bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P152: Video Self-instruction of Cardiopulmonary Resuscitation Skills for Hospital Personnel in Guatemala
Authors: Szyld Demian , Martinez Sergio N , Barriento Sonia , Leary Marion , Abella Benjamin S
Published: July 2021

Background:
Barriers to cardiopulmonary resuscitation (CPR) education are magnified in the developing world by relative cost and course availability. Though Guatemala has a developed medical infrastructure there are few certified CPR instructors and only one agency providing courses. An AHA video self-instruction (VSI) course is now available in Spanish but no data exist regarding its utility and implementation.

Objectives:
We sought to: (1) implement a pilot CPR education program in Guatemala using VSI and (2) assess attitudes and opportunities for secondary training by subjects (a “multiplier effect”).

Methods:
Hospital personnel were voluntarily trained using an established 30 min VSI program translated into Spanish, and subsequently demonstrated skill performance. CPR quality was recorded by direct observation and a CPR-sensing defibrillator without feedback. A post-training interview was performed to collect demographic information and attitudes regarding CPR training and the VSI program.

Results:
A total of 49 participants completed the study. Mean age (SD) was 36 (8), 86% were female, 65% were employees in clinical areas, and 29% had any prior CPR training. Regarding skills performance, 88% evaluated the patient prior to beginning CPR, 65% called for help, and 100% of participants gave 2 rescue breaths prior to each set of chest compressions. Mean (SD) chest compression depth was 38 mm (16) with adequate (>38 mm) depth noted in 51% of participants. Mean (SD) compression rate was 97 (17) per minute and adequate rate (80 –120 per min) was noted in 70%. Survey data revealed that 94% of participants (stated they would take the VSI kit home to practice and teach family and community members. Participants, blinded to actual price in the US ($25–30), stated they were willing to pay an average of $24 US dollars for the VSI kit (n=44).

Conclusions:
A previously-validated VSI CPR training kit, translated into Spanish, can be effectively implemented among hospital staff in a developing country in Latin America. Attitudes towards home training and dissemination were positive; further work will be required to establish rates of secondary CPR training.
Abstract P156: Health Care Professionals in Emergency Services and Hospitals Hesitate to Do CPR Because of Fear of Harming The Patient
Authors: Rosengren Ewa , Mäkinen Marja , Ponzer Sari , Kurola Jouni , Axelsson Christer , Kurland Lisa , Aune Solveig , Niemi-Murola Leila , Sopanen Marja , Christensen Erika , odegaard silje , Castrén Maaret
Published: July 2021

Introduction: Preliminary studies showed that 41,4 % of nurses in a Finnish hospital hesitate to perform defibrillation because of fear of injuring the patient and 22,9% because the patient might die and the nurse would feel guilty. Of 80 paramedics in Oslo 39 % stated that deep compressions could result in serious patient injury. These attitudes can result in bad quality CPR starting with a delay.

Hypothesis: We assessed the hypothesis that it is common among health care professionals to feel that CPR might harm the patient, to hesitate to do compressions and to hesitate to defibrillate.

Methods: Valid questionnaires were send to nurses working in Kuopio University hospital in Finland, Uppsala Akademiska sjukhuset, Sahlgrenska hospital, Göteborg and Södersjukhuset, Stockholm in Sweden and to paramedics working in Vantaa and Kuopio in Finland, Århus in Denmark and Gothenburg in Sweden. All together 811 nurses and 216 paramedics answered the questions about background data and attitudes towards resuscitation, defibrillation and guidelines.

Results: Of paramedics 60% felt that deep compressions could injure the patient and 32% felt the harm caused is bigger than the benefit of compressions. When arriving to the patient 21.5% prioritize intubation and infusion over everything else. Of the nurses 16% hesitate to start CPR because of anxiety, 26% did not feel competent to defibrillate and 21% hesitate to defibrillate because they fear to harm the patient. Still 54.2% had had CPR training at their hospital during the last six months. Nurses that hesitate to start resuscitation felt significantly more often that only doctors should defibrillate. This will delaye defibrillation and lives will be lost.

Conclusion: Major changes has to be done in training programs for CPR to ensure better confidence in performing the different tasks involved in resuscitation. Today too many health care professionals feel that CPR might do more harm than good.
Abstract P184: Novel SCN5A Mutations and SNP in Patients with Brugada Syndrome
Authors: Nakamura Kazufumi , Miura Daiji , Kobayashi Kaoru , Ouchida Mamoru , Shimizu Kenji , Ohmori Iori , Nishii Nobuhiro , Nagase Satoshi , Morita Hiroshi , Hata Yoshiki , Oka Takefumi , Kusano Kengo , Ohe Tohru
Published: July 2021

Background: Sodium channel alpha 5 subunit (SCN5A) mutations are important genetic abnormalities of Brugada syndrome (BS). Here we report novel SCN5A mutations in patients with BS.

Methods and Results: Genomic mutations in the SCN5A gene were analyzed by PCR and direct sequencing in 121 patients with BS (119 men and 2 women). Thirteen (11%) of the patients had SCN5A mutation and five (4%) of the patients had single nucleotide polymorphisms (SNP) associated with BS. Eleven patients had single point mutations, one patient had a deletion (1380 del N) and one patient had a mutation within the splicing junction (IVS21+1 g>a). The whole-cell patch clamp technique revealed that four novel mutations (F532C, R814Q, G833R, R878C) showed loss of function, and peak INa of a novel SNP (L1988R) transiently expressed in HEK 293 cells was significantly reduced (P<0.05). RT-PCR analysis revealed that the intronic mutation (IVS21+1 g>a) resulted in exon 21 deletion of SCN5A in cardiac biopsy specimens from the patient (Figure

).

