bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 242: Multi-omics Profiling of Rat Offspring Exposed to Gestational Diabetes Reveals Cardiometabolic Disease Development With Age
Authors: Kereliuk Stephanie M , Agarwal Prasoon , Brawerman Gabriel , Cole Laura , Xiang Bo , Fonseca Mario , Hatch Grant , McGavock Jonathan , Dolinsky Vernon
Published: October 2020

Through unknown mechanisms, fetal exposure to gestational diabetes mellitus (GDM) increases the risk for cardiovascular disease development later in life. We hypothesize that fetal exposure to GDM induces alterations in cardiomyocyte metabolism and induces left ventricular (LV) dysfunction with age. GDM was induced in female rats with a high fat (45% kcal) and sucrose diet prior to mating, throughout pregnancy and lactation. Lean control females received a low fat (10% kcal) diet. Fetal rat ventricular cardiomyocytes (FRVC) were isolated from e20.5 offspring for U-13C glucose metabolic flux analysis, mitochondrial respiration and calcium handling. The cardiac transcriptome and metabolome were measured in 3-month old offspring. LV morphology and function was assessed in the offspring from e18 to 12-months of age by transthoracic ultrasound. Offspring exposed to GDM exhibited increased LV posterior wall thickness across their life course (fetal to 12-months of age; p<0.05) and impaired LV filling beginning at 6-months of age (p<0.05). Consistent with the development of diastolic dysfunction in vivo, alterations in calcium flux and sarcoplasmic reticulum-dependent calcium re-uptake (1.5-fold and 1.6-fold greater, respectively) were observed in FRVC isolated from GDM offspring (p<0.05). When FRVC were treated with isoproterenol, U-13C glucose metabolic flux through glycolysis and the citric acid cycle was reduced in GDM offspring, compared to Lean controls. In 3-month old offspring serum metabolomics revealed reduced citric acid cycle intermediates and an altered acylcarnitine profile. These metabolic changes corresponded to alterations in gene expression patterns identified by RNASeq associated with glucose metabolism and fatty acid transport pathways (e.g.
Irs2, Slc2a4, Pfkfb2, Pdk4
and
Cpt1a)
. Large-scale profiling revealed GDM induced alterations in the cardiac gene expression profile leading to modified serum metabolite levels in the offspring. These alterations corresponded with mitochondrial dysfunction, impaired cardiomyocyte metabolic flux and contractility, in concert with LV hypertrophy and diastolic dysfunction in the rat offspring. Our findings identify several mechanisms that link early-life GDM exposure to the development of cardiovascular disease later in life.
Abstract 244: Post- But Not Pre-menopausal Female Smoothelin-like 1 Deficient Mice Develop Diastolic Dysfunction Unlike Their Male Counterparts
Authors: Murali Megha , Belke Darrell D , Cole William C , MacDonald Justin A
Published: October 2020

Diastolic dysfunction is a core characteristic of heart failure with preserved ejection fraction (HFpEF), which accounts for half of all HF cases. Our group revealed a novel impact of the smoothelin-like 1 (SMTNL1) protein which when subjected to genetic deletion drives alterations in endothelial and cardiovascular performances. Of note, previous cardiac hemodynamic profiling showed that young male SMTNL1 knockout (KO) mice developed diastolic dysfunction as evidenced by increased end diastolic pressure (EDP), left ventricular relaxation time constant (Tau) and steeper end diastolic pressure-volume relationship (EDPVR).

A majority of HFpEF patients are post-menopausal women, so this study examined the impact of SMTNL1 silencing on endothelial and cardiac function with increasing age in a novel female murine model. The cardiac function of SMTNL1 global KO mice (3-, 12- and 18-month old, male and female) was assessed by echocardiography and pressure-volume loop. Unlike young males, 3-month old female KO mice did not develop diastolic dysfunction. However, ageing provided progressive impairment of diastolic function in female KO mice with increased EDP (8.4±1.1 vs 12.2±0.8 mmHg, p<0.02) and EDPVR (0.22±0.05 vs 0.51±0.05, p=0.02) at 18 months; an elevated E/E′ (20.8±1.6 vs 28.3±2.2, p=0.02) and decreased E′/A′ (1.49±0.19 vs 0.97±0.14, p<0.05) at 12 months in comparison to their WT counterparts, along with a normal ejection fraction (>60%) for all age groups. Furthermore, while flow-mediated dilation was continuously impaired in male KO cohorts from 3-month age, female KO showed impairment only at 12-months. Aging was also associated with altered cardiac morphology and fibrotic levels in the aged female KO cohort.

Taken together, we have identified pathophysiologic effects of SMTNL1 silencing on vascular and cardiac function with ageing in female mice. The SMTNL1 KO model for the first time recapitulates the sex dimorphic onset of diastolic dysfunction that is central to HFpEF clinical phenotypes and represents a novel preclinical model to study the etiology of the HFpEF condition. Furthermore, the absence of diastolic dysfunction in KO female mice until post-menopausal age reveals a protective role of female sex hormones and warrants further study.
Abstract 246: YAP1 Activation Suppresses Heart Failure Through Regulation of DNA Repair
Authors: Jiang Jianming , Tan Chia Yee
Published: October 2020

Introduction:
The mammalian hearts including humans have a limited regenerative capacity. Activation of proliferation or related signaling pathways has been shown to reinstate cell renewal capacity of cardiomyocytes (CM) which is believed to ameliorate heart failure (HF). However, whether new CM are generated underlying cardiac regeneration in adult mammalian hearts upon damage is elusive. It is also unknown whether and by what mechanisms activation of CM proliferation benefits HF besides cardiac regeneration.

Hypothesis:
The protective role of cardiac cell cycle activation for damaged hearts could be independent of heart regeneration.

