bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 357: DNA Methylation Profiles of African American Val122Ile-Transthyretin Mutation Carriers Reveals Genes Involved in Amyloidosis Regulation
Authors: Pathak Gita , Wendt Frank , De Lillo Antonella , Nunez Yari , Goswami Aranyak , De Angelis Flavio , Fuciarelli Maria , Kranzler Henry , Gelernter Joel , Polimanti Renato
Published: October 2020

The
Transthyretin
(
TTR
) Val122Ile mutation causes a rare life-threatening disorder attributable to amyloid deposition. This mutation is mainly present in people of African descent; carriers have an increased risk of congestive heart failure and several other non-cardiac phenotypes such as carpal tunnel syndrome, peripheral edema, and arthroplasty. Cardiac disease in Val122Ile carriers may not depend solely on this mutation and other uninvestigated factors could contribute to clinical heterogeneity. One possible mechanism is through DNA methylation, the addition of a methyl group on CG-dinucleotides, which can result in altered gene expression. We investigated methylation changes contributing to heart disease in Val122Ile carriers to identify non-TTR regulatory mechanisms. We investigated 96 Val122Ile carriers of genetically-confirmed African descent using the Illumina EPIC array, which covers 850,000 methylation sites across the genome. We found changes in five methylated sites associated with heart disease. These map to
FAM129B, SKI, WDR27, GLS
, and an intergenic site near RP11-550A5.2 (p=1.6 to 4.6e-8), and a methylated region containing
KCNA6
and
GALNT3
(p=1.1e-12). Weighted methylated sites mapped to PPI network analysis identified
ABCA1
gene (p=0.001). We also found six cis-mQTLs associated with the
FAM129B
CpG site (p=4.1e-24 to 2.8e-14). We replicated two of the aforementioned CpG sites near RP11-550A5.2 (p=0.021) and in
FAM129B
(p=0.016) at nominal significance in a case-control analysis of confirmed cases of TTR amyloidosis.
GLS
encodes glutaminase, which catalyzes the conversion of glutamine to glutamate. Its expression is increased in amyloid-beta neurons and neurofibrillary tangles.
ABCA1
regulates cholesterol transport and interacts with
APOA1
and
APP
; its dysregulation increases amyloid deposition. Increasing
FAM129B
expression improves the clearance of amyloid deposits and rescues hippocampal neurons from apoptosis. The
SKI
expression modulates
TGF-beta
, resulting in cardiac fibrosis. Of note,
SKI
and
FAM129B
together are involved in the regularion of various muscle tissues. Collectively, these findings suggest that a complex amyloid-related gene circuit could explain diverse symptoms in Val122Ile carriers.
Abstract 376: Functional Significance of Anatomically Heterogeneous Distribution of Transversal-axial Tubule System in Mouse and Human Atrial Myocardium
Authors: Medvedev Roman Y , Ratajczyk Lucas , Deguire Frank C , Vasquez Alexander R , Lang Di , Glukhov Alexey
Published: October 2020

Background:
In cardiac myocytes, efficient and synchronous excitation-contraction coupling (ECC) occurs through a specialized network of membrane invaginations called transverse-axial tubule system (TATS). In contrast to an extensive TATS in ventricular myocytes, atrial cells show a significantly heterogeneous organization of TATS, varying from a ventricular-like organized TATS to absent/irregular TATS. However, anatomical distribution of atrial TATS and its functional significance remains unknown. We hypothesized that atrial myocytes with a different TATS organization have a specific anatomical location within the atria that determines their impact on atrial ECC and contraction.

Methods and Results:
In order to avoid the potential bias associated with cell isolation, we applied a mosaic imaging protocol at the whole-mount isolated mouse atrial preparation stained with a membrane dye RH-237. We developed an algorithm that can automatically remove the outer membrane and analyze TATS organization. This analysis showed that myocytes located within the inter-caval region (ICR, i.e., between the superior and inferior vena cava and between the crista terminalis in atrioventricular junction) have a lower TATS organization than cells located within the right atrial appendage (RAA): 0.21±0.01 A.U. vs. 0.50±0.02 A.U. in ICR vs. RAA, respectively, P<0.01. Fluorescent optical mapping of Ca
2+
transients from isolated mouse atrial preparations revealed that ICR region has a longer Ca
2+
transient rise up time compared to RAA (7.8±0.3 ms vs. 6.3±0.3 ms, P<0.01). Moreover, a similar region-specific TATS organization was found in human non-failing (donor) atrial tissue samples. The analysis of TATS density in specimens selected from ICR and RAA regions of the human hearts (n=4) showed a significantly lower density of TATS in ICR compared to RAA region (5.8±0.2% vs. 10.6±0.4%, P<0.01).

Conclusion:
Our findings show a region-specific structural and functional differences of myocytes from RAA and ICR regions of mouse and human heart that could represent a general organization of the mammalian atria. It may have important implications into establishment of different functional roles of cells with a specific TATS structure in atrial physiology and pathology.
Abstract 387: Role of Aldehyde Dehydrogenase (aldh) 2 in Angiotensin II-induced Decrease in Angiogenesis of Coronary Endothelial Cells
Authors: Roy Bipradas , Palaniyandi Suresh S
Published: October 2020