Conclusions: We detected five novel SCN5A mutations (1380 del N, F532C, R814Q, G833R, R878C) and a novel disease-associated SNP (L1988R) in patients with BS. We showed a novel analysis of splicing mutation (IVS21+1 g>a) that resulted in exon skipping in the heart.
Abstract P189: Method of Cold Saline Storage for Prehospital Induced Hypothermia
Authors: Kampmeyer Mitch , Callaway Clifton W
Published: July 2021

Recent research supports the use of cold IV fluid as a method for initiating therapeutic hypothermia in post-cardiac arrest resuscitation. However, prehospital care programs employing this treatment have encountered various difficulties. Barriers to prehospital induced hypothermia protocols include the lack of effective or economically reasonable methods to maintain cold saline in the field.

Objective.
Determine the time that a standard commercial cooler can maintain two 1-liter normal saline solution (NSS) bags below 4°C in 3 different environments.

Methods.
Environments simulating an ambulance compartment were created for the experiment. NSS temperatures were continuously recorded inside a standard commercial cooler with or without ice packs (IPs) under one of three scenarios: ambient room temperature (25°C) without (IPs), ambient room temperature with IPs and 50°C ambient temperature with IPs. Four trials under each condition were performed. Time to warm to 4°C was compared using Kaplan-Meier log rank test.

Results.
In a room temperature environment with IPs, the NSS warmed to 4°C in a mean interval of 29 hrs 53 mins versus in ambient room temperature without IPs (1 hr 21 mins) versus in constant hot environment of 50°C with IPs (10 hrs 50 mins). A significant difference was found between the three environments (log-rank =17.90, dF =2, p =0.0001).

Conclusions.
Low technology methods in the form of a cooler and IPs can provide cold NSS storage for longer than a full 24 hour shift in a room temperature ambulance. In hot ambient conditions, 4°C NSS can be maintained for nearly 11 hours using this method. This model exhibits an economical, easily deployable cold saline storage unit.
Abstract P197: Feasibility of Prehospital Pediatric King Laryngeal Tube Use
Authors: Ritter Seth , Cole John S , Guyette Francis
Published: July 2021

Introduction-
Prehospital endotracheal intubation (ETI) may lead to a worse outcome for pediatric patients. We evaluated prehospital use of the pediatric King laryngeal tube (KLT) as an alternative to ETI.

Hypothesis
- Prehospital providers can successfully place the KLT and ventilate a pediatric simulator in respiratory arrest.

Methods
- We studied the ability of forty-five (45) paramedics and flight nurses to place the pediatric KLT in a SimBaby manikin. We limited pediatric KLT training to selection of the correct size as the participants already had initial training and experience with the adult KLT. Outcomes included the rate of successful pediatric KLT placement, number of attempts to correctly place the KLT, and time to first ventilation. Subjects were evaluated on airway management using an 11-point skill test. Subjects indicated perceptions and preferences for the pediatric KLT using a 5-point Likert scale. Data were analyzed using descriptive statistics.

Results
- 96.5% (95%CI: 89.3–100) subjects successfully placed the pediatric KLT. Median number of attempts was one and mean time to placement was 34 seconds (95%CI: 26.4–67.3 sec). 90% of participants successfully completed the skill test and scored a mean score of 78.2% (95%CI: 73.6–82.7). Subjects strongly agreed (5, IQR: 4–5) that their previous training on the adult KLT and using it in the field adequately prepared them to use the pediatric KLT. Subjects agreed (4, IQR: 3–4) that the pediatric KLT was easier to place than a pediatric endotracheal tube; they strongly agreed (5, IQR: 5–5) that they would use the pediatric KLT as an alternative airway. Participants disagreed (2, IQR: 1–5) that they would prefer the pediatric KLT as a primary means of securing pediatric airways.

Conclusions
-The pediatric KLT was quickly and reliably placed. Providers perceived the pediatric KLT to be easier to use than pediatric ETI in this setting.
Abstract P198: Redox Monitoring Reveals Increased Susceptibility of Whole Blood to Oxidative Stress During Hemorrhagic Shock
Authors: White Nathan J , Collinson Maryanne M , Boe Richard A , Ward Kevin R
Published: July 2021

Introduction:
The oxidation/reduction (redox) chemistry of blood during resuscitation is not well defined. Improved understanding of whole blood redox behavior would assist in developing better resuscitation monitoring and possibly reducing free radical injury both of which are PULSE initiative priorities. We use direct electrochemical measurement of equilibrium redox potential to assess the hypothesis that the overall response of blood to strong oxidant/reductant challenge is altered during shock.

Methods:
Five swine underwent hemorrhage to an oxygen debt (OD) of 80 cc/kg. Arterial blood was tested at baseline (BL) and after hemorrhage when OD equaled 40 and 80 cc/kg. Native redox potential was measured as the equilibrium voltage potential recorded between a freshly polished 2mm Au and standard Ag/AgCl electrode submerged in whole blood (n=34). The oxidative and reductive stress responses at each level of OD were defined as the change from native voltage potential induced by the addition of a strong oxidant (KMnO4) (n=18) or reductant (Dithiotreitol) (n=18). Mean native redox potentials and mean redox stress responses were compared for differences at increasing levels of OD using repeated measures ANCOVA.

Results:
Lactate increased significantly with OD (mean diff BL vs. OD=80, +4.7 mmol/L [1.3, 8.1]). No effect of OD was found on native redox potential (p value =0.233). A significant effect of OD was found on redox stress response (p value =0.0276). The redox response to oxidative stress increased from BL with increasing oxygen debt, and became significantly greater at OD=80 cc/kg (mean diff =+31.9 Rmv [3.9, 59.9]).

