bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 220: miR-486 Mediates the Benefits of Exercise in Attenuating Cardiac Fibrosis
Authors: Lv Dongchao , Bei Yihua , Zhou Qiulian , Sun Qi , Xu Tianzhao , Xiao Junjie
Published: July 2021

MicroRNAs (miRNAs, miRs), a novel group of small non-coding RNAs, play important roles in cardiac fibrosis. Exercise-induced physiological cardiac growth is associated with hypertrophy and proliferation of cardiomyocytes. In addition, exercise has been shown to inhibit cardiac fibrosis. However, relative little is known about whether exercise could attenuating cardiac fibrosis via targeting miRNA. miR-486 is a muscle enriched miRNAs, however, its role in heart is relative unclear. The current study aimed to investigate the role of miR-486 in exercise-induced cardiac growth in a 3-week swimming training murine model as well as in the function of cardiac fibroblasts and production of extracellular matrix (ECM) using neonatal rat cardiac fibroblasts in primary culture. Our data showed that exercised mice displayed increased about three-fold expression of miR-486 in hearts as measured by microarray analysis and qRT-PCRs. EdU proliferation assays demonstrated that miR-486 mimics decreased (5.90%±0.57% vs 4.02%±0.27% in nc-mimics vs miR-486-mimics, respectively), while miR-486 inhibitor increased the proliferation of cardiac fibroblasts in vitro (5.87%±0.16% vs 9.60%±0.58% in nc-inhibitor vs miR-486-inhibitor, respectively). Although downregulation of miR-486 had no regulatory effect on α-sma and collagen-1 gene expression in cardiac fibroblasts, overexpression of miR-486 significantly reduced the mRNA level of α-sma (1.01±0.08 vs 0.28±0.04 in nc-mimics vs miR-486-mimics, respectively) and collagen-1(1.02±0.12 vs 0.58±0.09 in nc-mimics vs miR-486-mimics, respectively), indicative of attenuated activation of fibroblasts and reduced production of ECM. These data reveal that miR-486 is essentially involved in the proliferation and activation of cardiac fibroblasts, and might be a key regulator mediating the benefit of exercise in preventing cardiac fibrosis.
Abstract 229: Follistatin-like 1 Protects Heart From Post-infarction Rupture by Modulating Fibrogenesis
Authors: Maruyama Sonomi , Nakamura Kazuto , Walsh Kenneth
Published: July 2021

Rationale:
Stress injured heart secretes several cardiokines, including Follistatin-like 1 (Fstl1). Fstl1 is a member of SPARC (secreted protein, acidic and rich in cysteine) family. Fstl1 is strongly overexpressed in ischemic lesion and secreted to blood circulation in acute coronary syndrome. However, roles and mechanisms of Fstl1 in acute myocardial infarction have not been elucidated yet.

Methods and results:
To assess Fstl1 expression after myocardial infarction (MI), C57BL6 WT mice were underwent LAD permanent ligation. Heart and serum Fstl1 protein levels were increased after day 1 and peaked at 7 days after MI. Immunohistochemistry revealed that fibroblasts and myofibroblasts were the major Fstl1 expressing cells in infarcted area and border zone. However, neither cardiomyocytes nor leukocytes (neutrophil, macrophage) produced Fstl1. Fstl1 deleted mice in fibroblasts lineage (Fstl1-CKO mice) were generated by S100a4cre+/- xFstl1flox/flox mice. S100a4cre-/- xFstl1flox/flox mice were used as control. Fstl1 protein overexpression after MI was reduced 50% in Fstl1-CKO mice heart compared to control mice. Mortality rate after MI was significantly higher in Fstl1-CKO mice than control mice (50%;15 of 30 vs 34.8%; 8 of 23, respectively, log-rank test p=0.031). The majority of causes of death were cardiac rupture in acute period. Cardiac function, blood pressure and severity of inflammation were comparable between Fstl1-CKO and control mice. However, amount of myofibroblasts in Fstl1-CKO heart was significantly less than control mice. Overexpression of extracellular matrix proteins (collagen and fibronectin) in infarcted area was also diminished in Fstl1-CKO mice. Autocrine and paracrine functions of Fstl1 for fibroblasts were assessed in vitro study. Endogenous Fstl1 protein in neonatal rat cardiac fibroblasts (NRCFBs) was knocked down by siRNA fstl1. Migratory activity of NRCFBs was attenuated by Fstl1 ablation. Moreover, Fstl1 augmented TGF-β1 activated Smad2/3 signaling pathway in vivo and vitro.

Conclusions:
Increased Fstl1after MI prevents heart from cardiac rupture. This study revealed a novel function of Fstl1, promotes extracellular matrix generation to repair infarcted lesion.
Abstract 240: Strategic Distribution of the Mitochondrial Ca2+ Uniporter in Cardiac Mitochondria
Authors: de la Fuente Sergio , Vail Caitlin , Agra Elorm J , Holmstrom Kira , Sun Junhui , Mishra Jyotsna , Finkel Toren , Murphy Elizabeth , Sheu Shey-Shing , Csordas Gyorgy
Published: July 2021

Propagation of SR-derived Ca2+ signals to the mitochondrial matrix is an important tuning tool of cardiac energy metabolism (excitation-bioenergetics coupling). Effective activation of the low-affinity mitochondrial Ca2+ uniporter (mtCU) is supported by high [Ca2+] nanodomains where SR cisternae are tethered to mitochondria. Cardiac mitochondria have large inner membrane (IMM) surface with extensive cristae folding. However, recent comparative patch clamp studies found cardiac mtCU current density extremely low.

