bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 204: Temporal Assessment of Human iPSC-Derived Cardiomyocytes to Assess the Maturity for Cardiac Repair Applications
Authors: Kumar Naresh , Dougherty Julie A , Mergaye Muhamad , Angelos Mark G , Khan Mahmood
Published: February 2019

Cardiovascular disease is the major cause of heart failure following myocardial infarction (MI), which leads to death in acute condition. In the recent year, stem cell therapy has shown a great potential to repair the damaged heart tissue following an MI. Both adult and embryonic stem cells (ES) have potential to repair and regenerate tissue to compensate cell death during MI. Apart from ethical concern, there are several advantages of using human iPSC cardiomyocytes (hiPSC-CMs) over human ES. Most important, the hiPSC-CMs provides autologous cells source which one side eliminate the possibility of cellular rejection while on the other side enhance the possibility of its survival in damaged cardiac tissue. However, the lack of maturity in hiPSC-CMs is a major challenge to replicate and/or replace the adult cardiomyocytes with mature cardiomyocytes at the site of MI. Temporal assessment of cardiac maturity markers and structural gene in hiPSC-CMs cultured
in vitro
for up to four weeks and comparison with non-failing human heart (NF). The wk-4 hiPSC-CMs has shown an increase in cardiac transcription factor (2-fold, NKX-2.5); Myosin genes (2-3 fold, MYH7, MYHL2); cardiac troponin-T (3-4 fold, TNNI3 and TNNT3); K
+
channel genes (20-fold, KCNJ2); Ca
2+
related genes (10-fold, CASQ2); Gap-junction genes (2-fold, GJA1) and Na
+
Channel gene (5-fold, SCN5A). Further, TEM showed that the myofibrils in the wk-4 cardiomyocytes were more compact, aligned and organized. The Z and I-band and specifically M-band were also prominent in the wk-4 sarcomeres. Furthermore, the maturity of hiPSC-CMs was compared with the NF human heart. This study showed a time-course assessment of cardiomyocytes morphology, structural and expression of cardiac maturity genes in hiPSC-CMs cultured for up to four weeks
in vitro
. Based on the study of the cardiac transcription factor, Na-K channel-related gene and TEM analysis, wk-4 cardiomyocytes showed more maturity than week-1 cardiomyocytes. Studies are ongoing to compare the contractility and action potential of hiPSC-CMs. Finally, the mature hiPSC-CMs will provide autologous cell source and have a significant impact on the functional outcome of patients with acute MI.
Abstract 205: Cryopreserved Cardiac Tissue Graft (MyCardia) Improved Cardiac Function in Rats With Heart Failure
Authors: Lancaster Jordan , Koevary Jen , Goldman Steven
Published: February 2019

Introduction:
There are currently 2M patients with NYHA Class II-IV heart failure of ischemic etiology in the United States. Cell-based therapies hold tremendous potential to provide therapeutic benefit for treating degenerative diseases such as chronic heart failure (CHF). Therapeutic adoption of engineered grafts will require ease of use and demonstration of cost-effectiveness. Here we describe for the first time the ability to cryopreserve an engineered tissue (MyCardia™) and demonstrate functional recovery in a rat model of CHF.

Methods:
MyCardia grafts were cultured with human dermal fibroblasts (hNDF) and human iPSC derived cardiomyocytes (hiPSC-CMs) (FCDI) into a bioabsorbable mesh (Novus Scientific). Grafts were cryopreserved at -196C for 2-4weeks, thawed, reconstituted and implanted into rats with CHF. Myocardial infarction (MI) were generated in immune competent rats by ligating the left coronary artery. Three weeks after MI, rats received graft implants via median sternotomy. Echocardiography was performed at 3 and 7 weeks post graft implantation. In addition, terminal hemodynamics were performed.

Results:
MyCardia maintained spontaneous and synchronous contractions pre-freeze (65±10bpm) and post thaw (60±10bpm). Grafts were maintained to 40 days post thaw with no deterioration in composition or beat rate. MyCardia reversed maladaptive left ventricular (LV) remodeling by decreasing (p<0.05) LV volume systole/diastole, LV diameter systole/diastole with trending improvement in EF and FS from 3 and 7 weeks post implantation as compared to CHF controls. Grafts decreased LV end diastolic pressure, 24±6 mmHg vs. 18±3mmHg and showed trending improvements in LV dP/dt(-) and dP/dt(+) as compared to controls. In addition, MyCardia results in 36% reduction in LV EDP operating volume as demonstrated by a leftward shift of the diastolic pressure-volume relationship.

Conclusion:
MyCardia can be cryopreserved, reconstituted and implanted in rats with CHF. Treated rats demonstrated reversal of maladaptive LV remodeling and continued functional improvement from 3 to 7 weeks post treatment. We anticipate that the ability to cryopreserve will allow long-term storage after large batch manufacture, providing cost and utility advantages.
Abstract 215: Induced Pluripotent Stem Cell-Derived Cardiomyocyte Proliferation is Enhanced by Co-culture With Female Mesenchymal Stem Cells
Authors: Wanschel Amarylis B , Salerno Alessandro G , Hatzistergos Konstantinos E , Schulman Ivonne H , Balkan Wayne , Hare Joshua
Published: February 2019

Background:
Myocardial ischemia remains a leading cause of morbidity and mortality worldwide. Bone marrow-derived adult mesenchymal stem cells (MSCs) improve cardiac structure and function post-MI likely mediated in large part by paracrine factors. There is emerging evidence that female MSCs have greater therapeutic efficacy than male MSCs.

Objective:
We hypothesized that female MSCs promote cardiomyocyte differentiation, proliferation and contractility of induced pluripotent stem cells (iPSCs) better than male MSCs.