Methods and Results:
We show that cardiac specific activation of
YAP1
, the terminal effector of the Hippo signaling pathway, suppressed HF and cardiac fibrosis induced by
Lmna
insufficiency (
Lmna
HF). Although activation of
YAP1
promoted G1 and S phase of cardiac cell cycle in disease hearts, CM did not undergo mitotic phase, a critical step for heart regeneration. RNAseq followed by signaling pathway analysis and validation showed that Fanconi anemia (FA) DNA repair pathway was activated by
YAP1
. Cardiac DNA damage in
Lmna
HF was suppressed by
YAP1
activation. Importantly, reduction of
Fancd2
compromised the suppressive role of
YAP1
in
Lmna
HF. Mechanistically,
YAP1
promoted mono-ubiquitination of FANCD2 (ub-FANCD2) through promoting cell cycle.

Conclusions:
Our findings demonstrate that promoting cardiac DNA repair becomes a new therapeutic strategy against HF.
Abstract 251: Modeling LMNA Associated Cardiomyopathy Using Patient-Specific Human Induced Pluripotent Stem Cells
Authors: Fonoudi Hananeh , Wilcox Jane , Burridge Paul W
Published: October 2020

Mutations in LMNA are the most prevalent cause of dilated cardiomyopathy (DCM), accounting for up to 5-10% all the familial DCM.
LMNA
encodes the lamin A/C proteins which form filamentous structures that underline nuclear envelop. Sudden cardiac death and arrhythmia are common in patients with
LMNA
mutations. Despite recent advancement in the field, still the exact mechanism that link the mutation in
LMNA
to the formation of DCM and arrythmia is still largely unknown. In this study, we have generated an in vitro model of LMNA associated DCM using patient specific human induced pluripotent stem cells (hiPSCs). A family with pathogenic deletion in
LMNA
gene (c. 1142-1157 + 1del17) and history of DCM were selected. hiPSCs were generated from 4 affected individuals in the family and 5 healthy individuals. hiPSCs were then directly differentiated into cardiomyocytes and assessed at day 30 post differentiation. Cardiomyocytes derived from
LMNA
variant patients showed significantly higher level of nuclear deformation compared to control group. Moreover, after 2 days of mechanical stress cardiomyocytes derived from
LMNA
variant patients showed significantly higher level of nuclear dysmorphism while the control group were not affected. Field potential analysis of cardiomyocytes derived from
LMNA
patients compared to controls using multielectrode array revealed significantly higher beat rate irregularity in
LMNA
variant group which was consistent with the clinical symptoms of the patients. Furthermore, calcium transient of the cardiomyocytes derived from
LMNA
variant patients were significantly different from control group. Finally, patch clamp analysis also proved our previous findings and showed electrophysiological abnormalities in patients’ cells. In summary our finding thus far shows significant electrophysiological differences between cardiomyocytes derived from
LMNA
variant patients and control group which could help to unravel the cellular mechanism underlying formation of arrhythmia in
LMNA
variant patients.
Abstract 255: Cardiac Ischemia and Reperfusion Injury: The Emerging Role of Sialidase Neu3
Authors: Piccoli Marco , Canali Maria Elena , Rota Paola , La Rocca Paolo , Ghiroldi Andrea , Cirillo Federica , Anastasia Luigi
Published: October 2020

Reperfusion strategies, together with thrombolytic therapies, represent life-saving approaches to restore the blood flow in the cardiac tissue after acute myocardial infarction (AMI). However, they inevitably induce the so-called ischemia/reperfusion injury (IRI), resulting in increased cardiomyocytes damage and heart failure. In this context, many efforts have been made to clarify the molecular mechanisms involved in IRI, and the activation of pro-survival kinases, such as Akt and Erk, as well as of the hypoxia-inducible factor (HIF) has been recognized to be critical. Along this line, we discovered a novel mechanism of HIF-1α activation mediated by sialidase Neu3, which is PHDs independent, and that it increased muscle cells resistance to hypoxic stress, through the activation of Akt and Erk pathways. Moreover, an upregulation of Neu3 expression was observed under chronic hypoxia in cyanotic congenital cardiac patients.On these premises, this study aims at investigating the role of Neu3 in protecting cardiac cells against IRI. In particular, H9C2 rat cardiomyoblasts were exposed to an IRI model
in vitro
revealing a marked reduction in cell proliferation. This was accompanied by the modulation of Neu3 that was characterized by its progressive down-regulation during the ischemic phase, followed by its reactivation during reperfusion. These experiments resembled Neu3 modulation we observed in an IRI mouse model, obtained by the temporary occlusion of the LAD coronary vessel. Interestingly, overexpression of Neu3 significantly increased cardiomyoblasts resistance to IRI, both in terms of cell proliferation and resistance to apoptosis, as well as promoted HIF-1α and Akt/Erk activation. Remarkably, the treatment with Akt and Erk inhibitors completely reverted the beneficial effects mediated by Neu3 upregulation. Likewise, sialidase Neu3 inhibition reduced Akt/Erk activation, resulting in the complete loss of Neu3-mediated cardioprotection. In conclusion, our results demonstrate the role of sialidase Neu3 in counteracting the detrimental effects of IRI, calling for further studies to unveil its full potential as a therapeutic target to support current strategies to manage cardiac damage and to improve patients recover after AMI.
Abstract 257: Calreticulin Regulates Acetylation in Cardiac Cells During Endoplasmic Reticulum Stress
Authors: Manning Janet R
Published: October 2020

Background:
Myocardial infarction (MI) is exacerbated by endoplasmic reticulum (ER) stress, which contributes to apoptosis signaling and tissue death in cardiomyocytes. Although the calcium-handing protein calreticulin is upregulated during MI, the role of calreticulin in the progression of ER stress remains controversial. We hypothesized that calreticulin expression accelerates ER stress via regulation of the acetylation status of ER proteins, and knockdown reduces pro-apoptotic ER stress.