We hypothesized that efficient excitation-bioenergetics coupling would require strategic positioning of the mtCU. We thus analyzed the distribution of mtCU’s pore protein, MCU in rat and mouse cardiomyocytes and cardiac membrane fractions. Immunofluorescence (IF) using the commercial Sigma antibody showed significant non-mitochondrial (nuclear, cyto/sarcoplasmic) crossreactions as verified in MCU-KO MEFs and cardiomyocytes. To isolate mitochondrial labeling, we performed IF of isolated mitochondria (mtIF) and immunogold electron microscopy of freshly isolated cardiomyocytes. mtIF showed ~50% of MCU-positive structures colocalized with RyR2, in line with a location preference to SR-mitochondria associations. This distribution bias was also reflected in the immunogold TEM analysis. Moreover, WB showed both MCU and calsequestrin being enriched in the same sub-mitochondrial membrane fraction separated on sucrose gradient. The prevalence of MCU and even more the essential MCU regulator (EMRE) was higher in the mitochondria “contaminating” the heavy SR (SR-associated mito membranes) than in the crude mitochondrial fraction. [Ca2+] activation of mitochondrial Ca2+ uptake at similar IMM potentials was as effective in suspensions of SR-associated mito membranes as in the crude mitochondrial fraction.

Collectively, our data suggest a location bias of the functionally mandatory mtCU channel components MCU and EMRE toward SR-mitochondrial associations. As the bias is much stronger for EMRE, the component that is required for MCU to function as a channel, it raises the possibility that EMRE availability may be a physiological limiting factor in regional mtCU activity in the cardiac muscle mitochondria.
Abstract 260: Nanoparticle-mediated Simultaneous Targeting to Mitochondria and Inflammatory Monocytes Confers Additive Cardioprotection Against Myocardial Ischemia-reperfusion Injury
Authors: Ikeda Gentaro , Matoba Tetsuya , Ishikita Ayako , Egashira Kensuke
Published: July 2021

Background:
Targeting one mediator of myocardial ischemia-reperfusion (IR) injury failed to reduce infarct size in clinical trials, suggesting the necessity of the innovative strategy to target more than 2 mediators simultaneously. Previously, we have engineered poly(lactic acid/glycolic acid) nanoparticle containing cyclosporine A (CsA-NP) and pitavastatin (Pitava-NP), and reported that the former inhibits the opening of mitochondrial permeability transition pore (mPTP) and the latter reduces monocyte-mediated inflammation in IR hearts. Here we tested the hypothesis that nanoparticle-mediated simultaneous targeting to mPTP and monocytes confers additive cardioprotection against IR injury.

Methods and Results:
We produced mice deficient with both cyclophilin D (CypD, a key molecule for mPTP opening) and CCR2 (a receptor for monocyte chemoattractant protein-1), and found that the double-KO mice displayed dramatic reduction in myocardial IR injury model (Fig. A). Flow cytometric analysis and fluorescence molecular tomography showed that inflammation was markedly inhibited in CCR2-KO and CypD/CCR2-KO mice while residual inflammation was noted in CypD-KO mice. In CypD mice, Pitava-NP reduced recruitment of Ly6Chigh inflammatory monocytes and showed therapeutic effects (Fig. B). In contrast, CsA-NP reduced IR injury in CCR2-KO mice. Simultaneous treatment with CsA-NP and Pitava-NP at the time of reperfusion showed additive reduction in IR injury in wild-type mice (Fig. C).

Conclusions:
Nanoparticle-mediated simultaneous targeting to mitochondria and inflammatory monocytes can be developed as a novel therapeutic strategy in IR injury.
Abstract 276: Systemic Delivery of Microrna-146a Improves Endothelial Function and Ameliorates Atherosclerosis in Apolipoprotein E-deficient Mice
Authors: Ma Shuangtao , Tian Xiao Yu , Mu Chaofeng , Shen Haifa , Zhang Yunrong , Bismuth Jean , Wong Wing Tak
Published: July 2021

Rationale:
Endothelial inflammation is an early event in the development of atherosclerosis. The microRNA (miR)-146a showed anti-inflammatory effects in cultured endothelial cells. In this study, we investigated the therapeutic role of miR-146a in endothelial function and atherosclerosis in apolipoprotein E (ApoE)-deficient mice.

Methods and Results:
The miR-146a was packaged into a multistage vector (MSV) that was conjugated with an E-selectin-targeting thioaptamer (ESTA) to form an ESTA-MSV microparticle. The ApoE-deficient mice were fed with Western diet and injected through tail vein with 15μg of miR-146a loaded ESTA-MSV microparticles or vehicle vectors biweekly for 12 weeks. The expressions of miR-146a in aortic tissue was increased by five times at two weeks after injection. However, the expressions of miR-146a in heart, lung, liver, spleen, kidney, and skeletal muscle were not increased. The acetylcholine-induced endothelium-dependent relaxations in both carotid arteries and aortas were significantly improved in mice from miR-146a treated group compared with vehicle group. In addition, the endothelium-dependent contractions of carotid arteries were also improved by miR-146a treatment. The en face oil red O staining of whole aortas showed the plaque area was decreased in miR-146a-treated mice. Application of miR-146a also decreased the plaque size, macrophages, and T-lymphocytes, but increased the collagen deposition and vascular smooth muscle cells in the sections of aortic roots. The PCR results showed that expressions of chemokine (C-C motif) ligand (CCL)-2, CCL-5, and CCL-8 were decreased by miR-146a.

Conclusions:
E-selectin-targeting delivery of miR-146a improves endothelial function and inhibits atherosclerosis.
Abstract 28: Extracellular Matrix Coating Enhances Nanoparticle Uptake by Lung Epithelial Cells
Authors: Punnakitikashem Primana , Ravikumar Priya , Wu Jinglei , Nguyen Kytai , Hsia Connie , Hong Yi
Published: July 2021

Introduction:
Rapid uptake of drug-loaded nanoparticles (NPs) by lung cells is critical for effective pulmonary delivery of therapeutic agents because of the rapid pulmonary clearance mechanisms. We tested the possibility that coating NPs with extracellular matrix (ECM) derived from lung tissue enhances nanoparticles uptake by lung cells.

