bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P523: Time and Concentration Dependent Vascular Changes of Cancer Therapy Everolimus in Mesenteric Resistance Arteries
Authors: Martinez Quinones Patricia , Warner Alexander , Klee Nicole , Wenceslau Camilla F , McCarthy Cameron G , Webb Clinton
Published: March 2022

The leading cause of death in cancer survivors is cardiovascular disease, in part due to cancer therapy-induced cardiotoxicity. The short- and long-term cardiovascular side effects of a newer class of targeted chemotherapy, mTOR inhibitors, have not been well established as they have been with anthracyclines, anti-VEGF therapy and tyrosine kinase inhibitors. mTOR inhibitor Everolimus, used as an agent in transplant immunosuppression, is now indicated for the treatment of metastatic breast, colon, renal cell and pancreatic cancer. Retrospective clinical data indicate the possibility of heart failure and hypertension as cardiotoxic effects of Everolimus, although data are inconclusive and no vascular function studies are available. We studied the effects of Everolimus on the contractility and relaxation of mesenteric resistance arteries (MRA) of male Wistar rats (12-15 weeks old) at different drug incubation time frames (1, 12, and 24 hours) on a tension wire myograph. We hypothesized that the longer exposure to Everolimus (i.e. longer incubation time) leads to enhanced contractility to phenylephrine (PE) and impaired relaxation to acetylcholine (ACh). Two concentrations of Everolimus were evaluated, 0.1 μM and 0.1 nM. MRA exposed to Everolimus 0.1 μM showed a decreased contractile response to PE at 1 hour incubation (LogEC
50
, Ctrl: -5.627±0.07 vs. Drug: -5.390±0.10, *p=0.03) and at 12 hours incubation (LogEC
50
, Ctrl: -5.295±0.16 vs. Drug: -5.645±0.08, *p=0.04), while no difference was observed at 24 hours (p>0.05). MRA exposed to Everolimus 0.1 nM for 24 hours showed enhanced contractility to PE (LogEC
50
, Ctrl: -5.295±0.16 vs. Drug=-5.740±0.07, *p=0.005) while no difference at 1 hour or 12 hour incubation times (p>0.05). No differences were observed in the endothelium-dependent relaxation response to ACh for either drug concentration or any of the incubation times. Our data suggest that the effects of Everolimus on resistance arteries from normotensive rats are a time and concentration-dependent: short-term incubation (i.e., < 12hours) increases sensitivity of MRA to adrenergic stimuli. However, long-term treatment with this drug may lead to vascular dysfunction, which could be associated with cardiotoxicity observed in cancer survivals.
Abstract P528: Modifiable Factors Affecting BP in Identical Twins
Authors: Kidambi Srividya , Pan Xiaoqing , Li Yingchuan , Liu Pengyuan , Liettl Michelle , Kotchen Theodore , Cowley Allen , Kamassah Mawusi , Liu Yong , Burt S A , Klump Kelly , Rayamajhi Supratik , Rockwell Cheryl , Gernaat Eric , Donohue Mary , Reddy Rakesh , Watson Ralph , Mattson David , Liang Mingyu
Published: March 2022

Background:
Genetic factors determining blood pressure (BP) are largely unmodifiable. Understanding the effects of physiologic, environmental, and epigenetic (modifiable) factors on BP levels can improve strategies for hypertension (HTN) control. Monozygotic (MZ) twins with identical genetic background are a good model to study the effect of these factors on BP.

Methods:
Seventy pairs of MZ twins were from Milwaukee, WI and Michigan State University Twin Registry, East Lansing, MI (62% women; age of 44 ± 10 years). BPs (measured in triplicate and averaged), anthropometrics, physical activity (Rapid Assessment of Physical Activity), and sodium intake measurements (Block Sodium Screener) were obtained. DNA was obtained from T-lymphocytes for methylation (epigenetic) sequencing analysis. Differences in systolic (SBP) and diastolic (DBP) BPs between co-twins were used as continuous variables for these analyses.

Results:
Mean SBP was 124 ± 15 mm Hg (mean ± SD) and DBP was 78 ± 11 mm Hg. Average differences in SBP and DBP among co-twins were 8 ± 9 and 7 ± 6 mm Hg respectively. Twin pairs were considered concordant or discordant based on BP difference of 10 mm Hg between co-twins or one twin being hypertensive. Discordant twins as a group had higher BMI and waist circumference (WC) compared to concordant twins (p<0.05). Among discordant twins, the co-twin with higher BPs tended to have higher waist circumference (WC) (105 ± 15 vs. 98 ± 18 cm) and dietary salt intake (3900 ± 1437 vs. 3261 ± 1058 mg/d) even though they did not reach statistical significance. There were no differences in physical activity or education levels. Genome-wide methylation analyses revealed that 3 loci were associated with SBP difference and 2 were associated with DBP difference at an unadjusted significance level of p < 10
-4
. Two sites reached an adjusted significance level of 0.08 with SBP difference.

Conclusions:
In this study, we identified several differentially methylated sites of interest in relation to BP among MZ twins. In addition, higher central adiposity and sodium intake may contribute to BP levels among MZ twins. Understanding the relationship of modifiable risk factors such as WC and sodium intake with methylation changes may shed light into novel pathogenic pathways for HTN.
Abstract P548: Spontaneous Report of Ilicit Drug Use among Medical Students. A Social Problem in Healthy Professionals
Authors: Costa Lilian S , D’Angelo Fabiana S , Kobbaz Tiago M , Ribeiro Graziella I , Morgado Vanessa M , Bachour Karen K , Alvarenga Laura B , Concy Lucas V , Faria Daniela F , Pavan Ingrid S , Tavares Carolina P , Queiroz Fernanda T , Rocha Jéssica B , Banharo Mariana M , Magalhães Gabriela M , Ansel Jocasta C , Moreira Camilla S
Published: March 2022

Introduction:
According to the World Health Organization, licit and illicit drugs when used have the ability to change the processes of consciousness, mood and thinking through your acting mechanism in the brain, which regulate mood, functions of thought and motivation. Research has shown high levels of drug use among students of medical school, which constitute alternatives to psychological problems caused by the stressful routine of a full-time course, which requires changes in lifestyle and in the acquisition of greater responsibility.

