bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 349: Gene Regulatory Modules Regulate Cardiac Lineage Specification Revealed by Single-cell Rna Sequencing
Authors: Gong Wuming , Das Satyabrata , Rasmussen Tara L , Koyano-Nakagawa Naoko , Pan Wei , Garry Daniel J
Published: July 2021

Background:
The molecular definition of the differentiation process is of intense interest for cardiovascular developmental biologists. With the advent of single cell technologies, a number of a transcriptomic approaches have been developed to obtain a complete description of gene expression. While single cell RNA sequencing is a significant advance for describing gene expression at the cellular resolution, novel obstacles have emerged such as computational management of dropout events, discovery of the gene regulatory modules responsible for lineage specification and identification of novel lineage markers.

Methods:
Using Etv2- and Nkx2-5 EYFP transgenic embryos, from which progenitors of hematopoietic, endothelial and cardiac lineages can be isolated with high purity, we performed single cell transcriptome analysis. We developed a Bayesian statistical framework to (1) model the dropout events, (2) reconstruct the lineage specification pathways from mesodermal progenitors to hematopoietic, endothelial and cardiac lineages, (3) discover the core gene regulatory modules that govern lineage specification and (4) predict novel cell surface markers for cardiac components. We developed a structural stochastic variational inference method to efficiently estimate the model parameters for sequencing data with thousands of cells.

Results:
The analysis of 421 Etv2- and Nkx2-5 EYFP positive cells predicted the core regulatory networks involving transcription factors, signaling molecules and cell surface proteins, where the signaling cascades are significantly correlated the specification of the endothelial, hematopoietic and various cardiac lineages from the mesodermal progenitors. The method also predicted novel cell surface markers for the first and second heart fields.

Conclusions:
We report the analysis of complete gene expression profiles of mesodermal lineage specification at single cell resolution. We provide the first unified statistical framework to systematically analyze single cell RNA-seq data and quantitatively couple the network analysis and lineage specification. This strategy provides a platform for the identification of regulatory nodes and cascades that govern mesodermal and cardiovascular development.
Abstract 352: Dual Specificity Phosphatase 8 (dusp8) Regulates Cardiac Remodeling and Function by Affecting Erk1/2 Phosphorylation
Authors: Liu Ruijie , Molkentin Jeffery
Published: July 2021

Rationale:
Mitogen-activated protein kinases (MAPKs) are activated in the heart by various stress-inducing stimuli, where they participate in cardiac hypertrophy, contractility, and cell death. A family of dual-specificity phosphatases (DUSPs) directly inactivates each of the MAPK terminal effectors, thus providing a feedback mechanism to regulate the activity and recycling of MAPKs. How DUSPs regulate MAPK signaling to influence cardiac function is not well understood.

Objective:
To determine the role of DUSP8 in regulating MAPK signaling and the effect on cardiac disease.

Methods and Results:
We generated DUSP8 null (KO) and inducible cardiac specific DUSP8 transgenic mice to assess the effect on cardiac structure-function at baseline and with stress-responsiveness. Loss of DUSP8 did not alter cardiac structure-function or MAPK phosphorylation at baseline. However, with pressure overload or myocardial infarction injury, DUSP8 KO mice developed concentric hypertrophy with preserved cardiac function compared to wild type controls, suggesting a cardioprotective role for loss of DUSP8. DUSP8 transgenic mice developed cardiac hypertrophy at baseline as evidenced by elevated expression of hypertrophic marker genes as well as increased heart weights and cross sectional area of the myocytes. DUSP8 transgenic mice also had mild interstitial fibrosis and reduced fractional shortening. Biochemical analysis of MAPK phosphorylation demonstrated increased ERK phosphorylation in KO mice upon stress, suggesting a molecular mechanism underlying the increased concentric growth of DUSP8 KO cardiomyocytes.

Conclusions:
Taken together, these data demonstrate that DUSP8 modulates ERK phosphorylation to influence cardiomyocyte growth and consequent cardiac function with injury. Further analysis of MAPK phosphorylation in DUSP8 transgenic mice will provide more insight into DUSP8-ERK signaling in the heart.
Abstract 358: Pacemaker Induced Transient Asynchrony (PITA) Restores Contractile Reserve in Synchronous Heart Failure
Authors: Kirk Jonathan A , Chakir Khalid , Lee Kyounghwan , Pironti Gianluigi , Ranek Mark J , Tunin Richard S , de Tombe Pieter , Shenoy Sudha K , Rockman Howard A , Craig Roger , Kass David A
Published: July 2021

Heart failure (HF) with dyssynchrony treated with biventricular pacing (CRT) displays enhanced global and cellular function even compared to always synchronous HF. This suggests while HF is worsened by sustained dyssynchrony, it may paradoxically be improved by brief periods of Pacemaker Induced Transient Asynchrony (PITA). We tested this hypothesis in dogs tachypaced for 6 wks to induce HF. The HF group received atrial pacing and was compared to PITA (atrial pacing during day; right ventricular pacing, producing dyssynchrony, from 0000-0600). PITA blunted dilation (end diastolic and end systolic volumes reduced by 11 and 19%, respectively), reduced end-diastolic pressures from 22 to 13 mmHg, and improved the contractile response to dobutamine by 29%. Myocyte sarcomere shortening and calcium transient amplitude were depressed in HF and little improved by β adrenergic (βA) stimulation. PITA improved baseline function slightly, but virtually restored βA stimulated reserve. Membrane βA receptor density increased with PITA by 36% as well. Another contributor to the change in functional reserve was found in myofilament maximal calcium activated force (Fmax) normalized to cell cross sectional area (CSA). This declined ~40% in HF vs. Control, but was fully restored by PITA. However, as CSA was greater in HF and normalized by PITA, raw Fmax was similar despite hypertrophy in HF, suggesting HF myocytes had dysfunctional myofilaments, which PITA prevented. Electron microscopy confirmed normal myofilament structure in Control and PITA, whereas 40% of HF sarcomeres displayed deteriorated z-disks and loss of normal registration of the thick and thin filaments. In HF, 39% of HF isolated myofibrils produced virtually zero maximal force, whereas Control and PITA fibers functioned normally. Thus, there are two populations of myofibrils within HF hypertrophied cells, with ~40% structurally and functionally disrupted. PITA reverses this to restore force-calcium activation and with improved βA receptor signaling, restores functional reserve, suppressing chronic maladaptive remodeling. This surprising finding indicates PITA can ameliorate HF pathobiology and improve reserve function. Further studies are needed to test if such benefits translate to humans.
Abstract 36: Administration of 17β-Estradiol in C57Bl/6N Female Mice Leads to Cardiac Atrophy and Dysfunction via a β-Catenin Mechanism
Authors: Kararigas Georgios , Zelarayan Laura C , Toischer Karl , Hasenfuss Gerd , Jarry Hubertus , Regitz-Zagrosek Vera
Published: July 2021

