bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 802: Dynamic FRET-Based Ca-Calmodulin Measurements in Intact Ventricular Myocytes Uncover Differential Signal Integration Due to Ca-Calmodulin Affinity
Authors: Song Qiujing , Bossuyt Julie , Saucerman Jeffrey , Bers Donald
Published: July 2021

Background:
Myocyte [Ca]
i
varies beat-to-beat and calmodulin (CaM) transduces Ca signals to regulate many cardiac functions (e.g. via CaM-dependent kinase, CaMKII, and calcineurin, CaN). We hypothesize that the different Ca-CaM affinities of CaMKII (Kd ∼45 nM) and CaN (Kd ∼2 nM) will dictate their dynamic and tonic binding to Ca-CaM.

Methods:
Two adenoviral vectors encoding FRET (fluorescence resonance energy transfer) based biosensors with affinities similar to CaMKII and CaN (BsCaM-45 vs. BsCaM-2) were generated to monitor Ca-CaM dynamics in adult rabbit ventricular myocytes (with extra CaM expression via adenovirus).

Results:
Ca-CaM binding reduces FRET and increases the fluorescence emission ratio (F
480
/F
530
). Figure

shows the lower affinity sensor BsCaM-45 (like CaMKII) turns on and off completely with only slight diastolic increase at 1.0 Hz (5% ± 2.5%, vs. 0.1 Hz, n = 5). In contrast, the higher affinity sensor BsCaM-2 (like CaN) showed significant diastolic signal integration at 1.0 Hz (20% ± 3%, n = 5, p < 0.05). BsCaM-2 (n = 5) has slower kinetics vs. BsCaM-45 (n = 5): at 1.0 Hz, time to peak: 218 ± 11 vs. 244 ± 15 ms, ns; τ-decay: 647 ± 17 vs. 321 ± 25 ms, p < 0.05).

Conclusion:
CaM target proteins with different affinities (like CaMKII and CaN) respond very differently to the same Ca signal, (integrating vs. phasic), presumably tuned appropriately for their signaling pathway. This dynamic response shapes subsequent cellular processes in the heart. Indeed, altered diastolic [Ca]
i
or heart rate (as in pathophysiological states) can differentially influence downstream CaM-dependent pathways.

Beat-to-beat FRET signal changes in Rabbit Ventricular Myocytes
Abstract 854: Homing of Fetal Stem Cells to Damaged Maternal Hearts: A Potential Mechanism for Cardiac Repair in Peripartum Cardiomyopathy
Authors: Kara Rina J , Matsunaga Iwao , Dumlao Themy F , Tanweer Omar , Shachter Neil S , Naka Yoshifumi , Chaudhry Hina W
Published: July 2021

Fetal cells enter the maternal circulation during all pregnancies. They may persist in maternal blood and tissues for decades, creating a state of physiologic microchimerism. We hypothesize that fetal stem cells may be associated with a maternal response to cardiac injury and, therefore, might potentially contribute to cardiac recovery noted in peripartum cardiomyopathy. In order to examine whether fetal stem cells cross the placenta and participate in cardiac repair in the maternal heart, we created a mouse model of cardiac injury during pregnancy. We crossed wild-type B6CBA female mice (age 3– 6 months) with male EGFP transgenic mice. The female mice then underwent ligation of the left-anterior descending (LAD) artery in order to induce an anterolateral myocardial infarction (MI) at gestation day 12. Approximately 50% of the embryos were EGFP-positive. 13 experimental females underwent infarction; 9 female pregnant controls did not undergo surgery. Post-partum females were sacrificed at 1 week post-MI (8 experimental, 4 control) or 2 weeks post-MI (5 experimental, 5 control). Genomic DNA was extracted from each total heart. Using primers designed to amplify EGFP and an internal control gene, ApoB, real time PCR was done in triplicate. The ΔΔCt method was used to calculate the relative quantity of EGFP in experimental hearts compared to controls. Unpaired t-test was used to evaluate the ΔCt values generated from the PCR reactions. Experimental hearts harvested at 1 week post-MI contained 4.1 times more EGFP than controls (p=0.0477). Experimental hearts harvested 2 weeks post-MI contained 3.9 times more EGFP than controls (p=0.0312). In a separate group of experimental and control mice, immunofluorescence analysis with confocal microscopy was utilized to detect EGFP-positive cells in ventricular tissue sections of maternal hearts. EGFP-positive cells were noted in infarct zones of experimental maternal hearts only at both 1 and 2 weeks post-MI. At 2 weeks post-MI, EGFP-positive cardiomyocytes were noted in the infarct zones of maternal hearts, implicating differentiation of fetal stem cells to maternal cardiomyocytes. These data implicate fetomaternal stem cell transfer as an important mechanism in the maternal response to myocardial injury.
Abstract 195: Increases in Ca
2+
Influx through the L-type Ca
2+
Channel Induces Pathological Cardiomyocyte Hypertrophy
Authors: Chen Xiongwen , Nakayama Hiroyuki , Zhang Hongyu , Zhang Xiaoying , Harris David M , Berretta Remus M , Wilson Rachel M , MacDonnell Scott , Kubo Hajime , Weisser-Thomas Jutta , Eckhart Andrea D , Koch Walter J , Molkentin Jeffery D , Houser Steven R
Published: July 2021

Cardiac hypertrophy (LVH) is associated with the development of arrhythmia (sudden cardiac death) and congestive heart failure. While there is a clear role for activation of calcium (Ca) regulated signaling pathways in LVH, the specific role of Ca influx (I
Ca
) through the L-type calcium channel (LTCC) has not been directly examined and is the topic of this study.

Methods:
In vitro,
Ca influx into cultured feline (F) and neonatal rat (NR) ventricular myocytes (VMs) was increased by infecting VMs with an adenovirus (Ad) containing a fusion gene encoding the β2a subunit of the LTCC (β2a)-GFP with AdGFP as the control (CTR).
In vivo,
we established transgenic mouse lines with inducible (Tet-off ) overexpression of the β2a subunit (β2a TG). All evaluations of mouse hearts or VMs were done with 4-month old mice.