Objective:
To describe the prevalence of licit and illicit drug use in students of medical school to a private college in Rio de Janeiro, Brazil.

Method:
This subset study is part of an observational study with cross-sectional delineation performed on a sample from the first to the sixth year of medical students. The first stage of the study was designed to obtain data to prepare a socio demographic characteristics and evaluation of quality of life in these students. The data was collected from an anonymous questionnaire.

Results:
We evaluate 490 students: 84 (17.1%) of the first year, 119 (24.3%) of the second, 100 (20.4%) of the third, 66 (13.5%) of the fourth, 85 (17.3%) of the fifth and 36 (7.3%) of the sixth year. The average age was 21.7 years (16-42), 314 (64.1%) of female gender. In the total group, the percentage of alcohol consumption was 81.6%, there is no statistical significantly difference between the genders (81.3% male and 81.8% female) or between the basic cycles of first and second year course x internists from fifth and sixth years (79.3% x 80.8%, respectively). The consumption of illegal drugs found a percentage on the total group of 13.9%, with significant differences in both comparison groups (p < 0.001), 22.2% in male x 9.2% female gender and 12.8% basic cycle x 5.8% boarding school cycle.

Conclusion:
A sizable percentage of students reporting use of licit and illicit drugs, underscoring the increasing prevalence of alcohol use among women in the general population, especially the worrying percentage of use of illicit drugs among students. This underscores the need for strategies of University managers in implementing policies to reduce and control the consumption of drugs.
Abstract P549: Profile of Physical Activity in Medical Students - Constructing a Quality of Life Model
Authors: Costa Lilian S , Magalhães Gabriela C , Tavares Carolina P , Queiroz Fernanda T , Rocha Jéssica B , Banharo Mariana M , Moreira Camilla S , Marins Marina C , Morgado Vanessa M
Published: March 2022

Introduction:
According to the World Health Organization , performing physical activity (PA) results in less chance of coronary heart disease development, hypertension, diabetes, obesity, heart attack, colon and breast cancer and depression, in addition to promote quality of life (QOL) improvement. Some studies show that students’ perceptions about QOL during your college experience are essentially related to the levels of academic support. Thus, it is considered that the level of subject PA is crucial in promoting health and QOL in medical students and have important role to disseminate information in society, that could modify the community culture and habits where they are inserted.

Objective:
To identify the profile of PA on medical students of a private College in Rio de Janeiro, Brazil. Method: This subset study is part of an observational study with cross-sectional delineation, with data collected through the application of an anonymous questionnaire. The collection of these data was the basis of an instrument for assessing the QOL of the medical students of this College. For descriptive purposes, we analyze gender, age, BMI (body mass index), year, place and frequency of PA, type of exercise performed and time spent per week with PA.

Results:
Of the 490 students with an average age 21.7 years (16-42 years), 64.1% female, 75.1% practiced PA prior to entered the medical college, but 38.4% decreased the frequency after initiated the college, 22.2% perform regular PA three times a week, 44.5% practice anaerobic and aerobic PA and 28.6% are sedentary. Among the male participants, 83% x 70.7% female, practiced PA before starting the course of Medicine (p 0.003), both with reduced frequency after admission in college (38.1% x 38.5%, respectively, p NS). The combined aerobic and anaerobic activities predominates in both genders. Comparing the data obtained between the first and second years (Group A), with the fifth and sixth years students (Group B), statistically significant relative (1) sedentary (A 34.5% x 23.3% B) and reduction PA after admission in college (A 33% x 56.7% B).

Conclusion:
Studies confirm that the teaching model with full-time activities imposed in medicine schools, reduces the time required to adapt personal habits and maintain a good QOL.
Abstract P002: Pim-1 Engineering of Human CPCs Increases Their Ability to Repair the Heart After Myocardial Infarction
Authors: Mohsin Sadia , Khan Mohsin , Toko Haruhiro , Bailey Brandi , Siddiqi Sailay , Fischer Kimberlee , Gude Natalie , Truffa Silvia , Sussman Mark A
Published: March 2022

Rationale:
The efficiency of adoptively transferred human cardiac stem cells to repair damaged myocardium is extremely limited. There is an urgent need to improve the potency of human stem cells to engraft, proliferate and differentiate into cardiac phenotypes before launching them as viable therapeutic tool in clinics.

Objective:
Demonstrate that Pim-1 engineering of human cardiac stem cells (hCPCs) can improve their long term persistence in the damaged myocardium associated with significant augmentation of cardiac function.

Methods and Results:
hCPCs positive for the putative stem cell marker c-kit were isolated from heart biopsy samples from patients undergoing Left Ventricular Assist Device (LVAD) implantation. hCPCs were engineered to express Pim-1-GFP, a fused GFP version of the kinase by using a lentivirus expression system. Pim-1 engineering of hCPCs significantly increased their viability, proliferation and metabolic activity. Conversely, treatment with a Pim-1 specific inhibitor quercetagetin completely abrogated the observed proliferative response. Pim-1 engineered hCPCs were transplanted in immunocompromised mice with myocardial infarction to evaluate functional competency of the cells. Animals receiving Pim-1 CPCs showed increased hemodynamic performance as evidenced by dp/dt, LVEDP and LVDP measurements 10 weeks after transplantation. Cardiac function demonstrated sustained improvement even after 20 weeks of transplantation in Pim-1 CPC transplanted animals compared to control cell transplanted group. Concurrent with enhanced cardiac function, animals having Pim-1 CPCs demonstrated increased cellular engraftment, persistence and proliferation of human CPCs. In particular, a greater number of c-kit+ cells, improved vasculature and reduced infarct size were observed.

