bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P352: The Anticancer mTOR-Inhibitor Temsirolimus Produces Left Ventricular Dysfunction in Murine Hearts
Authors: Tocchetti Carlo G , Coppola Carmela , Quintavalle Cristina , Barbieri Antonio , Rea Domenica , Palma Giuseppe , Gala Marianna , Luciano Antonio , Giudice Aldo , Paolocci Nazareno , Arra Claudio , Iaffaioli Rosario V , Condorelli Gerolama , Maurea Nicola
Published: July 2021

Background:
Cardiotoxicity is a major drawback and social problem linked to many anticancer treatments. Early identification of signs of this adversity would certainly benefit the management of oncologic patients. The mTOR-inhibitor temsirolimus is currently being evaluated for anticancer efficacy in hundreds of clinical trials and is approved for treatment of advanced renal cell carcinoma. However, the PI3K/Akt pathway converges on mTOR, which is a central regulator of cell growth, including cardiomyocyte growth. Here, we aim at evaluating the cardiac effects of the anticancer mTOR-inhibitor temsirolimus in a mouse model in vivo.

Methods:
Left Ventricular (LV) fractional shortening (FS) was assessed by M-mode echocardiography in sedated C57BL/6 mice (2–4 mo. old) at day 0, and after 2, 7, 14, 21 days from a single i.p. injection of temsirolimus (0.1mg/kg, a dose comparable to the one used to treat cancer in humans) or vehicle. Doxorubicin (Doxo, 2.17 mg/kg/day for 7 days) was used as a positive control. With Speckle Tracking echocardiography (ST) we also evaluated radial myocardial strain (%), a very sensitive parameter which can detect subtle changes in cardiac function.

Results:
After 2 days, there was no change in FS with temsirolimus, but FS was already reduced with Doxo: 52±0.2%, p=.0000001 vs sham (60±0.4%). With temsirolimus, FS was reduced only after 21 days: 50±3%, p=.009 vs sham. Interestingly, with Speckle Tracking echocardiography we found that in the temsirolimus group radial strain was already decreased at 7 days: 42±5%, p=.01 vs sham (59±1%).

Conclusions:
The antineoplastic mTOR-inhibitor temsirolimus induces LV dysfunction in mice. Such dysfunction occurs later than the one observed with Doxo, but speckle tracking echocardiography is more sensitive than conventional echocardiography and can detect early signs of myocardial alteration that may prelude to overt LV dysfunction. The clear mechanisms of temsirolimus cardiotoxicity are to be elucidated in further experimental studies. We also plan to apply speckle tracking echocardiography to clinical studies, in order to evaluate the impact of early identification of temsirolimus cardiotoxicity in the treatment of renal cell carcinoma.
Abstract 101: Endothelial-Specific Fibroblast Growth Factor Receptor 1 and 2 Deletion Impairs Vascular Remodeling and Recovery in an In Vivo Closed-Chest Model of Cardiac Ischemia-Reperfusion Injury
Authors: House Stacey , Weinheimer Carla , Kovacs Attila , Ornitz David
Published: July 2021

Introduction:
Fibroblast growth factor (FGF) signaling is cardioprotective in various models of myocardial infarction. FGF receptors (FGFRs) are expressed in multiple cell types in the adult heart, but the cell type-specific FGFR signaling which mediates different cardioprotective endpoints is currently unknown.

Hypothesis:
We hypothesize that endothelial FGFR signaling is essential for vascular remodeling after cardiac ischemia-reperfusion (IR) injury.

Methods:
We have generated mice with a conditional ablation of the
Fgfr1
and
Fgfr2
genes in endothelial cells (

Tie2-Cre, Fgfr1
f/f
, Fgfr2
f/f

). These mice and littermate controls were subjected to a clinically relevant,
in vivo
, closed-chest model of cardiac IR injury which includes 90 minutes of occlusion of the left anterior descending artery followed by reperfusion for 7 days. Echocardiography was performed at baseline, day 1, and day 7 of reperfusion, and histological staining was performed on day 7.

Results:

Tie2-Cre, Fgfr1
f/f
, Fgfr2
f/f

mice had no baseline abnormalities in cardiac morphometry or function. When subjected to closed-chest, regional cardiac IR injury,

Tie2-Cre, Fgfr1
f/f
, Fgfr2
f/f

mice showed significantly increased infarct size compared to controls at 7 days (37±3% vs. 15±5%, p<0.05) but not 1 day after reperfusion.

Tie2-Cre, Fgfr1
f/f
, Fgfr2
f/f

mice also showed significantly worsened cardiac function (ejection fraction, stroke volume, and fractional shortening) compared to controls at 7 days (p<0.05) but not 1 day after reperfusion. Myocyte cross-sectional area measurement showed no impairment in the cardiac hypertrophic response in the

Tie2-Cre, Fgfr1
f/f
, Fgfr2
f/f

mice.

Tie2-Cre, Fgfr1
f/f
, Fgfr2
f/f

mice have normal vessel density in the non-injured state, but after cardiac IR injury,

Tie2-Cre, Fgfr1
f/f
, Fgfr2
f/f

hearts showed significantly decreased vessel density (both capillaries and smooth muscle actin containing vessels, p<0.05) compared to controls in the peri-infarct area.

Conclusions:
Ablation of FGFR1 and FGFR2 in endothelial cells results in impaired vascular remodeling, worsened cardiac functional recovery, and increased infarct size without affecting the cardiac hypertrophic response in an
in vivo
, closed-chest model of cardiac IR injury.
Abstract 114: Allogeneic Cardiosphere-Derived Cells Are Safe and Effective in a Pig Model of Myocardial Infarction
Authors: Smith Rachel R , Kreke Michelle , Malliaras Konstantinos , Kanazawa Hideaki , Huang Christene A , Valle Ileana , Blusztajn Agnieszka , Yee Kristine , Sachs David H , Terrovitis John , Marban Linda
Published: July 2021

Allogeneic cardiosphere-derived cells (CDCs) have proven safe and effective in a small animal model of myocardial infarction (MI), and have been shown to act primarily via paracrine mechanisms to stimulate endogenous regeneration. The present translational study tested allogeneic CDCs in a large animal model (mini-pigs).

