bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Exon Dosage Using Multiplex Ligation-Dependent Probe Amplification in Chinese Patients with Early-Onset Parkinson’s Disease
Authors: Lin Yu , Zeng Yi-Fang , Cai Nai-Qing , Lin Xiao-Zhen , Wang Ning , He Jin
Published: October 2019

Introduction: Several studies have identified a number of genes associated with Parkinson’s disease (PD). Genomic rearrangements (exon dosage variations) in these genes have emerged as significant, causing mutations. However, exon dosage variations in several PD genes were rarely investigated in Chinese patients. Objective: This study was aimed at determining the prevalence of PD-causing genes’ exon rearrangements in Chinese sporadic early-onset PD (EOPD) patients. Methods: A total of 150 Chinese sporadic EOPD patients and 100 healthy controls were enrolled. Multiplex ligation-dependent probe amplification (MLPA) was used to detect exon dosage in PD genes, including SNCA, PARKIN, UCHL1, PINK1, DJ1, LRRK2, and ATP13A2. Positive results were verified by real-time quantitative polymerase chain reaction. And exon sequencing was employed to screen for subtle mutations. Novel exon dosage variations were screened in families and controls. Results: PARKIN exon rearrangements were detected in 10 (6.7%) patients, including a novel heterozygous duplication of PARKIN exons 1–4. Clinical investigation showed that the percentage of individuals with PARKIN exon rearrangements was higher in the younger patients. Notably, the MLPA screening detected a heterozygous deletion of UCHL1 exon 1 in a patient. MLPA analysis in the family detected the deletion in an asymptomatic sister, indicating incomplete penetrance. Conclusion: Exon copy number variations (CNVs) in the PARKIN gene are relatively common among Chinese sporadic EOPD patients, whereas exon CNVs in other known PD genes can also be detected. Our findings demonstrate that it is important to perform exon dosage analysis for several known PD genes to obtain a better mechanistic insight into PD pathogenesis.
European Guidelines on the Organisation of Breast Centres and Voluntary Certification Processes
Authors: Biganzoli Laura , Marotti Lorenza , Cardoso Maria-Joao , Cataliotti Luigi , Curigliano Giuseppe , Cuzick Jack , Goldhirsch Aaron , Leidenius Marjut , Mansel Robert , Markopoulos Christos , Wyld Lynda , Rubio Isabel T.
Published: October 2019

Background: EUSOMA undertook the commitment of defining the requirements for a specialist breast centre, which has become the reference document for the implementation of breast centres. Summary: The EUSOMA requirements for a specialist breast centre give clear indications regarding the requisite caseload, dedicated team composition (core and non-core team), organisation, availability of services and equipment throughout the patient pathway, quality control, and application of a multidisciplinary approach. The minimum number of cases is 150 newly diagnosed breast cancer cases per year. Based on the EUSOMA requirements, a voluntary and accredited certification scheme has been developed. In Europe, other voluntary certification schemes are available, such as those developed by the German Cancer Society and German Society for Breast Disease, the National Cancer Peer Review Programme in the UK, and the “label de qualité” established by the Swiss Anticancer League and the Swiss Senology Society. The European Commission Initiative on Breast Cancer (ECIBC) has overseen the development of a European Quality Assurance Scheme. Key Messages: Nearly 20 years after the initial publication of the EUSOMA requirements, ensuring that all breast cancer patients in Europe are treated only in certified breast centres should be considered a high priority and eventually achieved through collaborative efforts.
Оценка качества жизни в отдаленном периоде у больных с послеоперационными вентральными грыжами
Authors: Davlatov S. S.
Published: October 2019

Цель работы: анализ качества жизни пациентов по усовершенствованной шкале у больных с послеоперационными вентральными грыжами.

Материалы и методы. Основу для исследования составили 217 больных с послеоперационными вентральными грыжами, которые находились под наблюдением и поступившие в хирургические отделения 1 и 2 клиники Самаркандского государственного медицинского института в сроки с 2009 по 2017 год. Больные были разделены на две группы, которые в зависимости от вида герниопластики (аутогерниопластика или аллогернипластика) были распределены ещё на две подгруппы: 1 – группа сравнения – 135 больных (2009–2014 гг.), которым применены стандартные меры по предоперационной подготовке; 2 – основная группа – 82 больных (2015–2017 гг.), при лечении которых применён усовершенствованный алгоритм выбора оптимального способа хирургического лечения.

Результаты исследований и их обсуждение. После применение предложенных разработок и инновационных подходов качество жизни больных с послеоперационными вентральными грыжами, перенесших герниопластику, в основной группе улучшилось в отличие от пациентов группы сравнения.
Клінічний випадок синдрому позиційного стиснення
Authors: Herasymchuk P. O. , Fira D. B. , Zghryvets Y. O.
Published: October 2019

У публікації описано випадок синдрому позиційного стиснення обох кистей і передплічь, які досить рідко трапляються в клінічній практиці. Хворій виконано фасціотомічні розрізи з метою декомпресії уражених ділянок. Також застосовано вакуумтерапію негативним тиском на ділянку ураження, що сприяло позитивному ефекту загоєння рани. Отже, використання вакуумтерапії для лікування синдрому позиційного стиснення дозволяє стабілізувати патологічний процес та попередити розвиток місцевих і системних ускладнень.
Abstract 101: Identification of Novel MICU1 Interactors Independent of the mtCU Complex
Authors: Tomar Dhanendra , Garbincius Joanne F , Kolmetzky Devin , Thomas Manfred , Jadiya Pooja , Elrod John W
Published: October 2019

