bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 158: Assessing the Paracrine Hypothesis in Cardiac Regeneration Through Endogenous c-Kit+ Cells
Authors: Vagnozzi Ronald J , Maillet Marjorie , Sargent Michelle A , Molkentin Jeffery D
Published: July 2021

Multiple studies have shown an innate capacity of the adult mammalian heart to partially regenerate following stress or injuries such as myocardial infarction (MI). However, this modest capacity is insufficient to resolve the significant damage that occurs post-MI, thus efforts have centered on bolstering this regenerative potential by delivery of various stem or progenitor cell types. To-date, over fifty clinical trials have evaluated cell-based therapeutics to induce regeneration in the setting of ischemic heart disease - mostly using bone marrow mononuclear cells (BM-MNCs) although c-Kit
+
cardiac progenitor cells (CPCs) and mesenchymal stromal cells (MSCs) from bone were also evaluated. Collectively, cell therapy has demonstrated clinical safety; yet overall effectiveness has been questioned, as evidence that transplanted cells differentiate into cardiomyocytes is lacking. Hence, studies showing some functional benefit have ascribed it to enhanced endogenous regeneration via paracrine factors secreted by the implanted cells. One hypothesis is that these factors stimulate resident c-Kit
+
progenitors to generate new myocardium. We recently developed an inducible genetic system to trace endogenous c-Kit
+
cells in the murine heart. We now use this system to evaluate whether delivery of different progenitor-like cell types can enhance c-Kit
+
-derived myocyte or vessel formation in the uninjured heart; using a dual-reporter strategy to differentially fate map transplanted cells versus endogenous c-Kit
+
cells. We observed that BM-MNCs induced a modest increase in c-Kit
+
-derived, CD31
+
vessels proximal to the site of injection, although no new myocytes were observed. There was no evidence for fusion between BM-MNCs and host myocardium, or for prolonged engraftment, as by 6 weeks virtually all injected cells were cleared. Taken together, this suggests that BM-MNCs are capable of inducing an acute c-Kit
+
-mediated vascular response in the uninjured heart. We are also currently evaluating CPCs, bone marrow or adipose-derived MSCs, and bone stromal cells for their capacity to stimulate c-Kit
+
cells, either without injury or after MI. Our goal is to better define the paracrine hypothesis, improving cell-based therapies for application in humans.
Abstract 166: Qiliqiangxin Attenuates Doxorubicin-induced Cardiomyopathy via Regulating Autophagy
Authors: Shen Shutong , Tao Lichan , Wu Xiaoting , Xiao Junjie , Li Xinli
Published: July 2021

Aims:
Doxorubicin-induced cardiomyopathy, as a common complication of cancer chemotherapy, greatly limits the clinical implication of doxorubicin in the treatment of cancer. Our previous clinical trial and animal studies have shown that Qiliqiangxin, a traditional Chinese medication, was protective for adverse cardiac remodeling. This study aimed at determine the therapeutic effect of Qiliqiangxin in mouse doxorubicin-induced cardiomyopathy.

Methods and results:
Three groups of mice (control, n=6; doxorubicin+saline, n=7; doxorubicin+Qiliqiangxin, n=7) were followed up for 4 weeks. Echocardiography was taken at the end of 4 weeks to evaluate cardiac function. Mice from the doxorubicin+saline group showed reduced ejection fraction (EF) and fractional shortening (FS) as compared with the mice from the control group. Qiliqiangxin could partly restore the reduction of EF and FS in doxorubicin-induced cardiomyopathy mice. Genes related with autophagosome formation (becline1, ATG4, ATG7, LC3) and autophagosome-lysosome fusion (Cathepsin B, Cathepsin D, Cathepsin L) were determined by quantitative reverse transcription polymerase chain reactions (RT-PCRs) in heart samples from mice of the three groups (control, doxorubicin+saline, doxorubicin+Qiliqiangxin) at the end of 4 weeks. All these 7 genes were found to be increased in doxorubicin-induced cardiomyopathy mice, implying doxorubicin may disturb the autophagy flux of mice hearts through promoting autophagosome formation and inhibiting autophagosome-lysosome fusion. Interestingly, the expression of these 7 genes were decreased in the doxorubicin+Qiliqiangxin group, implying that Qiliqiangxin may protect the cardiac function in doxorubicin-treated mice through regulating the autophagy flux of mice hearts. Moreover, autophagy-related proteins LC3 and P62 were determined by western blotting. Qiliqiangxin reversed the increase of LC3II level and the decrease of P62 level in the doxorubicin-treated mice hearts.

Conclusion:
Qiliqiangxin may protect the cardiac function of doxorubicin-induced heart failure mice through regulating autophagy in the heart. Qiliqiangxin might be a novel drug for doxorubicin-induced cardiomyopathy.
Abstract 168: Drp1 Accumulates in Mitochondria and Plays a Protective Role in the Heart in Response to Pressure Overload
Authors: Shirakabe Akihiro , Ikeda Yoshiyuki , Saito Toshiro , Zhai Peiyong , Sadoshima Junichi
Published: July 2021