To establish a robust allogeneic model, all pigs were swine leukocyte antigen (SLA) typed by PCR. A male donor and female recipients with full SLA I, II mismatch were used. Pigs underwent balloon occlusion of the LAD for 2.5 hours, followed by reperfusion. Two weeks later, 12.5 million CDCs (n=8) or vehicle (n=6) were infused. Animals were sacrificed 2 weeks or 2 months post-infusion to assess the cellular (histology using a clinical rejection scale) and humoral (donor-specific antibodies, complement-dependent cytotoxicity) immune responses, as well as cardiac function (left ventriculography, hemodynamics, morphometry). Numerous in-life assessments for safety were performed and CDC engraftment was assessed by FISH for Y chromosome.

All immunological assays indicated an undetectable response to CDCs. Cardiac enzymes and systemic inflammation showed no differences between groups. There were no systemic histological findings related to CDCs. CDCs did not permanently engraft, with <0.1% persisting 2 weeks post-infusion and none evident 2 months post. Despite evanescent engraftment, functional benefits were seen following infusion of CDCs. Ejection fraction (EF) and infarct size (IS) were significantly improved in CDC-treated animals compared to vehicle-treated 2 weeks post-infusion (EF: 48.6±2.1% vs 38.2±2.6%, p<0.05; IS: 9±2 vs 14±2%, p<0.05). At 2 months, EF still trended higher in CDC-treated animals (42±5 vs 32±10%, p=0.09), and IS still trended lower (14±4 vs 16±7%, p=0.60). Pressure-volume relationships revealed a trend for enhanced contractility in CDC-treated animals (Emax: 3.2±2.2 vs 1.0±1.1 mmHg/mL, p=0.26). The magnitude of the functional benefits was similar to that seen in a prior pig study using autologous CDCs.

Overall, results of the present study demonstrate that allogeneic CDCs are largely equivalent to autologous in terms of efficacy, and elicit no detectable immunological response or safety concern.
Abstract 115: The Changes in Cardioelectrical Activity of Rat With Myocardial Infarction Receiving Sarcoplasmic Reticulum Ca
2+
-ATPase Gene Modified Bone Marrow Stem Cell Transplantation by Microelectrode Array Technology
Authors: Ping Fan , Yuemei Hou , Lingpeng Wang , Huan Wang , Xiaoxia Xu , Xi Yang , Guangwei Zhang , Hongli Wang
Published: July 2021

0bjective
Comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca
2+
-ATPase (SERCA2a) gene modified BMSCs transplantation for therapy effects after acute myocardial infarction(AMI), as well as for assessment cardiac electrical activity by microelectrode array (MEA) technology.

Methods
Rats with myocardial infarction (
n
=24)and divided into 3 groups randomly: Sham group (
n
=8), BMSCs group(
n
=8) and SERCA2a gene modified BMSCs transplantation group (BMSCs+rAd.SERCA2a group,
n
=8). After 14 days, cardiac function was evaluated by echocardiography and heart electrical activity was evaluated by electrocardiogram and MEA technology.

Results
(1)The transduction ration of rAd.SERCa2a to BMSCs were 80% to 90%. (2)Compared to sham group, BMSCs group and BMSCs+rAd.SERCA2a group could significantly improve cardiac function on 14 days after therapy (
n
=6,
P
<0.05).(3)QT duration of BMSCs+rAd.SERCA2a group was significantly shorter than in the sham group(80.30 ms±6.53 ms versus 105.31 ms±21.89 ms),the mean value was significantly decreased 23.8%(
n
=7,
P
<0.05). Ventricular premature beats were also recorded in rats from sham group.(4)MEA results suggested that isolated heart beats were significantly slowed and ventricular arrhythmias and atrioventrocular block were recorded in sham group. The maximum field potential and field potential duration of infarcted myocardium area in BMSCs group and BMSCs+rAd.SERCA2a group were significantly longer than those in sham group(the maximum field potential: 0.51 mV±0.15 mV, 0.55 mV±0.16 mV, 0.23 mV±0.1mV; field potential duration: 104.5 ms±25.43 ms, 107.67 ms ±24.01 ms, 63 ms± 20.34 ms;
n
=6,
P
<0.05).(5)The conduction time was the shortest in BMSCs+rAd.SERCA2a group, the cardiac electrical conduction could keep consistency and improve in myocardial infarction tissue.

Conclusions
In short time, BMSCs and SERCA2a gene modified BMSCs transplantation could obviously enhance cardiac function. BMSCs+rAd.SERCA2a group could effectively improve electrical conduction of infarcted myocardium and block the occurrence of arrhythmia after myocardial infarction.
Abstract 116: Detrimental Role of Toll-Like Receptor 4 in Cardiovirus-Induced Myocarditis
Authors: Sato Fumitaka , Omura Seiichi , Martinez Nicholas E , Kawai Eiichiro , Chaitanya Ganta V , Alexander Jonathan S , Tsunoda Ikuo
Published: July 2021