Diabetes-induced coronary endothelial cell (CEC) dysfunction following defective angiogenesis is reported in cardiovascular diseases (CVD). Angiotensin II (Ang II), a vasoactive molecule, is upregulated in diabetes. However, the underlying molecular mechanisms of Ang II-induced CEC dysfunction are not fully understood. Aldehyde dehydrogenase (ALDH) 2 is cytoprotective in diabetic CVD. Thus, we hypothesize that ALDH2 improves Ang II-mediated defective CEC angiogenesis. To test our hypothesis, we treated the cultured mouse CECs with Ang II (0.1, 1 and 10 μM) for 2 and 4 hours. Next, we treated CEC with Alda-1 (10 μM), an ALDH2 activator or disulfiram (2.5 μM), an ALDH2 inhibitor, before challenging MCECs with Ang II. We found that Ang II attenuated tube formation (P<0.05 vs control) which indicates
in vitro
angiogenesis. Next, we found that Ang II have downregulated the mRNA expressions of vascular endothelial growth factor receptor VEGFR1 (p<0.05) and upregulated angiotensin II type-2 receptor (AT2R) (P<0.05) in cultured CECs compared to controls. ALDH2 inhibition with disulfiram potentiated Ang II-induced decrease in angiogenesis (P<0.005) by decreasing the expressions of VEGFR1 (P<0.0005) and increasing the expression of AT2R (p<0.05) relative to Ang II alone. Additionally, activation of ALDH2 activity with Alda-1 rescued Ang II-induced decrease in angiogenesis (P<0.05) by increasing the expression of VEGFR1 (P<0.05) and decreasing the expression of AT2R (P<0.05) relative to Ang II alone. Finally, we conclude that ALDH2 can be an important therapeutic target to improve coronary angiogenesis in diabetic CVD.
Abstract 394: Nicotinamide Phosphoribosyltransferase (Nampt) Potentiates Antioxidant Defense in Diastolic Heart Failure Associated With Diabetes
Authors: Oka Shinichi , Byun Jaemin , Huang Chun-yang , Imai Nobushige , Ralda Guersom , Zhai Peiyong , Xu Xiaoyong , Ikeda Yoshiyuki , Mizushima Wataru , Kashihara Toshihide , Sadoshima Junichi
Published: October 2020

More than half of diabetic patients develop diastolic heart failure. Nampt is a rate-limiting enzyme for NAD synthesis. Phosphorylation of NAD by NAD kinase (NADK) generates NAD phosphate (NADP), which is in turn converted to NADPH and provides the GSH and thioredoxin (Trx) systems with a reducing power. Whether Nampt is protective against diabetic cardiomyopathy (DCM) and, if so, whether NADK mediates the salutary function of Nampt are unknown. We employed mice with cardiac-specific Nampt overexpression (Nampt-cTG) and a mouse model of DCM induced by high fat diet (HFD) consumption. HFD-induced diastolic dysfunction, indicated by an elevated end-diastolic pressure-volume relationship (EDPVR), was ameliorated in Nampt-cTG mice (EDPVR: wild-type mice (WT) Normal Diet (ND): 0.045; WT HFD (1 month): 0.136; Nampt-cTG: 0.042: Nampt-cTG HFD: 0.055*, p<0.05 vs WT HFD). The protective effect was abolished by thionicotinamide, an NADK inhibitor (EDPVR: Nampt-cKO HFD with thionicotinamide 0.12*, p<0.05 vs Nampt-cKO HFD). Nampt overexpression also attenuated HFD-induced increases in GSSG/GSH and oxidation of Trx1 substrates, including peroxiredoxin 1 (Prdx1). Nampt overexpression upregulated not only NAD(H) (1.5 fold) but also NADP(H) (1.3 fold) in the heart and in cardiomyocytes. Nampt-induced NADPH upregulation was abolished in the presence of NADK knockdown. Nampt attenuated H
2
O
2
-induced oxidation of Trx1 substrates in an NADK-dependent manner in cardiomyocytes. Nampt overexpression inhibited H
2
O
2
-induced cell death, an effect that was attenuated in the presence of NADK inhibition. Systemic Nampt heterozygous knockout promoted HFD-induced diastolic dysfunction and oxidative stress in the heart
in vivo
. These results suggest that Nampt protects the heart against diastolic dysfunction through upregulation of the reducing system, which occurs through NADK-dependent upregulation of the reducing equivalent, namely NADPH.
Abstract 399: Tip60 Depletion in Adult Cardiomyocytes Promotes Proliferation and Preserves Cardiac Function After Myocardial Infarction
Authors: Wang Xinrui , Wan Tina C , Lauth Amelia , Patterson Michaela , Lough John W , Auchampach John A
Published: October 2020

Cardiac disease and injury are accompanied by significant cardiomyocyte (CM) loss. CMs are essentially non-regenerable, preventing re-muscularization and resulting in permanent dysfunction during the healing process. The identification of factors from multiple layers of inhibitors that induce and maintain CM’s pronounced state of proliferative senescence provides potential therapeutic targets. Tip60 (
T
at-
i
nteractive
p
rotein, 60 kD), a pan-acetylase tumor suppressor encoded by the
Kat5
gene, activates multiple anti-proliferative pathways in other cell types. Tip60 is known to acetylate Atm and p53, which respectively activate the DNA damage response and apoptosis. And, Tip60 regulates intracellular levels of p21 and Tert polymerase in a fashion designed to maintain proliferative senescence. Although these functions promote CM proliferative senescence and Tip60 is strongly expressed in the myocardium, whether Tip60 regulates these activities in CMs remains unknown. This study tests the hypothesis that Tip60 inhibits CM cell-cycle activation, promotes apoptosis in the myocardium, and prevents regeneration after cardiac injury. In adult mice containing
LoxP
-flanked
Kat5
alleles and a tamoxifen-inducible Cre-recombinase transgene driven by

