bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification
Authors: Byon Chang Hyun , McDonald Jay , Chen Yabing
Published: July 2021

The expression of receptor activator of nuclear factor
κ
B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC)
in vitro
. Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations
in vivo
, we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H
2
O
2
), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H
2
O
2
-induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H
2
O
2
responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H
2
O
2
. To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H
2
O
2
-induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H
2
O
2
induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.
Abstract 1393: Inhibition of Cardiac Remodeling by Pravastatin is Associated with Amelioration of Endoplasmic Reticulum Stress
Authors: Zhao Hui , Liao Yulin , Minamino Tetsuo , Asano Yoshihiro , Asakura Masanori , Kim Jiyoong , Asanuma Hiroshi , Takashima Seiji , Hori Masatsugu , Kitakaze Masafumi
Published: July 2021

Background
We previously reported that prolonged endoplasmic reticulum (ER) stress contributes to progression from cardiac hypertrophy to heart failure. Statins have an inhibitory effect on cholesterol synthesis, oxidative stresses, protein synthesis and production of inflammatory cytokines, all of which could be associated with ER stress. However, it is unknown whether statins can ameliorate ER stress in heart disease. This study was designed to investigate whether pravastatin could inhibit cardiac remodeling and ameliorate ER stress caused by pressure overload or tumor necrosis factor
α
(TNF
α
).

Methods and Results
Cardiac hypertrophy was induced by transverse aortic constriction (TAC) for four weeks in C57BL/6 male mice. Either pravastatin (5 mg/kg/d, n=20, TAC+prava group) or its vehicle (n=20) was orally administered to mice. The ER stress signaling pathway was also studied in pressure-overloaded mice hearts and in cultured cardiomyocytes treated with TNF
α
(10ng/ml) for 24 hours. Four weeks after TAC, both heart-to-body weight ratio (8.68 ± 1.23 in TAC group, 6.92 ± 1.11 in TAC+prava group) and lung-to-body weight ratio (11.08 ± 2.58 in TAC group, 7.92± 3.56 in TAC+prava group) became significantly lower in pravastatin-treated mice than in the TAC group. Left ventricular fractional shortening and left ventricular ejection fraction (LVFS and LVEF) were larger in TAC+prava group (48.0±1.9 % and 80±1.9% respectively) compared with TAC group (LVFS and LVEF, 34.8 ±1.4% and 65 ±3%; P<0.01 VS TAC group each). Markers of ER stress such as an increase in ER chaperones and CHOP expressions and enhanced phosphorylation of eIF2
α
were observed in the hearts of TAC mice, while pravastatin treatment significantly blunted these changes. Pravastatin-treated TAC mice also showed a decrease of cardiac apoptosis. Cardiac expression of TNF
α
was increased in TAC mice, and TNF
α
induced ER stress in cultured neonatal rat cardiomyocytes, either of which was significantly inhibited by pravastatin.

Conclusions
These findings indicate that pravastatin inhibits cardiac remodeling in mice subjected to pressure overload, and this action is associated with inhibition of the ER stress signaling pathway.
Abstract 1417: Stem Cell Based Gene Transfer of GATA4 Fused with Cell Penetrating Peptide VP22 for Cardiac Repair
Authors: Bian Jing , Popović Zoran B , Forudi Farhad , Kiedrowski Matthew , Penn Marc S
Published: July 2021

We have recently demonstrated that GATA4 fused with cell penetrating peptide VP22 is an efficacious strategy for delivering a cardiac transcription factor to the myocardium in ischemic cardiomyopathy. We have similarly demonstrated that the delivery of SDF-1 to the heart soon after myocardial infarction (MI) leads to stem cell recruitment and preservation of cardiac myocytes. We hypothesized that by combining VP22:GATA4 and SDF-1 delivery to the myocardium we could recruit stem cells to the heart and force their differentiation following MI. We used the infusion of genetically modified mesenchymal stem cells (MSC) to deliver the SDF-1 and VP22:GATA4 to the heart after MI. MSCs isolated from bone marrow and stably transduced with Lentiviral VP22:GATA4 or SDF-1 were infused through tail vein one week after LAD ligation in Lewis rat model. Experimental groups consisted of Saline, Control MSC, SDF-1 MSC, VP22:GATA4 MSC or combined VP22:GATA4 and SDF-1 MSC. In each situation 2 million MSC were infused, but no more than 1 million of any specific genetically modified MSC was infused. Ejection fraction was quantified by echocardgraphy 2 and 6 weeks after cell infusion. At 2 w after cell infusion we observed a 23 ± 1% and 40 ± 5% improvements in ejection fraction of MSC and SDF-1 groups relative to saline controls. We observed a 23 ± 4% decline in function in the VP22:GATA4 group and no improvement in the combination group. At 6 weeks after cell infusion, we observed no significant improvements in the MSC group and a 47 ± 10% and a 20 ± 4% improvements in the SDF-1 and combo groups, the function in the VP22:GATA4 group improved to have no significant difference compared to saline control. Intravenous infusion of MSC can be used to deliver natural and chimeric proteins to the myocardial tissue following MI. The initial decline in function in those animals that received VP22:GATA4 expressing MSC suggest that forced induction of cardiac differentiation soon after MI may have detrimental effects on myocardial tissue leading to increased cardiac myocyte death and adverse remodeling. These findings have implications in strategies being developed to induce myocardial regeneration soon after MI.
Abstract 1432: Cardiomyocyte Myostatin Contributes to Skeletal Muscle Wasting in Heart Failure
Authors: Heineke Joerg , Auger-Messier Mannix , Sargent Michelle , York Allen , Welle Stephen , Molkentin Jeffery D
Published: July 2021

Introduction:
Skeletal muscle wasting during heart failure constitutes a major therapeutic challenge. The TGFβ superfamily member myostatin is a negative regulator of skeletal muscle growth. For example, elimination of myostatin (MSTN) in adult mice through an inducible Cre/lox recombination strategy has been shown to increase skeletal muscle mass by about 25%. Previous studies identified skeletal muscle and to a lesser extent cardiac and fat tissue as the source of MSTN production in the body. MSTN is produced as a precursor protein, which has been suggested to constitute the main reservoir of the protein in skeletal muscle. In mouse serum, however, MSTN is abundantly present in its mature form, which consists of the C-terminal fragment of the precursor protein.

