bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 940: C-kit Positive Cardiac Progenitor Cells Contribute to the Embryonic Development of the Heart
Authors: Iaffaldano Grazia , Misao Yu , LeCapitaine Nicole , Bearzi Claudia , Gonzalez Arantxa , Hosoda Toru , Urbanek Konrad , Rota Marcello , Sonnenblick Edmund H , Ottolenghi Sergio , Meggs Leonard G , Leri Annarosa , Anversa Piero , Kajstura Jan
Published: July 2021

Recently, a compartment of c-kit positive cardiac progenitor cells (CPCs) has been identified in the adult mouse heart. Whether CPCs are restricted to the adult myocardium or are present throughout ontogenesis and contribute to heart development is unknown. For this purpose, a transgenic mouse in which EGFP was driven by the c-kit promoter was employed. C-kit-EGFP positive cells were present in the heart at all stages of embryonic and fetal development, and their number increased with time reaching 400 cells at E17. Symmetric and asymmetric division of c-kit-positive cells was identified respectively by the uniform and non-uniform localization of the endocytic proteins, Numb and α-adaptin. The presence of symmetric and asymmetric division was consistent with the rapid expansion of the pool of c-kit positive cells and the simultaneous generation of a committed progeny. A fraction of c-kit-EGFP positive cells expressed the myocyte transcription factors Nkx2.5 and MEF2C or specific sarcomeric proteins, α-sarcomeric actin, β-cardiac actinin, β-myosin heavy chain and troponin T. These findings demonstrated a linear relationship between CPCs and myocyte formation. Importantly, observations by two-photon microscopy showed that c-kit-EGFP positive cells in the forming heart exhibited morphogenic movements. Conversely, c-kit-EGFP positive cells from extracardiac regions or from the yolk sac did not translocate to the developing heart. Importantly, cardiac c-kit positive cells were negative for markers of hematopoietic stem cells CD34 and CD45. When c-kit-EGFP-positive cells were isolated from the embryonic heart and plated in vitro at limiting dilution, they formed single-cell derived clones. Almost all cells in the clones continued to express c-kit and EGFP. However, few cells located at the periphery of the clones were no longer positive for c-kit and EGFP and began to express sarcoplasmic proteins. In conclusion, c-kit positive cells in the developing myocardium exhibit the properties of stem cells; they are clonogenic and self-renewing and participate in the growth of the embryonic and fetal heart.
Abstract 944: Tight Binding Of Calmodulin To The Cardiac Ryanodine Receptor May Reduce The Spontaneous Ca
2+
Release Events Induced By Mutation-linked, Defective Inter-domain Interaction
Authors: Xu Xiaojuan , Yano Masafumi , Ohno Makoto , Uchinoumi Hitoshi , Tateishi Hiroki , Kobayashi Shigeki , Yamamoto Takeshi , Ikeda Yasuhiro , Matsuzaki Masunori
Published: July 2021

Interaction between N-terminal
1– 600
and central domains
2000 –2500
of ryanodine receptor (RyR2), where many mutations have been found in patients with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVC2) or catecholaminergic polymorphic ventricular tachycardia (CPVT), was recently found to play a critical role in channel regulation. Using the domain peptide approach, here, we investigated the role of calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), on Ca
2+
release kinetics. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) using DP
163–195
, which harbors a human mutation site in ARVC (R176Q), as a site-directing carrier. DP
163–195
mediated a specific MCA fluorescence labeling of central domain (60Kd) of RyR2. Addition of DP
163–195
to the MCA-labeled SR competitively induced the domain unzipping between N-terminal and central domains, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In saponin-permeabilized cardiomyocytes, the addition of DP
163–195
markedly increased the frequency of Ca
2+
sparks (SpF; s
−1
·100μm
−1
:13.1±0.9, p<0.01), compared with normal cells (6.9±0.3). Addition of recombinant CaM (100nM), in the presence of KN-93 (CaMKII inhibitor), inhibited the DP
163–195
-induced increase in SpF (7.2±0.5, p<0.01). This effect of CaM was, however, abolished by co-addition of the antibody against the binding site of CaM within RyR2 (3583–3603) (SpF:12.9±0.7, p=ns), strongly suggesting that the binding of CaM to RyR2 corrects the abnormal Ca
2+
release induced by DP
163–195
. The mutation made in the domain peptide, mimicking the same human mutation in ARVC (R176Q) abolished all of the effects that would have been produced by DP
163–195
. In conclusion, the mutation-linked defective inter-domain interaction between N-terminal and central domains within RyR2 (viz. domain unzipping) may increase spontaneous Ca
2+
release, perhaps by weakening CaM-binding to RyR2. Restored binding of CaM to RyR2 may correct defective channel gating of the mutant RyR2.
Abstract 953: Ca
2+
-permeable TRP Channels In Human Cardiac Fibroblasts
Authors: Du Jianyang , Silverman David , Liang Bruce , Yue Lixia
Published: July 2021