Methods & Results:
Human (h) iPSCs were co-cultured with male or female murine MSCs during or after differentiation into beating cardiomyocytes. Female MSCs were significantly more potent at stimulating ISL-1 and NKX2-5 expression in hiPSC-derived cardiac precursors and cardiomyocytes. Exosomes from female MSCs had lower levels of miR-17, a miR that directly represses
Isl1
. hiPSCs co-cultured with female MSCs, exhibited a 2-fold greater increase in proliferation compared to male MSCs. In addition, ß
2
-adrenergic receptor expression was up regulated 3.5-fold in hiPSCs incubated with female MSCs compared to 2.5-fold for male MSCs.

Conclusion:
Our findings illustrate that there are significant differences between female and male MSCs in their ability to affect cardiomyogenesis of hiPSCs and suggest that this effect is mediated, at least in part, by reduced levels of miR-17. The observation that female MSCs have greater potential to improve cardiomyocyte proliferation and contractility has important implications for stem cell therapy.
Abstract 216: Interleukin-10 Deficiency Impairs Reparative Properties of Bone Marrow-Derived Endothelial Progenitor Cell Exosomes
Authors: Yue Yujia , Garikipati Venkata N , Tang Yan , Cimini Maria , Cheng Zhongjian , Wang Chunlin , Troungcao May , Kishore Raj
Published: February 2019

EPC based therapy in clinical trials is largely benefited from paracrine effect such as exosomes. Exosomes mirror the behavior of parental cells and their function is dependent on RNAs and proteins packed inside. Systemic inflammation in MI patients greatly compromise the reparative performance of EPCs and relative exosomes. We hypothesized that EPCs under inflammatory stress produce dysfunctional exosomes with altered content, which compromise EPCs reparative benefit in ischemic heart disease. We choose interleukin-10 knockout (IL-10KO) mice as a model mimicking systemic inflammation. After EPC isolation and expansion from IL-10KO and wild-type (WT) mice, we isolated exosomes and compared their reparative properties both
in vitro
and
in vivo
. Our
in vitro
studies showed WT-EPC-Exo treatment enhanced endothelial cell proliferation and tube formation, and inhibited apoptosis, whereas IL-10KO-Exo exhibited impaired or even detrimental effects. We used MI mouse model to compare the
in vivo
function of two groups of exosomes, we found WT-EPC-Exo treatment significantly improved left ventricle cardiac function, inhibited cell death and promoted angiogenesis; while these benefits were lost in IL-10KO-EPC-Exo treated group. Both
in vitro
and
in vivo
studies suggested impaired exosome function under IL-10 deficiency. We checked the alteration of exosomal content using NGS RNAseq and mass spectrometry and found the RNA and protein expression pattern is drastically different in two groups of exosomes. Importantly, IL-10KO-EPC-Exo were highly enriched in microRNAs and proteins that promote inflammation and apoptosis and inhibit angiogenesis. We picked two candidates for further study, mir-375 and integrin linked kinase (ilk), both are highly enriched in IL-10KO-EPC-Exo. Through modulating the expression of mir-375 and ilk in exosomes, we partially rescued IL-10KO-EPC-Exo dysfunction. Thus, our study revealed that even the same type of cells, under different conditions, secrete exosomes with different function. The differences in exosomal function is caused by alteration in exosomal content, and the function can be enhanced or rescued by modulating specific targets in exosomes.
Abstract 221: Autophagy is Involved in the Protective Effect of Endophilin A2 on H
2
O
2
-induced Apoptosis in H9C2 Cardiomyocytes
Authors: Liu Yun , Liu Hai-Qi , Wang Xin-Qiu-Yue , Shen Huan-Jia
Published: February 2019

Apoptosis plays a critical role in normal embryonic development and tissue homeostasis regulation. EndophilinA2 (EndoA2) is widely reported to regulate endocytosis. Additionally, EndoA2 has been demonstrated to be involved in tumor metastasis, neuroregulation and vascular function. In this study, we used siRNA and Ad-EndoA2 transfection strategy to investigate whether EndoA2 provides a protective effect against apoptosis induced by H
2
O
2
in H9C2 cardiomyocytes and the underlying mechanisms. We found that EndoA2 siRNA knockdown promoted H
2
O
2
-induced apoptosis in H9C2 cardiomyocytes, evidenced by decreased cell number, increased apoptotic cells, and activation of caspase-3. In contrast, EndoA2 overexpression showed the opposite effects and inhibited H
2
O
2
-induced apoptosis in H9C2 cardiomyocytes. Further studies revealed that EndoA2 overexpression strengthened autophagy, evidenced by the increased LC3 II/I ratio and P62 degradation,whereas EndoA2 siRNA knockdown produced the opposite effects. Furthermore, we revealed that there was an interaction between Bif-1 and Beclin-1. Upon H
2
O
2
treatment, the association of Bif-1 and Beclin-1 remarkably increased. EndoA2 overexpression further promoted the binding of Bif-1 with Beclin-1, whereas EndoA2 siRNA knockdown reduced this association. These data strongly suggested that EndoA2 inhibited H
2
O
2
-induced apoptosis in H9C2 cardiomyocytes, possibly by promoting Bif-1 to form a complex with Beclin-1 and strengthening autophagy. This study provides a novel target for heart diseases.
Abstract 225: Comparison of Myocardial Infarction and Congestive Heart Failure Induced Through Interventional Ischemia Reperfusion Injury in Yorkshire (Domestic), Yucatan, and Göttingen Swine
Authors: Drummond Christopher A , Pielemeier Randy , Sweet Jenifer , DeVries Jennifer , Gesaman Janelle , LaBar Mark , Nugent Gayle , Johnson Mark D
Published: February 2019

