bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1905: Restoration of Normoglycemia Re-Establishes the Infarct-Sparing Effect of Postconditioning in Diabetic Mice
Authors: Przyklenk Karin , Maynard Michelle , Greiner Dale L , Whittaker Peter
Published: July 2021

Relief of ischemia in a stuttering manner (postconditioning: PostC) reduces infarct size in healthy adult populations, due in part to activation of ERK 1/2 during the early minutes of reflow. However, emerging evidence suggests that PostC fails to limit ischemia-reperfusion injury in the setting of diabetes. Our current aim was to establish whether this loss in efficacy of PostC reflects a permanent diabetes-associated defect in cardioprotective signaling, or whether therapy to restore normoglycemia can re-establish the PostC phenotype. To address this issue, isolated buffer-perfused hearts were obtained from 4 cohorts of C57BL/6J mice: healthy adults; animals injected 2 or 4 wks earlier with streptozotocin (STZ) to induce diabetes; and mice injected 4 wks earlier with STZ that, at 2 wks after being rendered diabetic, received islet cell transplantation (ICT). All hearts underwent 30 min global ischemia and, within each cohort, were assigned to receive either complete, abrupt reperfusion (controls) or PostC with 3 10-sec cycles of stuttered reflow. In hearts from healthy mice: (i) PostC was cardioprotective (infarct size [delineated by tetrazolium staining] was 27±3% vs 50±3% of the LV in PostC hearts vs controls; p<.05); (ii) PostC-induced protection was associated with significant, >2-fold increases in expression of phospho-MEK, phospho-ERK and downstream targets of ERK; and (iii) the benefits of PostC were abrogated by the MEK inhibitor PD98059. At both 2 and 4 weeks post-STZ injection, mice displayed: (i) low plasma insulin levels (0.18±0.08 and 0.07±0.02 vs 1.40±0.20 ng/ml) and hyperglycemia (blood glucose: 556±15 and 558±10 vs 178±7 mg/dl in healthy C57 mice); (ii) no up-regulation of ERK signaling in response to stuttered reflow; and (iii) no infarct-sparing effect of PostC (mean infarct sizes: 62– 63% in STZ-PostC groups vs 58 – 63% in STZ-controls). In contrast, ICT: (i) restored normal insulin and blood glucose levels (1.33±0.09 ng/ml; 189±7 mg/dl) and, most notably (ii) reinstated the up-regulation in ERK signaling and infarct-sparing effect of PostC (infarct size: 31±3% vs 51±5% in STZ±ICT controls; p<0.05). Thus, therapeutic control of insulin and blood glucose re-establishes the protective PostC phenotype in this mouse model of diabetes.
Abstract 1913: Distinct eNOS Regulation by Protease-Activated Receptor 1 and 2 Involving Gq, G12/13 and Y27632-Sensitive Non-Rho Kinase
Authors: Hinoki Akinari , Suzuki Hiroyuki , Eguchi Kunie , Higuchi Sadaharu , Kimura Keita , Motley Evangeline D , Watts Vabren , Eguchi Satoru
Published: July 2021

Protease-activated receptors (PARs), such as PAR1 and PAR2 have been implicated in the regulation of endothelial nitric oxide (NO) production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase (eNOS) through the selective phosphorylation of a positive regulatory site, Ser
1179
and a negative regulatory site, Thr
497
. In bovine aortic endothelial cells (BAECs), a PAR1 ligand, TFLLR, phosphorylated eNOS at Thr
497
. It had no effect on Ser
1179
phosphorylation or cyclic GMP (cGMP) production. In contrast, a PAR2 ligand, SLIGRL, phosphorylated Ser
1179
with no noticeable effect on Thr
497
. SLIGRL stimulated cGMP production that was blocked by L-NAME. Neither eNOS phosphorylation nor cGMP production was observed by a PAR4 agonist, AY-NH2. Thrombin has been shown to transactivates PAR2 through PAR1. Thus, thrombin stimulates eNOS phosphorylation at both sites as well as cGMP production in BAECs. Importantly, SLIGRL-induced Ser
1179
phosphory-lation and cGMP production were inhibited by a G
q
inhibitor, YM-254890. In addition, eNOS phosphorylation was not affected by over-expression of GRK2 C-tail or pertusis toxin pretreatment. By contrast, TFLLR-induced Thr
497
phosphorylation was selectively inhibited by a Rho-kinase inhibitor, Y27632, and over-expression of p115RhoGEF RGS domain, a selective G
12/13
inhibitor. However, other inhibitors (H-1152 and dnRho) that target Rho-kinase or a PKC inhibitor, GF109203X, failed to block TFLLR-induced Thr
497
phosphorylation. Taken together, these data suggest that PAR1-induced phosphorylation of eNOS at Thr
497
requires G
12/13
and subsequent activation of a Y27632-sensitive eNOS Thr
497
kinase that is distinct from Rho-kinase or PKC. We conclude that PAR1 and PAR2 distinctly regulate eNOS activity through G
q
and G
12/13
, respectively, delineating the novel signaling pathways by which proteases regulate eNOS activity. This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1951: Histamine H1 Receptor is Required for Atherogenesis
Authors: Rozenberg Izabela , Mocharla Pavani , Becher Burkhard , Hofmann Janin , Akhmedov Alexander , Boren Jan , Lüscher Thomas F , Tanner Felix C
Published: July 2021

Background and aims:
Histamine is an endogenous amine that plays a critical role as a mediator of inflammation and allergic response. Most of the proinflammatory actions of histamine are mediated via H1 receptor (H1R). Although histamine affects numerous inflammatory processes, its relevance in atherogenesis remains unknown.