Picornavirus infections have been known as a leading cause of viral myocarditis in humans. Theiler’s murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus, the family Picornaviridae and was reported to cause inflammation in the heart in one manuscript, while its pathomechanism is unclear. In viral myocarditis, viral replication in the heart and/or immune responses against virus as well as heart-antigen (autoimmunity) can contribute to the pathogenesis. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are important for recognizing pathogens as well as triggering innate immunity. Among TLRs, TLR4 has been demonstrated to play important roles in virus-mediated pathology: 1) TLR4 can contribute to viral entry in some viruses, 2) TLR4 may mediate tissue damage by anti-virus immune responses (immunopathology), 3) high levels of TLR4 expression were observed in the heart of patients with dilated cardiomyopathy following acute viral myocarditis, and 4) some viruses can bind to lipopolysaccharide (LPS), which is a TLR4 ligand. To determine the role of TLR4 in TMEV-induced myocarditis, we infected male C3H/HeJ (TLR4-deficient) and C3H/HeNtac (control TLR4+) mice with the DA strain of TMEV. We harvested the hearts and spleens on days 6 and 7 (acute phase) or days 63 and 64 (chronic phase) post-infection. Cardiac pathology was evaluated by hematoxylin and eosin staining and production of pro-inflammatory cytokines, interleukin (IL)-17A and interferon (IFN)-γ, from spleen cells was measured by an enzyme-linked immunosorbent assay (ELISA). In both mice, mild myocarditis was observed during the acute phase of TMEV infection. During the chronic phase, both mice developed severe pathology in the heart, including basophilic degeneration and calcification. However, the incidence of myocarditis was higher in control mice than TLR4-deficient mice. IL-17A and IFN-γ production was higher in control mice than in TLR4-deficient mice (control vs. TLR4-deficient mice, acute phase: IL-17A, 196 vs. 146 pg/ml; IFN-γ, 72 vs. 39 ng/ml; chronic phase: IL-17A, 290 vs. 229 pg/ml; IFN- γ, 142 vs. 88 ng/ml). These results suggest that TLR4 may be detrimental in TMEV-induced myocarditis by increasing pro-inflammatory cytokine production.
Abstract 143: Catalase, Oxidative Stress and Ischemic Injury: Role of c-Abl Tyrosine Kinase
Authors: Cabigas E Bernadette , Ding Guoliang , Chen Tao , Saafir Talib , Wagner Mary , Davis Michael E
Published: July 2021

Thousands of children with congenital heart disease are subjected to corrective surgery which involves stopping the heart and exposing it to ischemia reperfusion (IR) injury. Complications after cardiopulmonary bypass like low cardiac output can lead to inadequate organ perfusion and eventual organ dysfunction if not corrected in a timely manner. Newborn (2-4 days) hearts are more susceptible to ischemic injury than adult (>8 weeks) hearts as evidenced by their diminished ability to scavenge reactive oxygen species (ROS), specifically hydrogen peroxide (H
2
O
2
). Not only do newborn hearts exhibit decreased scavenging capability (compared to the adult) but they also contain reduced baseline levels of catalase (3.8 + 1.1 U/mg tissue in the LV and 4.3 + 2.3 U/mg tissue in the RV vs. 11.2 + 1.0 U/mg tissue in the adult LV and 11.6 + 0.3 U/mg tissue in the adult RV). We observed significantly increased levels of H
2
O
2
in newborn rabbit myocytes following IR injury (4.3-fold vs 2.2 fold; p<0.05) that was not accompanied by an increase in catalase activity as was seen in the adult myocytes. The tyrosine kinase c-Abl was shown to play a role in regulating phosphyorylation (and activation) of catalase and we hypothesized this pathway may be dysregulated in newborns, leading to differences in catalase activity. We performed immunoprecipitation with c-Abl and catalase as well as Western analysis of whole heart homogenates in both newborn and adult samples (n=3). Results demonstrated an increase in catalase phosphorylation in adult but not newborn hearts after IR. In the newborn, preliminary studies displayed diminished phosphorylation of c-Abl, indicating a potential mechanism. In addition, catalase levels seem to acutely rise following IR, too early for new protein synthesis. Catalase is known to undergo ubiquitination following release from the c-Abl complex and this may play a role in the failure of newborns to increase catalase. Elucidation of this pathway will lead to a greater understanding of the mechanisms controlling catalase activity in the newborn versus the adult heart and will prove beneficial in developing therapeutic interventions to minimize damage following ischemic injury in newborns.
Abstract 145: Apelin Reduces Myocardial Infarction Size and Promotes Angiogenesis by Increasing SDF-1/CXCR4 and AKT/eNOS/VEGF Pathways
Authors: Li Lanfang , Zeng Heng , Chen Jian-xiong
Published: July 2021

Background:
Apelin is an endogenous ligand for the angiotensin-like 1 receptor (APJ) and is emerging as a key player in the regulation of angiogenesis as well as ischemia/reperfusion injury. So far, little is known about the functional role of apelin in myocardial ischemia. We investigated the potential intracellular molecular mechanisms and protective role of apelin during myocardial ischemic injury.

Methods and Results:
Myocardial ischemia was achieved by ligation of the left anterior descending coronary artery (LAD) for 24 hours and 14 days. Myocardial apoptosis was detected by TUNEL staining. Akt, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), SDF-1 and CXCR4 expression were measured by western blot. The CD133+/cKit+/Sca1+, CD133/SDF-1+ and cKit/CXCR4+ cells were determined by immunostaining. Myocardial capillary and arteriole densities were analyzed in the border zone of infarcted myocardium at 14 d of ischemia. Treatment of C57BL/6J mice with apelin-13 (1 mg/Kg.d) by i.p. injection for 3 days before surgery results in significant decreases in TUNEL positive cells and myocardial infarct size at 24 hours of ischemia. Treatment with apelin increases the phosphorylation of AKT and eNOS and upregulates VEGF expression in the ischemic heart. Furthermore, treatment with apelin leads to the expression of SDF-1 and CXCR4 and increases in the number of CD133+/cKit+/Sca1+, CD133/SDF-1+ and cKit/CXCR4+ cells in ischemic hearts. Treatment with apelin also significantly increases myocardial capillary densities and arteriole formation together with a significant decrease in the ratio of heart weight to body weight at 14 days of ischemia. This is accompanied by a significant improvement of cardiac function after 14 days of ischemia.

Conclusions:
Our data demonstrate that apelin contributes to the protection of myocardial infarction and angiogenesis by the mechanisms involving in upregulation of SDF-1/CXCR4 and AKT/eNOS/VEGF pathways.
Abstract 146: Disruption of Nox2 and TNFRp55/p75 Eliminates Anisomycin-Induced Preconditioning Effect in the Heart
Authors: Zhao Ting C , Zhang Ling , Guo Tai-Liang
Published: July 2021

Background:
Transient activation of p38 through anisomycin is demonstrated to precondition the heart against myocardial injury. However, it remains unknown whether specific TNF-α receptor (TNFR) p55/p75 and Nox2, a subunit of NADPH-oxidase are involved in this event.

Objective:
We sought to investigate whether the genetic disruption of TNFRp55/p75 and Nox2 eliminates cardioprotection elicited by anisomycin and whether p38-dependent activation of Nox2 stimulates TNFR to ultimately achieve protective effects.