Conclusions:
Whole-blood redox potential did not change significantly even with severe shock, suggesting active redox buffering. However, our results suggest that the oxidative buffering capacity of blood may become impaired during severe shock as demonstrated by the significantly more positive redox potentials elicited by oxidative stress when OD was elevated. Impaired redox buffering during shock may exacerbate free radical injury induced by the oxidative stress of resuscitation. Monitoring the response of blood to oxidative stress may be a useful way to determine susceptibility to oxidative damage during resuscitation.
Abstract 243: Decreased Adiponectin Levels in Obese Mice with Polygenetic Susceptibility to Type 2 Diabetes Is Linked to Altered miRNA Levels of the ATF3 Gene In Visceral Adipose Tissue
Authors: Adi Nikhil , Adi Jennipher , Hollar Danielle , Kurlansky Paul , Agatston Arthur , Webster Keith A
Published: July 2021

INTRODUCTION:
Adiponectin encoded by the APMI gene is an adipocyte-expressed protein with insulin sensitizing, antiatherogenic and antiinflammatory properties. Activating transcription factor 3 (ATF3) is a member of the ATF/cAMP responsive element binding family that acts as a stress-inducible transcriptional repressor. ATF3 was recently shown to repress adiponectin gene expression.

HYPOTHESIS:
High fat diet with genetic susceptibility for T2D alters the miRNA profile for the adiponectin gene in visceral adipose (VA).

METHODS:
NONcNZO10 (NZ10) mice with polygenic susceptibility to T2D or NONLt controls (60 per group) were subjected to normal chow (NC; 6% fat), Western diet (WD; high fat/carb) or Mediterranean diet (MD; high protein/unsaturated fat) for 25 weeks. After a 16h fast, VA was isolated from all groups (5 mice per group) and subjected to gene-microarray, micro-RNA and protein analyses.

RESULTS:
NONLt (control) mice retained normal glycemic control and blood chemistry over 25 weeks with all diets. WD-fed NZ10 mice had increased plasma triglycerides (3-fold, p<0.001), insulin (1.8-fold. P<0.01) and severely impaired glucose tolerance relative to the MD or control groups (p<0.01). NZ10 mice on all diets accumulated 2.5-fold more VA than NONLt controls. Body weights of WD-fed NZ10 mice were 18% greater and fat depots were 22±3.1% larger than SB mice at 25 weeks (p<0.01). Serum adiponectin levels decreased >50% at 25–30 weeks in NZ10 mice fed WD compared with NONLt (p<0.01). The ATF3 gene was upregulated (5-fold, p<0.0001) in the NZ10 mice fed WD compared with MD and 2-fold (p<0.001) compared with NC. NONLt mice did not show any diet-related difference of ATF3 expression. Expression of ATF3 was increased 20-fold in NZ10 mice relative to NONLt mice independently of diet (p<0.001). MiR profiling of the ATF3 gene in VA revealed that miR-203, 685, 191 and 29a were markedly downregulated in NZ10-WD mice (all 2.5-fold, p<0.001). MiR expression in NONLt controls was unchanged and similar to that of NZ10 mice fed MD.

CONCLUSIONS:
Combined T2D susceptibility and a permissive diet cause selective repression of miR-203, 685, 191 and 29a and up-regulation of ATF3 gene expression. The consequences include defective adiponectin production and predisposition to T2D.
Abstract 307: Regulator of G-protein Signaling 2 (RGS2) Suppresses Pathologic but not Physiologic Cardiac Hypertrophy
Authors: Hsu Steven , Takimoto Eiki , El-Haddad Hazim , Bedja Djahida , Gabrielson Kathleen , Champion Hunter C , Kass David A
Published: July 2021

Regulator of G-protein signaling 2 (RGS2) is a GTPase activating protein thought to negatively regulate Gα
q/11
-protein coupled signaling.
In vitro
RGS2 over-expression blunts Gα
q/11
-stimulated hypertrophy, whereas its knockdown worsens it. Here we tested the impact of pathologic (aortic banding, AB) versus physiologic (swimming, SWM) cardiac stimulation on RGS2-cardiac regulation. Mice lacking RGS2 (RGS2
−/−
) develop modest hypertension but no baseline cardiac phenotype. RGS2
−/−
subjected to AB developed accelerated hypertrophy (93% vs 47% increase in heart weight/tibial length at 1 wk, p<0.05) and failure, and early mortality (40% vs. 0% mortality at 1 wk, p=0.012). Their hearts have enhanced activation of Gα
q/11
-coupled hypertrophic mediators such as calcineurin (Cn), extracellular response kinase 1/2 (ERK1/2), and calmodulin-dependent protein kinase II (CaMK II) compared to controls. In sharp contrast, after 6 weeks of exercise swimming, control and RGS2
−/−
mice developed similar increases in LV mass (17% and 15% increases in heart weight/tibial length; p<0.001 compared to sedentary controls, NS between genotypes). Both groups also had similar increases in relative wall thickness and LV mass by echocardiography. Invasive pressure-volume loop analysis revealed enhanced cardiac output in swimming mice but no differences between genotypes. The only disparities were that heart rate declined slightly (5%) in controls but was unchanged in RGS2
−/−
, and RGS2
−/−
mice swam more slowly (mean velocity 5.0±0.3cm/s vs 6.7±0.3cm/s, by video tracking). Control and RGS2
−/−
mice ± exercise had negligible changes in the Gα
q/11
mediators Cn, ERK1/2, and CaMK II. Thus, RGS2 plays a central role in blunting cardiac hypertrophic signaling and mortality in response to pathological stress, but not to physiological stimulation. This highlights its in vivo specificity to the regulation of Gα
q/11
signaling as opposed to the signaling of physiologic hypertrophy.

Changes in Heart Weight/Tibial Length in response to Aortic Banding (AB) vs Swimming (SWM)
Abstract 322: Acute Inhibition of GTP Cyclohydrolase 1 Uncouples Endothelial Nitric Oxide Synthase and Elevates Blood Pressure
Authors: Wang Shuangxi , Xu Jian , Song Ping , Wu Yong , Zhang Junhua , Choi Hyoung C , Zou Ming-hui
Published: July 2021

Objective:
GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in
de novo
synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) dictating at least partly, the balance of nitric oxide (NO) and superoxide (O
2
.−
) produced by this enzyme. The aim of this study is to determine the effects of acute inhibition of GTPCH1 on BH4, eNOS function, and blood pressure.