Methods:
We used both a pharmacological and a genetic approach to modulate H1R- or H2 receptor (H2R) -signalling in a mouse model of atherosclerosis. Apolipoprotein E (ApoE)-deficient mice were treated for 12 weeks either with the H1R antagonist mepyramine, the H2R antagonist ranitidine, or placebo. In parallel, we compared
H1R
−/−
ApoE
−/− and
H2R
−/−
ApoE
−/− to
ApoE
single knockout mice after 12 weeks of stimulation with high cholesterol diet (Clinton-Cybulski diet, 1.25% cholesterol).

Results:
ApoE-deficient animals treated with mepyramine, but not ranitidine, exhibited significantly reduced aortic plaque area compared with placebo-treated mice. Similarly, genetic deficiency of H1 receptor in ApoE−/− mice resulted in a 60% decrease in aortic plaque formation. Level of plasma lipids as well as permeability of the vessel wall was not affected by the absence of H1R. H1R-deficient animals showed significantly decreased levels of CCL5 and CD40 in the aorta. In parallel, atherosclerotic plaques of these animals revealed significantly decreased infiltration with macrophages and T-helper cells. Moreover, reduced blood lymphocyte number, spleen size, and decreased production of splenic Th1-specific cytokines (CCL5, IL-2, and IFNγ) were observed in the absence of H1R. Interestingly, bone marrow transplantation revealed, that the presence of H1R on bone marrow-derived cells is irrelevant for both atherosclerotic plaque formation and regulation of spleen size.

Conclusions:
We conclude that blocking the H1 receptor leads to decreased atherogenesis due to indirect alteration of leukocyte function.
Abstract 1962: CXCR4-mediated Bone Marrow Stem Cell Homing and Niche Maintenance Is Modulated by the Functional Status of c-kit
Authors: Cheng Min , Zhou Junan , Zhu Wuqiang , Tanaka Toshikazu , Zeng Qiutang , Kishore Raj , Losordo Douglas , Qin Gangjian
Published: July 2021

Objective:

The bone marrow (BM) “niche”, where the stem cells are dynamically maintained, is an attractive target for cell-based cardiovascular repair. Both CXCR4 antagonist and c-kit blocking antibody can rapidly mobilize BM stem cells; however, the functional interaction between CXCR4 and c-kit remains unclear. We hypothesize that CXCR4 crosstalk with c-kit to maintain BM stem cell niche.

Methods and Results:

We designed an
in vitro
adhesion assay to mimic BM niche. Mouse total BM cells were harvested and applied onto VCAM1-coated plates for 15 min, and followed by addition of CXCR4 agonist SDF-1 or antagonist AMD3100. The adhesion of BM cells to VCAM1 resulted in marked c-kit phosphorylation. Interestingly, AMD3100 significantly decreased while SDF-1 augmented the c-kit phosphorylation, suggesting that CXCR4 may regulate the functional status of c-kit. To determine the role of c-kit in CXCR4-regulated stem cell niche maintenance, we developed an
in vivo
homing assay. The c-kit mutant (c-kit
W/W-V
) and WT mice (n=5) were first treated with AMD3100 to disrupt CXCR4-mediated niche, and 2 h later, transplanted with eGFP-transgenic BM cells. After 3 h, BM cells were isolated and analyzed by FACS. The percentages of donor-derived eGFP+, eGFP+Lin+, and eGFP+Lin− cells were similar between the two groups. However, the donor-derived primitive stem cells, including eGFP+Lin-Sca1+ and eGFP+Lin-ckit+ cells, were significantly fewer in c-kit
W/W-V
mice than in WT mice (c-kit
W/W-V
vs. WT: 94.1% vs. 96.0%, p<0.05, and 33.82% vs. 55.27%, p<0.01, respectively), indicating that c-kit-deficient BM niche specifically affects primitive stem cell homing and maintenance. Importantly, although donor-derived CXCR4-expressing total (eGFP+CXCR4+) cells were similar in both groups, CXCR4-expressing stem cells, including eGFP+Lin-CXCR4+, eGFP+Lin-Sca1+CXCR4+, and eGFP+Lin-ckit+CXCR4+ cells, were much fewer in c-kit
W/W-V
than in WT mice (c-kit
W/W-V
vs. WT: 19.7% vs. 30.6%, p<0.01, 20.3% vs. 29.1%, P<0.05, and 6.7% vs. 17.9%, p<0.001, respectively), suggesting c-kit deficiency reduces the homing and maintenance of stem cells that express CXCR4.

Conclusion:

CXCR4-mediated BM stem cell homing and niche maintenance is modulated by the functional status of c-kit.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1963: Endothelial Cell-Derived Microparticles Interact with Regenerating Cells and Transfer Biological Information
Authors: Werner Nikos , Heiermann Nadine , Liu Hao , Horlbeck Fritz , Nickenig Georg
Published: July 2021

