bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1375: The Role of Nitric Oxide Synthase Uncoupling in Right Ventricular Hypertrophy and Failure
Authors: El-Haddad Hazim , Zaiman Ari L , Mozammel Shehzin , Kesari Priya , Hassoun Paul , Tuder Rubin M , Crow Michael T , Champion Hunter C
Published: July 2021

Right ventricular failure (RVF) is the leading cause of death in patients with severe pulmonary hypertension (PH). Despite its profound clinical consequences, little is known about right ventricular (RV) adaptation and failure within the context of PH. NOS uncoupling has been identified as a potential cause of LV failure. In this study, we hypothesized that NOS uncoupling plays an important early role in the RVH/RVF. We studied the mechanisms of RV failure in Dahl Salt-Sensitive (SS) rats using a RV pressure overload model produced by banding the main pulmonary artery (PAB). 11 days following PAB there was marked RV hypertrophy (0.24 vs 0.36) with marked reduction of both systolic and diastolic functions (RVESP 48 vs 22 mmHg, dP/dt IP 46 vs 80, Tau 29.7 vs 19.4 ms). Analysis of NOS activity and dimerization demonstrated that NOS uncoupling (shift from dimer to monomeric NOS with a concomitant drop in NOS activity) occurred within 4 days of PAB and persisted through day 11. To test the hypothesis that NOS uncoupling is secondary to BH4 oxidation, thus limiting NOS cofactor availability, we treated rats with supplemental BH4 (36 mg/kg/d po). We observed that NOS coupling was improved by BH4 treatment 4 days after PAB. Interestingly, we also observed that treatment with the phosphodiesterase 5 inhibitor sildenafil (100 mg/kg/d po) preserved NOS coupling at day 4 and the improvement persisted. Sildenafil treatment also resulted in a marked reduction in luminal chemiluminescence suggesting reduced ROS signaling and there was also a concomitant improvement in RV systolic (dP/dt/IP 74 vs 46) and diastolic (Tau 14.5 vs 29.7 ms) function and a reduction in cardiac hypertrophy (RV/(LV+septum) 0.28 vs 0.36). These data provide the first evidence that RV hypertrophy and failure are associated with NOS uncoupling that can be prevented by BH4 supplementation. Moreover, these data also provide the first evidence that NOS coupling can be prevented in the RV with sildenafil. These data suggest that sildenafil (likely via cGMP/PKG signaling) modulates NOS coupling, possibly via ROS reduction and preventing BH4 oxidation, or that BH4 synthesis itself is regulated by PKG signaling.
Abstract 1392: G Protein Coupled Receptor Kinase 6 Couples Stromal Cell Derived Factor-1
α
-CXCR4 Signaling to ERK/AKT Activation in Rat Cardiac Stem Cells
Authors: Sumanasekera Wasana , Dai Shujing , Anversa Piero , Bolli Roberto , Rokosh Gregg
Published: July 2021

Stromal cell derived factor-1
α
(SDF1) through the interaction with it receptor CXCR4 plays an important role in regulating the localization and function of hematopoietic stem cells. Recently, we have described SDF1-CXCR4 signaling in the heart and found it to regulate myocardial survival through the activation of ERK and AKT. CXCR4 is known to couple to the heterotrimeric G-Protein, Gi. The G-protein receptor kinase 6 (GRK6), may serve to uncouple the receptor from downstream signaling or to facilitate signaling via phosphorylation- dependent and independent mechanisms. To further understand cardiac SDF1-CXCR4 signaling and function we used rat clonal cKit
+
cardiac stem cells (rCSC) to assess the role of GRK6 in CXCR4 responses to SDF1. To specifically target GRK6 actions in SDF1-CXCR4 signaling and function in rCSCs we used siRNA to knockdown GRK6 in vitro. In untreated rCSCs, GRK6 specific siRNA significantly knocked down the expression of GRK6 by 50 – 60% which lead to significant decreases in levels of P-AKT, and P-ERK (about 70 - 90% respectively, P± 0.05) and increased levels of P-JNK, P-p38, and Caveolin1 in untreated rCSC. The treatment of rCSC with 25 nM SDF1 significantly increased GRK6 expression and this increase was attenuated by GRK6 specific siRNA. AKT, ERK, JNK, and p38 phosphorylation were significantly increased 5 min after rCSC treatment with 25nM SDF1 (0.5–3 fold increase, P<0.05) and GRK6 siRNA significantly attenuated SDF1activation (0.5 to 2 fold decrease, P<0.05). Our results indicate that in rCSC, GRK6 is necessary for SDF1-CXCR4 dependent ERK and AKT signaling Thus in rCSCs GRK6 serves predominantly in G-protein independent SDF1-CXCR4 signaling possibly through caveolar signaling complexes. In conclusion, in rCSCs SDF1-CXCR4 interaction activates downstream ERK and AKT activation via a GRK6 dependent mechanism. These findings will be important for the understanding of SDF1-CXCR4 regulation of rCSC function.
Abstract 1412: Sonic Hedgehog Gene Transfer Promotes Angiogenesis and Myocardial Regional Blood Flow via PKC Dependent Netrin Expression
Authors: Ahmed Rafeeq P , Haider Husnain K , Jiang Shujia , Afzal Muhammed R , Ashraf Muhammed
Published: July 2021

Background:
Hedgehog signaling effects endothelial and fibroblast cell migration and vascularization of various tissues during embryonic development. We propose here that stem cell based Shh gene delivery to the heart promotes neovascularization and enhances regional blood flow (RBF) in the infartced heart.

