bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 473: IL-7 Increases Fusion of Rat Bone Marrow Mesenchymal Stem Cells With Cardiomyocytes In Vitro and Improve Cardiac Function In Vivo
Authors: Salim Asmat , Haneef Kanwal , Ali Anwar , Khan Irfan , Naeem Nadia
Published: February 2019

Heart diseases are one of the main causes of morbidity and mortality all over the world particularly in the developing countries. Cardiac endogenous repair mechanisms have limited potential to regenerate myocardium following myocardial infarction. Mesenchymal stem cells (MSCs) hold significant promise as potential therapeutic candidates following cardiac tissue damage. Engrafted stem cells consistently experience programmed cell death and a large number of MSCs die in the ischemic area of the heart after transplantation. To ensure the survival of engrafted stem cells, it is beneficial to precondition these stem cells prior to their transplantation into the injured heart. In this study, we used interleukin-7 (IL-7), a hematopoietic growth factor having significant role in the cell survival, to transfect MSCs and observed its effect on cell-cell fusion
in vitro
and cardiac function
in vivo
. Cell fusion analyzed by co-culturing IL-7 transfected MSCs with neonatal rat cardiomyocytes (CMs) showed significantly (p<0.01) higher number of fused cells as compared to that in case of normal MSCs. Transplantation of IL-7 transfected MSCs into the rat model of myocardial infarction showed improvement in the cardiac function by significantly increasing the ejection fraction (EF) and fractional shortening (FS) (p<0.01), and significantly decreasing the diastolic and systolic left ventricular internal diameters (LVIDd and LVIDs) (p<0.001) and end systolic and diastolic volumes (ESV and EDV) (p<0.01 and p<0.001 respectively) as assessed by echocardiography after 4 weeks. IL-7 is able to enhance the fusogenic properties of MSCs and improve cardiac function. This Improvement in cardiac function may be attributed to the supportive action of IL-7 on cell proliferation and cell survival contributing to the regeneration of damaged myocardium.
Abstract 478: Small Molecule ICG-001, Sodium Butyrate, and Retinoic Acid Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes (iCMs)
Authors: Singh Vivek P , Pinnamaneni JayaPratap , Mathison Megumi , Sanagasetti Deepthi , Somvanshi Sonal , Pugazenthi Aarthi , Yang Lina , So Davis , Wang Kai , Yang Jianchang , Rosengart Todd K
Published: February 2019

Background:
Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs)
in situ
represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors Gata4, Mef2c and Tbx5 (GMT), and microRNAs can convert fibroblasts into iCMs, albeit with low efficiency
in vitro
. We aimed to increase the efficiency, maturation and speed of direct reprogramming using a unique combination of small molecules, in combination with previously identified reprogramming transcription factors, that we selected and combined in a cocktail specifically formulated to effect optimal impact on the aspects of reprogramming pathways thought to represent “roadblocks” or points of opportunity in these pathways.

Methods:
We screened a variety of such candidate compounds in primary rat/human cardiac fibroblast. Cardiac reprogramming was induced by lentivirus encoding Gata4, Mef2c and Tbx5 plus small molecules, and the presence of iCM was confirmed 10 days later by QRT-PCR, IFs and FACS analysis for the activation of endogenous cardiac troponin T (cTnT).

Results:
After validating and optimizing the dose and conditions for adding small molecules to the reprogramming cocktail, we treated GMT-overexpressing fibroblast with a combination of sodium butyrate (SB, 1mM; HDAC inhibitor), ICG-001(1μM; WNT inhibitor), and retinoic acid (RA, 1μM). We found that that a combination of SB, ICG-001, and RA significantly increased GMT-induced reprogramming efficiency to 22 % cTnT
+
iCMs from primary rat cardiac fibroblasts compared to 8% in the presence of single compounds and 3.5% without any added small molecule compound alone. QRT-PCR and IFs, analysis shows that a combination of the SB, ICG-001, and RA increased cTnT gene expression 4-6 fold when added to GMT-overexpressing rat cardiac fibroblasts. We also demonstrated that combined addition of SB, ICG-001, and RA increased cardiac fibroblast reprogramming efficiency 4 fold when added to GMTHM plus miR-590-overexpressing human cardiac fibroblasts.

Conclusions:
Thus, HDAC and WNT inhibitors along with RA, jointly accelerate GMT-induced cardiac reprogramming from rat/human cardiac fibroblasts and pave the way for new translational approaches for cardiac regeneration.
Abstract 479: The ER Unfolded Protein Response Effector, ATF6, Promotes Proliferation and Maintains Pluripotency in Cardiac Stem Cells
Authors: Stauffer Winston T , Doroudgar Shirin , Stephens Hailey N , Blackwood Erik A , Glembotski Christopher C
Published: February 2019

