bluuebunny/crossref_metadata_2025 huggingface dataset:
Associations of Skeletal Muscle Mass and Fat Mass With Incident Cardiovascular Disease and All‐Cause Mortality: A Prospective Cohort Study of UK Biobank Participants
Authors: Knowles Rebecca , Carter Jennifer , Jebb Susan A. , Bennett Derrick , Lewington Sarah , Piernas Carmen
Published: June 2021

Background

There is debate whether body mass index is a good predictor of health outcomes because different tissues, namely skeletal muscle mass (SMM) and fat mass (FM), may be differentially associated with risk. We investigated the association of appendicular SMM (aSMM) and FM with fatal and nonfatal cardiovascular disease (CVD) and all‐cause mortality. We compared their prognostic value to that of body mass index.

Methods and Results

We studied 356 590 UK Biobank participants aged 40 to 69 years with bioimpedance analysis data for whole‐body FM and predicted limb muscle mass (to calculate aSMM). Associations between aSMM and FM with CVD and all‐cause mortality were examined using multivariable Cox proportional hazards models. Over 3 749 501 person‐years of follow‐up, there were 27 784 CVD events and 15 844 all‐cause deaths. In men, aSMM was positively associated with CVD incidence (hazard ratio [HR] per 1 SD 1.07; 95% CI, 1.06–1.09) and there was a curvilinear association in women. There were stronger positive associations between FM and CVD with HRs per SD of 1.20 (95% CI, 1.19–1.22) and 1.25 (95% CI, 1.23–1.27) in men and women respectively. Within FM tertiles, the associations between aSMM and CVD risk largely persisted. There were J‐shaped associations between aSMM and FM with all‐cause mortality in both sexes. Body mass index was modestly better at discriminating CVD risk.

Conclusions

FM showed a strong positive association with CVD risk. The relationship of aSMM with CVD risk differed between sexes, and potential mechanisms need further investigation. Body fat and SMM bioimpedance measurements were not superior to body mass index in predicting population‐level CVD incidence or all‐cause mortality.
Standardizing the Definition and Analysis Methodology for Complete Coronary Artery Revascularization
Authors: Ali Ziad A. , Horst Jennifer , Gaba Prakriti , Shaw Leslee J. , Bangalore Sripal , Hochman Judith S. , Maron David J. , Moses Jeffrey W. , Alfonso Maria A. , Madhavan Mahesh V. , Dressler Ovidiu , Reynolds Harmony , Stone Gregg W.
Published: June 2021

Guideline‐based medical therapy is the foundation of treatment for individuals with coronary artery disease. However, revascularization with either percutaneous coronary intervention or coronary artery bypass grafting may be beneficial in patients with acute coronary syndromes, refractory symptoms, or in other specific scenarios (eg, left main disease and heart failure). While the goal of percutaneous coronary intervention and coronary artery bypass grafting is to achieve complete revascularization, anatomical and ischemic definitions of complete revascularization and their methodology for assessment remain highly variable. Such lack of consensus invariably contributes to the absence of standardized approaches for invasive treatment of coronary artery disease. Herein, we propose a novel, comprehensive, yet pragmatic algorithm with both anatomical and ischemic parameters that aims to provide a systematic method to assess complete revascularization after percutaneous coronary intervention or coronary artery bypass grafting in both clinical practice and clinical trials.
Electrocardiographic Imaging of Repolarization Abnormalities
Authors: Bear Laura R. , Cluitmans Matthijs , Abell Emma , Rogier Julien , Labrousse Louis , Cheng Leo K. , LeGrice Ian , Lever Nigel , Sands Gregory B. , Smaill Bruce , Haïssaguerre Michel , Bernus Olivier , Coronel Ruben , Dubois Rémi
Published: June 2021

Background

Dispersion and gradients in repolarization have been associated with life‐threatening arrhythmias, but are difficult to quantify precisely from surface electrocardiography. The objective of this study was to evaluate electrocardiographic imaging (ECGI) to noninvasively detect repolarization‐based abnormalities.

Methods and Results

Ex vivo data were obtained from Langendorff‐perfused pig hearts (n=8) and a human donor heart. Unipolar electrograms were recorded simultaneously during sinus rhythm from an epicardial sock and the torso‐shaped tank within which the heart was suspended. Regional repolarization heterogeneities were introduced through perfusion of dofetilide and pinacidil into separate perfusion beds. In vivo data included torso and epicardial potentials recorded simultaneously in anesthetized, closed‐chest pigs (n=5), during sinus rhythm, and ventricular pacing. For both data sets, ECGI accurately reconstructed T‐wave electrogram morphologies when compared with those recorded by the sock (ex vivo: correlation coefficient, 0.85 [0.52–0.96], in vivo: correlation coefficient, 0.86 [0.52–0.96]) and repolarization time maps (ex‐vivo: correlation coefficient, 0.73 [0.63–0.83], in vivo: correlation coefficient, 0.76 [0.67–0.82]). ECGI‐reconstructed repolarization time distributions were strongly correlated to those measured by the sock (both data sets,
R
2
≥0.92). Although the position of the gradient was slightly shifted by 8.3 (0–13.9) mm, the mean, max, and SD between ECGI and recorded gradient values were highly correlated (
R
2
=0.87, 0.75, and 0.86 respectively). There was no significant difference in ECGI accuracy between ex vivo and in vivo data.