Conclusion:
Genetic engineering of human CPCs with Pim-1 enhances their ability to repair damaged myocardium. Ex vivo gene delivery to enhance survival, proliferation and regeneration in damaged myocardium has emerged as a viable option addressing current limitations associated with stem cell therapy.
Abstract P008: Fibronectin Enhances Survival and Proliferation via Pim-1 and β1 Integrin in Cardiac Progenitor Cells
Authors: Konstandin Mathias , Völkers Mirko , Gastelum Grady , Gude Natalie , Sussman Mark A
Published: March 2022

Background:
Cardiac Progenitor Cells (CPC) are pivotally involved in cardiac repair. Extracellular matrix (ECM) components also contribute decisively in cardiac remodeling after myocardial infarction (MI). However, impact of ECM on CPC function and the involved signaling pathways have not been clearly elucidated. This study examines the relationship of ECM to cardioprotective signaling mediated by Pim-1, a serine/threonine kinase downstream of Akt, in CPC.

Methods:
Isolated mouse CPCs were plated on fibronectin (FN) or albumin coated dishes. FACS analysis and fluorescence based cell quantification have been used to determine viability and proliferation under stress and growth conditions. Signaling pathways were analyzed by immunoblotting, qRT-PCR and siRNA. Immunhistochemistry (IHC) has been used for in vivo studies after MI in mice.

Results:
FN inhibits starvation and staurosporine induced cell death in CPCs and promotes proliferation in conjunction with induction of Pim-1 expression. Protective and pro-proliferative effects of FN are abrogated by inhibition of Pim-1. Fibronectin signaling operates through the β1 integrin receptor that is crucial for FN-mediated signaling. Ongoing studies correlate these observations with in vivo responses to cardiomyopathic injury such as infarction challenge.

Conclusion:
FN provides pro-survival and pro-proliferative effects in CPCs in a Pim-1 kinase and β1 integrin dependent manner. Involved molecules colocalize in vivo in an infarction injury model. The causal contribution of FN in regeneration and wound healing after MI is to be confirmed by studies of a conditional FN knock-out mouse that is under construction.
Abstract P013: Canopy 2 Encodes for a Novel Secreted Proangiogenic Factor that Revascularizes and Regenerates Murine Hearts Post Myocardial Infarction
Authors: Guo Jian , Sun Zhuo , Mihic Anton , Au Sharon , Li Shu Hong , Hatta Kota , Zhang Yue Mei , Dhingra Sanjiv , Wu Jun , Weisel Richard D , Li Ren-Ke
Published: March 2022

Through genomic screening of 256 novel genes by RT-PCR in human smooth muscle cells, we discovered that Canopy 2 (CNPY2), previously named as MSAP, TMEM4 or ZSIG9, actually encodes for a secreted pro-angiogenic factor, which was differentially regulated by
in vitro
hypoxia. Tissue distribution profile reveals that CNPY2 has the highest expression levels in the heart, liver and lung. Surprisingly, CNPY2 was dramatically downregulated (∼2–5 fold) in the murine heart at day 1–7 post myocardial infarction. Recombinant CNPY2 proteins were purified both from
E.coli
to generate rabbit (blocking) antibodies, and from mammalian cells with proper folding and posttranslational modification to study signaling cascades. We demonstrate that recombinant CNPY2 protein binds to the extracellular domain of VEGFR2 and phosphorylates the intracellular Tyr
1175
and Tyr
1214
of VEGFR2, activates FAK and ERK which resulting in pERK nuclear translocation in human endothelial cells. On the other hand, CNPY2 binds to adhesion molecule desmoglein 1, activates Cdc42, PAK1 and FAK in human smooth muscle cells. CNPY2 reorganizes the dynamic vimentin structure in both cell types, resulting in an accelerated cell migration.
In vivo
delivery of recombination CNPY2 protein enhanced the blood capillary formation in a retinal ischemia/reperfusion mouse model, and dramatically revascularized blood vessel network, reduced infarct size and improved cardiac function in a rat myocardial infarction model. In line with the paracrine mechanism in (stem) cell-based therapy, our data suggests that CNPY2 might be employed as a promising therapeutic protein for ischemic tissue revascularization and regeneration.
Abstract P017: Plasmonic Photothermal Therapy of Atherosclerosis with the Use of Silica-Gold Nanoparticles and Mesenchymal Stem Cells Versus Ferromagnetic Approach
Authors: Kharlamov Alexander , Perrish Alexander , Gabinsky Jan , Ivanova Eugene
Published: March 2022

Background. Some modern angioplasty techniques generally just manipulate the form of the plaque and have some clinical and technical restrictions, relatively high complication rate and restenosis risk.

Methods. A total of 101 Yucatan miniature swines were assigned to the three groups (34 pigs into 60/15–70/40 nm silica-gold nanoparticles (NPs) nanogroup, 34 swines - into ferro-magnetic group with 100 nm NPs with delivery in hand of magnetic fields, and 33 - in sirolimus stenting control). Animals in nanogroup were split up to 4 subsets by delivery approach: 1) infused circulating progenitor cells; 2) with infused ultrasound-mediated albumin-coated gas-filled microbubbles; 3) CD73+CD105+ progenitor cells in the composition of bioengineered on-artery patch; 4) CD73+CD105+ progenitor cells transplanted by manual subadventitial injection.