Results:
We detected high levels of the mature MSTN protein (MM) in the mouse myocardium by western blotting. Interestingly, MM was significantly upregulated in the myocardium of mice subjected to long-term myocardial pressure overload (TAC, 12 weeks; protein levels: sham 100±22% vs. TAC 218±18%, p<0.01). In contrast, MM was barely detectable in mouse skeletal muscle. Immunhistochemical staining confirmed enhanced cardiomyocyte MSTN production after TAC. To determine the impact of cardiomyocyte MSTN on skeletal muscle growth during heart failure, we crossed cardiomyocyte specific Nkx2.5-Cre mice with mice in which the MSTN exon3 was flanked by loxp sites to eliminate expression of mature MSTN selectively in cardiomyocytes (CKO mice). While CKO mice did not have significant changes in skeletal muscle mass after a sham operation (e.g. quadriceps, normalized to tibia length: sham control 111±3.8 g/cm vs. sham CKO 106±4.3 g/cm), a 16% increase in skeletal muscle mass was observed in CKO mice after longterm TAC (quadriceps: TAC control 100±3.3 g/cm vs. TAC CKO 116±5.3 g/cm, p<0.05). In line with these results, mice with cardiomyocyte specific overexpression of MSTN (MSTN-Tg) showed a reduction in skeletal muscle mass (quadriceps: control 91±2.5 g/cm vs. MSTN-Tg 82±1.5 g/cm, p<0.05).

Conclusion:
Myocardial MSTN contributes to the development of skeletal muscle wasting in heart failure, most likely through an endocrine mechanism involving its secretion into the circulatory system.
Abstract 1456: Microrna-499 Regulates (cardio)myocyte Differentiation in Human-Derived Cardiomyocyte Progenitor Cells
Authors: Sluijter Joost P , van Mil Alain , van Vliet Piet , Metz Corina H , Doevendans Pieter A , Goumans Marie-José
Published: July 2021

To improve regeneration of the injured myocardium, it is necessary to enhance the intrinsic capacity of the heart to regenerate itself and/or replace the damaged tissue by cell transplantation. One of the potential sources for cell replacement therapy are heart-derived progenitor cells. Cardiomyocyte progenitor cells (CMPCs) are obtained from human fetal hearts and adult human biopsies, easily expanded in culture, and efficiently differentiated into beating cardiomyocytes. Recently, several studies have demonstrated that microRNAs (miRNAs) are important for transcriptional regulation during angiogenesis, heart development, and maintenance of stem cell populations by translational repression. We hypothesize that, since miRNAs regulate stem cell maintenance and differentiation, miRNAs are involved in differentiation of human cardiomyocyte progenitor cells
in vitro
.

Methods and results:
Human fetal CMPCs were isolated, cultured, and efficiently differentiated into beating cardiomyocytes. miRNA expression was profiled in proliferating and differentiated CMPCs. We observed that miRNA-499 was highly upregulated upon differentiation (>100 fold, microarray and qRT-PCR). Since tissue distribution of miRNA-499 was unknown, several mouse tissues were analyzed. qRT-PCR demonstrated that miRNA-499 is highly enriched in cardiac tissue. This was confirmed via in situ-hybridization, miRNA-499 was localized in cardiomyocytes in human fetal and adult hearts. Transient transfection of miRNA- 499 in undifferentiated CMPCs reduced their proliferation rate by 15% (MTT assay and FACS analysis). Moreover, upon miR-499 transfection spontaneous beating was observed earlier and expression levels of cardiac troponin T, alpha-cardiac actinin, and Mlc-2v was increased. This enhanced cardiomyocyte differentiation occurred via repression of Sox6.

Conclusion:
Our results demonstrate a strong regulatory role of miRNAs in human CMPC proliferation and differentiation into cardiomyocytes. By modulating miRNA-499 expression levels, the differentiation of CMPCs is affected, resulting in enhanced cardiomyogenesis.
Abstract 1470: The Kinase Activity of Cyclin-Dependent Kinase-9 is Required for Phosphorylation of p300 and its Histone Acetyltransferase Activity during Cardiomyocyte Hypertrophy
Authors: Sunagawa Yoichi , Morimoto Tatsuya , Takaya Tomohide , Kawamura Teruhisa , Wada Hiromichi , Shimatsu Akira , Fujita Masatoshi , Kita Toru , Hasegawa Koji
Published: July 2021

Introduction:
A zinc finger protein GATA4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy and forms a complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activity of GATA4 as well as for cardiomyocyte hypertrophy and the development of heart failure
in vivo
. By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9), a component of positive transcription elongation factor b, as a novel GATA4-binding partner. Cdk9 also formed a complex with p300 as well as GATA4. However, the precise functional relationships among p300, GATA4, and Cdk9 remain unknown.