Conclusions

ECGI reliably and accurately maps potentially critical repolarization abnormalities. This noninvasive approach allows imaging and quantifying individual parameters of abnormal repolarization‐based substrates in patients with arrhythmogenesis, to improve diagnosis and risk stratification.
Abstract 1: Abnormal Cardiac Differentiation Underlies Cardiac Hypertrophy in Noonan Syndrome With RAF1 Mutation
Authors: Jaffre Fabrice , Wang Gang , Roberts Amy , Pu William , Hahn Andreas , Kontaridis Maria
Published: June 2021

Noonan Syndrome (NS), an autosomal dominant RASopathy disorder, is caused by germ-line mutations that affect the canonical RAS-MAPK pathway. >95% of NS patients with an S257L mutation in
Raf1
exhibit cardiac hypertrophy (CH) at a very young age. However, the molecular and developmental mechanisms that elicit CH in these patients remain unknown. Hence, we aimed at modeling CH by differentiating human NS
Raf1
induced-pluripotent stem cells (iPSCs) towards the cardiomyocyte fate. We generated iPSCs from an NS pediatric patient with the S257L/+ mutation in the
Raf1
gene and corrected the mutation using CRISPR-Cas9 double nickase system. Differentiation of the mutant NS line led to a significantly increased number of cardiomyocytes (CMs) as measured by FACS sorting at day 20, as compared to isogenic corrected cells (5.71 million ± 0.38 of cTNT+ cells vs 3.77±0.54, n=6, p<0.01). Differentiation of mutant iPSCs into mesodermal cells was normal; however, differentiation into cardiac progenitor cells was enhanced by the mutation, as demonstrated by the increased number of NKX2.5+ cells in the mutant culture at this stage of development. In addition, differentiated S257L/+ CMs had a significantly increased cell surface area compared to the control CMs (3,331 μm
2
± 325 vs 1,638 μm
2
± 97, n=6, p<0.01). To uncover the aberrant signaling pathways underlying these phenotypes, we generated RAF1 S257L/+ knockin and RAF1 null (KO) Hela cell lines using CRISPR-Cas9n technology. Interestingly, we found that ERK1/2 activity was enhanced in KO cells, both at baseline and in response to growth factor (GF) stimulation. At the opposite, ERK1/2 activity was reduced in mutant cells, suggesting that the RAF1 S257L/+ mutant actually suppresses ERK1/2 activation. Finally, we found that AKT activation was upregulated in S257L/+ cells under GF stimulation, due to decreased ERK1/2-dependent p70-S6 kinase activity and subsequent increase in mTORC2 activity (n=6). Similar experiments are underway in S257L/+, corrected and KO RAF1 iPSC-derived CMs. In conclusion, we propose that the S257L/+ mutation modulates development of CH in NS RAF1 patients through a reduction in ERK1/2 activity and overactivation of the AKT/mTOR signaling pathway, leading to both an increased number and size of CMs.
Abstract 103: MCL-1
Matrix
Promotes Mitochondrial Fusion and Protects Against Mitophagy
Authors: Leon Leonardo J , Moyzis Alexandra G , Gustafsson Asa B
Published: June 2021

MCL-1 is a less well characterized anti-apoptotic BCL-2 protein which is highly expressed in the myocardium. We recently discovered that loss of MCL-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and development of heart failure. MCL-1 exists both in the outer mitochondrial membrane (MCL-1
OM
) and in the mitochondrial matrix (MCL-1
Matrix
). Because the

Mcl-1
-/-

knockout mice are missing both MCL-1
Matrix
and MCL-1
OM
, the functional importance of each form is still unknown. While studies have implicated MCL-1
OM
in regulating apoptosis, very little is known about the functional role of MCL-1
Matrix
. Here, we examined whether cardiac MCL-1 levels were altered in response to fasting. Our data show that the MCL-1
Matrix
/MCL-1
OM
protein ratio was significantly increased in mouse hearts after 24 and 48 h of fasting. This was mainly due to a decrease in MCL-1
OM
, while MCL-1
Matrix
levels were unchanged. We also confirmed that the reduction in MCL-1
OM
did not lead to a significant activation of apoptosis. Mitochondria are responsive to alterations in cellular metabolism and culturing cells in “oxidative” media containing galactose or acetoacetate led to a significant increase in the MCL-1
Matrix
/MCL-1
OM
ratio due to a reduction in MCL-1
OM
protein and to elongation of mitochondria. We also confirmed that the reduced MCL-1
OM
levels did not result in activation of apoptosis. This suggests that the MCL-1
Matrix
/MCL-1
OM
ratio is altered in response to changes in metabolism and energy demand without activating apoptosis. We have started dissecting how altering the MCL-1
Matrix
/MCL-1
OM
ratio affects mitochondrial morphology and turnover. We confirmed that MCL-1
Matrix
overexpression led to more fused mitochondria under baseline conditions. Interestingly, we also found that the elongated mitochondria were also more protected against FCCP-induced fission as well as degradation by autophagosomes. Thus, we propose that MCL-1
Matrix
protects mitochondria from unnecessary mitophagy to preserve the bioenergetic capacity when nutrients are limited. We are currently evaluating how cardiac restricted overexpression of MCL-1
Matrix
(αMHC-MCL-1
Matrix
) affects mitochondrial morphology, mitophagy and bioenergetics during fasting.
Abstract 108: Thioredoxin-interacting Protein Interacts with Glucose Transporters to Regulate Cardiac Glucose Metabolism
Authors: Wang Bing F , Myers Ronald B , Fomovsky Gregory M , Lee Samuel , Patwari Parth , Yoshioka Jun
Published: June 2021