Dynamin-related protein 1 (Drp1) plays an essential role in maintaining the quality control of mitochondria through mitochondrial (Mt) fission and mitophagy. We investigated how Mt function, autophagy and Drp1 are regulated in the heart during pressure overload (PO) and whether endogenous Drp1 plays an important role in regulating cardiac function. Mice were subjected to transverse aortic constriction (TAC) at multiple time points between 6 hours and 30 days. Left ventricular (LV) weight/tibial length (LVW/TL) was significantly elevated at Day 5 (TAC vs Baseline; 6.21 ± 0.28 vs 4.59 ± 0.36, p<0.05). Ejection fraction (EF) was maintained at Day 5 (79±5 vs 82±7%), but gradually decreased thereafter (30 days; 51±12%, p<0.05). LC3-II was decreased (-40.0%, p<0.05) while p62 accumulated (1.84 fold, p<0.05) significantly at Day 5. Both Mt ATP content (-65.6%, p<0.05) and production (-90.3%, p<0.05) were reduced significantly at Days 7 and 14, respectively, and thereafter. Mt mass, evaluated by electron microscopy, was also reduced (-19.9%, p<0.05) at Day 7. Drp1 accumulated in mitochondria at Day 7, and S616 phosphorylation of Drp1, associated with increased activity, was increased at Day 7. Thus, PO suppresses autophagy and induces Mt dysfunction by Day 7, at which time Drp1 accumulates in mitochondria and Mt mass is decreased. To examine the functional significance of endogenous Drp1 during PO, cardiac-specific heterozygous Drp1 knock out (Drp-hetCKO) mice were subjected to TAC. At Day 7, decreases in EF (57± 11 vs 80 ± 7%, p<0.05) and increases in LVW/TL (7.22 ± 0.26 vs 5.86 ± 0.65, p<0.05) and lung weight/TL (13.03 ± 1.09 vs 7.00 ± 1.31, p<0.05) were exacerbated in Drp-hetCKO compared to in control mice. LV end diastolic pressure was significantly higher (20.0 ± 5.7 vs 7.4 ± 3.1 mmHg, p<0.05) and myocardial fibrosis (14.1 ± 2.5 vs 6.2 ± 4.3 %, p<0.05) was greater, and Mt mass was also significantly greater in Drp-hetCKO than in control mice (relative Mt mass, 1.21 ± 0.46 vs 1.00 ± 0.02, p<0.05). These results suggest that PO inhibits autophagy and induces mitochondrial dysfunction by Day7, which coincides with Mt accumulation of Drp1. Drp1 plays an adaptive role in this condition, mediating decreases in Mt mass and protecting the heart from dysfunction.
Abstract 178: Blocking the Acetylcholine Activated Inward Rectifier Potassium Current but not the Rapid Delayed Rectifier Potassium Current Restores Sinus Rhythm in the Chronically Fibrillating Sheep Heart
Authors: Haburcak Marian , Bassil Guillaume , Takemoto Yoshio , Mehlenbacher Matthew , Slough Diana , Bou Abdallah Fadi , Jalife Jose , Karas Richard , Lin Yu-Shan , Noujaim Sami F
Published: July 2021

Background:
The acetylcholine activated inward rectifier potassium current (IKACh) was shown to be constitutively active in chronic atrial fibrillation. Its blockade has been proposed to be a possible antifibrillatory pharmacotherapy. We therefore tested the hypothesis that blocking IKACh with the bee venom peptide tertiapin, or the small molecule chloroquine, terminates chronic AF in the sheep heart.

Methods and Results:
We tested our hypothesis using biochemistry, electrophysiology and molecular modeling. In patch clamp, the IC50 of IKACh block by tertiapin, and chloroquine was 60 nM, and 710 nM respectively, while dofetilde, a currently used class III antiarrhythmic, did not block the current. On the other hand, dofetilide and chloroquine blocked the rapid delayed rectifier potassium current (IKr) with an IC50 of 50nM, and 2.3 microM respectively, while tertiapine had no effects. Furthermore, molecular modeling indicated that 1 chloroquine molecule blocks the intracellular ion permeation vestibule of the tetrameric Kir3.1, a molecular correlate of IKACh, by interacting with amino acids important for the channel’s rectification. Dofetilide did not interact with Kir3.1. In vitro fluorescence measurements of chloroquine titration into the purified Kir3.1 intracellular domain protein confirmed the computational results and indicated that chloroquine directly binds Kir3.1 with a stoichiometry of 1 chloroquine molecule per Kir3.1 tetramer. Finally, optical mapping of the chronically fibrillating sheep atria showed that while tertiapine, chloroquine, and dofetilide slowed down chronic AF and prolonged the action potential, only tertiapine and chloroquine (the IKACh blockers) restored normal sinus rhythm.

Conclusions:
IKACh could be a more powerful anti atrial fibrillation target compared to IKr. Pharmacological blockade of IKACh with a peptide, or a small molecule similar to chloroquine could terminate chronic AF and restore normal sinus rhythm.
Abstract 186: Increased STIM1 Expression Results in Altered Calcium Handling and Heart Failure
Authors: Correll Robert N , Goonasekera Sanjeewa A , van Berlo Jop H , Burr Adam R , Accornero Federica , York Allen J , Sargent Michelle A , Houser Steven R , Molkentin Jeffery D
Published: July 2021

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor that partners with Orai1, resulting in store-operated Ca2+ entry (SOCE) that is important for maintaining endoplasmic reticulum (ER) Ca2+ homeostasis. STIM1 is expressed in the heart and upregulated during disease, but its role in disease progression is unclear. In this study we used transgenic mice with STIM1 overexpression in the heart to model the known increase of this protein in response to cardiac disease. We found that STIM1 transgenic myocytes showed elevated Ca2+ entry following store depletion and STIM1 co-localized with the type 2 ryanodine receptor (RyR2) in the sarcoplasmic reticulum (SR). In addition, STIM1 transgenic mice exhibited sudden cardiac death as early as 6 weeks of age, while mice that survived past 12 weeks developed cardiac hypertrophy that progressed to heart failure, pulmonary edema, activation of the fetal gene program, alterations in mitochondrial structure, and reduced ventricular functional performance. When pre-symptomatic STIM1 transgenic mice were subjected to disease stimuli including pressure overload stimulation or neurohumoral agonist infusion, they showed greater pathology compared to control mice. STIM1 elevation also disrupted normal Ca2+ handling in cardiac myocytes, which showed spontaneous Ca2+ transients that could be inhibited by the SOCE blocker SKF-96265, as well as increased diastolic Ca2+ levels and elevated Ca2+ spark frequency. In keeping with this increase in Ca2+ cycling we also found that STIM1 elevation resulted in an increased baseline activity of cardiac nuclear factor of activated T-cells (NFAT) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). This increased CaMKII activity did not, however, translate into additional RyR2 phosphorylation, suggesting that the augmented Ca2+ spark frequency observed was likely due to an elevation in SR Ca2+ load. Our results suggest that increased STIM1 expression elicits augmented Ca2+ entry, SR Ca2+ load and Ca2+ spark frequency, that leads to mitochondrial pathology and the induction of Ca2+ sensitive hypertrophic signaling pathways that contribute to cardiac disease.
Abstract 193: RNA-sequencing of Disease-specific iPSC as a New Filter to Identify Genes Associated With Hypoplastic Left Heart Syndrome
Authors: Martinez Fernandez Almudena , Li Xing , Theis Jeanne L , Terzic Andre , Olson Timothy M , Nelson Timothy J
Published: July 2021