Results:

Overexpression of β2a (MOI = 5) in cultured FVMs increased I
Ca
(111%) and cell volume (16%), protein synthesis (25%) and ANF expression;

NFAT and HDAC translocations were increased in β2a FVMs (30% and 50% respectively);

Blockade of the LTCC (nifedipine, NIF), intracellular Ca (BAPTA), calcineurin (cyclosporine A (CsA) and FK506) and CaMK II (KN93) prevented β2a mediated increases in cell volume, protein/DNA ratio, NFAT and HDAC translocations;

The surface area of NRVMs infected with Adβ2a at 36 hours post infection was 52.4% larger than CTR, which was prevented by NIF, BAPTA, CsA, FK 506 and KN93;

In TG mice, I
Ca
(CTR: 13.7 ± 0.7pA/pF, n=6 vs. TG: 24.3 ± 2.6pA/pF, n=6), fractional shortening (CTR: 8.0 ± 0.5%, n=16 vs. TG: 11.9 ± 1.1%, n=11) and peak Ca transient (Fluo-3) (CTR: 2.25 ± 0.08%, n=19 vs. TG: 3.37 ± 1.58, n=11) were significantly (
p<0.05
) increased in TG-VMs than in CTR-VMs;

β2a TG mice had greater ejection fraction (CTR: 68.9 ± 1.3%, n=33 vs. TG: 74.4 ± 1.3%, n=34,
p<0.05),
and posterior wall thickness (CTR: 1.10 ± 0.04mm, n=33 vs. TG: 1.22 ± 0.03mm, n=34,
p<0.001
);

The heart weight to body weight ratio in TG mice (6.90 ± 1.05mg/g, n=17) was greater (
p<0.05
) than in CTR (6.04 ± 0.93mg/g, n=51) with significantly larger myocyte cross-section area.

Conclusion:
Increases in Ca influx through the LTCC are sufficient to induce cardiomyocyte hypertrophy both
in vitro
and
in vivo
through the calcineurin/NFAT and HDAC dependent pathways.
Abstract 875: Hematopoietic deficiency of Macrophage Inhibitory Cytokine-1 Affects CCR2 function and Attenuates Plaque Leukocyte Accumulation in LDL Receptor Knockout Mice
Authors: De Jager Saskia C , Bermudez Beatriz , Bot Ilze , Muriana Francisco J , van Berkel Theo J , Lee Se J , Abia Rocio , Biessen Erik A
Published: July 2021

Macrophage inhibitory cytokine-1 (MIC-1) is a distant member of the transforming growth factor β family with alleged anti-inflammatory activity that acts through an unknown receptor. Recently it was shown that elevated MIC-1 serum levels are an independent risk factor for atherothrombosis. In this study we have therefore addressed the involvement of hematophoietic MIC-1 in atherogenesis. LDLr−/− mice were lethally irradiated and 24 hours later transplanted with either MIC1−/− or littermate bone marrow. The animals were allowed to recover for 6 weeks and placed on a high cholesterol diet for 12 weeks after which lesions were analyzed. Contrary to our expectations, plaque burden in the aortic sinus of MIC1−/− chimeras was similar to that in littermate controls. However plaque cellularity was markedly decreased in MIC1−/− chimeras (−31%, P = 0.003), which was completely attributable to reduced macrophage infiltration (−32%, P = 0.047). As MIC1−/− chimeras also displayed reduced circulating monocytes levels (−37%; P = 0.04) we turned our attention to CCR2. Expression of CCR2 was significantly decreased in PBMC and peritoneal leukocytes, possibly explaining the reduced chemotactic capacity of these subsets in MIC-1 deficiency. In keeping with these in vivo findings, in vitro macrophage migration confirmed the chemotactic properties of MIC-1(+102%; P = 0.001). Interestingly MIC-1 did not augment MCP-1 induced migration (P = 0.7), nor did it induce chemotaxis in CCR2 deficient macrophages (P = 0.3). These findings point to a direct signaling of MIC-1 to CCR2. Besides chemotaxis, MIC-1 deficiency also resulted in a relative enrichment in circulating regulatory T cells in MIC1−/− chimeras. In conclusion, we are the first to demonstrate a regulatory role of MIC-1 in macrophage migration to sites of inflammation, such as the atherosclerotic plaque, possibly via modulating CCR2 function. Moreover MIC-1 appears to inhibit regulatory T cell responses, thereby prolonging already established immunogenic responses. While further characterization of the underlying pathways is still awaited, our data suggest that focal inhibition of MIC-1 signaling will improve plaque stability by impairing CCR2 activity and re-enforcing regulatory T cell responses.
Abstract 902: A Beta-2 Adrenergic Receptor Polymorphism Is Associated With Increased Left Ventricular Remodeling Following First ST-Elevation Myocardial Infarction
Authors: McLean Rhondalyn C , Hirsch Glenn A , Gerstenblith Gary , Schulman Steven P
Published: July 2021

Background: Prior studies demonstrate an association between specific β-adrenergic receptor polymorphisms and clinical outcomes in patients with chronic heart failure and following an acute coronary syndrome. The mechanism may relate to an effect on left ventricular (LV) remodeling. We hypothesized that β-adrenergic receptor polymorphisms predict LV remodeling after acute ST elevation myocardial infarction (STEMI).

Methods: We assessed the presence of β-1 and β-2 adrenergic receptor polymorphisms in 122 patients after their first STEMI enrolled in a single-center randomized, double-blind placebo controlled trial of L-arginine vs. placebo, 91% of whom received successful early reperfusion therapy. All patients were treated with a beta-1 receptor antagonist and underwent baseline (mean 5.9 days following STEMI) and 6-month LV volume evaluation using gated blood pool imaging. Univariate and multivariate linear and logistic regressions were used to assess the relationships between the polymorphisms, β1 Arg389Gly, β1 Ser49Gly, β2 Gly16Arg and β2 Gly27Glu and the six-month changes in LV volumes. The top quintiles of LV end-systolic (ESV) and end-diastolic (EDV) 6-month volume increases and LV ejection fraction decrease were compared to the lower quintiles in the logistic regression analyses.