Myh6 (Kat5
flox/flox;Myh6-merCremer

)
,
Tip60 depletion three days after myocardial infarction (MI) preserved cardiac function as assessed by echocardiography at 10, 21, and 28 days post-MI. Histologic evaluation revealed that depletion of Tip60 in CMs significantly diminished scarring and increased CM cell-cycle activation, as indicated by increased numbers of Ki67-, BrdU- and pH3-positive CMs at 28 days post-MI. This was accompanied by the presence of CMs in the infarct border zone expressing smooth muscle α-actin, indicative of CM de-differentiation, and by reduced apoptosis in the remote zone as assessed by TUNEL and cleaved caspase-3 staining, suggesting reduced cardiac remodeling. These findings suggest that genetic depletion of Tip60 protects CMs from ischemia-induced cell death while promoting CM proliferation, implicating Tip60 as a novel candidate for cardiac therapeutics.
Abstract 405: A Personalized, 3D Printed
in vitro
Model of Vascular Anastomosis in Single Ventricle Heart Defects
Authors: Tomov Martin L , Jing Bowen , Kumar Akaash , Do Katherine , Cetnar Alexander , Bhamidipati Sai Raviteja , Perez Lilanni , Panoskaltsis Nicki , SLESNICK Tim C , Avaz Reza , MANTALARIS Athanasios , Lindsey Brooks , Bauser-Heaton Holly , Serpooshan Vahid
Published: October 2020

Single ventricle physiology is a complex disease state requiring multiple open-heart surgeries to achieve stable hemodynamics. For patients with abnormalities in the pulmonary arteries (PAs), these must be remedied before the patient can be a candidate for such palliations. Transcatheter techniques could rescue this subset of single ventricle patients through intervascular PA connections, allowing a high-risk population to ultimately achieve stable pulmonary blood flow. However, there is currently no
in vitro
platform to model transcatheter processes for anastomosis, particularly to palliate single ventricle defects. This project utilizes 3D bioprinting and perfusion bioreactor technologies to develop a

functional
in vitro
biological device

to model severely stenotic PAs of single ventricle patients. Human endothelial (ECs) & smooth muscle (SMCs) cells embedded in extracellular matrix
bioink
are used in a multi-material bioprinting approach to create
3D bilayer vascular structures
with controlled geometry and flow.
In collaboration with CHOA Cardiac Catheterization Laboratory
, stent devices are deployed in the printed model to re-establish intervascular connection. Healthy, stenotic, and stented tissues are cultured via a bioreactor and analyzed for flow hemodynamics by echo PIV and 4D MR imaging. Cell viability, proliferation, and endothelialization of printed vessels, plus EC-SMC interplay were closely monitored pre- and post- anastomosis, to identify the effect of geometry and flow on cellular overgrowth. This advanced planning enables a subset of single ventricle patients, otherwise not eligible, to ultimately accept further palliative strategies.
Abstract 412: DJ-1 Deficiency Impairs Post-Ischemic Cardiac Fatty Acid Oxidation
Authors: Casin Kevin M , Pantner Yvanna , Shimizu Yuuki , Polavarapu Rohini , Chin Lih-Shen , Li Lian , Calvert John W
Published: October 2020

Background:
DJ-1 is a cytoprotective protein implicated in many cellular processes. We have recently shown that DJ-1 plays a protective role in the setting of acute myocardial ischemia-reperfusion injury and heart failure. However, specific mechanisms of action remain unknown. Here, we sought to determine if DJ-1 maintains fatty acid oxidation following myocardial I/R injury via the regulation of PPARalpha transcriptional activity.

Methods and Results:
WT and DJ-1 KO hearts were subjected to 45 minutes of ischemia via coronary artery ligation followed by up to 72 hours of reperfusion. Initial studies revealed that PPARalpha activity (PPARalpha Transcription Factor Assay Kit; abcam) was lower in DJ-1 KO hearts under both basal (sham) and I/R conditions. Next, we performed an analysis of PPAR genes using a qPCR array (Mouse PPAR Targets RT2 Profiler PCR Array from Qiagen) that profiled the expression of 84 key genes involved in PPAR activation and response. Marked differences in genes involved in FA metabolism were evident in the hearts of DJ-1 KO mice. Further studies using qPCR validated a significant decrease in the expression of acsl1 (protein ACSL1), acsl3, acsl5, cpt1b (protein CPT1), slc25a20 (protein CACT), and cpt2. The protein expression of ACSL1, CPT1, and CACT were also decreased in the hearts of DJ-1 KO mice following I/R injury. These changes were accompanied by higher cardiac lipid content and depressed mitochondrial fatty-acid β-oxidation and ATP synthesis.

Conclusion:
These data demonstrate that DJ-1 plays an essential role in regulating post-I/R cardiac FAO. Future studies will garner mechanistic insight for the impaired function of PPARalpha with the loss of DJ-1.
Abstract 413: Inhibition of Phospholipase A2 Preserves Endothelial Cell Migration in Presence of Lipid Oxidation Products
Authors: Putta Priya , Smith Andrew H , Chaudhuri Pinaki , Graham Linda M
Published: October 2020