The steroid hormone 17β-estradiol (E2) regulates several biological processes. In contrast to its anti-hypertrophic effects under pressure overload, we recently found that E2 induced physiological hypertrophic growth in healthy C57Bl/6J mice but not C57Bl/6N mice. Here, we aimed at the characterization of the effects of E2 in C57Bl/6N mice and tested the hypothesis that β-catenin mediates these E2 effects. Following ovariectomy, 2-month-old C57Bl/6N wild-type and cardiac-specific β-catenin-deleted (β-cat
Δex2-6
) mice were randomized to an E2-containing or soy-free (control, CON) diet (
n
= 7-13/group). Cardiac function was examined by echocardiography following established procedures. The 3-month physiological dose of E2 led to a higher relative uterus weight compared with CON (
P
< 0.001) in both WT and β-cat
Δex2-6
mice. The relative heart weight was significantly reduced by E2 compared with CON in WT mice (
P
< 0.001), while there was no significant effect in β-cat
Δex2-6
mice. Cardiomyocyte cross-sectional area was also significantly decreased by E2 (
n
= 5-7/group;
P
< 0.001) compared with CON in WT mice, while there was no significant effect in β-cat
Δex2-6
mice. Echocardiography revealed a significant decrease in septum width (
P
< 0.001) and posterior wall thickness (
P
< 0.01) in E2 treated WT mice compared with CON, while there was no significant effect in β-cat
Δex2-6
mice (
n
= 8/group). These E2-induced structural changes in WT mice were accompanied by a significant decrease in cardiac function, namely a 23% decrease in fractional shortening compared with CON (
P
< 0.05), while there was no significant effect in β-cat
Δex2-6
mice. Immunoblotting revealed a significant increase in the levels of the ubiquitin ligase and key regulator of proteasome-dependent protein degradation muscle-specific RING finger protein 1 (MuRF1) by E2 compared with CON in WT mice (
P
< 0.05), while there was no significant effect in β-cat
Δex2-6
mice. Although we hypothesized increased autophagic activity, we found no effect on the autophagy-related protein LC3 in WT or β-cat
Δex2-6
mice. In conclusion, our surprising findings show that E2 leads to cardiac atrophy and dysfunction in C57Bl/6N mice via a β-catenin mechanism seemingly in an autophagy-independent manner.
Abstract 360: Differential Analysis of Thrombospondin Proteins in Cardiac Disease
Authors: Schips Tobias G , Correll Nathan R , Sargent Michelle A , Molkentin Jeffery D
Published: July 2021

Thrombospondin (Thbs) proteins are multidomain, matricellular proteins comprised of 5 genes that each share similar domains and have been largely ascribed the same functional characteristics The Thbs protein family is divided in 2 subgroups based on multimerization as trimers, (group A: Thbs1 and 2), or as pentamers (group B: Thbs3, 4 and 5). Thbs proteins modulate aspects of cell- matrix interactions, and more recently we have shown that they can also serve an intracellular chaperone function along the secretory pathway in response to ER stress. Thbs1 and 2 can also alter angiogenesis and modulate MMP activity as well as TGFb activity. The overall aim of this study is to functionally compare the two Thbs subgroups and elucidate their involvement in cardiac homeostasis and disease. Hence we analyzed Thbs1 as a representative of group A and Thbs3 for group B. Cardiomyocyte specific overexpression of either Thbs1 or Thbs3 results in robust activation of an ATF6- dependent ER stress response, similar to our previous working published with Thbs4. However, Thbs1 overexpressing mice suffer from cardiomyopathy and die within 12 weeks. In contrast, Thbs3 overexpressing mice are completely normal at baseline and Thbs3 overexpression even increased survival rates after MI similar to Thbs4 from our previous work. However, Thbs3 hearts also showed increased chamber dilation after MI and lower heart function suggesting that Thbs3 also has maladaptive effects. In contrast to loss of Thbs4, which die within the first week of TAC mediated pressure overload, Thbs3 KO mice are protected from TAC induced hypertrophy. Moreover, the combination of Thbs3/4 dKO more closely phenocopies the effect of Thbs3 deletion, as these mice show a significant reduction in hypertrophy and lung congestion after long term TAC. This is the first study comparing the function of different Thbs isoforms in the heart, which suggests a novel role of Thbs3.
Abstract 361: Loss of Cardiac Retinoic Acid Receptor Alpha Promotes Diastolic Heart Failure With Preserved Ejection Fraction
Authors: Zhu Sen , Guleria Rakeshwar , Thomas Candice , Gerilechaogetu Fnu , Roth Amanda , Kumar Rajesh , Dostal David , Baker Kenneth , Pan Jing
Published: July 2021

Objectives:
We have previously demonstrated that the expression/activation of retinoic acid receptor (RAR) is inhibited in diabetic hearts, and that activation of RARα prevents diabetes-induced diastolic heart failure, suggesting that impairment of RARα signaling may be a critical mechanism in heart failure.