Hypoplastic Left Heart Syndrome (HLHS) is a complex multifactorial disease for which no definitive genetic causes have been found. Current genetic filtering strategies render lists of genes with unknown relevance in terms of pathogenesis. A complementary filter based on biological evidence would create a new approach to prioritize relevant candidate genes and mutations.

In our study, 5 members of a nuclear family including a child with HLHS were evaluated using echocardiography and their genetic information was obtained through whole genome sequencing (WGS). Data filtering including rarity, functional impact and mode of inheritance was implemented, resulting in identification of 34 genes with recessive or
de novo
variants potentially involved in the pathogenesis of HLHS.

Additionally, iPSC were derived from proband and parents and subjected to RNA-sequencing at the undifferentiated state and following spontaneous differentiation. Comparative transcriptional analyses identified genes differentially expressed in proband samples at each stage. These gene sets were used as an additional filter for the previously generated WGS data. This strategy revealed that out of 34 mutated genes originally identified, 10 displayed transcriptional differences in undifferentiated iPSC from the HLHS-affected individual while 16 out of 34 mutated genes showed significantly different expression levels in differentiated cells from proband. Furthermore, expression dynamics were studied during guided cardiac differentiation for the 9 genes fulfilling all applied criteria. Two genes not previously linked to HLHS, ELF4 and HSPG2 were found to behave significantly different in HLHS-iPSC when compared to control counterparts.

In summary, filtering WGS data according to a new layer of transcriptional information that leverages iPSC plasticity allows prioritization of genes associated with HLHS in an in vitro model of disease.
Abstract 197: Selective Destruction of Adrenergic Cells in Mice Leads to Severe Left-ventricular Dysfunction at Rest With Apparent Stress-induced Recovery
Authors: Owji Aaron P , Baker Candice N , Jacob Jeffrey , Ebert Steven N
Published: July 2021

The catecholamines epinephrine and norepinephrine play critical roles in the maintenance of cardiovascular function. Phenylethanolamine-N-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine to epinephrine and serves as a marker for adrenergic cells. We have previously shown that the selective destruction of Pnmt+ cells in the mouse produces severe left-ventricular dysfunction under anesthesia and that epinephrine deficiency alone does not recapitulate the phenotype. Here, we test the hypothesis that Pnmt+ cells are key modulators of the stress response to immobilization. Using a suicide reporter mouse model to ablate Pnmt+ cells (Pnmt-Cre/DTA), we achieve greater than 50% Pnmt+ cell reduction in the adrenal medulla and 97% reduction in Pnmt transcript. Remarkably, Pnmt+ cell destruction does not markedly diminish the cardiovascular response to restraint stress. At one hour of immobilization, heart rate and ejection fraction showed a similar increase in response to restraint in both groups, despite apparent heart failure in Pnmt-Cre/DTA mice at rest (Figure 1). Here, we show that, while Pnmt+ cells are required for the maintenance of basal cardiovascular function, loss of these cells does not diminish the cardiovascular response to restraint stress. These results suggest that left ventricular cardiac muscle has the capacity to respond to stress despite hypokinetic and poor ventricular function at baseline.
Abstract 203: Cardiomyocyte T-tubule Membrane Turns Over, Releasing BIN1 Containing Microparticles into Blood
Authors: Xu Bing , Fu Ying , Hong TingTing
Published: July 2021

Bridging integrator 1 (BIN1) is a cardiac muscle protein that folds cardiomyocyte T-tubule membrane. BIN1 is intrinsic to cardiac health, and is reduced in acquired heart failure. Interestingly, we have found that BIN1 is also blood available, and that plasma BIN1 correlates with cardiac function, suggesting cardiac origin of plasma BIN1. We found that low plasma BIN1 correlates with failing muscle and predicts ventricular arrhythmia. However, the paradigm does not exist for an intracellular membrane associate cardiomyocyte protein to be homeostatically turned over into blood. In this study, using a mouse model with cardiac specific deletion of
Bin1
gene, we identified with biochemical techniques that plasma BIN1 levels directly correlate with cardiac tissue BIN1 levels, indicating cardiac origin. Furthermore, investigations using both super-resolution fluorescent imaging and flow cytometry analysis revealed that adult ventricular cardiomyocytes constantly release BIN1 into blood via membrane microparticle production. Microparticles are small membrane vesicles shed from plasma membrane of a variety of cell types including platelets, leukocytes, and endothelial cells. Using super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we found similar to the blood cells, isolated adult mouse cardiomyocytes release Annexin V positive microparticles with diameters ranging between 0.1 to 1.0 μm. These microparticles also carry BIN1 protein. Flow cytometry was also used to detect and quantify microparticles <1.0 μm in size from medium bathing a pure population of adult mouse cardiomyocytes. BIN1 microparticle release is proportional to actin stability and amount of T-tubule membrane folds. Compared to wild type cardiomyocytes, microparticle release is significantly reduced from myocytes with heterozygous deletion of
Bin1
gene. These data indicate that cardiomyocyte membrane undergoes dynamic turnover, releasing T-tubule folds into blood as microparticles. Furthermore, plasma BIN1 can be used as a direct measure of cardiomyocyte health and reserve.
Abstract 206: microRNA-29b Contributes to Multiple Types of Muscle Atrophy
Authors: Li Jin , Cheng Liming , Bei Yihua , Zhou Qiulian , Yu Yan , Fu Siyi , Chen Lei , Xiao Junjie
Published: July 2021