During vascular intervention oxidized low-density lipoprotein (oxLDL) and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, which inhibits endothelial cell (EC) migration, and impedes endothelium healing. We have previously shown that lysoPC activates canonical transient receptor potential 6 (TRPC6) channel that leads to a prolonged [Ca2+]i influx, causing the inhibition in EC migration. We hypothesize that lysoPC activates phospholipase A
2
(PLA
2
) crucial for TRPC6 activation; PLA
2
acts on cellular membranes to release arachidonic acid that opens arachidonate regulated calcium channel to release the initial calcium required to trigger TRPC6, blocking PLA
2
will prevent the opening of TRPC6 channels, preserve migration and promote endothelium healing. After incubation of bovine aortic EC with ATK or Ax048 to block PLA2G4, or with BEL, FKGK11, or FKGK18 to block PLA2G6, the effect of lysoPC-induced TRPC6 externalization and EC migration was assessed. Reversible PLA2G6 pharmacological inhibitors maximally blocked lysoPC-induced TRPC6 externalization, arachidonic acid release and preserved EC migration; exemplified with biotinylation assay, arachidonate enzyme assay, and razor scrape migration assay respectively. Immunofluorescence microscopy for TRPC6 plasma membrane translocation and PLA
2
enzyme activity assay supported these findings. To further verify the specific isoforms involved in blocking TRPC6 externalization, siRNA mediated transient knockdown studies were performed with PLA2G4A, PLA2G4C, PLA2G6A, and PLA2G6B siRNAs in EA.hy926— human endothelial cell line. PLA2G6A and not PLAG6B/4A/4C downregulation completely blocked TRPC6 externalization and preserved migration comparable to control levels. These studies confirmed the role of PLA2G6A (a calcium-independent cytosolic phospholipase A
2
group VI β isoform) in blocking lysoPC-induced TRPC6 activation and preservation of EC migration. Additional studies are in progress to confirm
in vivo
relevance of these findings. Our results show the potential for developing PLA
2
targeted therapies to block TRPC6 activation and promote endothelium healing, thus improving the outcomes for patients undergoing cardiovascular intervention.
Abstract 417: Cardiac Akap12 Signalosome Overexpression Exacerbation of Heart Failure and β-arrestin Signaling Pathway
Authors: Qasim Hanan , Reyes Alcaraz Arfaxad , McConnell Bradley K
Published: October 2020

Background:
Heart failure (HF) is responsible for 1 out of 8 deaths per year in the U.S.A. andis the major cause of death globally. In HF, chronic β-adrenergic receptor (β-AR)stimulation leads to reduced cardiac function due in part to β-AR desensitization.β-arrestins are proteins that play a major role in desensitizing G protein-coupledreceptors (GPCRs) such as β-AR. Previously we reported enhanced cardiacfunction in mice lacking functional A Kinase Anchoring Protein 12 (AKAP12). Inthis study, we aim to investigate the impact of AKAP12 overexpression(oxAKAP12) on HF progression through assessing β-arrestins.
Our central hypothesis
is that cardiac AKAP12 overexpression potentiates HF developmentby influencing β-arrestin signaling downstream of the β-AR.

Methods:
HF was developed in WT and oxAKAP12-Tg mice (8-10) weeks old males andfemales, through chronic Isoproterenol (ISO) administration (60mg/kg/day for 14days). Left ventricular homogenates were used for gene expression analysis.Furthermore, AKAP12 was transiently overexpressed in AC16 cells (humancardiomyocytes cell line), to asses protein expression levels and Gαs pathwayactivity, upon treatment with 100 nM of ISO.

Results:
Cardiac oxAKAP12 in both males and females reduced left ventricular ejectionfraction (EF) by 14.5±2.5% and fractional shortening (FS) by 22.7±2% after 14-days of chronic ISO treatment when compared to control groups. β-arrestin-1gene expression levels were significantly lower (p=0.022) in oxAKAP12 malehomogenates treated with ISO (14 days) compared to control groups.Interestingly, female homogenates overexpressing AKAP12 showed significantlyhigher β-arrestin-1 gene expression levels (p<0.0001) with ISO treatment,compared to control groups. In AC16 cells overexpressing AKAP12 and treatedwith ISO, Gαs activity and β-arrestin-1 protein expression levels were bothreduced by 50% compared to AC16 ISO treated groups.

Conclusion:
Cardiac oxAKAP12 negatively influences systolic function in both male andfemale mice, potentially through affecting β-arrestin signaling pathway. Thus,designing novel drugs to inhibit AKAP12 is promising to ameliorate HF.
Abstract 418: Caveolar Disruption of L-type Calcium Channel and Ryanodine Receptor Facilitates Atrial Ectopy and Arrhythmogenesis in Heart Failure Mice
Authors: Lang Di , ratajczyk Lucas , Tyan Leonid , Turner Daniel , Alvarado Francisco , Kamp Timothy J , Glukhov Alexey
Published: October 2020

Atrial fibrillation (AF) often occurs during heart failure (HF). Ectopic foci that trigger AF, are linked to discrete atrial regions that experience the highest remodeling and clinically used for AF ablation; however, mechanisms of their arrhythmogenic propensity remain elusive. We employed
in vivo
ECG telemetry,
in vitro
optical mapping and confocal imaging of Ca
2+
transients (CaT) from myocytes isolated from the right atrial appendage (RAA) and inter-caval region (ICR) of wild type (WT, n=10), caveolin-3 knockout (KO, n=6) and 8-weeks post-myocardial infarction HF (n=8) mice. HF and KO mice showed an increased susceptibility to pacing-induced AF and enhanced ectopy originated exclusively from ICR. Optical mapping in isolated atria showed prolongation of CaT rise up time (CaT-RT) in HF ICR, which suggested a remodeled coupling between L-type Ca
2+
channels (LTCCs) and ryanodine receptors (RyRs) in this specific region. In WT mice, RAA consists of structured myocytes with a prominent transverse-axial tubular system (TATS) while ICR myocytes don’t have TATS. In RAA, CaT-RT depends on LTCCs in TATS triggering RyR, while in ICR, all the LTCCs are localized in surface caveolae where they can activate subsarcolemmal RyRs and lead to a slow diffusion of Ca
2+
inside the cell interior. Downregulation of caveolae was observed specifically in HF ICR. To mimic this, we used cav3-KO mice. Triggered activities were observed in myocytes isolated from HF and KO ICR, which presumably underlie the ectopic activities in tissue level. These myocytes presented significantly unsynchronized sarcoplasmic reticulum (SR) Ca
2+
releases (synchronization index: 10.8±0.9 in WT vs 38.3±4.1 in HF vs 21.5±2.1 in KO,
p
<0.01 for HF and KO vs WT respectively) especially at the subsarcolemmal space that prolongs CaT-RT (62.2±4.1 ms in WT vs 122.5±12.8 ms in KO,
p
<0.01). In addition, failing ICR myocytes showed a higher occurrence and size of spontaneous Ca
2+
sparks which were linked to CaMKII activity and associated phosphorylation of RyR. Our findings demonstrate that in HF, caveolar disruption creates “hot spots” for arrhythmogenic ectopic activity emanated from discrete vulnerable regions of the right atrium which are associated with desynchronized SR Ca
2+
release and elevated fibrosis.
Abstract 420: Mrs2 is the Authentic Mammalian Mitochondrial Mg
2+
Channel
Authors: Velusamy Prema , Santhanam Shanmughapriya
Published: October 2020