Methods:
We examined the expression of calreticulin in human cardiac cells in response to ischemia or thapsigargin-mediated ER stress. To test the effect of calreticulin modulation on cell mortality, we generated a stable partial calreticulin knockdown line in proliferating human cardiomyocyte-derived cells. We further examined the induction and acetylation of ER stress signaling proteins, including proteins in each of the three major pathways that account for ER-stress mediated cell death.

Results and Conclusions:
Calreticulin is upregulated in human ischemic heart failure cardiac tissue, as well as simulated hypoxia and reoxygenation and thapsigargin-mediated ER stress. It was found that partial knockdown protects against the expression of caspase 12, CHOP, and reduces thapsigargin-driven TUNEL staining. In addition, significant changes in the acetylation of ER stress proteins and the expression of the deacetylase SIRT1 were observed. These data shed light on the role that calreticulin plays in apoptosis signaling during ER stress in cardiac cells.
Abstract 259: Dysregulation of AMP-activated Protein Kinase-alpha Isoforms in Left Ventricular Myocardium of Dogs With Chronic Heart Failure
Authors: Gupta Ramesh C , Singh-Gupta Vinita , Sabbah Hani N
Published: October 2020

Background:
The adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) plays critical roles in regulating cellular growth and metabolism and is activated when cellular ATP levels decrease. The heterotrimeric structure of AMPK consists of a catalytic alpha (α) subunit and regulatory beta (β) and gamma (γ) subunits. The two isoforms of the catalytic α subunit have different cellular localization: AMPKα1 is predominantly found in the non-nuclear fraction and AMPKα2 is found in both the nuclear and the non-nuclear fractions. Both AMPKα isoforms reside in cardiomyocytes, increased expression of AMPKα1 during stress can trigger an increased inflammatory state whereas AMPKα2 has recently been recognized as a regulator of mitophagy, the selective degradation of damaged mitochondria by autophagy. Dysregulation in AMPKα1 and AMPKα2 has been reported in explanted failed human hearts. This study tested the hypothesis that mRNA and protein expression levels of AMPKα1 increase and that of AMPKα2 decrease in LV myocardium of dogs with chronic heart failure (HF) produced by microembolization.

Methods:
RNA was extracted and sodium dodecyl sulfate (SDS)-extract homogenate prepared from LV tissue of 6 dogs with microembolization-induced HF and 6 normal (NL) dogs. mRNA expression of AMPKα1 and AMPKα2 was measured using real-time PCR and normalized to GAPDH. Protein expression of AMPKα1 and AMPKα2 was measured by Western blotting and normalized to GAPDH. Bands were quantified in densitometric units (du).

Results:
mRNA and protein levels of GAPDH, used as internal control, were similar between NL and HF dogs. In HF dogs, mRNA expression of AMPKα2 was reduced by 5.09 fold and protein levels were reduced by 2.08 fold (1.02 ± 0.09 vs. 2.12 ± 0.18 du, p<0.05) compared to NL dogs. In HF dogs, AMPKa1 mRNA and protein levels were increased 5.67 fold and 2.09 fold (0.67 ± 0.10 vs. 0.32 ± 0.03 du, p<0.05), respectively compared to NL dogs.

Conclusions:
The results indicate that in LV myocardium of dogs with chronic HF, mRNA and protein levels for AMPKα1 are upregulated while AMPKα2 levels are downregulated compared to NL dogs. This AMPK isoform shift can trigger abnormalities of mitochondrial respiration and turnover that contribute to an abnormal energetic state characteristic of the failing heart.
Abstract 269: The Role of Neutrophils in Doxorubicin-induced Cardiotoxicity
Authors: Bhagat Anchit , kleinerman eugenie , Kleinerman Eugenie
Published: October 2020

Doxorubicin (Dox) is one of the most effective chemotherapy agents for treating childhood cancer. Unfortunately, Dox also causes damage to the heart which leads to reduced heart function and heart failure in >50% of survivors. Understanding the mechanisms by which Dox induces cardiotoxicity is crucial to identifying preventive interventions. Inflammation, in particular: neutrophils and monocytes has been linked to other form of cardiac disease. However, the role of these immune cells in Dox-induced cardiotoxicity has not been examined. We hypothesize that neutrophils are responsible for the early damage caused by Dox treatment. Using our mouse cardiotoxicity model, mice were treated with Doxorubicin for 2 weeks. Neutrophil infiltration in the heart 24 hours after therapy were evaluated by flow cytometry. There was an increase in neutrophils in heart tissue of Dox-treated mice as compared to controls and a decrease in heart function as quantified by echocardiography (ejection fraction [EF] and fractional shortening [FS]). Next, we depleted neutrophils using an anti-Ly6G antibody. Neutrophil depletion was confirmed by flow cytometry in the blood and hearts of Dox-treated mice. We found that neutrophil depletion prevented Dox-induced decrease in both ejection fraction and fractional shortening. Additionally, we also observed by flow cytometry that infiltration of monocytes was unaffected by the neutrophil depletion antibody. Based on this data we concluded that neutrophils and not monocytes are integral contributors to acute heart damage caused by Dox. These data indicate that targeting neutrophils may be a preventive strategy against Dox-induced cardiotoxicity.
Abstract 270: Micu1 Regulates Mitochondrial Structural Integrity and Cristae Function Independent of Mcu Calcium Uptake
Authors: Tomar Dhanendra , Thomas Manfred , Kolmetzky Devin , Salik Oniel , Garbincius Joanne , Jadiya Pooja , Carpenter April C , Elrod John W
Published: October 2020