Apoptosis of endothelial cells (ECs) leads to the development of endothelial dysfunction, which itself is strongly associated with poor cardiovascular prognosis. EC apoptosis can be quantified using flow-cytometry-based enumeration of circulating EC-derived microparticles (EMP) within peripheral blood. Bone marrow-derived endothelial progenitor cells (EPC) contribute to EC regeneration and are an important predictor for cardiovascular mortality. We postulate that EC apoptosis with concomitant release of EMP is an important signal for regenerating cells to initiate EC repair. EMP were generated from human coronary arterial endothelial cells (HCAEC). Proteomic analysis showed that the identified proteins represent cytoskeleton/cytoskeleton-binding proteins, proteins involved in intracellular transport/signalling, and protein folding. In addition, proteins related to apoptosis, proliferation, and migration were identified. HCAEC/EPC were able to incorporate EMP in an annexin I/ phosphatidylserine receptor-dependent way. Incubation of mononuclear cells with EMP lead to an enhanced conversion into early outgrowth EPC. EMP co-incubation also changed the phenotype of cultivated Langerhans-like dendritic cells into an immature phenotype. Co-cultivation of ECs and EPC with EMP prevented TNF-alpha induced cell apoptosis. Migration of EPC was enhanced in response to EMP. Finally, we measured EPC liberation from bone marrow into peripheral blood. Intravenous treatment of C57bl6 mice with EMP enhanced the number of sca-1/flk-1 positive EPC within peripheral blood compared to control. The number of CD31+/Annexin+ EMP and CD34
+
/KDR
+
EPC was determined in 40 patients with coronary artery disease. The number of circulating EMP correlated with EPC function (p<0.001, r=0.601). EMP and EPC seem to substantially interact in an annexin I/PSR dependent way. EMP influence EPC biology in vitro and induces EPC mobilization in vivo. We speculate that the described interaction of EMP with EPC enhance the homing process of EPC within the area of EC damage.
Abstract 3373: MRP-8/14 is Critical for the Biological Response to Vascular Injury
Authors: Croce Kevin , Gao Huiyun , Wang Yunmei , Sakuma Masashi , Mooroka Toshifumi , Shi Can , Sukhova Galina , Packard René R , Hogg Nancy , Libby Peter , Simon Daniel I
Published: July 2021

Background

: MRP-8 (S100A8) and MRP-14 (S100A9) are members of the S100-family of calcium-modulated proteins that regulate myeloid cell function and control inflammation, in part, through activation of toll-like receptor-4 and RAGE. Using a transcriptional profiling approach in patients with acute coronary syndromes, we made the novel discovery that MRP-14 is associated with atherothrombosis, and demonstrated that elevated plasma levels of MRP-8/14 heterodimer predict increased risk of first and recurrent cardiovascular events. Despite functioning as a pro-inflammatory mediator, the role of MRP-8/14 in vascular inflammation and disease are unknown.

Methods and Results

: We evaluated vascular inflammation in wild-type (WT) and MRP-14-deficient (MRP-14
−/−
) mice that lack MRP-8/14 complexes with experimental arterial injury, vasculitis, or atherosclerosis. After femoral artery wire injury, MRP-14
−/−
mice had a 33% reduction in leukocyte accumulation (
P
=.0004) and a 72% reduction in cellular proliferation (
P
=0.001) in the arterial wall followed by a 45% reduction in neointima formation (
P
=0.004) compared to WT mice. In a cytokine-induced local Schwartzman-like reaction that produces thrombohemorrhagic vasculitis, MRP-14
−/−
mice had a 59% reduction in lesion severity (
P
<0.0001), an 82% smaller hemorrhagic area (
P
=0.015), and a 45% decline in neutrophil accumulation (
P
=0.013). Finally, in response to high-fat feeding, mice doubly deficient in ApoE and MRP-8/14 complexes had attenuation in atherosclerotic lesion area by 34% (
P
=0.023) and a 60% decrease in macrophage accumulation in plaques (
P
=0.021) compared to mice deficient in ApoE alone.

Conclusions:

This study demonstrates that MRP-8/14 broadly regulates vascular inflammation and contributes to the biological response to vascular injury. Targeting MRP-8/14 may provide a novel therapeutic approach to modulate diverse forms of vascular injury, including restenosis, vasculitis, and atherosclerosis.
Abstract 3379: cGMP Specific Phosphodiesterase Type 5A Activity is Regulated by S-nitrosylation at Cys 181
Authors: Murray Christopher I , Gebska Milena A , Haile Azeb , Zhang Manling , Kass David A , Champion Hunter C , Van Eyk Jennifer E
Published: July 2021

Introduction: cGMP specific phosphodiesterase type 5A (PDE5A) has a key role in limiting the effect of the second messenger cGMP as demonstrated by the use of the specific inhibitor, sildenafil. However, the mechanism by which PDE5 activity is regulated is not fully understood. Recently, it has been reported that PDE5A specifically degrades the pool of cGMP produced by the nitric oxide (NO) stimulated soluble guanylate cyclase. NO is known to regulate other proteins in this pathway through S-nitrosylation, a redox modification where an NO group is covalently added to the sulfur atom of a cysteine. Here we test the hypothesis that PDE5 activity is regulated by S-nitrosylation and determine the critical modified cysteine residues.

Methods: Rates of cGMP hydrolysis were measured by an in vitro activity assay using purified mouse flag-PDE5 treated with the NO-donor, S-Nitrosoglutathione (GSNO, 1–100μM) or control. The S-nitrosylation status of PDE5A was determined by biotin switch assay of cell lysates over expressing flag-PDE5A using the equivalent conditions. After NO modifications were replaced with biotin moieties, labeled proteins or trypsin digested peptides were captured with streptavidin agarose and evaluated by western blot or tandem mass spectrometry, respectively.

Results: PDE5A activity was found to be decreased in a dose dependent manner with NO donor inducing a 25.0 ± 3.8% (n=4, p<0.01) decrease with 100μM GSNO treatment compared to controls. PDE5A was S-nitrosylated by the NO donors in a similar dose dependent manner when analyzed using the biotin switch assay. Subsequent mapping studies identified 2 potential sites of S-nitrosylation at C181 and C210, both of which are located in the N-terminal regulatory domain. Independent mutation of candidate cysteines to serine residues revealed that C181 is more receptive to NO modification than C210. Additionally, the C181S PDE5A was found to be resistant to the NO-donor dependent decrease (0.55 ± 2.3%, p=0.9) in activity while C210S activity was found to be depressed in the presence of 100μM GSNO (28.9 ± 1.7%, p<0.01) (n=4).

Conclusions: These data indicates that PDE5A activity is negatively regulated by S-nitrosylaion at C181 which may contribute to the overall regulation of cGMP signaling in the cell.