Objective:
Skeletal muscle atrophy has been observed in a significant proportion of heart failure patients while little is known about the mechanisms underlying muscle atrophy, especially in multiple types of muscle atrophy. MicroRNAs (miRNAs, miRs) are among key participants in gene regulatory networks and their dysregulation contribute to lots of diseases. However, miRNAs responsible for multiple types of muscle atrophy are unclear.

Methods and Result:
miRNAs were screened by miRNA arrays and were further verified by real-time reverse transcription polymerase chain reaction with the gastrocnemius sample from denervation -induce muscle atrophy. miR-130b, miR-212, miR-21, miR-221/222, miR-223 and miR-29b were upregulated >3 fold in gastrocnemius samples from denervation mice than that of sham group. However, only miR-29b presented a common change (upregulated >3) in other types of muscle atrophy models including dexamethasone-induced and fasting-induced muscle atrophy. Over-expression of miR-29b in gastrocnemius by intramuscular injection with agomiR-29b significantly promoted muscle atrophy, which is evidenced by the change in morphology (the diameter of myofiber was decrease by 60%) and increased atrophy-related genes including Atrogin-1 and MuRF-1 (upregulated for 1.5 folds and 1.4 folds, respectively). This in vivo data suggest that miR-29b is sufficient to induce muscle atrophy. In multiple types of muscle atrophy C2C12 cell models (dexamethasone, TNF-alpha and H2O2 induced), miR-29b was increased while inhibition of miR-29b could prevent that effects. Using TargetScan software, we found that IGF1 and PI3Ka were potential target genes for miR-29b, which was further validated by luciferase assays and western blotting. Moreover, over-expression of IGF1 or PI3Ka ameliorated muscle atrophy caused by miR-29b overexpression, indicating that IGF1 and PI3Ka are two target genes responsible for miR-29b’s pro-atrophy effects in muscle.

Conclusions:
Our data indicated that via targeting IGF1 and PI3Ka. Inhibition of miR-29b might represent a novel therapy for multiple types of muscle atrophy including in heart failure.
Abstract 207: Left Ventricular Assist Device (LVAD) Reverses Inhibition on Beta-adrenergic Receptor Resensitization
Authors: Martelli Elizabeth E , Chatterjee Arunachal , Starling Randall , Moravec Christine , Naga Prasad Sathyamangla V
Published: July 2021

Beta-adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Agonist occupied βAR undergo desensitization through phosphorylation by G-protein coupled receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by protein phosphatase 2A (PP2A) in the endosomes and we have shown previously shown that PP2A activity is regulated by phosphoinositide3-kinaseγ (PI3Kγ). Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and an integral contributor to heart failure. To test whether resensitization mechanisms are altered in heart failure and could play a role in recovery of βAR function post-LVAD, we used paired pre- and post-LVAD human heart samples along with non-failing hearts. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from these human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of recovery in βAR function by G-protein coupling. Significant PI3Kγ activity was observed in the endosomes of pre-LVAD compared post-LVAD and non-failing human heart samples while plasma membrane PI3K activity remained similar across all samples. Similarly, AC activity was markedly reduced in plasma membranes and endosomes of pre-LVAD samples compared to post-LVAD and non-failing showing reduced ability of receptors to couple to G-proteins indicating reduced recovery of receptor function in post-LVAD heart samples. Consistent with the reduced recovery in βAR function in pre-LVAD samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of pre-LVAD compared to post-LVAD and non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in the pre-LVAD samples which was remarkably reversed in post-LVAD samples similar to the activity in non-failing. These studies suggest that resensitization is inhibited in end-stage human heart failure and may critically contribute to cardiac remodeling and hypertrophic response upon cardiac stress.
Abstract 216: Genetic Deletion of Cardiomyocyte Mineralocorticoid Receptors Prevents Cardiac Dysfunction and Premature Death in Mice Lacking Glucocorticoid Receptors in the Heart
Authors: Oakley Robert H , Cruz-Topete Diana , Foley Julie F , Myers Page H , Chambon Pierre , Willis Monte S , Cidlowski John A
Published: July 2021

Heart failure is one of the leading causes of death in the Western world, and stress is increasingly associated with adverse cardiac outcomes. Glucocorticoids are primary stress hormones, but their direct role in heart physiology and pathology is poorly understood. The actions of glucocorticoids are mediated classically by the glucocorticoid receptor (GR); however, in cardiomyocytes glucocorticoid occupancy and activation of the mineralocorticoid receptor (MR) may also contribute to the observed glucocorticoid response. To elucidate the
in vivo
function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO). The cardioGRKO mice spontaneously develop cardiac hypertrophy by 2 months of age and left ventricular systolic dysfunction and dilatation between 3-6 months of age, and they die prematurely from heart failure. The mean survival age of the cardioGRKO mice was 9.3 months, and 97% (38 of 39) of the mice died prior to reaching 12 months of age. Loss of GR in the heart was not accompanied by an increase in fibrosis. To investigate whether cardiomyocyte MR signaling contributes to the pathology in the cardioGRKO mice, we generated for the first time mice lacking both GR and MR in the heart (cardioGRMRKO). Despite showing increased expression of classic hypertrophic marker genes, the cardioGRMRKO mice were protected from the development of cardiac hypertrophy, and no major alterations were observed in the function and chamber size of the left ventricle. Moreover, the cardioGRMRKO mice were protected from premature death as 94% (15 of 16) of the mice survived past the 13 month study endpoint. Microarray analysis revealed marked differences in gene expression profiles between the cardioGRKO and cardioGRMRKO hearts. These findings reveal that cardiomyocyte MR signaling, when unopposed by GR signaling, plays a major role in the progression of cardiac disease. Moreover, they suggest that combining GR agonists with MR antagonists may represent an improved therapeutic approach for treating heart failure.
Abstract 225: Pulmonary Hypertension Induced Right Ventricle Fibrosis is Associated With Male Gender
Authors: Rafikova Olga , Rafikov Ruslan , Meadows Mary L , Kangath Archana , Black Stephen M
Published: July 2021