Recent studies have suggested that multipotent stem cells residing in the adult heart, called cardiac stem cells (CSCs), mitigate damage in the infarcted or failing heart. Investigating the factors governing CSC proliferation and differentiation is key to understanding what role these cells play in the heart and in future therapeutic strategies. Additionally, activating transcription factor 6 (ATF6), an effector of the endoplasmic reticulum (ER) unfolded protein response (UPR), plays critical roles in development, as well as in the differentiation of certain stem cell types, though it has not been studied in this regard in the heart. Our lab has demonstrated that ATF6 in cardiac myocytes is cardioprotective
in vivo
during ischemia/reperfusion partly by virtue of its ability to induce an antioxidant gene program that reduces damaging reactive oxygen species (ROS). However, ATF6, and its involvement in antioxidant gene induction, have not been studied in CSCs. Therefore, here we hypothesized that activation of the ATF6 branch of the UPR in CSCs is important for their proliferation and differentiation, given that ROS is known to be essential for these processes. To address this hypothesis, we subjected cultured mouse CSCs to simulated ischemia and observed increased ATF6 target gene mRNA levels. This demonstrates that, despite their undifferentiated status, CSCs have a functional UPR, which can be activated in response to ischemic stress. ATF6 loss of function (LOF) in CSCs, via RNAi or chemical inhibitor, yielded a basal decrease in cell viability and an increase in several differentiation markers, similar to the effect of dexamethasone differentiation stimulus. Increased ROS was also observed in an ATF6 LOF model. Strikingly, cotreatment with a chemical ROS inhibitor significantly rescued cell viability and reduced markers of differentiation in CSCs with reduced ATF6 function. These results suggest that CSCs require a basal level of ATF6 activity to maintain their proliferation and pluripotentcy
in vitro
and that this is mediated by the role of ATF6 in the mitigation of ROS. This is an important finding given that stem cell expansion
in vitro
is a critical step in the characterization of stem cells and their use in many therapeutic treatment strategies.
Abstract 486: Role of the Adenine Nucleotide Translocator Family in the Mitochondrial Permeability Transition Pore
Authors: Karch Jason , Bround Michael J , Parks Randi J , Terada Naohiro , Wallace Douglas C , Murphy Elizabeth , Molkentin Jeffery D
Published: February 2019

Cardiomyocyte necrosis is dependent on the prolonged opening of the mitochondrial permeability transition pore (MPTP) which has escaped molecular identification for decades. Once thought to be comprised of the adenine nucleotide translocator (ANT) family, this hypothesis has fell by the way side when it was shown that liver mitochondria devoid of ANT 1 and 2 still display MPTP opening, even though these mitochondria were desensitized to MPTP opening and required a greater calcium bolus for it to engage. Thus, the ANT family has been labeled as a critical regulator of the MPTP and not the pore forming component. However, mice express three isoforms of ANT and only two of the three isoforms where deleted when ANTs were deemed to be non-essential. We hypothesize that the third ANT isoform may have compensated for the loss of the other two ANT family members during this analysis. Here, we have generated mice lacking all isoforms of ANT in a heart-, liver-, or cell-specific manner. Surprisingly, mice lacking all isoforms of ANT specifically in the liver are viable and are highly resistant to MPTP opening. Indeed, the amount of calcium required to engage the MPTP in mitochondria lacking all ANTs is 11 fold higher than wild type mitochondria and greater than 2 fold higher than wild type mitochondria treated with the MPTP inhibitor cyclosporine A (CsA). Furthermore, the treatment of CsA on ANT null liver mitochondria completely blocks MPTP opening suggesting that the ANT family is the primary pore in the liver. However, when subjected to extreme levels of calcium, a cyclophilin D (CypD) dependent pore engages. CypD is the MPTP trigger located in the matrix of the mitochondria which is inhibited by CsA and in wild type mitochondria high levels of calcium are enough to overcome the inhibition of CypD to open the pore. But, in ANT depleted liver mitochondria, extremely high levels of calcium do not override CypD inhibition which is suggestive of the presence of an additional pore. Further experimentation is underway to determine if MPTP-conductance is detectable by patch clamping mitoplasts lacking all isoforms of ANT.
Abstract 488:
Clock
Regulates Autophagy and Cell Survival of Cardiac Myocytes During Hypoxia Stress
Authors: Rabinovich-Nikitin Inna , Minuk Illana , Margulets Victoria , Aguilar Floribeth , Martino Tami A , Kirshenbaum Lorrie A
Published: February 2019

Circadian rhythms play a fundamental role in cell metabolism and tissue homeostasis and are influenced by the diurnal oscillation of cellular circadian clock genes. Zeitgebers influence the activation of the circadian rhythm by serving as molecular switches for the intrinsic cellular clock. Herein, we provide new evidence that hypoxia is a Zietgeber for the circadian rhythm and regulator of
clock
gene expression in cardiac myocytes. We further show that clock gene regulation promotes survival of cardiac myocytes by a mechanism that bi-directionally influences autophagy. Cardiac myocytes exhibited phasic oscillations in
clock
gene expression under basal conditions. Interestingly, in contrast to control cells, a marked time dependent decline in
clock
gene transcription was observed in cardiac myocytes subjected to hypoxia with maximal decline occurring at 18hrs. This coincided with a reciprocal increase in mitochondrial autophagy genes including
beclin-1
. Loss of function mutations of
clock
that disrupted nuclear localization or DNA binding promoted wide-spread cell death. Hence, the findings of the present study provide the first direct evidence that hypoxia is a Zeitgeber for regulating
Clock
gene expression and cell survival of cardiac myocytes during hypoxic injury.
Abstract 499: Identification and Characterization of Lamin-Associated Domains in Cardiac Myocytes Isolated From Human Patients With Dilated Cardiomyopathy Caused by LMNA Pathogenic Variant and Their Effects on Gene Expression and DNA Methylation
Authors: Marreddy Sirisha C , Gurha Priyatansh , Hu Hin , Sweet Mary , Taylor Matthew , Mestroni Luisa , Coarfa Cristian , Marian Ali J
Published: February 2019

Rationale:
Lamin A/C (LMNA) is a nuclear inner membrane protein that interacts with genome through Lamin-Associated Domains (LADs) and regulates gene expression. Mutations in the
LMNA
gene causes a diverse array of diseases that are collectively referred to as laminopathies. Dilated cardiomyopathy (DCM) is a major component of laminopathies and the leading cause of premature death.