A change in basal glucose transport into skeletal muscle, heart, and adipose is a critical feature of insulin resistance and diabetes. Glucose transport is tightly regulated by a protein family of glucose transporters (GLUTs). Thus, defining how cardiomyocytes adapt to changes in extracellular and intracellular glucose concentrations by recruiting GLUTs is crucial to understanding cardiac metabolism under both normal and diabetic conditions. Thioredoxin-Interacting Protein (Txnip), originally characterized as a binding partner of antioxidant thioredoxin, is now known to be a member of the arrestin protein superfamily. We have previously shown that glucose uptake is robustly enhanced in Txnip-knockout hearts; however, the precise mechanism for this effect remains elusive. Here, we present a novel feedback mechanism by which Txnip controls glucose metabolism. High glucose levels induced Txnip expression in rat cardiomyocytes in vitro and in the myocardium of streptozotocin (STZ)-induced diabetic mice in vivo. Our proteomic and functional analyses found that Txnip directly interacts with GLUT1 and GLUT4 and strikingly inhibits cellular glucose uptake through direct interactions with GLUTs. Using inducible cardiac-specific deletion of Txnip (Txnip-CKO), we further demonstrated that Txnip plays a functional role in STZ-induced diabetic cardiomyopathy. While β-adrenergic challenge revealed a blunted myocardial inotropic response in wild type diabetic animals, Txnip-CKO diabetic mice retained a greater cardiac response to β-adrenergic stimulation. An ex vivo analysis of perfused hearts further demonstrated that the enhanced functional reserve afforded by deletion of Txnip was associated with myocardial glucose utilization during β-adrenergic stimulation. Thus, a high extracellular glucose concentration represses glucose uptake into the cytoplasm via Txnip-mediated inhibition of GLUTs. The metabolic alterations affected by Txnip deletion promote myocardial glucose uptake, directing cardiomyocyte towards enhanced functional reserve under diabetic conditions. These results provide a novel link between GLUTs and Txnip and highlight a fundamental regulatory mechanism of glucose homeostasis in the heart.
Abstract 112: Microrna-532-5p Protects the Mouse Heart Against Myocardial Infarction by Repressing a Positive Regulator of Endothelial-to-mesenchymal Transition, Prss23
Authors: Bayoumi Ahmed S , Teoh Jian P , Kim Il-man
Published: June 2021

Myocardial infarction (MI) leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after MI has been identified as a critical event in this process. MicroRNAs (miRs), negative regulators of gene expression, have emerged as important players in heart diseases including MI. We previously showed that miR-532-5p (miR-532) is up-regulated by the β-arrestin-biased β-blocker carvedilol (Carv), which activates protective pathways in the heart independent of G protein-mediated second messenger signaling (Figure A-B). Here, we test whether a β2-adrenergic receptor (β2AR)/β-arrestin-responsive miR, miR-532 functions as a cardioprotective miR against MI. Using cultured cardiac endothelial cell (CEC) and
in vivo
approaches in mice, we demonstrate that knockdown of miR-532 induces CEC apoptosis when subjected to simulated ischemia-reperfusion.
In vivo
blocking of miR-532 via intramyocardial injection of its locked nucleic acid-anti-miR results in post-MI abnormalities in cardiac function and remodeling. Mechanistically, miR-532 promotes cardiac vascularization after MI in part by repressing a predicted target, Prss23 and subsequently activating endothelial cell markers such as CD-31 (Figure C). Prss23, which is a vascular protease and modulates Snail transcription to promote endothelial-to-mesenchymal transition (EndoMT), is also down-regulated by Carv in mouse hearts. In conclusion, our novel findings indicate that miR-532-Prss23 axis acts as an important regulator of CEC function during MI and can be suitable for therapeutic intervention in the setting of ischemic heart disease.
Abstract 114: Downregulation of Small GTPase Rnd3 in Cardiomyocytes Leads to Hyperinflammatory Response in Post-myocardial Infarction Heart
Authors: Dai Yuan , Yang Xiangsheng , Yue Xiaojing , Yang Tingli , Yi Xin , Lin Xi , Wang Fei , Chang Jiang
Published: June 2021

Background:
Hyperinflammatory response leads to maladaptive healing and remodeling after acute myocardial infarction (MI), while a balanced inflammatory response is essential for post-MI repair and remodeling. Most of the studies have focused on inflammatory cells, extracellular matrices and myofibroblasts in the regulation of acute MI-induced inflammatory response, the investigation of crucial role of cardiomyocytes in the initiation and regulation of the responsive inflammation in heart has been overlooked.

Subjects and Methods:
To demonstrate that small G protein Rnd3 functions as a negative regulator to refine the inflammatory response by preventing excessive inflammation in cardiomyocytes after acute MI. Genetic mouse models with cardiomyocyte-specific knockout and overexpression of Rnd3 were studied along with multiple cell culture approaches. An acute MI was introduced by ligation of the left anterior descending artery.

Results:
Mice with cardiomyocyte-specific deletion of
Rnd3
(heterozygous,
Rnd3
+/-
) showed a significantly higher mortality, worse cardiac function, and increased infarct size compared to wild-type control mice after MI. Massive inflammatory cell infiltration along with a profound increase in the expression levels of cytokines, MMP2 and MMP9 were observed in the infarcted and remote areas in
Rnd3
+/-
heart. Mechanistic studies revealed that Rnd3 bound to NFκB complex and attenuated NFκB activity by 1) physically interacting with NFκB component p65; 2) competing with NFκB component p50 for p65 binding; 3) blocking p65 nuclear import. The mechanisms were demonstrated
in vitro
and
in vivo
. Cardiomyocyte-specific overexpression of
Rnd3
in mice inhibited hyperactivation of NFκB signaling, attenuated excessive inflammatory response, reduced infarct areas and ameliorated post-MI cardiac function.