Methods and Results:
Cardiac RARα gene deletion (KO) was achieved by tamoxifen injection at 6-weeks old RARαfl/fl α-MHC-MerCreMer mice, RARαfl/fl mice were used as control (WT). Heart function was monitored by echocardiograph for 64 wks. Mice were sacrificed at 20 or 64 wks post gene deletion, respectively. A significant decrease in E/A ratio and TDI E’ and increase in IVRT (isovolumic relaxation time) and DT (deceleration time) suggested diastolic dysfunction after 16 wks of gene deletion in KO mice, which was confirmed by cardiac catheterization (decreased dP/dtmin and increased tau). Concentric hypertrophy developed in KO mice after 56 wks of gene deletion, as confirmed by increased thickness of left ventricular wall and interventricular septum and elevated heart weight/tibia length ratio. However, no significant difference was observed in LVEF (LV ejection fraction), FS (fraction shortening) and dP/dtmax between KO and WT mice. Significantly increased gene expression of NOX2 (NADPH oxidase 2) and NOX4, decreased SOD1 and SOD2 levels and increased intracellular reactive oxygen species (ROS) were observed in KO mouse hearts, along with a significantly decreased protein expression of SERCA2a and CaMKIIδ, decreased phosphorylation of PLB, Akt and CaMKIIδ. Overexpression of RARα in cardiomyocytes rescued RARα deletion-induced changes in SERCA2a, PLB, Akt and CaMKIIδ. Deletion of RARα in cardiomyocytes impaired intracellular calcium reuptake into SR and cardiomyocyte relaxation. Inhibition of ROS by N-acetyl cysteine abolished RARα deletion-induced calcium mishandling and cardiomyocyte relaxation.

Conclusion:
Cardiac specific deletion of RARα induces diastolic heart failure with preserved ejection fraction (HFpEF), by promoting intracellular ROS and disrupting SERCA2a-mediated calcium reuptake and cardiomyocyte relaxation. Our study suggests that deficiency in RARα signaling is a novel mechanism leading to HFpEF.
Abstract 362: Cardioprotective Effects of Adiponectin in Volume Overload Induced Electrophysiological Remodeling
Authors: Zhong Juming , Wang Lili , Miller Dori , Schwartz Dean , Amin Rajesh , Judd Robert
Published: July 2021

The electrophysiological hallmark of the failing heart is the prolongation of action potential duration that induces arrhythmia and sudden death. Depressed outward potassium currents (Ito) has been implicated as the major cause of altered action potential during ventricular remodeling. The molecular mechanism underlying depressed Ito in the diseased heart is still poorly understood. Recent studies have demonstrated that adiponectin (APN) has a cardio-protective effect in response to various pathological stimuli; however, little information is available regarding the potential effects of APN on electrophysiological remodeling under pathological conditions. The present study were to determine the effect of adiponectin treatment on ventricular potassium channel function in a rat model of volume overload induced heart failure. Volume-overload was induced by surgical creation of an infrarenal aorta-vena cava fistula. Rats were administrated with or without adenovirus-mediated overexpression of adiponectin (Ad-APN) at 2-, 5- and 8- weeks post-fistula. In vivo ECGs were used to evaluate changes in QT interval in rats at 10 weeks post-fistula. Ventricular myocytes were isolated at 10 weeks post-fistula. Western blots were used to measure cytoplasmic and membrane protein expression of potassium channels Kv4.2 and Kv 4.3, as well as, KChIP2. Whole cell patch clamp was used to evaluate action potential and Ito currents. Results showed that adiponectin levels in serum and myocytes were significantly reduced following fistula. The duration of action potential was prolonged in ventricular myocytes following 10-week fistula, which was correlated with the in vivo QT interval prolongation, as well as a depression of functional Ito and decreased protein expression of Ito channel subunits in ventricular myocytes. In vivo supplementation of Ad-APN increased the protein levels of Ito channel subunits and prevented Ito depression in ventricular myocytes following 10-week fistula. This further restored the duration of action potential and the QT interval on the ECG back to the normal. These results indicate that adiponectin was able to prevent volume overload-induced ventricular electrophysiological remodeling.
Abstract 368: Biomechanical Properties of Scar ECM: from the Acute to Chronic Stages of Myocardial Infarction
Authors: Brazile Bryn , Butler J. R , Patnaik Sourav , Xu Yanyi , Claude Andrew , Prabhu Raj , Williams Lakiesha , Guan Jianjun , Liao Jun
Published: July 2021

Introduction:
Myocardial infarction (MI) affects more than 8 million Americans, causing massive heart cell death and heart function decrease. To better understand the scar biomechanics, we characterized the mechanical properties of pure scar ECM, obtained by decellularizing the MI tissues.

Materials and Methods:
Infarcted rat hearts were generated by a permanent left coronary artery ligation (PLCAL) and harvested at 15 min, 1, 2, and 4 weeks (per acute to chronic stages of MI)(N = 6 each). Scar ECM were obtained by decellularizing the infarcted hearts in 0.1% sodium dodecyl sulfate (SDS) solution for 3 weeks. Scar ECM specimens were trimmed into square shape, and then subjected to biaxial testing with one edge aligned with the circumferential direction and the other edge aligned with the longitudinal direction of the rat heart. After 10 cycle preconditioning, an equibiaxial tension protocol of

T
circ

:

T
rad

= 30:30 N/m was performed to capture the tissue biaxial behavior.

Results and Discussion:
Scar ECM 15 minutes through 4 weeks post infarction showed a stiffening biaxial behavior along with the time (Fig.1). The decrease of extensibility along longitudinal direction was more noticeable than circumferential direction, which led to a decrease in degree of anisotropy.