Methods:
Adult wild type and p55/p75
-/-
and Nox2
-/-
mice received intraperitoneal injections of anisomycin (0.1mg/kg), a potent activator of p38. The hearts were subjected to 30 min myocardial ischemia /30 min reperfusion in the Langendorff perused heart after twenty four hours. Left ventricular function was measured and infarct size was determined by triphenyltetrazolium chloride. Myocardial TNF-α protein, Nox2 and superoxides releases were detected. Gel kinase assay was employed to detect the effect of p38 on Nox2 phosphorylation.

Results:
Activation of p38 through anisomycin produces marked improvements in the recovery of left ventricular end diastolic pressure, rate pressure products, and the reduction of myocardial infarction, which were completely abrogated by disruption of Nox2 and TNFR p55/p75. Genetic disruption of Nox2 and TNFR p55/p75 abolished the effect of anisomycin-induced reduction of infarct size. Ansiomycin induced the production of TNFα, which was abrogated in Nox2
-/-
mice. Notably, activation of p38 resulted in the phosphorylation of Nox2.

Conclusion:
Taken together, these results indicate that stimulation of the Nox2 and TNFR p55/p75 pathway is a novel approach to anisomycin-induced cardioprotection.
Abstract 148: Sterol Regulatory Element-Binding Protein-1 Is Involved in the Development of Angiotensin II-Induced Cardiac Fibrosis
Authors: Sakai Satoshi , Nakagawa Yoshimi , Shimojo Nobutake , Kimura Taizo , Tajiri Kazuko , Keisuke Kuga , Homma Satoshi , Miyauchi Takashi , Aonuma Kazutaka , Shimano Hitoshi
Published: July 2021

Sterol Regulatory Element Binding Protein (SREBP)-1 is a transcription factor for triglyceride synthesis. SREBP-1 is shown to contribute to the organ damages such as pancreatic beta cell, liver, and kidney; however, it is unclear whether SREBP-1 also contributes to the cardiac pathogenesis. We made cardiac dysfunction and fibrosis model by 2-week infusion of angiotensin II (A-II, 1.44 mg/kg BW/day). Mice were divided into followings (n=5∼6 in each group): wild with vehicle (WC), wild with A-II (WA), SREBP-1 knockout mice (SREBP-KO) with vehicle (SC), and SREBP-KO with A-II (SA). WA clearly demonstrated cardiac dysfunction and severe perivascular fibrosis compared to WC; however, these findings were not observed in SA compared to SC. We analyzed gene expression by DNA microarray using the software DAVID and quantitative RT-PCR to find gene clusters mostly illustrative for these phenotypes. Gene expression of extracellular matrix (Col1a, 3a, periostin) was increased in WA. Highly scored annotations in WA were chemokines (CCL5, CXCL10) and their receptors (CCR5, CXCR3), and Th2 cytokines (IL-13 and TGFb), suggesting that chronic inflammatory and repairing responses occurred. These changes were normalized in SA compared to SC. Expression of NOX4, a component of NADPH oxidase, was significantly increased in WA and SA compared to each control in a similar extent, suggesting that the Ang II-induced oxidative stress to the heart did not differ. To elucidate why the cardiac fibrosis differed between WA and SA, we analyzed the expression of transcription factors. Nrf2, a transcription factor for detoxification and anti-oxidant gene against to reactive oxygen species (ROS), was significantly decreased in WA compared to WC; however, it did not differ between in SA and SC. Furthermore, expression of the Nrf2-inducible genes HO-1 and NQO1, antioxidant genes, was significantly decreased in WA compared to WC; meanwhile, there were no differences between in SA and SC. [Conclusion] SREBP-1 may positively contribute to the A-II-induced cardiac fibrosis via the involvement of chronic inflammatory responses, which is induced partly by the reduction of antioxidant activity.
Abstract 154: Generation of Purified Cardiomyocytes from Pluripotent Stem Cells Using Molecular Beacons
Authors: Ban Kiwon , Wile Brian , Kim Sangsung , Byun Jaemin , Saafir Talib , Wagner Mary , Bao Gang , Yoon Young-Sup
Published: July 2021

Background:
While various methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been reported, all available methods are only allowed to produce heterogeneous population of CM mixed with non-CM cells. Therefore, strategies to enrich pure CMs for scientific and clinical applications have been highly required. Hence, we developed a novel system in which CMs can be purified by cardiac specific molecular beacons (MBs). MBs are dual-labeled antisense nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. We hypothesized that MBs targeted to CM specific mRNAs can identify CMs and allow isolation of purified CMs by fluorescence-activated cell sorting (FACS).

Methods and results:
Five MBs targeting distinct sites on either cardiac troponin T (cTNT) or α/β myosin heavy chain (α/βMHC) were designed and characterized in various cell types. To find the optimal MB that can selectively identifying CMs, each MB was delivered into HL-1 CMs, an immortalized mouse CM cell line, smooth muscle cells, endothelial cells, mouse ESCs and fibroblasts and its specificity was determined by flow cytometry. As a result, two MBs identified MB+ cells up to 98% from HL-1 CMs but lower than 10% of the non-CM cells suggesting these MBs are CM specific. Subsequently, the selected MBs were delivered into both mouse and human PSCs derived CMs and 41 to 49% of the cells were identified as an MB+ population. Interestingly, the rate of MB+ cells was similar to CM quantification determined by cTNT intracellular flow cytometry. Finally, we determined whether cell sorting with cardiac-specific MBs can enrich CMs from the heterogeneous mouse and human PSC cultures and found that ∼97% of MB-based sorted CMs expressed cTNT. These enriched cells were further cultured and their CM identity was verified by immunocytochemistry and qRT-PCR analysis. Ca2+ transient analysis further confirmed that these purified CMs displayed functional CM characteristics

Conclusion:
Using cardiac specific MBs, we were able to obtain highly purified CMs. These purified CMs and the system can be highly useful for clinical applications as well as drug discovery.
Abstract 166: Interaction Between LDL and Sympathetic Adrenoreceptors
Authors: Grün Daniel , Lendner Janna , Krohn Stephan , Jumar Agnes , Zakrzewicz Andreas , Ermilov Eugeny , Siegel Günter
Published: July 2021

Introduction:

LDL is a significant risk factor for cardiovascular disease and is known to impair flow-dependent vasodilatation. Furthermore, it has been demonstrated that LDL can even directly cause vasoconstricton. Recent studies suggested that lower LDL levels, achieved through administration of statins, are correlated with lower blood pressure, independent from pleiotropic statin effects. Our objective was to investigate whether LDL-induced impairment in flow-dilatation was due to an interaction of LDL with sympathetic adrenoreceptors.