Objective:
To identify and characterize LADs in cardiac myocytes, cardiac DNA methylation status, and transcriptomes and integrate the findings to find the pathogenic pathways in DCM

Methods:
We performed ChIP-Seq, reduced representative bisulfite sequencing (RRBS), and RNA-Seq in 5 control and 5 DCM hearts, the later with defined pathogenic variants. ChIP-Seq was performed using an anti-LMNA antibody on protein extracts from FACS-isolated myocyte nuclei, identified by expression of PCM1. RRBS was performed on whole heart DNA extracts to identify methylation state of ~ 500,000 CpG sites. RNA-Seq was performed on whole heart ribosome-depleted RNA extracts. LADs were identified using Enriched Domain Detector (EDD).

Results:
We identified 370±50 LADs with an average size of ~ 1Mbp (range: 0.5 to 10 Mb), collectively covering about 12,000 genes and 12% of the genomic regions in the control myocytes. The number and size of the LADs were unchanged in DCM. However, genomic locations of LADs had shifted, leading to loss of 3,302 genes from the LADs. Integration of LADs, CpG methylation, and transcript levels showed CpG hypermethylation and suppressed transcript levels in the LADs both in control and in DCM hearts. However, the effects were differential and LADs were associated with increased hypermethylation and further suppression of gene expression in DCM hearts. Pathway analysis of integrated omic datasets identified TGFβ1 and cell cycle pathways as the most activated and MPAK and glycolytic pathways as the most repressed pathways in DCM hearts.

Conclusions:
LADs, CpG methylation and transcriptomes are dysregulated in cardiac myocytes in patients with DCM caused by
LMNA
pathogenic variants. The dysregulated biological pathways provide potential targets for interventions in DCM caused by
LMNA
mutations.
Abstract 500: Low Aldehyde Dehydrogenase 2 (ALDH2) Potentiates Obesity in Leptin Receptor Mutant Mice
Authors: Pan Guodong
Published: February 2019

Obesity aggravates type 2 diabetes and thus cardiometabolic complications. Aldehyde dehydrogenase 2 (ALDH2), a cardiac mitochondrial enzyme is known to be cardioprotective. ALDH2*2 is the most common genetic mutation in East Asians (~800 million carriers), which causes a low ALDH2 activity. We hypothesize that ALDH2*2 mutation aggravates obesity and diabetes in leptin receptor deficient db/db mice and consequentially cardiometabolic complications. To test our hypothesis, we cross db/db mice with ALDH2*2 knock-in mutant mice. We obtained a new breed of mice which had leptin receptor mutation and ALDH2*2 mutant gene. We termed this mice as ALDH2*2-Fat. The body weight gain of ALDH2*2-Fat mice was extremely faster than their non-obesity littermates (ALDH2*2-Lean, the mice only carrying ALDH2*2 mutant gene). At 30 weeks old, the body weights of ALDH2*2-fat mice (84.15±6.75 g) was 2.88 folds higher than ALDH2*2-Lean mice (29.32±3.75 g, p<0.001). Fasting blood glucose and serum insulin concentration were higher in ALDH2*2-Fat mice than ALDH2*2-Lean mice (blood glucose: 506.20±88.27 vs. 192.83±21.32 mg/dl, p<0.001; insulin: 13.94±6.28 vs. 2.46±0.58 ng/ml, p<0.001 respectively). Echocardiographic measurements revealed a progressive cardiac dysfunction in ALDH2*2-Fat mice (Fractional shortening, 48.00±3.17 vs. 56.90±3.11 % at 6 months, p<0.05, and 39.11±7.50 vs. 56.98±1.55 % at 9 months, p<0.01). Body temperatures were significantly lower in ALDH2*2-Fat mice (35.5±0.94 vs. 38.86±0.49 °C, p<0.01). The interscapular brown adipose tissues in ALDH2*2 fat mice were shrunk to invisible at 9 months old. Western-blotting found a decreased PGC-1 α and increased PPAR-γ expression in white adipose tissues from ALDH2*2-fat mice. Cultured primary bone marrow cells showed a significantly decrease in colony forming units in ALDH2*2-Fat mice (7.67±4.50 vs., 26.33±3.33 p<0.05). Under adipogenic medium, the adipocytes differentiation was increased in ALDH2*2 fat mice. In conclusion, low ALDH2 activity aggravates obesity and consequentially cardiometabolic complications in leptin receptor deficiency-induced obesity and diabetes.
Abstract 502: Identifying Tetraspanins and Integrins Relevant to Cardiovascular Conditions Using Bioinformatics
Authors: Sun Ge
Published: February 2019

Objectives:
Our study aims at mining the bioinformatic databases to find potential cardiovascular (CV) regulators from tetraspanins and integrins.

Methods:
The Genotype-Tissue Expression (GTEx) was used to identify the tissue-specific mRNA level of each gene in CV system. The database of Genotypes and Phenotypes (dbGaP) was used to identify the association between genes and CV phenotypes through SNPs. Gene Expression Omnibus (GEO), was used for human gene correlation and functional prediction. GeneNetwork was used to find gene-phenotype or gene-gene correlation in mice.

Results:
The GTEx results are shown in the heatmaps (panel A). In dbGaP, we found ITGA8 has impact on cholesterol, along with ITGA9 on arterial pressure, ITGA2, ITGAE and ITGAM on platelet function tests, all from exon missense mutations. TSPAN5, TSPAN15, ITGA2 and ITGA6 are worth mentioning for their multiple intron SNP associations. GEO analysis showed that CD9, CD82, CD151, TSPAN18, UPK1B and most of the integrins are associated with genes related to CV phenotypes. In GeneNetwork, numerous associations were found in mice (table). Combining the data from both species: TSPAN5 and ITGA8 are associated with blood metabolic chemistry; TSPAN9 is associated cardiac ischemic conditions; TSPAN11 with fat/weight; CD82 and ITGA9 with blood pressure; ITGAM, ITGA2 and ITGB6 with platelet functions. The network graphs in the panel C show the correlated genes of these 9 final candidates in mouse aorta.