This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).
Abstract 3380: Acetylation-dependent Regulation Of Notch1 Signaling In Endothelial Cells
Authors: Guarani Virginia , Dequiedt Franck , Zeiher Andreas M , Dimmeler Stefanie , Potente Michael
Published: July 2021

The Notch signaling pathway is a versatile regulator of cell fate decisions and plays an essential role for embryonic and postnatal vascular development. As only modest differences in Notch pathway activity suffice to determine dramatic differences in blood vessel development, this pathway is tightly regulated by a variety of molecular mechanisms. Reversible acetylation has emerged as an important post-translational modification of several non-histone proteins, which are targeted by histone deacetylases (HDACs). Here, we report that specifically the Notch1 intracellular domain (NICD) is itself an acetylated protein and that its acetylation level is tightly regulated by the SIRT1 deacetylase, which we have previously identified as a key regulator of endothelial angiogenic functions during vascular growth. Coexpression of NICD with histone acetyltransferases such as p300 or PCAF induced a dose- and time-dependent acetylation of NICD. Blocking HDAC activity using the class III HDAC inhibitor nicotinamid (NAM), but not the class I/II HDAC inhibior trichostatin A, resulted in a significant increase of NICD acetylation suggesting that NICD is targetd by class III HDACs for deacetylation. Among the class III HDACs with deacetylase activity (SIRT1, 2, 3, 5), knock down of specifically SIRT1 resulted in enhanced acetylation of NICD. Moreover, wild type SIRT1, but not a catalytically inactive mutant catalyzed the deacetylation of NICD in a nicotinamid-dependent manner. SIRT1, but SIRT2, SIRT3 or SIRT5, associated with NICD through its catalytic domain demonstrating that SIRT1 is a direct NICD deacetylase. Enhancing NICD acetylation by either overexpression of p300 or inhibition of SIRT1 activity using NAM or RNAi-mediated knock down resulted in enhanced NICD protein stability by blocking its ubiquitin-mediated degradation. Consistent with these results, loss of SIRT1 amplified Notch target gene expression in endothelial cells in response to NICD overexpression or treatment with the Notch ligand Dll4. In summary, our results identify reversible acetylation of NICD as a novel molecular mechanism to control Notch signaling and suggest that deacetylation of NICD by SIRT1 plays a key role in the dynamic regulation of Notch signaling in endothelial cells.
Abstract 3410: FAK is Required for Neural Crest Cell Remodeling of the Cardiac Outflow Tract
Authors: Vallejo-Illarramendi Ainara , Reichardt Louis F
Published: July 2021

Background: Cardiac neural crest cells (NCCs) participate in the remodeling of the outflow tract and pharyngeal arch arteries during cardiovascular development. Abnormal growth factor and integrin signaling in cardiac NCCs leads to cardiovascular malformations. Focal adhesion kinase (FAK) is a ubiquitous protein kinase that transduces integrin and growth factor signaling pathways. However, a direct role for FAK in cardiac NCC morphogenesis has not been demonstrated. Here we have used a Cre/loxP method for NCC-specific deletion of FAK in mouse embryos.

Results: We found that targeted deletion of FAK in NCC results in craniofacial and cardiovascular malformations, leading to early postnatal lethality. Mutant mice display cleft palate together with several cardiovascular defects, including persistent truncus arteriosus, transposition of the great arteries, overriding aorta, ventricular septal defect and type B interruption of the aortic arch, all of which resemble common forms of human congenital heart disease. We found that NCC counts in the outflow tract of FAK mutant and control littermates are similar at E11, suggesting that FAK is not required for cardiac NCC migration to the outflow tract. Furthermore, NCCs appear to differentiate normally into smooth muscle cells in the outflow tract of FAK conditional mutant embryos at E11 and E12.5. In contrast, NCCs in the aortic arch arteries of FAK conditional mutants show impaired differentiation into smooth muscle cells in these embryonic stages. Additionally, we observed that rotation of the outflow tract fails to occur by E11 in FAK mutants, resulting in a dextroposed aortic root. Moreover, FAK-deficient NCCs form a less compact mesenchyme in the conotruncal cushions and show disorganized F-actin stress fibers in the aorticopulmonary septum.

Conclusions: Our results indicate that FAK plays an essential role in the development of the cardiac outflow tract and in the remodeling of aortic arch arteries. However, it is dispensable for migration and differentiation of cardiac NCC in the outflow tract. Further studies with this novel model of congenital heart disease may help elucidate the NCC signaling pathways that orchestrate septation of the outflow tract.
Abstract 3412: Molecular and Cellular Characterization of Splice Site Mutations in
MYBPC3
-encoded Myosin Binding Protein C
Authors: Theis Jeanne L , Bos J M , Ommen Steve R , Ackerman Michael J
Published: July 2021

Background:
Mutations in
MYBPC3
-encoded myosin binding protein C underlie the most common genotype in hypertrophic cardiomyopathy (HCM). Compared to most HCM-associated mutations which are primarily missense mutations, MYBPC3-HCM is often caused by insertions, deletions, nonsense mutations or mutations involving the canonical splice site. However, the effects of most mutations that prematurely truncate MYBPC3 are unknown. Access to cardiac specific mRNA and tissue has permitted a molecular and cellular elucidation of the consequences of two splice site mutations.

Methods:
Mutational analysis of
MYBPC3
revealed 2 HCM-susceptibility mutations involving the splice donor sites of exons 7 and 30. Reverse transcription was performed on cDNA generated from RNA extracted from cardiac tissue obtained following surgical myectomy. Western blot analysis and immunohistochemistry (IHC) of the myofilaments were performed to further assess the impact of the splice-site mutations.