Methods and Results:
The pCMVShh plasmid was constructed and used for non-viral transfection of primary mesenchymal stem cell (MSC) culture. Successful transfection and expression of Shh in transfected MSCs (
Shh
MSCs) was confirmed by immunoflourescence, real-time PCR and Western blotting. Real-time PCR based array showed upregulation of angiogenic factors in
Shh
MSCs including netrin (>62-fold) and iNOS (>100-fold). The expression of netrin and iNOS was PKC dependent and was abolished in the presence of 2.5
μ
M chelerythrin. Transduction of iNOS gene into MSCs increased netrin expression implying a role for iNOS downstream of PKC. Western blot showed that 45-KDa PKM proteolytic subunit of PKC was involved in this activity.
Shh
MSCs conditioned medium was biologically active and caused greater migration and tube formation of human vascular endothelial
in vitro
in comparison with untransfected MSCs (
non-Shh
MSCs) conditioned medium. For
in vivo
studies, 70
μ
l DMEM without cells (group-1) or containing 1x10
6
male
non-Shh
MSC (group-2), or
Shh
MSCs (group-3) were transplanted into female rat model of acute coronary artery ligation. S
ry
-gene studies on day-4 after cell engraftment showed higher survival of
Shh
MSCs (
p
<
0.05
vs
non-Shh
MSC). Immunostaining for vWillebrand Factor-VIII and smooth muscle actin showed robust angiogenesis with distinctly large vessels in group-3 (
p
<
0.001
vs group-2). RBF measured by fluorescent microspheres was increased (
p
<
0.01
) and infarction size was attenuated (
p
<
0.05
) in group-3 as compared with control groups. Left ventricle ejection fraction (52.3±2.6%) and fractional shortening (21.8±1.2%) were more preserved in group-3 as compared with control groups.

Conclusions:
Ex-vivo Shh gene delivery improves angiogenesis and RBF in the infarcted heart to preserve global heart function through the upregulation of multiple angiogenic cytokines including iNOS and netrin.
Abstract 1414: Pim-1 Stimulates Cardiac Progenitor Cell Proliferation And Asymmetric Division
Authors: Cottage Christopher T , Tuck Savilla , Fischer Kimberlee , Gude Natalie , Muraski John , Sussman Mark A
Published: July 2021

Cardiac progenitor cells (CPCs) blunt cardiomyopathic damage and increase survival following adoptive transfer into hearts subjected to myocardial infarction (MI), but the initial survival, persistence, and long term engraftment of the donated cell population remains problematic. Previous studies from our group have demonstrated that transgenes driven by the
α
-myosin heavy chain gene promoter are expressed in the CPC population allowing for enhanced proliferation and survival. This study details a genetic engineering strategy to augment the salutary effects of CPCs through the use of a serine/threonine kinase named Pim-1 that promotes cellular proliferation and survival. Transgenic mice created with cardiac-specific Pim-1 overexpression (Pim-wt) exhibit enhanced Pim-1 activity in both cardiomyocytes and CPCs, both of which show increased proliferative activity assessed using BrdU or Ki-67 markers relative to non-transgenic (NTG) controls. However, CPC population number was not increased in the Pim-wt hearts during normal postnatal growth or after infarction challenge, suggesting that Pim-1 expression promotes asymmetric division resulting in maintenance of the CPC pool as well as expansion of the cardiomyocyte population. Localization and quantitation of cell fate determinants Numb and
α
-adaptin by confocal microscopy were employed to assess levels of asymmetric division in the CPC population. Polarization of Numb in mitotic phospho-histone positive cells demonstrates asymmetric division in 65% of the CPC population in hearts of Pim-wt mice versus 26% in NTG hearts after infarction challenge. Similarly, Pim-wt hearts had fewer cells with uniform
α
-adaptin staining indicative of symmetrically dividing CPCs, with in 36% of the CPCs versus vs. 73% in NTG sections. These findings define a mechanistic basis for enhanced myocardial regeneration in transgenic mice overexpressing Pim-1 kinase in the myocardial lineage cells.
Abstract 1461: Wnt2 Plays a Key Role in Cardiac Development Via a Non-Canonical Pathway
Authors: Onizuka Takeshi , Yuasa Shinsuke , Shimoji Kenichiro , Fukuda Keiichi , Ogawa Satoshi
Published: July 2021

Embryonic stem (ES) cells are a promising source of cardiomyocytes, but their clinical application has been hindered by the lack of selective differentiation methods. Although several signals are involved in heart development, the precise signals that mediate cardiomyocyte differentiation remain undetermined. Wnt family has a potent effect on the various organ development. To address this issue, we investigated the expression of wnt genes in the embryonic heart. Then we applied these findings to establish an efficient protocol to induce cardiomyocytes
in vitro
. (1) We analyzed TOP-EGFP mice to clarify whether canonical wnt signal pathway is important in the developing heart. TOP-EGFP mice are transgenic mice in which the EGFP gene is located under the β-catenin binding site so that the EGFP protein expresses when the canonical pathway is activated. They did not reveal any GFP in early stage heart, indicating that the canonical pathway is not involved. (2) Expression of non-canonical wnt was screened. Whole mount
in situ
hybridization of wnt2 and nkx2.5 (positive control) was performed at mouse embryo. Wnt2 was strongly expressed in the the heart-forming area in stages from E7.5 to E9.0. (3) We applied this embryonic wnt2 expression pattern to ES cell differentiation. Using siRNA we knocked-down wnt2 protein in various phases, which inhibited formation of beating EB, and decreased cardiac muscle genes only during the primitive stage (day 2– 4). Also adding wnt2 protein to embryonic cells in the appropriate phase led to a marked induction of cardiac specific genes. But wnt2 did not affect the mesodermal gene expression, Brachyury T and Mesp1, suggesting that Wnt2 does not affect the primitive development of the mesodermal progenitor cells. However, Wnt2 critically promotes cardiac specification after mesodermal induction and increases the eventual cardiac musculature. Wnt2 was strongly expressed in the heart-forming region. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by adding wnt2 protein and also to inhibit generation of cardiomyocytes by inhibition of wnt2 signaling. We concluded that wnt2 plays a key role in cardiac development via a non-canonical pathway.
Abstract 1462: Lineage Analysis of
Mesp1
Expressing Cells in Cardiac Conduction System
Authors: Rafie Reza , Hajrasouliha Amir R , Tavakoli Sina , Kotak Kamal , Pashmforoush Mohammad
Published: July 2021