Conclusions:
1) This study demonstrates cardiomyocytes as an intrinsic and crucial component in the initiation of the inflammatory cascade in acute MI; 2) discover Rnd3 as a new “fine-tuning” factor situated at the nexus of the inflammatory response; 3) reveal a novel molecular mechanism through Rnd3-mediated NFκB signaling axes responsible for a balanced inflammatory response after acute MI.
Abstract 119: Tempol Pre-treatment Restores Isoflurane Preconditioning in the Aged Cardiomyocytes: Involvement of Pink1/Parkin-mediated Mitophagy
Authors: Ma Li , Gao Qun , Rebecchi Mario J , Liu Lixin
Published: June 2021

Background:
Aging is associated with chronic oxidative stress and impaired cardiac preconditioning. Four-week pre-treatment with TEMPOL, an antioxidant, improves mitochondrial functions and restores isoflurane (ISO) preconditioning in the aging rat heart. Because mitophagy is implicated in cardiac preconditioning and declines with age, this study was designed to investigate how age influence mitophagy in response to preconditioning and whether TEMPOL pre-treatment improves it.

Methods:
Male Fischer 344 old (22-24 months) rats treated with or without 4-week TEMPOL (2 mM in drinking water) and young (4-6 months) untreated rats were used. Cardiomyocytes isolated from these rats were preconditioned with ISO and then collected for Western blot analyses of mitochondrial PINK1, Parkin and ubiquitination, or fixed for IF staining of co-localization of PINK1/Parkin with mitochondria. Autophagic flux was determined by IF staining of LC3 puncta, membrane-associated LC3-II and p62 in the presence and absences of bafilomycin A1, an autophagy inhibitor. ISO preconditioning was assessed by LDH release in a simulated hypoxia/reoxygenation model.

Results:
ISO protected young but not old cardiomyocytes. Four-week pre-treatment with TEMPOL
in vivo
restored ISO preconditioning in the old cardiomyocytes. In addition, ISO increased mitochondrial PINK1 and Parkin levels and promoted mitochondrial protein ubiquitination in the young, but not the old cardiomyocytes. TEMPOL pre-treatment improved these responses in the old cardiomyocytes. Aging impaired both baseline and ISO-induced autophagic flux, which was restored by TEMPOL pre-treatment. Inhibition of autophagy by bafilomycin abolished ISO preconditioning in the cardiomyocytes from the young and TEMPOL-pre-treated old rats.

Conclusion:
Aging reduces PINK1/Parkin-mediated mitophagy in response to ISO and impairs baseline as well as preconditioning-induced autophagic flux. A 4-week pre-treatment of TEMPOL reverses these changes and restores ISO preconditioning in the old cardiomyocytes. Our results may explain why cardiac preconditioning has failed in some clinical trials, because most patients were with pathologies, such as diabetes and advanced age, in which mitophagy is disrupted.
Abstract 122: Pkialpha Protects Hearts From Beta-adrenergic Overstimulation
Authors: Zhang Xiaoying , Li Ying , Ai Xiaojie , Tang Mingxin , Szeto Christopher , Hua Xiang , Chen Xiongwen
Published: June 2021

Introduction:
Protein kinase inhibitor peptide α (PKIα) isoforms are abundantly expressed endogenous inhibitors of PKA in the heart. The physiological and pathophysiological relevance of PKIα has not been studied.

Hypothesis:
PKIα inhibits PKA activity in normal and stressed (post-myocardial infarction (MI)) hearts.

Methods:
PKIα knockout (KO) and c57bl/6 control (CTR) mice were used to test if PKIα affected cardiac β-adrenergic response and if played a role in post-MI hearts.

Results:
Protein abundance of PKIα was significantly increased in failing human and post-MI mouse ventricular tissue. The knockout of PKIα in mice did not affect the expression of the other two PKI isoforms. Nor did it change the expression of PKA regulatory and catalytic subunits. PKIα KO mice did not show alterations in basal cardiac morphology, function and life span of mice. Intra-LV hemodynamic measurements in response to different doses of isoproterenol showed that the loss of PKIα sensitizes the heart in response to isoproterenol (ISO) with a lower EC50 and the maximum response was increased more in KO mice than in control mice. ISO (10nM) increased myocyte contraction and Ca2+ transient amplitudes more in KO myocytes than control myocytes though 100nM ISO increased myocyte contraction and Ca2+ transients amplitude to the same. At the ISO concentration of 10nM, ISO increased more phosphorylation of the Ser16 site (PKA site) on phospholamban. In post-MI animals, the loss of PKIα enhances cardiac function at 1 week after MI but caused more myocardial infarction and functional depression at 8 weeks after MI.