Methods and Results:
Fresh adult porcine lung tissue obtained from a local slaughterhouse was decellularized using detergent (sodium dodecyl sulfate) and then enzymatically digested into a soluble solution. The double emulsion method was utilized to fabricate core-shell poly(lactic-co-glycolic) (PLGA) nanoparticles loaded with bovine serum albumin (BSA) for protein release studies, 6-coumarin for cellular uptake studies, or human erythropoietin receptor (hEPOR) cDNA co-expressing green fluorescent protein (GFP) for
in vivo
gene expression studies. The ECM was coated onto the nanoparticle surface by physical adsorption using the ECM solution (100 μg/ml). There is no significant difference in the diameter, blood compatibility or cell toxicity between coated and uncoated NPs. ECM-coated NPs showed slower protein release rate than uncoated NPs as the ECM coating hindered protein diffusion into the solution. ECM-coated NPs showed significantly higher cellular uptake by human lung epithelial cells than collagen-coated or uncoated NPs. In addition, ECM-coated and uncoated NPs loaded with hEPOR-GFP cDNA were aerosolized and delivered by inhalation into rat lung. Following single inhalation using uncoated NPs, GFP expression in lung tissue progressively increased for up to 21 days. Using the ECM-coated NPs EPOR expression peaked at 14 days, then declined thereafter.

Conclusions:
Coating NPs with lung-derived ECM markedly enhances NP uptake by lung cells, delays the release of encapsulated protein or DNA, and shortens the duration of peak tissue gene expression compared to uncoated NPs. This NP formulation may be useful where more precise timing of delayed payload release is desired.
Abstract 3: Human CD34+ Cells Deficient in miR-377 Attenuates Cardiac Dysfunction and Repair After Ischemia-reperfusion Injury
Authors: Joladarashi Darukeshwara , Thandavarayan Rajarajan A , Suresh Babu Sahana , Jeyabal Prince , Krishnamurthy Shashirekha , Garikipati Venkata Naga Srikanth , Verma Suresh , Mackie Alexander , Khan Mohnsin , Vaughan Erin , Kishore Raj , Krishnamurthy Prasanna
Published: July 2021

microRNAs (miRNA/miR) dysregulation has been implicated in cardiac remodeling after injury or stress, however its effects on Human CD34
+
cells (hCD34
+
) biology and function, particularly in the context of cell-based therapy for cardiomyopathy is not fully understood. miRNA array data analysis indicates that miR-377 is a potential interest. pre-miR-377 transfection in EPCs inhibits their migration and vascular tube formation ability in HUVECs. Furthermore, hCD34
+
cells treated with miR-377 mimic showed decrease in expression of STK35 (a novel serine/threonine kinase). Moreover, STK35 is predicted as a potential target gene of miR-377 by computational analysis. Interestingly, in a relevant mouse model of ischemia reperfusion, intramyocardial transplantation of miR-377-silenced hCD34
+
cells promotes neovascularization leading to improvement in myocardial function and repair. Echocardiography showed LV function was significantly improved in mice receiving miR-377-silenced hCD34
+
cells compared to control-miR-transfected hCD34
+
cells. Taken together, these data suggest that inhibiting miR-377 in hCD34
+
cells promotes their angiogenic ability after transplantation into ischemic myocardial tissue, potentially through activation of STK35 signaling.
Abstract 304: Efficient Generation of Cardiac Purkinje Fiber-like Cells From ESCs by Activating cAMP Signaling
Authors: Tsai Su-Yi , Maass Karen , Lu Jia , Fishman Glenn I , Chen Shuibing , Evans Todd
Published: July 2021

Dysfunction of the cardiac conduction system (CCS) significantly impacts pathogenesis of arrhythmia, a major cause of morbidity and mortality. Strategies to derive cardiac conduction cells including Purkinje fiber cells (PC) would facilitate models for mechanistic studies and drug discovery, and also provide new cellular materials for regenerative therapies. A high-throughput chemical screen using CCS:lacZ and Contactin2:eGFP (Cntn2:eGFP) reporter embryonic stem cell (ESC) lines was used to discover a small molecule, sodium nitroprusside (SN), that efficiently promotes the generation of cardiac cells that express gene profiles and generate action potentials of PC-like cells. Imaging and mechanistic studies suggest that SN promotes the generation of PC from cardiac progenitors initially expressing cardiac myosin heavy chain, and that it does so by activating cAMP signaling. These findings provide a novel strategy to derive scalable PC, along with insight into the ontogeny of CCS development.
Abstract 32: Mitral Valve Interstitial Cells With the Serotonin Transporter (SERT)-LL Polymorphism are More Efficiently Activated by Serotonin
Authors: Driesbaugh Kathryn H , Grau Juan B , Bavaria Joseph E , Alkhaleel Farrah , Lai Eric K , Shaw Richard E , Gorman Robert C , Gorman Joseph H , Levy Robert J , Ferrari Giovanni
Published: July 2021

Prolapse of the Mitral Valve (MV) leaflet is a hallmark of several cardiac disorders such as Myxomatous MV Disease (MMVD) or ischemic MV regurgitation (MR), and is only treated surgically. Elevated levels of serotonin (5HT) have been associated with valvulopathies. Ongoing studies in our laboratory suggest that 5HT signaling plays an important role in the progression of MMVD. In addition, a 44 base polymorphism in the promoter region of the 5HT transporter (SERT) gene, designated as short (S) or long (L) allele, has been reported with Mendelian distribution, with LL resulting in increased 5HT reuptake compared to SS.

We hypothesized that patients with MMVD have enhanced 5HT signaling due to a high frequency of SERT-LL. DNA was extracted from 152 MMVD surgical patients, genomic fragment analysis performed to determine allelic frequencies, and a chi-square statistical test completed. We show that surgical patients with MMVD have a higher than expected frequency of SERT-LL (34% (52 of 152) vs. 25%). Notably, the frequency of SERT-LL is particularly enhanced (51% (21 of 41) vs. 25%) (p=0.009) in MMVD patients under 60 years old.

Based on these findings, we tested whether MV interstitial cells (MVICs) expressing SERT-LL have increased susceptibility to 5HT-mediated activation. We demonstrate that SERT-LL MVICs respond to 5HT with a 4-fold increase in smooth muscle actin (SMA) mRNA expression, a marker of VIC activation, compared to a less than 1-fold increase in SERT-LS and SERT-SS cells. The SMA response is abrogated by specific pharmacological inhibitors of the 5HT receptors 5HTR2A and 5HTR2B, and SERT, as well as siRNA knockdown. Studies of 5HTR expression in cells of each genotype are in progress.