Conclusions:
Scar ECM biomechanics showed a stiffening behavior with a marked reduction in extensibility (longitudinal) with time. This change in biomechanical properties can be correlated to the collagen structure changes with progression of MI. Knowledge of the structural-mechanical relationship of scar ECM will help us understand MI progression and help formulate regenerative therapies.
Abstract 370: MicroRNA-19b Contributes to Cardiac Fibrosis
Authors: Chen Ping , Lv Dongchao , Xu Jiahong , Zhou Qiulian , Sun Qi , Xiao Junjie
Published: July 2021

Fibrosis is one of the most important characteristics of cardiac remodeling during heart failure. The accumulation of extracellular matrix (ECM) within myocardium is the major feature of cardiac fibrosis. microRNA (miR)-19b, a key functional member of miR-19-72 cluster family, has been suggested to be involved in aging-induced heart failure through regulating ECM-related proteins, such as connective tissue growth factor (CTGF), thrombospondin-1 (TSP-1), collagen-1A1, and collagen-3A1. In the current study, we aimed to investigate the role of miR-19b in cardiac fibroblast function and ECM production using neonatal rat cardiac fibroblasts in primary culture. We found that overexpression of miR-19b increased, while inhibition of miR-19b decreased the proliferation and migration of cardiac fibroblasts, using Cell Counting Kit-8 (CCK-8) (0.660±0.019 vs 0.720±0.014 in nc-mimic and miR-19b mimic, 0.506±0.009 vs 0.454±0.008 in nc-inhibitor and miR-19b inhibitor, respectively), EdU incorporation assay (0.059±0.002 vs 0.096±0.006 in nc-mimic and miR-19b mimic, 0.059±0.006 vs 0.040±0.003 in nc-inhibitor and miR-19b inhibitor, respectively), and wound healing assay (0.528±0.024 vs 0.896±0.027 in nc-mimic and miR-19b mimic,0.520±0.028 vs 0.174±0.019 in nc-inhibitor and miR-19b inhibitor, respectively), respectively. Meanwhile, the inhibition of miR-19b downregulated the mRNA levels of α-SMA (0.556±0.048 vs 1.038±0.137 in nc-inhibitor and miR-19b inhibitor, respectively) and collagen-1 (1.023±0.116 vs 0.551±0.033 in nc-inhibitor and miR-19b inhibitor, respectively) in cardiac fibroblasts, indicating a reduction in fibroblast activation and ECM production via miR-19b inhibition. Furthermore, we found that PTEN was negatively regulated by miR-19b in cardiac fibroblasts using western blot analysis. PTEN, a well-known tumor-suppressor gene, has been known to inhibit cell proliferation and migration. However, it remains to be further clarified whether PTEN could mediate the effect of miR-19b in the proliferation, migration and activation of fibroblasts. These data might provide important evidence suggesting that miR-19b could be a potential therapeutic target for cardiac fibrosis.
Abstract 379: Inhibition of Bcl-x Phosphorylation Prevents Cardiomyocyte Death, Cardiac Remodeling and Heart Failure After Myocardial Infarction
Authors: Nakamura Michinari , Zhai Peiyong , Re Dominic D , Sadoshima Junichi
Published: July 2021

Cardiac remodeling promotes heart failure (HF). Cardiomyocyte (CM) death is one of the mechanisms to develop cardiac remodeling. We recently reported that Mst1 phosphorylates Bcl-xL at Ser14, which promotes apoptosis by inducing dissociation of Bcl-xL from Bax and consequent activation of Bax in CMs. Its phosphorylation is increased in response to ischemia-reperfusion (IR) in an Mst1-dependent manner. However, the functional significance of endogenous Bcl-xL phosphorylation remains unclear in vivo. To address this question, knock-in (KI) mice with alanine mutation at Ser14 in Bcl-x were generated. At baseline, cardiac function was similar between wild-type (WT) and heterozygous KI (HKI) mice (EF 76% and 79%, respectively). HKI mice exhibited smaller % infarct area (30%) than WT (43%) (p=0.016) upon IR, suggesting that phosphorylation of endogenous Bcl-xL at Ser14 plays an essential role in mediating IR injury. In order to test the role of Bcl-xL phosphorylation in the development of HF, HKI and WT mice were subjected to permanent ligation of LAD for 4 weeks. During progression of cardiac remodeling, Mst1 was activated in both WT and HKI mice. Phosphorylation of Bcl-xL and Bcl-xS, an alternative transcriptional variant of Bcl-x, both at Ser14, were increased in WT mice, which were abrogated in HKI mice. The infarct area evaluated with TTC staining at Day 1 was similar in WT and HKI mice (59.1% and 61.2%, p=0.65). Four weeks after myocardial infarction (MI), WT mice exhibited lower cardiac contraction (EF 46.5%) and higher LVEDP (10.8mmHg) than those in HKI mice (EF 68.9% and LVEDP 7.0mmHg) (both p<0.05). Scar area and TUNEL-positive CMs were greater in WT (49.0% and 1.6%, respectively) than in HKI mice (29.2% and 0.4%, respectively) (both p<0.05). Cleaved caspase 3 and 9 were significantly increased (3.2- and 5.7-fold, respectively) in WT but not in HKI mice. In vitro experiments with overexpression of phospho-mimicking mutant (Bcl-xS-S14D) showed 13% reduction in cell viability compared with that of phospho-resistant mutant (Bcl-xS-S14A) (p=0.01%). Our results suggest that phosphorylation of Bcl-xL and Bcl-xS at Ser14 contributes to CM death in response to IR and chronic MI in vivo, thereby promoting cardiac remodeling and HF.
Abstract 381: Prolonged Renal Failure Leads to Reduced Number of Active Cardiac Mitochondria in a Rat Model for Long Term Chronic Kidney Disease
Authors: Hertzberg-Bigelman Einat A , Entin-Meer Michal , Aharon-Hananel Genya , Saada Ann , Levy Ran , Cohen Lena , Rozenbaum Zach , Keren Gad
Published: July 2021

Objectives
- Cardiorenal syndrome type 4 is characterized by primary chronic kidney disease (CKD) leading to an impairment of cardiac function. We recently showed a reduced expression of several cardiac mitochondrial genes in short-term CKD rat model. We aimed to evaluate whether cardiac mitochondrial structure and function is modified in long-term CKD and if so, to characterize the potential associated mechanisms.