In our laboratory, recent single cell electrophysiologic studies have demonstrated the absence of voltage-gated Ca
2+
channels in human cardiac fibroblasts. The more positive membrane potential found in these cells suggests that Ca
2+
entry occurs through a different mechanism. We hypothesized that non-voltage-gated Ca
2+
-permeable TRP channels are responsible for Ca
2+
entry in human cardiac fibroblasts. With informed consent, right atrial biopsies were obtained from patients undergoing cardiac surgery (n=4:.3M, 1F; mean age 65±8 yrs, EF 63±5%, LVEDP 24±4 mm Hg). Fibroblasts were dissociated and cultured for 7 to 10 days. We found that TRPC1, TRPC4, TRPC6, TRPV4, TRPV5, TRPV6, TRPM4 and TRPM7 were detectable at message levels by RT-PCR. Functional expression of these channels was evaluated by patch-clamp technique. An outward rectifying current with typical I–V relation of TRPM7 was readily recorded in the fibroblasts. The averaged current density was 14.5±0.8 pA/pF (mean±SEM, n=60 from four patients). These currents exhibit similar properties to those of heterologues expressed TRPM7, including permeation to Ca
2+
and Mg
2+
, single channel conductance, and inhibition by 2-APB. Moreover, the TRPM7-like current in human fibroblasts was activated by acidic pH conditions, suggesting that this channel function may be enhanced under ischemic conditions. To study whether this TRPM7-like current promotes fibroblast proliferation, we designed siRNA specifically targeting human TRPM7. Fibroblasts treated with TRPM7 siRNA for five days showed a 76.3% decrease in current density, indicating that transcription of trpm7 produces the message for the Ca
2+
-pemeable channel in human cardiac fibroblasts. Consistent with the decreased current amplitude, Ca
2+
influx in siRNA treated fibroblasts was significantly smaller than those of control fibroblasts, further suggesting that TRPM7-like current contributes to calcium signaling in these cells. Taken together, our results indicate that TRPM7 provides the molecular basis of the Ca
2+
-permeable cation channel in human cardiac fibroblast, and that TRPM7 may play a crucial role in the fibrogenesis cascade in human heart.
Abstract 955: Molecular Mechanisms of Ca
2+
and Mg
2+
Permeability and pH Sensitivity of the Bi-functional Channel Kinase TRPM7
Authors: Du Jianyang , Li Mingjiang , Jiang Jianmin , Ratzan William , Su Li-Ting , Runnels Loren , Yue Lixia
Published: July 2021

The channel-kinase TRPM7 have recently been discovered to play important roles in Mg
2+
and Ca
2+
homeostasis, which is critical to both human health and cell viability. TRPM7 is highly expressed in both cardiac myocytes and fibroblasts. We recently found that both heterologous expressed TRPM7 currents and endogenous TRPM7-like currents in the heart are sensitive to changes of extracellular pH. Acidic pH significantly potentiates inward current of TRPM7. Given the importance of TRPM7 in Ca
2+
and Mg
2+
homeostasis and potential pathological role of TRPM7 under acidosis conditions, this study was designed to elucidate the molecular basis underlying TRPM7’s unique Mg
2+
and Ca
2+
permeability and pH sensitivity. We created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the channel’s divalent permeability and pH sensitivity. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I–V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than that of TRPM7, respectively. The binding affinity of Ca
2+
and Mg
2+
was decreased by 50-to 140-fold in E1052Q and E1047Q, respectively. Ca
2+
and Mg
2+
currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca
2+
and Mg
2+
permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E 1047Q and largely diminished in E1052Q, indicating that E1047 and E1052Q are critical to TRPM7’s Ca
2+
and Mg
2+
permeability, and its pH sensitivity. Our results indicate that these two glutamate residues are key determinants of both channels’ divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/ pathological functions of TRPM7, and provide molecular insight of the pore architecture of the bi-functional channel kinase TRPM7.
Abstract 956: Acid-Sensing Ion Channels (ASICs) Contribute to Transduction of Extracellular Acidosis in Rat Carotid Body Glomus Cells
Authors: Tan Zhi-Yong , Lu Yongjun , Whiteis Carol A , Benson Christopher J , Chapleau Mark W , Abboud Francois M
Published: July 2021

The molecular mechanism of pH sensing by chemoreceptors is not clear, although it had been proposed to be mediated by a drop in intracellular pH of carotid body glomus cells, which inhibits a K
+
current. Recently, pH-sensitive ion channels have been described in glomus cells that respond directly to extracellular acidosis. In this study, we investigated the possible molecular mechanisms of carotid body pH-sensing by recording the responses of glomus cells isolated from rat carotid body to rapid changes in extracellular pH using whole-cell patch-clamping technique. Extracellular acidosis evoked transient inward currents in glomus cells that were evident at pH 7.0 and half-activated (pH 50) at 6.3. The current had the characteristics of ASICs. It averaged 40.7±15.7 pA (n=5) at pH 5.0 and was blocked by the ASIC channel blocker amiloride (200 μm) to 2.5±1.6 pA. Na
+
free bathing solution eliminated the current and a Ca
2+
free buffer enhanced (P<0.05) the current at pH 6.0 from 18.5±2.2 to 86.0±12.5 pA (n=5). Enhancement of the current was also seen with the addition of lactate. In the current clamp mode extracellular acidosis evoked both a transient and sustained depolarization. The initial transient component at pH 6.0 averaged 18.2±2.6 mV and was blocked by amiloride to 2.1±2.1 mV supporting the contribution of ASICs. However, the sustained depolarization was not blocked by amiloride but was eliminated by removal of K
+
from the pipette solution which reduced significantly intracellular K
+
. This sustained depolarization was partially blocked by the TASK channels blockers anandamide (from 14.9±1.6 mV to 9.3±2.2 mV at pH 6.0, n=5) and quinidine (from 27.5±2.2 mV to 11.3±2.3 mV at pH 6.0, n=3). The results provide the first evidence that ASICs may contribute to chemotransduction of low pH by carotid body chemoreceptors, and that extracellular acidosis directly activates carotid body chemoreceptors through both ASIC and TASK channels.
Abstract 974: Endothelial NOS (eNOS) Regulates Mitochondrial Oxidative Stress in Fetal Pulmonary Artery Endothelial Cells (PAEC)
Authors: Konduri Girija , Eis Annie , Bakhutashvili Ivane
Published: July 2021

Background
: Endothelial NOS regulates pulmonary vasodilation at birth by synthesis and release of NO. Exposure of fetal lung to O
2
results in increased oxidative phoshporylation and ATP release at birth. ATP stimulates NO release from eNOS. Superoxide (O
2
−
) is generated by mitochondria as a byproduct of oxidative phosphorylation. The regulation of mitochondrial O
2
during this transition and its role in impaired vasodilation that occurs in persistent pulmonary hypertension of newborn (PPHN) remain unknown.