Due to the enhanced frequency of the SERT-LL genotype among MMVD patients, particularly within the younger subset, and increased susceptibility of SERT-LL MVICs to 5HT, we postulate that the SERT-LL genotype may contribute to an increased risk of rapid disease progression by increasing the efficiency of 5HT signaling and uptake in MVICs resulting in extracellular matrix production leading to prolapse and impaired valve function. The SERT-genotype may constitute a novel means of identifying patients who may benefit from pharmacotherapy that alter 5HT-related mechanisms.
Abstract 342: Lack of Lipid Phosphate Phosphatase 3 Promotes Cardiac Dysfunction and Early Mortality in Mice
Authors: Panchatcharam Manikandan , Chandra Mini , Maxey Benjamin , Day Alicia , Escalante-Alcalde Diana , Miriyala Sumitra
Published: July 2021

Lysophosphatidic acid (LPA) is a naturally occurring glycerophospholipid and has been reported to increase heart rate and left ventricular pressure in the heart
in vivo
. The primary route of circulating LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin (ATX). Lipid phosphate phosphatase-3 (LPP3) is a plasma membrane enzyme that regulates the availability of LPA by dephosphorylation. We made the novel discovery that tissue-specific deficiency of
Ppap2b
(gene that encodes LPP3) leads to embryonic lethality (endothelial LPP3), exaggerates vascular inflammation, increases heart rate, enhances endothelial permeability, and promotes the development of the neointima after vascular injury. Based on our earlier reports, we have generated mice that specifically lack LPP3 in cardiomyocytes. These mice showed early mortality ~8 months due to cardiac dysfunction. Whereas lack of LPP1 or LPP2 (global knockouts) didn’t had any obvious phenotypic effect. Lack of LPP3 accounts for less than 10 percent activity in cardiomyocytes purified from the Myh6-Ppap2b
Δ
, which augments our previous finding that the other two LPP isoforms have a lesser role in the cardiovascular system. Blood pressure was similar in Ppap2b
fl/fl
(96 ± 9 mmHg; n = 19) and Myh6-Ppap2b
Δ
mice (92 ± 7 mmHg; n = 19), although heart rates were significantly higher in Myh6-Ppap2b
Δ
3-month old mice (642 ± 21 bpm, compared to Ppap2b
fl/fl
with 600± 17 bpm; P<0.001). Knockdown of LPP3 enhanced cardiomyocyte hypertrophy induced by LPA based on analysis of sarcomere organization, cell surface area, levels of fetal genes ANP and BNP, and ANF release from nuclei, which are hallmarks of cardiomyocyte hypertrophy, indicating that LPP3 negatively regulates cardiac dysfunction caused by LPA. We observed an increase in ATX levels accompanied by a decrease in LPP3 expression following infarction in the myocardium of Ppap2b
fl/fl
mice. Infarction induced expression of IL-6 and KC, were 3 ± 0.5-fold and 2 ± 0.6-fold higher, respectively, in Myh6-Ppap2b
Δ
mice. Analysis of plasma by cytokine antibody array confirmed the elevation in IL-6 and KC, whereas G-CSF and sICAM-1 appeared lower than in Ppap2b
fl/fl
.
Abstract 35: Pseudo-knockin Mice Expressing JPH2-A399S Develop Cardiac Hypertrophy by Magnetic Resonance Imaging
Authors: Quick Ann P , Beavers David L , Showell Jordan , Philippen Leonne E , Landstrom Andrew P , Morris Shaine A , Pautler Robia G , Wehrens Xander H
Published: July 2021

Background:
Dysfunctional intracellular Ca2+ handling has been implicated in adverse cardiac remodeling leading to hypertrophy and failure. Recent evidence has linked mutations in the Ca2+ handling protein Junctophilin-2 (JPH2) with the development of hypertrophic cardiomyopathy (HCM). However, the mechanism remains unknown.

Objective:
To use advanced in vivo imaging modalities in conjunction with biochemical techniques to determine the mechanism of hypertrophic remodeling in a murine model hosting a novel JPH2 mutation.

Methods and Results:
1. Pseudo-knockin (PKI) Mice: transgenic mice with the JPH2-A399S mutation (or WT JPH2) containing inducible shJPH2 were dosed with tamoxifen to knock down the combined levels of JPH2 to near WT expression. 2. MRI: A399S and WT PKI controls were imaged at 2 months post injection at which time body weight was similar for mutants (29.8±.81g) and controls (31.8±1.29g). Intragate (Bruker) was used to obtain FLASH cine images and EKG-gated tagged images were obtained to determine strain. MRI post-processing and measurements were performed using Amira and Diagnosoft software. A399S PKI mice exhibited significantly increased left ventricular mass (2.96±.21g/kg; n=4) compared to controls (2.27±.08g/kg; n=3) and max diastolic septal thickness (1.39±.05mm; n=9 versus 0.97±.00mm; n=4; P<0.01). Mutants trended toward decreased septal strain (-8.97±1.04; n=4) compared to the control (-10.28; n=1) indicative of reduced regional contractility 3. Biochemistry: stress markers were measured by qPCR. Average BNP was increased over 3 fold. Larger sample size is needed to reach significance. Western blot showed no significant change (p=.78) in phosphorylated Ca2+/calmodulin-dependent protein kinase II, which is often activated by aberrant Ca2+ signaling.