Results. A change of the PV (mm3) immediately after the laser irradiation/ in 6 months in groups were −28.9/ −56.8%, −30.8/ −59.1% and 0/ +4.3% (p<0.01) respectively, and in the subsets in 6 mns reached −54.5/ −45.9/ −74.7/ −61.8% (p<0.05) respectively. Some cases of atherothrombosis (3 cases in S2 subset) and distal embolism (8/34, 23.5% in nanogroup, 2/33, 6% in control) were revealed while first 4 weeks. Restenosis confirmed in 3 (9%) swines of stenting group only. An impact over the non-organic part of the plaque was predominated in nanogroup (−43.4% vs −12.1% and +3.7% respectively, p<0.005). Coronary flow-mediated vasodilation was observed after hyperemia and injection of nitroglycerine (+10.2 and +16.6%, +8.2 and +9.6%, +8.1 and +9.8% in groups respectively, p<0.05). The highest level of the infiltration with NPs was achieved in the liver that equal to 612 units per cm3 without signs of fibrosis or allergic responses. There were 344, 201 and 99 units per cm3 in the heart, brain, and lungs respectively without any morphological dynamics.

Conclusion. Nanoburning is the novel promising technique to demolish and melt the plaque especially in combination with stem cell technologies challenging functional restoration of the vessel. Plasmonics is the high-effective and safe alternative to stenting for angioplasty.
Abstract P022: Functional and Transcriptomic Recovery of Infarcted Mouse Myocardium Treated with Bone Marrow Mononuclear Cells
Authors: Carvalho Adriana B , Lachtermacher Stephan , Esporcatte Bruno , Rocha Nazareth , Costa Patricia , Iacobas Dumitru , Iacobas Sanda , Spray David C , Goldenberg Regina , Campos de Carvalho Antonio Carlos
Published: March 2022

Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue ten days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 new genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a new metric calculated using a formula that incorporates both recovery and side effect of treatment, was 74.5%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice.
Abstract P035: The Nucleolar Protein Nucleophosmin Regulates Mitosis Progression in Cardiac Progenitor Cells
Authors: Avitabile Daniele , Sundararaman Balaji , Truffa Silvia , Sussman Mark A
Published: March 2022

Besides its notorious function during ribosome biogenesis, emerging evidence suggests a central role for the nucleolus in controlling many other important cellular processes such as stress response, proliferation and mitosis. Here we show that the nucleolar protein Nucleophosmin (NPM) is necessary for the correct progression of mitosis in primary cultures of cardiac progenitor cells (CPCs).

Methods:
CPCs were cultured on Permanox chamber slides at high confluence and fixed with 4% PFA. CPCs were blocked in cytokinesis with 2μ M cytochalasin B (CytB) for 18 hours. Silencing of NPM (ShNPM) or scramble (ShSc) was performed by specific shRNA expressing lenti viral vectors. Confocal microscopy, co-immunoprecipitation and proximity ligation assay (PLA), FACS analysis and Cyquant assay were employed to study protein localization and interaction, cell death and proliferation respectively.

Results:
ShNPM infected CPCs showed reduced proliferation with no apparent cell death compared to scramble. Cells in prometa and metaphase significantly increased after NPM knock down. ShNPM-CPC showed 50% decrease in the number of cells that complete mitosis as assessed by counting binucleated cells after CytB treatment. NPM localized in the nucleolus during interphase as assessed by co-staining with nucleostemin (NS), a highly expressed nucleolar protein. NS and NPM decorate the chromosome periphery from prometaphase to anaphase and return to the newly forming nucleolus at the end of telophase. The phosphorylated form of NPM at threonine 199 (pNPM) was found to co-localize with spindle components such us NUMA and α-tubulin specifically at methaphase. NPM was also found to co-precipitate and directly interact with α-tubulin as shown by in-situ protein interaction assay (PLA) in the cytoplasm during interphase and at the centrosome and spindle during mitosis.

Conclusion:
This is the first study to detail the role of nucleolar proteins during CPCs mitosis. Our findings demonstrate a central role for NPM during metaphase transition, possibly thorough interaction with components of the spindle and in particular with α-tubulin.
Abstract P036: Regional Characterization of Myocardial Renewal in Humans
Authors: Bergmann Olaf , Zdunek Sofia , Salehpour Mehran , Bernard Samuel , Sjöström Staffan , Håkansson Karl , Alkass Kanar , Druid Henrik , Possnert Göran , Frisén Jonas
Published: March 2022

We and other groups have reported cardiomyocyte renewal in adult human hearts. However, there is no consensus about magnitude and regional heterogeneity in myocardial turnover. Important requirements to quantify cellular turnover are the accurate identification of cardiomyocytes and ploidy level. We present a regional analysis of turnover in the adult myocardium, using the integration of atmospheric radiocarbon (14C) into genomic myocardial DNA. Cardiomyocyte nuclei were labeled with pericentriolar protein 1 (PCM-1) and isolated by flow cytometry. In order to improve estimates on cardiomyocyte turnover, nuclei were isolated according to their DNA content. A substantial fraction of cardiomyocyte nuclei in the right and left ventricle were polyploid: 28.9%±10.6% diploid, 58.9%±8.5% tetraploid and 12.1%±7.4% octaploid in the left ventricle and 47.9%±10.6% diploid, 46.6%±8.3% tetraploid and 5.6%±3.2% octaploid in the right ventricle. Cardiomyocyte turnover was analyzed according to their ploidy distribution in the left and right ventricle. Data from this ongoing study indicates a limited regenerative potential in both ventricles. A comprehensive mathematical analysis of regional turnover distribution will be presented. Non-cardiomyocytes were in average 18.0±2.3 years younger than the respective subjects, indicating a substantial turnover in the whole myocardium. The human heart has the capability to regenerate cardiomyocytes. However, the magnitude of this process does not allow for the renewal of the whole myocardium under homeostatic condition in a lifetime. Understanding the underlying mechanisms of cardiac renewal will open up new avenues to treat cardiac injuries.
Abstract P038: Cardiomyocyte Proliferating Chemicals: Activation of Proliferation of ESC/iPSC-Derived Cardiomyocytes
Authors: Uosaki Hideki , Yamashita Jun K
Published: March 2022