Results: The polymorphisms β1 Arg389Gly, β1 Ser49Gly, β2 Gly16Arg were not associated LV remodeling. However, the 25% of patients homozygous for the β2 Glu27 variant were 5.2 times more likely to have an increase in 6-month ESV than those who had the Gln27 variant (OR 5.2, 95%CI 1.4 –19.0). Multiple linear regression analyses demonstrated that absolute ESV at six months was 19 ml greater (p = 0.02) and EDV was 21 ml greater (p = 0.01) in post STEMI patients with the β2 Glu27 polymorphism compared to the wild type or heterozygous patients.

Conclusions: Increased LV volumes post-STEMI are associated with an increased risk of heart failure and death. The common β2 receptor polymorphism, Glu27Glu, is associated with increased odds of adverse LV remodeling in patients treated with a beta-one receptor antagonist. Whether treatment with a non-specific β-adrenergic receptor blocker guided by this genetic polymorphism ameliorates the effect requires further study.
Abstract 924: Aging Induces Titin Oxidation And Increases Passive Cardiomyocyte Stiffness
Authors: Lim Chee , Zhong Lin , Helmes Michiel , Sawyer Douglas B
Published: July 2021

One of the hallmarks of aging is an increase in ventricular passive stiffness leading to diastolic heart failure, however, the molecular basis for this is unclear. The giant elastic protein titin, working as an entropic spring, is the major determinant of passive cardiomyocyte stiffness. We hypothesized that accumulation of oxidized titin in the aging heart results in increased cardiomyocyte passive stiffness and thereby contributes to diastolic dysfunction. We further hypothesized that an age-assocated disturbance in the ubiquitin/proteasome pathway plays a role in the accumulation of oxidized titin.

Methods:
Cardiomyocytes from adult (4 month) and aging (34 month) mouse hearts were isolated and chemically skinned and the passive force-sarcomere length relationship in single cells were obtained. Post-translational modification of titin was assessed in whole heart homogenates by determining the content of 1) titin carbonyls, a marker of irreversible protein oxidation, and 2) titin ubiquitinylation, a marker for proteasomal degradation.

Results:
Cardiomyocyte passive tension was significantly higher in the aging group (at SL=2.45 μm, 1008.4 ± 45.5 vs. 871.6 ± 48.3 μg; p<0.05). While the total amount of titin was unchanged between both groups, the carbonyl content of titin was increased 3-fold with aging (p<0.05). High molecular weight ubiquitin-conjugated proteins were increased with aging, which is consistent with a decrease in proteasomal activity with age. Surprisingly, titin ubiquitinylation was decreased with aging suggesting an impairment upstream of the proteasome, in titin-specific ubiquitin ligase activity. Furthermore, the muscle specific ubiquitin ligase MURF-2, which has been shown to interact with titin and play a role in muscle protein turnover, showed a significant shift from a high to low molecular weight isoform with aging.

Conclusion:
Our data suggests that an age-associated impairment in titin turnover contributes to accumulation of oxidatively modified titins with consequent impairment of cardiomyocyte stiffness. We speculate that the oxidation of titin will change the elastic properties of titin, directly accounting for the change in passive tension and contributing to diastolic dysfunction associated with aging.
Abstract 930: NADPH Oxidase 2 is Responsible for α1-Adrenergic Receptor-Dependent Reactive Oxygen Species Production in Adult Rat Ventricular Myocytes
Authors: Lancel Steve , Weinberg Ellen O , Valsky Eugene B , Jeong Mark Y , Farukhi Zareen , Pimentel David R , Mahadev Kalyankar , Goldstein Barry J , Pagano Patrick J , Siwik Deborah A , Colucci Wilson S
Published: July 2021

Background:
Five NAPDH oxidase (NOX) isoforms have been implicated in reactive oxygen species (ROS) generation in a cell type- and agonist-specific manner. We previously showed that α
1
-adrenoreceptor (α
1
-AR) stimulation in cultured adult rat ventricular myocytes (ARVM) leads to NADPH-dependent ROS-production, suggesting that a NOX may be responsible. The goal of this project was to determine whether NOX mediates α
1
-AR-stimulated ROS production in ARVM, and if so, which isoform(s) is responsible.

Methods and Results:
ROS production, assessed by dichlorofluoresceine (DCF) fluorescence, was increased by 51±7 % in ARVM subjected to α
1
-AR stimulation (norepinephrine 1μM + propranolol 2 μM; 30 min; p=0.002, n=3/group). Apocynin (Apo, 50 μM), a broad-spectrum NOX inhibitor, prevented α1-AR-mediated ROS generation (+19±8% vs Apo-treated/α
1
-AR-unstimulated cells, n=4, NS), suggesting a role for NOX. Expression of NOX1, 2 and 4, as well as regulatory proteins p22
phox
, p47
phox
, Rac1 and Rac2, but not NOX3, was detected by RT-PCR in ARVM. Neither the inhibitory Nox4-tat peptide (+56±7% vs NOX4-tat-treated/α
1
-AR-unstimulated cells) nor adenovirus-mediated overexpression of NOX4 dominant-negative protein (+84±17% vs NOX4DN-infected/α
1
-AR-unstimulated cells) reduced α
1
-AR-induced DCF fluorescence increase (n=4/ group, both p<0.01 vs α
1
-AR-unstimulated cells). In contrast, adenovirus-mediated overexpression of dominant negative p47
phox
, p67
phox
and Rac1 proteins prevented α
1
-AR-mediated ROS production, suggesting that the responsible NOX requires each of these proteins. NOX2-tat inhibitory peptide (+18.4±7.3% vs NOX2-tat-treated/α
1
-AR-unstimulated cells, n=6, NS), but not NOX1-tat inhibitory peptide (+51±8% vs NOX1-tat-treated/α
1
-AR-unstimulated cells, n=3, p<0.01), prevented α
1
-AR-mediated ROS production.