Conclusions:
Our data show that the JPH2-A399S mutation leads to septal hypertrophy in PKI mice. This suggests that defects in JPH2 are sufficient to induce pathological cardiac remodeling. Despite the role of JPH2 in Ca2+ handling, this form of hypertrophy does not appear to be mediated by traditional Ca2+ signaling. Further studies will focus on alternative Ca2+-dependent pathways to elucidate the molecular mechanisms of these hypertrophic changes.
Abstract 367: Lysyl Oxidase Expression by Cardiac Fibroblasts is Regulated by Integrin-mediated Signaling
Authors: Gao Albert , Black Lauren D
Published: July 2021

Cardiac fibrosis following myocardial infarction (MI) leads to reduced cardiac function, and contributes to heart failure and mortality. Recent studies shown the extent of adverse remodeling may be mitigated by therapeutic strategies which regulate cardiac fibroblast mediated-remodeling. Since cross-linking by lysyl oxidase (LOX) increases following MI and alters the mechanical properties of the infarct, it is critical to characterize how its expression is regulated by CFs post-MI. While LOX expression is attributable to TGF-β1 signaling, we hypothesize that changes in the stiffness and composition of the ECM can also alter LOX expression via integrin-mediated signaling. To investigate this, we isolated CFs from healthy left ventricle (LV) and infarcted cardiac fibroblasts (ICFs) from 1 week post-MI LV and cultured them on tissue culture plastic (TCP) and collagen I-coated plates (COL) in serum-free media for 48 hours to assess the expression of genes associated with LOX signaling, fibrosis, and myofibroblast activation. Our results show an upregulation of LOX gene expression in both CFs and ICFs when cultured on COL and this is further emphasized with the presence of TGF-β1 (Fig. 1A). Gene expression of col1α1, integrin β1 subunit and αSMA (Fig. 1B-D) also exhibit similar upregulation. Ongoing studies will investigate how altered substrate stiffness and composition affect gene expression of LOX and other genes associated with fibrosis. By understanding the effect of the physical microenvironment on the expression of fibrotic genes including LOX, we aim to develop novel therapeutic strategies to attenuate cardiac fibrosis and thus improve cardiac recovery following MI.
Abstract 380: Resident Cardiac Fibroblasts Give Rise to Periostin+ Myofibroblasts Which are the Primary Mediators of Cardiac Fibrosis
Authors: Kanisicak Onur , Khalil Hadi , Karch Jason , Brody Matthew , Lin Suh-Chin , Sargent Michelle , Molkentin Jeffery D
Published: July 2021

Resident cardiac fibroblasts (CFs) are potential therapeutic targets in treating or preventing heart failure since they play a critical role in cardiac remodeling and fibrosis after injury or with prolonged stress stimulation. Heterogeneity among activated fibroblasts within the heart has been noted by a number of previous studies in the literature. In addition to resident CFs, many cell types such as endothelial, perivascular and bone marrow cells have been suggested to go through a mesenchymal transition and acquire a myofibroblast-like phenotype during disease conditions. Hence, the cellular origin of the activated myofibroblast within the heart remains uncertain, in part because of a lack in reliable genetic strategies to define cellular lineage. Recent studies suggest that epicardial precursor cells expressing transcription factor 21 (Tcf21) give rise to resident CFs in the adult heart. In addition, the secreted matricellular protein periostin (Postn), appears to be expressed only within activated fibroblasts (myofibroblasts) within the heart. Here we used Tcf21-MerCreMer (Tcf21MCM) knockin mice and Postn-MerCreMer (PostnMCM) knock-in (KI) mice to lineage trace resident CFs and myofibroblasts with injury stimulation. To account for other potential cellular lineages giving rise to fibroblasts in the heart we also performed lineage tracing with the mouse genetic models including LysM-Cre (macrophage), ckit-Cre (bone marrow), Tie2CreERT2 (endothelial) and Myh11-CreERT2 (smooth muscle) in conjunction ROSA26 (R26) locus based loxP inactivated reporter alleles. Results of this study indicate that the Tcf21+ resident CFs are the predominant source for the activated periostin+ MFs which are the key mediators of extracellular matrix (ECM) production and ECM stability in heart whereas the contribution of other lineages to MFs are minimal. Additionally, we have performed single cell RNA sequencing on TCF21+ and Postn+ isolated CFs pre and post myocardial injury in order to define the fibroblast lineage itself at greater molecular depth.
Abstract 384: Leucine, Serine and Methionine Differentially Affect Protein Synthesis in Rat Heart
Authors: Karlstaedt Anja , Vasquez Hernan , Taegtmeyer Heinrich
Published: July 2021

Alternating flux through metabolic pathways enables the heart to rapidly adapt to stress. As a consequence, compensatory mechanisms are often associated with changes in mitochondrial function and cellular redox state. The collective importance of these metabolic changes to energy provision and protein turnover remains unknown. We therefore integrated radioactive tracer studies with a mathematical model of cardiac metabolism - CardioNet. We assessed through flux rate analysis the impact of altered amino acid supply on mitochondrial metabolism. In combination with leucine, both serine and methionine resulted in a two- to threefold increased variance of mitochondrial flux rates. Next we used the isolated working rat heart to determine whether supply of glucose and leucine, in combination with either serine or methionine at different concentrations, affects cardiac protein turnover and energy provision. While glucose remained the major energy source, oxidation of leucine contributed to ATP production. Methionine, but not serine, markedly increased both myocardial oxygen consumption and cardiac power at normal physiologic concentrations. Protein synthesis rates (units given in μmol/min/g dry wt) did not significantly differ from leucine alone (0.037±0.001) or leucine in combination with serine (0.038±0.003) or methione (0.045±0.007). However, with increased serine supply, protein synthesis significantly increased (0.0907±0.009) compared to methionine (0.059±0.006, p=0.024) or leucine only (0.015±0.002, p=0.031). The results suggest that myocardial protein synthesis is differentially regulated by specific amino acids.
Abstract 391: Endogenous Drp1 Modulates Cardiac Respiration Through mPTP and Independent of Fission
Authors: Zhang Huiliang , Bisetto Sara , Sheu Shey-Shing , Wang Wang
Published: July 2021

Background:
The cardiac mitochondria exhibit a stable morphology with a rather low level of dynamic changes. However, fission and fusion proteins, such as dynamin-related protein 1 (DRP1) are abundant in the heart. Whether these proteins bear other functions in the heart than mitochondrial dynamics regulation are largely unknown. We hypothesize that endogenous DRP1 in the heart regulates mitochondrial respiration independent of fission.