Mesp1
is the earliest transcription factor detected in cardiac precursor cells considered to play a key role in their early specification. The lineage analysis, using
Mesp1-
Cre transgenic mouse line, has shown that there is a minor contribution of
Mesp1
non-expressing cells to the formation of ventricular conduction system. The purpose of the present study is to evaluate the contribution of
Mesp1
-expressing and non-expressing cells to the development of sinoatrial node, atrioventricular node and His bundle in embryonic, neonatal and adult heart. In this study, the contribution of
Mesp1
-expressing cells was evaluated by a lineage analysis using the cross of
Mesp1
-Cre and ROSA26R reporter mice. Different components of the cardiac conduction system were identified by a combination of histological (hematoxylin/eosin), acetylcholinesterase, and immunofluorescent stainings for specific markers (HCN4 and connexin40). Sinoatrial and atrioventricular nodes as well as His bundle are derived almost exclusively from
Mesp1-
expressing cells as evident by β-gal activity. The contribution of
Mesp1
non-expressing cells becomes prominent, though still less than
Mesp1
-expressing cells, in the distal components of cardiac conduction system (i.e. bundle branches). This pattern was consistent in embryonic and neonatal stages as well as adult hearts. Unlike the distal components of cardiac conduction system, which is composed of a mixed population of
Mesp1
-expressing and non-expressing cells, sinoatrial node, atrioventricular node and His bundle are derived almost exclusively from
Mesp1
-expressing cells.
Abstract 1465: Cardiomyocyte Hypertrophy Induces O-linked-β-N-acetylglucosamine Modification of PGC-1α and Augments its Transcriptional Activity
Authors: Facundo Heberty T , Pratridge Charles R , Prabhu Sumanth D , Jones Steven P
Published: July 2021

Background and Hypothesis:

PGC-1α (peroxisome proliferator activated receptor-gamma coactivator-1α) coordinately regulates fatty acid metabolism. The O-linked β-N-acetylglucosamine post-translational modification (O-GlcNAc) of proteins is a glucose-derived metabolic signal. We hypothesized that metabolic changes during cardiomyocyte hypertrophy might involve interaction between glycolysis and fatty acid metabolism, specifically via O-GlcNAc modification of PGC-1α.

Methods and Results:

Mechanical stretch (24 h at 4%; Flexercell FX-4000) in neonatal rat cardiomyocytes (n > 4/group) induced a significant (p<0.05) increase (113 ± 35% over No Stretch) in ANP mRNA, confirming induction of hypertrophy. Mechanical stretch significantly augmented (p<0.001; n = 5) global O-GlcNAcylation of several proteins, which was completely reversed by adenoviral overexpression of the deglycosylating enzyme (O-GlcNAcase). Mechanical stretch also augmented mRNA levels of O-GlcNAc transferase (OGT: adds O-GlcNAc to proteins) and glutamine:fructose aminotransferase (GFAT: rate-limiting step for the O-GlcNAc sugar donor), further indicating recruitment of O-GlcNAc signaling. Immunoprecipitation identified PGC-1α as an O-GlcNAc target in this cardiomyocyte hypertrophy model. Real-time (q)-PCR revealed that O-GlcNAc modification of PGC-1α correlated with elevated mRNA levels (n=4/group) of MCAD and COXIV-5b, implying transcriptional activation of PGC-1α.

Conclusions:

Cardiomyocyte hypertrophy induces O-GlcNAcylation of PGC-1α and represents a surprising and novel potential regulatory interaction between glycolytic and fatty acid metabolism.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1477: Identification and Functional Analysis of Protein Interaction Partners of the Estrogen Receptor Alpha in the Human Heart
Authors: Mahmoodzadeh Shokoufeh , Pham Hang , Davidson Mercy M , Regitz-Zagrosek Vera
Published: July 2021

Background and aims:
Estrogens (E2) are important regulators of physiological and pathological processes in various target tissues, including the human heart. Most of the biological actions of E2 are mediated by nuclear steroid receptors, the estrogen receptor alpha (ERα) and beta (ERα), which function as hormone-inducible transcription factors. ERs act in concert with other regulatory elements to mediate estrogenic effects. So far, only few cofactors of ERs have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and functionally analyzed the interaction partners of ERα.

Methods:
The yeast two hybrid screen (YTH) was performed in the presence of E2 with ERα as bait in a human heart cDNA library. Co-immunoprecipitation analysis (Co-IP) with human atrial proteins was carried out to confirm the interaction in a mammalian system. Immunofluorescence (IF) methods were employed on human atrial tissue samples as well as in cotransfected AC16 cells to analyze the co-localisation of ERα and its binding partners. Promoter reporter assays were performed to show the regulation of the identified interaction partner by ERα.

Results:
Yeast two hybrid screening of a human heart library revealed that ERα interacts with the atrial natriuretic peptide precursor A (NPPA). Retransformation experiments showed that NPPA can interact with ERα full-length and the ERα-EF domain in an E2-dependent manner. The LxxLL-motif of NPPA was found to interact with the ERα-EF domain in yeast. The interaction of NPPA and ERα was also confirmed using proteins derived from human atrium by Co-IP. IF analysis showed that E2 treatment of AC16 cells led to an increased translocation of NPPA and ERα in the nucleus of AC16 cells and also their co-localization there. Furthermore, expression analysis with NPPA luciferase promoter constructs showed a significant increase in the promoter activity in the presence of E2/ERα.