Objective
: We investigated the hypothesis that targeting of eNOS to mitochondrial outer membrane regulates O
2
−
in fetal PAEC in response to ATP and O
2
.

Methods
: Studies were done in isolated PAEC and pulmonary arteries (PA) from fetal lambs. PPHN was induced by prenatal ductal constriction for 8 days. Association of eNOS with outer membrane protein, porin and chaperone, hsp90 was determined by immunoprecipitation and double label immuno-fluorescence. Mitochondrial NO and O
2
−
levels were determined by DAF-2 and mito-HE reagents. Relaxation responses of PA rings to ATP and NO were determined in the presence/absence of decoy peptides that inhibit eNOS association with hsp90 and porin and mitochondrial targeted ubiquinones.

Results
: Mitochondria from PAEC showed presence of eNOS on outer membrane but no nNOS. Exposure of PAEC to ATP or O
2
increased the association of eNOS with porin and hsp90 in the mitochondria. ATP increased mitochondrial NO release. Inhibition of eNOS-hsp90 association or eNOS-porin association with decoy peptides that mimic specific eNOS domains required for these interactions resulted in increased mitochondrial O
2
−
and attenuated the relaxation response of PA rings to ATP and NO. PAEC from PPHN lambs showed increased basal O
2
consumption, decreased NO-regulation of O
2
consumption and increased mitochondrial O
2
−
. PA rings from PPHN lambs showed impaired relaxation responses to ATP and NO and improved relaxation when treated with mitochondrial targeted ubiquinone.

Conclusion
: Interaction of eNOS with mitochondria plays an important role in the regulation of O
2
−
in PAEC during postnatal transition to the O
2
rich environment. Pulmonary hypertension is associated with impaired targeting of eNOS and increase in mitchondrial O
2
−
.
Abstract 988: Tissue Inhibitor Of Metalloproteinase-3 Regulates Myocardial Fibrosis By Regulating The TGFβ-TNF Interaction
Authors: Kassiri Zamaneh , Hariri Mehrdad , Anthwal Shalini , Oudit Gavin Y , Dawood Fayez , Liu Peter P , Khokha Rama
Published: July 2021

Fibrosis is the outcome of excess deposition of extracellular matrix components and underlies diastolic and systolic heart failure. Metalloproteinases (MMPs and ADAMs) and their inhibitors (TIMPs) are critical regulators of ECM turnover and fibrosis. TIMP3 is a potent inhibitor of MMPs and ADAMs, whereby it regulates tissue proteolysis and cytokine bioavailability. TIMP3 levels are reduced in human heart disease, and we found that mice lacking Timp3 (KO) exhibit severe dilated cardiomyopathy (DCM), extensive interstitial fibrosis and early heart failure (HF) following pressure overload compared to wild type (WT) mice. We showed upregulated TNF signaling within 6 hrs of aortic banding (AB) which when blocked, significantly improved DCM and prevented HF. We then investigated the mechanism underlying the extensive fibrosis despite the increased MMP activity in the TIMP3KO mice. Comparative microarray analyses show a significant upregulation of the TGFβ1 signaling pathway within 6 hrs of AB in KO vs WT hearts.
In vivo
treatment of KO-AB mice with a TGFβ1-neutralizing antibody (1D11) completely abolishes fibrosis, markedly improves left ventricular dilation, systolic and diastolic heart function (LVEDD(mm): 4.3 ± 0.1 in WT, 5.4 ± 0.1 in KO, 4.7 ± 0.1 in KO+ 1D11; fractional shortening(%): 40.2 ± 1.7 in WT, 19.6 ± 2.2 in KO, 36.2 ± 0.7 in KO+ 1D11; E/Ac ratio: 2.5 ± 0.3 in WT, 1.8 ± 0.3 in KO, 2.4 ± 0.2 in KO + 1D11; n = 22, P < 0.05). Interestingly, blocking TGFβ1 also prevented the early rise in TNF in KO hearts. Using primary neonatal culture systems, we found that cardiomyocyte-fibroblast interaction is essential for a fibrotic response to agonists (Ang II, PE). Further, KO co-cultures generate a 5-fold greater fibrosis (P < 0.05) through activation of Smad2/3 pathway compared to WT. TGFβ also regulates TNF transcription more potently in KO cells since a 3-fold higher TNF induction occurred in response to recombinant TGFβ1 in KO vs WT co-cultures (P < 0.05). In conclusion, TIMP3 is a critical regulator of TGFβ1 and TNF, and its deficiency induces parallel dysregulations in the signaling of these cytokines very early after pressure overload, leading to severe interstitial fibrosis and DCM, hence, TIMP3-based therapies can simultaneously impact fibrosis and DCM.
Abstract 996: Growth Differentiation Factor-5 (GDF-5) Dictates Cardiac Remodelling in a Mouse Model of Myocardial Infarction
Authors: Zaidi Syed , Riazi Ali M , Huang Qingling , Momen Md A , Husain Mansoor
Published: July 2021

Background:
Bone Morphogenetic Proteins (BMPs) regulate diverse cellular functions during foetal development and postnatal life. Growth Differentiation Factor 5 (GDF5 a.k.a. BMP-14) is a BMP, which is expressed in a variety of tissues including heart. We previously showed that cardiac GDF5 mRNA levels are elevated after experimental myocardial infarction (MI) caused by permanent left anterior descending coronary artery (LAD) ligation. However, the significance of this finding was not known.