Cardiomyocytes derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are promising cell sources for cardiac regeneration. Previously, we established a novel systematic cardiovascular cell induction system with mouse ESCs and iPSCs. ESC/iPSC-derived cardiomyocytes rarely proliferate after differentiation, similar to neonatal and adult cardiomyocytes. Cardiomyocyte proliferation is highly restricted and the regulating machineries of proliferation and growth arrest of cardiomyocytes are long-standing mysteries of cell biology. In order to establish cardiac regenerative medicine, it is critical to dissect and manipulate the machineries involved. Here we devised a novel approach using small molecules in an attempt to unravel the mystery and to manipulate ESC/iPSC-derived cardiomyocyte proliferation.

We screened a chemical library containing well-established kinase inhibitors with high content screen to enhance mouse ESC-derived cardiomyocyte proliferation, and identified two novel chemical groups, extracellular signal-regulated kinase activators and Ca2+/calmodulin-dependent protein kinase II inhibitors, and two previously reported chemical groups, glycogen synthase kinase-3 inhibitors and a p38 mitogen-activated protein kinase inhibitior. Each chemical increased actual cardiomyocyte cell numbers two- to three-fold compared to control. An optimal combination of these chemicals strongly enhanced proliferation of ESC-derived cardiomyocytes and ESCM number was reached up to 14-fold. Expanded cells retained various functional and structural features of cardiomyocytes. These chemicals are robustly effective on cardiomyocytes from various sources including human iPSCs.

Efficient combination of stem cell and chemical biology demonstrated a novel molecular mechanism of cardiomyocyte proliferation and offered a critical technological basis for cardiac regeneration.
Abstract P039: Encapsulation of Human Mesenchymal Stem Cells in Alginate Microspheres to Enhance Paracrine-Mediated Vascular Recovery
Authors: Landázuri Natalia , Levit Rebecca D , Safley Susan A , Weber Collin J , Sambanis Athanassios , Taylor W Robert
Published: March 2022

Bone marrow cells migrate to ischemic areas and facilitate revascularization mostly through paracrine mechanisms. As a result, therapeutic strategies for vascular repair have focused on increasing the concentration of these cells at the site of ischemia by direct injection. These strategies have led to encouraging results in clinical trials, yet the number of cells that remain at the site of delivery is very low.

The goal of this study is to design an effective cell delivery system to enhance revascularization by maintaining large numbers of cells at the ischemic site, thus increasing the local delivery of angiogenic factors. To achieve this goal, we encapsulated bone marrow derived human mesenchymal stem cells (hMSCs) in non-degradable, barium-gelled alginate spheres prior to delivery to the ischemic site. Our microspheres are biocompatible, do not generate a fibrotic response, and are stable for extended periods of time. They prevent incorporation of cells into the neighboring tissue and protect implanted cells from immune mediated clearance or wash out by the host capillary and lymphatic systems. The microspheres allow diffusion of molecules as large as 80 kDa (which includes most angiogenic factors) but not of larger molecules such as IgG. To test this cell delivery system in vivo, we used a model of murine hind limb ischemia in athymic nude mice. We delivered encapsulated hMSCs, or, as a control, empty capsules, to the ischemic muscle. At various time points, we retrieved the capsules by gentle scooping, then assessed the viability of the encapsulated cells and their protein secretion profile. Approximately 70% of the cells remained viable within the first week and 10-20% within the first month after implantation. In addition, encapsulated cells secreted angiogenic factors (VEGF, bFGF, HGF, angiogenin) in vitro and in vivo. We also assessed recovery of perfusion in the hind limb and found that delivery of encapsulated cells enhanced vascular recovery as compared to empty capsules.

The results obtained from this study show that microcapsules are an effective delivery vehicle of therapeutic cells for vascular repair. Encapsulation represents a powerful and highly translatable tool to enhance cell-based therapies in cardiovascular disease.
Abstract P042: Loss of Mcl-1 in the Heart Leads to Rapid Mitochondrial Dysfunction and Development of Heart Failure
Authors: Thomas Robert L , Lee Youngil , Huang Chengqun , Rikka Shivaji , Fisher Kimberlee , Andreyev Aleksander , Murphy Anne N , Sussman Mark A , Gustafsson Åsa B
Published: March 2022