Swine are important translational research models for developing and gaining regulatory approval for novel therapies for heart failure. Three swine species in particular are utilized in these studies, the Domestic Yorkshire (DY), Yucatan Minipigs (Yuc), and Göttingen minipigs (Gott). Selecting the right swine breed is important to the translational success of novel therapies and is the source of debate in the field. The current study compared interventionally induced Ischemia-reperfusion injury (IRI), a method that mimics clinical myocardial infarction and subsequent heart failure, in these breeds. Interventional IRI was induced by occluding the Left Anterior Descending Coronary Artery for 90 minutes using a PTCA balloon placed distal to the first diagonal branch. Yuc have the highest rate of survival of the three breeds following a 90 min occlusion (81%; 56 out of 69), Gott survival was 70% (28 out of 40), and DY survival was 57% (75 out of 131). Assessment of serum levels of Cardiac Troponin I (CTnI) revealed significant increases in this parameter that peak at 3 hrs following occlusion. Gott have the highest levels at 122ng/mL, followed by Yuc (99ng/mL), and DY (85ng/mL) at this time point. Functionally, all three breeds displayed an Ejection Fraction assessed by echocardiography of less than 40%, with Gott having the lowest mean, followed by Yuc, and DY. These levels remain low or decrease slightly over 1 to 4 months. Magnetic Resonance imaging demonstrates infarct sizes are approximately 25% of the left ventricle following 1 month and decreasing over time to 22% at 2 months post and 13% 4 months post. Additionally, all three species lose their hemodynamic responsiveness to dobutamine challenge following IRI. All three breeds respond similarly to IRI functionally and structurally, and they develop the clinical features of congestive heart failure, the main distinguishing factor in the current study was survival of the IRI procedure. Based on this data, factors such as rate of body weight gain in relation to the ability to obtain functional endpoints, availability, and other practical considerations should drive the selection of breed for translational studies.
Abstract 233: Molecular and Electrophysiological Analyses of Gene Variants in Bilateral Adrenal Hyperaldosteronism
Authors: Hattangady Namita G , Lerario Antonio M , Ponce-Balbuena Daniela , Monticone Silvia , Mulatero Paolo , Else Tobias
Published: February 2019

Primary aldosteronism (PA) is the most common cause of secondary hypertension with a high prevalence amongst patients with resistant hypertension. Recently somatic mutations have been defined in aldosterone-producing adenomas (APAs)-associated PA. Herein, we analyzed a single hereditary case and in 30 sporadic cases of bilateral adrenal hyperaldoteronism (BHA) using whole exome sequencing and generated a candidate gene list using an allelic frequency ≤1%. Unique variants were identified in the gene
ATP2B4
[coding for a Ca-ATPase Pump, PMCA4], in the affected individuals in the familial case and in two sporadic BHA samples. These variants were chosen for
in vitro
analyses due the potential role on PMCA4 in regulating intracellular calcium and due to the causal role of mutations in the family member
ATP2B3
in APA-related PA. The
ATP2B4
variants were functionally analyzed testing the hypothesis that it causes an inability to pump out intracellular Ca
2+
, causing elevated intracellular Ca
2+
, net cell depolarization and increased aldosterone synthase (
CYP11B2
) expression. Results from
in vitro
molecular analyses: 1) Real time quantitative PCR (qPCR) confirmed
ATP2B4
mRNA in the various tissue including the human adult adrenal gland, APAs, and the human adrenocortical cell line HAC15. 2) Immunohistochemistry indicated PMCA4 expression throughout the adrenal cortex. 3) Knock down of
ATP2B4
in HAC15 exhibited reduced Angiotensin II-stimulation in one of four shRNA clones. 4) Stable HAC15 cell lines for doxycycline (dox) - inducible wildtype and variant forms of
ATP2B4
, were generated. Q-PCR and immunostaining confirmed dox-induced upregulation of ATP2B4 mRNA and protein. However, upregulation of the PMCA4 variants did not have a differential effect on basal or agonist-stimulated
CYP11B2
expression. Whole cell recordings in HAC15 and HEK cells indicated robust PMCA4 conductance in native cells, but reduced conductance in the WT and variant PMCA4. In conclusion, while this study did not define a pathogenic role for
ATP2B4
variants in BHA, we describe an approach of WES analysis for familial and sporadic BHA and outline a template for the thorough
in vitro
characterization of gene variants in the pathogenesis of BHA.
Abstract 240: Predictive and Experimental Modeling of Bag3-associated Cardiomyocyte Dysfunction
Authors: McDermott-Roe Christopher , Lv Wenjian , Shehu Amarda , Benjamin Ivor , Geurts Aron , Musunuru Kiran
Published: February 2019

High throughput sequencing continues to uncover novel potentially pathogenic variants in cases of familial cardiomyopathy. Clinical interpretation of such variants is challenging, and hence definitive assignment of causality frequently remains unestablished. A case in point is provided by BAG3, a critical regulator of protein quality control in cardiomyocytes, which has been recurrently linked to dilated cardiomyopathy (DCM). Using theoretical and experimental approaches, we assessed the impact of a functionally-uncharacterized DCM-associated variant in BAG3 (c.1430G>A; p.Arg477His). Molecular dynamics simulations of wildtype and mutant forms of BAG3 suggested the residue substitution perturbs a crucial interaction between BAG3 and HSC70 by altering electrostatic field distribution, conformational rigidity and side-chain orientation. Acute and stable expression of the mutant BAG3 protein led to reduced HSC70 engagement and protein aggregation in HEK293 and HL1 cells. Using the CRISPR-Cas9 system, we then introduced the variant into a healthy induced pluripotent stem cell line line and upon differentiation into cardiomyocytes, observed protein aggregation and a sensitivity to pro-apoptotic stressors. In conclusion, disease-in-a-dish modeling was used to study the molecular and cellular consequences of a potentially pathogenic BAG3 variant. We define what we believe is the primary molecular lesion and propose ectopic protein quality control and potentiated stress sensitivity as possible pathogenic drivers and potential therapeutic entry points.
Abstract 265: TGF Beta Signaling and Fibrosis in cMyBP-C-dependent Cardiac Disease
Authors: Meng Qinghang , Bhandary Bidur , Osinska Hanna , James Jeanne , Shay-Winkler Kritton , Gulick James , McNamara James , Bhuiyan Md. Shenuarin , Sadayappan Sakthivel , Robbins Jeffrey
Published: February 2019

Background:
Mutations in cardiac myosin binding protein C (
MYBPC3
) account for more than one third of identified hypertrophic cardiomyopathy (HCM). Data from both human patients and mouse models suggest that cardiac fibrosis preceeds hypertrophy and cardiac dysfunction, implying that fibrosis related signaling may be one of the initial signs of developing cardiac disease. We wished to determine if cardiomyocyte-autonomous TGFβ signaling is sufficient to initiate pathogenic fibrosis in the heart in the context of a cMyBP-C-induced disease.