Magnesium (Mg
2+
) is an important cation critical for cellular functions and tissue integrity. Mitochondria have been demonstrated to be capable of both accumulate and release Mg
2+
. However, the exact molecular machinery associated with mitochondrial Mg
2+
(mMg
2+
) influx has not yet been delineated. In the present study we characterized the mammalian mMg
2+
channel, Mrs2 and comprehensively studied its role in energy metabolism. Protein flux, membrane fractionation and STED microscopy studies revealed Mrs2 to localize on the inner mitochondrial membrane with its N and C-terminus in the matrix. Western blot and qPCR analysis confirmed the ubiquitous distribution of Mrs2 in all metabolically active tissues. We adopted lentiviral based strategy to stably knock down (KD) Mrs2
in vitro
. Primarily, the use of FRET-based mMg
2+
sensor,
MitoMario
showed a decreased influx of Mg
2+
into mitochondria in Mrs2 KD cells. This was further confirmed by patch clamping the mitoplasts of the control and Mrs2 KD cells. Because Mg
2+
is an important co-factor in the machineries that replicate, we next assessed the mitochondrial copy number. The decreased influx of mMg
2+
impacted the mitochondrial copy number and electron transport chain (ETC) complex assembly. The defective ETC assembly was marked by increased generation of mitochondrial reactive oxygen species, increased proton leak, decreased ATP levels, and also prompted a metabolic switch from mitochondrial oxidative phosphorylation to glucose oxidation in Mrs2 KD cells. Additionally, Mrs2 KD cells had an increased sensitivity to mROS-induced mitochondrial permeability transition pore opening. To further study the role of Mrs2 in cardiac mitochondrial metabolism and cellular energetics, we have successfully adopted the CRISPR/Cas9 mediated gene targeting strategy to generate the cardiac-specific Mrs2 knock out mouse model. Our study is the first of its kind to characterize the mitochondrial Mg
2+
channel and its impact on mitochondrial copy number and cell viability. Our findings not only identify Mrs2 as an authentic mitochondrial Mg
2+
channel, but also validates the critical role of mMg
2+
in maintaining the bioenergetic state of the cell.
Abstract 424: Loss of Sirt2 Protects Against Pressure Overload- and Ischemic Reperfusion Injury-induced Cardiac Dysfunction
Authors: Sato Teruki , Yan Xiaoyan , Chang Hsiang-Chun , Chunlei Chen , Shapiro Jason S , Ardehali Hossein
Published: October 2020

Introduction:
Sirtuins are NAD+ dependent deacetylases and critical regulators of energy metabolism and response to oxidative stress. Sirtuin2 (SIRT2) is a cytoplasmic member of the sirtuin family, and has been shown to regulate cellular iron homeostasis through deacetylation of nuclear factor erythroid-derived 2-related factor 2 (NRF2). However, whether SIRT2-NRF2 pathway is involved in the development of heart failure remains unknown.

Methods and results:
To investigate the functional role of SIRT2 in the response to cardiac stress, SIRT2 knockout (KO) mice and their littermate controls were subjected to pressure overload by transverse aortic constriction (TAC). SIRT2 KO had normal appearance and cardiovascular parameters at baseline. However, in response to TAC,
Sirt2
-/-
mice displayed resistance to the pathological hypertrophic response, whereas wild type (WT) mice developed cardiac hypertrophy and heart failure. In addition, SIRT2 KO mice displayed less cardiac damage after /reperfusion injury. SIRT2 knockdown in neonatal rat cardiomyocytes (NRCM) reduced reactive oxygen species (ROS) production and cell death after H2O2 treatment. Since cellular oxidative stress is one of major contributor of cardiac dysfunction caused by both I/R injury and pressure overload, we examined whether NRF2 is associated with SIRT2-mediated cardiac response to oxidative stress. Levels of NRF2 was upregulated in NRCM with SIRT2 knockdown and treated with H2O2 compared to wild type (WT) cells. Moreover, NRF2 is translocated into the nucleus and its anti-oxidant target proteins are upregulated in NRCM with SIRT2 knockdown. SIRT2 was also found to bind and deacetylate NRF2 directly as determined by co-immunoprecipitation studies. This led to a reduction of its nuclear translocation and transcriptional activity. Finally, knockdown of both SIRT2 and NRF2 diminished the effects of SIRT2 knockdown on ROS production and cellular damage.