Pulmonary hypertension (PH) is a rare but incurable disease. The primary reason of PH mediated mortality is right ventricle (RV) failure. There is a well-established sexual dimorphism in regard to PH, with females generally associated with higher susceptibility to develop PH, while males possess lower survival rate. We hypothesized that sex difference in PH mediated mortality is associated with sex difference in mechanisms of RV failure development, with males being more prone to develop RV dysfunction. The angioproliferative PH was induced in 8 week old male and female rats by bolus injection of VEGF receptor 2 antagonist, SU5416 (20 mg/kg, s.c.) followed by 4 weeks of exposure to hypoxia (10%±0.5 O2) and 10 weeks of normoxia (21% O2). We found that PH induced a significantly higher mortality in males comparing to females, although the increase in RV peak systolic pressure (RVPSP) was similar in both genders (95.8±13.5 vs. 84.6±18.2; p=0.636; N=5). At the early stage of PH (4 weeks) both males and females had possessed the similar levels of PH induced increase in RV free wall (RVFW) thickness, assessed by Doppler echocardiography. However, starting from 8 week RV hypertrophy continues to progress in males only, while RVFW thickness in females was found to be preserved (RVFW at 12 week, mm: 2.1±0.2 vs. 1.2±0.1; p=0.003; N=5-6). After 14 weeks of study there was a significant impairment of RV relaxation and RV diastolic function in male group only (RV peak diastolic pressure (RVPDP), mmHg: -0.6±0.9 vs. -5.4±0.9; p=0.008; N=4-5). Finally, Masson’s trichrome staining revealed severe fibrosis in the RV of male, but not female rats with PH (fibrotic score: 2.5±0.3 vs. 0.7±0.2; p=0.0007; N=5-6), which was associated with endothelial nitric oxide synthase (eNOS) uncoupling, assessed by measuring caveolin expression (fold male Cont: 0.3±0.003 vs. 1.9±0.3; p=0.002; N=4) and eNOS dependent increase in superoxide generation. We conclude that males with PH have a high risk of mortality due to increased cardiac fibrosis development, which, in turn, leads to RV failure. The severe oxidative stress with subsequent injury of myocardial tissue may be responsible for RV fibrotic changes in males, which are lack of female sex hormones known to exert strong anti-oxidant protection.
Abstract 235: Sarcolemmal Membrane Associated Protein Isoform 1: a Unique Regulator of Glucose Uptake and Metabolism in the Myocardium
Authors: Dewan Aaraf , Salih Maysoon , Triggle Christopher , Ding Hong , Tuana Balwant
Published: July 2021

As one of the leading causes of heart disease, diabetes is a problem which needs a solution. Regulation of glucose uptake and metabolism within skeletal and cardiac muscle has proven capable of altering systemic glucose levels and impacting metabolism to potentially improve patient outcomes. Unfortunately, to date, very few muscle specific metabolic regulators are known which can allow us to achieve blood glucose uptake and metabolism. Sarcolemmal Membrane Associated Protein Isoform 1 (SLMAP1) is a novel protein expressed predominantly within muscle tissue. It has been linked to diabetes through animal models, although its role in metabolism remains to be defined. Here we describe a novel role for SLMAP1 in glucose metabolism within the myocardium. We engineered a transgenic (TG) mouse with cardiac specific expression of SLMAP1. Using neonatal cardiomyocytes (NCMs) collected from these mice we performed glucose uptake assays with 2-deoxy-glucose (2DG), measured glycolytic rate using an Extracellular Flux XF24e Bioanalyzer, and analyzed glucose transporter 4 (GLUT4) trafficking using Western Blots, qPCR, and immunofluorescence imaging. NCMs extracted from TG hearts expressing SLMAP1 displayed increased levels of 2DG uptake (93% ± 25%, n=5, P<0.01), basal glycolysis (glycolysis (92 ± 40%, n=5, P<0.05), and maximal glycolysis (75 ± 31%, n=5, P<0.05) compared with wildtype littermates. Confocal microscopy revealed both increased localization of glucose transporter 4 (GLUT4) at the cell surface as well as an expansion of GLUT4 early endosomes in TG NCMs. The data here indicates SLMAP1 as a novel regulator of glucose uptake and metabolism in the myocardium. The targeted expression of SLMAP1 in a muscle specific manner may enhance systemic glycemic control and serve to limit cardiovascular disease in metabolic disorders such as diabetes.
Abstract 245: Assessing Proarrhythmic Potential of Drugs Using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes and Optical Recordings With Voltage-sensitive Dyes
Authors: Blinova Ksenia , Stohlman Jayna , Chan Dulciana , Hortigon Maria , Rodriquez Victor Z , Smith Godfrey , Crumb William , Pang Li , Lyn-Cook Beverly , Vicente Jose , Johannesen Lars , Stockbridge Norman , Stockbridge Norman , Strauss David
Published: July 2021

Background:
Drug-induced arrhythmias have been a leading reason for withdrawing drugs from the market and abandoning the development of new drugs. For the past decade FDA has required new drugs to be tested for block of the hERG potassium channel and QT interval prolongation. While these requirements have prevented arrhythmia-related drug recalls, some new drugs affecting multiple ion channels are being dropped from development inappropriately. Human induced pluripotent stem cell derived cardiomyocytes (iPS-CMs) are a new technology for preclinical risk assessment; however they have not been fully characterized or validated.