Conclusion:
The NOX2 complex, including the catalytic subunit gp91
phox
, and the regulatory proteins p47
phox
, p67
phox
and Rac1, is directly responsible for α
1
-adrenergic-receptor induced ROS production in adult rat ventricular myocytes.
Abstract 934: Contributory Role of VEGF Overexpression in Endothelin-1-induced Cardiomyocyte Hypertrophy: Involvement of Hyopoxia Inducible Factor
Authors: Shimojo Nobutake , Jesmin Subrina , Hattori Yuichi , Maeda Seiji , Miyauchi Takashi , Aonuma Kazutaka
Published: July 2021

Although endothelin-1 (ET-1) stimulates vascular endothelial growth factor (VEGF) expression in a variety of cells, including endothelial cells and vascular smooth muscle cells, the effect of ET-1 on expression of VEGF and its receptors in cardiomyocytes is unknown. In the present study, we found that treatment of neonatal rat cardiomyocytes with ET-1 for 24 h resulted in upregulation of VEGF and its two principle receptors, fetal liver kinase (flk)-1 and fms-like tyrosine kinase (flt)-1, in a concentration-dependent manner (10
−12
-10
−6
M). ET-1 treatment also caused significant cardiomyocyte hypertrophy, as indicated by increases in cell surface area (2.0-fold compared to control) and
14
C-leucine uptake (1.8 fold) by cardiomyocytes. And this ET-1 mediated upregulation of VEGF in cardiomyocytes was associated with the induction of hypoxia inducible factor (HIF)-1β and HIF-2α, not HIF-1α. Treatment with TA-0201 (10
−6
M), an ET
A
selective blocker, eliminated ET-1-induced overexpression of VEGF and its receptors as well as cardiomyocyte hypertrophy. Treatment with VEGF neutralizing peptides (5–10 μg/ml) partially but significantly inhibited ET-1-induced cardiomyocyte hypertrophy. Both TA-0201 and VEGF neutralizing peptides also significantly prevented the increase of phosphorylated KDR, which implies the activation of VEGF system in ET-1 induced hypertrophied cardiomyocyte. These results suggest that ET-1 treatment of cardiomyocytes promotes overexpression of VEGF and its receptors via activation of ET
A
receptors, and consequently the upregulated VEGF signaling system appears to contribute, at least in part, to ET-1-induced cardiomyocyte hypertrophy.
Abstract 936: Inhibition of the Endoplasmic Reticulum Calcium ATP-ase Affects the Early Steps of Left-Right Patterning in Zebrafish Embryos.
Authors: Kreiling Jil A , Balantac Zaneta L , Crawford Andrew , Toure Jamal , Celik Alper , Kochilas Lazaros K , Creton Robbert
Published: July 2021

Vertebrates develop well defined and conserved left-right (L-R) asymmetries of the heart, gut and brain. Calcium signals play a role in the specification of L-R asymmetry, by translating cilia-dependent fluid flow into asymmetric patterns of gene expression. We aimed to determine the role of early calcium signals on the L-R patterning in zebrafish embryos. Calcium signals were manipulated in zebrafish embryos using thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium ATP-ase pump. The embryos were treated with 0.5 μM thapsigargin during early gastrulation (4 – 6 hpf), mid-gastrulation (6 – 8 hpf), late gastrulation (8 –10 hpf) or early somitic stages (10 –12 hpf). The phenotype was analyzed with subtractive imaging, immunolabeling and
in situ
hybridization (ISH). At 30 hpf, the heart was centralized or reversed in 53% of the early thapsigargin-treated embryos (n=72) vs 5% of the DMSO-treated control embryos (n=75). The embryos were most sensitive to thapsigargin during early and mid-gastrulation with subsequent decrease in heart laterality defects. The incidence of heart laterality defects correlated with decreased or absent
no tail
expression in the dorsal forerunner cells and disruption of Kupffer’s vesicle formation in 73% of the early treated embryos (n=203), vs 2% of the control embryos (n=198) (p<0.01). Deviation from the normal morphological L-R asymmetry in the habenular nucleus of the diencephalon was found in 54% of the thapsigargin treated embryos (n=17) and in 88% of the embryos with L-looped heart (n=9) vs none control embryos. Analysis of the expression pattern of the “left-sided” marker
pitx2
α in the dorsal diencephalon by ISH revealed this to be reversed or bilateral in 56% of the treated embryos (n=97) vs only 10% of the control embryos (p<0.05). In addition 47% of the thapsigargin treated embryo displayed reversed gut-looping (n=49) vs none control embryos. Our data suggest that inhibition of the ER calcium pump by thapsigargin during gastrulation impairs development of the Kupffer’s vesicle and disrupts the concordant heart, brain and gut L-R asymmetry. These results suggest an additional role of calcium in L-R asymmetry determination well before its previously recognized role in sensing fluid flow in the Kupffer’s vesicle.
Abstract 985: BAC Transgenic Analysis of the Smooth Muscle Calponin Gene
Authors: Long Xiaochun , Georger Mary A , Liu Vivian , Miano Joseph M
Published: July 2021

Regulatory elements controlling gene expression are often found in separate, sometimes remote, regions around gene loci. Artificial chromosomes offer a means to capture all regulatory elements for study of gene regulation in a near-correct genomic context. The smooth muscle calponin gene (CNN1) encodes for a multifunctional protein involved in signaling, contractile force generation, and growth regulation. While CNN1’s physiology has been studied extensively, its transcriptional regulation has proven to be intractable to conventional
in vivo
assays. Four evolutionarily-conserved serum response factor (SRF) binding sites (called CArG boxes) are present in the first intron of CNN1 and appear to be required for full transcriptional competence of CNN1 in cultured smooth muscle cells (SMC). To assess the functionality of CNN1 CArG elements
in vivo
, we exploited a 103-kb bacterial artificial chromosome (BAC) containing the human CNN1 locus shown previously to completely recapitulate endogenous mouse CNN1 expression
in vivo
. We now show similar SMC-restricted expression of human CNN1 from only 38-kb of BAC sequence. BAC recombineering studies in which the first intron (containing four CArG elements) of CNN1 is replaced with a bacterial selection gene (
galK
) reveal complete loss of human CNN1 expression in vascular SMC, with residual expression in some visceral SMC-containing tissues such as bladder. Transgenic mice carrying the 38-kb BAC with point mutations in all four intronic CArG elements show similar findings suggesting that SRF directly mediates
in vivo
expression of CNN1 primarily through intronic CArG elements. Bioinformatic, luciferase, and ChIP assays reveal the presence of distal, conserved CArG elements, including a previously unrecognized one located more than 3-kb upstream of the CNN1 transcription start site. The latter CArG-like element is responsive to the SRF coactivator myocardin and is essential for full CNN1 promoter activity
in vitro
. Moreover, this distal CArG-like element is embedded in a
cis
regulatory module with conserved binding sites to other distinct transcription factors. We propose that full
in vivo
CNN1 gene expression requires multiple elements, including five CArG boxes, spread over 14-kb of genomic sequence.
Abstract 991: The Novel BTB/POZ Protein, RhoBTB3, Regulates Mitochondrial Function Via The Ubiquitin Proteasome System
Authors: Yuasa Shinsuke , Onizuka Takeshi , Kenichiro Shimoji , Ohno Yohei , Katayama Masaharu , Arai Takahide , Hattori Fumiyuki , Murata Mitsushige , Hakuno Daihiko , Kimura Kensuke , Makino Shinji , Sano Motoaki , Ogawa Satoshi , Fukuda Keiichi
Published: July 2021