Methods and Results:
A series of GST pull-down assays revealed that the N-terminal of Cdk9 bound to the N-terminal zinc finger domain of GATA4 and C/H-3 domain of p300, respectively. By chromatin immunoprecipitation and DNA pull-down assay, we showed that GATA4 recruited Cdk9 onto GATA elements within the endothelin-1 promoter. Immuno-precipitation followed by Western blotting demonstrated that intact p300 induced not only the acetylation of GATA4, but also the interaction between GATA4 and Cdk9. Furthermore, p300 induced the hyperphosphorylation of RNA Pol II, suggesting that p300 is involved in the regulation of Cdk9 kinase activity. All of these effects were inhibited by the co-expression of a dominant-negative form (DN-) of p300. Conversely, Cdk9 induced not only the hyperphos-phorylation of RNA Pol II, but also the phosphorylation of p300. Notably, Cdk9 induced the acetylation and DNA binding of GATA4. However, DN-Cdk9, which loses its kinase activity by a single amino acid substitution, was unable to achieve these, suggesting the requirement of Cdk9 kinase activity for p300 HAT activity. Finally, a Cdk9 kinase inhibitor, DRB, inhibited phenylephrine-induced hypertrophic responses as well as the acetylation of GATA4 in cardiomyocytes.

Conclusion:
These findings demonstrate that Cdk9 is required for the phosphorylation of p300 and its HAT activity, and forms a functional complex with p300/GATA4 during cardiomyocyte hypertrophy.
Abstract 1508: Stim1-Mediated Store-Operated Calcium (SOC) Influx in Calcineurin-Dependent Cardiac Hypertrophy
Authors: Luo Xiang , Tandan Samvit , Jiang Nan , Rothermel Beverly A , Hill Joseph A
Published: July 2021

Introduction
: Calcineurin is activated by Ca
2+
/calmodulin and promotes pathological remodeling of the heart. In many cell types, store-operated calcium (SOC) entry, a process triggered by depletion of ER Ca stores, plays an obligate role in repleting Ca stores, and hence, in subsequent sustained increases in intracellular Ca. Little is known regarding SOC Ca influx in heart or its role in stress responsiveness.

Methods and Results
: Using RT-PCR, immunofluorescence staining, and Western blot methods, we found that Stim1, a recently identified molecular component of the SOC channel, is expressed in both neonatal rat ventricular myocytes (NRVMs) and adult myocytes. Working with isolated cells, we used Fura-2 to measure SOC Ca influx, finding that this mechanism does, in fact, exist in cardiomyocytes. However, SOC Ca influx was substantially more robust in neonatal myocytes than adult. Given that SOC current was prominent in neonatal cells, we tested ventricular myocytes isolated from calcineurin-transgenic mice, a model where the fetal gene program is prominently activated. Here, we found that Stim1 is up-regulated (1.9±0.4-fold, p<0.05). Treatment of NRVMs with angiotensin II (100nM) elicited similar increases in Stim1 (2.3±0.2-fold, p<0.01). To probe the molecular basis of cardiac SOC current, we expressed mutant (D76A, constitutively active; ΔERM, dominant negative) forms of human Stim1 in NRVMs. D76A markedly increased SOC Ca influx triggered by thapsigargin (2μM) or cyclopiazonic acid (50μM). In contrast, ΔERM blocked SOC Ca influx.

Conclusion:
SOC Ca entry exists prominently in neonatal cardiomyocytes but is down-regulated in adult cells. Stim1 participates in SOC current and is up-regulated during hypertrophic transformation of the myocardium. We conclude that SOC Ca currents are part of the fetal gene program in heart and may play a significant role in the sustained Ca overload of heart failure.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 1515: Regional Effects of Katp Channel Openers Reflect Distinct Atrial and Ventricular Katp Channel Structure in the Murine Heart
Authors: Glukhov Alexey V , Flagg Thomas P , Fedorov Vadim V , Efimov Igor R , Nichols Colin G
Published: July 2021

Classically, cardiac sarcolemmal KATP channels are thought to be composed of Kir6.2 (inward-rectifier potassium channel 6.2, KCNJ11) and SUR2A (sulfonylurea receptor type 2A, ABCC9) subunits. However, the evidence is strong that SUR1 (sulfonylurea receptor type 1, ABCC8) subunits are also expressed in the heart and that they play a significant functional role in the atria. To examine this further, we have estimated the effects of potassium channel-opening drugs diazoxide (specific to SUR1 > SUR2A) and pinacidil (SUR2A > SUR1) in intact hearts from wild type (WT, n=6) and SUR1−/− mice (KO, n=3) that lack SUR1 subunits. Action potential duration (APD) in both atria and ventricles were estimated by optical mapping of posterior surface of Langendorff-perfused hearts using the voltage sensitive dye RH237 and high spatiotemporal resolution CMOS camera (100x100 pixels; 3,000 frames/sec). In WT hearts, diazoxide (300 μM) decreased APD in atria (from 33.6±2.1 ms to 25.5±1.0 ms, p<0.001) and did not change it in ventricles (60.0±8.5 ms vs 61.2±8.3 ms, NS). The absence of SUR1 in KO mice resulted in loss of efficacy of diazoxide in atria (37.5±0.7 ms vs 36.5±0.7 ms, NS). In contrast, pinacidil (300 μM) significantly decreased ventricular APD in both type of mice (from 60.0±8.5 ms to 30.5±4.2 ms in WT, p<0.001; and from 62.0±1.4 ms to 30.5±6.4 ms in KO, p<0.001) and did not change atrial APD in either WT or KO hearts. Glass microelectrode recordings from isolated superfused atria confirmed the optical data. In both WT and KO hearts, the APD in left ventricle was significantly longer and the effect of pinacidil was significantly greater than in right ventricle (APD decreasing by 56.3±4.2% and 62.3±12.0% in left ventricle vs 49.4±3.4% and 50.9±4.1% in right ventricle for WT and KO mice respectively, p<0.05). Similar differences between APDs in right and left atria were not observed. Collectively, these results indicate that in the intact mouse heart, significant differential KATP pharmacology in atria and ventricles results from SUR1 predominance in forming the atrial channel, highlighting an unappreciated heterogeneity of KATP function in the heart.
Abstract 1516: Role of Oxidative Stress in Atrial Tachycardia Remodelling
Authors: Qi Xiao-Yan , Villeneuve Louis R , Ordog Balazs , Chartier Denis , Van Wagoner David R , Nattel Stanley
Published: July 2021