Conclusion:
Bioinformatic analyses revealed CVS-related candidates: TSPAN5, TSPAN9, TSPAN11, CD82, and ITGAM, A2, A8, A9, B6. These results require further confirmation by experimental methods.
Abstract 504: LAMP-2B Regulates Human Cardiomyocyte Function by Mediating Autophagosome-lysosome Fusion
Authors: Chi Congwu
Published: February 2019

Autophagy plays a crucial role in cell homeostasis and function. Two types of autophagy have been well studied. Macroautophagy (referred to as autophagy hereafter) is mediated by double-membrane autophagosomes that enclose cytosolic cargoes, followed by fusion with late endosomes/lysosomes for degradation. Chaperone-mediated autophagy (CMA) is a process of chaperon-dependent selection of cytosolic proteins that are translocated into the lysosome for degradation. LAMP-2A, the A isoform of LAMP-2, is an essential receptor for CMA. However, alternative splicing of pre-LAMP-2 mRNA produces another two isoforms, LAMP-2B and LAMP-2C, with unclear functions. In non-cardiomyocytes, STX17 is required for autophagosome-lysosome fusion by interacting with SNAP29 and VAMP8. Whether cardiomyocytes (CMs) utilize the STX17-dependent mechanism to mediate autophagic fusion remains elusive. Using CMs derived from induced human pluripotent stem cells (hiPSC-CMs) and genome editing approaches, we identified previously undefined biological and molecular roles for LAMP-2B in the control of autophagosome-lysosome fusion. Remarkably, LAMP-2B functions independently of STX17. Instead, LAMP-2B interacts with ATG14 and VAMP8 through its C-terminal coiled-coil domain to promote autophagic fusion. Knockout of
LAMP-2B
in hiPSC-CMs decreased colocalization of ATG14 with VAMP8, and autophagosome-lysosome fusion. Forced expression of LAMP-2B in LAMP-2 deficient hiPSC-CMs restored autophagic signaling. In addition,
LAMP-2B
-deficient hiPSC-CMs recapitulated the metabolic and contractile defects observed in hiPSC-CMs derived from Danon patients. Danon disease represents a severe form of cardiomyopathy and has been associated with mutations in X-linked
LAMP-2
gene. However, mechanisms by which LAMP-2 deficiency leads to cardiomyopathy are poorly understood. Given the similarity of phenotype between Danon and
LAMP-2B
knockout hiPSC-CMs, we conclude that LAMP-2B deficiency is sufficient and necessary to cause Danon cardiomyopathy. Therefore, our findings not only reveal a STX17-independent autophagic fusion mechanism mediated by LAMP-2B in human CMs, but also provide a molecular mechanism underlying Danon cardiomyopathy.
Abstract 512: Regulation of Neutrophil Extracellular Traps (NETs) by Mesenchymal Stromal Cell Derived Adenosine
Authors: Levit Rebecca , Shin Eric , Xu Kai , Wang Lanfang
Published: February 2019

Introduction:
Mesenchymal stromal cells (MSCs) reduce the infiltration of leukocytes into the heart after ischemia reperfusion injury (MI/R). Inhibition of the surface ectonucleotidase CD73 prevents this anti-inflammatory effect. CD73 is a key step in a pathway that converts adenine nucleotides (ATP, ADP, AMP) into adenosine, which in turn exerts vasodilatory and immunosuppressive effects. Neutrophil extracellular trap (NET) formation is a thrombotic and inflammatory process involved in coronary occlusion and myocardial damage. We hypothesized that adenosine may be a key regulator of NET formation and that CD73-dependant adenosine production by MSCs reduces NETs after MI/R.

Methods:
Neutrophils isolated from venous blood of healthy donors by percoll gradient density centrifugation were cultured with MSCs for in vitro studies. MSCs were encapsulated in aliginate by electrostatic encapsulation and secured to the epicardium with PEG-maleimide hydrogel at the time of reperfusion. This biomaterial strategy supports and maintains viable cells in the vicinity of injury for 1 week. 24 hours after injury, hearts were perfused, excised, and digested and NETs quantified by flow cytometry.

Results:
Treatment of neutrophils with adenosine or the adenosine receptor agonist NECA reduces NET formation

in vitro

. Co-culture of MSCs with neutrophils also prevents NET formation. Inhibition of CD73 activity on MSCs with the antagonist APCP prevents the anti-NET activities of MSCs.

In vivo

, encapsulation and placement of MSCs on the epicardial surface at the time of MI/R reduces myocardial NETs 24 hours after MI/R. Treatment of MSCs with APCP prior to implantation reduces the anti-NET effects of MSCs, as well as functional benefits.