Conclusion:
These results indicate that SIRT2 contributes to pressure overload and I/R injury induced heart impairment in mice, and promotes oxidative stress injury in cardiomyocytes via deacetylating NRF2 and altering its activity.
Abstract 431: Probing the Pathogenic Mechanisms Underlying Ca
V
1.2 Channelopathies
Authors: Bamgboye Moradeke , Traficante Maria , Herold Kevin , Owoyemi Josiah , Dick Ivy
Published: October 2020

The Ca
V
1.2 channel is essential to cardiac function. It is the sentinel that translates the electrical signal on the surface of the cell to the intracellular calcium cascade that leads to contraction. To allow the precise tuning of that is essential for cardiac function, Ca
V
1.2 is subject to two forms of feedback regulation, voltage dependent inactivation (VDI) and calcium dependent inactivation (CDI). The first mutations described in Ca
V
1.2 were causative of Timothy Syndrome (TS), a multisystem disorder with neurological, developmental and life-threatening cardiac symptoms, defined as long-QT type 8 (LQT8). These single point mutations occur in the S6, a region commonly associated with channel activation. In fact, the original TS mutations were shown to perturb channel activation as well as inactivation. Since this first description of TS, multiple additional mutations have been discovered in Ca
V
1.2 and are associated with either TS-like or cardiac specific phenotypes. This phenotypic variance suggests that these mutations disrupt channel mechanisms in divergent ways. Moreover, for patients harboring mutations in Ca
V
1.2, the cardiac symptoms have proven recalcitrant to conventional therapy including calcium channel blockers (CCBs). This therapeutic inadequacy reinforces the need for a better understanding of how mutations in the S6 disrupt channel function and how this affects therapeutic options for patients that carry them. Here we show that LQT8 mutations that cluster in the S6 of Ca
V
1.2 do indeed have a variety of mechanistic underpinnings. This may account for the gradient of symptoms seen in patients with these mutations. In addition, we demonstrate that many of these mutant channels are resistant to block by CCBs, and this resistance strongly correlates to specific mechanistic perturbations of the channel. Thus, reliance on a CCB based therapeutic strategy is likely to leave many in a growing population of patients without a viable treatment option.
Abstract 438: Downregulation of Ca
2+
/calmodulin-dependent Protein Kinase Type II Delta in the Heart by Antisense Oligonucleotides Mitigates Development of Post-infarct Ventricular Arrhythmias
Authors: Yeh Ting-yuan T , Yeap Xin Yi , Mullick Adam E
Published: October 2020

Pathological activation of Ca2+/calmodulin-dependent protein kinase type II delta (CaMK2d) has been implicated in the development of heart failure (HF) and arrhythmia. In preclinical models, inhibition or deletion of cardiac CaMK2d has shown a benefit to prevent HF progression or arrhythmia occurrence. We hypothesized that reduction of CaMK2d in the heart by antisense oligonucleotides (ASO) can mitigate the inducibility of MI-associated ventricular arrhythmias. Eight-week-old female C57BL6/N mice, which received permanent ligation of the left anterior descending coronary artery, were randomized to receive PBS, control ASO or two different mouse CaMK2d ASOs five days after the surgery. The ASOs were administered subcutaneously at 50 mg/kg weekly for four weeks. At the end of study, programmed electrical stimulation (PES) was performed to evaluate ventricular effective refractory period (VERP) and inducibility of ventricular arrhythmias (VA). PCR results showed 70% reduction of cardiac CaMK2d in the CaMK2d ASO groups and no changes of other CaMK2 isoforms. There is no significant change of VERP (VERP expressed as mean±SEM, PBS: 35.2±3.0 ms, control ASO: 36.2±3.8 ms, CaMK2d ASO1: 35.7±2.3 ms and CaMK2d ASO2: 33.8±1.4 ms) while the inducible arrhythmias by PES are declined after CaMK2d ASO treatment (VA inducibility index expressed as mean±SEM, PBS: 1.21±0.27, control ASO: 1.21±0.52, CaMK2d ASO1: 0.54±0.22, CaMK2d ASO2: 0.47±0.22). Echocardiography and invasive hemodynamic studies were unable to demonstrate significant functional improvements in the CaMK2d ASO treated groups, suggesting that the anti-arrhythmia effects of CaMK2d ASO treatment were not secondary to improved cardiac mechanics. In conclusion, ASO-mediated reduction of cardiac CaMK2d in a murine MI model ameliorates MI-associated ventricular arrhythmias, providing
in vivo
proof-of-concept support of the pathological role of CaMK2d activity in MI-related arrhythmia.
Abstract 439: Trajectory Analysis of Left Ventricular Dimensions From Biobank Data Uncovers Novel Genetic Associations
Authors: Pottinger Tess , Puckelwartz Megan J , Pesce Lorenzo L , Gacita Anthony , Salamone Isabella , Kearns Samuel , Pacheco Jennifer A , Nobrega Marcelo , Rasmussen-Torvik Laura J , Smith Maureen E , Chisholm Rex , McNally Elizabeth M
Published: October 2020

Background:
Approximately 6 million adults in the United States have heart failure. The progression of heart failure is variable arising from differences in sex, age, genetic background including ancestry, and medication response. Many population-based genetic studies of heart failure have been cross-sectional in nature, failing to gain additional power from longitudinal analyses. As heart failure is known to change over time, using longitudinal data trajectories as a quantitative trait will increase power in genome wide association studies (GWAS).

Methods:
We used the electronic health record in a racially and ethnically diverse medical biobank from a single, metropolitan US center. We used whole genome data from 896 unrelated participants analyzed, including 494 who had at least 1 electrocardiogram and 324 who had more than 1 echocardiogram (average of 3 observations per person). A mixture model based semiparametric latent growth curve model was used to cluster outcome measures used for genome-wide analyses.

Results:
GWAS on the trajectory probability of QTc interval identified significant associations with variants in regulatory regions proximal to the
WLS
gene, which encodes Wntless, a Wnt ligand secretion mediator.
WLS
was previously associated with QTc and myocardial infarction, thus confirming the power of the method. GWAS on the trajectory probability of left ventricular diameter (LVIDd) identified significant associations with variants in regulatory regions near
MYO10
, which encodes unconventional Myosin-10.
MYO10
was previously associated with obesity and metabolic syndrome.