Atrial tachycardia remodeling (ATR) promotes atrial fibrillation (AF). Oxidant stress (OxS) occurs in atria of AF patients and antioxidants may be beneficial in AF. This study used a previously validated
in vitro
paced canine cardiomyocyte model to assess the potential role of OxS in ATR. Cultured canine atrial cardiomyocytes were paced at 1 or 3 Hz (P1, P3) for 24 hrs. I
CaL
was recorded with whole cell voltage clamp. Single cell superoxide production was assessed by dihydroethidium fluorescence (DHEF) imaging. ATR (3 Hz pacing x 24 hrs) decreased I
CaL
(Fig. A

) and induced OxS (Fig. B

). Short term induction of OxS (H
2
O
2
100 μM x 10 mins) increased I
CaL
(Fig. C

) and enhanced Ca
2+
loading (Indo-1 AM). 24-Hr H
2
O
2
100 μ M increased DHEF in P1 cells by 250%* (*P<0.05) and mimicked ATR, decreasing I
CaL
by 51%* (Fig. C

). H
2
O
2
-mediated DHEF changes were suppressed by inhibiting calmodulin (W7) or CaMKII (KN93). H
2
O
2
-induced I
CaL
suppression at 1 Hz was prevented by: decreasing Ca
2+
i
loading by I
CaL
blockade (nimodipine) or Ca
2+
chelation (BAPTA-AM); W7 or KN93; antioxidants (2-MPG or N-acetylcysteine, NAC); or suppression of free radical generation
via
NADPH-oxidase (apocynin). 2-MPG, NAC, and apocynin prevented I
CaL
downregulation by 3-Hz pacing and W7, KN93 and apocynin abolished ATR-induced DHEF increases. H
2
O
2
mimics ATR by causing Ca
2+
i
loading and CaMKII activation coupled to NADPH-oxidase stimulation. ATR-induced I
CaL
-downregulation is mediated in part by OxS generation. These findings provide insights into the pathways by which OxS contributes to ATR and present a mechanistic framework for understanding the effects of antioxidant interventions in AF.
Abstract 1517: Remodeling of Ion-Channel Expression and Cellular Electrophysiology of Cardiac Purkinje Fibers by Heart Failure
Authors: Maguy Ange , Lebouter Sabrina , Comtois Philippe , Chartier Denis , Villeneuve Louis , Nattel Stanley
Published: July 2021

Purkinje fibers (PFs) play a key role in cardiac conduction and arrhythmogenesis. Heart failure (HF) causes extensive electrical remodeling. HF-induced changes in atrial and ventricular ion channel subunit expression have been well characterized, but little is known about HF-induced ion channel subunit remodeling and functional consequences in PFs. This study assessed ion channel subunit expression, action potential (AP) properties and conduction in cardiac PF false tendons from control and HF dogs. HF was induced by 2 wk ventricular tachypacing (240 bpm). Control and HF PFs were fast-frozen for ion channel subunit mRNA (RT-qPCR) and protein (Western Blot, immunohistochemistry) assessment. APs were studied with standard micro-electrodes. HF significantly downregulated mRNA expression of subunits involved in AP propagation (Nav1.5, by 56%**, **P<0.01; Cx40, by 66%**, Cx43, by 56%**), automaticity (HCN2, by 75%**; HCN4, by 78%**) and repolarization (Kv4.3, by 43%*, *P<0.05; minK, by 31%*). No significant changes occurred in KChIP2, KvLQT1, ERG, Kir2.1 or Kir3.1/3.4 mRNA. At the protein level, significant downregulation was seen for Nav1.5 (by 38%**), Kv4.3 (by 42%**), HCN4 (by 74%*), Cx40 (by 53%**) and Cx43 (by 30%**). Immunohistochemistry revealed reduced Cx40 and Cx43 expression at PF intercalated disks. AP analysis showed changes consistent with observed decreases in I
to
and I
Na
subunits: HF decreased phase 1 slope (by 56%**), AP overshoot (by 32%*) and dV/dt
max
(by 35%*). AP properties associated with unchanged subunits (eg, resting potential, overall AP duration) were unaltered. Because of consistently significant changes in subunits governing impulse propagation (Nav1.5, Cx40, Cx43), we examined HF effects on AP propagation in PF false tendons with dual microelectrodes: conduction velocity decreased from 2.2±0.1 m/s (control) to 1.5±0.1 m/s* (HF). We have characterized for the first time HF effects on ion-channel subunit expression in cardiac PFs, finding prominent alterations in a variety of important subunits that control AP repolarization and propagation. These changes in PF ion-channel subunits likely contribute to conduction disturbances and arrhythmogenesis in the failing heart.
Abstract 1523: A Novel
SCN5A
Gain-of-Function Mutation M1875T Associated with Familial Atrial Fibrillation
Authors: Makiyama Takeru , Akao Masaharu , Shizuta Satoshi , Doi Takahiro , Nishiyama Kei , Yuko Oka , Nishio Yukiko , Tsuji Keiko , Itoh Hideki , Kimura Takeshi , Kita Toru , Horie Minoru
Published: July 2021

Background:
Mutations in the cardiac sodium (Na
+
) channel gene,
SCN5A
, have been associated with a variety of inherited arrhythmias, but the gain-of-function type modulation in
SCN5A
is associated with only one phenotype, long-QT syndrome type3 (LQTS3).