Conclusions:
PKIα is an important regulator of cardiac β-adrenergic responses by antagonizing PKA. This mechanism could be protective to avoid β-adrenergic overstimulation, especially in post-MI patients and animal models.
Abstract 128: Impaired Beta Adrenergic Receptor Resensitization Contributes to Human Heart Failture
Authors: Martelli Elizabeth E , Chatterjee Arunachal , Mohan Maradumane M , George John , Starling Randall , Moravec Christine , Naga Prasad Sathyamangla V
Published: June 2021

Beta adrenergic receptors (βARs) are one of the most powerful regulators of cardiac function, and βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization by G-protein coupled receptor kinase phosphorylation leading to βAR internalization. Desensitized βARs are resensitized by dephosphorylation in the endosomes by PP2A; however, less is known about role of resensitization in heart failure. Although we have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on
in silico
predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also tested whether resensitization as a mechanism is altered in endstage human heart failure samples. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of βAR function. Significant PI3Kγ activity was observed to increase in heart failure samples while AC activity was reduced in plasma membranes. Consistent with the reduced recovery in βAR function in heart failure samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of human heart failure samples compared to non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in heart failure samples versus controls suggesting inhibited resensitization occurs in human heart failure. Together our studies suggest that resensitization is inhibited in human heart failure and targeting I2PP2A may provide preservation of receptor function and beneficial cardiac remodeling.
Abstract 129: Transient Activation of AMPK Prior to Cardiac Pressure Overload Alleviates Fibrotic Accumulation and Functional Decline
Authors: Reineke Erin L , Taffet George E , Kaelber Jason T , Taegtmeyer Heinrich , Entman Mark L , O'Malley Bert W
Published: June 2021

The multiple adaptive pathways activated during cardiac stress must communicate with each other for an efficient response; however, little is known about the molecular mechanisms underlying this coordination. During left ventricular pressure overload induced by transverse aortic constriction (TAC), an increase in metabolic flux to meet the ATP demand is the first molecular change observed in the heart. Following initial metabolic changes, there is genetic remodeling of the metabolic machinery and activation of other acute and long-term adaptive pathways to control hypertrophy, fibrosis, and contraction. In order to better understand how the early metabolic changes affect the activation and magnitude of the downstream pathways, we treated mice with the AMPK activator AICAR for 6 days prior to TAC and then monitored effects on the cardiac stress response for 4 weeks. This treatment was performed in both WT mice and in mice lacking cardiomyocyte expression of steroid receptor coactivator-2 (SRC-2 CKO), a model we have previously shown to be genetically similar to a stressed mouse and whose function declines rapidly in response to TAC. Interestingly, we found that this small transient treatment with AICAR is sufficient to blunt hypertrophy (20% reduction) and fibrotic accumulation (56% reduction) and prevent left ventricular dilation and pleural edema. Furthermore, AICAR treatment in the SRC-2 CKO animals was able to rescue the functional decline observed post-TAC. We are currently investigating the molecular pathways underlying these changes. Our results strongly suggest that there are very early events during cardiac stress that are key determinants in the ability of the heart to adapt and maintain function under stress, even in late stages post-stress. Disruption of these determinants can lead to rapid failure, whereas their promotion could hold a key for therapeutic intervention.
Abstract 130: Arginine Methylation of Smad6 Defines Bone-morphogenetic-protein (BMP) Signaling Duration and Intensity
Authors: WU JIAN , Jackson-Weaver Olan , Zhang Tingwei , Gou Yongchao , Xu Jian
Published: June 2021

Bone-morphogenetic-protein (BMP)/Smads signaling pathway plays crucial role during heart development and vessel angiogenesis. BMP signaling is induced by the binding of BMP ligands (eg. BMP4) to their receptors, which recruit and phosphorylate receptor-Smads (R-Smads, eg. Smad1, Smad5) that form nuclear-transporting complexes with Smad4 for transcriptional regulation. Smad6 is an inhibitory Smad expresses predominantly in atria-ventricular cushion and outflow tract of the developing mouse heart, and expands to valves and great vessels. At the cell surface level, Smad6 binds to BMP type I receptor to block R-Smads recruitment to the receptor. At cytosolic level, Smad6 block Smad1/Smad4 complex formation. In the nucleus, Smad6 represses transcription. How these three levels of regulation are coordinated to inhibit BMP signaling is not known. We previously showed that BMP ligand induces an acute Smad6 methylation at arginine 74 (R74) at the cell surface level by a methyltransferase PRMT1, and methyl-Smad6 dissociates from receptor to allow receptor-induced Smad1/5 phosphorylation and activation. We further identified a delayed methylation on arginine 81 (R81) of Smad6 in the cytosol by PRMT1. We found that R81 methylation is required for BMP signaling-induced recruitment of Smad6 to phosphor-Smad1; it is also required for Smad6 to disrupt phosphor-Smad1/Smad4 complex formation and the following nuclear transportation, as well as for Smad6 to suppress Smad1 targeting gene transactivation. Previous findings indicate that Smad6 binds to type I receptor and Smad1 through its C-terminal region. We examined how arginine methylation in the N-terminal region, regulates the binding properties of C-terminal Smad6. We found that N-terminal Smad6 stabilizes the interaction between C-terminal Smad6 and Smad1 and enhances Smad6 inhibitory function. Disruption of R81 methylation results in loss of inhibitory function because of an increase in binding between N-term and C-term Smad6 that results in a "closed" conformation. In summary, R81 methylation controls Smad6 activity and R81 methylation of Smad6 defines the duration and intensity of BMP-induced Smad1/5 signaling.
Abstract 132: A Novel Hydrogen Sulfide (H
2
S) Prodrug, SG1002, Protects Against Myocardial Oxidative Damage and Hypertrophic Signaling via Induction of Cystathionine Β-Synthase (CBS) and Antioxidant Proteins
Authors: Islam Kazi N , Donnarumma Erminia , Donnelly Erinn , Lefer David J
Published: June 2021

Background:
Endogenously produced H
2
S is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H
2
S levels have proven cardioprotective in models of acute myocardial infarction and heart failure (HF). The present study was under taken to investigate the effects of a novel H
2
S prodrug, SG1002, on stress induced hypertrophic signaling in murine HL1 cardiomyocytes.