Methods and Results:
Neonatal rat ventricular cardiomyocytes (NRVMs) expressing either wild-type or
Mybpc3
mutations were mediated by adenovirus for 48 hours. Both cells and culture medium (conditional medium) were harvested. The fibrotic effects of the conditional medium on neonatal rat ventricular fibroblasts (NRVFs) were determined using “fibrotic” gene promoters driving luciferase (luc) expression
(
αSMA-luc
,
postn-luc
and
col1a1-luc
). TGFβ levels in the conditional medium were determined using ELISA assays. Expression of
cMybpc3
mutations activated TGFβ promoter activity (
Tgfb1-luc
,
Tgfb2-luc
and
Tgfb3-luc
) in a TGFβ receptor(s) (TβRs) dependent manner. Upregulation of TGFβ was also confirmed in transgenic mice expressing a fragment of cMyBP-C (cMyBP-C
40kDa
) whose expression results in significant fibrosis and cardiac disease. To establish causality, we genetically ablated TGFβ receptors (
Tgfbr1
and
Tgfbr2
) in the cardiomyocytes of the

cMybpc3
40kDa

mice. TGFβ receptors ablation blocked the induction of TGFβ expression, alleviating cardiac fibrosis and decreasing cardiac hypertrophy. Cardiac function was significantly improved and survival of the cMyBP-C
40kDa
mice was increased.

Conclusions:
Cardiomyocytes are a critical source of TGFβ in cMyBP-C
40kDa
hearts during the initial stages of pathogenesis. Thus, TβRs ablation at the early stage completely prevent the progression of the pathological process
in vivo
.
Abstract 285: Glycogen Receptor Antagonism Ameliorates Progression of Heart Failure
Authors: GAO Chen , Ren Shuxun (Vincent) , Yu Junyi , Thai Dung , Lu John , Cai Zheqing , Zeng Cunyu , Yan Hai , Wang Yibin
Published: February 2019

Glucagon is an important hormone for glycemic control with counter-balancing function vs. insulin, and treatment of glucagon receptor antibody is shown to be efficacious for type I and type II diabetes. It was reported recently that systemic treatment of glucagon aggravated myocardial injury and pathological remodeling in heart in a glucagon receptor dependent manner, while cardiomyocyte specific inactivation of glucagon receptor protected heart from ischemic injury and remodeling, implicating the therapeutic potential of targeting glucagon receptor for heart failure. In this report, we investigated the functional outcome of glucagon receptor antagonist treatment in mice following myocardial infarction (MI) and pressure-overload using a humanized monoclonal glucagon receptor antagonist antibody REMD2.59. Mice treated with REMD2.59 after myocardial infarction showed significantly reduced scar sizes, blunted cardiac hypertrophy and fibrotic remodeling, and attenuated contractile dysfunction at four weeks after myocardial infarction comparing to mice treated with vehicle as controls. In contrast, treatment of glucagon aggravated these pathological manifestations triggered by myocardial infarction. Furthermore, treatment with REMD2.59 at the onset of pressure-overload significantly suppressed the development of cardiac hypertrophy and chamber dilation with marked preservation of cardiac systolic and diastolic function. More remarkably, initiation of REMD2.59 treatment two weeks after pressure-overload significantly blunted the progression of cardiac pathology with remarkable recovery of hypertrophic gene induction. These results provide the first
in vivo
proof-of-concept evidence that glucagon receptor antagonism is a potentially efficacious therapy to block both onset and progression of heart failure.
Abstract 294: Investigating the Small Heat Shock Proteins Impact on Aggregate Inhibition
Authors: Roth Andrew , Rye Hays
Published: February 2019

The small heat shock proteins (sHsp) are a highly conserved protein chaperone family needed for ischemia/reperfusion recovery and mitigation of protein aggregation. Without them, environmental stressors, like hypoxia and oxidative stress, can cause highly toxic protein aggregation, leading to cardiomyopathy. Nevertheless, the mechanisms by which sHsps protect cell viability are poorly understood.

sHsp mechanism remains elusive due to their highly heterogeneous and dynamic nature making them difficult to study with current methods. To solve this problem, we use a single particle technique called Burst Analysis Spectroscopy (BAS). BAS provides a powerful approach, permitting the real-time observation of size population-resolved kinetics under non-perturbing, free solution conditions. This method allow us to observe events like protein aggregation as well as the interacting sHsp complexes.

To study the mechanism of aggregate inhibition by sHsps, we utilize the
E. coli
sHsps IbpA and IbpB (IbpAB) with two established protein substrates, RuBisCO from
R. Rubrum
and PepQ from
E. coli
. Prior work,
in vivo
and
in vitro
, has established that IbpAB play a similar role in
E. coli
as sHsp do in higher eukaryotes suggesting all sHsps may share common mechanistic properties.