MICU1 is a Ca
2+
-sensor regulating the mitochondrial Ca
2+
uniporter channel (mtCU) and mitochondrial Ca
2+
uptake. MICU1 contains two Ca
2+
- binding EF-hand domains and directly interacts with the mtCU pore-forming subunit, MCU. MICU1-null mouse and fly models display perinatal lethality with disorganized mitochondrial architecture. Interestingly, these phenotypes are distinct from other mtCU loss-of-function models (
MCU, MICU2, EMRE, MCUR1
) and thus are likely not explained solely by changes in matrix Ca
2+
content, suggesting MICU1 has cellular functions independent of the mtCU. To discern novel MICU1 molecular interactions we utilized a proximity-based biotinylation proteomic approach, BioID2, and expressed a MICU1-BioID2 fusion construct in wild-type and

MCU
-/-

cells. LC-MS analysis of purified biotinylated proteins identified Mitochondrial Contact Site and Cristae Organizing System (MICOS) components MIC60, CHCHD2, and CHCHD3 as MICU1 interacting partners. Loss of MCU did not affect the MICU1-MICOS interaction, suggesting that MICU1 could be regulating the MICOS independent of the mtCU. Fast protein liquid chromatography (FPLC), blue native-PAGE, co-immunoprecipitation, live-cell Ca
2+
imaging, electron microscopy, confocal and super-resolution imaging confirmed that MICU1 (independent of matrix Ca
2+
) is regulating mitochondrial membrane dynamics, membrane potential, and cell death signaling. Further, the deletion of
Chchd2, a core component of the MICOS,
resulted in disruption of cristae structure without any observable effect on mitochondrial

Ca
2+
uptake. In addition, RNA sequencing of murine models of heart failure revealed a correlation between the expression of MICU1 and MICOS components and disease progression. These results are the first to identify how Ca
2+
regulates cristae structure and function and suggests MICU1, at the MICOS, regulates mitochondrial membrane remodeling in the context of cardiac stress and disease independent of the mtCU.
Abstract 273: Utilization of Proximity-labeling to Identify Sarcomere Assembly Dependent Interactions
Authors: Ladha Feria , Thakar Ketan , PETTINATO Anthony , Legere Nick , Cohn Rachel , Romano Robert , Meredith Emily , Chen Yu-sheng , Hinson John
Published: October 2020

The cardiac sarcomere is a multiprotein complex that generates pulsatile contractile force, enabling the heart to circulate vital nutrients, but its molecular components and dynamic interactions are incompletely understood. Cardiac alpha-actinin-2 (encoded by
ACTN2
; referred to as actinin) functions as the structural backbone of the Z-disc and is prone to mutations that result in human heart failure. We hypothesize that through identifying actinin PPIs at the Z-disc, we can identify new sarcomere functions and gain insights into heart failure pathogenesis. Until now, studying sarcomere PPIs has been challenging due to limitations in purifying intact sarcomere complexes and the inability to detect dynamic molecular interactions. To overcome this, we adapted BioID proximity proteomics to comprehensively study actinin PPIs in human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes (iCMs).We engineered an isogenic hiPSC line carrying an in-frame knock-in of
BirA*
, which encodes a promiscuous biotin ligase, at the
ACTN2
locus. Upon differentiation into iCMs, the Actinin-BirA* fusion construct localizes to the Z-disc and, upon biotin supplementation, biotinylates vicinal proteins. We immunoprecipitated and analyzed Z-disc PPIs via quantitative tandem mass spectrometry. Among the >100 PPIs detected by this method, we identified known interactions including sarcomere and focal adhesion proteins, but also new interactions including proteins involved in translation and metabolism. Furthermore, we wanted to better understand which of these protein interactions are dependent on sarcomere assembly and function, potentially elucidating interactions involved in normal cardiomyocyte biology and disease states. Through disruption of sarcomere assembly in our Actinin-BirA* model, we were also able to identify sarcomere-dependent actinin interactions including specific proteins involved in translation, the unfolded protein response, and glycolysis. Further investigation will allow us to better understand the proteomic network around actinin and provide novel insights into human sarcomere biology.
Abstract 276: NOTCH1 is Essential for Ventricular Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells
Authors: Zhao Mingtao , Ye Shiqiao , Zhang Joe , Shao Ningyi , Liu Chun , Zhou Yang , Nishiga Masataka , Yang Yang , Cui Bianxiao , Garg Vidu , Wu Joseph C
Published: October 2020

Pathogenic variants in
NOTCH1
have been implicated in multiple types of congenital heart defects, such as bicuspid aortic valve, Tetralogy of Fallot, and hypoplastic left heart syndrome (HLHS). However, the mechanisms by which
NOTCH1
pathogenic variants cause abnormalities in human embryonic heart development are largely unknown. Here, we used CRISPR/Cas9-mediated genome editing to genetically delete
NOTCH1
in human induced pluripotent stem cells (iPSCs). We found that
NOTCH1
was dispensable for mesodermal and vascular endothelial differentiation of human iPSCs. Disruption of NOTCH activity promoted venous-specific gene expression but suppressed arterial-specific gene expression in iPSC-derived endothelial cells (iPSC-ECs). Intriguingly,
NOTCH1
deletion significantly impaired the cardiac differentiation efficiency. In
NOTCH1
homozygous knockout (
NOTCH1
-/-
) iPSC-derived cardiomyocytes (iPSC-CMs), atrial-specific genes (
NR2F2, KCNJ3
, and
MYL7
) were upregulated whereas ventricular-specific genes (
MYL2, IRX4
, and
MYH7
) were downregulated. Electrophysiological analysis by patch clamp and optical mapping indicated that atrial-like cardiomyocytes were dominant whereas the percentage of ventricular-like iPSC-CMs was dramatically reduced (<1%) in
NOTCH1
-/-
iPSC-CMs. In addition, mitochondrial respiration was reduced in NOTCH1 deficient iPSC-CMs compared to wild-type controls, which was likely attributed to the reduction of ventricular cardiomyocytes in
NOTCH1
-/-
iPSC-CMs. As
NOTCH1
is primarily expressed in endothelial cells rather than cardiomyocytes, we conclude that
NOTCH1
affects ventricular cardiomyocyte lineage commitment possibly through controlling cell fate determination of cardiac progenitors during human iPSC differentiation. Our study may provide novel insights into the mechanisms by which
NOTCH1
mutations lead to left ventricular hypoplasia in HLHS patients.
Abstract 279: A Defective Mechanosensing Promotes Impaired Fibroblast-to-myofibroblast Maturation in the Aging Mouse Heart
Authors: Angelini Aude , Trial JoAnn , Cieslik Katarzyna A
Published: October 2020