Materials and Methods:

Flow-dependent isometric tension, intracellularly recorded membrane potential and cAMP-cGMP were measured in segments of 22 coronaries from heart transplantations.

Results:

As

compared to Krebs solution, LDL affected flow-dilatation and caused a relative contraction [Δ(T
3
-T
100
): Krebs 0.459 g; LDL 0.278 g]. Complete blockade of both α- (phentolamine 10
-7
mol/L) and β-receptors (propranolol 10
-7
mol/L) resulted in a ∼50% reduction of flow-dilatation impairment induced by LDL. Blockage of either α- (1) or β-receptors (2) showed smaller effects which added up to the measured effect under blockade of both receptors (4). Similar effects were apparent in the recorded membrane potential of the vascular smooth muscle cells.

Discussion:

We conclude that the LDL-induced impairment in flow-dependent dilatation is partially due to the interaction between lipoprotein and both α- and β-adrenoreceptors. Since blockade of each receptor resulted in a relative dilatation, we assume that the LDL effect is caused by a stimulation of α-adrenoreceptors and an inhibition of β-adrenoreceptors.
Abstract 168: Does Calcium Influx Through TTCC Induce Cardiomyocyte Proliferation?
Authors: Wang Fang , Gao Hui , Kubo Hajime , Fan Xiaoxuan , Berretta Remus , Chen Xiongwen , Zhang Hongyu , Molkentin Jeffery , Houser Steven R
Published: July 2021

T-type Ca²[[Unable to Display Character:
+
]] channels (TTCC) are primarily expressed in fetal/neonatal cardiomyocytes and their functional role is not well defined. Here we explored the idea that Ca
2+
influx through TTCC regulates myocyte proliferation. The fact that Mibefradil (TTCC and L-type calcium channel (LTCC) antagonist) inhibits smooth muscles proliferation supports this idea. Our hypothesis is: Blocking TTCC will reduce neonatal cardiomyocyte proliferation.

Wild type (WT) and α1G TTCC knockout (α1G-/-) neonatal mice ventricular myocytes (NMVMs) were used. Patch clamp was used to measure TTCC. Flow cytometry was used to determine cell cycle distribution.
1.
On day 1 (after birth), TTCC was detected in 35% WT NMVMs (n=31), whereas only 4% α1G-/- NMVMs have TTCC (n=25). On day 7, no TTCC was detected in both WT (n=16) and α1G-/- (n=27).
2.
In vivo: On day 1 there’s no significant difference in cell cycle distribution: [88.2%±2.7% in G1/G0, 8.1%±2.8% in G2/M, 3.2%±0.1% in S; n=12] in WT vs. [90.6%±1.6% in G1/G0, 5.7%±1.5% in G2/M, 3.7%±0.3% in S; n=20] in α1G-/-. On day 7 there’s a significant difference: [52.3%±1.6% in G1/G0, 47.7%±1.6% in G2/M; n=28] in WT vs. [68.2%±2.1% in G1/G0, 31.9%±2.1% in G2/M; n=20] in α1G-/-, p<0.05.
3.
In vitro: Mono and binucleated myocytes percentage was measured: On day 1 there’s no significant difference. On day 7 there were more binucleated cells in WT: 51%±4% mono and 49%±4% binucleated in WT (n=495) vs. 80%±3% mono and 20%±3% binucleated in α1G-/- (n=1107), p<0.05. Cell surface area (CSA) (um²) in α1G-/- (n=164) was smaller than WT (n=109): 860.3±323.3 in WT vs. 716.7±274.9 in α1G-/- in mono (p<0.005), 1333.9±534.0 in WT vs. 1016.6±315.4 in α1G-/- in binucleated (p<0.001). In 2-month old mice there’s a difference in percentage of mono and binucleated myocytes: [7.3%±3.7% mono, 92.7%±3.6% binucleated; n=687] in WT vs. [30.8%±6.1% mono, 69.2%±6.1% binucleated; n=793] in α1G-/-, p<0.05. CSA in α1G-/- was smaller than in WT: 2267.4±1119.7 in WT vs. 1784.6±683.9 in α1G-/- in mononulceated (p<0.0001), 2419.2±712.4 in WT vs. 2048.3±671.1 in α1G-/- in binucleated (p<0.0001).

There is large amount of DNA synthesis in NMVMs after birth, by day 7 most of the WT NMVMs are arrested in G2 and become binucleated. Myocyte without α1G TTCCs did not exit from the cell cycle normally.
Abstract 176: β-Adrenergic Receptor--Mediated Regulation of Cardiac Gene Expression Is Gefitinib Sensitive
Authors: Tilley Douglas G , Talarico Jennifer A , Grisanti Laurel A , Carter Rhonda L
Published: July 2021