Conclusions:
There is growing evidence of the importance of NETs in cardiac ischemia-reperfusion injury but little is known of the regulation of NETs
in vivo
. Our data suggests that adenosine exerts a strong anti-NET effect and that CD73-generated adenosine from MSCs may be capable of
in vivo
anti-inflammatory and anti-NET regulation. Future studies are focused on genetic techniques to knock down CD73 activity on MSCs, direct implication of NETs on cardiovascular pathophysiology, and non-cellular methods to deliver CD73 in MI/R.
Abstract 513: Lower Nadir Cd4 T-cell Count is a Strong Predictor of Endothelial Dysfunction in HIV Infected Patients
Authors: Mogadam Emad , King Kevin , Shriner Kimberly , Chu Karen , Sondergaard Anders , Royal Darlene , Young Kristal , Kloner Robert
Published: February 2019

Background:
Nadir CD4 T-cell count is a predictor of negative outcomes in HIV -infected patients and strongly correlates with WHO disease staging. There is lack of data pertaining the relationship between vascular function and nadir CD4 in patients without risk factors of hypertension and diabetes. Therefore we investigated the association between low nadir CD4 T-cells and endothelial function in these otherwise cardiovascular healthy patients.

Methods:
We conducted a study of 19 HIV-infected men on antiretroviral therapy with undetectable plasma RNA levels without hypertension or diabetes. Vascular reactivity index (VRI), a marker for vascular endothelial function, was measured via VENDYS technique using temperature rebound in response to hyperemic blood flow and vasodilation to the forearm following a 5-minute cuff occlusion of the brachial artery.

Results:
VRI showed a significant correlation with low nadir CD4 count (p = 0.019) (Figure 1). Lower CD4 counts were associated with significant endothelial dysfunction. We examined the effect of potential mediators on such association. No significant correlation was found with LDL-C (p=0.60), HDL-C (p=0.59), systolic and diastolic blood pressures (p=0.12 and 0.13) or history of smoking (p=0.55) with VRI in the studied HIV cohort.

Conclusion:
Low nadir CD4 count remained independently associated with vascular reactivity index and thus a significant clinical predictor of endothelial dysfunction in HIV infected patients with no history of hypertension or diabetes even before clinical evidence of cardiovascular disease. Vascular Reactivity Index vs CD4 Nadir(Figure1).
Abstract 517: Pathogenic Mechanism of a Catecholaminergic Polymorphic Ventricular Tachycardia Causing-Mutation in Cardiac Calcium Release Channel RyR2
Authors: Liu Zheng
Published: February 2019

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition that is characterized by an abnormal heart rhythm in response to physical or emotional stress. The majority CPVT patients carry mutations in the
RYR2
gene that encodes the calcium release channel/ryanodine receptor (RyR2) in cardiomyocytes. The pathogenic mechanisms that account for the clinical phenotypes of CPVT are still elusive. We have identified a
de novo
mutation, A165D, from a CPVT patient. A knock-in mouse model that expresses an A165D point mutation was generated using CRISPR/Cas9 technology. Echocardiogram, electrocardiography, and histological analysis were performed on mutant mice. We found that ventricular tachycardia was readily induced by exercise and pharmacological challenges. Functional properties of the mutant RyR2 channel were evaluated in isolated cardiomyocytes. The mutant RyR2 channels enhanced SR Ca
2+
release, triggered delayed afterdepolarization in cardiomyocytes. These recapitulated CPVT phenotypes in both animal models and cardiomyocytes indicated that RyR2 A165D is a CPVT-causing mutation. Furthermore, we pinpointed the 3D localization of the mutation site in the structures determined by cryo-electron microscopy. It revealed that the A165D mutation change the electric charge of the residue, disrupted conformational stability of the RyR2 tetramer structure, and promoted a closed-to-open state transition. Moreover, the location of the A165D mutation is in a loop that harbors several other mutations, and, like A165D, most these mutations result in changes in the electrostatic charge of the side chains of the residues. This demonstrated a common molecular mechanism of CPVT-causing RyR2 mutations. Our data illustrated disease-relevant functional defects and provide a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.
Abstract 518: Hemodynamic Instability in Atrial Flutter With Slow Ventricular Response
Authors: Nwafo Nnamdi A
Published: February 2019

Background:
The commonest atrioventricular (AV) conduction ratio in atrial flutter is 2:1, with corresponding ventricular response of about 150 beats/min. Higher degree of AV block with resulting lower ventricular conduction may indicate use of AV nodal blocking drugs, intrinsic conducting system disease, electrolyte abnormalities or hypothermia.

Case:
An 86-year-old with history of hypertension, hyperlipidemia, hypothyroidism, coronary and carotid artery disease was admitted after a syncopal episode while on a cross country trip. He was hypotensive and profoundly bradycardic(heart rate initially in the 30s). Examination revealed irregular rhythm and right carotid bruit. EKG showed atrial flutter with variable ventricular response ranging from 3:1 to 5:1 (or more) block, with ventricular rate in the 50s and 60s. Echocardiogram showed low normal left ventricular systolic function without regional wall abnormalities, right ventricle enlargement with global systolic dysfunction, and severe left atrial enlargement. Thyroid function was normal.

Decision-making:
With hemodynamic instability in the setting of atrial flutter, he was started on anticoagulation and intravenous hydration, and after left atrial thrombus was ruled out with a transesophageal echocardiogram, he underwent successful urgent cardioversion. He was then noted to have intermittent Mobitz type II second degree and third degree AV block, prompting urgent temporary transvenous pacemaker (PM) placement, followed by permanent dual chamber PM implantation. He had bleeding from the PM placement site but this was controlled with pressure. Therapy with beta blocker was re-initiated, and electrolytes were supplemented as needed. Vitals signs improved and he was discharged in stable health to follow up with his cardiologist.