Methods and Results:
We studied a Japanese family with autosomal dominant hereditary atrial fibrillation (AF), multiple members of which showed onset of AF or frequent premature atrial contractions at a young age. The 31-year-old proband received radio-frequency catheter ablation, during which time numerous ectopic firings and increased excitability throughout the right atrium were documented. Mutational analysis identified a novel missense mutation, M1875T, in
SCN5A
. Further investigations revealed the aggregation of this mutation in all of the affected individuals (Figure A

). Functional assays of the M1875T Na
+
channels using whole-cell patch-clamp demonstrated a distinct gain-of-function type modulation; a pronounced depolarized shift (+16.4 mV) in V
1/2
of the voltage dependence of steady-state inactivation (Figure B

), and no late Na
+
current which is a defining mechanism of LQTS3. These biophysical features of the mutant channels are potentially associated with increased atrial excitability and normal QT interval in all of the affected individuals.

Conclusions:
We identified a novel
SCN5A
mutation associated with familial AF. The mutant channels displayed a gain-of-function type modulation of cardiac Na
+
channels, which is a novel mechanism predisposing increased atrial excitability and familial AF. This is a new phenotype resulting from the
SCN5A
gain-of-function mutations and is distinct from LQTS3.
Abstract 1534: Structural and Functional Remodeling of the Coupling between Sarcolemmal Ca
2+
Channels and the Ryanodine Receptor in Atrial Fibrillation
Authors: Lenaerts Ilse , Bito Virginie , Holemans Patricia , Heidbüchel Hein , Sipido Karin R. , Willems Rik
Published: July 2021

Background:
Permanent atrial fibrillation (AF) in humans has been associated with structural and electrical remodelling. Data on contractile remodelling and control of sarcoplasmic reticulum (SR) Ca
2+
release via the ryanodine receptor (RyR) in permanent AF are however limited.

Methods
: Ewes were atrially paced at 600 bmp for a median of 23 weeks resulting in permanent AF (mean of 89.6±6 days, N=13) and compared to matched, non-instrumented control animals (CTRL, N=17). Atrial myocytes were isolated and cell shortening (field stimulation), membrane currents (whole cell voltage clamp) and [Ca
2+
]
i
(Fluo-3) were measured. Protein expression was analyzed by immunoblotting. T-tubule density was quantified from confocal Z-stack images of cells stained with di-8-ANEPPS. Data are shown as mean±SEM of at least 17 cells and 5 animals.

Results:
Myocyte shortening and underlying [Ca
2+
]
i
transients were profoundly reduced in AF (by 54.8% and 62%, p<0.01). This reduced cell shortening could be corrected by increasing [Ca
2+
]
i
during caffeine-induced Ca
2+
release from the SR (L/L
0
9.7±0.5% in AF vs. 10.8±0.5% in CTRL). SR Ca
2+
content (integrated Na
+
/Ca
2+
exchange current during caffeine application) was not different (CTRL 1.9±0.3 pC/pF vs. AF 2±0.4 pC/pF), but calculated fractional SR Ca
2+
release during a depolarizing step to +10 mV was reduced in AF (by 20.6%, p<0.05). Peak Ca
2+
current density was modestly decreased (at <10 mV by 23.9%, p<0.01). T-tubules were present in the CTRL atrial myocytes though their density was much lower than in ventricular cells (11.4% vs. 20.6%, p<0.05). T-tubule density was robustly reduced in AF vs. CTRL (by 45%, p<0.01) with a reduction of myocyte surface:volume ratio (by 26%, p<0.01). The organization of RyR was apparently unchanged but protein expression was reduced (by 18.6%, p<0.05).

Conclusion:
In permanent AF, reduced SR Ca
2+
release is a major factor in the reduced cell contraction. Loss of T-tubules contributes to uncoupling of sarcolemmal Ca
2+
channels to RyR and reduced fractional SR Ca
2+
release despite preserved SR Ca
2+
content.
Abstract 1581: Extension of Titin as a Function of Filling Pressure and Sarcomere Length in the Porcine Left Ventricle
Authors: LeWinter Martin M , Popper Joseph , Nyland Lori , Bell Stephen B , Granzier Henk
Published: July 2021

The giant sarcomeric protein titin is a molecular spring that is the chief source of cardiomyocyte passive tension and a major determinant of myocardial stiffness. The spring portion is located in the I band and consists of PEVK, Ig repeat and N2B and N2A elements. Titin occurs as two isoforms. N2B is a smaller and stiffer isoform that contains only the N2B element and predominates in the left ventricle (LV) of rodents. N2BA titin contains both N2A and N2B elements. N2B and N2BA are co-expressed in the sarcomere of large mammals (~60:40 ratio). As a result, passive cardiomyocyte and myocardial stiffness in large mammals is less than in rodents. Details of titin extension as a function of sarcomere length (SL) have been elucidated in rodents but not in large mammals, where the presence of both isoforms would be expected to modify extension and passive tension. Accordingly, we studied titin extension in miniswine. We first established the relation between filling pressure and SL in the anterior LV wall of the in situ, freshly arrested (KCl) heart. SL was determined over a range of filling pressures using a light microscopic method that minimizes shrinkage. At equilibrium volume (transmural pressure 0 mmHg), SL was between 2.00–2.10 μm, longer than slack length of ~1.85 μm in muscle strips. SL reached a maximum of ~2.50 βm when the LV was over-distended (filling pressure >40 mmHg). We then examined extension of titin in myocardial strips using electron microscopy and immuno-labeling of selected epitopes. The chief difference between isoforms was that the N2B-Us epitope segment in N2B titin lengthened ~four times more than the N2B-Us segment in N2BA titin over SLs from ~1.80 to ~2.50 μm. This difference remained large over the SL range present in the in situ LV. Linear fits of the measured end-to-end length of N2B-Us segments were used to estimate the force-SL relation of single N2B and N2BA molecules. This analysis predicted a much steeper relation for N2B titin. Thus, over the range of SLs present in the in situ LV the most prominent difference in extension of N2B and N2BA titin is greater lengthening of the N2B segment of N2B titin. This predicts a much greater in situ stiffness for N2B titin and demonstrates how passive stiffness can be exquisitely controlled by varying isoform expression.
Abstract 1588: Midkine Gene Transfer Ameliorates Ischemic Cardiomyopathy through Enhancements of Neovascularization via Akt/PI3 Kinase and ERK Pathways and Anti-Apoptosis by Regulating Bcl-2 and Bax
Authors: Sumida Arihiro , Horiba Mitsuru , Ishiguro Hisaaki , Takenaka Hiroharu , Ueda Norihiro , Lee John-Kook , Murohara Toyoaki , Kadomatsu Kenji , Kodama Itsuo
Published: July 2021

Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, e.g. neural survival, carcinogenesis and tissue repair. We have demonstrated recently that MK ameliorates acute myocardial injury by ischemia/reperfusion through prevention of apoptotic reactions. Chronic effects of MK on the ventricular remodeling after myocardial infarction (MI) remain to be confirmed. MI was created in Wistar rat by ligation of the left anterior discending coronary artery. Rats of MK overexpression were prepared by intramyocardial injection of adenoviruses encoding MK (AdMK, n=12) at the onset of MI. Control rats were injected with adenoviruses encoding beta-galactosidase (AdLacZ, n=12). In echocardiography 4w after MI, AdMK showed smaller LVESD and LVEDD than AdLacZ (LVESD: 6.7±0.7 vs. 9.3±0.7mm, LVEDD: 8.7±0.7 vs. 10.7±0.7mm, p<0.01). AdMK had better FS than AdLacZ (FS: 23.0±2.8 vs. 12.4±2.1%, p<0.01). Better preservation of systolic/diastolic function in AdMK than AdLacZ was also recognized in the LV catheterization 6w after MI (dP/dt
max
: 3092±619 vs. 2171±763mmHg/s, dP/dt
min
: −2525±878 vs. −1700±722mmHg/s, LVEDP: 3.3±0.9 vs. 8.3±2.2mmHg, p<0.01). Morphological study on AdMK 6w after MI revealed less fibrosis (collagen deposition area: 10.2±5.3 vs. 15.3±4.5%, p<0.05) and a higher vWF-positive capillary density in the perinfarct zone (32±11 vs. 21±5/mm
2
, p<0.01) compared with AdLacZ. In Western blotting, the levels of Akt and ERK phosphorylation and PI3K were elevated in AdMK compared with AdLacZ (pAkt; 2.08-fold increase, pERK; 1.43-fold increase, PI3K; 2.36-fold increase, relative to AdLacZ, p<0.05). In addition, the level of Bcl-2 increased and the level of Bax decreased in AdMK compared with AdLacZ (Bcl-2; 2.15-fold increase, Bax; 3.63-fold decrease, relative to AdLacZ, p<0.01). Overexpression of MK has long-term beneficial effects to reduce the progression of LV remodeling and dysfunction after MI. This amelioration is attributable in part to an enhancement of neovascularization via PI3K/Akt and ERK pathways, and in part to anti-apoptotic actions regulating Bcl-2 and Bax. MK gene transfer might be a new therapeutic strategy for treatment of ischemic heart disease.
Abstract 1605: T-Type Calcium Current Activates Pro-Survival Signaling Pathways after Myocardial Infarction
Authors: Jaleel Naser , Kubo Hajime , Gao Erhe , MacDonnell Scott , Chen Xiongwen , Nakayama Hiroyuki , Angert David , Flynn Timothy , Berretta Remus , Zhang Hongyu , Koch Walter , Molkentin Jeffery , Houser Steven
Published: July 2021

Ca
2+
influx through L-type Ca
2+
channels (I
Ca,L
) is essential for cardiac contraction and for activation of a variety of Ca
2+
dependent signaling pathways. However, Ca
2+
can also enter cardiac myocytes through T-type Ca
2+
channels (TTCCs), which are found in the fetal heart and become re-expressed in adult ventricular myocytes (VMs) during cardiac pathology. While excess I
Ca,L
promotes VM death and dysfunction, the role of Ca
2+
influx through TTCCs (I
Ca,T
) in cardiac stress responses is unknown and is the topic of this study.

METHODS:
Transgenic (TG) mice with inducible, cardiac-specific expression of α1G-TTCCs and control (CTR) mice were used. Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. Mice were sacrificed at 1 week and 7 weeks post-MI.

RESULTS:
TG hearts had greater in vivo fractional shortening (FS) than CTR (TG: 39.6±3.2%, N=16 vs CTR: 32.2±1.2%, N=14; p<0.05). VMs isolated from TG hearts showed peak I
Ca,T
of −32.8±3.0 pA/pF. I
Ca,L
was smaller in TG than in CTR VMs (−9.6±0.7 pA/pF vs −12.0±0.4 pA/pF, p< 0.05). Compared to I
Ca,L
, I
Ca,T
triggered smaller Ca
2+
transients (1.5±0.1 vs 2.1±0.1, n=10; p<0.01) and contractions (2.6±0.7% vs 8.7±1.2%, n=10; p<0.01). Despite higher Ca
2+
influx than CTR hearts, TG hearts showed no cardiac histopathology or premature death, and TG hearts had higher levels of phosphorylated Akt (p<0.05) and dephosphorylated NFAT (p<0.05), which are known to be cytoprotective. After 1 week of MI, TG and CTR hearts displayed similar reductions in FS (25.3±1.7%, N=14 vs 25.7±1.8%, N=19) and increases in cardiac size (HW/BW, mg/g: 4.4±0.1, N=14 vs 4.5±0.3, N=19). However, TG hearts had smaller infarcts than CTR hearts (19.9±1.8%, N=7 vs 28.4±2.3%, N=6, p<0.01). At 7 weeks post-MI, TG mice showed a higher survival rate than CTR mice (66.7% vs 47.6%). Also, at 7 weeks post-MI, TG hearts were larger than CTR (HW/BW: 10.4±1.0, N=12 vs 7.7±0.5, N=10; p<0.05) and had similar FS to CTR hearts (28.7±2.7, N=8 vs 28.0±2.8) and isolated VM contractility (TG: 5.6±0.7%, n=15 vs CTR: 5.3±0.5%, n=14).