Results:
Both splice donor mutations produced abnormal RNA splicing of
MYBPC3.
The c.821+1 g>a mutation in the splice donor site of exon 7 generated two alternatively-spliced mutant transcripts resulting in two frame-shifted, premature truncations (H257 fs/37 and H257 fs/15). The c.3330+2 t>g mutation in exon 30’s splice donor site produced a single alternatively-spliced mutant transcript that translated into a frame-shifted premature truncation (V1063 fs/37). Expression levels of wild type myosin binding protein C were decreased in the myectomy specimen from the patient with the exon 7 splice donor site mutation. By IHC, the spatial organization of myosin binding protein C was disrupted severely in both patients.

Conclusions:
This study provides the first molecular and cellular characterization of HCM-causing mutations involving canonical splice-site motifs within the intron. Loss of function of the splice site resulted in exon skipping and generation of frame-shifted and prematurely truncated myosin binding protein C and disruption of the distribution and organization of myosin binding protein C in the heart tissue.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 3422: Asymmetric Division of Human Cardiac Progenitor Cells Involves Immortal DNA Strand Cosegregation
Authors: Bardelli Silvana , Bearzi Claudia , Carrillo-Infante Cynthia , Carvalho Adriana Bastos , D’Amario Domenico , Gonzalez Arantxa , Bolli Roberto , D’Alessandro David A. , Michler Robert E. , Quaini Federico , Hosoda Toru , Moccetti Tiziano , Anversa Piero , Kajstura Jan
Published: July 2021

The objective of this study was to determine whether asymmetric division of human cardiac progenitor cells (hCPCs) occurs by:

random DNA template segregation;

selective retention of the old template DNA strand; or

a combination of both processes.

Myocardial samples were enzymatically dissociated and hCPCs were sorted for the stem cell antigen c-kit. During in vitro expansion, hCPCs were exposed to BrdU for 36 hours to reach a 90% degree of labeling. BrdU-tagged hCPCs were plated at limiting dilution to obtain single cell-derived clones. Sixty clones comprising 10 –125 c-kit-positive hCPCs developed in 7–10 days. In four cases, one single BrdU-bright hCPC was identified while the remaining clonogenic cells were negative for the halogenated nucleotide. In these clones, the number of BrdU-negative hCPCs was 25, 85, 95, and 111. Conversely, in 56 clones hCPCs were uniformly labeled and showed very low levels of BrdU. In a second group of experiments, hCPCs in late-anaphase initial-telophase were identified and the distribution of BrdU in the two clusters of chromosomes was analyzed. In 5% of mitotic cells, three-dimensional reconstruction by confocal microscopy documented that BrdU-labeling was restricted at one pole only of the dividing hCPCs. PCNA which is highly expressed in newly synthesized DNA was restricted to the BrdU-negative chromosomes. In a third set of studies, hCPCs were loaded with quantum dots, cultured for 36 hours in the presence of BrdU and examined 96 hours later. Quantum dots are progressively diluted by cell division independently from the modality of DNA template segregation. Thus, hCPCs with minimal levels of quantum dots and bright BrdU localization were interpreted as replicating cells which retained the old DNA strand. By this approach, 5% hCPCs displayed these two critical properties. The uneven distribution of the cell fate determinants Numb and α-adaptin confirmed that hCPCs underwent asymmetric division. In conclusion, these data support the hypothesis that immortal DNA strand cosegregation participates in asymmetric kinetics of hCPCs although random-segregation of DNA template is the prevailing mechanism of hCPC growth.
Abstract 3433: Cardiac Myocyte-specific Caveolin-3 Overexpression Modulates ERK/EGFR Signaling and Attenuates Cardiac Hypertrophy
Authors: Horikawa Yousuke T , Tsutsumi Yasuo M , Patel Hemal H , Insel Paul A , Roth David M
Published: July 2021

Introduction: The localization and organization of signaling molecules involved in cardiac hypertrophy and remodeling are poorly understood. Caveolae, invaginations of the sarcolemma and enriched in the protein caveolin (Cav), localize signaling molecules involved in cardiac hypertrophy and remodeling. Cav-3, which is unique to myocyte caveolae, inhibits extracellular signal-regulated kinase (ERK) phosphorylation in vitro and binds the epidermal growth factor (EGF) receptor. We hypothesized that cardiac myocyte-specific overexpression of Cav-3 (Cav-3 OE) would modulate ERK signaling via direct interaction and alteration in EGF receptor signaling and would attenuate cardiac hypertrophy.

Methods: Transgenic mice with cardiac myocyte-specific Cav-3 OE (generated using an α-myosin heavy chain promoter) and littermate controls (C) were subjected to transverse aortic constriction (TAC) for 4 weeks. Echocardiography, histology and molecular analysis were performed. Cardiac myocytes were isolated and stimulated with phenylephrine (Phe) and EGF to assess ERK activity via activation of Gq and EGF receptors, respectively.

Results: Cav-3OE mice had reduced heart weight/tibia length ratio post-TAC compared to C (9.9±0.8 vs. 12.9±0.5 mg/mm; n=8; P<0.05) and decreased myocyte cross sectional area compared to C (203±8 vs. 327±17 μm2/cell; n=7; P<0.001). Cav-3 OE and C mice had similar fractional shortening (FS) pre-TAC but post-TAC, Cav-3 OE had greater FS compared to C (29.2±3.2 vs. 20.8±3.0%; n=8–9; P<0.05). Fibrosis, as assessed by trichrome stain, was decreased in Cav-3 OE compared to C mice. Post-TAC, pERK expression in cardiac homogenates was decreased in Cav3-OE mice vs. C. In vitro, pERK had, similar expression in the two groups in response to Phe, but pERK was expressed at higher levels in Cav3-OE myocytes in response to EGF receptor simulation.