Methods
- Lewis rats underwent 5/6 nephrectomy for induction of CKD. Upon necroscopy, eight months later, cardiac sections were analyzed by histology and electron microscopy (EM). Mitochondrial DNA content was determined by the mitochondrial gene, cytochrome B. Mitochondrial content was assessed by citrate synthase (CS) activity in tissue homogenate and respiratory chain function was determined by the activity of complexes I-IV in isolated mitochondria. The levels of PGC1a, a transcription factor for mitochondrial biogenesis, Angiotensin II type 1 receptor and cytosolic cytochrome C were assayed by western blot. Cytokine serum profile was determined by microarray.

Results
- Long-term CKD leads to cardiac hypertrophy and increased interstitial fibrosis. EM analysis revealed a massive spatial disarrangement accompanied by a considerably increased volume of swollen-damaged mitochondria in CKD hearts (32±3%, n=5, 48±6%, n=4; respectively; p<0.05). Total mitochondrial DNA content was decreased in cardiac tissue of CKD rats. Concomitantly, active mitochondrial content was significantly reduced. Conversely, no differences were observed in respiratory chain enzymes’ functions (complexes I-IV) in isolated active mitochondria. Moreover, inflammatory response and activation of Renin-Angiotensin-Aldosterone-System (RAAS) were detected in the CKD setting.

Conclusion
- CKD results in a marked reduction of active mitochondria in the heart. Inflammatory cytokines and RAAS, may set a deleterious environment to cardiac mitochondria, as suggested in non-CKD models. The data may represent a significant milestone in the personalized medicine strategy for treating CKD patients who present with normal cardiac function accompanied by positive biomarkers for cardiac mitochondria damage.
Abstract 385: D-2-hydroxyglutaric acid Acutely Impairs Oxidative Decarboxylation in the Heart
Authors: Karlstaedt Anja , Zhang Xiaotian , Vasquez Hernan , Goodell Margaret A , Taegtmeyer Heinrich
Published: July 2021

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1, IDH2) have been described in low-grade glioma and in acute myeloid leukemia. Accumulation of the oncometabolite D-2-hydroxyglutaric acid (2HG) and its release into the blood is associated with dilated cardiomyopathy. The mechanisms leading to changes in cardiac metabolism and contractile function are unknown. We studied in the isolated working rat heart preparation metabolic consequences of increased 2HG supply and its impact on cardiac energy provision. In combination with physiological levels of glucose (5mM) and lactate (0.5mM), hearts were perfused at different concentrations with 2HG (0.5mM, 1.0mM). We confirmed the uptake and enrichment of 2HG in the tissue through liquid chromatography followed by mass spectrometry (LC/MS). 2HG markedly decreased cardiac power and cardiac efficiency in a concentration dependent manner. At the same time glucose oxidation increased significantly (1.4±0.1 μmol/min/g dry wt, p<0.05) at higher workloads. The alpha-Ketoglutarate Dehydrogenase activity was reduced by two-fold (2.84±0.56 μmol/min/g dry wt, p<0.01) and production of reactive oxygen species (e.g. H2O2) increased by three-fold (1.06±0.07 μmol/min/g dry wt, p<0.01) in presence of 2HG. Consistent with this reduction in oxidative decarboxylation, the cytosolic NAD+/NADH redox state increased (3.1±0.1, p<0.001), while the cellular energy charge declined (AMP:ATP ratio, 0.15±0.1, p<0.001). Together, our results demonstrate a direct impairment of cardiac energy substrate metabolism with a resulting decline in contractile function. The data suggests that 2HG directly suppresses cardiac function by specific metabolic alterations in a range of pathologies.
Abstract 389: Alterations of Mitochondrial Calcium Handling in Heart Failure
Authors: Wei An-Chi , Liu Ting , O’Rourke Brian
Published: July 2021

Heart failure (HF) and sudden cardiac death (SCD) are major public health concerns that are increasing in incidence, yet the mechanisms underlying SCD in patients with HF are poorly understood. In a novel guinea pig model of HF/SCD, we showed that in vivo treatment with a mitochondrial Na+/Ca2+ exchanger (mNCE) inhibitor attenuates cardiac remodeling, preserves cardiac contractile function, and improves survival, supporting a critical role for altered mitochondrial Ca2+ dynamics in the pathophysiology. Here, we investigate whether the intrinsic mitochondrial Ca2+ transport rates are altered in this HF model.

Methods:
Ascending aortic constriction, combined with daily i.p. injection of isoproterenol (ISO), were used to induce HF (ACi) with acquired long QT. This group was compared with animals subjected to aortic constriction alone (AC), or sham-operated animals with (SHAMi) or without (SHAM) ISO treatment. Ca2+ Green-5N was used to measure total mitochondrial Ca2+ uptake and to quantify mitochondrial Ca2+ influx and efflux rates in isolated cardiac mitochondria.

Results:
Both the total mitochondrial Ca2+ load and the Ca2+ capacity prior to triggering permeability transition pore (mPTP) opening were reduced in HF mitochondria (5mM NaCl present). Mitochondrial Ca2+ fluxes, individually measured with sequential additions of 15μM free Ca2+, 10nM Ru360 and 5mM NaCl, showed that initial Ca2+ uptake rate through the mitochondrial Ca2+ uniporter (mCU: 0.55 nmol/sec/mg) was not significantly changed in HF; however, the Ca2+ extrusion rate through mNCE was larger in HF (AC:0.022 nmol/sec/mg; SHAM:0.018; ACi:0.013; SHAMi:0.009), but with a lower affinity for Na+. Interestingly, Na+-independent efflux via mPTP increased in HF (AC:0.0040 nmol/sec/mg; SHAM:0.0022; ACi:0.0013; SHAMi:0.012). Mitochondria from failing hearts also showed decreased respiration and increased ROS emission.

