in pregnant mice at mid-gestation. The maternal heart was analyzed 4 weeks post-MI for the presence of Cdx2-eGFP-derived cardiomyocytes. Additionally, Cdx2 cells were isolated from late-gestation placenta and assayed for cardiac differentiation
in vitro
followed by live cell imaging. Phenotypic and whole-cell proteomic analysis, clonal and vascular lineage differentiation and immune profiling were carried out subsequently. We observed that Cdx2 cells migrated to injured maternal hearts and differentiated into cardiomyocytes highlighting the functional significance of fetal-maternal stem cell transfer. Additionally, isolated Cdx2 cells from the late placenta differentiated into spontaneously beating cardiomyocytes and expressed structural proteins cardiac troponin T(cTnT), α-sarcomeric actinin and gap junction protein Cx43. These cells underwent clonal expansion and differentiated into endothelial and smooth muscle lineages in culture indicative of their multipotent nature. Low expression of MHC molecules and other components of the immune-response, infer that these cells possess the ability to evade host immune surveillance. Proteomic analysis demonstrated that 145 proteins were uniquely identified in the Cdx2 cells compared to embryonic stem cells. These protein networks reflected an increased activation of functions involving migration, fertility, homing, and chemotaxis. Our study is the first to demonstrate that Cdx2 may play a role in cardiac differentiation and delineate multipotent cells in placenta with an inherent “homing” ability. These findings point to a potential role for Cdx2 cells in cardiac regenerative therapies using allogeneic cells.
Abstract 2: MANF, A Structurally Unique Redox-Sensitive Chaperone, Restores ER Protein Folding in the Ischemic Heart.
Authors: Arrieta Adrian , Blackwood Erik A , Stauffer Winston T , Santo Domingo Michelle , Pentoney Amber N , Thuerauf Donna J , Doroudgar Shirin , Glembotski Christopher C
Published: July 2021

Rationale:
In cardiomyocytes, most secreted and membrane proteins are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that during myocardial ischemia, decreased oxygen creates a reducing environment in the SR/ER, preventing protein disulfide isomerases (PDIs) from forming disulfide bonds in nascent proteins, causing ER stress, i.e. the toxic accumulation of unfolded proteins which contributes to cardiomyocyte death. In response to ER stress, the transcription factor, ATF6 induces chaperones that restore SR/ER protein folding. We found that ATF6 also induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function cannot be inferred from other proteins. Since MANF is induced by ATF6, is ER-localized, and contains a conserved redox-sensitive motif found in PDIs, we hypothesized that MANF is a redox-sensitive chaperone that optimizes cardiomyocyte viability during ischemia.

Methods:
The redox status of MANF during reductive ER stress and the ability of MANF to bind misfolded proteins during ischemia were assessed in neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF to suppress aggregation of misfolded proteins was examined in an
in vitro
chaperone assay. Finally, the effects of MANF loss-of-function in the ischemic heart,
in vivo
, were determined by generating a transgenic mouse model that expresses a cardiomyocyte-specific MANF-targeted microRNA.

Results:
In NRVM subjected to ER stress MANF was as sensitive to changes in ER redox status as the sentinel PDI, PDIA1. Moreover, MANF formed disulfide-linked complexes with misfolded proteins during ischemia-mediated ER stress. Under reducing conditions, recombinant MANF suppressed aggregation of model misfolded proteins,
in vitro
. MANF knockdown in the heart,
in vivo
, increased damage from myocardial infarction, and an AAV9-based gene therapy approach rescued the effects of MANF deficiency,
in vivo.