Conclusion:
We showed the interaction of ERα and NPPA in an E2-dependent manner. Our findings suggest that the E2-activated ERα and NPPA complex binds to specific regions on the NPPA promoter leading to transcriptional regulation of the NPPA gene.
Abstract 1509: Inducible TBX3 Overexpression As A Tool For Biopacemaker Engineering
Authors: Boink Gerard J , Bakker Martijn L , Verkerk Arie O , Bakker Diane , de Bakker Jacques M , Seppen Jurgen , Christoffels Vincent M , Tan Hanno L
Published: July 2021

Introduction: Currently constructed biopacemakers based on single gene transfer strategies function suboptimally, with periods of slow heart rates and instabilities during rest. In the sinoatrial node (SAN), the dominant native pacemaker, multiple genes are required for proper impulse formation and impulse propagation. TBX3 is an important regulator of the SAN gene program during heart development. We examined the effects of inducible TBX3 overexpression in adult hearts and in vitro we explored whether lentiviral TBX3 overexpression may be used in biopacemaker engineering.

Methods: In vivo atrial and ventricular expression levels of the connexin isoforms Cx43 and Cx40 (impulse propagation) and SCN5A were studied in mice with tamoxifen inducible overexpression of TBX3 using quantitative PCR analysis. Single neonatal rat cardiac myocytes were transduced with TBX3 expressing lentivirus to analyze the effects of TBX3 on action potentials and membrane currents (impulse formation) using the perforated patch-clamp technique.

Results: In vivo, Cx43, Cx40 and SCN5A, which are not or only moderately expressed in the native SAN, were severely down-regulated to 20%, 15%, and 40%, respectively, by TBX3 (n=12; p<0.01). Single neonatal cardiac myocytes overexpressing TBX3 exhibited faster spontaneous beating rates, along with decreased maximum diastolic potential, inward rectifier potassium current (I
K1
), and fast sodium current (I
Na
). These properties are typical of SAN pacemaker cells.

Conclusions: TBX3 can act as a strong repressor of the working myocardium gene program in the adult heart. Overexpression of TBX3 might be a useful tool in biopacemaker gene and cell therapy.
Abstract 1514: RXP-E: A Connexin43-Binding Peptide that Prevents Action Potential Propagation Block
Authors: Lewandowski Rebecca , Procida Kristina , Vaidyanathan Ravi , Jalife José , Nielsen Morten S , Taffet Steven M , Delmar Mario
Published: July 2021

Gap junctions (GJ’s) provide a low resistance pathway for cardiac electrical propagation. The role of GJ regulation in arrhythmias is unclear, partly due to the limited availability of pharmacological tools. Recently, we showed that a peptide called “RXP-E” binds to the carboxyl terminal of connexin43 (Cx43) and prevents chemically-induced uncoupling in Cx43-expressing N2a cells. Here, we used pull-down experiments to show that RXP-E binds to adult cardiac Cx43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes (NRVM’s). Separately, RXP-E was concatenated to a cytoplasmic transduction peptide for translocation into the cytoplasm (construct CTP-RXP-E). The effect of RXP-E on action potential (AP) propagation was assessed by high resolution optical mapping in monolayers of NRVMs, containing approximately 20% of randomly distributed myofibroblasts. Conduction velocity (CV) was 164 ± 8mm/sec (avg±SEM; n=12; pacing frequency 2Hz) in untreated cells, and 158±10mm/sec, (n=6) and 180±7mm/sec (n=10) in monolayers treated with CTP-RXPE or a scrambled version of the peptide (CTP-Scr), respectively (pNS). Exposure of either untreated, or CTP-Scr-treated monolayers to heptanol caused propagation block. However, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, AP propagation was maintained, albeit at a slower velocity (87±5mm/sec;n=4; P<0.001). Similarly, intracellular acidification (pHi=6.2) caused a loss of AP propagation in control or CTP-Scr monolayers; however, propagation was maintained in CTP-RXP-E treated cells, though at a slower rate (CV=93 ± 28mm/sec; n=4). Consistent with these results, patch clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium or calcium currents, nor did it alter the voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to:

bind cardiac Cx43;

prevent heptanol and acidification-induced uncoupling of cardiac GJ’s and

preserve AP propagation among cardiac myocytes.

RXP-E can be used to characterize the role of GJ’s in the function of multicellular systems, including the heart.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 1525: Atrial Rhythm Acutely Modulates Connexin43 Expression and Phosphorylation State in Human Atrial Fibrillation
Authors: Ram Rashmi , Ruehr Mary L , Bunnell Beth S , Gillinov A M , Van Wagoner David R
Published: July 2021

Background:
Adjacent areas of heterogenous conduction in the atria form a substrate for atrial fibrillation (AF). Connexin proteins (Cx) underlie conduction between myocytes; conduction is modulated by connexin expression and phosphorylation. Here we tested the hypothesis that left atrial (LA) expression and/or phosphorylation of Cx43 is distinctly regulated by atrial rhythm.

Methods:
Cx43 expression and phosphorylation was evaluated in LA tissue homogenates from 3 groups:

cardiac surgery patients with history of AF presenting for surgery in AF (AFAF, n=8);

history of AF but in sinus rhythm (AFSR, n=8);

patients with no history of AF (CTL, n=8). Groups were matched for age, LA size, EF (>50%), CAD, valve disease, and hypertension.