Methods & Results:
GDF5 knock-out (KO; n = 18 for MI) and wild-type (WT; n = 18 for MI) littermate controls were subjected to chronic LAD ligation in order to investigate the consequences resulting from the loss of GDF5 signalling following MI. At 28 days post-LAD ligation or sham (n = 12 for KO; n = 10 for WT), invasive hemodynamic parameters of cardiac function were examined just prior to sacrifice. Histopathology was assessed by morphometric analyses of perfusion fixed hearts and subsequent immunostaining. At 28 days post-MI, GDF5-KO mice exhibited decreased left ventricular systolic pressure and peak positive- and negative-
dP/dt
, and increased heart rate as compared to WT littermates (
P < 0.005
for each parameters). GDF5-KO mice also exhibited a significant increase in the area, length and transmural expansion of the infarct, scar thinning and cardiac dilatation (
P < 0.05
for each parameter). In addition, GDF5-KO mice displayed significantly fewer myocardial vessels in the infarct and peri-infarct regions as compared to WT littermates (
P < 0.05)
. To explore mechanisms underlying this phenotype, we assessed gene expression levels of relevant potential downstream targets of GDF5. At 7d post-MI, quantitative RT-PCR revealed a significant reduction (35%) in VEGF mRNA levels in hearts of KO (n = 6) as compared to WT mice (n = 5, P = 0.033).

Summary & Conclusion:
These data suggest that increased GDF5 expression observed in hearts after MI plays an important role in cardiac remodelling. Absence of GDF5 expression in KO mice confers detrimental effects on healing and repair of myocardial and vascular tissues after MI. Regulated levels of GDF5, a BMP family member, play an important role in the repair process following cardiac injury.
Abstract 1016: Regenerative Cellular Therapy: How To Enrich The Mesenchymal Stem Cell Content In The Periinfarction Zone After Myocardial Infarction
Authors: Schrepfer Sonja , Deuse Tobias , Stein William , Reichenspurner Hermann , Fischbein Michael P , Robbins Robert C , Pelletier Marc P
Published: July 2021

Background:
The pluripotent capacity of adult mesenchymal stem cells (MSCs) makes this cell type a potential candidate for tissue regeneration after myocardial infarction (MI). However, the most effective route of MSC application and the impact of contributory cytokine enhancement remains to be elucidated.

Methods and Results:
Murine CD90 + CD45-CD34-sca-1 + c-kit-MSCs were culture-expanded, stably transfected with firefly luciferase for bioluminescence imaging (BI), and labeled with CSFE for histology (H). High-throughput proteomics and a battery of
in vitro
and
in vivo
assays were used to profile 20 key cardiac-specific cytokines upregulated following MI for their effects on the biologic performance of MSCs. VEGF and HGF showed most effective enhancement of MSC proliferation, migration, and trans-membrane invasion. After LAD ligation, syngeneic mice received intraperitoneal (IP), intravenous (IV) or intramyocardial (IC) MSC injections. The development of cardiac MSC content was closely monitored by
in vivo
BI and H. Cytokine enhancement (CE) protocol: VEGF 8ng/ml and HGF 40ng/ml were injected into the infarction border zone to attract MSCs to the heart (after IV- or IP-injection) or to retain them in the heart (after IC-injection). 1 day after IP injection, a maximum of only 0.4 ± 0.5 cells/HPF was detected in the heart. Most IP-injected MSCs failed to be absorbed and enter the circulation. They rapidly decayed to <10% within the peritoneum over 4 ± 2 days. Due to their large size (19um), huge amounts of IV-injected MSCs were trapped within the pulmonary capillaries, causing hemodynamic alterations if >10
6
cells were used. After 1 day, a maximum of 1 ± 0.5 cells/HPF was found in the heart and CE significantly increased the cell content to 6 ± 3 cells/HPF (p=0.020). IC-injected MSCs vanished rapidly and cell counts dropped to 32 ± 18% at 6 days and 9 ± 6% at 12 days. Interestingly, the CE protocol significantly prolonged intramyocardial MSC survival and preserved higher cell counts (125 ± 21% at 6 days, p<0.001 and 87 ± 31% at 12 days, p<0.001).

Conclusions:
The most effective route of MSC application seems to be direct injection into the heart combined with cytokine enhancement in the border zone to create a beneficial microenviroment for cell survival.
Abstract 1021:
In Vivo
Immune Response To Allogeneic Porcine Mesenchymal Stem Cell (msc) Transplantation: A 12-day Course Of Fk-506 Abbrogates Humoral Response.
Authors: Poncelet Alain J , Vercruysse Johnatan , Saliez Alain , Gianello Pierre
Published: July 2021

Purpose
Several reports have highlighted the low immunogenic profile of MSCs. Therefore, we decided to test
in vivo
the humoral response to allogeneic MSCs transplantation and the effect of transient immunosuppression in a porcine model.

Methods/Material
MHC-controlled mini-swine SLA
cd
and SLA
dd
were used as donor and recipients, respectively. Two sites of transplantation were selected: subcutaneous and intracardiac. In our control group (n = 5), animals received no immunosuppression. In the study group (n = 11), nearly 1 x 10
6
allogeneic MSC/kg were injected. FK-506 was given from day 0 –12 and adjusted to therapeutic blood levels. Sera were serially collected up to one year after transplantation. The presence of specific anti-donor IgM and IgG was tested by flow cytometry and by a complement-mediated cytotoxicity assay.

Results
In the control group, all animals developed humoral responses in both IgM/G classes that persisted up to ten weeks. When transplanted subcutaneously, a single injection failed to elicit a complement-mediated cytotoxic response but subsequent re-challenge did. In the study group, only 2/11 FK-treated animals developed a transient humoral response, both in IgM and IgG, whereas all others failed to develop donor-specific antibodies. However, none of the sera tested from those 11 animals could elicit a complement-mediated cytotoxic response.

Conclusion
Allogeneic MSCs injected subcutaneously or intra-cardially can elicit prolonged humoral responses despite their putative low immunogenic profile. As already shown for experimental allogeneic organ transplantation, a transient immunosuppressive regimen can overcome the initial B cell response. Our result suggests that
in vitro
and
in vivo
characteristics of MSCs might differ and emphasizes the importance of pursuing research on allogeneic stem cell transplantation.
Abstract 1047: Infusion of Reconstituted High Density Lipoproteins Into Humans Enhances the In Vitro Anti-Inflammatory Capacity of the High Density Lipoprotein Fraction.
Authors: Patel Sanjay , Drew Brian , Duffy Stephen , Nahkla Shirley , Rye Kerry-Anne , Barter Philip , Celermajer David , Kingwell Bronwyn
Published: July 2021

Background:
Intravenous infusions of reconstituted high density lipoproteins (rHDL) have been shown to promote regression of coronary atheroma. Here we investigate the effects of rHDL infusion on the anti-inflammatory capacity of the isolated HDL fraction.