CONCLUSION:
Ca
2+
influx via TTCCs has small effects on cardiac contraction, but is associated with reduced infarct size and increased survival, suggesting that it induces cytoprotective signaling.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 1606: A Novel Cardiac-Specific Isoform of the Cell Cycle-Related Kinase Provides Cardioprotection during Myocardial Ischemia
Authors: Qiu Hongyu , Tian Bin , Shen You-Tang , Vatner Dorothy E , Vatner Stephen F , Depre Christophe
Published: July 2021

Since heart failure (HF) is characterized by the decreased activity of pro-survival signaling pathways, we reasoned that examining genomic changes in a clinically relevant model of HF would uncover novel genes involved in protecting the heart that were downregulated in HF. Accordingly, we examined a genomic profile by DNA microarray in a primate model of myocardial infarction (MI) followed by three weeks pacing to develop HF, and compared to sham animals (n=5/group). In this array, the transcript of the gene encoding the cell cycle-related kinase (CCRK), for which no function is known in adult heart, was down-regulated by 50% in HF versus sham (P<0.05), which was confirmed by quantitative PCR. Upon sequencing, the cardiac CCRK (cCCRK) showed a conservation of the N-terminal kinase domain, but differed in its C-terminal half from the “generic” CCRK cloned in other tissues, due to alternative splicing. Although the “generic” CCRK phosphorylated its substrate, the cyclin-dependent kinase 2, and doubled the number of myocytes in the S phase of the cell cycle, these effects were not observed with cCCRK (P<0.05 between groups). Rather, overexpression of cCCRK in cardiac myocytes induced a 36% increase in protein/DNA ratio and a 50% decrease in apoptosis upon chelerythrine treatment (both, P<0.05 vs. LacZ). A tetracycline (Tet)-dependent transgenic (TG) mouse model with cardiac-specific overexpression of cCCRK was generated, which showed a 3-fold increase in cCCRK abundance compared to wild type (WT) mice (P<0.01 vs. WT) upon Tet removal. Both WT and TG mice (n=3/group) underwent 45 min no-flow ischemia of the left anterior descending artery and 24h reperfusion, followed by measurement of the area-at-risk (AAR) and infarct size (IS). The AAR/left ventricular mass was similar in WT and TG. However, IS/AAR was reduced (P<0.01) by more than 50% in TG (29±3%) compared with WT (61±5%). Therefore, a variant of CCRK is expressed in the heart, which is down-regulated in HF, which promotes cell growth and survival, when overexpressed
in vitro
, and which provides major protection against myocardial ischemia when overexpressed
in vivo
. Thus, CCRK is a novel mechanism for cardioprotection in ischemia, and may be a new approach to HF treatment.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1613: Activation of a Novel Estrogen Receptor, GPR30, is Cardioprotective in the Rat
Authors: Deschamps Anne M , Murphy Elizabeth
Published: July 2021

It has been well documented that pre-menopausal females have a lower incidence of cardiovascular disease than their male counterparts. However, post-menopausal women reach the rates of disease in men suggesting a protective role of estrogen. Numerous animal studies have also demonstrated that estrogen is cardioprotective. Historically, estrogen has been thought to signal through two nuclear receptors, ERa and ERb; however, a third, membrane bound receptor, GPR30, has been identified and shown to bind estrogen with high affinity. GPR30 is a G protein coupled receptor localized to the plasma membrane and endoplasmic reticulum. To date, there is little information on GPR30 in the heart and no study has looked at the effect of GPR30 activation during myocardial ischemia and reperfusion. Therefore, the goal of this study was to determine whether activation of GPR30 results in a cardioprotective phenotype in rats. A highly specific GPR30 agonist, G-1, was administered to Sprague Dawley (200–300g) rat hearts 10 minutes prior to 20 minutes of ischemia followed by 120 minutes of reperfusion using a Langendorff model. Recovery of post-ischemic function was significantly better in G-1 treated hearts compared to controls (39.4±5.5 vs 23.6±2.5% of pre-ischemic rate pressure product, p<0.05). Additionally, infarct size was reduced in the G-1 treated animals when compared to control (16.5±4.4 vs 30.6±3.3% p<0.05). The protection afforded by G-1 was blocked by co-administration of a PI3K inhibitor (wortmannin 100nM). Through western blot analysis, it was demonstrated that G-1 induced the activation of both Akt and Erk1/2 and was blocked by PI3K inhibition. Taken together, the data show that G-1 activation of GPR30 improved functional recovery and reduced infarct size in isolated rat hearts following ischemia and reperfusion through a PI3K dependent mechanism.
Abstract 1656: Vegf Induces vWF Release via A VEGFR2/Tyr1175-PLC- gamma1 Pathway
Authors: Xiong Yan , Huo Yingqing , Chen Chao , Lu Xiaofan , Luo Jincai
Published: July 2021