Conclusions:
The data indicate that an increase of intrinsic Ca2+ efflux and the increase in cytoplasmic Na+ in HF could both contribute to blunted mitochondrial Ca2+ in HF, which will affect cardiac energetics and ROS balance. Inhibitors of mNCE or mPTP are thus proposed to be therapeutic interventions that would improve mitochondrial Ca2+ balance and function in HF.
Abstract 395: Transient Inactivation of Retinoblastoma Induces De-Novo Cardiomyogenesis From Human Myocardial Progenitors but Not Pre-Existing Cardiomyocyte Replication
Authors: Hatzistergos Konstantinos E , Sage Julien , Conklin Jamie F , Bellio Michael , Valasaki Krystalenia , Margitich Irene S , Premer Courtney , Balkan Wayne , Hare Joshua M
Published: July 2021

Introduction:
Activation of cardiac cell cycle re-entry is considered the primary therapeutic strategy for cardiomyocyte (CM) regeneration. However, the role of cardiac cell-cycle control in cardiomyogenesis remains elusive. Here, we combined RNA interference and stem cell modeling to investigate the role of Retinoblastoma (RB) in human cardiomyogenesis.

Hypothesis:
RB regulates proliferation and differentiation of cardiac progenitors (CPCs) but not CM replication.

Methods:
H9 human embryonic stem cells (hESCs) stably expressing tetracycline (tet)-inducible shRNAs against RB (hESCshRB) or hemagglutinin-tagged RB (hESCHA-RB) were tet-induced at selected time-points during or after CM differentiation.

Results:
Analysis of ser-608 illustrated stage-specific differences in the degree of RB inactivation during normal hESCs-cardiogenesis. Transient shRB knockdown in hESCshRB-derived embryoid bodies (EBs) during the CPC-stage (EB-days 5-8), significantly upregulated GATA4, ISL1, CTNNI, and cKit transcription (p<0.05), while increasing the yield of beating EBs by 2.4-fold (n=6/group, p<0.0001 vs. vehicle). Gene-expression arrays of 22 RB-related genes, illustrated that shRB-knockdown upregulated CCND1, CCND2, CCND3, and CDK4, CDK6 (p<0.05), followed by a 3.6-fold increase in E2F3 (p<0.05) expression. Moreover, expression of p107 and p130, p27, p57, ARF and CDKN3 were also significantly increased (p<0.05), whereas TP53 and MDM2 remained unchanged. Ectopic HA-RB in CPCs did not significantly affect cardiogenesis (n=18). Conversely, shRB knockdown in EB-day 60-derived CMs (n=15) did not stimulate cell cycle re-entry, as assessed by analysis of EdU incorporation and Aurora-B kinase (AurB). Remarkably, co-culture of hESCHA-RB-derived CMs with adult cardiac (CSCs) and/or mesenchymal (MSCs) stem cells (n=15/group), increased cell-cycle re-entry ~2.8-fold, assessed by ser-10 Histone H3 (p=0.0002) and AurB (p<0.0001).

Conclusions:
These findings suggest that RB regulates proliferation and differentiation of human CPCs in a cell-autonomous manner, via a CCND-CDK4/6-E2F3 mechanism. Conversely, CM replication may be enhanced via cell-cell interactions with MSCs and/or CSCs, but not cell-autonomously via RB inactivation.
Abstract 396: Uncontrolled Endothelial Migration In Hereditary Hemorrhagic Telangiectasia: Disease Modeling With Ips Cell
Authors: Takei Makoto , Yuasa Shinsuke , Kusumoto Dai , Kunitomi Akira , Kashiumura Shin , Yodu Gakuto , Shimojima Masaya , Motoda Chikaaki , Tanaka Atsushi , Kuroda Yusuke , Tohyama Shugo , Seki Tomohisa , Fukuda Keiichi
Published: July 2021

Introduction:
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant heritable disease caused by mutation of Activin like receptor-1 (ALK1), an endothelial specific TGF-β family receptor. The histological hallmark in HHT is abnormal atriovenous communication, caused by abnormal migration of endothelial cells (ECs). These findings suggest that the enhanced migration of ECs would have a role in the development of HHT. However, how EC migration is regulated in HHT remains unclear. Hence, the aim of this study is to develop an in vitro HHT model with ECs derived from patient-specific induced pluripotent (iPS) cells.

Methods:
We generated iPS cells from a HHT patient with ALK1 mutation (ALK1mt) and two control subjects (WT). ECs were derived from these iPS cells with prespecified differentiation method. We compared the effects of BMP-9, a selective agonist of ALK1, between ALK1mt- and WT-ECs. Also, microarray analysis comparing transcriptome of ALK1mt- and WT ECs with or without BMP-9 stimulation was conducted.

Results:
Migration capacity was significantly reduced with BMP-9 in WT-ECs, but not in ALK1mt-ECs. With BMP-9 stimulation, elevation of genes associated with vascular stabilization and maturation was observed in WT-ECs but not in ALK1mt-ECs.

Conclusion:
With ECs derived from iPS cells, modeling of HHT phenotype, uncontrolled endothelial migration, was achieved.

Clinical implications:
By elucidating the biological process with which BMP-9 controls the migration of endothelial cells, new drug targets for HHT may be found. Also, these result may lead to the development of a new experimental drug screening system for HHT.
Abstract 398: Histone Demethylase Kdm6a Contributes To X Chromosome Dependent Ischemia/reperfusion Injury Via Epigenetic Regulation
Authors: Li Jingyuan , Itoh Yuichiro , Chen Xuqi , Arnold Arthur , Eghbali Mansoureh
Published: July 2021

Introduction:
Sex differences in susceptibility to ischemia/reperfusion (I/R) injury have been mostly attributed to sex hormones. Recently we examined the role of sex chromosomes in sex differences in myocardial I/R injury. We discovered that gonadectomized mice with two X chromosomes (XX or XXY) have ~50% larger infarct size after I/R injury, compared to mice with one X chromosome (XY or XO). Only few X genes escape X inactivation and are expressed higher in XX than XY individuals. Here we examined the role of “X escapee” histone demethylase Kdm6a which is important in cardiac development.