Conclusion: These results show that cardiac-specific Cav-3 OE alters ERK/EGFR signaling in cardiac myocytes, producing a phenotype with blunted cardiac hypertrophy and preserved cardiac function post-TAC. This suggests that cardiac myocyte-specific Cav-3 OE may provide a novel approach to improve cardiac function in pathological cardiac hypertrophy and remodeling.

This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Abstract 3435: Left Ventricular Hypertrophy is Resistant to Inhibition of Expression of the R403Q Alpha-Myosin Heavy Chain Cardiac Hypertrophy-Inducing Mutant Protein
Authors: Cannon Leah , Marciniec Tadeusz , Mearns Bryony , Graham Robert M , Fatkin Diane
Published: July 2021

Left ventricular hypertrophy (LVH) develops as a compensatory response to myocardial dysfunction due to diverse causes, but is nonetheless a major risk factor for premature cardiovascular morbidity and mortality. It is thus unclear if regressing LVH is beneficial or may worsen patient outcome. To evaluate the effects of LVH regression, we developed a transgenic mouse model in which the expression of a familial hypertrophic cardiomyopathy (FHC)-inducing mutation (R403Q alpha-MHC) can be regulated in a temporal and dose-dependent manner. In this model, transgene expression can be shut off by feeding with a tetracycline analogue (doxycycline). Serial echocardiography and histology studies were performed in a cohort of mice expressing the FHC mutant (“gene-on”) and in wildtype (WT) littermates. A second cohort of WT and 403/+ mice was randomised to placebo or doxycycline (“gene off”) from 6 (Dox6) or 20 weeks (Dox20) and evaluated at 40 weeks of age. Compared to WT littermates, “gene on” 403/+ mice showed increased LV mass, LV end-diastolic diameter (LVDD) and left atrial diameter (LAD), and reduced fractional shortening (LVFS), with changes evident from 12 weeks of age. LV sections from 403/+ mice showed typical features of FHC: myofibre disarray and interstitial fibrosis. LV mass, LV function and myocardial histology were unchanged in both male and female placebo- vs Dox6 or Dox20 mice at 40 weeks (Table 1

). Thus, consistent with the major LV thickening in FHC humans occurring in adolescence, overexpression of R403Q for only 6 weeks is sufficient to trigger the complete LVH phenotypic response. Moreover, switching off the genetic trigger for LVH in 403/+ mice at 6 weeks (prior to overt disease manifestation) or 20 weeks (established disease) does not induce regression of LVH or exacerbate contractile dysfunction. Interventions to induce LVH regression may, therefore, need to be directed at downstream factors in hypertrophic pathways.

Table 1.

Echo data for male WT and 403/+ mice aged 40 weeks
Abstract 3441: Paraoxonase-192 Polymorphism Interaction with Low HDL Cholesterol in Coronary Artery Risk
Authors: Pereira Andreia M , Mendonca Isabel M , Freitas Ana I , Sousa Ana C , Gomes Susana , Faria Paula , Freitas Sonia , Ornelas Ilidio , Brehm Antonio , Cardoso Antonio , dos Reis Roberto Palma
Published: July 2021

Background: Increased lipid peroxidation is associated with accelerated progression of atherosclerosis. Paraoxonase (PON1) is an antioxidative enzyme in high density lipoproteins (HDL), which protects against lipid per oxidation and Coronary Artery Disease (CAD). PON 1 activity is under genetic control and its molecular basis is a polymorphism in the PON 1 gene that shows two common isoforms: Q (192 Gln) with high ability to protect LDL from lipid peroxidation in vitro, and R (Arg) with lower ability. Aim: To explore the interaction of the R allele paraoxonase gene and low HDL cholesterol concentrations, in CAD risk emergence.

Methods: 818 individuals participated in the study, 298 coronary patients, 55.0±10.3 years, 78.9% male sex, and 520 healthy controls, 47.3±12.6 years, 57.5% male sex, age and sex matched. Low HDL-C was defined as <0.90 mmol/L in men and < 1.11 mmol/L in the women. Comparisons of genotypes between cases and control subjects were performed by a chi-square test. Statistical significance was accepted at p<0.05. Odds ratio as well as their 95% confidence intervals for the RR genotypes and HDL deficient subjects were computed using univariate analysis (2x2 tables). To determine the interaction between the RR paraoxonase genotype and HDL-deficient subjects, we used the epidemiologic tables 4x2 and the synergy measures: the additive model (Rothman’s synergy index) and the multiplicative model (Khoury’s synergy index). We calculated the relative excess risk (RERI) and the attributable proportion (AP) due to interaction (Rothman).

Results: The PON 1 RR192 is associated with coronary heart disease [OR=1.61; p=0.043] in whole population. The HDL-deficient subjects 192 RR homozygotes showed an increased risk of CAD (OR=17.38; p< 0.0001) compared to normal HDL 192 RR (OR=1.39; p=0.348) and HDL-deficient subjects not carrying RR genotype (OR=7.79; p<0.0001). The genotype PON 192 RR increases the risk of CAD in the HDL-deficient populations (SI=2.3, SIM=1.6). The attributable proportion due to this interaction (AP) was 0.53, meaning that 53% of CAD was explained by this interaction.