Many endothelial inflammatory and prothrombotic mediators are stored in and rapidly released from Weibel-Palade bodies (WPBs), endothelium-specific storage organelles upon stimulation. The von Willebrand factor (vWF), a major component inside WPBs, mediates the initial contact of platelets with the injured vessel wall and thus plays an important role in haemostasis and thrombosis. It has previously been shown that vascular endothelial growth factor (VEGF) triggers a rapid release of vWF. However, specific VEGF receptors and their potential downstream pathways involved in this process have not been carefully determined. To dissect the role of VEGF receptors in vWF release activation, we utilized two approaches: one is to use receptor-specific ligands and the other is to use a chimeric receptor approach. The ligands for VEGF receptor 2 (VEGFR2), but not VEGF receptor 1 (VEGFR1), stimulated vWF release. The predominant role of VEGFR2 in vWF release regulation was further confirmed by using a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR1 or VEGFR2, and was then expressed in early-passage HUVECs. Further, the knockdown of phospholipase C-γ1 (PLCγ1) suppressed VEGF-triggered intracellular calcium increase and blocked VEGF-induced vWF release. In addition, the two products of PLCγ1 hydrolysis, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), are both required for VEGF-induced vWF release. Finally, combined with point mutagenesis, the responsible binding sites for PLCγ1 and their importance in VEGFR2-activated vWF release have been determined. Point mutation of a single tyrosine residue Tyr1175, a putative binding site for PLCγ1 on the C-terminus, abolished VEGFR2-activated vWF release. This study presents the first evidence that the PLCγ1 is essential for VEGF-triggered vWF release mediated through a VEGFR2/Tyr1175 pathway.
Abstract 1690: Pre-Beta HDL and Lecithin:Cholesterol Acyltransferase Levels Are Strong Positive Diagnostic Risk Markers for Ischemic Heart Disease in Subjects with Both High and Low HDL-C Levels in the Copenhagen City Heart Study
Authors: Sethi Amar A , Sampson Maureen , Warnick Russel , Nordestgaard Boerge G , Tybjaerg-Hansen Anne , Remaley Alan T
Published: July 2021

Objective:
We hypothesize that patients with high HDL-C levels and Ischemic Heart Disease (IHD) may have dysfunctional HDL or unrecognized non-conventional risk factors.

Methods:
Subjects with IHD and either high HDL-C (n = 53; women:≥ 73.5mg/dL; men: ≥ 61.9mg/dL) or low HDL-C (n = 42; women: ≤ 38.7mg/dL; men: ≤ 34.1mg/dL) were compared with subjects without IHD and matched HDL-C levels (n = 110). All subjects had normal LDL-C (< 160mg/dL) and triglyceride levels (< 150mg/dL) and none were treated with lipid-lowering medications. Pre-beta HDL and Phospholipid Transfer Protein (PLTP) levels were measured using commercial kits and Lecithin:Cholesterol Acyltransferase (LCAT) activity, using a proteoliposome cholesterol esterification assay.

Results:
Pre-beta HDL levels were 2–3 fold higher in subjects with IHD in the high (p < 0.0001) and low HDL-C (p = 0.001) groups. Interestingly, low LCAT activity was found in the same subjects with IHD and high (p = 0.002) or low (p = 0.03) HDL-C. Furthermore, higher PLTP levels were observed in the group with high HDL-C and IHD when compared to individuals without disease (p < 0.0001). Receiver operating characteristic curve for pre-beta HDL in the high HDL-C groups yielded an area under curve of 0.71 for predicting IHD, which increased to 0.92 when LCAT values were included. Similar results were obtained for the low HDL-C groups. A strong inverse correlation between LCAT activity and pre-beta HDL-C (
r

2

= 0.30, p < 0.0001), which was especially strong in the low HDL-C group (
r

2

= 0.56, p < 0.0001) was observed. Individuals with high HDL-C and IHD had approximately 15% more apoA-I (p = 0.001) and apoA-II (p = 0.002) and nearly 10% more phospholipid (p = 0.007) than subjects with similar HDL-C and no IHD. Likewise, the group with low HDL-C had 30% more apoE (p = 0.035) and approximately 10% more phospholipid (p = 0.047) than the group with similar HDL-C levels and no IHD.

Conclusions:
IHD was associated with high pre-beta HDL and low LCAT levels. Measuring both gave correct classification in 92% of cases with IHD, thus making pre-beta HDL and LCAT activity a powerful diagnostic tool for IHD, particularly in commonly found patients wtih isolated low HDL-C levels with low to intermediate risk of IHD based on conventional risk factors.
Abstract 1715: Prognosis of Patients Under Chronic Clopidogrel Therapy while Suffering an Acute Coronary Syndrome
Authors: Bonello Laurent , de Labriolle Axel , Lemesle Gilles , Roy Probal , Steinberg Daniel H , Slottow Tina L Pinto , Zue Zhenyi , Torguson Rebecca , Suddath William , Satler Lowell , Kent Kenneth , Pichard Augusto , Waksman Ron
Published: July 2021

Background:
Chronic clopidogrel therapy (CCT) has been shown to be beneficial in decreasing the frequency of major adverse cardiovascular events (MACE) in coronary artery disease. However, some patients suffer an acute coronary syndrome (ACS) despite CCT.

Objective
: To assess the prognosis of patients suffering an ACS while on CCT compared to patients naive to clopidogrel.

Method:
Retrospective analysis of propensity matched cohorts of patients undergoing PCI for an ACS was performed. Patients under CCT before the ACS were matched 1:2 with patients not under CCT before the ACS. The primary endpoint was a composite of cardiac death and myocardial infarction (MI) at 1-year follow-up. A cohort of 2325 patients undergoing PCI for an ACS was studied. Among them, 256 patients were taking CCT for > 1 month at the time of the ACS and 2069 weren’t taking clopidogrel prior to the ACS. After propensity score matching 1:2, the 2 groups included, respectively, 84 and 168 patients.

Results:
Patients in both groups had similar rates of previous MI (no CCT vs CCT 47.6 vs 48.8%; p = 0.86) or PCI (40.5 vs 40.5%; p = 1). There was no difference in drug use on discharge between the 2 groups; in particular for thienopyridines (94 vs 96.4%; p = 0.6). At 1-year follow-up, patients under CCT before the ACS exhibited a worse prognosis than patients who were not under CCT prior to the ACS, with a higher rate of cardiac death and MI (14.2 vs 5.5%; p = 0.015).

Conclusions:
This study suggests that patients suffering an ACS while under CCT have a poor long-term prognosis, which could be linked to clopidogrel low response.