Methods:
Mitochondrial respiration was determined by measuring the OCR with Seahorse assay or Clark type electrode in adult rat cardiomyocytes or mitochondria isolated from adult mouse heart. Confocal imaging was used to quantify mitochondrial morphology in adult cardiomyocytes and H9C2 myoblasts. To evaluate the role of mitochondrial permeability transition pore (mPTP), we monitored superoxide flashes (SOF) and laser-induced mPTP openings, and used cyclophilin D knockout mice (CypD KO). Mitochondrial ROS and Ca2+ were also monitored.

Results:
Inhibiting the DRP1 GTPase activity by Mdivi-1 or overexpression of the dominant-negative mutant (DRP1-K38A) induced mild mitochondrial morphological changes in adult cardiomyocytes, and inhibited mitochondrial respiration. Modulation of fission/fusion by overexpressing DRP1 or treating cells with S3, a compound facilitates fusion, exhibited significant morphological changes, but failed to influence respiration. Therefore, endogenous DRP1 activity may regulate respiration in the heart and this effect is dissociated with morphological changes. Further, inhibiting DRP1 activity attenuated the frequency of SOF, indicating decreased transient mPTP openings, delayed laser-induced permanent mPTP opening, and increased mitochondrial Ca2+. Inhibiting DRP1 activity decreased mitochondrial ROS levels. The role of DRP1 inhibition on respiration absents in CypD KO myocytes, suggesting the involvement of mPTP in the modulation of respiration by endogenous DRP1.

Conclusion:
These results suggest that endogenous DRP1 positively regulates respiration in the heart. This effect is likely independent of its role in mitochondrial fission. DRP1 regulation of respiration may involve transient opening of mPTP and contribute to mitochondrial Ca2+ and ROS signaling.
Abstract 393: MCL-1 Promotes Survival and Influences Mitochondrial Dynamics in Cardiac Myocytes
Authors: Moyzis Alexandra G , Thomas Robert L , Kuo Jennifer , Gustafsson Åsa B
Published: July 2021

The BCL-2 family proteins are important regulators of mitochondrial structure and integrity. MCL-1 is an anti-apoptotic BCL-2 protein that is highly expressed in the myocardium compared to the other anti-apoptotic proteins BCL-2 and BCL-X
L.
Recently, we reported that MCL-1 is essential for myocardial homeostasis. Cardiac-specific deletion of MCL-1 in mice led to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Surprisingly, MCL-1 deficient myocytes did not undergo apoptotic cell death. Instead, the cells displayed signs of mitochondrial deterioration and necrotic cell death, suggesting that MCL-1 has an additional role in maintaining mitochondrial function in cardiac myocytes. Similarly, deletion of MCL-1 in fibroblasts caused rapid mitochondrial fragmentation followed by cell death at 72 hours. Interestingly, the MCL-1 deficient fibroblasts retained cytochrome
c
in the mitochondria
,
confirming that the cells were not undergoing apoptotic cell death. We have also identified that MCL-1 localizes to the mitochondrial outer membrane (OM) and the matrix in the myocardium and that the two forms respond differently to stress. MCL-1
OM
was rapidly degraded after myocardial infarction or fasting, whereas MCL-1
Matrix
levels were maintained. Similarly, starvation of MEFs resulted in rapid degradation of MCL-1
OM
, whereas MCL-1
Matrix
showed delayed degradation. Treatment with the mitochondrial uncoupler FCCP led to rapid degradation of both forms. This suggests that the susceptibility to degradation is dependent on its localization and the nature of the stress. Our data also suggests that these two forms perform distinct functions in regulating mitochondrial morphology and survival. Overexpression of MCL-1
Matrix
promoted mitochondrial fusion in fibroblasts under baseline conditions and protected cells against FCCP-mediated mitochondrial fission and clearance by autophagosomes. Thus, our data suggest that MCL-1 exists in two separate locations where it performs different functions. MCL-1
Matrix
promotes mitochondrial fusion, which protects cells against excessive mitochondrial clearance during unfavorable conditions.
Abstract 394: Transient Bone Morphogenic Protein Antagonism Directs Differentiation of iPSCs into the Cardiac Neural Crest and cKit+ Myocardial Progenitor Lineages
Authors: Hatzistergos Konstantinos E , Takeuchi Lauro M , Saur Dieter , Seidler Barbara , Dymecki Susan M , Mai Jia Jia , Kanashiro-Takeuchi Rosemeire , Balkan Wayne , Hare Joshua M
Published: July 2021

Introduction:
The capability of cKit+ cardiac progenitor cells (CPCs) to participate in cardiomyocyte regeneration remains controversial, despite basic and clinical studies supporting such a role.

Hypothesis:
A non-permissive cardiac milieu minimizes the generation of cardiomyocytes from CPCs.

Methods:
We lineage-traced CPCs using novel dual-recombinase responsive indicator mice (cKitCreERT2;Wnt1::Flpe;RC::Fela) and iPSCs derived from cKitCreERT2;IRG (iPSCKit) mice.

Results:
Intersectional genetic fate-mapping of cKitCreERT2;Wnt1:: Flpe;RC::Fela embryos supported that cKit marks Wnt1-expressing cardiac neural crest (CNC) progenitors, emerging at ~E9.5 and contributing a limited number of cardiomyocytes. To decipher the mechanisms underlying cardiomyocyte differentiation of CPCs, we lineage-traced CPCs during stage-specific cardiogenic differentiation of iPSCKit. Ascorbate treatment promoted differentiation of cKit+ iPSC-derived embryoid bodies (EBs) into Nkx2.5+ myocardium, 45.5%±6.7% of which co-expressed the Cre-reporter EGFP (n=154 EBs; 12 preparations), suggesting that CPCs encompass fully competent cardiomyogenic progenitors. Noggin (or Dorsomorphin), a BMP antagonist transiently expressed in the heart at E7.5-E8.5 but not during CNC invasion, directed the differentiation of iPSCkit-EBs into Mesp1+/Isl1+/Nkx2.5+ cardiac mesoderm progenitors (p≤0.0001). Remarkably, the same signaling pathway subsequently directed EBs into the cKit+/Wnt1+/Pax3+/Mitf-H+/Isl1+/Nkx2.5+ CNC lineage (p≤0.0001), while suppressing the generation of WT1+/Tbx18+ epicardium (p<0.05). Stage-specific induction of Cre-recombination delineated that iPSCkit-derived CPCs encompass Mesp1–/cKit+/Nkx2.5+ CNC progenitors which contributed EGFP+ CNC derivatives, including Nkx2-5+ cardiomyocytes, to 60.7%±7.3% of spontaneously beating EBs (n=147 EBs; 12 preparations).