Methods:
The biopterin content was detected by HPLC. O
2
.−
and NO productions were assayed by using DHE and DAF fluorescence respectively. The vessel relaxation was assayed by organ chamber. The blood pressure in wild-type (WT) or eNOS
−/−
mice was determined by a carotid catheter method.

Results:
Exposure of bovine or mouse aortic endothelial cells to GTPCH1 inhibitors (10 mM DAHP or 1 mM NAS) for 24 hours or GTPCH1 siRNA transfection significantly reduced both BH4 and NO levels, but increased O
2
.−
levels. This increase was abolished by 10 μM L-sepiapterin (BH4 precursor) or 1 mM L-NAME (non-selective NOS inhibitor). Incubation of isolated WT mice aortas with DAHP or NAS for 24 hours impaired acetylcholine-induced endothelium-dependent relaxation, but not endothelium-independent relaxation. Aortas from GTPCH1 siRNA-injected mice, but not their control-siRNA injected mice, also exhibited impaired endothelium-dependent relaxation. Furthermore, GT-PCH1 siRNA injection in mice reduced BH4 levels in aortas, associated with increased aortic levels of O
2
.−
, 3-nitrotyrosine, and adhesion molecules (ICAM1 and VCAM1). In addition, an elevated mean, systolic, and diastolic blood pressure was induced by GTPCH1 siRNA injection
in vivo
, but not control siRNA (mean blood pressure: 114.28±4.48
vs
. 136.81±2.45 mmHg) in WT mice. GTPCH1 siRNA was unable to elicit the similar effects in eNOS
−/−
mice, including increased oxidative stress (O
2
.−
, 3-nitrotyrosine, ICAM1, VCAM1) and blood pressure. Finally, sepiapterin supplementation, which had no effect on high blood pressure in eNOS
−/−
mice, partially reversed GTPCH1 siRNA-induced elevation of systemic blood pressure in WT mice.

Conclusion:
GTPCH1 via BH4 maintains normal blood pressure and endothelial function by preserving eNOS-dependent NO biosynthesis.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 352: Juvenile Doxorubicin Treatment Impairs Recovery and Promotes Apoptosis in Adult Hearts via failure of up-regulation of Mcl-1 after Myocardial Infarction
Authors: Huang Chengqun , Quinsay Melissa N , Kim Lucy , Kubli Dieter A , Gustafsson Åsa B
Published: July 2021

The anthracycline doxorubicin (DOX) is an effective chemotherapeutic agent used to treat pediatric cancers. However, it is associated with cardiotoxicity which can manifest many years after the initial exposure. Very little is known about the mechanisms of this late-onset cardiotoxicity. To understand this problem, we have developed a model of pediatric DOX cardiotoxicity where mouse pups were injected with 4 low doses of DOX or saline at 5, 10, 15, and 20 days of age. After completion of treatment, all mice were healthy and had normal cardiac function as adults. However, when mice were subjected to myocardial infarction (MI), DOX treated mice developed severe cardiac fibrosis and had increased mortality compared to saline treated mice (67% vs. 20%), suggesting that these hearts do not tolerate stress well. Assessment of infarct size revealed that infarct size was greater in DOX-treated hearts compared to saline. Anthracyclines are known to affect the Bcl-2 family proteins which are also important regulators of cell death in the heart. We found that Mcl-1, an anti-apoptotic Bcl-2 protein, is expressed in the heart and localizes to the mitochondria. Overexpression of Mcl-1 in HL-1 myocytes reduced simulated I/R-mediated cell death (58±8% vs. 26±7%, n=3, p<0.05), suggesting that Mcl-1 promotes survival of cardiac cells in response to stress. Immunostaining analysis of Mcl-1 expression in mouse hearts revealed that Mcl-1 was significantly increased in myocytes in the border zone after acute MI. Myocytes with enhanced Mcl-1 expression were negative for apoptosis as assessed by TUNEL staining, whereas cells with reduced Mcl-1 expression were TUNEL positive. Since anthracyclines have been shown to reduce Mcl-1 expression in cancer cells, we investigated whether juvenile DOX treatment would suppress Mcl-1 expression in adult heart in response to stress. We found that cardiac myocytes in the border zone of DOX treated hearts failed to upregulate Mcl-1 in response to MI which likely impaired recovery and contributed to increased injury and mortality. These studies suggest upregulation of Mcl-1 is an important salvage response after MI and that juvenile DOX treatment affects Mcl-1 expression in adulthood resulting in a heart that is more susceptible to stress.

This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Abstract 372: The Smad3 Pathway Critically Regulates Myofibroblast Proliferation And Synthetic Activity In Healing Myocardial Infarcts
Authors: Dobaczewski Marcin , Bujak Marcin , Gonzalez Carlos , Li Na , Wang Xiao-Fan , Frangogiannis Nikolaos G
Published: July 2021