Although BTB/POZ family proteins play a critical role in the development of various organs, and are recently known as a substrate adaptor of CUL3 ubiquitin ligases, the involvement of these proteins in heart development remains unknown. We recently isolated the novel protein, RhoBTB3, an 83-kD protein which comprises an N-terminal Rho GTPase resembling domain and two tandem BTB domains. The purpose of this study was to investigate the molecular and functional characteristics of this protein in mice heart.

[Methods and Results]

RhoBTB3 was faintly expressed in the embryonic heart, but was strongly expressed in the adult heart. It was weakly expressed in adult brain and testis.

RhoBTB3-GFP fusion protein cDNA was transfected into primary cultured rat cardiomyocytes using adenovirus. Fluorescence microscopy revealed that RhoBTB3 was located at the cytoplasm of living cardiomyocytes.

HA-tagged RhoBTB3 and MYC-tagged CUL family proteins (1 – 5) were co-transfected into COS7 cells. RhoBTB3 was co-immunoprecipitated with CUL3
in vitro
, but not with other CUL proteins.

The various mutant RhoBTB3 and wild type CUL3 protein were examined by immunoprecipitation assay. Both the first BTB domain and second BTB domain can bind to CUL3 protein independently.

It was difficult to detect RhoBTB3 in protein analysis. To determine whether or not RhoBTB3 itself could be the Cul3 ubiquitin target, RhoBTB3 protein was measured under the presence of proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide. The RhoBTB3 protein was immediately degraded and dropped below the detection level within 2 hours.

The transgenic mice which overexpressed the heart-restricted RhoBTB3 were healthy at a young age.

But after 3 months, they gradually showed heart failure and finally fell into sudden death. Interestingly before heart failure, electron microscopy demonstrated that mitochondria dramatically increased in number and showed deformity in RhoBTB3 transgenic hearts.

[Conclusions] RhoBTB3 is mainly expressed in the adult heart, and its protein was rapidly degraded with the CUL3-mediated ubiquitin proteasome system in the heart. The overexpression of RhoBTB3 caused a marked increase in abnormal mitochondria, followed by severe heart failure.
Abstract 994: Cardioprotection Against Myocardial Infarction by Ischemic Pre- and Post-Conditioning are Both Completely Abrogated by Acute Hyperglycemia
Authors: Yang Zequan , Xu Yaqin , Kron Irving L , French Brent A
Published: July 2021

Introduction:
Acute hyperglycemia (HG) is associated with larger infarct size, impaired cardiac function and increased mortality, independent of diabetes. We hypothesized that the cardioprotective effects of both ischemic pre- and post-conditioning (pre-C & post-C) would be impacted by acute HG.

Methods:
A total of 37 C57BL/6 mice were assigned to 6 groups. All mice underwent 40 min of LAD occlusion followed by 60 min of reperfusion. Acute HG (~400 mg/dl) was induced in 3 groups with an IP injection of dextrose (2g/kg body weight) given 40 min prior to the start of index ischemia. The ischemic pre-C protocol (2 groups) was 2 cycles of 5 min ischemia and 5 min reperfusion prior to index ischemia. The post-C protocol (2 groups) was 3 cycles of 5 sec reperfusion and 5 sec ischemia following index ischemia.

Results:
Risk region (RR, % of LV) as determined by Phthalo blue staining was similar among study groups (Fig

, p = NS). In euglycemic control mice, infarct size (by TTC staining) without cardioprotection was 51.6 ± 2.0 % of RR (Fig

). Early pre-C reduced infarct size to 25.5 ± 3.7 (a 50% reduction, p < 0.05 vs control) while post-C reduced it to 37.9 ± 0.5 (a 27% reduction, p < 0.05 vs control). In HG mice, infarct size was significantly exacerbated to 63.1 ± 2.9 (a 22% increase, p < 0.05 vs euglycemic controls). Cardioprotection by pre-C was completely abrogated in HG mice (55.9 ± 3.6 of RR, p = NS vs HG controls). Similarly, post-C was completely ineffective in protecting HG mice against infarction (59.4 ± 2.5, p = NS vs HG controls).

Conclusion:
Acute hyperglycemia in non-diabetic mice completely abolishes the cardioprotective effects of both ischemic pre-C and post-C against myocardial infarction.
Abstract 1015: Human Heart Contains A Stem Cell Population With Mesenchymal Properties
Authors: Rossini Alessandra , Scopece Alessandro , Pozzoli Ombretta , Pompilio Giulio , Gaetano Carlo , Capogrossi Maurizio C. , Germani Antonia
Published: July 2021

Background
Mesenchymal Stem Cells (MSC) were initially isolated from the bone marrow (BM) and identified as plastic adherent cells with a typical immunophenotype (negative for CD45, CD34, CD117 and HLA-DR and positive for CD105, CD90, CD44, CD73 and CD13), and able to differentiate into mesodermal lineages
in vitro
. Recently, MSC have been isolated from different adult tissues, i.e. liver, fat, kidney and thymus. Aim of the present study was to search for Cardiac Mesenchymal Stem Cells (C-MSC) in the adult human heart.