Conclusions:
Collectively, our data show that CPCkit are fully competent CNC-derived cardiomyogenic progenitors, whose differentiation to cardiomyocytes is minimized by a latent Noggin-mediated signaling pathway. Therefore exploiting CPCkit therapeutically, provides an important strategy for maximizing myocardial regeneration.
Abstract 405: Inhibition of Class I Histone Deacetylases at Reperfusion Attenuates Ischemia-reperfusion Injury and Modifies Mitochondrial Acetylation
Authors: Herr Daniel J , Aune Sverre E , Menick Donald R
Published: July 2021

Although rapid reperfusion of ischemic tissue is the treatment of choice for myocardial infarction, much of the resultant damage occurs as a consequence of reperfusion itself. Previously, we have shown that pretreatment with MS-275, a selective class I histone deacetylase (HDAC) inhibitor, preserves left-ventricular (LV) function and substantially reduces the area of infarcted tissue in isolated rat hearts subjected to ischemia-reperfusion (IR) injury. Here, we tested the hypothesis that MS-275 treatment at reperfusion reduces LV tissue damage and improves post-ischemic LV contractile function. To do this, hearts from male Sprague-Dawley rats were isolated and perfused ex vivo on a Langendorff perfusion apparatus. A saline-filled balloon was inserted into the left ventricle of the heart to monitor ventricular pressure development throughout the experiment. Hearts were subjected to 30 minutes of ischemia, followed by 60 minutes of reperfusion. MS-275 was administered during the entire reperfusion phase, and resultant functional data were compared to untreated hearts. There was no difference in any metric of pre-ischemic contractile function between groups. 10nM MS-275 administered at reperfusion significantly improved multiple measures of LV function, including dP/dtmax, -dP/dtmax, developed pressure and rate pressure product. We also observed a significant reduction in infarct area of treated hearts compared to control, as measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Unexpectedly, mass spectrometry analysis revealed significant changes in acetylation state of multiple mitochondrial enzymes. Administration of MS-275 during the reperfusion phase of IR is sufficient to partially rescue LV function from reperfusion-induced damage. This study emphasizes the importance of exploring class I HDAC inhibitors for protection against ischemia-reperfusion.
Abstract 412: Dissection of Thrombospondin-4 Domains Involved in Adaptive ER Stress Responsive Signaling and Secretory Pathway Functions
Authors: Brody Matthew J , Schips Tobias G , Kaniscak Onur , Karch Jason , Blair N. S , Molkentin Jeffery D
Published: July 2021

Thrombospondins are a family of stress-inducible secreted glycoproteins with well-characterized roles in the extracellular matrix and tissue remodeling. We recently reported critical intracellular functions for thrombospondin-4 (Thbs4) by activation of an adaptive endoplasmic reticulum (ER) stress pathway in cardiomyocytes, in part by promoting activation of activating transcription factor 6α (Atf6α). Here, we dissect the domains of Thbs4 that mediate interactions with ER proteins and activation of this adaptive ER stress response, and determine the domains of Thbs4 involved in secretory pathway functions. We show that the N-terminal laminin G like domain (LamG) of Thbs4 exhibits intracellular localization almost exclusively at the Golgi apparatus and is robustly secreted in cultured cardiomyocytes, indicating rapid flux through the secretory pathway. We also generated a full length Thbs4 with mutations in calcium-binding motifs within the type III repeat (T3R) domain, including mutations homologous to human disease-causing mutations identified in THBS5/COMP. While wildtype Thbs4 localized to the ER and post-ER vesicles and was actively secreted in cardiomyocytes, the Thbs4 calcium-binding mutant localized predominately to the ER and was not secreted. We identified BiP (Grp78) as a novel binding partner of Thbs4 in cardiomyocytes in vitro and in vivo, and utilized adenoviruses overexpressing Thbs4 domain mutants in cardiomyocytes to map the TSP-C domain (L-type lectin domain) of Thbs4 as the BiP-interacting motif. Additionally, the TSP-C domain of Thbs4 bound Atf6α and overexpression of the TSP-C domain alone was sufficient to induce the adaptive ER stress pathway in cardiomyocytes, activate Atf6α, and protect against ER stress-induced cell death. These studies characterize the functional domains of Thbs4 and aid in parsing out the intracellular and extracellular roles of thrombospondins. Taken together, data indicate critical functions for the TSP-C and LamG domains of Thbs4 in ER stress-responsive and secretory pathway functions, respectively.
Abstract 430: Circadian Control of Heart Rate
Authors: D’souza Alicia , Wegner Sven , Johnsen Anne Berit , Gill Eleanor , Cox Charlotte , Dobrzynski Halina , Wisløff Ulrik , Hart George , Piggins Hugh D , Boyett Mark R
Published: July 2021

Background:
Bradyarrhythmias occur more frequently at night. On the basis of heart rate variability this is attributed to high vagal tone. Here we tested the alternative hypothesis that an intrinsic circadian clock-driven remodelling of pacemaking ion channels underlies fluctuations in heart rate (HR).