We have recently demonstrated that the Transforming Growth Factor (TGF)-β/Smad3 pathway is activated in healing infarcts and plays an essential role in the pathogenesis of cardiac remodeling. Smad3 −/− mice were protected from the development of ventricular dilation following infarction and exhibited markedly reduced fibrosis of the peri-infarct area and the remodeling non-infarcted heart. Accordingly, we hypothesized that Smad3 signaling plays an essential role in regulating cardiac fibroblast function and gene expression in myocardial infarction. Surprisingly, Smad3 −/− infarcts exhibited increased peak infiltration with myofibroblasts, associated with evidence of enhanced proliferative activity. Smad3 −/− mice had a higher density of Ki-67-positive proliferating myofibroblasts in the infarcted myocardium in comparison with wildtype (WT) animals (Smad3−/− 917±291 cells/mm
2
vs. WT 614±115 cells/mm
2
, p<0.05). In vitro experiments suggested that TGF-β inhibits murine cardiac fibroblast proliferation in a concentration-dependent manner and that the antiproliferative effects of TGF-β are abrogated in Smad3 −/− fibroblasts. On the other hand Smad3 signaling was essential for extracellular matrix protein synthesis by cardiac fibroblasts. TGF-β-mediated induction of procollagen type III and of the matricellular protein tenascin-C in cardiac fibroblasts was dependent on Smad3. In addition, TGF-β-induced Tissue Inhibitor of Metalloproteinases (TIMP)-1 and -2 upregulation was also abrogated in Smad3 −/− fibroblasts, suggesting that Smad3 signaling regulates matrix metabolism. In vivo, Smad3 −/− infarcts exhibited attenuated tenascin-C and collagen deposition in the infarct and in the remodeling non-infarcted heart. Our findings suggest that the Smad3 pathway critically regulates fibroblast function in healing myocardial infarction. In Smad3 −/− mice, the healing infarct contains abundant myofibroblasts that exhibit enhanced proliferative activity, but have markedly decreased ability to synthesize extracellular matrix proteins and to produce TIMPs. In the absence of Smad3, attenuated matrix deposition in the remodeling non-infarcted heart results in decreased dilation and ameliorated diastolic dysfunction.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 402: Endothelial Specific Jagged-1/Notch Signal in Bone Marrow Niche Regulates Endothelial Progenitor Cell Development
Authors: Kwon Sang Mo , Kamei Naruske , Yokoyama Ayumi , Horii Miki , Masuda Haruchika , Asahara Takayuki
Published: July 2021

The developmental cascade of endothelial progenitor cells (EPCs) in response to environmental signals is dynamically regulated by tight-communication between stem cells and stromal cells in bone marrow niches. We have recently reported that Jagged-1 dependent signals specifically regulated EPC-mediated vascularization using mice of Mx-cre dependent inactivation of jagged-1. However, the specific contribution of EPC development via modulating endothelial Jagged-1/Notch axis remain unclear. EPC colony forming capacity of CD34(−)/c-kit(+)/Sca-1(+)/Lin(−) stem cells in tamoxifen-treated Tie2cre-Jagged-1 null mice are drastically enhanced, although the CFU-EPC in CD34(+)/c-kit(+)/Sca-1(+)/Lin(−) progenitor cells was significantly impaired in Jagged-1 null mice. Moreover, the impaired EPC development was also confirmed in single cell-derived EPC colony forming assay. Notably, the development of primitive EPC, an early stage of EPC development, was significantly increased in stem cells, while progenitors derived from BM niche did not give rise to form definitive EPC, a late stage of EPC development. Similarly, we observed markedly decreased cell number and CFU-EPC of progenitor subset (Sca-1+/c-Kit+) in cultured EPCs. Isolated Jag-1 deficient progenitors in cultured EPCs never gave rise to definitive EPC, while WT-derived progenitor did. When co-culture with stromal cells derived from endothelial specific Jagged-1 null mice, stem cells could not develop functional EPCs. EPCs from endothelial specific Jagged-1 deficient niche showed impaired in vitro migration capacity in response to VEGF, SDF-1a, SCF or TPO, as well as the decreased incorporation ability into ischemic neo-vessel. In hind limb ischemia models, we observed the significantly reduced blood vessel regeneration with the decreased capillary density in endothelial specific Jagged-1 null mice. In conclusion, the present report clearly showed for the first time that the Jagged-1/Notch axis plays a pivotal role in EPC development in special niche, suggesting that modulation of EPC development via regulating Jagged-1/Notch axis provide a clue of therapeutic tissue regeneration.
Abstract 412: Pentaerithrityltetranitrate Does Not Deteriorate Endothelial Dysfunction In Patients With Coronary Artery Disease. A Prospective, Randomized, Double-blind, Placebo-controlled, Monocentric Clinical Trial (PENTA)
Authors: Schiewe Robert , Schnorbus Boris , Ostad Mir Abolfazl , Medler Christoph , Wenzel Philip , Daiber Andreas , Münzel Thomas , Warnholtz Ascan
Published: July 2021

Purpose: Chronic nitrate therapy remains controversial in patients with coronary artery disease (CAD) due to induction of tolerance and deterioration of endothelial dysfunction. Pentaerithri-tyltetranitrate (PETN) differs from other organic nitrates by the lack of tolerance induction and by antioxidative properties. We hypothesized that treatment with PETN improves endothelial dysfunction in patients with CAD.

Methods: In a prospective, double-blind trial, we randomly assigned 80 patients to double-blind treatment for 8 weeks with oral PETN 80mg t.i.d. (PETN) or placebo (C), respectively. The primary endpoint was the absolute change in flow-mediated dilation of the brachial artery (FMD) from baseline to follow-up. Secondary endpoints were the changes in nitroglycerin-mediated dilation (NMD), peripheral arterial tonometry (EndoPAT2000) index as a marker of endothelial function of small arteries, the augmentation index (AI) as a marker of arterial stiffness and the activity of the PETN bioactivating enzyme aldehydedehydrogenase-2 (ALDH-2) in monocytes from baseline to follow-up. Raw data entry, data monitoring and statistic analysis were independently performed by the coordination centre of clinical trials (KKS), Mainz. The trial was registered as ISRCTN 14741768.

Results: Both treatment groups were comparable regarding age, gender, distribution of cardiovascular risk factors and concomitant medication. There was no difference in the mean (±SD) change in FMD between the two treatment groups (PETN: +1.6±3.3% vs. C: +1.4±4.1% p=0.7). NMD significantly rose after treatment with PETN compared to C (PETN: +3.8±5.5% vs. C: +0.6±4.2% p=0.0036). Mean PAT-Index, AI and ALDH-2 activity remained unchanged in both treatment groups.