In the aging heart, fibroblasts have a reduced ability to mature into myofibroblasts (expressing contractile actin a-SMA), which is necessary for scar contraction in wound healing. The mechanosensing pathway bridges the extracellular matrix (ECM) with the actin cytoskeleton and is altered during aging. We emphasize that changes in this pathway may affect myofibroblast activation. Using various methods (mass spectrometry, qPCR, flow, IF staining, and western), we found several abnormalities in the fibroblasts derived from old mouse hearts: 1) ECM deposited was altered with an increased quantity of collagens and the presence of fibronectin variant Anastellin that opposes myofibroblast maturation; 2) there was a 50% reduction of Kindlin-2, a protein that promotes integrin activation; 3) Polymerized (F-) to monomeric (G-) actin ratio was decreased by 65%, possibly due to a 5-fold increase in pERKs1/2 level, a negative regulator of F-actin; and pERKs inhibition (by 2.5μM, PD-0325901) increased by 2-fold F/G actin ratio; 4) we found an increased cytoplasmic retention of MRTF-A (by 44%), an actin-sensitive co-transcription factor (necessary for a-SMA) that shuttles into the nucleus in response to F-actin formation; 5) pERKs inhibition increased a-SMA transcription by 78%. Thus, the disrupted mechanosensing pathway contributes to the impaired myofibroblast maturation in the aging heart.

All experiments were performed in young (3-4 month-old) and old (24-30 month-old) hearts or primary cardiac fibroblasts isolated from male C57BL/6J mice. Unpaired Student’s t-test was used for statistical analysis, n=2 for mass spectrometry and n=4-10 for all other biological repeats.
Abstract 284: Loss of Micu3 Confers Cardioprotection Against Cardiac Injury
Authors: Puente Bao , Sun Junhui , Fergusson Maria , Liu Julia , Kosmach Anna , Parks Randi , Springer Danielle , Liu Chengyu , Murphy Elizabeth
Published: October 2020

Background:
Mitochondrial calcium flux and signaling is integral to cardiac function and contraction. However, during pathologic conditions such as ischemic/reperfusion injury, mitochondrial calcium overload can induce the opening of the mitochondrial permeability transitioning pore (PTP), resulting in the collapse of mitochondrial membrane potential, ATP depletion, and generation of reactive oxygen species, all together leading to cell death. Hence, modulation of mitochondrial calcium and inhibition of the PTP is a promising target for cardioprotection and prevention of cardiomyocyte death. The mitochondrial calcium uniporter (MCU) complex mediates rapid mitochondrial calcium uptake. MICU3 is a regulator of the MCU complex and has been shown to be a highly potent stimulator of MCU-dependent calcium uptake in neuronal cells. We found that MICU3 is expressed in hearts and we therefore investigated the role of MICU3 in the heart. We examined the role of MICU3 in the development of hypertrophy and in a separate study we examined the response to ischemic-reperfusion (I/R) injury. Given its role in regulating mitochondrial calcium uptake, we hypothesized that loss of MICU3 confers protection against cardiac injury.

Methods:
Mice with global deletion of Micu3 (Micu3

-/-

) were created utilizing CRISPR-Cas9 technology. Adult knockout and littermate wild type mice were treated with Isoproterenol (15mg/kg/day) for two weeks to induce hypertrophy. Echocardiograms were performed at baseline and after treatment to assess changes in left ventricular size and function. I/R injury was studied using Langendorff ex vivo perfused heart system, exposing knockout and wild type hearts to 20 minutes of ischemia and 90 minutes of reperfusion. Hemodynamic data and infarct size were collected and compared. Student t-test and 2-way ANOVA were used for statistical analysis.

Result:
Micu3
-/-
mice had normal cardiac function at baseline. There was no sex difference in cardiac function. Micu3
-/-
mice continued to show normal function after 2 weeks of treatment with Isoproterenol, whereas wild type mice exhibited depressed function (median FS 35% vs. 24% p = 0.0001, EF 64% vs. 50% p = 0.0001). Wild type mice developed LV dilation from baseline (median 4.15mm vs. 4.57mm, p = 0.0014), whereas LV dimension remained stable in Micu3

-/-

mice (median 4.12mm vs. 4.18mm, p= 0.9892). Micu3
-
/-
mice were also protected from I/R injury. Compared to wild types, Micu3
-/-
hearts demonstrated less contractile dysfunction at end reperfusion (median rate pressure product 62% vs. 41%, p = 0.002), and significantly smaller infarct size (median 33% vs. 53%, p = 0.0001).

Conclusion:
Loss of MICU3 confers cardioprotection against ischemic reperfusion injury and Isoproterenol induced cardiac dysfunction.
Abstract 285: High Throughput Profiling of Gsk3α Regulated Fibroblast Kinome Reveals Raf as a Mediator of Fibrosis in Failing Heart
Authors: Umbarkar Prachi , Tousif Sultan , Singh Anand P , Anderson Joshua C , Zhang Qinkun , Lal Hind
Published: October 2020

Background:
Heart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK3β as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK3α in cardiac pathophysiology is unknown.