Using BAS, we established PepQ and RuBisCO form large heterogeneous amorphous aggregates, with RuBisCO also capable of forming fibril-like aggregates under different aggregation conditions. When IbpAB are present during aggregation, both aggregate types decrease in size and form a very specific homogenous aggregate size distribution suggesting IbpAB activity is independent of protein aggregate substrate or structure. Remarkably, binding of IbpAB to RuBisCO aggregates differs dramatically depending on the aggregate structure. The amorphous aggregates appear to bind IbpAB at a RuBisCO monomer-to-IbpAB ratio of 1:1 while the fibril-like RuBisCO aggregates display a bifurcation, where two, distinct aggregate sub-populations bind IbpAB at very different stoichiometries. This data has uncovered the unknown capability of IbpAB/sHsps to inhibit ongoing aggregation of two substrates and structures through two different mechanistic processes all to the same homogenous aggregate size.
Abstract 296: Cd38 Deficiency Protects Heart From Lipid Overload-induced Oxidative Stress via Activating Sirt3/foxo3 Pathway
Authors: Wang Ling Fang , Huang Cong Cong , Xiao Yun Fei , Guan Xiao Hui , Wang Xiao Nv , Cao Qing , Liu Yu , Huang Xuan , Deng Li Bin , Deng Ke Yu , Xin Hong Bo
Published: February 2019

Background/Aims:
Our and other studies showed that CD38 deficiency protected heart from ischemia/reperfusion injury and high fat diet (HFD)-induced obesity in mice, respectively. However, the roles of CD38 in lipid overload-induced heart injury were not evaluated. In the present study, we investigated the preventive effects and mechanisms of CD38 deficiency on mouse heart injury induced by HFD.

Methods:
The discrepant metabolites in heart from wild type (WT) and CD38 knockout (CD38
-/-
) mice fed with HFD were examined using metabolomics analysis. The cell viability, the lactate hydrogenase (LDH) release, super oxide dismutase (SOD) activity, reactive oxygen species (ROS) production, triglyceride concentration and the expressions of various corresponding genes were examined for evaluating the effects of CD38 on oleic acid (OA)-induced cell injury using CD38 knockdown H9C2 stable cell line.

Results:
Our results revealed that CD38 deficiency significantly elevated the intracellular glutathione (GSH) concentration and GSH/GSSG ratio, decreased the contents of various free fatty acids and increased intracellular NAD
+
level in heart of CD38
-/-
mice fed with HFD. Knockdown of CD38 significantly attenuated OA-induced cell injury, ROS production and lipid synthesis, seen as enhancing cell viability, reducing LDH release, elevating SOD activity, and decreasing triglyceride content and expressions of FASN in the CD38 knockdown H9C2 stable cell line. In addition, we also observed that the mitochondrial deacetylase Sirt3 and its target genes FOXO3 and SOD2 were upregulated in the stable cell lines after OA stimulation. Furthermore, the expressions of NOX2 and NOX4 were attenuated, accompanied by decreasing Bax expression and increasing Bcl-2 expression in the cells after OA stimulation.

Conclusions:
Our results demonstrated that CD38 deficiency protected heart from HFD-induced oxidative stress via activating Sirt3/FOXO3-mediated anti-oxidative stress pathway.
Abstract 303: Fast Skeletal Myosin Binding Protein-c Expression in Heart Failure
Authors: McNamara James W , Lynch Thomas L , Song Taejong , Rubinstein Jack L , Sadayappan Sakthivel
Published: February 2019

Background:
Heart failure is a devastating disease affecting nearly 5 million Americans. We recently identified the upregulation of fast skeletal myosin binding protein-C (fsMyBP-C) in models of heart failure. fsMyBP-C is a homologue of cardiac MyBP-C, which is normally expressed in the heart. This family of proteins regulate contractility by modulating thick and thin filament interactions. However, the functional role(s) of fsMyBP-C expression in the heart is unknown.

Methods:
To understand the role of fsMyBP-C expression in the heart, we generated cardiac specific transgenic mice expressing fsMyBP-C under the control of the alpha myosin heavy chain promoter. Mice underwent serial echocardiography to monitor cardiac function. Western blotting and qRT-PCR were performed to determine transgene expression levels. Additionally, fsMyBP-C localization in cardiomyocytes was established by immunofluorescence. Furthermore, to understand the temporal expression profiles of the MyBP-C homologues, we performed qRT-PCR on 9 muscle groups at different developmental stages in healthy mice.

Results:
qRT-PCR demonstrated an upregulation of the slow MyBP-C transcript in the developing heart, while fsMyBP-C transcript levels increased steadily following birth in WT mice. At 3 months of age, transgenic mice displayed a significant increase in ventricular dimensions compared to NTG littermates by echocardiography (LVID
d
: 3.72 ± 0.07 vs. 4.17 ± 0.09 mm, p<0.01; LVID
s
: 2.44 ± 0.09 vs. 2.81 ± 0.09, p<0.05), with no changes in wall thickness. This was accompanied by a 25% increase in stroke volume (p<0.01) and a 30% increase in cardiac output (p<0.05). fsMyBP-C correctly localized to the C-zone of the sarcomere. Furthermore, protein levels showed mild expression of fsMyBP-C, in line with the small upregulation seen in diseased hearts, while qRT-PCR confirmed overexpression of fsMyBP-C in the ventricles.