Methods:
Eight fasting Type 2 Diabetics (mean age 52 ± 2 years) received IV saline or apolipoprotein A-I (apoA-I)/phosphatidylcholine complexes (rHDL) at an apoA-I dose of 80mg/kg, on separate occasions in random order. The HDL fraction was isolated from subjects at baseline, 4 & 72hrs after infusion and assayed for chemical composition. The anti-inflammatory capacity of HDL preparations was assessed by incubation for 16hrs with human coronary artery endothelial cells. Cell expression of vascular cell adhesion molecule-1 (VCAM-1) and inter-cellular adhesion molecule (ICAM-1) 5 hrs after TNF-α stimulation was quantitated by flow cytometry.

Results
: Saline infusion had no effect on concentration or anti-inflammatory capacity of isolated HDL. rHDL infusion increased the concentration of HDL apoA-I (at baseline, 4h & 72h, [apoA-I] = 966 ± 105, 2562 ± 330 & 1701 ± 180 (ug/mL) respectively), as well as HDL cholesterol and HDL phospholipids. The addition of equivalent volumes of HDL (100uL) isolated at each time point inhibited VCAM-1 (Panel A) and ICAM-1 (Panel B) expression in proportion to the apoA-I concentration of the isolated HDL (p<0.01 ANOVA across time points for both VCAM-1/ICAM-1 expression and apoA-I concentration) (see figure

).

Conclusions:
Infusion of rHDL in humans increases the concentration of HDL apoA-I. The anti-inflammatory capacity of isolated HDL is enhanced in proportion to its apoA-I concentration.
Abstract 213: The Late Phase of Ischemic Preconditioning Is Abrogated by a Specific Prostaglandin I2 Receptor Antagonist
Authors: Guo Yiru , Jones Steven P , Tan Wei , Zhu Xiaoping , Wu Wen-Jian , Mu Jingyao , Bolli Roberto
Published: July 2021

Previous studies have shown that cyclooxygenase-2 (COX-2) mediates the late phase of ischemic preconditioning (PC) by producing various cardioprotective prostanoids including prostacyclin (PGI
2
) and prostaglandin (PG) E
2
(PGE
2
). Prostanoids are known to act via activation of specific receptors. However, the specific PG receptors responsible for the salubrious actions of COX-2 in PC and myocardial ischemia/reperfusion injury remain unclear. The PGI
2
receptor (IP) mediates the actions of PGI
2
and PGE
2
, the two major COX-2-derived prostanoids with cardioprotective properties. Accordingly, the goal of this study was to determine the role of the IP in late PC. C57BL/6 mice underwent a 30-min coronary occlusion (O) followed by 24 h of reperfusion (R). Administration of the IP receptor selective antagonist, RO 3244794 (group III, 10 mg/kg i.p.), 30 min prior to the 30-min O had no appreciable effect on infarct size compared with untreated and vehicle-treated groups (68.4 ± 1.2 % vs. 63.3 ± 3.1 % and 65.6 ± 3.9 % of the risk region, respectively). When mice were preconditioned with six cycles of 4-min O/4-min R 24 h prior to the 30-min O, infarct size was markedly reduced (group IV, 33.2 ± 2.9 %), indicating a late PC effect. In the vehicle-treated late PC group, infarct size (group V, 37.2 ± 4.9 %) was similar to the late PC group, indicating the vehicle had no effect. However, the protective effect of late PC was completely abrogated by administration of RO 3244794 30 min before the 30-min O (group VI, 63.8 ± 4.5 %). We conclude that the IP receptor is an obligatory mediator of the late phase of ischemic PC
in vivo
, suggesting that pharmacologic manipulations of this receptor may be therapeutically useful.
Abstract 1054: Widely Used Endothelial Progenitor Cell Assays Define Different Classes of Circulating Progenitor Cells
Authors: Povsic Thomas J , Ulanch Stacie , Lambeth Katherine , Kelly Francine , Peterson Eric D
Published: July 2021

Introduction:
Interest in the assessment of circulating endogenous progenitor cells (EPCs) is growing as evidence of their role in vascular repair mounts. Multiple measures of EPCs have been described, but there has been limited study of the comparability of these assays.

Methods:
We sought to determine the correlation between and reproducibility of alternative EPC assay methodologies. The precision of each EPC assay was determined by comparing EPC numbers in duplicate samples. Temporal stability of EPCs was assessed in samples obtained 24 hours apart from healthy volunteers. We then assessed the correlation of EPCs identified using the two most commonly used culture techniques: endothelial cell outgrowth and colony forming unit outgrowth (CFUs) (n=110), as well as EPCs based on cell surface marker expression (CD133, CD34, and VEGFR-2) and aldehyde dehydrogenase (ALDH) activity (n=77) in patients with suspected coronary disease.

Results:
EPC enumeration based on flow cytometry (FACS) based techniques was more precise (r
2
= 0.84 vs. 0.70) and temporally stable (r
2
= 0.82 vs. 0.21) than culture assays. There was limited correlation in EPC numbers determined using the two common culture based assays, either with each other (p=NS) or with EPCs enumerated based on CD34/CD133 expression (p=NS); however, endothelial CFUs correlated with VEGFR-2 (p<0.0001, Spearman r = 0.60) and CD34/VEGFR-2 (p<0.05, Spearman r = 0.31) expressing cells. EPCs defined by expression of CD133, CD34 and CD133/CD34 correlated with each other (CD133
+
vs. CD34
+
cells, p<0.05, r = 0.27; CD34
+
vs. CD133
+
CD34
+
cells, p<0.0001, r
2
= 0.52) and with ALDH
br
cells (ALDH
br
vs. CD133
+
CD34
+
cells, p<0.05, r
2
= 0.27), but not with VEGFR-2
+
cells (p=NS).