Methods:
HL1 cells were maintained either in serum starvation (1%) or serum containing (10%) media followed by treatment either with SG1002 or H
2
O
2
, or endothelin-1 (ET-1)/phenylephrine (Phe) or in combination. Treated cells were analyzed for specific experimental needs.

Results:
SG1002 significantly increased cellular levels of the H
2
S producing enzyme, CBS, as well as production of H
2
S and nitrosothiol in HL1 cells cultured both in serum starvation or serum containing media. SG1002 significantly inhibited H
2
O
2
and ET-1/Phe induced oxidative stress in both culture media as measured by advanced protein oxidation products and MDA levels. Expression of ANP and BNP were markedly attenuated by SG1002 treatment. Cells cultured in media supplemented with serum containing H
2
O
2
/(ET-1 or Phe) or in 1% serum exhibited decreased levels of CBS, SOD1, and catalase. When the HL1 cells were coincubated with SG1002 cellular damage from oxidative stress was significantly attenuated accompanied by an increase in CBS expression.

Conclusion:
Our data clearly demonstrate that SG1002 attenuates myocardial cellular damage via increasing antioxidant proteins. SG1002 directly increases H
2
S levels and upregulates CBS. Studies are currently underway to evaluate the clinical utility of SG1002 in HF.
Abstract 135: Thoracic Aortic Wall Tension Regulates microRNA-133a Abundance
Authors: Akerman Adam W , Nadeau Elizabeth K , Stroud Robert E , Mukherjee Rupak , Ikonomidis John S , Jones Jeffery A
Published: June 2021

Background:
MicroRNA-133a (miR133a) is a small non-coding RNA, which represses the translation of multiple mRNAs. This laboratory has reported an inverse relationship between aortic diameter and miR133a abundance in aortic tissue from patients with thoracic aortic aneurysm (TAA); as diameter increased, the abundance of miR133a decreased. Given that wall tension at a given pressure increases with increasing vessel diameter (Law of LaPlace), this study tested the hypothesis that elevated aortic wall tension results in a loss of miR-133a.

Methods/Results:
TAA was induced in wild type mice using an established murine model (0.5M CaCl
2
application, 15 min). MiR133a abundance (QPCR) was reduced in TAA tissue (3-wk TAA, 42.1±8.6% p<0.05 vs mice without TAA (100%)). In two
in vivo
models of elevated wall tension (
simulated hypertension
): 1) ANGII (angiotensin II infusion; 1.44mg/kg/day), and 2) BPH2 (spontaneously hypertensive mice, The Jackson Laboratory, Stock #003005), miR133a levels were decreased compared to normotensive controls (ANGII: 53.0±4.3%; BPH2: 51.7±7.0%; p<0.05 vs normotensive control (100%)). Aortic rings from wild type mice were hung on parallel wires in an
ex vivo
tissue myograph at 0.7 g, then ANGII (100nM) was added to the tissue baths, which generated increased tension (1.21±0.15g) and resulted in reduced tissue miR133a abundance (46.0±12%; p<0.05 vs no AngII,). Furthermore, increased tension alone (1.5g, 3 hr) resulted in decreased tissue miR133a abundance (39.0±7.0%; p<0.05 vs 0.7 g tension). Isolated primary aortic cell lines (fibroblasts (FB) and smooth muscle cells (SMC)) were exposed to biaxial cyclic stretch for 3 hr. FB miR133a was reduced (62.8±8.3%; p<0.05 vs unstretched control (100%)), while SMC miR133a abundance remained unchanged.

Conclusion:
The significance of these unique findings is 2-fold: First, tension alone was sufficient to decrease miR133a abundance in aortic tissue. Second, increased tension reduced miR133a abundance in FB, a cell type that is responsible for extracellular matrix remodeling. These findings suggest changes in wall tension alone (
hypertension
) may be associated with pathological extracellular matrix remodeling, in part, through the loss of miR133a in fibroblasts.
Abstract 137: Ctcf is Essential for Transcriptome Regulation in the Healthy Heart
Authors: Rosa-Garrido Manuel , Kimball Todd , Chapski Douglas J , Shih Tsai-Ting , Soehalim Elizabeth , Balderas Enrique , Galjart Niels J , Wang Yibin , Vondriska Thomas M
Published: June 2021