Conclusions: The three main findings of our trial are: In contrast to other organic nitrates PETN does not deteriorate endothelial dysfunction (1), does not impair ALDH-2 activity (2) and does not induce cross tolerance to nitroglycerin (3). We conclude that chronic PETN therapy in patients with CAD may be established for symptomatic treatment without adverse effects on endothelial function.
Abstract 471: Paradigm Shift to Epigenetic Memory for the Pathological Understanding of Chronic Heart Failure
Authors: Asano Yoshihiro , Takashima Seiji , Liao Yulin , Kitakaze Masafumi
Published: July 2021

Background-

Epigenetic modification becomes a popular regulatory mechanism of gene expression in development or tumor progression. However its role in cardiovascular diseases has not been elucidated. Since reactivation of cardiac fetal gene expression in chronic heart failure (CHF) is more remarkable than that expected by the increased transcriptional factors, the concept of epigenetic modification may be required for the explanation of such a phenomenon. We tested this idea.

Methods-

To test the epigenetic potency in cardiomyocytes, we focused on the expression of both BNP and ANP genes. As epigenetic markers, nucleic chromatin structure by electron microscopy and the entire-regional histone modifications were evaluated using our newly developed
in vivo
chromatin immunoprecipitation (ChIP) assay.

Results-

In the developmental stage of hearts, we observed a parallel movement of fetal gene expression and epigenetic markers. On the other hand, in the short time stimuli of GPCR agonists in murine cardiomyocytes, fetal gene expression was controlled only by the amount of transcription factors, Gata4 and Nkx2.5, but epigenetic maker was not altered. In contrast, in
in vivo
murine model of CHF both Gata4 and Nkx2.5 depressed despite elevated expression of both BNP and ANP. Nucleic chromatin structure in failing cardiomyocytes was changed to less condensed forms and increased accumulation of both histone H3K9-acetylation and H3K4-trimethylation was observed across these gene loci. Also in human failing heart, similar changes of nucleic chromatin structure were observed as that in murine CHF model and interestingly these changes was reversed when the heart function was recovered along with proper treatment.

Conclusions-

We for the first time clarified that both BNP and ANP reactivation mechanisms in chronic heart failure link to the alteration of histone modification by the
in vivo
ChIP method. Different from acute stimuli, this change was coincided with nucleic chromatin structure and was explicable of paradoxical depression of transcription factors, suggesting epigenetic potency. Epigenetic mechanism may play a pivotal role in the nuclear memory-mediating failing myocardium, raising the hope for a novel pathological understanding for human CHF.
Abstract 473: Genetic Fate Mapping Identifies Second Heart Field Epicardial Progenitor Cells as a Source of Adipocytes in Arrhythmogenic Right Ventricular Cardiomyopathy
Authors: Lombardi Raffaella , Dong Jinjiag , Rodriguez Gabriela , Bell Achim , Leung Tack Ki , Schwartz Robert J , Willerson James T , Brugada Ramon , Marian Ali J
Published: July 2021

The pathological hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), a genetic disease of desmosomal proteins, is fibro-adipocytic replacement of cardiac myocytes. The cellular origin of adipocytes in ARVC is an enigma. In desmosomal ARVC the impetus has to instigate from cells that express the mutant protein. Cardiac myocytes are the only cells in the heart known to express the desmosomal proteins. Adult myocytes are terminally differentiated, and hence, an unlikely source. In contrast, cardiac progenitor cells can differentiate to different lineages. Thus, we conditionally deleted Dsp, encoding desmoplakin, and concomitantly expressed enhanced yellow fluorescent protein (EYFP) using the Cre-LoxP technique, regulated by Nkx2.5, an early cardiac lineage promoter. We also screened the human hearts with ARVC for co-expression of markers of cardiac cell lineages and adipocytes. We generated Nkx-2.5-Cre:Dsp
W/F
and Nkx-2.5-Cre:Dsp
W/F
:R26-EYFP
F/F
mice, which showed enlarged hearts, depressed cardiac systolic function and patchy areas of fibro-adiposis, particularly at the epicardium. We detected expression of EYFP by immunoblotting (IB) and immunofluorescence (IF) and expression of cardiac progenitor cells and adipocytes markers by IF. A subset of epicardial cells in the Nkx-2.5-Cre:Dsp
W/F
:R26-EYFP
F/F
mice co-expressed adipogenic transcription factors C/EBP-α and PPAR-γ along with EYFP and the second heart field markers IsI1 and Mef2c. Likewise, we detected co-expression of C/EBP-α and IsI1 or Mef2C in the fibro-adipocytic regions in human hearts with ARVC. To determine the specific cardiac cell lineage that differentiates to adipocytes, we co-stained sections of human and lineage trace mouse hearts and detected co-expression of SM actin, a marker of smooth muscle cells (SMCs) and C/EBP-α as well as co-expression of cardiac α-actin, a marker of cardiac myocytes, and C/EBP-α but not co-expression of PECAM1, a marker of endothelial cells and C/EBP-α. We conclude excess adipocytes in desmosomal ARVC originate from second heart field epicardial SMCs/myocyte progenitor cells.
Abstract 501: The Measurement of Pulmonary Flow Reserve for Early Detection of Pulmonary Vascular Disease
Authors: Ilsar Rahn , Chawantanpipat Chirapan , Chan Kim H , Waugh Richard , Hennessy Annemarie , Celermajer David S , Ng Martin K
Published: July 2021