Conclusion:
Identification of EPCs using flow cytometry can be performed more precisely and reliably than using culture techniques. EPCs can be broadly grouped into two classes:

VEGFR-2 expressing cells which are correlated with the numbers of endothelial CFUs, and

cells identified based on expression of CD133/CD34 or ALDH activity.

These observations underscore the need for better assay standardization and a more precise definition of EPCs in cellular therapy research.
Abstract 1066: Cardiac-restricted Disruption of Supressor of Cytokine Signaling-3 Leads to Dilated Cardiomyopathy in a gp130 Independent Manner
Authors: Yajima Toshitaka , Yasukawa Hideo , Zhou Hanbing , Yajima Tomoko , Lim Byung-Kwan , Kirk Knowlton
Published: July 2021

We recently reported that cardiac-restricted overexpression of suppressor of cytokine signaling-3 (SOCS3) markedly increases cardiac myocyte susceptibility to virus infection. This suggests that endogenous SOCS3 also has significant biological effect in the cardiac myocyte. To understand physiological role of SOCS3 in the cardiac myocyte, we generated cardiac-specific SOCS3 knockout mice. The mice were born at the expected Mendelian ratio, but developed cardiac dysfunction (%FS; wt: 45±2.6 vs. ko: 31±4.5, mean±SE, n=8, p=0.017, 5-month-old) estimated by echocardiogram from 5 months of age and died with signs of dyspnea by 7 months of age. Postmortem analysis revealed significant pleural effusions and ascites, consistent with the presence of heart failure. Histological analysis showed that the heart has a thin left and right ventricular walls with chamber dilatation; however, typical findings of a myopathic heart such as myocyte disarray, inflammation and fibrosis were only rarely observed. Ultrastructure analysis of the dilated heart samples showed intact myofibrils, mitochondria, intercalated discs and gap junctions, suggesting that the myocyte had non-structural abnormalities that led to functional impairment. Unexpectedly calcium transients in the isolated adult myocytes from the failing hearts were significantly increased as compared to those from age-matched normal hearts (Fura-2 intensity ratio 340/380 nm; wt: 0.44±0.01 vs. ko: 0.54±0.01, mean±SE, n=3, p<0.0002), suggesting the decreased calcium sensitivity of SOCS3 deficient myocytes. We also observed spontaneous ventricular tachycardia in the knockout mice by telemetry analysis. Since SOCS3 has been reported to be a crucial negative-feedback regulator of gp130 signaling in vivo, we sought to determine whether the phenotype is due to overactivation of cardiac gp130 signaling. While gp130 downstream signaling such as STAT3, ERK and AKT pathways were significantly activated in the knockout heart, cardiac-specific knockout of both gp130 and SOCS3 did not rescue the dilated cardiomyopathy phenotype.

Conclusion: These results indicate that a gp130-independent mechanism is responsible for the dilated cardiomyopathy that occurs in SOCS3 deficient hearts.
Abstract 1071: Expression and Pharmacological Correction of a LQT2 Mutant (hERG) Channel in Native Cardiomyocytes
Authors: Holzem Katherine M , Balijepalli Ravi C , Lin Eric C , Wang Jing , Kamp Timothy J , Delisle Brian P , January Craig T
Published: July 2021

Mutations within the
human Ether-a-go-go Related Gene (hERG)
encoding for the hERG K
+
channel can lead to long QT syndrome type 2 (LQT2). Previous investigations have been performed using non-cardiac heterologous expression systems, and in mammalian systems most LQT2-linked missense mutations are trafficking deficient, with the channel protein failing to express at the cell surface membrane. Furthermore, the majority of these mutations can be induced to traffic to the cell surface by incubation in drugs (pharmacological correction), which has suggested that pharmacological correction might be developed as a clinical therapy. To further establish the feasibility of pharmacological correction in the heart, we demonstrated it in cardiomyocytes. We studied expression of WT-hERG and the trafficking-deficient LQT2-linked N470D-hERG mutation in HEK293 cells (using Superfect) or cardiomyocytes (using electroporation) isolated from 2-day-old mice. Using the patch clamp method to measure I
hERG
, peak tail current amplitude for N470D-hERG was reduced by 74% compared to WT-hERG in HEK293 cells (n=7–13 cells, p<0.05) and 94% in cardiomyocytes (n=6 –9 cells, p<0.05). HEK293 cells or cardiomyocytes expressing N470D-hERG were incubated with or without E-4031 (10 μM for 24 hrs) and E-4031 was washed out prior to recording I
hERG
. Incubation in E-4031 increased I
hERG
by 447% in HEK293 cells (n=9 –13 cells, p<0.05) and 1300% in cardomyocytes (n=6 –9 cells, p<0.05). We also measured the activation properties for I
hERG
. The potentials for half-maximal activation (V
1/2
) of WT-hERG expressed in HEK293 cells or cardiomyocytes were −7.5±1.8 and −12.9±1.1mV with slope factors of 6.7±0.2 and 6.2±0.3mV/
e
-foldΔ, respectively (n=5 cells each). The V
1/2
for N470D-hERG after pharmacological correction in E-4031 was -38.1±2.4mV for HEK293 cells and -50.6±5.3mV for cardiomyocytes with slope factors of 7.1±0.3 and 9.9±1.1 mV/
e
-foldΔ, respectively (n=5 cells each). Our results demonstrate functional expression of WT- and N470D-hERG in a native cardiomyocyte system and that hERG channel properties are similar between expression systems. These are the first data to show pharmacological correction in cardiomyocytes.
Abstract 1088: Prevention of Connexin 43 (Cx43) Dephosphorylation is Associated with Reduced Ventricular Tachycardia in Cardiac-Specific Angiotensin Converting Enzyme (ACE) Overexpression Mice
Authors: Iravanian Shahriar , Xiao Hong D , Witham Emily A , Bernstein Kenneth E , Dudley Samuel
Published: July 2021

Introduction:
Connexin 43 (Cx43) is a major component of the ventricular gap junctional complex, which provides a low resistance pathway for electrical conduction between myocytes. Reduction of Cx43 amount of phosphorylation has been implicated in the pathogenesis of ventricular tachycardia (VT) and fibrillation. Cardiac-specific angiotensin converting enzyme overexpression mice (ACE 8/8) have slow conduction, show conduction blocks, are prone to VT and sudden death, and show profound reductions in Cx43 levels and phosphorylation. Therefore, we hypothesized that reversal of Cx43 changes, by preventing the renin-angiotensin system (RAS) activation, would reduce the arrhythmic risk in these animals.