Conclusions:
This is the first study to show an association with variants in or near
MYO10
and left ventricular dimension changes over time. Further, we found that using trajectory probabilities can provide increased power to find novel associations with longitudinal data. This reduces the need for larger cohorts, and increases yield from smaller, well-phenotyped cohorts, such as those found in biobanks. This approach should be useful in the study of rare diseases and underrepresented populations.
Abstract 446: Exploring the Myosin-dependence of Mechanical Control of Relaxation
Authors: Bukowski Melissa J , Matus Alexandra R , Hunter Dlugas , Wang Madison P , Pittman Lindsay A , Czeiszperger Teresa L , Chung Charles S
Published: October 2020

Impaired relaxation is a prevalent form of diastolic dysfunction, present in nearly all cases of heart failure and in many asymptomatic adults. To date, there are no accepted treatments for impaired relaxation, despite its biochemical control by calcium reuptake, thin filament deactivation, and crossbridge kinetics. Mechanical modification of relaxation was previously theorized to occur through afterload; we, however, have recently shown that relaxation was actually modified by the strain rate of myocardial lengthening. We termed this Mechanical Control of Relaxation, or the sensitivity of the relaxation rate to the strain rate. The mechanisms underlying Mechanical Control of Relaxation are unknown, but computational models and our preliminary data suggest a dependence on myosin detachment. The objective of this study was to evaluate whether myosin was a modifying factor of Mechanical Control of Relaxation. Intact cardiac trabeculae and cardiomyocytes were obtained from rats (Sprague Dawley both treated and untreated with propylthiouracil, Spontaneously Hypertensive, and Wistar Kyoto strains) and underwent load-clamp studies. Mechanical Control of Relaxation could be improved by reducing preload (length) 5%, increasing the sensitivity of the relaxation rate to strain rate by 28±16%. Treatment with 400μM Omecamtiv Mecarbil, a myosin-ATPase specific drug, induced similar increases. Myosin isoform differences were also studied. Collagenase treatment of intact trabeculae increased Mechanical Control by 21±3%; intact myocyte studies show no collagen-dependence. These data provide evidence that the properties of myosin’s response to strain rate are major factors that modify Mechanical Control of myocardial Relaxation.
Abstract 449: Myristic Acid Conjugated Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Cardioprotective Effects in Rat Myocardial Ischemia-Reperfusion Injury
Authors: Baker Alison M , Beale Melinda , Metellus Daphne , Madison Ian , Singh Sunit , Castro Jeremy , Lloyd Michael , Gjoka Redona , Martorana Rose , McIntyre Anahi , Dang Jennifer , Humayun Annam , Dean Tameka , Chen Qian , Barsotti Robert , Young Lindon
Published: October 2020

Reactive oxygen species (ROS) induced ischemia-reperfusion (I/R) injury is a phenomenon causing paradoxical myocardial damage after cardio-angioplasty, coronary bypass and organ transplantation. Previous studies show that a cell-permeable myristic acid (myr-) conjugated PKCβII peptide inhibitor given at reperfusion prevents PKCβII translocation (N
-
myr-SLNPEWNET; myr-PKCβII-) and significantly attenuates ROS mediated I/R injury. We included a scrambled myr-PKCβII- (N-myr-WNPESLNTE; myr-PKCβII-scram) to examine the effects of myr separately. We hypothesize that myr-PKCβII- will improve and myr-PKCβII activator peptide (N-myr-SVEIWD; myr-PKCβII+) will exacerbate infarct size and post-reperfused cardiac function compared to myr-PKCβII-scram and untreated controls. Hearts isolated from male Sprague-Dawley rats (~300g) were perfused with Krebs’ buffer at a constant pressure of 80mmHg and subjected to 30 min of global ischemia and 50 min reperfusion. Myr-PKCβII-, myr-PKCβII+, myr-PKCβII-scram (all 20μM), or untreated control were given during the first five minutes of reperfusion. Left ventricular (LV) dP/dt
max
and dP/dt
min
(mmHg/s) were measured using a pressure transducer, and infarct size was determined using 1% triphenyltetrazolium chloride staining comparing infarcted tissue vs. total tissue weight. Data were evaluated using ANOVA with Student-Newman-Keuls post-hoc analysis. Myr-PKCβII- (n=17) significantly improved LV dP/dt
max
and dP/dt
min
to 1535±107 and 1063±83 at 50 min post-reperfusion compared to untreated control (815±107 and 722±89; n=15); myr-PKCβII-scram (513±78 and 433±66; n=12), and myr-PKCβII+ (860±118 and 694±81; n=14) (all p<0.05). Myr-PKCβII- significantly reduced infarct size (%) to 13±2 compared to untreated control (24±4); myr-PKCβII-scram (22±2), and myr-PKCβII+ (21±3) (all p<0.05). Unexpectedly, myr-PKCβII-scram significantly depressed post-reperfused LV dP/dt
max
and LV dP/dt
min
compared to untreated control and other treated groups (p<0.05). Results suggest that myr-PKCβII- exerted significant cardioprotective effects compared to untreated controls, myr- PKCβII+ and myr-PKCβII-scram and would improve clinical outcomes after cardio-angioplasty or organ transplantation.
Abstract 467: Exosomes Derived From Podoplanin Positive Cells Alter Physiology and Structure of Healthy Mouse Heart
Authors: Cimini Maria , Garikipati Venkata , Elia Andrea , Wang Chunlin , TRUONGCAO MAY , Huang Grace , Mallaredy Vandana , Benedict Cynthia , Kishore Raj
Published: October 2020