Heart failure is a syndrome resulting from complex genetic predisposition and multiple environmental factors. It is well established that global changes in gene expression accompany the transition through hypertrophy and on to overt failure producing deterioration of function at the organ level. Unknown, however, are the global changes in chromatin structure that allows for pathological gene expression. The epigenetic factor CTCF is a ubiquitous protein thought to be involved in maintenance of chromatin microenvironments. We found that hypertrophic stimulation down-regulates CTCF in the heart, and CTCF expression is significantly correlated with heart size in disease across genetic backgrounds. To test this relationship further, we have generated an inducible cardiac-specific CTCF knockout animal. These animals quickly develop severe heart failure characterized by a dilated left ventricle, decreased ejection fraction, and reactivation of the fetal gene program. Our analysis of published CTCF ChIP-seq data performed in mouse heart has also shown CTCF binding peaks flanking the fetal genes. Furthermore, we have found that CTCF abundance at these sites changes after hypertrophic agonist isoproterenol treatment, suggesting that CTCF protects from the development of cardiac disease by regulating gene expression. We also used RNA-seq to compare the transcriptome of adult myocytes isolated from control, CTCF KO mice, and mice that underwent transverse aortic constriction (TAC) to induce heart failure. These analyses showed that CTCF depletion remodels gene expression in a manner that mimics aspects of the pathological transcriptome in heart failure. Our data demonstrate that precise control of CTCF levels is required for normal transcriptome regulation in the adult heart and that alterations in CTCF levels (either via genetic, pharmacologic or surgical techniques) lead to cardiac pathology.
Abstract 138: Inhibition of Stress-inducible Genes That Distinguish Pathological Cardiac Hypertrophy via Targeting Acidic Nuclear Protein 32e
Authors: Shin Hyewon , Yang Zhi , He Minzhen , Sayed Danish , Abdellatif Maha
Published: June 2021

Cardiac hypertrophy is a manifestation of an increase in the workload imposed on the heart. The cause, duration, and extent of the workload dictate the physiological vs. pathological nature of the hypertrophy and its likelihood to transition into cardiac failure. Our goal is to identify differences between these 2 forms of hypertrophy by examining the binding patterns of key basic transcription factors and regulators in a genome-wide fashion. Our data show that a pause-release mechanism of RNA polymerase II (pol II) bound to the transcriptional start site (TSS) induces housekeeping/essential genes (~ 63% of expressed genes), in a synchronous and incremental fashion that is proportional to the increase in myocyte size and does not require
de novo
pol II binding. This mechanism is responsible for the increase in cell size and mass that constitute hypertrophy in both physiological and pathological forms. Superimposed on this is a second and critical group of genes (~3.5%) that are stress-induced in the pathological form of hypertrophy but are minimally, if at all, expressed in the postnatal and adult heart (e.g.
Acta1, Ankrd1,
Xirp2, Nppa, Col1a1
..etc). In contrast to the housekeeping genes, these genes require
de novo
pol II recruitment and exhibit a robust increase in expression. To further identify the transcriptional mechanisms that distinguish these sets of genes, we performed chromatin immunoprecipitation-deep sequencing (ChIP-Seq) analysis for cyclin-dependent kinase 9 (Cdk9) and histone H2A.z. The results reveal an increase in Cdk9 at the TSS of paused genes vs. an increase at TSS and throughout the gene body of inducible genes. In contrast, H2A.z levels remained constant. However, we found that Acidic Nuclear Protein 32e (Anp32e), a nuclear protein phosphatase 2A (PP2A) inhibitor, interacted with H2A.z and regulated phosphorylation of Cdk9. Interestingly, knockdown of Anp32e during pressure overload hypertrophy resulted in 70-90% inhibition of stress-inducible genes, but only an incremental inhibition of the increase in cardiac size. Thus, we propose that targeting Anp32e may partially revert pathological hypertrophy to a more physiological form via specifically inhibiting the stress-induced genes while preserving the increase in cardiac mass.
Abstract 139: Role of Histone Lysine Demethylase PHF8 in Epigenetic Regulation of Embryonic Stem Cell Cardiac Differentiation
Authors: Tang Yan , Yue Yujia , Verma Suresh K , Khan Mohsin , Kishore Raj
Published: June 2021

Epigenetic control mechanisms play a key role in the regulation of lineage commitment of stem/progenitor cells, while the epigenetic regulators involved in the determination of cardiomyogenic lineage are incompletely defined. Using in vitro cardiac differentiation system of mESCs with Brachyury and Nkx2.5 selection, we analyzed expression profiles of epigenetic regulators at critical stages of cardiomyogenesis by RT
2
profiler PCR arrays. To identify the potential epigenetic regulators in the cardiac lineage decision, we compared their expression levels in Brachyury
+
mesodermal cells with Brachyury
-
cells, and in Nkx2.5
+
cardiac progenitor cells with the Nkx2.5
-
cells, respectively. To further understand the role of these epigenetic regulators in cardiac lineage commitment, we knockout these genes and investigate their function. For example, deletion of the H3K9me2 demethylase PHF8 in mESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis, without significant differences between differentiating wide-type (ph8
+/Y
) and ph8
-/Y
ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. These results reveal the new epigenetic control mechanism in mesodemal and cardiac differentiation and establish a link between the apoptosis and cell lineage decision as well as cardiogenesis.
Abstract 2: Cortical Bone Stem Cells Derived Exosomes as Potent Modulator of Cardiac Immune Response and Repair After Injury
Authors: Mohsin Sadia , Troupes Constantine D , Khan Mohsin , Yang Yijun , Johnson Jaslyn , Petovic Jennifer L , Starosta Timothy , Berretta Remus M , Kubo Hajime , Houser Steven R
Published: June 2021

Rationale:
Cortical bone derived stem cells (CBSCs) are known to have improved growth kinetics and myocardial repair properties that are superior to other known stem cell types used. Salutary effects of CBSCs in large are mediated by paracrine secretion. Since exosomes represent an active component of released factors we tested if CBSC derived exosomes (CBSCs-Exo) can recapitulate the beneficial reparative effects of CBSCs.

Objective:
Determine CBSCs derived exosomes and their contents for myocardial repair.