Methods and Results:
GSK3α was deleted specifically from cardiac fibroblasts or myofibroblasts with tamoxifen-inducible TCF21- or periostin- promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury, and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In both settings, GSK3α deletion restricted fibrotic remodeling and improved cardiac function. To investigate underlying mechanisms, we examined the effect of GSK3α deletion on myofibroblast transformation and pro-fibrotic TGFβ1-SMAD3 signaling
in vitro
. WT and KO mouse embryonic fibroblasts (MEFs) were treated with TGFβ1. Indeed, a significant reduction in cell migration, collagen gel contraction, and α-SMA expression in TGFβ1 treated KO MEFs confirmed that GSK3α is required for myofibroblast transformation. Surprisingly, GSK3α deletion had no effect on SMAD3 activation, indicating the pro-fibrotic role of GSK3α is SMAD3 independent. At 4 weeks post-injury, total proteins were isolated from CFs of WT and KO animals, and kinome profiling was performed by utilizing PamStation®12 high throughput microarray platform. The upstream kinase analysis identified the downregulation of RAF family kinase activity in GSK3α-KO-CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. These findings are consistent with previous studies that implicated ERK in fibrotic diseases across multiple organs.

Conclusion:
CF-GSK3α plays a causal role in the cardiac pathophysiology that could be therapeutically targeted for future clinical applications.
Abstract 293: Functional Recovery After Gene Therapy and Hippo Pathway Knock Down After Myocardial Infarction in a Pig Model
Authors: Liu Shijie , Li Ke , Heallen Todd , Tang Li , Wang Yidan , Wagner Leonardo , Willerson James T , Perin Emerson C , Zhang Sui , Martin James F
Published: October 2020

Heart failure (HF), a devastating disease and a leading cause of death worldwide, is an unmet clinical need. The Hippo pathway, an inhibitory kinase cascade, represses adult cardiomyocyte (CM) proliferation post-myocardial infarction (MI) in rodent models. Here, we investigated an Adeno Associated Virus 9 (AAV9) gene therapy that knocks down the Hippo pathway gene,
Sav,
in a pig model of MI after ischemic reperfusion (I/R), to closely model the human condition. Two weeks post-MI, when pigs had established left ventricular (LV) systolic dysfunction, we delivered an AAV9-
Sav
-shRNA or control virus by subendocardial injections to border zone (BZ) CMs. Three months post viralinjections, AAV9-
Sav
-shRNA injected pigs had improved LV systolic function, evidence of CM division, and reduced scar size. In addition, AAV9-
Sav-
shRNA treated hearts had more capillaries and reduced CM ploidy. AAV9-
Sav
-shRNA therapy was well tolerated without any mortality and pathologic examination of liver and lungs failed to uncover evidence for tumor formation revealing that AAV9-
Sav
-shRNA gene therapy is safe within the timeframe of this study. Our findings indicate that gene therapy, knocking down the Hippo pathway post-MI, in a pig model improves heart function and may have utility for humans.
Abstract 294: Microrna-208a: A Marker of No-Reflow in STEMI Patients Undergoing Primary Percutaneuos Coronary Intervention
Authors: Salama Abou Bakr M , Khalil Wael A , Al-Zaky MAnar M , Abdallah Somia H , Kandil Nader T , Abdelsabour Ahmed , Shaker Ahmed M , Hasanein Mesbah T , Luciani Giovanni B , Azzazy Hassan
Published: October 2020

Background:
Biomarkers are the corner stone for diagnosis of myocardial infarction (MI). MicroRNA-208a (miR-208a) is known to be heart specific. So, this study was designed to explore its role as a marker for diagnosis of MI as well as a predictor of outcome of primary percutaneuos coronary angiography especially no-reflow phenomenon.

Methods:
This study was carried out between January 2018 and August 2019 in Cardiology Department, Zagazig University Hospitals, Egypt. Approval was obtained from institutional review board adhering to the guidelines of the Declaration of Helsinki. Patients (n=75) presented by chest pain were recruited into two groups. Group 1 (n=40) had STEMI and underwent primary PCI: 21 patients with sufficient reperfusion and 19 with no-reflow. Group 2 (n= 35) had negative troponin T (cTnT). Plasma miR-208a expression was assessed using quantitative polymerase chain reaction and patients were followed for occurrence of in-hospital (3 days average) major adverse cardiac events (MACE).

Results:
Analysis of relative expression values shows that miR-208a has reasonable sensitivity and specificity for MI diagnosis (AUC= 0.92, sensitivity = 92.5, Specificity = 80) which is comparable to the routine cardiac biomarkers; creatine kinase-MB (CK-MB) (area difference 0.0439, P= 0.235) and cTnT (area difference 0.0614, P= 0.06). The no-reflow group (b) had higher expression compared to subgroup (a). ROC analysis shows it to be a good predictor of no-reflow (AUC= 0.88, sensitivity = 73.7, Specificity = 95.2) which is significantly superior to cTnT (AUC difference of 0.231, P= 0.0233). As well, it was superior to cTnT as a predictor of in-hospital MACE (AUC difference of 0.367, P= 0.0053).