Pulmonary vascular disease (PVD) is an insidious, fatal disorder, characterized by progressive microvascular obstruction. Current pulmonary artery pressure (PAP) based diagnosis of PVD is inadequate, as PAP only rises once >50% of the microvasculature is destroyed. We propose a novel index of pulmonary flow reserve
(PFR = maximal hyperemic divided by basal pulmonary blood flow)
that may detect microvascular disease
prior
to rises in PAP. We aimed to validate Doppler flow velocity (v

d

) use for PFR assessments, define the optimal maximal hyperemic stimulus for PFR assessments and compare PFR & PAP during progressive microcirculatory obliteration, in a primate model. Doppler sensor guidewires (DSG) were placed in segmental pulmonary arteries (SPA) of 11 ketamine-anaesthetized baboons. Vessel diameter (
D
), v

d

& hemodynamics were recorded at baseline & after intra-SPA administration of saline, adenosine (50–500μg/kg/min), acetylcholine (ACh, 10
−8
–10
−6
M) and papaverine (3– 60mg). Increasing amounts of intra-SPA microspheres (diameter 40–120μm) progressively obliterated the microvasculature. Intra-SPA saline did not alter
D
or v

d

(p>0.1) validating the use of local drug delivery. Adenosine induced dose-dependent increases in v

d

(22.5±2.3
v
32.7±4.8cm/s, baseline
v
400–500μg/kg/min, p<0.0001, plateau at 200μg/kg/min) and heart rate (HR, 92±4
v
100±4bpm, p<0.04) while causing systemic hypotension (106±6
v
77±3mmHg, p<0.0001). Papaverine induced increases in v

d

(23.9±1.1
v
34.6±4.0cm/s, baseline
v
24mg, p<0.0001) and HR (92±7
v
104±4bpm, p>0.6). Neither agent affected mean PAP or
D
(p>0.3). ACh induced small reductions in v
d
(p<0.0001) possibly due to ketamine-interaction. Normal PFR values were 1.35±0.1 & 1.39±0.1 using adenosine & papaverine, respectively. Microsphere delivery significantly reduced PFR (1.54±0.26
v
1.18±0.09, baseline
v
10
6
microspheres, p<0.02) without affecting resting mean PAP (21±6
v
22±3mmHg, p>0.7). Doppler based PFR assessments are feasible using adenosine or papaverine. PFR is capable of detecting microvascular obliteration of magnitude insufficient to raise PAP making it potentially valuable for diagnosis of early PVD.
Abstract 518: Cyclophilin A is a Novel Pro-Inflammatory Cytokine that Accelerates Development of Atherosclerosis
Authors: Satoh Kimio , O’Dell Michael R , Liao Duan-fang , Cui Zhaoqiang , Nigro Patrizia , Mohan Amy , Alexis Jeffrey , Goldman Bruce , Berk Bradford C
Published: July 2021

Background -

Cyclophilin A (CyPA), a member of the immunophilin family, is highly expressed in vascular smooth muscle cells (VSMC) in mice and humans. CyPA is secreted in response to reactive oxygen species (ROS) and also is a positive regulator of helper T (Th) profile promoting differentiation of Th0 cells into Th1 lymphocytes (increased IFN-γ The development of atherosclerosis involves vascular inflammatory cytokine release, inflammatory cell migration/activation, and altered Th1/Th2 profile. Based on the role of CyPA in ROS signaling and Th profile we hypothesized that CyPA would promote atherosclerosis.

Methods and Results -

We compared atherosclerosis development in ApoE
−/−
and ApoE
−/−
CyPA
−/−
mice using two different models.

In ApoE
−/−
mice fed a high-fat diet for 16 weeks, atherosclerosis assessed by oil-red O (ORO) staining was significantly greater in ApoE
−/−
mice compared to ApoE
−/−
CyPA
−/−
mice (%ORO, en face aorta; 23.1±5.5 vs. 6.8±3.2,
P
<0.01). Immunostaining of atherosclerotic lesions in the aortic cusp revealed the co-localization of CyPA, CD45, and MCP-1, suggesting the importance of CyPA for inflammatory cell recruitment and cytokine production.

Angiotensin II infusion (AngII, 1000 ng/min/kg, 4 weeks) significantly increased atherosclerosis area in ApoE
−/−
mice, which was significantly less in ApoE
−/−
CyPA
−/−
mice (%ORO, en face aorta; 11.1±4.6 vs. 4.0±2.8, P<0.01).

ApoE
−/−
mice showed significant increases in vascular MCP-1 expression, VCAM-1 expression, and inflammatory cell migration to the aortic wall, all of which were significantly less in ApoE
−/−
CyPA
−/−
mice. Cytokine/chemokine array analysis revealed that AngII significantly increased secretion of numerous cytokine/chemokines from ApoE
−/−
spleno-cytes, which was significantly less in ApoE
−/−
CyPA
−/−
splenocytes. Mechanistic studies in vitro revealed that AngII enhanced CyPA secretion from mouse aortic VSMC. Finally, expression of CyPA was significantly increased in atherosclerotic plaques of patients with acute coronary syndromes.

Conclusion -

CyPA is a novel mediator of atherosclerosis by promoting inflammatory cytokine/chemokine secretion, adhesion molecule expression, and inflammatory cell migration.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 1357: PGC-1
α
as a Novel Regulator for Angiotensin II-induced Reactive Oxygen Species Production and Hypertrophy in Vascular Smooth Muscle Cells
Authors: Xiong Shiqin , Ahmad Mushtaq , Patrushev Nikolay A , Hilenski Lula , Alejandra San Martin Almeyda , Ushio-Fukai Masuko , Alexander Wayne R
Published: July 2021