Methods and Results
: Auricle fragments were obtained from adult patients undergoing cardiac surgery. From these specimens we isolated a high proliferating plastic adherent cell population that, by FACS analysis, expressed mesenchymal markers (CD105, CD44, CD73, CD13 ) and was negative for hematopoietic markers (CD45, CD34) and HLA-DR. Further, in appropriate differentiating media, these cells exhibited osteogenic and adipogenic differentiation as cultures were positive for Oil Red O and Von Kossa staining used to mark intracellular lipid droplets and calcium salts deposits, respectively. Quantitative Real Time (qRT) -PCR analysis revealed that cells were negative for the early cardiac markers Nkx2.5 and Tbx5 and for the adult cardiac markers α-Myosin Heavy Chain and Myosin Light Chain-2a. Interestingly, in standard culture conditions, cells were positive for the early cardiac marker GATA4; in contrast, BM-MSC expressed GATA4 only after dexamethasone treatment. Additionally, when injected into the zebrafish blastula (about 100 –200 cells, n=70), C-MSC specifically homed in the cardiac region, as found at 24 –28 hpf (hours post fertilization). In contrast, transplanted BM-MSCs, were found not only in proximity of the heart, but throughout several mesodermal derivatives of the embryo (n=25).

Conclusions:
The adult human heart contains C-MSCs that can be easily expanded
in vitro
and may represent a useful population for autologous cell therapy of some cardiac diseases.
Abstract 1031: First Human Experiments: Aggressive Statin Therapy Reduced Key Inflammatory Factors including c-Jun N-terminal kinase (JNK) and Dendritic Cell and Matrix Metalloproteinase Expression in Human Abdominal Aortic Aneurysmal Wall
Authors: Kajimoto Kan , Miyauchi Katsumi , Shimada Kazunori , Kasai Takatoshi , Kojima Yuko , Shimada Akie , Sumiyoshi Katsuhiko , Niinami Hiroshi , Amano Atsushi , Daida Hiroyuki
Published: July 2021

Abdominal aortic aneurysms (AAA) accumulate feature of a chronic inflammatory disorder and irreversible destruction connective tissue. Recent experimental study demonstrated that c-Jun N terminal kinase (JNK) was a proximal signaling molecule in the pathogenesis of AAA and vascular dendritic cells (DC) were the key molecular in inflammatory reaction and the degradation of the extracellular matrix. Statins can inhibit cell proliferation and vascular inflammation, which might contribute to prevent AAA progrssion. But supporting clinical data from human studies are lacking. We hypothesized that atorvastatin might inhibit JNK and DC, resulting in suppression of inflammatory cells and matrix metalloproteinase (MMPs) in human tissue of AAA.

Methods
: Patients with AAA were randomized to the atorvastatin (20 mg/day, 10 patients) group or control (10 patients) group. After treatment of 4 weeks, patients underwent abdominal aorta replacement, tissue specimens were obtained, and the tissue composition was assessed with special stains and immunocytochemistry with quantitative image analysis.

Results
:Atorvastatin significantly reduced JNK and DC expression (46% and 72%) compared to control. T cells, macrophages (60%, 72%,
P<
0.05), and MMP-2 and MMP-9 immunoreactivity (47%, 43%,
P<
0.05) were also suppressed in atorvastatin group. However, tissue expression of metalloproteinase 1 (TIMP-1) immunoreactivity and a collagen content by sirius red staining were similar in both groups. In atorvastatin group, serum LDL-C level was significantly decreased by 40%.

Conclusion:
Atorvastatin treatment reduced the JNK expression and DE, resulting in inflammatory cells content and the expression of MMPs in human abdominal aortic aneurysm wall.
Abstract 1042: Viral Chemokine Modulating Protein, M-T7, Interrupts Chemokine : Glycosaminoglycan (GAG) Interaction: M-T7 Inhibitory Activity is Dependent upon Y46 and V210 aminoacid residues
Authors: Dai Erbin , McIvor Dana , Liu Liying , Munaswamy-Ramanujam Ganesh , Sun Yunming , Esko Jeffrey , Charo Israel F , McFadden Grant , Lucas Alexandra
Published: July 2021

Background: Chemokines bind to glycosaminoglycans (GAGs) forming gradients that direct inflammatory cell invasion. The viral chemokine modulating protein (CMP), MT-7 binds the C terminal, GAG-binding domain of chemokines and has been previously reported to significantly reduce cell invasion and plaque growth in rat aortic and renal transplant models. Two other viral CMPs, M-T1 and M3 CMPs bind the N terminal domain of chemokines that bind to cell surface receptors. To determine the role of CC chemokine receptor 2 (CCR2) and GAGs for M-T7 anti-inflammatory activity, effects of M-T7 on plaque growth were assessed after mouse CCR2 deficient (CCR2−/−) or GAG deficient (NDST1−/−) aortic allograft transplant. Mononuclear cell migration in response to MCP-1 or RANTES into mouse ascites was also tested. Active sites necessary for M-T7 inhibition of chemokine function and monocyte activation, were assessed by infusion of in the mouse cell migration and human monocyte membrane fluidity assays.

Results: M-T7 significantly reduced cell migration and intimal hyperplasia in wild type CCR2+/+ (p<0.009), and CCR2−/− aortic transplants (p<0.026). M-T1 and M3 inhibited cell invasion and plaque in CCR2+/+, but not CCR2−/− mice. M-T7 inhibited plaque growth and CC chemokine (MCP-1 and RANTES)-induced cell migration in wild type mice (P<0.01), but not in NDST1−/− mice (P=0.34). Selected M-T7 point mutations Ty (Y)46A, and Val (V) 210A no longer block chemokine-induced cell migration nor monocyte activation, whereas Asn (N) 40, Asn (N) 63 and Val (V)129 retain inhibitory activity.

Conclusions:

M-T7 but not M-T1 nor M3, blocks cell migration and plaque growth in CCR2 deficient (CCR2−/−) mouse aortic transplant models.

M-T7 loses the ability to block cell migration and plaque growth in NDST1−/−, GAG (heparan sulfate) deficient mice.