Methods:
Prevention of RAS activation was accomplished by one of three methods:

ACE 8/8 receiving an ACE inhibitor (captopril),

ACE 8/8 mice treated with a specific angiotensin receptor type-1 blocker (losartan), and

ACE 8/8 mice crossed with homozygote angiotensinogen null mice.

Pooled results were compared to untreated ACE 8/8 littermates. Surface electrocardiography and invasive electrophysiological study with programmed ventricular stimulation were performed on ten-week old mice, and the hearts were extracted for immunoblotting.

Results:
VT was induced in 10 out of 12 untreated ACE 8/8 mice, but only in 3 out of 11 treated mice (p<0.02). Total Cx43 was not changed significantly in treated animals as compared to controls, but the ratio (P/NP) of phosphorylated Cx43 (bands P1 and P2 in immunoblot) to non-phosphorylated Cx43 (band P0) showed a trend toward increase in the treatment group (0.78±0.18 vs. 1.25±0.19; P=0.09). Dividing the mice into two groups, high-risk (inducible and/or spontaneous VT) versus low-risk (no VT), we observed a significant difference in P/NP ratio (0.75±0.12 in high-risk mice vs. 1.78±0.32 in low-risk, p<0.01).

Discussion:
Unexpectedly, disruption of RAS signaling did not increase total Cx43 levels in ACE 8/8 mice. On the other hand, the treatments increased phospho-Cx43, suggesting that RAS controls Cx43 phosphorylation but not total amount. Even in the presence of significantly depressed levels of Cx43, small changes in Cx43 phosphorylation were associated with a significant reduction in VT inducibility.
Abstract 215: Effect of Adipose Tissue-derived Stromal Cell Transplantation in Mouse Model of Acute Myocardial Infarction
Authors: Yu Long-Hao , Kim Moo H , Kum Dong S , Park Tae H , Cha Kwang S , Kim Young D , Seo Soo Y , Jung Jin S
Published: July 2021

Background:
Adipose tissue has been identified as a source of multipotent cells that have the capacity to differentiate into cardiac myocytes, endothelial cells, similar to bone marrow cells. We sought to investigate the effect of adipose tissue-derived stromal cells (ADSCs) therapy on cardiac contractility and remodeling in C57BL/6 mouse model of acute myocardial infarction (AMI).

Materials and Methods:
Total 30 adult male C58BL/6 mice (12 weeks of age, 28~30g body weight) were used for the study. Mice were randomized into two groups (MI + media, n = 15 and MI + ADSCs, n= 15) after producing AMI by ligation of left anterior descending coronary artery. Intramyocardial injection of 1 × 10
6
ADSCs cells was compared to the injection of media alone. Ten animals were excluded from further analyses because of death. Most of them died 7 to 10 days after AMI, a mouse died duing surgical procedure following LAD ligation. All survived mice (n = 20) were received echocardiograpic analysis before and 2 weeks after cell transplantation, and then sacrificed for histologic analysis.

Results: The fractional shortening and left ventricular ejection fraction improved significantly in ADSCs transplant group at 2 weeks compared to control group (
20.9
± 3.48% vs 29.9 ± 8.6%,
p
=
0.006
and 46.4 ± 7.6% vs 63.1 ± 12.8%,
p
=
0.002
). Left ventricular end dialstolic dimension in ADSCs transplant group showed a little decrease from 4.65 ± 0.63 mm (control) to 4.14 ± 0.53 mm (ADSC), but there was no statistical difference (
p
< 0.072), whereas left ventricle end-systolic diameter decreased significantly in cell transplantation group (
p
< 0.05). Also, there were significant difference in injury size, infarct area, wall thickness or scar formation in ADSC group. In histochemical analysis, ADSCs have been shown to migrate into the injured sites and to integrate into the scar areas with some of transplanted ADSCs expressing endothelial marker.

Conclusion:
ADSCs improved left ventricular function and showed favorable effect on remodeling. They could be a good candidate for the source of cell therapy after myocardial injury.
Abstract 1096: Predictors Of Carotid Intimal-medial Thickness In Subjects With Hyperalphalipoproteinemia
Authors: West Michael , Asztalos Bela , Pollin Toni , Post Wendy , Rodriguez Annabelle
Published: July 2021