Conclusion:
Mice expressing fsMyBP-C in the heart display a mild hypertrophic phenotype at 3 months, with preserved function, possibly indicating compensated hypertrophy. Ongoing studies investigate cardiac function at later stages and cardiac specific conditional knockout of fsMyBP-C to determine the long-term effect of fsMyBP-C regulation in the heart.
Abstract 306: Identifying Downstream Effectors of the Extracellular Signal-related Kinase 1/2-controlled Directional Growth Response in Cardiomyocytes
Authors: Grimes Kelly M , Maillet Marjorie , Molkentin Jeffery D
Published: February 2019

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the lengthening and widening of cardiomyocytes, respectively. To-date the only known proteins found to control this directional growth of cardiomyocytes are MEK1, mitogen-activated protein kinase kinase 1, and ERK1/2, extracellular signal-related kinase 1/2. The only known role of MEK1 is to phosphorylate ERK1/2 after MEK1 itself becomes phosphorylated through RAS/RAF signaling. Upon phospho-activation, ERK1/2 can interact with targets in the cytoplasm or translocate to the nucleus to induce cell growth and proliferation pathways. We previously found that cardiac overexpression of activated MEK1 causes cardiac hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins in the heart causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 (a MEK1 inhibitor), or GFP adenovirus (control). For proteins with the greatest change in phosphorylation upon MEK1 activation or inhibition, we have verified the screen results using Phos-tag gels and western blots. Many of these hits have cytoskeletal or actin regulatory functions, and a few have also been implicated in stress-sensing in previous literature in other cells types. Furthermore, many hits showed increased protein levels in addition to augmented phosphorylation in NRVMs treated with activated MEK1 adenovirus. Given our nearly 20 interesting candidate proteins, we are currently conducting an
in vivo
screen in which each gene is individually expressed in mice using AAV9. After injection of virus into the thoracic cavities of P5 wild type mice, we are assessing if cardiac hypertrophy/dilation or cardiomyocyte dimensional growth are present at 4 months of age. Any novel candidates identified in this manner will be presented.
Abstract 310: Mechanistic Insights into Duchenne Muscular Dystrophy-Associated Cardiomyopathy
Authors: Tassin Tara , Huang Jian , Gemelli Terry , Kanchwala Mohammed , Punnen Lisa M , Chung Emily , Rivas Dylan , Goetsch Sean , Daniel Juan A , Shelton John , Richardson James A , Schneider Jay , Xing Chao , Singh Sarvjeet , Mammen Pradeep P
Published: February 2019

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder resulting from a mutation in the dystrophin gene. Loss of dystrophin function leads to progressive muscle wasting and cardiomyopathy. In 2018, advanced cardiomyopathy is the primary mode of death, despite the application of standard of care CHF therapies. Our group has recently undertaken a cardiac MRI study demonstrating that adult DMD patients have small hearts with very low LV mass as compared to patients with non-ischemic cardiomyopathy or healthy patients. The central hypothesis is DMD-associated cardiomyopathy develops secondary to dysregulation of various cardiac signaling pathways that modulate cardiac growth, namely cardiomyocyte proliferation. Utilizing the
mdx
mouse, a model of DMD, decreased heart/body weight ratios were noted from birth (P1: Hrt/BW 5.6 ± 0.16 vs. 4.8 ± 0.18
NTg
vs
mdx
p<0.005 n=32).
NTg
and m
dx
hearts have similar cardiomyocyte cell size (WGA staining: 0.08 ± 0.001 vs 0.08 ± 0.002
NTg
vs
mdx
p<0.05 n=3), suggesting there are fewer cells in
mdx
hearts. FACS analysis indicated
mdx
hearts have fewer cardiomyocytes than
NTg
hearts (35% reduction; 51.7e
4
± 4.9e
4
vs. 33.8e
4
± 3.5e
4
NTg
vs
mdx
p<0.05 n=6). Transcriptome profiling demonstrated that molecular pathways governing cardiac proliferation were significantly reduced in
mdx
hearts (Ki67: 4.8 ± 0.3 vs 2.0 ± 0.1
NTg
vs
mdx
p<0.005 n=3), with a corresponding increase in cardiac atrophy gene expression at later time points (Foxo3 1.4 ± 0.2 vs 2.8 ± 0.3
NTg
vs
mdx
p<0.005 n=3). Furthermore, immunohistochemical analyses showed significantly reduced proliferation in
mdx
hearts (Ki67: 13.7 ± 1.2 vs. 6.0 ± 1.0
NTg
vs
mdx
p<0.005, n=3; pH3: 118 ± 5.3 vs 59 ± 4.5 p<0.005 n=3). Finally, RNA-Seq data revealed a disruption of the YAP signaling pathway, an important mediator of cardiomyocyte proliferation, in
mdx
P4 hearts. Cell cycle targets of YAP were reduced in P4
mdx
hearts (20/27 genes, n=3) suggesting a disruption of the Hippo-Yap pathways in
mdx
hearts. Collectively, the current study provides a unique insight into the mechanism leading to the development DMD cardiomyopathy and directs investigation into potential therapeutic targets for the amelioration of DMD-associated cardiomyopathy.
Abstract 312: Cardiomyocyte-secreted Signaling Factors Modulate Adipocyte Differentiation And Function
Authors: Gresham Kenneth S , Koch Walter J
Published: February 2019

Heart failure (HF) alters cardiomyocyte metabolism by altering energy substrate utilization and mitochondrial function, which has potential implications for global metabolic health. Recent studies demonstrated that cardiomyocyte-released signaling factors communicate with peripheral organ systems to alter metabolic function and that signaling pathways activated by HF control the release of these factors. However, the identity of these factors is still under investigation. Conditioned media (CM) collected from neonatal rat ventricular myocytes (NRVMs) was used to treat 3T3-L1 pre-adipocytes to 1) identify signaling molecules released by cardiomyocytes that could regulate global metabolic function and 2) determine how HF signaling pathways alter their release. NRVM-CM slowed adipocyte differentiation as measured by lipid accumulation using the fluorescent probe BODIPY 493/503 (57±8% decrease in normalized fluorescence compared to control media) and reduced the expression of adipocyte-related proteins. NRVM CM treatment also increased basal mitochondrial oxygen consumption (78 ± 3 vs 62±4 pmol/min) and maximal respiration (270±12 vs 220 ± 16 pmol/min) in mature adipocytes. CM protein fractionation revealed that released-factor activity was retained in >100kDa fractions and this activity was abolished by trypsin digestion, suggesting a novel, high-MW cardiomyocyte-released signaling protein that targets adipocytes. To determine if NRVM-released factor activity is altered by HF signaling, the ability of G-protein kinase 2 (GRK2, upregulated by HF) signaling activity to alter NRVM-released factor activity was investigated. GRK2 overexpression in NRVMs increased the activity of this released-factor as measured by further decreased 3T3-L1 differentiation (lipid accumulation measured by BODIPY fluorescence was 70±8% compared to CM from GFP-overexpressing NRVMs), while overexpression of the GRK2 inhibitor βARKct increased adipocyte differentiation (140±13% compared to control NRVM-CM). These results suggest a novel, high-MW cardiomyocyte-released signaling protein under the control of HF signaling pathways communicates with adipocytes to alter adipose tissue function and potentially alter global metabolic function.
Abstract 318: MicroRNA Regulation of G Protein-Coupled Receptor Kinase 2 After Cardiac Injury
Authors: Lieu Melissa , Chuprun Kurt , Gao Erhe , Koch Walter J
Published: February 2019