Conclusion: These data suggest the existence of a synergistic effect of the RR 192 PON 1 genotype (with lower ability) and HDL-deficient subjects in CAD emergence.
Abstract 3442: Normal Circadian Rhythms In Calcineurin-dependent Signaling And Protein Phosphorylation Are Disrupted In Heart Failure
Authors: Dey Asim , Sachan Nita , Oh Misook , Hill Joseph A , Rothermel Beverly A
Published: July 2021

Many important cardiovascular factors, including metabolism, heart rate, blood pressure, and hormone release, oscillate over a 24-hour period. In humans, the incidents of adverse cardiac events, such as myocardial infarction, and ventricular tachycardia vary according to the time of the day. Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways involved in normal cardiac function and heart failure. Here we show that there are large circadian oscillations in calcineurin-dependent activities in normal, healthy mouse hearts. This finding is remarkable because activation of the protein phosphatase calcineurin has primarily been thought of as a pathological process driving cardiac hypertrophy and failure. In the hearts of wild type C57BL6 mice there was a 17-fold change in mRNA levels for the calcineurin/NFAT target gene
RCAN1.4
that peaked in the early morning at the beginning of the animal’s sedentary phase. Nuclear occupancy of calcineurin-regulated transcription factor NFAT coincided with the changes in mRNA suggesting that circadian changes in calcineurin activity underlie these oscillations. Phosphorylation of the protein phosphatase 1 inhibitor 1 (I-1), a direct calcineurin substrate, and phospholamban (PLB), an indirect target, oscillated directly out of phase with calcineurin-dependent signaling. We propose that temporal separation of peak kinase and phosphatase activities form interdependent feedback loops helping coordinate cardiac function. In pressure overload-induced failing hearts, oscillations in calcineurin-dependent transcription persisted, however both the peak and trough activities were markedly elevated compared to control hearts. In contrast, oscillations in PLB and I-1 phosphorylation were largely lost in failing hearts where phosphorylation of I-1 could no longer be detected and PLB phosphorylation remained elevated throughout. Together, our results suggest that the normal relationships between circadian oscillations in protein phosphorylation and dephosphorylation are disrupted in heart failure.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 3470: Deletion of Glycogen Synthase Kinase-3α (GKS-3α) Causes a Glycogen Storage Cardiomyopathy
Authors: Zhou Jibin , Kerkela Risto , Harris David , MacAulay Katrina , Kocheritz Lisa , Doble Bradley W , Schneider Timothy , Woulfe Kathleen , Gao Erhe , Lei Yonghong , Woodgett James R , Huggins Gordon S , Force Thomas L
Published: July 2021

Background:
GSK-3 and its targets play critical roles in a wide array of processes including development and cancer. In mammalian cells there are two isoforms, α and β. GSK-3β is purported to be a negative regulator of cardiac hypertrophy, but this is based solely on over-expression approaches, and virtually nothing is known of the functions of GSK-3α.

Methods:
We generated mice deleted for GSK-3α. Heart development, as well as postnatal cardiac growth, glycogen metabolism, morphology, physiology, and ECG conduction invervals were examined. Age-matched wild type (WT) mice served as controls.

Results:
Heart development was normal, consistent with full compensation by GSK-3β for loss of GSK-3α during development. However, echocardiographic LV mass (in mg) was significantly increased in the KO compared to WT: 187.95 ± 35.05 vs 143.52 ± 23.94*. Heart weight (HW, mg) and HW/body weight ratio were also significantly increased in the KO: 186.73 ± 15.3* and 4.96 ± 0.38* for KO (n = 19); 146.33 ± 14.92 and 4.12 ± 0.27 for WT (n = 10). Thus deletion of GSK-3α leads to significant cardiac hypertrophy with aging. The underlying mechanism appears to be marked glycogen deposition seen with both Periodic acid-Schiff staining and transmission electron microscopy. Although LV function as assessed by echocardiography was normal at 4 months of age, invasive hemodynamic evaluation demonstrated a depressed response to isoproterenol infusion. ECG revealed a significantly shortened PR interval without pre-excitation.

Conclusions:
We demonstrate for the first time a striking isoform-specific role for GSK-3α in the heart, and that role is as a critical regulator of glycogen metabolism. Deletion of GSK-3α leads to a glycogen storage cardiomyopathy, sharing some features with that seen with mutations in AMP-activated protein kinase. These data suggest the possibility that mutations in, or alterations in activity of, GSK-3α could account for some cases of hypertrophic cardiomyopathy. (*, P < 0.01, KO vs. WT)
Abstract 3474: Cardiac -Specific SOCS3 Dificient Mice Shows Resistance to Lipopolysaccharide-Induced Cardiac Dysfunction
Authors: Nobuyoshi Futamata , Yasukawa Hldeo , Ohba Toyoharu , Mawatari Kazutoshi , Fukui Daisuke , Hoshijima Masahiko , Imaizumi Tsutomu
Published: July 2021

Background
: Lypopolysaccharide (LPS)-induced left ventricular (LV) dysfunction is a well-established model for sepsis-induced acute heart failure. STAT3 signaling in the heart has been shown to promote cardiomyocyte survival during LPS-induced LV dysfunction. Little is known, however, about the role of negative regulation of STAT3 signaling during LPS-induced LV dysfunction. Suppressor of cytokine signaling 3 (SOCS3) is an intrinsic negative regulator of gp130 cytokine-induced STAT3 signaling that plays an important role in cardiomyocyte survival. In this study, we determined whether STAT3 signaling and its negative regulator SOCS3 would play a role in LPS-induced LV dysfunction.

Methods and Results
: We examined the activation of STAT3 and inductions of gp130 cytokines and SOCS3 in the wild-type (WT) mice hearts after LPS injection by western blot and real-time PCR (RT-PCR). RT-PCR revealed that gp130 cytokines were markedly increased after AMI. Western blot revealed that STAT3 was markedly phosphorylated and SOCS3 was induced in WT mice hearts after LPS injection. To investigate the role of STAT3 signaling and SOCS3 in LPS-induced LV dysfunction, we generated cardiac-specific SOCS3 knockout mice (SOCS3-CKO). Left ventricular ejection fraction (LVEF) of SOCS3-CKO mice was similar to that of WT mice at baseline (64.2 ± 6.1 vs. 62.4 ± 4.4%). LPS (30mg/kg) elicited a significant and robust reduction of LVEF in both SOCS3-CKO mice and WT mice 3 hr after LPS injection (18 ± 4.5 vs. 16 ± 5.2%,
p
<0.01). LVEF in WT mice was further reduced 6 hr after LPS injection. On the other hand, interestingly, LVEF was restored to the baseline in SOCS3-CKO mice 6 hr after LPS injection (10.4 ± 3.9 vs. 62.2 ± 8.1%,
p
<0.01). Also the duration and intensity of STAT3 phosphorylation after LPS injection was greater in SOCS3-CKO mice than WT mice. Furthermore, SOCS3-CKO mice showed greater survival rate than WT mice after LPS injection (
p
<0.01).