Conclusion:
This study highlights the value of miR-208a as a diagnostic and prognostic marker in STEMI context. To the best of our knowledge, this is the first study to investigate the value of miR-208a in no-reflow and almost the second to investigate the role of any known miRNA in no-reflow. Although still far from clinical application due to long time needed for the assay, the future introduction of new fast nucleic acids assays could support its clinical application. The study points to a possible mechanism for no-reflow development that needs to be thoroughly investigated.
Abstract 295: Genome-wide CRISPR Screen Identifies Keap1-Nrf2 Pathway as a Crucial Regulator for Fibrosis
Authors: nishiji toshiyuki , hoshino atsushi , taminishi shunta , hato daichi , matoba satoaki
Published: October 2020

Cardiac fibrosis is the pathological feature of myocardial remodeling and clinically involved in various cardiovascular diseases including heart failure with preserved ejection fraction (HFpEF) and atrial fibrillation. Accumulating evidence revealed the molecular mechanism behind organ fibrosis, however, no effective anti-fibrotic therapy has been developed. To find novel anti-fibrotic molecular targets, we performed genome-wide CRISPR library screening with focus on myofibroblast activation. Since a feedforward autocrine loop accelerates myofibroblast differentiation, we employed connective tissue growth factor (CTGF), a center of profibrotic secretome, as a target factor for screening assay. We firstly generated a CTGF-GFP knock-in reporter cell in mesenchymal progenitor cells. The reporter cells were transduced with lentivirus CRISPR library and treated with TGF beta, then high and low GFP signal cells were sorted and sequenced. This screening showed that Keap1-NRF2 pathway was one of the top hits and a novel regulator for CTGF expression. Keap1 knockout attenuated TGF beta-mediated CTGF expression and additional NRF2 knockout cancelled it. Keap1-NRF2 pathway also regulated broadly other profibrotic factors, alpha SMA, periostin, and fibronectin. An activator of NRF2, CDDO-Im treatment had similar effect on TGF beta-mediated profibrotic response. CTGF knockout had little effect on these profibrotic factors, indicating that Keap1-NRF2 is better therapeutic target, as compared with CTGF. Keap1-NRF2 originally reported as a transcriptional regulator for antioxidant and detoxification genes. In silico analysis indicates CTFG promoter has NRF2 response element and ChiP-qPCR experiment confirmed that NRF2 directly regulated CTFG transcriptional expression, not indirectly through antioxidant gene expression. In conclusion, genome-wide screening identified Keap1-NRF2 pathway as a crucial regulator for myofibroblast activation and NRF2 activation might be novel therapy for fibrosis-related diseases.
Abstract 297: Development of Molecular Targeted Therapy Against Right Ventricular Failure: Involvement in a Network of Immunocompetent Cells
Authors: Ito Shogo , Yuasa Shinsuke , Komuro Jin , Kimura Mai , Kusumoto Dai , Hashimoto Hisayuki , Aoki Hiroki , Fukuda Keiichi , FUKUMOTO Yoshihiro , Ito Kei , Yada Hirotaka
Published: October 2020

Backgrounds:
Right ventricular (RV) failure plays a critical role in right heart failure and left heart failure. However, there is no specific therapy developed for RV failure. To elucidate a novel therapeutic target against RV failure, we focus on RV specific genes to develop novel therapeutics for RV failure.

Methods:
Microarray analysis using several parts of adult murine heart was conducted and differentially expressed genes (DEGs) were applied to pathway analysis. Molecular mechanism was examined by using neonatal rat ventricular cardiomyocyte (NRVM) in vitro. To understand the function of target molecule in vivo, we induced RV failure by pulmonary artery constriction (PAC) in mice and inhibition experiments were performed using RV failure model.

Results:
In microarray analysis for RV, left ventricle and ventricular septum, 995 genes were extracted as DEGs in RV. Pathway analysis showed that alternative complement pathway-related genes were significantly up-regulated in RV. Moreover, complement factor D (Cfd) and C3a was a potential upstream factor attributable to unique feature of RV. Administration of C3a recombinant protein to NRVM phosphorylated several MAP kinases. In vivo, in C3KO PAC mice, RV dysfunction was significantly suppressed, and histological study showed that RV fibrosis was significantly suppressed. In wild type mice with PAC, administration of C3a receptor antagonist dramatically improved RV dysfunction and reduced RV fibrosis. Additionally, in vivo electrophysiological study revealed that the inducibility of ventricular arrhythmia was increased in wild type PAC mice, but ventricular arrhythmia was significantly attenuated in C3KO PAC mice. Furthermore, the expression of chemokine Ccl5 was enhanced in RV of wild type PAC mice, while Ccl5 was significantly attenuated in C3KO PAC mice.

Conclusion:
We revealed that complement C3a was highly produced in RV and chemical or genetical blockade of C3a ameliorates RV dysfunction and RV fibrosis in PAC mice. C3a was to be a potent bioactive protein for immunocompetent cells that played an important role in modulating cardiac function. Accordingly, we demonstrated that the blockade of C3a had a potential role for novel therapeutic target to RV failure.
Abstract 303: Decreasing Microenvironment Stiffness Improves Exogenous Extracellular Matrix Induced Heart Regeneration in Neonatal Mouse
Authors: Wang Xinming , Senyo Samuel
Published: October 2020

Introduction:
Transplanting cardiac extracellular matrix (ECM) has been demonstrated to influence healing in post-ischemic hearts. We propose that altering mechanical properties can stimulate a regenerative response in ECM-treated hearts. In this study, we investigate the role of mechanical unloading and solubilized ECM to modulate matrix-induced heart regeneration in low-regenerative P5 neonatal mice after acute myocardial infarction and mouse ventricle explants.

Methods:
P5 neonatal mouse heart stiffness was lowered by inhibiting formation of new collagen crosslinks. Solubilized fetal ECM was injected immediately after myocardial infarction (MI). Heart function and histology were conducted at week 3 post-MI. Cardiac ventricle explants were also used to investigate relevant signaling pathways.