The increase in myocardial expression and activity of G Protein-Coupled Receptor (GPCR) Kinase 2 (GRK2) during the development and progression of heart failure (HF) has been documented to be pathological. Upregulation of GRK2 that is involved with deactivating agonist-activated β-adrenergic receptors (βARs) results in weakened contractile responsiveness through decreases in βAR density, coupling, and sensitivity. Non-GPCR activity of GRK2 has also been shown to be involved in cardiac dysfunction. Studies with knockout or blockade of GRK2 are able to significantly reduce and/or prevent HF phenotypes. Conversely, models with over-expressed GRK2 produce increased injury and accelerated HF development. Being such an important kinase in cardiac disease, regulation of GRK2 expression and activity is critical. Of interest, there is not much known about how GRK2 is regulated (mRNA or protein) in the heart and mechanisms for its up-regulation after cardiac injury are not well understood. There is evidence of GRK2 being regulated by microRNAs (miRNAs) in endothelial and blood cells. We believe cardiac GRK2 may also be regulated in a similar miRNA-mediated fashion. MiRNA profiles become dysregulated with the onset of stress/injury and contribute to orchestrating many classical HF phenotypes, including βAR aberrations. We hypothesize that the expression of a GRK2-regulating miRNA is altered in HF, which contributes to changes in GRK2 regulation resulting in its pathological increase. MiRNA microarray analysis was done on 2-week post-myocardial infarction (MI) murine hearts to examine miRNA profiles compared to sham-operated. Cardiac miRNA species were examined for their expression levels, seed sequence, and TargetScan prediction and alignment to mouse and human GRK2 3’UTR. Candidate miRNAs underwent 3’UTR analysis against GRK2 3’UTR and transfection into AC16 cardiac cells. Two miRNAs were identified to significantly reduce GRK2 protein and/or RNA levels as well directly bind to the 3’UTR of GRK2. Further studies will be done to elucidate the mechanism these miRNAs are able to regulate GRK2 levels. Results from these studies can be applied towards GRK2 inhibition via miRNA-based therapeutics.
Abstract 319: Mir-222 is Required for Physiological Hypertrophy but Inhibits Pathological Hypertrophy and Heart Failure
Authors: Liu Xiaojun , Li Haobo , Xiao Chunyang , Damilano Federico , Platt Colin , Lerchunmuller Carolin , Zhu Han , Wei Paul , Yeri Ashish , Most Patrick , Rosenzweig Anthony
Published: February 2019

Background:
Physiological cardiac hypertrophy occurs commonly in response to exercise and can protect against pathological stress. In contrast, pathological hypertrophy occurs in disease and often precedes heart failure . Although physiological and pathological hypertrophy often involve distinct signaling mechanisms, miR-222 is an exercise-induced microRNA that is required for physiological hypertrophy but is also induced in pathological hypertrophy. Here, we sought to define the role of miR-222 in pathological hypertrophy.

Methods and Results:
We generated miR-222 gain-of-function (GOF) models through cardiac-specific constitutive transgenic miR-222 expression (TgC-miR-222) or somatic expression with AAV9-miR-222. Loss-of-function (LOF) was achieved using a locked nucleic antimiR (LNA) specific for miR-222. miR-222 GOF and LOF models manifested normal cardiac structure and function at baseline. After transverse aortic constriction (TAC), TgC-miR-222 had less pathological hypertrophy as well as better cardiac function (FS = 37.9% vs 28.7%, p<0.01) and survival (median survival = 120 days vs 54 days, p<0.05). Similarly, AAV9-miR-222 gene transfer immediately after TAC also better preserved cardiac function. Conversely, LNA-anti-miR-222 dramatically exacerbated pathological hypertrophy, cardiac dysfunction, and heart failure (FS = 24.4% vs 35.3%, p<0.01) after TAC. PUMA, a pro-apoptotic Bcl-2 family member and two transcription factors, Hmbox1 and NFATc3, were identified as novel, direct miR-222 targets contributing to its roles in this context.

Conclusions:
Although miR-222 is upregulated in both physiological and pathological hypertrophy, its roles in these conditions are quite different. In response to pressure overload, miR-222 inhibits pathological hypertrophy, adverse remodeling, and cardiac dysfunction. These findings reinforce the conceptual model that physiological and pathological hypertrophy are qualitatively distinct, rather than differing only in degree. Further, they suggest that miR-222 may hold promise as a potential therapeutic target in pathological cardiac hypertrophy and heart failure.
Abstract 341: Prolonged Post-Differentiation Culture Influences the Biophysics of Na
+
- And Ca
2+
Channels in Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Authors: Ross Gracious , Emelyanova Larisa , Rizvi Farhan , Tajik A Jamil , Jahangir Arshad
Published: February 2019

Background:
Cardiomyocytes (CM), derived from human induced-pluripotent stem cells (iPSC-CM), are promising tools for
in vitro
proarrhythmia screening for drug safety. However, among various challenges with interpretation of iPSC-CM experimental results, the influence of varied culture periods post-differentiation on the electrophysiological properties of iPSC-CMs is unclear.