Conclusion:
Urgent or emergent cardioversion is indicated for hemodynamic instability in atrial flutter, even with slow ventricular response. In the absence of use of AV nodal blocking agents, lower rate of ventricular response may indicate intrinsic conducting system disease, and as in the case above, may require PM placement.
Abstract 519: Mitochondrial-mediated Oxidative CaMKII Activation Induces Early Afterdepolarizations in Guinea Pig Cardiomyocytes: An
in Silico
Study
Authors: Yang Ruilin , Ernst Patrick , Song Jiajia , Liu Xiaoguang M , Zhang Jianyi , Zhou Lufang
Published: February 2019

Background:
Oxidative stress-mediated CaMKII phosphorylation of cardiac ion channels has emerged as a critical contributor to arrhythmogenesis in cardiac pathology. However, the link between mitochondrial-derived reactive oxygen species (mdROS) and increased CaMKII activity in the context of cardiac arrhythmias has not been fully elucidated and is difficult to establish experimentally.

Methods:
We hypothesize that pathological mdROS can cause erratic action potentials through the oxidation-dependent CaMKII activation pathway. We further propose that CaMKII dependent phosphorylation of sarcolemmal slow sodium channels alone is sufficient to elicit early afterdepolarizations. To test the hypotheses, we expanded our well-established guinea pig cardiomyocyte
e
xcitation-
c
ontraction coupling,
m
itochondrial
e
nergetics and
R
OS-
i
nduced-
R
OS-
r
elease model by incorporating oxidative CaMKII activation and CaMKII-dependent Na
+
channel phosphorylation
in silico
.

Results:
Simulations show that mdROS mediated-CaMKII activation elicits early afterdepolarizations (EADs) by augmenting the late Na
+
currents (

I
Na,L

), which can be suppressed by blocking L-type Ca
2+
channels or Na
+
/Ca
2+
exchangers. Interestingly, we found that oxidative CaMKII activation-induced EADs sustain even after mdROS has returned to its physiological levels. Moreover, mitochondrial-targeting antioxidant treatment can suppress the EADs, but only if given in an appropriate time window. Incorporating concurrent mdROS-induced RyRs activation further exacerbates the proarrhythmogenic effect of oxidative CaMKII activation.

Conclusions:
We conclude that oxidative CaMKII activation-dependent Na channel phosphorylation is a critical pathway in mitochondria mediated cardiac arrhythmogenesis.
Abstract 523: Pioglitazone and Glucagon-like Peptide 1 Receptor Agonist Stimulate Mitochondrial Turnover in the Heart When Administered After the Infarct in Obese Mice
Authors: Andres Allen M , Andres Allen M , Germano Juliana , Huang Chengqun , Tucker Kyle C , Song Yang , Sin Jon , Gottlieb Roberta A , Mentzer Robert M
Published: February 2019

Randomized clinical trials have demonstrated that pioglitazone (PIO), a thiazolidinedione, and liraglutide and semaglutide, glucagon-like peptide-1 receptor agonists (GLP1Ra), are effective in treating Type 2 diabetes mellitus. Of considerable interest is the additional observation that they may also be effective in reducing the incidence of major adverse cardiovascular events (MACE) independent of glycemic control. The mechanism underlying this protection is unknown. To test the hypothesis that enhanced mitochondrial turnover (mitophagy and mitochondrial biogenesis) might play a role, obese mice with insulin resistance were subjected to permanent coronary artery ligation (PCAL). Two hours later they were administered an i.p. bolus of vehicle (50μL DMSO), PIO (10mg/kg), or a GLP1Ra (10nmoles/25g) (Sigma). This was repeated every other day for 2 weeks. Hearts were probed by western blot for markers of mitochondrial biogenesis and mitophagy. As shown, PIO was a strong inducer of mitochondrial biogenesis evidenced by increased mitochondrial markers in the whole lysate (mitochondrial biogenesis) whereas GLP1Ra stimulated mitophagy, indicated by Parkin, p62 and optineurin translocation to the mitochondrial fraction. These findings suggest that enhanced mitochondrial biogenesis and mitophagy may be important therapeutic targets for the prevention of adverse cardiovascular outcomes. If confirmed, enhanced mitochondrial turnover may be one of the mechanisms underlying the reduced incidence of MACE that was observed in the PROactive (PIO) and the LEADER and SUSTAIN-6 (GLP1Ra) clinical trials.
Abstract 536: Increased O-linked Glycosylation in Diabetic Myocardium of Mice and Human
Authors: Agbortoko Vahid , Liu Yuhong , Shi Guangbin , Usheva Anny , Singh Arun K , Sellke Frank W , Feng Jun
Published: February 2019

Objectives:
The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Our focus was to establish the difference of O-linked glycosylation in diabetic (DM) and Non-diabetic (ND) heart of mouse and human in the presence or absence of cardioplegic ischemia and cardiopulmonary bypass (CP/CPB).

Methods and results:
Right atrial tissue was harvested pre- and post-CP/CPB from the ND patients and patients with type-2 DM undergoing cardiac surgery (n = 4/group).The post-CP/CPB tissue samples were from a atrial tissue segment exposed to cold, hyperkalemic blood cardioplegia (ischemia) and a brief period (10 minutes) of reperfusion. Ventricular tissue samples were also obtained from mice with and without type-2 DM (n =3/group). Thin Layer Chromatography (TLC) was used to measure the contents of acetyl coenzyme A (acetyl-CoA) in the tissue lysates of mouse and human myocardium. Western blotting and immunohistochemistry analysis were employed to determine the expression/localization of O-Linked β-N-acetylglucosamine (O-GlcNAc) in the tissue lysates and paraffin-embedded tissue sections. Diabetes tends to increase the amounts of acetyl-CoA and O-GlcNAc (20% increase vs. ND, P<0.05) in the myocardium compared with ND. Post-CP/CPB further induced more acetyl-CoA and this effect was more pronounced in the diabetic atrial myocardium than that of ND. Immunofluorescence experiments with O-GlcNAc-specific antibody and paraffin-embedded tissue sections from human atria and mouse heart show a significant number of cells stain positively with the antibody in the diabetes sections (3 folds increases in optical density vs. ND, P <0.01). The protein oxidation and ratio of NADH/NAD in the DM myocardium were significantly increased compared with ND (P<0.05).