Conclusions:
MANF is a redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and decreases tissue damage in the ischemic heart.
Abstract 20: Caridomoyocyte Biology Revealed by Fluorescence Ubiquitination-based Cell-cycle Indicators
Authors: Alvarez Roberto , Quijada Pearl J , Wang Bingyan J , Amheh Lilian , Navarrette Natalie , Shaytrit Maya , Ho Thi , Gude Natalie A , Sussman Mark A
Published: July 2021

Existing myocyte contribution to new myocyte formation remains an active area of investigation. Novel experimental methodology is needed to faithfully label cardiomyocyte cell-cycle activity after birth and following injury. The Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) system can be used to aid visualization of cell cycle activity and progression by monitoring the inverse oscillation dynamics of fluorescently tagged cell cycle fusion proteins AzG-hGeminin and mKO2-hCdt1. Using this system, we
hypothesize that cardiomyocytes retain the capacity to cycle throughout postnatal development and re-enter the cell cycle following acute myocardial infarction injury (MI).

A novel cardiac specific FUCCI transgenic mouse model, αMHC-FUCCI, was developed to study cell-cycle dynamics of cardiomyocytes. αMHC-FUCCI hearts were collected throughout postnatal (PN) development to examine cardiomyocyte cell-cycle. Similarly, adult αMHC-FUCCI mice were subjected to MI, injected daily with BrdU and harvested at 3, 7, 10, 14 and 21 days post-MI for further analysis.

Peak incidence of single mKO2-hCdt1 (7%, G
1
) and AzG-hGem (2%, S/G
2
/M) fluorescence in cardiomyocytes occurs at PN7 and decreases over time as as confirmed by colocalization with BrdU and/or mitotic marker phospho-histone 3. Interestingly, continued mitotic activity exists at PN14 as observed by AzG+/pH3+ myocytes and concurrent mKO2+/AzG+ fluorescence is observed in 60% of adult myocardium by at one month. Together, these results indicate cardiac myocytes remain active at least two weeks after birth and transition into a G1/S phase as opposed to a mitotic exit (G0) as adults. Intriguingly, BrdU+ label is only detected in the non-myocyte interstitial population in and around the border zone through the first two weeks post-MI. BrdU+/AzG+ and or/mKO+ myocytes are detectable at 21days post-MI, indicating a lag in cardiomyocyte cell cycle re-entry. These results suggest myocytes retain the ability to re-enter the cell cycle at low levels three weeks post-MI. Future studies will analyze cardiomyocyte cell-cycle biology in response to diffuse injury and will further elucidate the mechanism behind myocardial regeneration.
Abstract 200: Nanofibrous Microsphere Mediated High Engraftment of hESC Derived Cardiomyocytes Improves Rat Heart Function After Infarction
Authors: Wang Zhong
Published: July 2021

Cell-based therapy is a promising strategy to regenerate the injured heart after myocardial infarction. However, the current strategies only show modest therapeutic benefits largely due to the low engraftment and poor regeneration of functional cardiac muscle tissue. To address these critical issues in heart regeneration, we applied poly (L-lactic acid) nanofibrous hollow microspheres (NF-HMS) as cell carriers to transplant human embryonic stem cell (hESC) derived cardiomyocytes (CMs) into infarcted rat hearts. Our studies showed that, 28 days after cell transplantation, graft size of the CM+NF-HMS group (1.85 ± 0.72 mm
2
) was significantly higher than that of the CM only group (0.49 ± 0.34 mm
2
) (P<0.01), resulting in a 3.78 fold increase of CM engraftment by application of NF-HMS. Consequently, the reduction of infarct size in CM+NF-HMS group was significantly greater than that of the CM only group (42.58% versus 12.07% reduction). Furthermore, the grafted cells could couple with host cells as indicated by connexin 43 connections between grafted and host cells. Functionally, the left ventricular ejection fraction and fractional shortening in CM+NF-HMS group was significantly improved compared with CM only and PBS control groups. The increased engraftment of CMs in CM+NF-HMS group also promoted a higher density of vasculature in the infarction border zone. Thus, the highly engrafted transplantation of CMs using NF-HMS as a cell carrier resulted in significant improvement of CM engraftment, revascularization, and functional performance, providing an exciting strategy for heart regeneration.