Objective:
To test the hypothesis that prolonged post-differentiation culture of iPSC-CMs will influence the biophysical characteristics of Na
+
- and Ca
2+
-channels.

Methods:
Using whole-cell patch clamp, we compared the electrophysiological properties of differentiated ventricular-like iPSC-CMs (APD
30-40
/APD
70-80
>1.5) cultured for 10 to 15 days (D10-15) to those cultured for more than 30 days (≥ D30) both under current- and voltage- clamp configurations. Quantitative real-time PCR was used to quantify the mRNA expression of voltage-gated Na channel subunits SCN1A, SCN5A and Ca channel subunits CACNA1C, CACNA1G and CACNA1I. Fold change was calculated by 2
-ΔΔCt
method. Data were analyzed by Student’s t test, and
P
value <0.05 was considered significant.

Results:
Post-differentiation prolonged culture imparts increased cellular excitability with high frequency spontaneous action potentials, robust increase in the magnitude of peak Na
+
-current level, relatively shallow inactivation kinetics of Na
+
channels, faster recovery from inactivation, and augmented Ca
2+
current density compared to early cultured iPSC-CM. This was associated with a greatly enhanced mRNA expression of α-subunit transcripts of SCN1A (7.5 fold) and SCN5A (38 fold) Na channel subtypes while less robust elevation in the mRNA expression of CACNA1C (0.5 fold), CACNA1G (2 fold) and CACNA1I (2 fold) subunits in prolonged culture compared to early cultured iPSC-CM.

Conclusion:
Prolonged culture of ventricular-like iPSC-CMs affects the excitability, single-cell electrophysiological properties and differential ion channels subunit expression. Standardization of post-differentiation period for ventricular-like iPSC-CMs culture is crucial for
in vitro
proarrhythmia screening, health/disease modelling to study cellular mechanisms or test high-throughput drugs’ efficacy and toxicity.
Abstract 346: A Standard Flow Cytometry Protocol for Assessing Human Pluripotent Stem Cell-derived Cardiomyocyte Identity by Troponin Positivity
Authors: Waas Matthew , Weerasekera Ranjuna , Kropp Erin M , Romero Marisol , Poon Ellen , Boheler Kenneth R , Burridge Paul W , Gundry Rebekah L
Published: February 2019

Directed differentiation of human pluripotent stem cells (hPSC) into cardiomyocytes (hPSC-CM) offers an inexhaustible supply of cells for basic science research and translational applications. However, despite significant advancements in defining the factors most critical for differentiation, the resulting cultures remain a heterogeneous mixture of cells. Ultimately, as heterogeneity can pose challenges to interpreting functional data, the ability to accurately and precisely assess cell identity in differentiation cultures is paramount to well-defined and reproducible studies. To date, a standardized flow cytometry protocol that is broadly accepted among laboratories has not been established for assessing cell type heterogeneity within hPSC-CM cultures, posing challenges to evaluating outcomes generated among laboratories and studies. A survey of studies published over the past seven years (1/2010-10/2017) reveals the wide range of antibodies and experimental conditions reported for flow cytometry-based assessment of hPSC-CM. Although our literature survey revealed that a preponderance of studies relied on TNNT2 as a marker of cardiomyocyte identity, TNNI3 is more specific to cardiomyocytes than TNNT2 throughout development. We applied targeted mass spectrometry to confirm the presence of TNNI3 in our hPSC-CM. We then investigated five commercially available anti-TNNI3 and three sample preparation techniques for use in the assessment of heterogeneity of hPSC-CM cultures by flow cytometry. Results demonstrate two of the five anti-TNNI3 clones appear suitable for marking hPSC-CM and reveal differential susceptibility of antibody clones to sample preparation conditions. To further test the rigor and utility of our flow cytometry protocol, it was shared with two collaborating laboratories and applied to heterogeneous mixtures of positive and negative cell types. Results from these two laboratories demonstrate the protocol successfully distinguishes positive and negative cell types similarly, despite using different cell lines and differentiation protocols. We propose a workflow for establishing the fit-for-purpose use of antibodies and a standard protocol for assessing hPSC-CM identity by troponin positivity.
Abstract 362: Aortic Valve Hamartoma: A Rare Valvular Tumor Mimicking Infective Endocarditis
Authors: Salagundla Nethuja , Merza Nooraldin , Asm Islam , Masoud Alzeerah , Alsharif Muath
Published: February 2019

A 46 YO female with PMH of DM2, HTN, Morbid obesity presented to hospital with chief complaint of chest pain, fever and SOB. She had recent hospitalization and was managed for abdominal wall abscess, MRSA bacteremia and diagnosed with infective endocarditis with aortic valve vegetation by TEE, and was discharged with antibiotics. With her current presentation: Temp was 100.8F, blood cultures grew Klebsiella Oxycota, and on TTE there was concern of increased size of aortic valve vegetation with super infection. Repeat TEE showed increase in size of vegetation. CT surgeon suggested AVR with mechanical valve. Biopsy results of the aortic valve came back as Lipomatous hamartoma. Patient recovered well post procedure and antibiotics were discontinued. Discussion: Prevalence of valvular hamartoma is as rare as 0.01 to 0.02% contributing to low diagnosis rates. If patient does not have symptoms of inflammation or infection and does not meet clinical diagnostic criteria, tumors should be included in the differential as medical management is not appropriate for hamartomas and other valvular tumors, which needs urgent surgery to prevent life threatening embolization. Cardiac valve lipomatous hamartomas are very rare lesions with preferential localization on the pericardium followed by endocardial surface. Most common histologic type of cardiac valve tumor is papillary fibroelastoma, myxoma, fibroma and hamartoma. Even if they are asymptomatic, surgical treatment should be considered to prevent embolization to brain and coronary circulation. To the best of our knowledge, we are reporting the second case of native aortic valve hamartoma so far.