Point mutations Tyr46 and Val 210 lack inflammatory for mouse and human inflammatory monocyte responses indicating that these amino acid residues on the M-T7 CMP protein are required for inhibitory activity.
Abstract 1044: Entpdase1 (CD39) Downregulates Monocyte And Neutrophil MAC-1 Expression
Authors: Hyman Matthew C , Petrovic-Djergovic Danica , Mazer Sean P , Pinsky David J
Published: July 2021

The ecto-enzyme ENTPDase1 (CD39) has been shown to limit neutrophil and monocyte influx following brain, gut, and kidney ischemia-reperfusion injury, yet the exact mechanism remains unclear. Although it has been proposed this increased leukocyte-recruitment may be related to an effect of endothelial CD39 on endothelial barrier function, here we sought to explore a different hypothesis, one of intrinsic regulation of leukocyte trafficking by CD39 bound to the surface of the leukocytes themselves. Flow cytometry was used to analyze whole blood for adhesion molecule expression and all values were expressed relative to wild-type (WT) or vehicle injected controls. A specific upregulation of the purine responsive adhesion molecule CD11b (the μM subunit of MAC-1) was seen in CD39
−/−
monocytes, compared to WT controls (1.83± .25, n=6, p<0.01). The neutrophil population, conversely, did not display differences in cell surface MAC-1 at baseline (0.94 ± .14, n=6, p>0.05). However, following stimulation with the bacterial mimetic peptide fMLP, CD39
−/−
neutrophils showed a 34% greater increase in expression of MAC-1 compared to fMLP treated WT neutrophils (2.58 ± 0.50 vs. 1.92 ± 0.13, n=6, p<0.03). This elevated CD11b expression on fMLP stimulated CD39
−/−
neutrophils was highly reproducible. There was no change in CD11b expression following fMLP stimulation in WT or CD39
−/−
monocytes. Treatment of CD39
−/−
animals with a soluble apyrase (a functional analogue of CD39 which has been shown to mitigate platelet desensitization in CD39
−/−
mice) resulted in restoration of a near wild-type expression of CD11b on monocytes relative to vehicle injected wild-type animals (1.1 ± 0.2, p>0.05). By comparison, treatment of a wild-type animals with apyrase resulted in a 50% reduction in monocyte surface CD11b expression relative to vehicle (0.45 ± 0.03, p<0.01). These findings reveal a mechanism of leukocyte autoregulation whereby CD39 modulates leukocyte trafficking to sites of vascular injury through a mechanism of intrinsic leukocyte catabolism of nucleotides and possibly via intrinsic and localized generation of adenosine.
Abstract 1046: The ApoA-I Mimetic Peptide, 4F, Inhibits LPS-induced Inflammatory Reactions Through Dysregulation Of The NFkB Activation Pathway.
Authors: Datta Geeta , White C. R , Chaddha Manjula , Palgunachari M. N , Anantharamaiah G. M , Smythies Lesley
Published: July 2021

Background: The apoA-I mimetic peptide 4F possesses potent anti-inflammatory properties. We previously reported that 4F inhibits lipopolysaccharide (LPS)-induced cytokine, chemokine and adhesion molecule expression
in vivo and in vitro
. Other data show that 4F reduces macrophage content in atheroma in susceptible mice.

Objective: In this study, we explored anti-inflammatory mechanisms of 4F action under basal conditions and in the presence of LPS using THP1 monocytes/macrophages and primary human blood monocytes.

Methods and Results: Blood monocytes isolated by Ficoll and CD14+ MACS separation were analyzed by FACS for CD14, TLR-4 and p-NFκB expression. Cytokines, chemokines and adhesion molecules were measured by the Searchlight multi-analyte analysis (Pierce, MA, USA). 4F treatment (3 days) of both THP-1 and blood monocytes enhanced the secretion of GM-CSF (25-fold), a potent inducer of monocyte to dendritic cell differentiation. 4F also inhibited (by 80%) the PMA-induced phagocytosis of FITC-labeled beads by THP-1 cells. In subsequent studies, effects of 4F on LPS-induced inflammatory responses in monocytes were tested. Pre-treatment with 4F significantly inhibited LPS-induced MCP-1 expression (by 50%) in both THP-1 cells and blood monocytes. Under these conditions, the expression of the anti-inflammatory cytokine IL-10 was increased by 200%. The effects of 4F on the activation of NFκB, a downstream mediator of LPS action, were studied using flow cytometry. 4F pre-treatment significantly inhibited LPS-induced NFκB phosphorylation (NFκBp65) compared to vehicle treatment. Protective effects of 4F in LPS-treated monocytes were associated with a 50% reduction in the expression of TLR-4 and CD14, cell surface receptors for LPS.

Conclusions: These data provide evidence that 4F modulates functional properties of monocytes and show for the first time that 4F inhibits LPS-induced inflammatory responses by inhibiting the expression of CD14, TLR-4 and phosphorylation of NFκB. Enhanced GM-CSF expression and inhibition of monocyte phagocytic activity also suggest that 4F may favor the differentiation of monocytes to a dendritic cell rather than a macrophage phenotype.
Abstract 1068: Genetic Evidence for a Central Role of cGMP-Dependent Protein Kinase I Alpha in Regulating Cardiac Hypertrophy
In Vivo
Authors: Blanton Robert M , Aronovitz Mark , Dabreo Alexandra , Karas Richard H , Mendelsohn Michael E
Published: July 2021