Mcl-1 is an anti-apoptotic Bcl-2 family protein that is expressed at high levels in the heart. However, the functional importance of Mcl-1 in the heart has not been investigated. To study the function of Mcl-1 in cardiac myocytes, we generated inducible, myocyte-specific Mcl-1 knock-out mice using a tamoxifen-driven loxP/α-MHC-Cre-recombinase system. We found that deletion of Mcl-1 by tamoxifen injections resulted in rapid development of cardiac hypertrophy, pulmonary edema and cardiac dysfunction in adult mice. Although Mcl-1 is known to inhibit apoptosis in cells, Mcl-1 deficient hearts were TUNEL negative and showed no activation of caspase-3. The anti-apoptotic proteins Bcl-2 and Bcl-X
L
have also been reported to inhibit autophagy, but we found no increase in autophagy upon loss of Mcl-1. Western blot analysis of other Bcl-2 family members revealed that Bcl-2 was significantly increased in Mcl-1 deficient hearts, suggesting a compensatory mechanism. Interestingly, ultrastructural analysis revealed the presence of swollen mitochondria, ruptured cells, and intracellular contents in the extracellular space. These signs are characteristics of necrotic cell death. Mcl-1 deficient hearts also exhibited reduced mitochondrial respiration and significantly lower cellular ATP levels compared to wild type, suggesting that the loss of Mcl-1 impairs mitochondrial function. Moreover, mitochondria isolated from Mcl-1-/- hearts showed pronounced swelling at baseline that could be alleviated by polyethylene glycol-induced shrinkage. Calcium release experiments also showed higher baseline Ca
2+
levels in mitochondria from Mcl-1-/- hearts compared to wild type, suggesting that Mcl-1 deletion increases permeability to Ca
2+
. Our data suggest that loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial impairment and necrotic cell death. Thus, in addition to its anti-apoptotic role, Mcl-1 has an essential but unidentified role in maintaining mitochondrial function in cardiac myocytes.
Abstract P044: Activation of Interferon Regulatory Factor-3 Prolongs Stress-Induced Cardiomyocyte Apoptosis and Decreases Survival Post Myocardial Infarction
Authors: de Couto Geoffrey , Dawood Fayez , Sun Mei , Chen Manyin , Heximer Scott , Stanford William , Medin Jeffrey , Liu Peter
Published: March 2022

Background. The innate immune response is critical in mediating host protection from foreign antigen or stress. It is evident that multiple innate immune pathways also tightly regulate the remodeling process post-myocardial infarction (MI), however it is not clear how this process is mediated. In order to further elucidate the underlying mechanism of this process, we screened both mouse and human post-MI microarray databases and identified a common, significantly upregulated transcription factor of interest, interferon regulatory factor-3 (IRF3). To assess its function in vivo, we tested the hypothesis that activation of IRF3 post-MI coordinates maladaptive repair by promoting cardiomyocyte apoptosis.

Methods/Results. We randomly allocated IRF-3 knockout (IRF-3-/-) mice and their wild-type (WT) counterparts to either left anterior descending (LAD) coronary artery ligation or sham operation. Four weeks post-MI, deficiency in IRF3 led to improved survival [88% (44/50) vs. 29% (14/49) in WT, p<0.0001], preserved cardiac function via echocardiography [Fractional Shortening: 37% vs. 22.9% in WT, LVEDD: 5.15mm vs. 5.85mm in WT, LVESD: 4.15mm vs. 5.14mm in WT; p<0.057#x005D;, and attenuated infarct size [27% vs. 44% in WT, p<0.05]. Assessing the stress response 1-week post-MI, we found that NFkB signaling (IKKa, Ikba, p65) as well as pan-ubiquitination of proteins, were both significantly downregulated in the border infarct zone of IRF3-/- mice. These findings were complemented with increased protein and mRNA expression of de-ubiquitinating enzyme A20. Interestingly, in IRF3-/- mice these results were linked with decreased apoptosis (Caspases, TUNEL staining), however with a more specific attenuation of the mitochondrial-specific apoptotic pathway (Bid, Cytochrome C, Caspase9).

Conclusions. These data establish a novel link between the innate immune transcription factor IRF3 and maladaptive regulation of cardiomyocyte apoptosis through mitochondrial apoptotic pathway activation. As an acute responder coordinating maladaptive remodeling, IRF3 may prove to be an efficacious therapeutic target to improve outcome following MI.
Abstract P047: A Novel Antiapoptotic Function of Thioredoxin-1 Is Mediated by Interaction with Caspase 3
Authors: Eckers Anna , Altschmied Joachim , Haendeler Judith
Published: March 2022

Thioredoxin-1 (Trx-1) is one of the most important redox regulators in endothelial cells (EC). It is an oxidoreductase with anti-oxidative and anti-apoptotic properties. In EC not only the active site cysteines 32 and 35 but also the cysteine 69 are required for its anti-apoptotic function. Trx-1 has several interaction partners, but this list is far from complete. The underlying molecular mechanisms in apoptosis protection by Trx-1 are not completely understood. The most important apoptosis executor protein in EC is Caspase 3. Therefore, the aim of this study was to investigate whether Trx-1 interferes with Caspase 3 function. We first analyzed if Trx-1 is associated with Caspase 3. Coimmunoprecipitation revealed an interaction between both proteins, which was only found under non-reducing conditions, suggesting involvement of a disulfide bridge. To get insights into the functional relevance of the association between Trx-1 and Caspase 3, we investigated whether Trx-1 can directly interact with the catalytic subunit p17 of Caspase 3. After coexpression of p17 and Trx-1, coimmunoprecipitation experiments under non-reducing conditions revealed that p17 associates with Trx-1. Analysis of Trx-1 mutants demonstrated that the interaction depends on cysteine 32 and/or 69 in Trx-1. To assess the functional relevance of the Trx-1/Caspase 3 interaction in EC, we induced apoptosis by overexpression of p17 and investigated the influence Trx-1 wildtype (wt) and mutants in which single or multiple cysteine residues were replaced by serines thereby preventing disulfide bridge formation. Apoptosis induction by p17 was significantly reduced by Trx-1 wt (p17+lacZ 16.99±1.35% vs. p17+Trxwt 8.60±1.31% apoptotic cells). Interestingly, overexpression of Trx-1 with mutations of cysteines 32 and 69 enhanced apoptosis induction by Caspase 3 p17 (23.42±2.84% apoptotic cells). On the contrary, no increase in apoptosis was observed when only cysteine 32 or 69 were mutated, suggesting that both residues can scavenge p17 via disulfide bridge formation thereby preventing its association with the second Caspase 3 subunit p12 and thus activation of the enzyme. These data suggest that Trx-1 has a novel anti-apoptotic function by binding the apoptosis executing Caspase 3.
Abstract P051: Induction of Cardiac Nitrosative Stress and Cell Death by Chronic Alcohol Consumption in an Angiotensin II-, PKC-, and NOX-Dependent Manner
Authors: Tan Yi , Zhou Zhanxiang Zhou , Cai Lu
Published: March 2022