Results:
Western blot analysis revealed increased total Cx43 expression (t-Cx43) in AFAF (2.03±0.38) compared to AFSR (0.85±0.15, p=0.01) and SR (0.98±0.16, p=0.02) groups. Blots re-probed with an antibody detecting non-phosphorylated Cx43 at S368 (Np-Cx43) revealed a significant decrease in the Np-Cx43/t-Cx43 ratio, indicating increased S368 phosphorylation in the AFAF group (by 23% in AFAF vs. AFSR, p=0.046; by 12% in AFAF vs. CTL, p=0.051). Subcellular compartmentation of t-Cx43 was assessed in 3 samples per group using tissue fractionation and immunoblotting. Cytoskeletal t-Cx43 levels were increased in AFAF vs. AFSR and vs. CTL, ANOVA, p=0.016); t-Cx43 expression was not different between groups in membrane, cytoplasmic or nuclear fractions. Confocal microscopy revealed greater Cx43 lateralization in both AF groups compared to CTL.

Conclusion:
Our results (increased Cx43 expression and phosphorylation) suggest that AF rhythm
at the time of surgery
, more than the history of AF, has a
distinct
impact on LA Cx43 remodeling. Further the dynamic increase restricted to the cytoskeletal compartment suggests that cytoskeletal changes in AF may modulate connexin trafficking to the cell surface. This may affect the anisotropic properties of atrial conduction. Additional electrophysiological studies are needed to determine the functional contribution of lateralized Cx. Our study provides the first evidence linking acute rhythm status with regulation of Cx43 expression and phosphorylation in human atria.
Abstract 1532: Rare Genetic Variant in the hH1 cDNA Clone, R1027Q, Dramatically Alters the Cellular Electrophysiological Phenotype Ascribed to the Common Polymorphism, H558R, and a Brugada Syndrome Mutation, R1512W: Implications for Functional Studies
Authors: Tan Bi-Hua , Ackerman Michael J , Makielski Jonathan C
Published: July 2021

Background:
Previous studies in the
SCN5A-
encoded Na channel showed that amino acid substitutions at one position affected function of a Na channel containing a pathogenic mutation elsewhere. The first and most commonly used SCN5A cDNA clone (hH1) is the minor alternatively spliced transcript with an extra glutamine at position 1077 (Q1077), and also differs from the Na channel found in nearly all humans by virtue of a rare amino acid substitution (R1027Q). We hypothesized that R1027Q in previous studies using hH1 may have increased expression levels of the Brugada syndrome (BrS)-associated mutation (R1512W) and the common polymorphism (H558R).

Methods: We generated mutations in the alternatively-spliced
SCN5A
transcript containing Q1077 (as in the hH1 clone) as the wild type (WT) channel to generate channels with the following amino acid substitutions in addition to Q1077ins: H558R, H558R/R1027Q, R1512W, and R1027Q/R1512W. These channels were expressed in HEK-293 cells and current densities measured by whole cell voltage clamp.

Results: The current density for H558R was reduced dramatically (−7±2 pA/pF) whereas H558R/R1027Q exhibited normal current density (−297±56 pA/pF) comparable to WT (−310±52 pA/pF). The BrS mutation, R1512W-hH1, was characterized originally as having only subtle inactivation defects but normal current density. However, when we expressed R1512W in the patient’s true WT background (i.e. without R1027Q), the current density was attenuated significantly (−79±19 pA/pF) whereas the double mutant (R1027Q/R1512W) which replicated R1512W-hH1 showed nearly normal density (−180±21 pA/pF, −253±35 pA/pF for hH1, n=10–25 cells).

Conclusions: R1027Q, the rare genetic variant present in the hH1 cDNA clone, rescues expression of Na current for channels with H558R and the BrS1-associated mutation, R1512W. Using the hH1 clone which contains R1027Q as background (as is the case for nearly all previous studies of SCN5A mutations) may yield erroneous conclusions regarding the functional impact assigned to putative pathogenic mutations. It may be critical to study mutations in either true WT clones or in the patient’s own genetic background.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 1614: Inducible Nitric Oxide Synthase Upregulates Heme Oxygenase-1 (HO-1) in Myocardium by Enhancing Nrf2 Phosphorylation and Binding to the HO-1 Promoter
Authors: Li Qianhong , Ou Qinghui , Tan Wei , Wu Wen-Jian , Zhu Xiaoping , Lanceta Lilibeth B , Rokosh Gregg D , Guo Yiru , Bolli Roberto
Published: July 2021

Previous studies have shown that iNOS gene transfer leads to elevated expression of HO-1, which in turn protects against ischemic injury. However, the mechanism of this effect is unknown. Recently, Nrf2 has emerged as a key transcription factor in response to stress. Thus, we hypothesized that the upregulation of HO-1 by iNOS gene therapy involves an Nrf2-mediated pathway. Mice received injections in anterior LV wall of Av3/LacZ or Av3/iNOS; 3 days later, they were subjected to a 30-min coronary occlusion and 4 h of reperfusion. At 24 h after iNOS gene transfer (confirmed by iNOS immunoblotting), phosphorylated Nrf2 increased significantly in the myocardial nuclear fraction (+ 220% vs. LacZ group, n=6, P<0.05; Fig

); chromatin Nrf2-immunoprecipitation (Nrf2-ChIP) analysis in vivo showed that Nrf2 was specifically recruited to the HO-1 gene promoter (+ 4.7-fold vs. LacZ group, n=4, P<0.05; Fig

). At 3 days after iNOS gene transfer, myocardial HO-1 protein expression increased markedly (+ 2.6-fold vs. LacZ group, n=6, P<0.05; Fig

), but HO-2 did not change (n=3). The infarct-sparing effects of iNOS gene therapy were completely abrogated by disruption of Nrf2 (Nrf2−/−) (62.8±2.1% in Nrf2−/−+Av3/iNOS group, n=5, vs. 15.3±4.3% in WT+Av3/iNOS group, n=7; Fig

). We conclude that

iNOS upregulates HO-1 by enhancing Nrf2 phosphorylation and binding to the HO-1 promoter; and

targeted disruption of the Nrf2 gene completely abrogates cardioprotection after iNOS gene transfer.