Methods:
Female mice with a heterozygous global knockout of Kdm6a (Kdm6a+/-) and with 2 copies of Kdm6a (Kdm6a+/+, regular WT) were used. Isolated mouse hearts were subjected to 30 min global normothermic ischemia followed by 60 min reperfusion. RNA-Seq analysis was performed by comparing gene expression in hearts of Kdm6a+/+ vs. Kdm6a+/- females at baseline before ischemia. We calculated an unbiased composite score of relevance in which the level of significance of the Kdm6a effect on expression (p value) was integrated with the size of the KDM6A effect on expression (fold change), and with the amount of H3K27me3 mark found on the genes in the heart based on online ChIP-Seq data. Two way ANOVA was used for statistical analysis. P<0.05 was considered statistically significant. Values are expressed as mean± SE.

Results:
Kdm6a+/+ female mice had significantly lower heart functional recovery compared to their littermate Kdm6a+/- (LVDP: 46.7±9.8% vs. 79.8±3.5%; RPP: 44.1±10.5% vs. 76.2±8.5%, n=6-8 mice/group, p<0.01). The integration of our RNA-Seq data using the hearts of female mice with 2 vs. 1 copy of Kdm6a (n=4 samples per group) with online datasets measuring the H3K27me3 mark, sex differences in expression in humans and mice, and involvement in ischemic heart failure, revealed carbonic anhydrase-3 (Car3) as the most interesting candidate (upregulated ~7 fold in the hearts of Kdm6a+/+ vs. Kdm6a+/- female mice) at baseline. Car3 encodes one isoform of carbonic anhydrase, involved in pH regulation, which is a critical part of I/R injury.

Conclusion:
Histone demethylase KDM6A contributes to X chromosome dependent I/R injury via epigenetic regulation
Abstract 399: Reactive Oxygen Species in Myocardial Ischemic Injury and Protection
Authors: Yang Huang-Tian , Tan Ji-Liang , Liu Jin-Long , Wu Lan , Wang Zhi-Hua
Published: July 2021

Myocardial injury following ischemia/reperfusion (I/R) is a common clinical scenario in patients suffering from ischemic heart disease. An excessive production of reactive oxygen species (ROS) during the early phase of reperfusion following myocardial ischemia has been proposed to contribute to reperfusion injury. Paradoxically, the ROS has also been recognized as a trigger of pro-survival signaling pathways mediating cardioprotection at a low level. However, the precise mechanisms for the dual role of ROS have not yet been fully clarified. To address this question, using intermittent hypobaric hypoxia (IHH) as a cardioprotective model, combining with H2O2 pre- and post-conditioning, we studied the level and roles of ROS during early reperfusion following ischemia in I/R injury and protection and explored the regulatory mechanisms underlying. Our results reveal that the elevated ROS generated during early reperfusion are injurious but insufficient to reach the threshold to trigger protective signaling pathways. The moderate level of ROS, higher than that elevated by I/R, during early reperfusion is critical for triggering cardioprotection against I/R injury via alleviating intracellular Ca2+ overload and preserving mitochondrial function through the efficient activation of Akt, PKCε[[Unable to Display Character: ]]and JAK2/STAT3 pathways. We then identified the downstream target of ROS-JAK2/STAT3 signals that interacts with the sarcoendoplasmic reticulum Ca2+-ATPase 2 (SERCA2) to improve the activation of SERCA2 and subsequently regulate intracellular Ca2+ homeostasis. When the heart is exposed to excessive ROS, the activation of protective mechanisms reaches a plateau and fails to counteract the severe nonspecific oxidative stress. These findings provide a new angle to interpret the controversial roles of ROS in myocardial I/R and demonstrate that the differential effects of ROS in myocardial I/R are derived from a quantity-dependent wrestling between its detrimental and signaling roles.
Abstract 408: Myocardial Knockdown of Mir-375 Attenuates Post-mi Inflammatory Response and Left Ventricular Dysfunction via Pdk-1-akt Signaling Axis
Authors: Garikipati Venkata N , Verma Suresh K , Khan Mohsin , Gumpert Anna M , Zhou Jibin , Cheng Zhongjian , Benedict Cindy , Nickoloff Emily , Johnson Jennifer , Yuan Ancai , Gao Erhe , Kishore Raj
Published: July 2021

MicroRNAs are known to be dysregulated in the ischemic heart disease and have emerged as potential therapeutic targets for treatment of myocardial infarction (MI). Recently MicroRNA-375 has been shown to be up-regulated in humans with MI. In this study, we assessed whether inhibition of the miR-375 using an i.v.-delivered locked nucleic acid (LNA)-modified anti-miR (LNA-antimiR-375) can provide therapeutic benefit in mice with pre-existing pathological cardiac remodeling and dysfunction due to myocardial infarction (MI).After the induction of acute myocardial infarction, mice were treated with either control or LNA based miR-375 inhibitor, and inflammatory response, cardiomyocyte apoptosis and LV functional and structural remodeling changes were evaluated. Anti-miR-375 therapy significantly suppressed infiltration of inflammatory cells, expression of proinflammatory cytokines in the myocardium and cardiomyocyte apoptosis. These changes were associated with miR-375 mediated activation of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) and downstream AKT phosphorylation on Thr-308. LNA anti-miR-375 therapy significantly improved LV functions, reduced infarct size, and attenuated infarct wall thinning. Moreover, LNA based miR-375 therapy significantly increased capillary density in the infarcted myocardium. Further, our in vitro studies demonstrated that miR-375 negatively regulates the expression of PDK-1 by directly targeting the 3’UTR of the PDK-1 transcript. Taken together, our studies demonstrate that anti miR-375 therapy suppresses inflammatory response, cardiomyocyte death and contributes to improved LV function and enhanced angiogenesis via activation of PDK-1/AKT signaling.
Abstract 410: Interleukin-21 Promotes Pulmonary Arterial Hypertension Through M2 Macrophage Polarization
Authors: Nakaoka Yoshikazu , Hashimoto-Kataoka Takahiro , Shirai Mikiyasu , Sakata Yasushi
Published: July 2021