BetaAR-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to promote cardioprotection in a mouse model of heart failure, however the mechanism(s) responsible for this pro-survival response are not known. We hypothesized that this transactivation event could impact a number of processes in the heart, including survival, via regulation of gene expression. To test the capacity of BetaAR-mediated EGFR transactivation to regulate this process, acute changes in cardiac gene expression were assessed via RNA sequencing in the hearts of C57BL/6 mice given i.p. injections of the BetaAR agonist isoproterenol (ISO, 1mg/kg) in the presence or absence of the EGFR antagonist gefitinib (Gef, 5mg/kg) for 1 hour. The total RNA from 4 hearts per treatment group (Control, ISO, Gef, Gef/ISO) were combined and underwent DNA library generation and SOLiD sequencing analysis, which revealed a substantial number of genes and isoforms regulated by each of the treatments. Interestingly, Gef alone significantly altered the expression of 270 genes compared to control suggesting potential Gef-dependent alterations in the heart during clinical use. ISO alone and Gef/ISO significantly altered 401 and 723 distinct genes compared to control, respectively. Further statistical analysis was performed between the ISO and Gef/ISO groups to assess true Gef sensitivity of ISO-regulated genes in the heart, confirming 173 genes significantly altered between the groups. Classification of these genes revealed 4 categories: ISO-dependent gene upregulation (1) or downregulation (2) antagonized by Gef, and ISO-dependent gene upregulation (3) or downregulation (4) promoted in presence of Gef. Identified within these categories were several genes known to be involved in the regulation of cardiac hypertrophy, apoptosis, sarcomeric structure and Ca2+-handling, which were selected for validation via qPCR. In conclusion, BetaAR-mediated EGFR transactivation induces rapid modulation of cardiac gene expression in response to catecholamine stimulation in vivo, with potential functional impacts on a number of cellular processes, while simultaneously acting to antagonize gene expression changes mediated via distinct BetaAR-mediated signaling pathways.
Abstract 180: Alcohol Consumption Negatively Competes with Estrogen-Mediated Myocardial Repair in Ovariectomized Mice: Ethanol-Mediated Suppression of Estradiol-Induced Survival Signaling in Endothelial Progenitor Cells
Authors: Mackie Alexander R , Lambers Erin , Thal Melissa A , Verma Suresh , Krishnamurthy Prasanna , Hoxha Eneda , Ramirez Veronica , Hamada Hiromichi , Losordo Douglas W , Kishore Raj
Published: July 2021

Studies in post-menopausal women and ovariectomized (OVX) animals have suggested a cardio-protective (CP) role of estrogen replacement therapy. However, randomized clinical trials have not confirmed this finding with hormone replacement (HRT). Although HRT initiation and duration might explain part of this disparity, other factors that mask the CP effect of estradiol (E2) require scrutiny. We and others have reported that E2-supplementation (E2sup) with 17β-E2 pellets enhances voluntary alcohol consumption in OVX mice. In this study, we examined the effect that enhanced ethanol consumption with E2sup has on post-infarct myocardial function and repair. E2sup OVX BALB/c mice, consumed significantly more 10% ethanol for 6 weeks compared to those implanted with placebo pellets. Following acute myocardial infarction (AMI), LV functions were consistently depressed in mice consuming ethanol (E2sup+Etoh) compared to mice receiving only E2sup. Additionally, the E2sup+Etoh group also displayed a loss of the improvements in both fibrosis area and capillary density observed in the E2sup mice following AMI. E2sup+Etoh mice also displayed a diminished number of circulating endothelial progenitor cells (EPCs) as compared to the E2sup group. In vitro, exposure of EPCs to ethanol suppressed E2-induced EPC proliferation and survival. At the molecular level, estrogen response element (ERE) DNA binding activity and ERE-dependent reporter gene transcription were markedly repressed in ethanol treated EPCs. Ethanol also attenuated E2-induced eNOS expression and phosphorylation. Furthermore, ethanol blocked E2-induced activation of cell survival signaling pathways, Akt and ERK, and activated the pro-apoptotic JNK signaling via direct association with mixed lineage kinase 3. These data suggest that E2 modulation of alcohol consumption and the ensuing EC dysfunction may negatively compete with the beneficial effects of estrogen on the heart.
Abstract 183: A Genomewide Screen to Identify Novel NFAT Regulators
Authors: Maillet Majorie , Blair N Scott , Molkentin Jeffery D
Published: July 2021

NFAT (Nuclear Factor of Activated T cells) transcriptions factors are directly activated by the Ca2+ activated serine-threonine protein phosphatase calcineurin. Following dephosphorylation, NFAT factors translocate to the nucleus. They share a Rel/NFkB homology DNA binding domain that functions in concert with other transcription factors such as AP-1, GATA4, NFkB, MEF2, CREB, Myc, and IRF-1 to alter the expression of genes involved in tissue and cellular differentiation as well as adaptation to extracellular stimuli. Although the fundamental aspects of how NFAT factors are regulated by phosphorylation and dephosphorylation, as well as some interacting factors, have been described; much remains to be understood about the function of these factors in cardiac myocytes and other cell types. To identify potential new NFAT regulators that could influence nuclear shuttling or its trans-activating properties, we performed a genome wide screen with a NFAT-dependent luciferase reporter and a library of cDNA expression plasmids. Roughly 20,000 cDNA expression plasmids from the Mammalian Genome Collection were individually co-transfected with a NFAT-luciferase reporter in a human immortalized cell line in 384-well plates. Approximately 2% of the genes of the library gave a positive result, including well-known NFAT partners, calcium sensitive genes and transcriptions factors. Among them, twenty potential NFAT activators were selected and NFAT-Luciferase activation was validated in a secondary screen which normalized for transfection efficiency and cell number. The results of the screen will be presented along with functional characterization of the more interesting interacting NFAT co-activators.
Abstract 184: TNF-TNFR1/p55 or TNFR2/p75 Receptor-Ligand Interactions Inhibit Early and Increase Delayed Radio-Biological Bystander Responses in BM-Derived EPCs
Authors: Muralidharan Sujatha , Sasi Sharath , Yan Xinhua , Enderling Heiko , Goukassian David
Published: July 2021