These results provide unequivocal molecular genetic evidence for a necessary role of Nrf2 in the protection afforded by iNOS. Thus, activation of Nrf2 is a key molecular event that links iNOS to HO-1.
Abstract 1632: Tie2-Deficient Mice Exhibit Increased Susceptibility to Endothelial Apoptosis, Arteriolar Muscularization, and Pulmonary Arterial Hypertension in Response to Interleukin-6
Authors: Kugathasan Lakshmi , Ray Julie Basu , Deng Yupu , Han Robin N , Dumont Daniel , Stewart Duncan
Published: July 2021

Introduction: We have demonstrated downregulation in lung Tie2 receptor expression and activity in experimental models of PAH, implicating reduced Tie2 survival signaling in the pathogenesis of this disease. Therefore, we hypothesized that Tie2 deficiency would predispose to PAH due to increased endothelial cell (EC) apoptosis in response to environmental triggers.

Methods: Adult male Tie2± or littermate WT mice received saline or interleukin-6 (IL-6) at a rate of 200ng/kg/day via subcutaneous injections for 1 or 2 weeks.

Results: Significant elevation in RVSP (Fig.1

) was observed in IL-6 treated Tie2± mice (31 ± 3mmHg) compared to saline-treated Tie2 ± mice (25 ± 1mmHg) and IL-6 treated WT mice (22 ± 1mmHg). Following 1 week of IL-6 treatment, increased pulmonary microvascular EC apoptosis was observed by TUNEL in Tie2± mice compared to WT (6.3 ± 0.3% vs. 0.9 ± 0.1%, respectively; p < 0.01). Furthermore, after 2 weeks, a significant increase in pulmonary microvascular muscularization by SMA staining (Fig.2

) was seen in IL-6 treated Tie2±/mice compared to WT (36.4 ± 1.5% vs. 22.5 ± 1.1%, respectively; p < 0.01). Interestingly, Western blot analysis revealed a significant decrease in Angiopoietin-1 (Ang-1) level in the lung after 2 weeks of IL-6 treatment in Tie2± mice, and ELISA demonstrated a 60% decrease in Ang-1 secretion in vitro after incubation of pulmonary artery smooth muscle cells with IL-6 (p < 0.01).

Conclusions: Tie2± mice demonstrated enhanced susceptibility to pulmonary EC apoptosis, increased muscularization, and PAH following chronic exposure to IL-6, supporting an important protective role for endogenous Tie2 signaling in the lung microvasculature.

Figure 1.

Increased RVSP in Tie2
+/+
mice following 2 weeks of IL-6 treatment (*P < 0.05 compared to Saline Tie2
+/+
; † P < 0.01 compared to IL-6 WT)

Figure 2.

Increased pulmonary microvascular muscularization in Tie2
+/+
mice following 2 weeks of IL-6 treatment.
Abstract 1708: Decreased S-Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness
Authors: Tuday Eric C , Kim Jae Hyung , Dowzicky Phillip , Cortes Helena , Ryoo Sungwoo , Vandegaer Koenraad , Nyhan Daniel , Halushka Marc , Flavahan Nicholas A , Berkowitz Dan E , Santhanam Lakshmi
Published: July 2021

Vascular aging is marked by an increase in vascular stiffness. Tissue-type transglutaminase (tTG) enzymatically forms crosslinks between extracellular matrix proteins and may contribute to this pathobiology. Recently, it has been demonstrated that s-nitrosylation of cysteine residues within the enzyme leads to its inhibition. We hypothesize that tTG is more active in the aging vasculature due to decreased bioavailability of nitric oxide (NO) and impaired S-nitrosylation. tTG activity in aorta measured by the incorporation of biotin labeled pentylamine, a tTG substrate, into crosslinks was markedly increased in old (O) animals compared to young (Y) (3.2 fold increase, n = 6, p = 0.0018). S-nitrosylation of tTG, as determined by the biotin switch assay, was significantly reduced in O vs. Y rat aorta. tTG specific (ϵ-(γ-glutamyl)lysine) crosslinks measured by immunofluorescent staining was significantly increased in O vs Y rat aortic rings. Denitrosylation of tTG in Y rats with DTT increased tTG activity by 41% as did the NOS-inhibitor L-NAME. Treatment of O rats with cystamine (tTG inhibitor) lead to a decrease in vascular stiffness as measured by pulse wave velocity (5.55 ± 0.11 m/s to 4.99 ± 0.22 m/s, n = 6, p = 0.038). Given that tTG nitrosylation is likely NOS-3 dependent, we measured tTG activity and function in NOS-3 −/− mice. tTG activity was significantly increased in NOS-3 −/− vs. WT mice (18.94 ± 0.70 RU vs. 31.63 ± 2.06 RU, n = 4, p = 0.015). Treatment of NOS-3 mice with cystamine decreased aortic stiffness and increased carotid artery compliance. Finally, tTG activity in old human aortic tissue (collected post-mortem) is increased by 30% compared to young despite unchanged protein abundance. These data suggest that NOS-3 dependent NO constrains the activity of tTG by S-nitrosylation of the enzyme. Furthermore decreased NO bioavailability contributes to tTG mediated increases in vascular stiffness. Finally tTG promises to be a critical target in age-related vascular stiffness.
Abstract 1810: Vitamin K Epoxide Reductase Complex Subunit 1 (vkorc1) Gene Polymorphism is Associated with Atherothrombotic Complication after Drug-Eluting Stent Implantation : Two Center Prospective Cohort Study
Authors: Suh Jung-Won , Park Jin-Shik , Kang Hyun-Jae , Chae In-Ho , Choi Dong-Ju , Park Hun-Jun , Kim Pum-Joon , Baek Sang-Hong , Seung Ki-Bae , Kim Hyo-Soo
Published: July 2021