Superseding fibrosis through paracrine signals enhances the ventricular dysfunction aftermyocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein,positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells couldindependently alter healthy heart physiology and structure. PDPN+ cells were isolated 2 daysafter MI, cultured expanded and activated with TNFα and AngiotensinII. Exosomes derivedfrom activated PDPN+ cells conditioned media were used in vitro treatment of mouse cardiacendothelial cells (mCECs), mouse embryonic fibroblast (MEF) and monocytes and in vivo forthe treatment of healthy mouse hearts. PDPN+ cells derived exosomes (PDPN-exo)reprogramed mCECs to the lymphatic phenotype enhancing the expression of the majorlymphatic lineage markers and upregulated the expression of fibrotic markers suggesting anendothelial-mesenchymal transition. Furthermore, PDPN-exo drove the MEF to myo-fibroblastphenotype and monocytes toward pro-inflammatory phenotype. Proteomic analysis of PDPN-exo suggest these transitions may depend on NOTCH cleavage trough β-γSecretase andSerum Amyloid A3 protein accumulation/mis-folding. In vivo, PDPN-exo were initially injectedinto the left ventricle of healthy mouse hearts followed with exosomes boosters delivered byretro-orbital vein injection. Treated mice developed an extended epicardial fibrosis andamyloidosis with a subsequent impairment in the contractility and increase of the end diastolicand systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. In conclusionthese data suggest that PDPN-exo alter the biology of mCECs, fibroblast and monocytes andparticipate in adverse remodeling after MI; their specific cargo may represent a cohort oftargets for the treatment of cardiac fibrosis.
Abstract 468: Previously Unrecognized Intronic Variants in the Dystrophin Gene Identified as Possible Contributors to Dilated Cardiomyopathy
Authors: Gyftopoulos Alex , Ashvetiya Tamara , Chen Yi-Ju , Wang Libin , Williams Charles H , Chun Young Wook , Perry James A , Hong Charles C
Published: October 2020

Nonischemic dilated cardiomyopathy (DCM) often has a genetic etiology, however, its prevalence and etiologies are not completely understood. The UK Biobank comprises clinical and genetic data for greater than 500,000 individuals with enrollees 40-69 years of age. Our group created a custom phenotype of heart failure using ICD-10 codes for several subtypes of heart failure diagnoses including DCM. We then compared the individuals included in the custom heart failure phenotype to control individuals in a 20-to-1 fashion to identify genetic differences. Data were compared using Mixed Model Analysis for Pedigrees/Populations (MMAP) mixed-model regression. We identified 8 unlinked intronic variants in the dystrophin gene (
DMD
) that, when separated by self-identified race, occurred with a combined minor allele frequency of 0.15 in individuals with heart failure who identified as being of African descent. The combined minor allele frequency of these variants was 0.05 in individuals who self-identified as being of European descent. One variant of
DMD
in particular (rs139029250), was identified with a minor allele frequency of 0.05 in African British with DCM. The unadjusted odds ratio of a diagnosis of heart failure in individuals with rs129029250 was 4.65. When separated by gender, the unadjusted odds ratios are 2.02 for females and 6.44 for males.
DMD
is most notably known for its role in Duchenne and Becker muscular dystrophy, both of which are known to cause dilated cardiomyopathy in affected individuals. However, none of the individuals (36 female and 43 male) identified in our analysis with rs129029250 have been diagnosed with Duchenne muscular dystrophy, Becker muscular dystrophy, or a primary disorder of muscle (ICD code G70). Additionally, these individuals have an intronic variant of
DMD
, while Duchene and Becker muscular dystrophy are both due to exonic mutations. These findings suggest a possible common variant in the DMD gene that may contribute to DCM in individuals of African descent.
Abstract 476: Mitochondrial DNA Preservation Preserves Cardiac Function Following Sudden Cardiac Arrest
Authors: Rutledge Cody A , Redding Kevin , Dezfulian Cameron , Kaufman Brett A
Published: October 2020

Background:
Sudden cardiac arrest (SCA) affects over 350,000 Americans yearly with greater than 70% mortality. Survivors frequently develop cardiomyopathy after SCA. Cardiac reperfusion causes mitochondrial ROS production, which is known to damage mitochondrial DNA (mtDNA), but the physiologic consequences of mtDNA damage are unclear. We investigated the role of mtDNA damage by altering the expression of TFAM, a nuclear-encoded transcription factor that protects mtDNA from ROS, in a mouse model of SCA.

Methods:
WT and transgenic mice featuring cardiac-specific TFAM overexpression (TFAM-OE) and under-expression (TFAM Flox) underwent either 8 min of SCA or sham surgery followed by cardiopulmonary resuscitation. Survivors were assessed by echocardiography at 1-day, 1-week, and 4-weeks. Tissues were collected for assessment of mtDNA copy number and damage and assessment of mitochondrial morphology, protein expression, and function.

Results:
WT, TFAM-OE, and TFAM Flox mice had no significant changes to baseline body weight or ejection fraction (EF). There were no changes in time to return of spontaneous circulation or body temperature between groups. 1 day after SCA, WT mice have reduced EF (38.49±3.76%) compared to sham WT mice (59.73±1.42). EF is protected in TFAM-OE mice (51.11±2.95%) and exacerbated in TFAM-UE mice (29.36±5.40%). TFAM-OE have significantly higher survival at 4 weeks (80%, 8 of 10) when compared to WT mice (38%, 5 of 13), but there is no change in TFAM-Flox mice (43%, 3 of 7). TFAM OE mice have higher mtDNA copy number and lower mtDNA damage when compared to WT mice.

Conclusions:
TFAM OE protects cardiac function 1-day after SCA and improves 4-week survival. This is likely driven by TFAM-mediated protection of mtDNA. TFAM-Flox mice have lower EF at one day but no change to survival. This work suggests a role for mtDNA damage as a mechanism and potential therapeutic target of cardiomyopathy after SCA.