Methods and Results:
Exosomes were isolated from murine CBSCs by ultracentrifugation and had typical size (30-100nm), as validated by electron microscopy and dynamic light scattering. To determine cardiac therapeutic value, CBSCs- Exo (60μg) were injected into the border zone of the mouse heart after myocardial infarction (MI). Animals injected with CBSCs-Exo had reduced infarct size and increased myocyte survival after MI injury. Interestingly, serum levels of pro-inflammatory cytokines were significantly reduced along with decreased expression of CD68+ cells in animals receiving CBSCs-Exo versus control animals. Long term analysis of CBSC-Exo animals showed improved cardiac function and contractility compared to saline treated animals concurrent with enhanced angiogenesis 6 weeks after MI. Salutary effects of CBSC-Exo were confirmed in vitro. CBSCs-Exo increased cardiac protection in NRVMs after hypoxic challenge and enhanced tube formation in HUVECs. Simultaneously, treatment of bone marrow-derived macrophages stimulated with lipopolysaccharide (LPS) and treated with CBSCs-Exo showed increased polarization towards the M2 phenotype, demonstrating an immunomodulatory capacity of CBSCs-Exo. The underlying mechanism for beneficial effects was linked to increased packaging of cardioprotective miRs including miR125, miR20 and miR18a in CBSCs-Exo confirmed by MiRNA array analysis.

Conclusion:
Exosomes derived from CBSCs provide a cell free system that retains the reparative power of CBSC. CBSCs-Exo augment cardiac function after myocardial injury recapitulating earlier findings with CBSCs. The packaging of cardioprotective and immune-modulatory miRs in CBSCs-Exo appears to enhance their reparative effects after MI.
Abstract 200: Role of Silent Mating Type Information Regulation 2 Homolog - 6 in Wound Healing in Diabetes
Authors: Amirthalingam Thandavarayan Rajarajan , Joladarashi Darukeshwara , Jeyabal Prince , Suresh Babu Sahana , K Verma Suresh , Venkata Naga Srikanth Garikipati , Khan Mohsin , Kishore Raj , Krishnamurthy Prasanna
Published: June 2021

Delayed wound healing is one of the major complications in diabetes and is characterized by chronic proinflammatory response, and abnormalities in angiogenesis and collagen deposition. Sirtuin family proteins regulate numerous pathophysiological processes, including those involved in promotion of longevity, DNA repair, glycolysis and inflammation. However, the role of sirtuin 6 (SIRT6), a NAD+-dependent nuclear deacetylase, in wound healing specifically under diabetic condition remains unclear. To analyse the role of SIRT6 in cutaneous wound healing, paired 6-mm stented wound was created in diabetic db/db mice and injected siRNA against SIRT6 in the wound margins (transfection agent alone and nonsense siRNA served as controls). Wound time to closure was assessed by digital planimetry, and wounds were harvested for histology, immunohistochemistry and Western blotting. SIRT6-siRNA-treated diabetic wound showed impaired healing, which was associated with reduced capillary density (CD31-staining vessels) when compared to control treatment. Interestingly, SIRT6 deficiency decreased vascular endothelial growth factor expression and proliferation markers in the wounds. Furthermore, SIRT6 ablation in diabetic wound promotes nuclear factor-κB (NF-κB) activation resulting in increased expression of proinflammatory markers (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor-α and interleukin-1β) and increased oxidative stress. Collectively, our findings demonstrate that loss of SIRT6 in cutaneous wound aggravates proinflammatory response by increasing NF-κB activation, oxidative stress and decrease in angiogenesis in the diabetic mice. Based on these findings, we speculate that the activation of SIRT6 signalling might be a potential therapeutic approach for promoting wound healing in diabetics.
Abstract 203: A Shh Coreceptor Cdo is Required for Efficient Cardiomyogenesis of Pluripotent Stem Cells
Authors: Jeong Myong-Ho , Leem Young-Eun , Kim Hyun-Ji , Kang Kyungjin , Cho Hana , Kang Jong-Sun
Published: June 2021

Sonic hedgehog (Shh) signaling plays an important role for early heart development, such as heart looping and cardiomyogenesis of pluripotent stem cells. A multifunctional receptor
Cdo
functions as a Shh coreceptor together with Boc and Gas1 to activate Shh signaling and these coreceptors seem to play compensatory roles in early heart development. Thus in this study, we examined the role of
Cdo
in cardiomyogenesis by utilizing an in vitro differentiation of pluripotent stem cells. Here we show that
Cdo
is required for efficient cardiomyogenesis of pluripotent stem cells by activation of Shh signaling.
Cdo
is induced concurrently with Shh signaling activation upon induction of cardiomyogenesis of P19 embryonal carcinoma (EC) cells.
Cdo
-depleted P19 EC and
Cdo
–/–
mouse embryonic stem (ES) cells display decreased expression of key cardiac regulators, including Gata4, Nkx2.5 and Mef2c and this decrease coincides with reduced Shh signaling activities. Furthermore
Cdo
deficiency causes a stark reduction in formation of mature contractile cardiomyocytes. This defect in cardiomyogenesis is overcome by reactivation of Shh signaling at the early specification stage of cardiomyogenesis. The Shh agonist treatment restores differentiation capacities of
Cdo
-deficient ES cells into contractile cardiomyocytes by recovering both the expression of early cardiac regulators and structural genes such as cardiac troponin T and Connexin 43. Therefore
Cdo
is required for efficient cardiomyogenesis of pluripotent stem cells and an excellent target to improve the differentiation potential of stem cells for generation of transplantable cells to treat cardiomyopathies.