Conclusion:
Diabetes and cardioplegic ischemia are associated with an increase in O-linked glycosylation in the mouse and human myocardium, suggesting that diabetes and cardioplegic ischemia affect myocardial metabolism via hexosamine biosynthetic pathway.
Abstract 537: Identifying Novel Regulators of Mitochondrial Complex I Biogenesis
Authors: Garcia Christian J
Published: February 2019

Mitochondrial Complex I (CI) is composed of 44 distinct subunits, that are assembled together with eight Fe-S clusters and a single flavin mononucleotide, to form a functioning enzyme. Chaperone proteins referred to as assembly factors assist with the assembly process; and a dozen or so
bona fide
CI assembly factors (CIAFs) have been characterized. However, about half of CI disorders cannot be traced to mutations in any of the 44 CI subunits or known assembly factors, suggesting that additional regulators of CI biogenesis remain to be characterized. Studies from our lab have shown that the mechanism of CI assembly is conserved between humans and
Drosophila
, allowing us to exploit our system to find novel regulators of CI biogenesis. In our studies, we use a genetic and proteomic approach to identify novel regulators of CI assembly that interact with NDUFS3 (a CI subunit); and have tested these candidate regulators in both
Drosophila
and humans cell lines. The ease of isolating copious amounts of mitochondria from flight muscles, extensive arsenal of tools for genetic analyses, relatively short generation time, and limited gene redundancy in
Drosophila
are assets that should facilitate the discovery of new regulators of CI assembly.
Abstract 538: Reduced Protein and mRNA Levels of Velosin Containing Protein in Left Ventricular Myocardium of Dogs With Advanced Heart Failure Are Restored Following Chronic Therapy With Elamipretide
Authors: Gupta Ramesh C , Gupta Vinita S , Sabbah Hani
Published: February 2019

Introduction:
The failing heart manifests abnormalities of mitochondrial function characterized by reduced respiration, reduced membrane potential and opening of permeability transition pore (mPTP). Recent studies have shown that velosin containing protein (VCP), an ATPase associate protein, preserves mitochondrial respiration and prevents opening of the mPTP. Long-term therapy with elamipretide (ELAM), a novel mitochondria-targeting tetra-peptide, was previously shown improve mitochondrial respiration, membrane potential and prevent opening of the mPTP in dogs with intracoronary microembolization-induced heart failure (HF).

Hypothesis:
This study tested the hypothesis that mRNA and protein levels of VCP are decreased in left ventricular (LV) myocardium of dogs with HF and that chronic therapy with ELAM reverses this dysregulation.

Methods:
Studies were performed using LV tissue from 14 HF dogs randomized to 3 months monotherapy with subcutaneous injections of ELAM (HF+ELAM, 0.5 mg/kg once daily, n=7) or no therapy at all (control, HF-CON, n=7). LV tissue from 6 normal (NL) dogs was used for comparisons. Protein levels of VCP and GAPDH, an internal loading control, were determined in LV SDS-extracts using Western blotting coupled with chemiluminescence detection and band intensity was expressed in densitometric units (du). Using isolated RNA and specific primers, mRNA expression of VCP normalized to GAPDH.

Results:
mRNA and protein levels of GAPDH in LV tissue remained unchanged among the 3 study groups. Compared to NL, mRNA expression of VCP was reduced by 3.67 fold in HF-CON. Compared to NL, protein level of VCP was significantly reduced compared to HF-CON (0.30 ± 0.04 vs. 1.77 ± 0.31 du, p<0.05). In HF+ELAM treated dogs, mRNA expression of VCP was reduced by only 1.07 fold compared to HF-CON (p<0.05). Similarly, in HF+ELAM, protein levels of VCP were significantly higher than in HF-CON (1.03 ± 0.16 du, p<0.05).

Conclusions:
VCP mRNA and protein levels are significantly reduced in LV myocardium of dogs with chronic HF. Long-term therapy with ELAM reverses this dysregulation. The findings support the observations of improved mitochondrial respiration and reduced mPTP opening in dogs with advanced HF following chronic therapy with ELAM.
Abstract 542: Manipulation of Beta Adrenergic Receptor in Pressure-Overloaded Murine Hearts Mimics Adverse Cardiac Remodeling and Reverse Remodeling
Authors: Nishimura Koichi , Asakura Masanori , Okuhara Yoshitaka , Naito Yoshiro , Hirotani Shinichi , Ishihara Masaharu , Masuyama Tohru
Published: February 2019

Background:
Adverse cardiac remodeling (ACR) during pressure overload is a major concern in development of heart failure (HF). Cardiac reverse remodeling (CRR) normally occurs following induction of beta-receptor blockade and is linked to improved prognosis of HF. However, the detailed and regulatory mechanisms of both ACR and CRR remain to be fully elucidated. To solve these issues, a novel highly reproducible model that mimics these pathophysiological conditions is needed.