HDL cholesterol (HDL-C) is inversely associated with risk for CVD in the general population. However, the metabolic pathways leading to high HDL (hyperalphalipoproteinemia) are complex, and some patients with high HDL-C are not protected against atherosclerotic disease. The purpose of this study was to determine the associations between CVD risk factors, especially HDL subpopulations, and subclinical vascular disease in patients with high HDL-C. Community-dwelling adult subjects with and without CAD between the ages of 18 – 80 years with HDL-C levels above 60 mg/dl were recruited for this study (n=46, 34 women and 12 men, mean age 58.7 ± 9.7 years). After an overnight fast, blood was obtained for measurement of lipids, inflammatory markers and lipoproteins by NMR spectroscopy (Liposcience, NC) and 2D-gel analysis. Far wall common carotid artery intimal-medial thickness (IMT) was measured using carotid ultrasonography. None of the subjects were taking statins. The lipid levels (mean ± SD) were total cholesterol 225.6 ± 37.9 mg/dl, triglycerides 77.9 ± 29.4 mg/dl, HDL 90.4 ± 22.5 mg/dl and LDL 119.6 ± 33.4 mg/dl. Carotid IMT values were normally distributed, with a range of 0.46 –1.1 mm. Unadjusted pairwise-correlations showed that carotid IMT was significantly associated with LDL particle number (
p<
0.03), HDL subpopulations by 2D-gel analysis [α-3:
p<
0.005; α-4:
p<
0.04; and preα-1:
p<
0.03], C-reactive protein (
p<
0.03), sVCAM-1 (
p<
0.02), age (
p<
0.0008), systolic blood pressure (
p<
0.02), heart rate (
p<
0.02) and corneal arcus (
p<
0.04). Carotid IMT was not associated with any traditional lipid level, gender or history of CAD. In a multiple regression model, age (
p
=0.0007), α-3 (
p<
0.008) and CRP (
p<
0.03) were significant independent predictors of carotid IMT in this population. Thus, in subjects with hyperalphalipoproteinemia, increased small, dense HDL (α-3) and CRP levels were associated with increasing carotid IMT, suggesting heterogeneity in the role of HDL particles on subclinical atherosclerosis.
Abstract 1103: Integrin-linked Kinase and Laminin alpha 4 Mutations Cause Human Cardiomyopathy
Authors: Knöll Ralph , Postel Ruben , Krätzner Ralph , Hennecke Gerrit , Vacaru Andrei M , Vakeel Padmanabhan , Knöll Gudrun , Schäfer Katrin , Bos Erik , Hertog Jeroen den , Peters Peter J , van Eeden Fredericus , Schaper Jutta , Schaper Wolfgang , Hasenfuss Gerd , Schultheiss Heinz-Peter , Chien Kenneth R , Bakkers Jeroen
Published: July 2021

Background - Dilated Cardiomyopathy (DCM) is a syndrome characterized by ventricular dilation, contractile dysfunction and symptoms of congestive heart failure. Extracellular matrix proteins such as laminins as well as endothelial cells are known to influence cardiomyocyte performance, however a molecular link between mutations in the ECM and DCM has not been provided.

Methods and Results - Using a forward genetic screen in zebrafish to identify novel genes required for myocardial function we were able to identify the lost-contact (loc) mutant, encoding a nonsense mutation in the integrin-linked kinase (ILK) gene. This loc/ilk mutant is associated with both, a severe defect in cardiomyocytes as well as in endothelial cells leading to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between the ECM component laminin alpha 4 (LAMA4), integrin and ILK, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy (DCM). We identified two novel amino acid residue altering mutations (2828C>T (Pro943Leu), 3217C>T (Arg1073X)) in the integrin interacting domain of the LAMA4 gene and one mutation (785C>T (Ala262Val)) in the ILK gene. BIAcore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin binding capacity in case of the Pro943Leu (KD=5 +/− 3 uM) and Arg1073X lama 4 (KD=1 +/− 0.2 uM) mutants in comparison to the wildtype lama 4 protein (KD = 440 +/− 20 nM). Cell attachment assays point to a defect in endothelial cell adhesion when LAMA4 mutants have been expressed in vitro and used as a substrate. 785C>T (Ala262Val) ILK did not change its affinity, as measured by GST pull down assays as well as by a yeast 2 hybrid analysis, to a well known interacting protein such as b-parvin but revealed a 63% decrease in kinase activity. Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, leading ultimately to heart failure.

Conclusions - We report the first human mutations in the LAMA4, integrin and ILK system and provide a new mechanistic basis for DCM in man, which involves endothelial cells as well as cardiomyocytes.
Abstract 1112: Re-isolation and Functional Characterization of Cardiomyocytes after Implantation of Bone Marrow Stromal Cells or Neonatal Cardiomyocytes in Rat Heart
Authors: Sato Motoki , Carr Carolyn A , Stuckey Daniel J , Watt Suzanne M , Clarke Kiernan , Harding Sian E
Published: July 2021

Background:
Replacement of cardiac muscle is a key goal of cell therapy, but uncertainty exists about whether cell implantation strategies actually generate true functioning cardiomyocytes. We therefore aimed to re-isolate implanted bone marrow stromal cells (BMSCs) to determine their functional characteristics
ex vivo
. Neonatal cardiomyocytes (NCMs), in which the myocyte phenotype was established before implantation, were used as a gold standard.

Methods:
NCMs from GFP-transgenic rats were injected into the hearts of nude rats (n=4), and re-isolated after 1, 2 or 4 weeks. These were compared with adult myocytes, and with myocytes from 2 and 4 week-old (2W-O and 4W-O) rats. labelled myocytes detected 2 weeks after implantation of DiI and iron-labelled BMSCs (2W-BI) into infarcted rat hearts (n=4) were studied.

Results:
GFP-labelled myocytes approaching the adult morphology (rod-shaped, with defined sarcomeres) were not detected until 2 weeks after implantation of NCMs (2W-I), and were significantly shorter than adult myocytes in cell length (52 ± 5μm, n=10 vs. 99 ± 9μm, n=9, p<0.001), but the similar length to those from 2W-O (56 ± 3μm, n=15). Contraction amplitude (% shortening) was significantly lower and time to 50 % relaxation (R50) longer for the re-isolated cells compared with adult. By 4 weeks after implantation (4W-I), re-isolated myocytes were close to the adult phenotype, although cell length (64 ± 5μm, n=20, p<0.01) showed immature characteristics. After implantation of BMSCs, DiI-labelled myocytes were detected in only 4/10 rats and then at low levels (0.4–21 per 10,000). Labelled myocytes, isolated 2 weeks after implantation of BMSCs, had similar length (101 ± 20, n=21), contraction amplitude and beat duration to adult myocytes.

Conclusions:
Implanted NCMs mature within the heart over a period of at least 4 weeks. The adult phenotype of labelled myocytes after BMSC implantation is inconsistent with a process of transdifferentiation and maturation.