Single nucleotide polymorphisms (SNPs) of Vitamin K epoxide reductase complex subunit 1 (VKORC1) affect warfarin dose response. Recently, SNP of
VKORC1 +2255
was reported to have association with arterial vascular disease. However, it is not known whether SNP of VKORC1 affects clinical outcomes among patients who underwent drug-eluting stent (DES) implantation. We prospectively collected genomic DNA in patients who underwent DES deployment from Sep 2003 to Dec 2006 and compared clinical outcomes according to their VKORC1 genotype. The primary end-point was composite of atherothrombotic events [cardiac death, myocardial infarction, and non-hemorrhagic stroke]. Mean follow-up duration was 631±251 days. Genotyping was completed in 764 cases (TT genotype (n=640, 83.8%) vs. non-TT (CC or CT) genotype (n=124, 16.2%)). Non-TT group showed more composite events events than TT group (7.3% vs, 3.0%, p=0.032). In the Cox regression analysis, non-TT genotype of VKORC gene was a significant predictor of atherothrombotic events (Hazard ratio 2.56, 95% confidence interval 1.14±5.78). In the event-free survival analysis, non-TT group also showed significantly poorer cardiovascular events-free survival rate than TT group (p=0.02). VKORC1 genotype is associated with cardiovascular events in patients with DES implantation, suggesting the role of coagulation system.
Abstract 1818: Wnt5a Increases the Histone Demethylase Jmjd2b and Induces Epigenetic Remodelling in Adult Progenitor Cells
Authors: Koyanagi Masamichi , Iwasaki Masayoshi , Boeckel Jes-Niels , Zeiher Andreas M , Dimmeler Stefanie
Published: July 2021

Wnt signalling controls the balance between stem cell proliferation and differentiation and patterning throughout development. Non-canonical Wnts, such as Wnt5a or Wnt11, enhance cardiac gene expression of endothelial progenitor cells (EPC), mesenchymal stem cells (MSC) and bone marrow mononuclear cells suggesting that non-canonical Wnts regulate cardiac commitment. However, the underlying mechanism is unclear. Using microarray analysis of Wnt5a-induced gene expression in EPC, we discovered that Wnt5a (1.0 μM, 24hours) significantly increased a variety of enzymes, which play key roles in epigenetic remodelling by modifying histone acetylation and methylation. Specifically, JMJD2B (205%), REST (311%), HDAC5 and HDAC7A (716 and 381%), YY1 (304%), and PCGF2 (352%), were significantly increased by Wnt5a. Wnt5a induced up-regulation of JMJD2B in EPC and MSC was further confirmed by real-time RT-PCR. The member of the JmjC-domain containing family, JMJD2B was recently shown to demethylate trimethylated histone H3 at lysine 9 (H3K9me3), thereby removing repressive histone modifications. Therefore, we hypothesized that increased JMJD2B might epigenetically modify gene expression, open chromatin structures at silenced promoters and modulate the differentiation of adult progenitor cells. Indeed, Wnt5a treatment reduced the repressive chromatin mark H3K9me3 as shown by Western blot and immunohistochemistry whereas global active chromatine marks (acetyl-H3 and trimethyl- H3K4) remained unchanged indicating that Wnt5a shifts the balance towards active gene expression. Furthermore, chromatin immunoprecipitation revealed that Wnt5a treatment increased the binding of JMJD2B at the promoter of the cardiac gene ANF. In line with removal of repressive chromatin marks, Wnt5a significantly increased differentiation of progenitor cells to cardiomyocytes. These data demonstrate that non-canonical Wnt5a induces epigenetic remodelling by removing repressive chromatin marks and may, thereby, sensitize adult progenitor cells for acquiring a cardiac cell fate. Further studies will investigate the specific contribution of the novel epigenetic modulator JMJD2B for adult stem cell functions.
Abstract 1838: Neural Crest Stem Cells Supply Intrinsic Cardiac Adrenergic Cells and Contribute to Reinnervation after Myocardial Infarction in Mice
Authors: Tamura Yuichi , Ieda Masaki , Matsumura Keisuke , Kanazawa Hideaki , Kimura Kensuke , Ieda Yasuyo , Endo Jin , Arai Takahide , Kawaguchi-Manabe Haruko , Tomita Yuichi , Yuasa Shinsuke , Sano Motoaki , Kobayashi Kazuto , Ogawa Satoshi , Fukuda Keiichi
Published: July 2021