Angiotensin II (Ang II) increases H
2
O
2
production and vascular smooth muscle cell (VSMCs) hypertrophy, in part through redox-sensitive PI3K/Akt, which is inhibited by catalase overexpression. The relevant molecular mechanism remains unclear. Peroxisome proliferator-activated receptor gamma coactivator-1
α
(PGC-1
α
) is reported to protect from oxidative stress by regulating expression of antioxidant enzymes such as catalase. We thus hypothesized that PGC-1
α
may be important mediator for Ang II-induced H2O2 production and vascular hypertrophy. Here we show that Ang II stimulation increases serine phosphorylation of PGC-1
α
(2.2 folds) with a peak at 15 min, which is inhibited by LY294002, a specific PI3 kinase inhibitor (98% decrease), and by Akt inhibitor-2/Triciribine (95% decrease). Ang II promotes PGC-1
α
phosphorylation mainly at Ser 570 in an Akt-dependent manner. Ang II significantly suppresses Gal4-fused PGC-1
α
transcriptional activity in a dose dependent manner, which is partially reversed by PI3K/Akt inhibition. Chromatin immunoprecipitation (ChIP) assay shows that PGC-1
α
associates with the catalase promoter and this association is blocked by Ang II in a PI3K/Akt-dependent manner. Consistent with these results, Ang II stimulation time-dependently decreases endogenous catalase expression at both messenger RNA and protein levels. Ang II-induced downregulation of catalase at protein level at 24 hrs is prevented by Akt inhibitor (86%) and by overexpression of PGC-1
α
S570A, an Akt phosphorylation site mutant, (75%). Moreover, overexpression of PGC-1
α
S570A significantly inhibits Ang II-induced increase in H2O2 production (>80%) and leucine incorporation (>90%) as measured at 12 and 24 hrs, respectively. In summary, Akt-dependent serine phosphorylation of PGC-1
α
by Ang II plays an important role for Ang II-induced downregulation of catalase, thereby increasing H2O2 production, which may contribute to ROS-dependent VSMC hypertrophy. These findings provide insight into a novel mechanisms by which Ang II promotes long-term H2O2 production to increase oxidative stress via targeting PGC-1alpha, and mediates metabolic abnormalities.
Abstract 1371: Overexpression Of Mitochondrial Transcription Factor A Protects Failing Myocardium From Oxidative Stress
Authors: Yoshida Masayoshi , Ide Tomomi , Yamato Mayumi , Ikeuchi Masaki , Tsutsumi Takaki , Inoue Takahiro , Ando Makoto , Todaka Koji , Kang Dongchon , Utsumi Hideo , Tsutsui HIroyuki , Sunagawa Kenji
Published: July 2021

Background:
Reactive oxygen species (ROS) from mitochondria play a pivotal role in the pathogenesis and progression of heart failure. Mitochondrial transcription factor A (TFAM), a nucleus-encoded protein, in mitochondria promotes transcription of mitochondrial DNA (mtDNA), maintains mtDNA, and increases mtDNA copy number. We previously reported that overexpression of human TFAM ameliorated cardiac remodeling and improved survival by maintaining mitochondrial function. We investigated whether those beneficial effects result from the increase of antioxidative properties both in vivo and in vitro.

Methods and Results: [in vivo study]
We created myocardial infarction (MI) in wild type (WT) mice and human TFAM transgenic (TG) mice as a model of heart failure. We evaluated free radical generation by in vivo ESR on 28th day after the operation. MI size did not different between WT and MI. Overexpression of TFAM ameliorated MI-induced cardiac hypertrophy (histology) and LV dysfunction (2D echo and cath). Furthermore, the rate of signal decay in
in vivo
ESR, indicative of oxidative stress, was accelerated in WT-MI (0.10 ± 0.01 vs 0.19 ± 0.02/min, n=4–5, P<0.01) whereas decelerated in TG-MI (0.12 ± 0.02/min; n=4, P<0.01). DNA microarrays analysis of myocardium (8 weeks old, male) indicated that TG overexpressed (double or more) antioxidant relevant genes mostly encoded in nuclear DNA, such as Mthfd2 and Adh1 without affecting mitochondrial respiratory enzyme activities.
[in vitro study]
We measured superoxide in Hela cells using Dihydroethidium in the presence of rotenone, a complex I inhibitor. Overexpression of TFAM in Hela cells significantly reduced superoxide production (72.8 %).

Conclusion:
Overexpression of TFAM suppresses ROS. Such an antioxidative property of TFAM may contribute to its powerful anti-remodeling effect in failing heart.
Abstract 1374: A Novel eNOS-Independent Protective Action of Statin against Angiotensin II-Induced Atrial Remodeling Via Attenuating Rac-1-Mediated Oxidative Stress
Authors: Yagi Shusuke , Akaike Masashi , Aihara Ken-ichi , Ishikawa Kazue , Ise Takayuki , Yosihda Sumiko , Sumitomo Yuka , Ikeda Yasumasa , Iwase Takashi , Soeki Takeshi , Matsumoto Toshio , Sata Masataka
Published: July 2021

Activation of the renin-angiotensin system exacerbates atrial remodeling, leading to atrial fibrillation (AF) and thrombosis, especially in a condition with decreased nitric oxide bioavailability. Although antiarrhythmic and anticoagulation agents are used under these pathological conditions, these drugs are not able to ameliorate atrial remodeling. Recently, it has been reported that statins reduce the incidence of AF through attenuating atrial remodeling; however, the mechanisms have not been completely elucidated. This study was designed to determine the beneficial effect of pitavastatin (Pit) against angiotensin II (Ang II)-induced atrial remodeling and to elucidate its mechanism. eNOS knockout mice were sham-operated or infused with Ang II by an osmotic mini-pump for 2 weeks, and Ang II-infused mice were divided into 3 treatment groups: Pit, tempol, a free radical scavenger, or a vehicle. Echocardiography and electrocardiography showed that Ang II infusion increased left atrial volume and caused a high incidence of AF, whereas Pit and tempol treatment prevented Ang II-induced left atrial enlargement and AF. Histological analysis showed that acceleration of Ang II-induced interstitial fibrosis, perivascular fibrosis and cardiomyocyte hypertrophy in the atrium were all attenuated by Pit and tempol treatment. Immunohistochemistry showed that Ang II down-regulated thrombomodulin and up-regulated tissue factor and plasminogen activator inhibitor-1 in the left atrium and that Pit and tempol treatment corrected the Ang II-induced thrombogenic condition. Moreover, Pit and tempol reduced Ang II-induced atrial superoxide production, detected by dihydroethidium staining, and atrial TGF-
β
1 expression and Smad 2/3 phosphorylation. Activity of Rac-1-GTPase involved in the activation of NADPH oxidase in the atrium was attenuated by Pit but not by tempol. Pit exerts eNOS-independent protective actions against Ang II-induced atrial structural and electrical remodeling with enhanced thrombogenicity through suppressing Rac-1-mediated oxidative stress, leading to suppression of the TGF-
β
1/Smad pathway.