Methods:
We studied 26 drugs and 3 drug combinations that block multiple cardiac ion channels with two commercially available iPS-CM lines from Axiogenesis and Cellular Dynamics using optical recordings of action potentials with voltage-sensitive dyes. Drug-induced action potential duration (APD) prolongation was compared to clinical QT prolongation from two FDA-sponsored clinical trials. The effects of the drugs on multiple individual cardiac ion channels were assessed using manual patch clamp of overexpressed cell lines. Cardiac ion channel gene expression in the iPS-CMs was quantified and compared to primary human heart cell controls.

Results:
Of 19 drugs with an FDA label of clinical QT prolongation, 15 exhibited iPS-CM APD prolongation after acute drug exposure. None of the 6 drugs without clinical QT prolongation caused iPS-CM APD prolongation. Of 14 drugs with arrhythmia risk on the FDA label, nine caused arrhythmias in iPS-CMs. iPS-CM response had good correlation with clinical QT data for drugs that block hERG and calcium channels, while drugs blocking the late sodium current had variable response. This was in line with gene expression data, which showed most robust expression of hERG and calcium channels.

Conclusion:
Optical recordings from iPS-CMs with voltage sensitive dyes is a promising technology for high-throughput toxicity assessment for drug-induced arrhythmias. This study provides a comprehensive characterization of the cardiac ion channel properties of multiple commercially available iPS-CMs to support a potential new paradigm for assessing the arrhythmia risk of all new drugs.
Abstract 248: Aberrant TGFβ Signaling as an Etiology of Left Ventricular Non-compaction Cardiomyopathy
Authors: Kodo Kazuki , Ong Sang-Ging , Jahanbani Fereshteh , Termglinchan Vittavat , InanlooRahatloo Kolsoum , Ebert Antje D , Shukla Praveen , Abilez Oscar J , Churko Jared M , Karakikes Ioannis , Jung Gwanghyun , Snyder Michael P , Bernstein Daniel , Wu Joseph C
Published: July 2021

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and has a unique phenotype with characteristically extensive hypertrabeculation of the left ventricle, similar to the embryonic left ventricle, suggesting a developmental defect of the embryonic myocardium. However, studying this disease has been challenging due to the lack of an animal model that can faithfully recapitulate the clinical phenotype of LVNC. To address this, we showed that patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from a family with LVNC history recapitulated a developmental defect consistent with the LVNC phenotype at the single-cell level. We then utilized hiPSC-CMs to show that increased transforming growth factor beta (TGFβ) signaling is one of the central mechanisms underlying the pathogenesis of LVNC. LVNC hiPSC-CMs demonstrated decreased proliferative capacity due to abnormal activation of TGFβ signaling (Figs A-B). Exome sequencing demonstrated a mutation in TBX20, which regulates TGFβ signaling through upregulation of ITGAV, contributing to the LVNC phenotype. Inhibition of abnormal TGFβ signaling or genetic correction of the TBX20 mutation (Figs C-D) using TALEN reversed the proliferation defects seen in LVNC hiPSC-CMs. Our results demonstrate that hiPSC-CMs are a useful tool for the exploration of novel mechanisms underlying poorly understood cardiomyopathies such as LVNC. Here we provide the first evidence of activation of TGFβ signaling as playing a role in the pathogenesis of LVNC.
Abstract 251: Circulating Mir-21, -378, and -940 Increase in Response to an Acute Exhaustive Exercise in Patients With Chronic Heart Failure
Authors: Xu Tianzhao , Zhou Qiulian , Che Lin , Li Guanghe , Xiao Junjie
Published: July 2021

Background:
Exercise training is recommended as a useful intervention in cardiac rehabilitation for patients with chronic heart failure. There has been emerging evidence indicating that circulating microRNA (miRNA, miR) could mediate the beneficial effects of exercise in both healthy persons and patients with cardiovascular diseases. The current study was aimed to investigate the regulation of circulating miRNAs in response to an acute exhaustive exercise in patients with chronic heart failure (CHF).

Methods:
Twenty-eight CHF patients (mean age=59.07±1.79 years, 14 in NYHA Class II and 14 in NYHA Class III) performed a symptom-limited incremental cardiopulmonary exercise test on a bicycle ergometer according to a standardized exercise protocol of revised Ramp10 programs (acute exercise). Serum samples were collected before and immediately after exercise session. The cardiac or muscle specific/enriched miRNAs including miR-1, miR-133a, miR-133b, miR-499, miR-208a, miR-208b, miR-378, miR-486, and miR-940 were determined. In addition, other miRNAs involved in angiogenesis (miR-20a, miR-328, miR-126, miR-221), inflammation (miR-21, miR-146a, miR-155), and ischemia adaptation (miR-210, miR-21, miR-146a) were also investigated. Moreover, inflammatory and muscle damage markers including creatine kinase (CK), creatine kinase MB isoenzyme (CK-MB), troponin T (Tn-T), N-terminal pro-brain natriuretic peptide (NT-ProBNP) and high sensitive C reactive protein (hs-CRP) were determined by enzyme linked immunosorbent assays (ELISA).

Results:
Serum miR-21, -378, and -940 were found to be significantly increased in response to acute exercise in CHF patients. In addition, no robust correlation was identified between changes of these miRNAs and muscle damage or inflammation, indicating a distinct adaptation by miRNAs. However, a linear correlation between the change of miR-21 and VO2max was observed (R=0.42, P=0.03).