Alcohol induced cardiac cell death is causative of cardiomyopathy, but the mechanisms responsible for alcoholic cardiac cell death remain largely unknown. We hypothesize that alcohol-induced Ang II plays a critical role in cardiac nitrosative stress, cell death and cardiomyopathy. To test this hypothesis, male C57BL/6 mice were pair-fed an alcohol or isocaloric maltose dextrin liquid diet for 2 months. Alcohol feeding caused significant cardiac cell death and nitrosative damage (shown by TUNEL positive cells, caspase-3 activation and protein nitration) and cardiac remodeling (shown by hypertrophy and fibrosis) along with increases in serum Ang II and cardiac AT1 expression. Mechanistic study in which cardiac H9c2 cells were treated with 100–400 mM alcohol for 24h revealed that alcohol exposure induced cell death, nitrosative damage and fibrosis, along with NADPH oxidase p47
phox
(NOX) activation in a dose-dependent manner. Pre-treatment of alcohol-treated cells with NOX inhibitor (apycinin), peroxynitrite scavenger (urate), NOS inhibitor (L-NAME) and superoxide dismutase mimic (MnTMPyP) significantly abolished alcohol induced cell death. Furthermore, exposure to alcohol significantly up-regulated the expression of angiotensin II (Ang II) and its type 1 receptor (AT1) in these cultured cardiac cells. Either PKCα/β1 inhibitor or AT1 blocker completely prevented alcoholic NOX activation, and AT1 blocker inhibited the expression of PKCβ1, implying that alcoholic NOX activation is dependent of PKCα/β1 activation via AT1. To validate the
in vitro
findings, alcohol-fed and pair-fed mice were treated with MnTMPyP (5 mg/Kg) daily for 2 months, which did not change blood pressure, alcohol-induced cardiac PKC and NOX activation, but significantly prevented alcohol-induced cardiac nitrosative damage and cell death, along with a prevention of cardiac fibrosis and dysfunction. These results suggest that alcohol-induced cardiac cell death is mediated by up-regulated Ang II that via AT1 induces PKCα/β1-dependent NOX activation. The cardiac cell death plays a critical role in the development of alcoholic cardiomyopathy.
Abstract P052: Parkin Deficiency Results in Disarray of the Mitochondrial Network and Increased Susceptibility to Myocardial Infarction
Authors: Kubli Dieter , Zhang Xiaoxue , Gustafsson Asa
Published: March 2022

Parkin is an E3 ubiquitin ligase that was recently discovered to be involved in mitochondrial turnover via autophagy. Although Parkin has been characterized in the setting of neurodegenerative diseases, it is expressed in many other tissues including the heart. In this study, we have investigated the functional role of Parkin in the myocardium using global Parkin knockout (PKO) mice. We found that PKO mice have no overt cardiac phenotypes under normal conditions. Echocardiography showed normal left ventricular fractional shortening (38.82 3.55% for PKO vs. 39.89 1.19% for WT) and ejection fraction (68.53 4.21% for PKO vs. 71.27 1.60% for WT) at 12 weeks of age. Cardiac mitochondria isolated from PKO mice had normal respiratory rates and displayed normal coupling. Additionally, Parkin deficient mitochondria were as susceptible to mitochondrial permeability transition pore (mPTP) opening as WT mitochondria. However, mitochondria from PKO mice had higher baseline absorbance values than WT mitochondria (0.66 0.02 for PKO vs. 0.59 0.02 for WT), suggesting a smaller morphology. Ultrastructural analysis by transmission electron microscopy (TEM) confirmed a disorganized mitochondrial network with the presence of clusters of small mitochondria at 12 weeks of age in PKO hearts. At 6 months, PKO mice displayed a decline in cardiac function, and TEM revealed the presence of degenerating mitochondria. Although PKO mice have normal cardiac function at 12-weeks, they were very susceptible to stress. The PKO mice developed more severe dilated cardiomyopathies with reduced survival rates compared to WT in response to myocardial infarction. These data demonstrate that Parkin is involved in the maintenance of the mitochondrial network in the myocardium, and that Parkin deficiency exacerbates stress-induced heart failure. Given the role of Parkin in mitochondrial turnover, Parkin deficiency may result in a gradual decline of mitochondrial quality, the effects of which may surface under conditions of acute stress.
Abstract P065: Hypercholesterolemia Induced Atherosclerosis via the Mechanism of Autophagy Pathway
Authors: Tan Chunjiang , Chen Wenlie , Wu Yanbin , Lin Ruhui , Lin Jiumao
Published: March 2022

Objectives:
To investigate the mechanism of hypercholesterolemia in the pathogenesis of atherosclerosis (AS).

Background:
As the main risk factor of AS, the mechanism of hypercholesterolemia (HC) in the pathogenesis of AS remains unclear.

Methods:
White New Zealand male rabbits were fed a high cholesterol diet (H) for 14 weeks to induce AS with normal diet fed rabbits as a control(N). In vitro, human aortic vascular endothelia (HAVED) and smooth muscle cells (HSMC) were incubated with different concentrations of HC in the comparison with the cells that the specific gene of Beclin1 or LC3-I/II were knocked down by siRNA transfection. Blood lipids, and vascular histologic structure, mRNA and protein expressions of Becline1, microtubule-associated protein 1 light chain 3(LC3-I/II) and autophagosomes were determined in the rabbit abdominal aorta or in the cultured cells.