Interleukin-6 (IL-6) is a multifunctional proinflammatory cytokine that is elevated in the serum of pulmonary arterial hypertension (PAH) patients and can predict the survival of idiopathic (I)PAH patients. Previous animal experiments and clinical human studies indicate that IL-6 is important in PAH; however, the molecular mechanisms of IL-6-mediated pathogenesis of PAH have been elusive. Here we identified IL-21 as a novel downstream target of IL-6-signaling in PAH. First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Furthermore, the hypoxia-induced upregulation of IL-17 and IL-21, which are primarily produced by Th17 cells, was also ameliorated by IL-6 blockade in mice. Whereas IL-17 blockade with an anti-IL-17 neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. Consistently, IL-21 indeed promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Moreover, significantly enhanced expressions of IL-21 and M2 macrophage markers were detected in the lungs of IPAH patients who underwent lung transplantation. Collectively, these findings suggest that IL-21 promotes PAH through M2 macrophage polarization, downstream of IL-6-signaling. IL-6/Th17/IL-21-signaling axis may be a novel potential target for treating PAH.
Abstract 413: Engineering Targeted Growth Factors for Selective Pro-survival Signaling in Apoptotic Cardiomyocytes Following Ischemic Injury
Authors: Stowe Timothy R , Jennings-Antipov Laura D , Kuchenbecker Kristopher M , Zhang Yan , Eshghi Shawdee , Millard Bjorn L , Nickerson Andrea D , Nielsen Ulrik B , Onsum Matthew D
Published: July 2021

Background:
Despite the benefits of reperfusion therapy after myocardial infarction (MI), most patients still progress to heart failure. MI patients suffer irreversible loss of heart function due to cardiomyocyte (CM) death and tissue scarring. Growth factors (GFs) reduce scar size and limit CM apoptosis in preclinical studies, however native GFs have poor drug-like qualities and have not been successful in the clinic. Silver Creek Pharmaceuticals is developing a new class of targeted, growth factor-based therapeutics (Smart Growth Factors) that are engineered to have optimized pharmacokinetics, dynamics and safety profiles. Our first generation of SGFs use annexin-V (AnxV) to target IGF-1 to damaged CMs and selectively activate pro-survival signaling.

Methods and Results:
We used biophysical simulation to design an IGF-1-based SGF that selectively activated the PI3K pathway in damaged cells. SGFs were engineered to target IGF-1 to damaged cells through the binding of AnxV to phosphatidylserine exposed on the surface of apoptotic cells. These bispecific proteins were constructed with half-life modulators and linkers, then screened for their ability to selectively increase pAKT levels in apoptotic iPSC-derived human CMs. We identified a subset of SGFs that were able to selectively increase pAKT levels in apoptotic cells as compared to healthy cells (p<0.05). Based on these data, we tested the ability of SGFs to activate pro-survival signaling and reduce infarct size in a rat ischemia/reperfusion model of acute MI (AMI). SGFs were able to selectively prolong pAKT in the infarcted region of the left ventricle without activating signaling in remote healthy tissue out to 2 hours post-reperfusion (n=3-6/group, p<0.05). For efficacy studies, rats were subjected to 60 minutes of ischemia followed by 72 hours of reperfusion. A single IV dose of SGF (600 pmol/kg) administered at time of reperfusion was able to significantly reduce infarct size relative to the area-at-risk (infarct/AAR%) as compared to controls (p<0.05; SGF 12.7±5% n=10; wt IGF-1 20.3±3% n=8 and vehicle 27.6±7% n=12).

Conclusions:
This work demonstrates that SGFs selectively activate pro-survival signals in distressed CMs and lead to reduced infarct size in vivo without off-target effects.
Abstract 415: Cardioprotective Effects of Interleukin-15 in Immortalized Human Ventricular Cardiomyocytes
Authors: McBride Marin Jane , Durham Kristina , Trigatti Bernardo L
Published: July 2021

Interleukin-15 (IL-15) is a pleotropic cytokine that has a profound effect on the proliferation, survival and differentiation of many distinct cell types. The IL-15 receptor complex has 3 subunits: the unique receptor chain IL-15 receptor alpha (IL-15Rα), and two receptor chains shared with interleukin-2 (IL-2) and/or other cytokines, referred to as IL-2 receptor beta (IL-2Rβ) and IL-2 receptor gamma/gamma common chain (IL-2Rγ/γc), respectively. To our knowledge, this is the first study to examine the effects of IL-15 in immortalized human cardiomyocytes. Data collected by RT-PCR shows mRNA expression of IL-15Rα, IL-2Rβ and IL-2 Rγ/γc in these cells. Additionally, western blotting for IL-15Rα, IL-2Rβ and IL-2 Rγ/γc confirms the presence of all three IL-15 receptors. Early experiments examining the effect of IL-15 on cardiomyocyte cell survival show a statistically significant protective effect of IL-15 on the survival of cells exposed to tunicamycin, a pharamacological endoplasmic reticulum (ER) stress inducing agent. These findings suggest that IL-15 signaling may be an important cardioprotective pathway that is involved in the cardiac ER stress response. As ER stress is a major component of multiple different cardiac pathologies, such as myocardial infarction, heart failure and diabetes, uncovering the molecular mechanism by which IL-15 protects the heart will allow for deeper understanding of the cardiac ER stress response.