Conclusion:
Our data suggest that circulating miR-21, miR-378, and miR-940 might be potential biomarkers for the improvement of exercise capacity in patients with chronic heart failure.
Abstract 254: 14, 15-EET Protects Heart Pericytes by Delaying I/R Injury-induced Calcium Overloading
Authors: Cao Zhiping , Davis Catherine M , Kaul Sanjiv , Alkayed Nabil J
Published: July 2021

Background
- Pericytes are an important cellular component of the blood vessel wall of the arteries, arterioles, and microvessels of the heart; they provide structural integrity and regulate vessel diameter by contracting and relaxing dynamically in response to vasoactive stimuli. It has been suggested that pericytes contribute to coronary no-flow due to pericyte constriction following myocardial infarction, thus worsening outcome. It has also been demonstrated that intracellular calcium is involved in perciyte constriction. Our previous findings indicate that cardiomyocytes are protected following ischemia/ reperfusion injury (I/R) by the eicosanoid 14,15-EET. Since 14,15-EET is protective following I/R and a vasodilator, we tested the hypotheses that I/R injury induces calcium overloading, which injures peciytes, and that 14, 15-EET is able to block this process.

Methods and Results
- We isolated and cultured pericytes from the mouse heart ventricle by 3G5 antibody Dynabead sorting. Pericytes were characterized by multiple immunocytochemical markers for contractile proteins, cytoskeletal protein, and cell surface receptors (alpha-smooth muscle actin, calponin-1, NG2, vimentin, CD31,smoothlin, and fibroblast protein-1). Cultured pericytes were subjected to 5 hours of oxygen and glucose deprivation, with or without 14,15-EET, followed by 15 hours of re-oxygenation in the absence of 14,15-EET. Calcium imaging and cell death during re-oxygenation were assessed by Fluo-4 and propidium iodide respectively. Digital images were taken with confocal microscope (Nikon Eclipse Tie-A1RSi). The brightness of the green fluorescent signals represents the relative level of intracellular calcium and the red fluorescent signals represent the cell death. We found that calcium signal peak (overloading) occurred during re-oxygenation, immediately followed by cell death. This process was delayed by 14,15-EET treatment during oxygen and glucose deprivation. The cell death at 5h, 10h, and 15h of re-oxygenation was 57.2%, 71.1%, and 85.3% in control group, and 19.9%, 35.3%, and 58.3% in 14,15-EET treated group.

Conclusions
- Our data suggests that 14,15-EET-induced protection in pericytes is mediated through the calcium signaling pathway.
Abstract 257: Berbamine Postconditioning Protects the Heart From Ischemia/Reperfusion Injury Through the Regulation of Autophagy During Reperfusion
Authors: Zheng Yanjun , Li Xuxia , Wang Jinxi , Tan Jiliang , Zhang Caimei , Yang Huangtian
Published: July 2021

Myocardial infarction resulting from coronary atherosclerosis is the leading cause of death in modern society. Reperfusion is an essential treatment to salvage ischemia myocardium from necrosis, while it also leads to additional damage. Therefore, exploring effective medicines to protect the heart from post-ischemic injury is one of the major objectives of cardiovascular research. Berbamine, a nature compound of bisbenzylisochinoline alkaloids from Barberry, protects the heart from ischemia/reperfusion (I/R) injury when given before I/R, but it is unknown whether it has cardioprotective effects when given at the onset of reperfusion (postconditioning, PoC), a protocol with more clinical impact. Therefore, the present study was designed to determine whether berbamine PoC (BMPoC) is able to protect the heart from reperfusion injury by using perfused I/R rat hearts and simulated I/R cardiomyocytes. We found that BMPoC added during the first 5-min of reperfusion concentration-dependently improved post-ischemic myocardial function and limited infarcted area. A similar protection was observed in isolated rat cardiomyocytes characterized by the attenuation of I/R-induced depression of cell contraction and loss of mitochondrial membrane potential. Moreover, autophagy (an intracellular bulk degradation process for proteins and organelles) was significantly stimulated by myocardial I/R, but it was suppressed by BMPoC. Next, we examined how berbamine regulates autophagy and found that BMPoC inhibited the expression of beclin 1, a critical regulator for autophagosome initiation. Then, adenoviral overexpression and knockdown of beclin 1 in ventricles conformed its roles in the BMPoC-mediated cardioprotection. Further, Western blot analysis revealed that BMPoC-suppressed beclin 1 expression was mediated via the activation of PI3K/Akt signaling pathway. Our results suggest that BMPoC confers cardioprotection against I/R injury at least in part by the inhibition of beclin1-dependent autophagy through the activation of PI3K/Akt signaling pathway. These findings reveal new roles and mechanisms of berbamine in the cardioprotection against I/R injury.
Abstract 259: Xenon-loaded Echogenic Liposomes Provide Cardioprotection in Acute Myocardial Infarction
Authors: Yin Xing , Ren Yuping , Klegerman Melvin E , Moody Melanie R , Peng Tao , Kim Hyunggun , McPherson David D , Huang Shao-Ling
Published: July 2021

Background:
Acute myocardial infarction (AMI) is a leading cause of sudden cardiac death. Following AMI, left ventricular (LV) remodeling plays an important pathophysiologic role in progression to heart failure (HF). Effective therapeutic approaches to decrease the size of MI can reduce progression to HF. Xenon (Xe), a non-toxic, easily diffusible, noble gas can provide cardiovascular protection. However, delivery of Xe is challenging. Carrier delivery of Xe may be an effective way to deliver this gas to the ischemic area. We have developed Xe-loaded echogenic liposomes (Xe-ELIP) for intravascular delivery of the bioactive gas to the infarct area. Here, we investigate the effect of Xe-ELIP on MI size and LV remodeling in an AMI model.