TNF-α (TNF) binds two receptors TNFR1-p55 and TNFR2-p75. Ionizing radiation (IR) increases tissue TNF levels. TNF signaling regulates numerous cyto- and chemokines, known to mediate radiation-induced non-targeted effects (NTE), a phenomenon where cells that are not directly “hit” by IR exhibit IR effects as a result of signals received from nearby or distant IR cells. Little is known about the role of p55 or p75 in regulating NTEs in bone marrow (BM)-derived EPCs. We hypothesized that inhibition of TNF signaling either via p55, or p75 may alter TNF-mediated inflammatory responses, inducing NTEs. Medium transfer experiments (MTE) were performed in WT, p55 knockout(KO) and p75KO BM-derived EPC ex-vivo, where one set of EPCs was irradiated with 1Gy of γ-IR, then IR-conditioned medium (CM) was collected from these dishes at 1, 5, 24h and 3, 5d post-IR. Filtered (0.22µm) IR-CM was transferred to naïve non-IR same genotype EPCs and 24h post-incubation naïve EPCs were processed for p-y-H2AX staining for presence and decay of double strand breaks (DSB). CM from IR EPCs were processed for ELISA profiling (16 genes) and IR EPCs were processed for transcriptional profiling. In WT EPC the peak of detectable mean p-y-H2AX foci/cell were at 24h, whereas in p55 and p75KOs p-y-H2AX were the lowest at 24h (9±0.8 vs 4.8±0.6 and 3.7±0.5, p<0.01 and 0.001, WT vs p55 and p75KOs). This finding indicates that altered TNF signaling inhibits early NTEs (hours) in EPCs. Compared to WT, delayed (5 days) NTEs were amplified in naïve p55 and p75KO EPCs (3.8±0.4 vs 8.5±1 and 5.9±0.8, p<0.02 and 0.01, WT vs p55 and p75KOs), suggesting significant role for TNF signaling in mediating delayed NTEs. ELISA profiling of 16 proteins in CM over 5 days post-IR showed 200-1600% increases (p<0.002, p55KO vs WT) in cumulative levels of TNF, IFNr, IL6, EGF, MIP-1α, GM-SCF, Rantes, and 200-1000%, increases (p<0.05, p75KO vs WT) in IL1α, IL1β, G -CSF, MCP-1, SCF. Transcriptional profiling of γ-IR EPC revealed 139 genes (>2 fold) up or down regulated, clustered into 9 groups. Other aspects of gene array data are being considered. We conclude that TNF ligand-receptor axis regulates NTEs in naïve EPCs and suggest that restoring TNF signaling balance could be used to prevent delayed NTEs in distant from primary IR target tissues.
Abstract 186: AMP-Activated Protein Kinase Mediates Cardioprotection in the Regenerative MRL Mouse Strain
Authors: Varghese Susan T , Holley-Cuthrell Jenan , Warren Chad M , Kuenster Ann , Heydemann Ahlke
Published: July 2021

Introduction:
The

Murphy Roths Large (MRL/MpJ) mouse model is well-known for its healing and regenerative properties. The actual mechanisms promoting regeneration are still unknown. AMP activated protein kinase (AMPK) has been lauded for its role as a cardioprotective sensor of cellular metabolic stress, cell growth, and ATP depletion. Based on our preliminary data, we hypothesize that increased levels of AMPK in the MRL hearts is primarily responsible for its altered mitochondrial biogenesis and metabolism, reduced oxidative stress, and altered cardiac function in the MRL mice.

Methods:
Adult C57BL/6 (control) and MRL mice (n=5/group) were studied pre- and post-7 days of exercise, and post-exercise with 3 days injection of either
5-amino
-imidazole carboxamide riboside (AICAR, AMPK stimulator, 250ug/g, IP), or Compound C (AMPK inhibitor, 20ug/g, IP).

Results:
Compared to control at baseline, the MRL hearts had higher AMPK protein levels (9 fold increase, p<0.001), equivalent AMPK mRNA levels by qRT-PCR, and higher AMP to ATP ratios by HPLC (3 fold increase, p>0.05, n.s.). The MRL hearts also had increased mitochondria by electron microscopy (by 65 ± 4.5%, p<0.05), reduced HW to BW ratio (48% decrease, p<0.001), reduced cellular and mitochondrial reactive oxygen species (by DHE and Mitosox stains respectively), and increased glycolysis (Glut-4 sarcolemmal translocation). Hypertrophic response to exercise was greater in the MRL mice (HW/BW increased by 40% vs. 2% in control, p<0.001). Mimicking injury, Compound C reduced active phosphorylated AMPK protein levels in control (by 26%, p<0.05), while increased it in MRL mice (by 30%, p<0.05). AICAR injection significantly increased pAMPK protein in both strains (p=0.008). Baseline MRL mice echocardiographic analysis (vs. control) indicated higher systolic function, but increased diastolic E/A and E/E’ ratios (p<0.05). In the MRL hearts, Compound C treatment reduced %EF and %FS, while AICAR reduced post-exercise hypertrophy (p=0.02).

Conclusions:
This study highlights the cardioprotective role AMPK plays in the MRL mice, through its modulation of mitochondrial function and reduction of oxidative stress. This can in turn be used to develop new therapeutic treatment for heart failure.
Abstract 194: Increasing Expression of Connexin43 in Bone Marrow Stem Cells Enhances Their Protective Effects on the Infarcted Heart
Authors: Duran Jason M , Sharp Thomas E , Taghavi Sharven , Berretta Remus M , Gaughan John , Rabinowitz Joseph E , Kubo Hajime , Houser Steven R
Published: July 2021

Introduction:
Coupling of stem cells (SCs) injected into the infarcted heart to endogenous cells that survived injury may enhance SC survival and engraftment and improve their protective effects. Connexin43 (Cx43) gap junctions are some of the first proteins expressed by differentiating SCs transplanted into the heart after myocardial infarction (MI), suggesting that cell coupling is critical for SC survival and differentiation. Hypothesis: Overexpression of Cx43 in SCs will enhance their ability to engraft, differentiate, and improve cardiac function and survival after transplant into the infarcted heart.

Methods:
Adult mouse SCs isolated from compact bone were sorted for c-kit surface marker using magnetic beads. SCs were transduced with PLL3.7 lentivirus encoding the Cx43 gene (GJA1) at MOI=300 to produce overexpression, which was confirmed by Western analysis on SC lysates. MI was induced in 12-week-old male C57BL/6 mice by ligating the left anterior descending coronary artery, and 40,000 SCs in 20 uL saline were administered immediately after MI via intramyocardial injection. A total of 69 animals were used: 18 received Cx43 SCs, 30 received non-transduced SCs (NTSC), 11 received only saline, and 10 underwent sham surgery. Echocardiography was performed 1 and 2 weeks post-MI.