Results:
Serum levels of total cholesterol,triglyceride and low density lipoprotein cholesterol were significantly increased in H than those in N (
P
<0.05). Histologic structure of abdominal aorta showed more aggravated in wall thickness, lumen narrowness and atheroma in H than those in N (
P
<0.05). The expressions of Becline1, LC3-I/II in mRNA or proteins were strongly increased in H than those in N (
P
<0.05), which was consistent with the expressions of autophagosomes in the two groups.At the cap or the shoulder of atheromas, the expressions of Becline1, LC3-I/II and autophagosomes were far more enhanced than those in non-atheromatous areas in the same sample of H group (
P
<0.05). In vitro, HAVED or HSMC that preincubated with HC revealed more expressions of Becline1, LC3-I/II and autophagosomes, which were relevant to the concentrations and the preincubation time of HC, compared to the cells that incubated with N serum. Further, the autophagosomes in the cultured cells were correlated with the number of survive cells. Compared to the intact cells, the expressions of autophagosomes in the cells that the gene of Beclin1 or LC3-I/II was knocked down were greatly suppressed, and the number of survive cells was relatively increased, even they were exposed to HC incubation.

Conclusions:
The current experiments suggested that HC mediated the pathogenesis of AS partially via the pathway of autophagy.
Abstract P072: Blunted β-Adrenergic Response in R92L Cardiac Troponin T Mutant Hearts Occurs via Decreased Accessibility to PKA-Mediated Phosphorylation Sites at Serine 22/23 Residues of Cardiac Troponin I
Authors: Jimenez Jesus , Tardiff Jil C
Published: March 2022

While diastolic dysfunction due to impaired relaxation is a classic finding in patients with Familial Hypertrophic Cardiomyopathy (FHC), the primary cellular mechanisms remain undefined. We have previously demonstrated impaired relaxation in our transgenic mouse models of FHC carrying the cTnT R92L mutation. We hypothesize that the impaired relaxation in RL is a result of an allosterically mediated, diminished structural accessibility to the PKA phosphorylation sites on cTnI. Protein levels of phosphorylated TnI at baseline and following stimulation with isoproterenol were significantly lower for RL compared to non-transgenic (NT) mice while protein levels of phosphorylated phospholamban (PLB), TnI, and PLB were the same between both groups. These results indicate that PKA signaling is intact and uncoupling occurs at the myofilament level. Next, we crossed RL mice with phosphomimetic mice that have had their cTnI residues S
22
S
23
changed to D
22
D
23
to generate the double transgenic RL/DD. Preliminary isovolumic studies demonstrated improved contractility and relaxation for RL/DD compared to RL alone but did not reach levels observed for NT following graded doses of dobutamine (Figures A and B). In Ca
2+
transient measurements of isolated adult cardiac myocytes, DD cells demonstrated enhanced peak rates of Ca
2+
rise and fall as well as accelerated times to 50% and 90% Ca
2+
declines compared to NT and RL. However, RL/DD mice did not show improvement in any of these parameters. Collectively, these results indicate that the diastolic dysfunction observed in R92L is caused by direct impairment of the myofilament axis in the beta adrenergic signaling cascade.
Abstract P074: Loss of Function of TRPC4 Protects Against Cardiac Dysfunction Progression After Myocardial Infarction
Authors: Zhang Hongyu , Makarewich Catherine , Gao Erhe , Kubo Hajime , Gao Hui , Wang Fang , Berretta Remus , Koch Walter , Molkentin Jeffery , Houser Steven
Published: March 2022

The source of Ca
2+
to hypertrophic signaling after myocardial infarction (MI) is not clearly defined. Transient Receptor Potential Canonical (TRPC) channels could be an important source of hypertrophic Ca
2+
after MI. The objective of this study was to determine if TRPC 4 is a major source of Ca
2+
influx mediating cardiac dysfunction after MI.

Methods:
Cardiac-specific transgenic mice that express a dominant-negative (dn) TRPC4 that reduces the activity of the TRPC1/4/5 subfamily of channels in the heart were used. MI was produced and in-vivo cardiac function was measured with ECHO. Myocytes were isolated and isoproterenol (ISO) effects on LTCC Current (
I
Ca-L
), fractional shortening (FS) and Ca
2+
transients were measured 6 weeks after MI.

Results:
Baseline ejection fraction (EF) and fractional shortening (FS) were greater in (dn) TRPC4
vs.
WT mice. Two weeks after MI, EF and FS were significantly decreased in all animals (WT: 37.1% and 18.2%; (dn) TRPC4: 41.7% and 20.5%), but there was no significant difference between WT and (dn) TRPC4 mice. Six weeks after MI, EF and FS were significant greater in (dn) TRPC4 compared with WT mice (WT: 37.4% and 18.2%; (dn) TRPC4: 52.2% and 27.4%). Heart weight and lung weight were significantly increased after 2 weeks MI, but there were significant lower heart and lung weight in (dn) TRPC4 vs. WT mice after 6 weeks MI.
I

Ca-L
[[Unsupported Character - Codename ­]]

after 6 weeks MI was smaller than that in sham myocytes, and there was no significant difference between (dn) TRPC4 and WT myocytes. Contractions and Ca
2+
transients were significantly greater in sham and post-MI (dn) TRPC4 vs. WT myocytes. ISO increased contractions and Ca
2+
transients to a similar extent in all myocytes.

Conclusions:
(dn) TRPC4 mice have greater baseline cardiac and myocyte function. While initial effects of MI were similar to control, there was improved function in these mice by 6 weeks. These results suggest that blocking TRPC4 after MI may reduce pathological cardiac remodeling.