Methods:
Xe-ELIP were prepared by a pressurization-freeze method. Rats were randomly divided into 4 groups; sham, AMI control, empty ELIP, and Xe-ELIP at 45 minutes following AMI. AMI was induced by ligating the left anterior descending artery. Liposomes were intravenously administrated immediately after reperfusion. Cardiac function was evaluated by ultrasound at baseline, after AMI, and on day 7. Infarct size was evaluated by TTC staining.

Results:
Gas chromatography-mass spectrometry demonstrated that 1 mg lipid contains 190 μl Xe. Xe-ELIP treatment decreased end-systolic LV volume (Fig. 1A), improved cardiac output, and reduced infract volume (Fig. 1B, 1C).

Conclusion:
This novel carrier system allows a therapeutic dose of Xe to reach the ischemic myocardium with resultant all preservation. This methodology can be easily translated for intracoronary injection of Xe-ELIP at the time of coronary intervention at AMI for cellular protection.
Abstract 264: Nitrite Therapy Ameliorates Myocardial Injury via H2S and Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2)-Dependent Signaling in Chronic Heart Failure
Authors: Islam Kazi N , Donnarumma Erminia , Bhushan Shashi , Lefer David J
Published: July 2021

Background:
Nitric oxide (NO) and hydrogen sulfide (H
2
S) are reduced in congestive heart failure. Recent studies suggest cross-talk between NO and H
2
S signaling. We previously reported that sodium nitrite (NaNO
2
) significantly ameliorates myocardial ischemia-reperfusion injury and heart failure. Nrf2 regulates the expression of antioxidant protein genes and is upregulated by H
2
S. We examined the effects of NaNO
2
therapy on endogenous H
2
S bioavailability and Nrf2 activation in mice subjected to ischemia-induced heart failure.

Materials and Methods:
Mice underwent 60 min. of left coronary artery occlusion and 4 weeks (WKS) of reperfusion. NaNO
2
(165 μg/kg) or saline vehicle (VEH) was administered at reperfusion and then in drinking water (100 mg/L) for 4 wks. Left ventricular ejection fraction (LVEF) was determined at baseline and 4 wks of reperfusion. Myocardial tissue was collected and analyzed for oxidative stress status and respective gene/protein levels.

Results:
NaNO
2
therapy preserved LVEF (47 ± 4% vs. 32 ± 4%, p < 0.01) and LV diastolic and systolic dimensions (LVEDD/LVESD; 4.0/3.1 mm vs. 4.5/3.9 mm, p < 0.05) at 4 wks. MDA and protein carbonyl contents were significantly reduced in NaNO
2
treated mice as compared to VEH. NaNO
2
markedly increased expression of CuZn-superoxide dismutase and catalase at 4 wks. Furthermore, NaNO
2
increased mRNA levels of H
2
S producing enzymes and H
2
S bioavailabilty. Cardiac Nrf2 activation was also observed with NaNO
2
therapy.

Conclusions:
Our results demonstrate that NaNO
2
therapy significantly improves left ventricular function via by increasing H
2
S bioavailability, activation of Nrf2, and increased antioxidant defenses.1
Abstract 266: Screening and Validation of Small Molecule Inhibitors of Serum and Glucocorticoid Regulated Kinase-1 as Novel Therapy for Cardiac Arrhythmia Disorders
Authors: Bezzerides Vassilios J , Simonson Bridget , Shanmugasundaram Kumaran , Phyllis Ottaviano , Lynch Stacey , Hessler Katherine , Milan David , Rigby Alan , Das Saumya , Rosenzweig Anthony
Published: July 2021

Alterations in sodium flux (INa) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. Recent data demonstrates a critical role for the serum and glucocorticoid regulated kinase-1 (SGK1) by modulation of INa in the heart, by regulating the voltage-gated sodium channel NaV1.5. To better understand and pharmacologically probe the significance of SGK1 in cardiac dysrhythmias, we have used computer aided drug discovery (CADD) to identify small molecule inhibitors of SGK1.

Expression of a constitutively active form of SGK1 (SGK1-CA) increased INa (1.7 fold, p <0.005) in a stable line of HEK cells expressing NaV1.5. Conversely, expression of a dominant negative form (SGK-DN) decreased NaV1.5 channel activity (2.8 fold, p <0.005). We examined the effects of SGK1 inhibition in a LQT model, by quantifying the ability of SGK1 inhibition to rescue the 2:1 AV block phenotype of the potassium channel zebrafish mutant, breakdance (bkd). Morpholino injection or expression of SGK1-DN significantly rescued the 2:1 AV block phenotype as compared to controls (p < 0.05).

Using CADD partnered with iterative empirical screens we identified several hit chemical scaffolds. Our lead compound inhibits the phosphorylation of the SGK1 target gene, GSK3-β in a dose dependent manner in cardiomyocytes (CMs) expressing SGK1-CA at the lowest effective concentration of 0.5μM. There was no significant inhibition of AKT dependent phosphorylation of GSK3-β up to a concentration of 50μM, demonstrating specificity of the inhibitor for SGK1. Incubation of bkd zebrafish mutants with the inhibitor rescued the 2:1 AV block in a dose dependent manner (60% rescue with 45μM, p < 0.05). Acute application of the inhibitor dramatically inhibited INa with either expression of SGK1-CA (90.8% reduction, p <0.05 ) or with RFP only (77.5% reduction p < 0.005). The half-time of inhibition was 200s with resulting current densities that were not statistically different than those observed with genetic inhibition by expression of SGK1-DN.

We conclude SGK1 activity regulates INa and speculate that structure activity relationship (SAR) derivatives of our lead compound might have a role in treatment of human cardiac arrhythmias.