Results:
Animals receiving Cx43 SCs had improved survival (90.0%) versus NTSC (76.7%) and saline controls (70.0%). The Cx43 SC group displayed improved ejection fraction versus NTSC and saline controls at 1 week (48.7% v. 43.3%, p=0.04; 30.1%, p<0.0001) and 2 weeks post-MI (45.7% v. 40.4%, p=0.08; 26.8%, p=0.0004). Similar improvements in fractional shortening, stroke volume, and cardiac output were observed. Administration of Cx43 SC and NTSC attenuated remodeling with no difference between the two groups. Both decreased end-diastolic and systolic volumes, increased thickness of the infarct-related anterior wall, and decreased heart weight (HW), HW/body weight and HW/tibia length ratios at 1 and 2 weeks post-MI.

Conclusion:
All SCs improved survival and cardiac function, and attenuated remodeling. Transplantation of Cx43 SCs improved survival and cardiac function even more than NTSCs, suggesting that Cx43 may play a crucial role in engraftment and differentiation.
Abstract 216: Angiogenic Factor AGGF1 Promotes Therapeutic Angiogenesis in a Mouse Limb Ischemia Model for Peripheral Arterial Disease
Authors: Wang Qing , Lu Qiulun , Tu Xin , Wang Qing
Published: July 2021

Introduction:

Peripheral arterial disease (PAD) is caused by atherosclerosis. PAD is a common disease that affects 12% of the adult population and causes significant morbidity and mortality. The existing treatments for PAD, including pharmacological, interventional and surgical procedures, are not sufficient and many patients are opt for limb amputation. Therapeutic angiogenesis using angiogenic factors has been considered to be a potential treatment option for PAD patients.

Hypothesis:

In this study, we assessed the potential of a new angiogenic factor AGGF1 for therapeutic angiogenesis in a critical limb ischemia model in mice for PAD.

Methods and Results:

We generated a unilateral hindlimb ischemia model in mice by ligation of the right common iliac artery and femoral artery. Ischemic mice with intrasmuscular administration of DNA for an expression plasmid for human
AGGF1
(
AGGF1
group) resulted in increased expression of both
AGGF1
mRNA and protein after the administration compared with control mice of hindlimb ischemia with injection of the empty vector (control group). The blood flow in ischemic hindlimbs was significantly increased in the
AGGF1
group compared to control mice at time points of 7, 14, and 28 days after DNA administration. Four weeks after the injection of DNA, the blood flow was approximately 2.19 fold higher in
AGGF1
group than that in control. With the increased blood flow in the
AGGF1
group, ambulatory impairement were inhibited compared with the control group. Increased
AGGF1
expression decreased necrosis in muscle tissues injected with
AGGF1
DNA compared with the control tissue injected with the empty vector. CD31-positive capillary vessel density was significantly higher in
AGGF1
-injected limbs than in empty vector-injected limbs.

Conclusions:
These data establish that plasmid DNA-based gene transfer of
AGGF1
into mouse skeletal muscles is efficient in increase the expression levels of AGGF1 mRNA and protein. Increased
AGGF1
expression increases blood flow by increasing density of CD31-positive vessels and by decreasing necrosis in muscle tissues. These findings suggest that
AGGF1
may be used as a candidate therapeutic agent for therapeutic angiogenesis to treat PAD.
Abstract 217: Hexokinase II Binding to Mitochondria Suppresses Irreversible Ischemia Reperfusion Injury in the Beating Heart by Respiratory Inhibition and Reduced ROS Levels
Authors: Nederlof Rianne , Xie Chaoqin , Gúrel Ebru , Koeman Anneke , Hollmann Markus W , Southworth Rick , Akar Fadi G , Zuurbier Coert J
Published: July 2021

Introduction:
The glycolytic enzyme hexokinase II (HKII) can be mitochondrial-bound (mtHK) or free in the cytosol. Increased mtHKII protects the heart against ischemia-reperfusion (I/R) injury. The mechanism of protection is not yet elucidated.

Hypothesis:
We hypothesized that mtHKII protects the heart against I/R injury by respiratory inhibition and reducing reactive oxygen species (ROS).

Methods:
Langendorff perfused rat hearts were exposed for 20min to 1 μ M TAT-only, 1 μ M TAT-HK or 200 nM TAT-HK, followed by 15 min ischemia and 30 min reperfusion. TAT-HK contains the binding motif of HKII and dislodges HKII from mitochondria. ROS was measured using DHE fluorescence (n=6/group). Lactate was measured after peptide treatment, necrosis (LDH release) was determined during reperfusion and oxygen consumption (MVO
2
) and heart function (rate pressure product (RPP)) were monitored (n=12-15/group).

Results:
Disruption of mtHKII binding resulted in increased MVO
2
/RPP values during both baseline and reperfusion in the 1 μ M TAT-HK group. Effluent lactate increased from (mean±SEM) 0.024±0.0041 μ mol/ml effluent in the TAT-only group to 0.042±0.0072 and 0.091±0.016 μ mol/ml effluent in the 200 nM and 1 μ M TAT-HK treated hearts, respectively. TAT-HK treatment had no effect on ROS production during baseline conditions. However, during both ischemia (normalized values (mean±SEM) 1.14±0.05 and 1.17±0.08 versus 1.09±0.06) and reperfusion (1.58±0.24 and 1.84±0.15 versus 1.30±0.17) TAT-HK significantly and dose dependently increased ROS when compared to TAT-only treatment. This was accompanied by an increased LDH release during reperfusion of (mean±SEM) 27.1±4.1 and 42.9±4.2 μ mol/30min/g heart for the 200 nM and 1 μ M TAT-HK treated hearts, respectively. There was no cell necrosis in the TAT-only group.

Conclusion:
>Our results show for the first time that disruption of the mtHK binding affects cardiac MVO
2
and causes an increase in ROS production during ischemia and reperfusion in the intact heart. These data indicate that mtHKII is a master switch turning reversible ischemia into irreversible ischemia. Our data also suggest that mtHKII is a determinant of glycolysis/glucose oxidation coupling.
Supported by the Dutch Heart Foundation (NHS 2010B011)