Cardiac nerve sprouting and heterogeneous reinnervation after myocardial infarction (MI) lead to arrhythmogenesis and sudden cardiac death. Despite its importance, little is known about the contribution of the intrinsic cardiac nervous system on reinnervation. We previously reported that neural crest stem cells (NCSC) reside in the heart, and can differentiate into neuron, glia, smooth muscle cells, and cardiomyocytes. The present study scrutinized the developmental origin of intrinsic cardiac adrenergic (ICA) cells, and investigated whether NCSC supply ICA and contribute to reinnervation after MI. (1) We analyzed two distinct double transgenic mice, encoding protein 0-Cre/Floxed-EGFP (P0-EGFP) and dopamine
β
-hydroxylase-Cre/Floxed-EGFP (DBH-EGFP), to map neural crest-derived cell fate and catecholaminergic cell fate, respectively. ICA cells, demarcated with tyrosine hydroxylase (TH, catecholaminergic marker)-immunopositive cells within ventricles, co-expressed EGFP in both P0-EGFP and DBH-EGFP mice. Most ICA cells were nucleated and round-shaped, although 5% of the cells extended nerve fibers. (2) NCSC were marked with EGFP in P0-EGFP mice, expressed nestin, musashi-1 and p75 nerve growth factor (NGF) receptor, and showed sphere formation. They frequently resided in the outflow tract, but also existed in all parts of the heart. (3) ICA cell number and TH mRNA expression within ventricles were increased in transgenic mice overexpressing NGF in the heart. (4) P0-EGFP mice were subjected to MI by coronary artery occlusion. EGFP-positive cells were markedly increased after MI, and more so in the border zone area than in the non-infarcted area. (5) Similarly, DBH-EGFP subjected to MI revealed an increase in EGFP-positive cells at the border zone area, and some of them extended neurites among the sprouting nerves. (6) TH and EGFP mRNA were increased in DBH-EGFP ventricles, concomitant with cardiac NGF upregulation. These findings demonstrated that ICA cells were derived from neural crest cells, and dependent on NGF synthesized in the heart. NCSC were mobilized to the infarcted myocardium, differentiated into TH-positive nerves, and contributed to reinnervation after MI.
Abstract 1880: Molecular Basis of Inward-Rectifier Potassium Current Upregulation in Human Atrial Fibrillation: Contrasting Mechanisms for I
K1
versus Constitutive I
KACh
Authors: Girmatsion Zenawit , Biliczki Peter , Kuehbacher Angelika , Scherer Mirella , Wimmer-Greinecker Gerhard , Nattel Stanley , Hohnloser Stefan H , Ehrlich Joachim R
Published: July 2021

Introduction:
Upregulation of Kir-based inward rectifiers I
K1
and constitutively active acetylcholine-regulated current (I
KACh,c
) is a prominent contributor to atrial fibrillation (AF) maintenance. This study assessed occurrence and molecular basis of altered I
K1
and I
KACh,c
in man.

Methods:
Left atrial samples from mitral valve repair patients (n = 21 AF; n = 20 SR) were studied. Patch-clamp was applied to record currents. Immunoblots and confocal microscopy were used to study protein content and localization. Quantitative real-time PCR was performed to study microRNA and mRNA levels.

Results:
I
K1
and I
KACh,c
densities were greater in cells from AF than SR patients (at −120 mV: 3.5 ± 2.6 vs. 2.1 ± 1.1 pA/pF for I
K1
and 1.3 ± 0.8 vs. 0.4 ± 0.3 pA/pF for I
KACh,c
). Kir2.1 protein (underlying I
K1
) was increased by AF (Fig.

) but Kir2.3 was unchanged. Kir2.1 mRNA was increased and Kir2.3 mRNA reduced. Kir3.1 and Kir3.4 (underlying I
KACh,c
) protein was unaffected. The inhibitory regulatory G-protein G
αi-3
was downregulated, potentially explaining increased free G
αγ
and I
KACh,c
. No change in other regulatory proteins (RGS4, calmodulin) or subcellular channel and regulatory proteins localization occurred with AF. Kir2.1 protein expression is known to be downregulated by the microRNA
miR-1: miR-1
expression was reduced by AF (Fig.

).

Conclusions:
Both inward rectifier currents I
K1
and I
KACh,c
are increased in human AF. I
K1
changes are caused by increased protein expression through transcriptional and post-transcriptional modifications related to
miR-1
downregulation, whereas the increase in I
KACh,c
is functional, possibly mediated by decreased inhibitory G-protein expression.
Abstract 1946: Regulation OF Entpdase1 (CD39) Expression by Fluid-phase Shear Forces
Authors: Hyman Matthew C , Ni Chih-Wen , Visovatti Scott H , Nam Douglas , Neral Mithun K , Jo Hanjoong , Pinsky David J
Published: July 2021

Sudden death due to thrombotic occlusion of a ruptured plaque is the leading cause of mortality in patients with atherosclerosis. Little is known about how the apyrase ENTPDase1 (CD39), a primary regulator of platelet activation
in vivo
, is affected at the plaque surface. By examining coronal sections of atherosclerotic plaques from
apoE
−/−
mice, we determined that CD39 is lost specifically in endothelium overlying the plaque surface, but not in adjacent endothelium. This led to the hypothesis that CD39 expression in the vascular wall may be driven by laminar, fluid-phase shear forces. To distinguish the effects of hyperlipidemia from that of shear forces, coronal aortic arch sections from wild type mice were examined at the greater curvature (an area defined by laminar shear stresses) and the ostia of the left subclavian artery (an area defined by non-laminar blood flow). Fluorescent intensity quantification revealed a near 6-fold induction of CD39 staining in areas exposed to laminar shear stresses. This finding was confirmed by confocal microscopy of
en face
aorta preparations as well.
In vitro
studies with human umbilical vein endothelial cells in a cone plate viscometer showed a 10.8-fold induction of CD39 mRNA by laminar shear stress (~15 dyne/cm
2
) when compared to fluid stasis (p<0.001). By comparison oscillatory flow (0 ± 5 dyne/cm
2
, 1 Hz) did not induce a significant change in CD39 mRNA when compared to static control. Western blotting revealed that the CD39 mRNA induced by laminar shear manifested as a more than 4.3-fold induction of protein (p<0.01). Flow cytometry confirmed the western blotting results showing a 3.8-fold induction of CD39 protein on the surface of cells exposed to laminar shear. These are the first data to show that CD39 is regulated by shear, and that it is lost from endothelium overlying atherosclerotic plaque in regions of turbulent flow. These results could be of great clinical relevance as they demonstrate that CD39, the enzyme that prevents aberrant platelet activation, is lost on the plaque surface. These data support the hypothesis that regional vascular zones are athero- and thrombo-resistant due to local ectonucleotidase expression modulated by dynamic/shear forces in flowing blood.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).