Results:
We observed that lowering tissue stiffness increased the regenerative influence of fetal ECM treatment on heart function, fibrosis, and cardiomyocyte proliferation. Decrease heart stiffness inhibits fibrosis and better preserves heart function in fetal ECM treated hearts (Figure 1). We further provide evidence that yes-associated protein (Yap) signaling pathway plays a role in ECM-induced cardiomyocyte proliferation possibly through cytoskeleton polymerization.The results suggest that the native microenvironment stiffness, particularly with aging or post-ischemia, affects the therapeutic efficacy of drugs for heart disease.

Figure 1
. Fetal ECM treatment P5 mouse hearts showed a higher ejection fraction in comparison with the control hearts at 3-weeks post-MI. Decreasing heart stiffness in P5 mouse heart further promoted increased ejection fraction in fetal ECM treated animals. (n=5, two-way ANOVA test and Tukey’s test, *p<0.05, ****p<0.0001.)
Abstract 304: Adaptive Sarcomeric Remodeling of Left Atrial Cardiomyocytes in Non-failing Non-valvular Atrial Fibrillation
Authors: Desai Parth V , Martin Thomas , Stachowski Marisa , Papadaki Maria , Kirk Jonathan A
Published: October 2020

Contractile remodeling in sustained atrial fibrillation (AF) has been analyzed by limited studies whose results were confounded by either coexisting systolic heart failure or valve disease (valvular AF) or origin of tissue (right chamber instead of left, atrial appendage instead of main wall). We sought to assess the structural changes in contractile apparatus and its physiological implications on single cardiomyocyte mechanics in patients with non-failing non-valvular AF. We utilized left atrial wall tissue from rejected donor hearts from 5 subjects in sinus rhythm (SR) and 3 with AF (age 50, 2♀ vs 60, 1♀), all with no signs of cardiovascular or valvular disease. Isolated single skinned myocytes were mounted to a force transducer and length controller and set to an initial sarcomere length of 2.1 μm. Isometric active and passive forces were recorded using custom software during [Ca2+] solution switching (0.79 - 46.8 μM). Surprisingly, we found that isometric maximal calcium-activated force (Fmax) was almost two times higher in AF compared to SR patients (n: SR = 15 cells, AF = 9 cells, p < 0.0001). This was unexpected, as previous studies found that AF patients had depressed contractile function, although these were confounded by heart failure and valve disease. There were no differences in calcium sensitivity, hill coefficient, or cell cross-sectional area (CSA) between SR and AF. We next performed 1D SDS-PAGE electrophoresis to compare myosin heavy chain (MHC) isoforms. In SR patients, atrial expression of β-MHC was very low (14% of total MHC expression), but this was significantly elevated in AF patients (37%, p = 0.05). The observed rise in contractile force might be a compensatory adaptation to sustain ventricular filling in initial stages of non-valvular non-failing AF patients. Or it may be a maladaptive response to atrial unloading resulting in wasted energy utilization. The contribution of this cellular increase in contractility to whole organ function is unclear. There is strong evidence that fibrotic remodeling and inflammation play an important role in AF, but the clinical challenge is still significant. Conversely, there has been very little work done on the contractile apparatus in AF, and whether it may represent a possible therapeutic target.
Abstract 316: Influence of Aging and Na Channels on Sinus Rhythm and Heart Rate Variability
Authors: Comelli Martina , Meo Marianna , Cervantes Daniel O , Pizzo Emanuele , Plosker Aaron , Hund Thomas J , Jacobson Jason T , meste olivier , Rota Marcello
Published: October 2020

Heart rate variability (HRV) is an index of cardiovascular health and reflects the ability of the heart to adjust sinus rhythm in response to autonomic regulation, a process affected by age. Importantly, Na channels have emerged as key components of discharge and conduction of the sino-atrial node, but whether altered Na channels affect HRV remains to be established. Thus, we studied effects of aging and altered Na channels on heart rate and HRV using wild type (WT) mice and mice with phosphomimetic mutation of Na channel Nav1.5 at Ser571 (S571E mice). Electrocardiograms (ECG) were obtained in male conscious mice at ~4, ~12, ~18, and ~24 months of age (n=13-51). The mean RR interval of the ECG was similar in WT mice at ~4, ~12, and ~18 months (83±4, 81±2, 83±2 ms, respectively), but increased at ~24 months (85±5 ms). In contrast, mean RR interval in S571E mice was maximal at ~4 months (86±6 ms) and progressively decreased at ~12 (83±9 ms), ~18 (79±2 ms), and ~24 months (80±3 ms). Standard deviation of RR intervals (SDRR), an indicator of RR variability, was maximal in WT at ~4 months (3.8±1.6 ms) and was reduced at ~12, ~18, and ~24 months (2.1±0.9, 2.4±1, and 2.6±1.2 ms). Similarly, SDRR was maximal in S571 at ~4 (5.1±2.6 ms) and ~12 months (4.3±1.5 ms), and decreased at ~18 and ~24 months of age (2.6±1.0 and 3.1±1.1 ms). Standard deviation of instantaneous (SD1) and long-term (SD2) RR interval variability, indicative of parasympathetic and sympathetic influence, respectively, were derived from Poincaré plots of RR
i
and RR
i+1
intervals. Overall, SD1 was preserved for the two groups of aging mice, whereas SD2 was maximal at ~4 months and decreased at ~18 and ~24 months. In the presence of complete autonomic block (propranolol and atropine), RR interval increased in young (~4 months, +10%) and old (~24 months, +16%) WT mice. Similarly, autonomic block increased RR interval in young (~4 months, +12%) and old (~24 months, +14%) S571E, but it abrogated RR interval differences between young and old. For the two genotypes, autonomic block reduced SDRR and SD2 exclusively in young animals, abolishing age-related differences in HRV. Overall, these data suggest that Na
+
channel function conditions heart rate and its age-related adaptations, but does not interfere with the decline of HRV occurring with age.