Methods and results:
The occurrence of a circadian rhythm in HR was tested by placing nocturnal C57BL6/J mice under a strict 12/12h light-dark cycle and telemetry-based ECG intervals measured every 2 h for 48 h. Under these conditions, the R-R interval was rhythmic (n=10). To test whether this is caused by circadian rhythms in the expression of ion channels controlling HR, sinus node (SAN) biopsies were collected at time points corresponding to the minima (ZT0, subjective day) and maxima (ZT12, subjective night) of HR, as determined by ECG recordings. Real-time PCR normalised to
28s
demonstrated an elevated expression of the key pacemaking ion channel HCN4 that carries the pacemaker current
I
f
and genes encoding the Ca
2+
-handling proteins SERCA2a and RYR2 at ZT12 (
P
<0.05, n=10). Presence of clock machinery (essential transcription factors involved in setting intrinsic circadian rhythms) as potential regulators of ion channel oscillation were investigated in the SAN of

mPer1
Luc

mice which carry the 5’ upstream region of the
mPer1
gene (a key core clock component) fused to a luciferase gene.
mPer1-luc
bioluminescence was recorded in the isolated SAN using a light-tight photomultiplier tube assembly to reveal a circadian rhythm with a periodicity of 24 h (n=3). Disruption of the molecular clock by global knockout of core clock components
Cry1 and Cry2
abrogated the circadian cycling of
mPer1-luc
in the SN of

Cry1
-/-
/Cry2
-/-

double knockout

mice (n=3). Examination of 10[[Unable to Display Character:  ]]kb of the
Hcn4
promoter revealed a conserved consensus binding site for CLOCK and its heterodimer BMAL1, other essential transcription factors involved in setting intrinsic circadian rhythms.

Conclusions:
This is the first demonstration of a peripheral circadian clock in the cardiac pacemaker and circadian oscillations in key pacemaker mechanisms. Data reveal a novel regulator of SN function and the occurrence of bradyarrythmias at night.
Abstract 49: Mir-21 Contributes to Cardiac Aging by Targeting Pten
Authors: Wu Xiaoting , Wang Xiuzhi , Tao Lichan , Bei Yihua , Xiao Junjie , Li Xinli
Published: July 2021

Background—
Aging is among predominant risk factors for cardiovascular diseases and it also contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs, miRs) have been indicated in aging. However, the regulatory mechanisms of miRNAs which govern the aging progress are poorly understood.

Objective—
To identify miRNAs responsible for cardiac aging besides miR-34 family members.

Methods and Results—
miRNA arrays were used to determine the dysregulated miRNAs in cardiac aging using cardiac ventricles from mice at 8 weeks and 15months of age. Besides miR-34 family members, miR-21 was also found to be elevated 3-fold in ventricles from mice aged 15 months compared to mice aged 8 weeks. Over-expression of miR-21 in the neonatal rat cardiomyocytes (NRCM) shortened the telomere length, attenuated and strengthened the activity of telomerase and senescence-related beta[[Unable to Display Character: –]]galactosidase, respectively, which was similar to the aging caused by doxorubicin (DOX). Using the quantitative PCRs and Western blotting, phosphatase and tensin homologue (PTEN) gene was found to be negatively regulated by miR-21 in NRCM. Moreover, knockdown of PTEN using siRNA mimics the effects of miR-21 in pro-aging in NRCM. Interestingly, knockdown of miR-21, as well as up-regulation of PTEN, repressed NRCM aging induced by DOX.

Conclusions—
These findings suggest that ectopic up- or down-modulation of miR-21 may be an important regulatory mechanism governing cardiac aging by targeting PTEN. Inhibition of miR-21 represent a novel therapy for cardiac aging.
Abstract 70: Novel Multi-target Compound, Agonist of Adenosine Receptor and Inhibitor of Phosphodiesterase 5, Ameliorates Monocrotaline-induced Pulmonary Hypertension in Rats
Authors: Zapata-Sudo Gisele , Alencar Allan K , da Silva Jaqueline S , Barreiro Eliezer J , Fraga Carlos A , Ferraz Emanuelle B , Nascimento Jose H , Sudo Roberto T
Published: July 2021

Aims:
Pulmonary hypertension (PH) is a disease that results in right ventricular (RV) dysfunction and premature death. This work investigated the effects of LASSBio-1386, a compound with dual target, activation of the adenosine A
2A
receptor and inhibition of phosphodiesterase 5 in rats with monocrotaline (MCT)-induced PH.

Methods and Results:
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Male Wistar rats received a single intraperitoneal injection of MCT (60 mg/kg) for PH induction. Experimental groups were: control, MCT + vehicle (DMSO), MCT + LASSBio-1386 (100 μmol/kg/day p.o.) and MCT + Sildenafil (100 μmol/kg/day p.o.). Animals were treated with vehicle or drug for 14 days after the onset of disease (n = 6 per group). Treadmill test and transthoracic echocardiography were performed to access exercise capacity and cardiac function, respectively. Right ventricular systolic pressure (RVSP) and ratio between RV and body weight (RV/BW) were analyzed. Exercise capacity (m.kg) was reduced from 1336.2 ± 82.2(control) to 479.7 ± 75.5 (MCT+ vehicle) and recovered to 1357.0 ± 87.8 and 1221.0 ± 96.8 after treatment with LASSBio-1386 and sildenafil, respectively. Pulmonary acceleration time (PAT) (ms) was reduced from 45.3 ± 0.8 (control) to 21.9 ± 0.3 in MCT + vehicle group (
P
< 0.05) and restored to 42.9 ± 0.7 in MCT + LASSBio-1386 group, but it was only partially restored to 37.8 ± 1.3 (ms) in MCT + Sildenafil-treated group. RVSP (mmHg) was increased from 27.1 ± 0.7 (control) to 52.9 ± 1.5 in the PH rats and was reduced to 29.1 ± 1.0 and to 31.8 ± 0.6 after treatment with LASSBio-1386 and sildenafil. PH induced an increase of RV/BW (mg/g) from 0.62 ± 0.03 (control) to 1.81 ± 0.20 which was reduced to 0.70 ± 0.07 after administration of LASSBio-1386. Sildenafil treatment did not reverse the RV hypertrophy in PH rats (RV/BW = 1.52 ± 0.10 mg/g).

Conclusions:
LASSBio-1386 was effective to prevent RV dysfunction and exercise intolerance indicating important implications for ongoing clinical evaluation of multi-target drugs for the treatment of PAH.