Conclusion
: Our data show that the deletion of SOCS3 in cardiomyocytes prevents the LPS-induced LV dysfunction in mice, possibly by augmenting the STAT3-mediated gp130 cytokine signaling.
Abstract 3481: A Novel Mechanism Contributing to End Organ Damage in Endotoxic Shock: Role of TRPV1
Authors: Wang Youping , Wang Donna H
Published: July 2021

We previously showed that transient receptor potential vanilloid type 1 (TRPV1) channels play a vital role in the survival of animals during endotoxic shock. To examine effects of TRPV1 on systemic hemodynamics, inflammatory responses, and renal and liver function/damage during endotoxic shock, lipopolysaccharide (LPS, 10 mg/kg) was injected i.p. into wild type (WT) and TRPV1-null mutant (TRPV1
−/−
) mice. Mean arterial pressure (MAP, mmHg) measured with radiotelemetry in conscious mice were severely decreased 11 hours after LPS injection in WT and TRPV1
−/−
mice, with no difference between the two strains (76±6 vs. 71±4, p>0.05). The serum levels of alanine aminotransferase (ALT, indicator of liver dysfunction, IU/L) or creatinine (indicator of renal dysfunction, mg/dL) were increased 6 and 24 h after LPS injection in WT and TRPV1
−/−
mice, with a greater magnitude in the latter strain (ALT, 79±11 vs. 105±16; creatinine, 0.7±0.09 vs. 1.2±0.08, p<0.05). Serum cytokine (pg/ml) and chemokine (ng/ml) levels were higher 6 h after LPS injection in TRPV1
−/−
compared to WT mice (TNF-α, 718±43 vs. 428±44; IL-1β, 736±39 vs. 420±46; KC, 100±8 vs. 60±7; MIP-2, 2.2±0.4 vs. 1.3±0.3, p<0.05). LPS-induced neutrophil infiltration (cells/mm
2
) was greater in the kidney and liver in TRPV1
−/−
than in WT mice (kidney, 93±6 vs. 38±4; liver, 247±13 vs. 156±12, p<0.05). VCAM-1 or ICAM-1 expression was increased 6 and 24 h after LPS injection in the kidney and liver in WT and TRPV1
−/−
mice, with a greater magnitude in the latter strain (p<0.05). Renal glomerular hypercellularity and hepatocellular injury were apparent 24 h after LPS injection in WT and TRPV1
−/−
mice, with greater cellular density or injury in the latter strain. These data demonstrate that deletion of TRPV1 had no effect on LPS-induced fall in MAP, but accelerated organ damage during endotoxic shock. Exaggerated organ damage was accompanied by increased inflammatory responses as evident by enhanced neutrophil infiltration, proinflammatory cytokine production, and adhesion molecule expression. These data indicate that activation of TRPV1 attenuates organ damage possibly via its anti-inflammatory action without affecting systemic hemodynamics during endotoxic shock.
Abstract 3503: Hydrogen Peroxide-Induced Afterdepolarizations are Dependent on Calmodulin Kinase II Signaling
Authors: Xie Lai-Hua , Chen Fuhua , Weiss James N
Published: July 2021

Background:
In the heart, hydrogen peroxide (H
2
O
2
) has been shown to cause early afterdepolarizations (EADs) and triggered activity by impairing Na current (I
Na
) inactivation. Since H
2
O
2
has been recently shown to activate Ca
2+
/calmodulin kinase II (CaMKII), and since CaMKII activation has also been reported to impair I
Na
inactivation and predispose to EADs, we hypothesized that CaMKII activation by H
2
O
2
may be an important factor in the genesis of EADs induced by oxidative stress.

Methods and Results:
Patch-clamped Fluo-4 AM-loaded rabbit ventricular myocytes were exposed to H
2
O
2
(0.1–1mM), which induced spontaneous EADs after 5–15 min. Both the I
Na
blocker tetrodoxtin (TTX, 10 μM) and the I
Ca,L
blocker nifedipine shortened AP duration (APD) and suppressed EADs. H
2
O
2
increased both peak and steady-state I
Ca,L
under square-pulse voltage clamp, and enhanced I
Ca,L
to a greater extent during the AP plateau than during the AP upstroke under AP clamp conditions. In addition, by prolonging the AP plateau and increasing Ca influx via maintained I
Ca,L
, H
2
O
2
-induced EADs frequently caused DADs delayed afterdepolarizations (DADs) due to spontaneous SR Ca release waves after repolarization. KN-93(1 μM), a CaMKII inhibitor, prevented H
2
O
2
-induced EADs (n=4), whereas the inactive analogue KN-92 did not (n=5).

Conclusion:
These findings indicate that H
2
O
2
-induced EADs depend on both impaired I
Na
inactivation to reduce repolarization reserve and enhanced I
Ca,L
to reverse repolarization. Intact CaMKII signaling is necessary for EAD generation in this setting, presumably via its actions on I
Na
and I
Ca,L
, although direct redox effects on other ion channels/transporters may also be important. Our observations support a link between increased oxidative stress, CaMKII activation and afterdepolarizations as triggers of lethal ventricular arrhythmias in diseased heart.

This research has received full or partial funding support from the American Heart Association, AHA National Center.