Methods and results:
We explored suitable experimental conditions for establishing a highly reproducible model that mimics adverse cardiac remodeling. To examine suitable durations of transverse aortic constriction (TAC) before one-week infusion of ISO (3mg/kg/day), mice were distributed into three groups according to TAC durations. Proportion of adverse cardiac remodeling was 44.4% (4 of 9) in one-week, 60.0% (6 of 10) in two-week, and 91.7% (11 of 12) in three-week TAC groups. Next we examined adequate dose of ISO infusion after three weeks of TAC. Proportion of adverse cardiac remodeling was 37.5% (3 of 8) in 1mg/kg/day, 83.3% (5 of 6) in 3mg/kg/day, and 100% (8 of 8) in 10mg/kg/day group. But infusion of 10mg/kg/day ISO caused an increase in systolic BP. Thus we identified 3-week TAC and subsequent one-week ISO (3mg/kg/day) infusion as a suitable model for ACR. To identify molecular signatures of failing hearts of our ACR model, we performed RNA-Seq analyses. We found the up-regulated genes were mainly related to fibrosis including
Fbn1
,
C1qtnf6
and
Loxl2
and the down-regulated genes associated with mitochondrial functions including,
Uqcrc1
,
Ndufs3
, and
Idh2
. Next, we followed the changes in cardiac function after ceasing ISO to investigate CRR. Decreased LVFS gradually recovered to baseline within 7 weeks after cessation of ISO and exhibited CRR. Gene expression signature of CRR hearts was almost identical to that of TAC hearts without ISO infusion.

Conclusions:
Our model exhibits a transition to adverse cardiac remodeling and subsequent reverse remodeling with high reproducibility. This novel mouse model might add new insights into these cardiac remodeling.
Abstract 547: Pharmacologic ATF6 Activation Confers Global Protection in Widespread Disease Models by Reprogramming Cellular Proteostasis
Authors: Blackwood Erik A , Azizi Khalid , Thuerauf Donna J , Paxman Ryan J , Plate Lars , Kelly Jeffery W , Wiseman Rockland L , Glembotski Christopher C
Published: February 2019

Pharmacologic activation of stress-responsive signaling pathways provides a promising approach for ameliorating imbalances in proteostasis associated with diverse diseases. However, this approach has not been employed
in vivo
. Here, using a mouse model of myocardial ischemia/reperfusion, we showed that selective pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) during reperfusion, a typical clinical intervention point after myocardial infarction, transcriptionally reprograms proteostasis, ameliorates damage and preserves heart function. These effects were lost upon cardiac myocyte-specific
Atf6
deletion in the heart, demonstrating the critical role played by ATF6 in mediating pharmacologically activated proteostasis-based protection of the heart. Pharmacological activation of ATF6 was also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a first-in-class proteostasis-based therapeutic strategy for ameliorating ischemia/reperfusion damage, underscoring its unique translational potential for treating a wide range of pathologies caused by imbalanced proteostasis.
Abstract 550: Exosomes Released by Human Induced Pluripotent Stem Cell-Cardiomyocytes Induce In-Vitro Angiogenesis: A New Strategy for Cell-Free Therapeutics
Authors: Dougherty Julie A , Kumar Naresh , Noor Mohammad , Angelos Mark G , Chen Chun-An , Khan Mahmood
Published: February 2019

Due to the poor regenerative properties of cardiac tissue, acute ischemic events can lead to progressive terminal heart failure unless cardiac regeneration can be stimulated. Recent evidence using stem cell therapy in the heart demonstrates a greater role of their paracrine signaling potential, as cell survival post-transplantation is very low. The main objective of this study was to characterize exosomes secreted by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and evaluate their
in vitro
angiogenic potential. HiPSC-CMs were cultured under normal conditions with exosome-free serum. Isolated exosomes were characterized by TEM, NTA, and immunoblotting. Angiogenic potential was assessed by wound-healing, proliferation, and tube formation assays using bovine aortic endothelial cells (BAECs). TEM imaging showed secretion of exosomes for hiPSC-CMs, as confirmed by the size and shape of the isolated particles. The size of exosomes was determined to be 71.4 nm (mode), within the characteristic exosomal range. Immunoblot analysis of exosome lysate demonstrated enrichment of known exosome-specific protein markers (CD63 and Hsp70). Most importantly, BAECs treated with exosomes showed significant (p < 0.05) increases in migration, proliferation, and tube formation, as compared to the no exosome control. Overall, our results demonstrate that treating BAECs with exosomes isolated from hiPSC-CMs enhances angiogenesis. Overall, this study demonstrates the angiogenic potential of
human
iPSC-CM- derived exosomes. The cell-free therapeutics using exosomes constitutes a novel innovative approach for future clinical therapy to induce angiogenesis in the post-ischemic heart.
Abstract 552: Harnessing the Versatility of PLGA Nanoparticles for Targeted Cre-Mediated Recombination
Authors: Nguyen Ngoc , Chen Cheng-Han , Zhang Yulong , Zhao Peng , Wu Benjamin , Ardehali Reza
Published: February 2019

The Cre/LoxP recombinase system has emerged as an important tool for the generation of conditional somatic mouse mutants. This method enables spatial control of gene activity in almost any tissue, providing new avenues for studying gene function and establishing animal models of human diseases. A major technical advance was the development of ligand-dependent Cre recombinases that are activated by tamoxifen. While this advance has paved the way for temporally-controlled developmental studies previously deemed impractical, the generation of tissue-specific transgenic mouse lines can be time-consuming and costly. Herein, we design a ‘smart’, biocompatible, and biodegradable nanoparticle system that can be actively targeted to specific cell types in vivo through surface conjugation of antibodies. We provide proof-of-principle for successful delivery of macromolecules and plasmid DNA. Importantly, we demonstrate that these ‘smart’, tamoxifen-loaded nanoparticles can be precisely targeted to cells of interest in vivo to bind and activate Cre recombinase, resulting in tissue-specific Cre activation. This system provides a simple, yet powerful approach to induce recombination in a ubiquitious Cre system for a time- and cost-effective strategy to generate new transgenic mouse lines.