The nitric oxide-cGMP-dependent protein kinase pathway is a central regulator of cardiovascular physiology. Protein Kinase G I (PKGI), a principle mediator of this pathway, has been implicated as a negative regulator of cardiac hypertrophy, but the specific mechanisms involved are unknown. To test the hypothesis that PKGIα regulates cardiac hypertrophy, we characterized the cardiac phenotype of mice homozygous for a mutant form of PKGIα in which critical amino acids in the N-terminal leucine zipper (LZ) motif have been substituted to disrupt PKGIα LZ binding to specific downstream effector proteins. In the unstressed state, male PKG Iα leucine zipper mutant ( LZM) mice develop progressive LV hypertrophy compared with wild type (WT) littermates with LV mass/tibia length 12.3% greater at 30 weeks of age (p=0.05), and 27% greater at 60 weeks (p=0.001). Compared with WT littermates, the hearts of 30 week old PKGIα mutants are hypercontractile with decreased end systolic diameter (p=0.02) and increased fractional shortening on echocardiography (p=0.03). Invasive hemodynamics demonstrate that LZM mice also have increased LV systolic pressure (p=0.04), developed pressure (p=0.05), and LV dP/dt max (p=0.13). To evaluate the response to hemodynamic stress, cardiac hypertrophy was induced by transaortic constriction (TAC) in male WT and LZM mice. TAC resulted in early mortality in the LZM mice (60%) compared to the WT (19%) mice at 21 days post procedure (p=0.008), with evidence of accelerated LV hypertrophy in the mice that died early (LV mass/tibia length 9.8 mg/mm in the early LZM deaths vs 7.2 mg/mm in WT survivors, p<0.001). Additionally, the LZM early deaths had evidence of congestive heart failure with increased RV mass (RV mass/ tibia length 1.8 mg/mm in LZM early deaths vs 1.2 mg/mm in WT survivors, p<0.05), and increased lung mass (23.1 mg/mm LZM early deaths vs 12.5 mg/mm in WT survivors, p<0.005). These findings support that the N-terminal LZ domain of PKGIα is required for suppression of cardiac hypertrophy. The early mortality following TAC in the LZM mice also suggests a critical role for PKGIα in attenuating pathologic cardiac remodeling, identifying PKGIα as an attractive candidate drug target for prevention of cardiac hypertrophy and failure.
Abstract 1076: Conditional Fkbp12.6 Overexpression In Mouse Cardiac Myocytes Protects From Triggered Ventricular Arrhythmia Through Specific Alteration In Ec Coupling
Authors: Gellen Barnabas , Gomez Ana-Maria , Le Quang Khai , Briec Francois , Vinet Laurent , Rouet Patricia , Benitah Jean-Pierre , Pezet Mylene , Fernández-Velasco María , Palais Gael , Pellegrin Noemie , Zhang Andy , Perrier Romain , Escoubet Brigitte , Richard Sylvain , Jaisser Frederic , Charpentier Flavien , Mercadier Jean-Jacques
Published: July 2021

In cardiac myocytes, Ca2+ release from the sarcoplasmic reticulum (SR) into the cytoplasm via the ryanodine receptor (RyR2) activates cell contraction. During diastole, RyR2 are closed and prevent Ca2+ efflux from the SR. One of the major regulators of RyR2 function is FKBP12.6. Binding of FKBP12.6 stabilizes RyR2 in the closed formation in diastole and contributes to synchronized RyR2 opening in systole. Beta-adrenergic stimulation dissociates FKBP12.6 from RyR2, leading to diastolic Ca2+ leak that can trigger ventricular tachycardias (VT). We tested the hypothesis whether FKBP12.6 overexpression in the myocardium can reduce the susceptibility to VT in stress conditions. We developed a mouse model with conditional cardiac specific overexpression of FKBP12.6 using the Tet-Off system. Transgenic (TG) mice and controls (CT) were examined by echocardiography, PV-catheterization, ECG, and underwent intracardiac stimulation to trigger VT before and after pre-treatment with isoproterenol. In isolated cardiac myocytes, SR Ca2+ load, Ca2+ sparks and Ca2+ transient were measured using confocal microscopy, and L-type Ca2+ current was determined by the patch-clamp technique. Echocardiography, PV-catheterization and ECG recording did not reveal differences between Tg (n=11) and CT (n=13) mice. Burst pacing (figure 1) could induce TV in 4 of 24 controls and in 0 of 14 TG mice (n.s.). Following pre-treatment with isoproterenol, TV could be induced in 10 of 23 controls, but only in 1 of 14 TG animals (figure 2, p<0.05). In isolated myocytes, decreased Ca2+ spark frequency, increased Ca2+ spark size, unchanged SR Ca2+ load and decreased Ca2+ transient were observed in TG cells (n=19) as compared to controls (n=48, p<0.05). L-type Ca2+ channel current was found to be decreased in Tg myocytes (n=29 vs n=32, p<0.01). We conclude that myocardial FKBP12.6 overexpression has a protective effect against VT induced by rapid pacing after pretreatment with catecholamines. This antiarrhythmic effect is probably, at least in part, linked to decreased diastolic SR Ca2+ leak. Our results support the hypothesis that increased FKBP12.6 binding to RyR2 might represent a potential therapeutical target in the prevention and treatment of ventricular arrhythmias.
Abstract 1102: Cardiac-Specific Myosin Light Chain Kinase (MLCK), a Third Member of MLCK Family, Potentiates Sarcomere Formation and Cardiac Contraction
Authors: Chan Jason Y , Takeda Morihiko , Briggs Laura E , Lu Jonathan T , Horikoshi Nobuo , Weinberg Ellen O , Aoki Hiroki , Chien Kenneth R , Kasahara Hideko
Published: July 2021

Background: Two myosin light chain kinase (MLCK) proteins, skeletal (encoded by mylk2 gene) and smooth muscle MLCK (encoded by mylk1 gene) have been shown to be expressed in mammals. Human mylk2 has been mapped as a disease locus for familial cardiac hypertrophy (OMIM
606566
), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. While phosphorylation of the putative substrate of skeletal MLCK, myosin light chain 2 (MLC2), is recognized as a key regulator of cardiac contraction, the abundance of skeletal MLCK in the heart is controversial, suggesting the existence of an additional MLCK that is preferentially expressed in cardiac muscle.

Methods and Results: We characterized a new kinase named cardiac MLCK that is encoded by a gene homologous to mylk1 and 2 and is specifically expressed in the heart in both atrium and ventricle. Expression of cardiac MLCK was highly regulated by the cardiac homeobox transcription factor, Nkx2.5, in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK including putative catalytic and adjacent Ca2+/calmodulin binding domains at the carboxyl-terminus. The amino-terminus is unique without significant homology to other known proteins. Cardiac MLCK phosphorylated MLC2v with a catalytic value of Km=4.3 micro M (Lineweaver-Burk analysis) indicating high affinity of cardiac MLCK to MLC2v, similar to the affinity of skeletal muscle MLCK to skeletal muscle MLC2 and smooth muscle MLCK to smooth muscle MLC2. Adenoviral-mediated overexpression of cardiac MLCK and knockdown of cardiac MLCK using RNAi in cultured cardiomyocytes revealed that cardiac MLCK regulates MLC2v phosphorylation, sarcomere organization and cardiac myocyte contraction. Expression of cardiac MLCK protein was significantly decreased in severe heart failure in vivo (post-myocardial infarction heart failure mouse model).

Conclusion: Cardiac MLCK is a new key regulator of cardiac contraction and sarcomere organization. Reduction of cardiac MLCK function leading to decreased phosphorylation of MLC2v may contribute to compromised contractile function in the failing heart.