MICU1 is an EF-hand containing mitochondrial protein that gates the mitochondrial Ca
2+
uniporter complex (mtCU) and directly interacts with the pore-forming subunit MCU. Previous studies have shown perinatal lethality and altered mitochondrial architecture in MICU1 knockout (
Micu1
-/-
) mice, phenotypes that are distinct from other knockout models of mtCU components, such as MCU, and thus are likely not explained solely by changes in matrix [Ca
2+
] uptake. Further, our proteomic studies suggest that MICU1 exists in mitochondrial complexes void of MCU. This suggests that MICU1 may have cellular functions independent of mtCU regulation. To discern novel MICU1 molecular interactors we employed a biotinylation-based proteomic approach in

Mcu
-/-

and
Micu1
-/-
cells to detect proteins interacting with MICU1 using fusion protein containing BioID2, a small biotin ligase for proximity-dependent labeling. Expression of MICU1-BioID2 in

Mcu
-/-

cells allowed the identification of mtCU-independent interactors. Fast protein liquid chromatography (FPLC), blue native-PAGE, co-immunoprecipitation, live-cell Ca
2+
imaging, confocal and super-resolution imaging methods were used to confirm novel roles for MICU1 in mitochondrial biology. LC-MS analysis of biotinylated proteins after avidin-based purification identified the Mitochondrial Contact Site and Cristae Organizing System (MICOS) components IMMT, CHCHD2, and CHCHD3 as interacting with MICU1 (MICU1-BioD2 expressed in Micu1
-/-
cells to avoid aberrant expression). These same MICOS components were identified in MCU
-/-
cells, suggesting that MICU1 could be involved in the regulation of MICOS independent of the mtCU. Further, the deletion of CHCHD2 resulted in the loss of MICOS and cristae disorganization without any observable effect on
m
Ca
2+
uptake. RNA sequencing revealed correlative expression changes in MICU1 and MICOS components in response to heart failure progression during transverse aortic constriction and myocardial infarction. These results suggest MICU1 likely serves cellular functions independent of the mtCU and may serve as a key regulator of Ca
2+
-dependent signaling (EF-hands) in other cellular processes that are dysregulated during heart failure.
Abstract 104: Identification and Characterization of a
Titin
Enhancer using CRISPR/Cas9 Genome Editing and hiPSC-Derived Cardiomyocytes
Authors: Neyazi Meraj , Schmid Manuel , Sharma Arun , Toepfer Christopher N , Kim Yuri , Wasson Lauren K , Kim Seong Won , DeLaughter Daniel M , Willcox Jon A , Agarwal Radhika , Tai Angela , Gorham Joshua M , DePalma Steven , Seidman Jonathan G , Seidman Christine E
Published: October 2019

Dilated cardiomyopathy (DCM) is a leading cause for heart failure and is associated with a rate of mortality of 20% within 5 years of diagnosis. The most common genetic causes for DCM are mutations of the sarcomere protein titin (encoded by
TTN
), which occurs in 10-20% of DCM cases. Dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency. Thus, strategies to increase the expression of the wild type
TTN
allele could attenuate damaging effects of TTNtv. Utilizing bioinformatic tools, we identified a putative enhancer for
TTN
in its intron 1. We deleted a 658 bp region from intron 1 which encompasses the region of interest in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing to validate its function. Utilizing RNA sequencing and qPCR of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs), we demonstrated that a homozygous deletion in this region leads to a drop in
TTN
expression compared to the wild type (WT) control (0.344-fold change, p < 0.001). To further characterize this region, we subdivided it into three parts which we called E1 (296 bp), E2 (206 bp), and E3 (139 bp). E1 includes a highly conserved region and a region of open chromatin as identified by the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) performed on hiPSC-CMs. A homozygous E1 deletion resulted in a decreased
TTN
expression of 0.63-fold compared to the WT control (p < 0.001) when performing RNA sequencing on hiPSC-CMs. Both homozygous E2 and E3 deletions resulted in an increased
TTN
expression (1.56-fold change, p < 0.001; 1.19 fold change, p < 0.001). Utilizing a published sarcomere tracking platform, SarcTrack, to investigate hiPSC-CM physiology, we saw a decreased contractility of 6.6% in hiPSC-CMs carrying a homozygous E1 deletion compared to 10.1% in the WT control (p < 0.001). Cells carrying homozygous E2 or E3 deletions were hypercontractile (13.8%, p < 0.001; 13.7%, p < 0.001). Given our results, we hypothesize that
TTN
expression depends on the E1 region. If confirmed, we expect that increasing the activity of this enhancer using small molecules may provide a novel therapeutic target for DCM caused by TTNtv.
Abstract 105: MCL-1 Promotes Drp1-Mediated Mitochondrial Fission as an Adaptive Response to Stress
Authors: Moyzis Alexandra G , Lally Navraj S , Najor Rita A , Leon Leonardo J , Gustafsson Åsa B
Published: October 2019

The anti-apoptotic BCL-2 family member, Myeloid Cell Leukemia-1 (MCl-1), is highly expressed in myocardium and plays a critical role in maintaining mitochondrial homeostasis. We have previously found that cardiomyocyte-specific MCL-1 knockout mice develop rapid cardiac dysfunction and cardiomyopathy but show little activation of apoptotic cell death. Instead, loss of MCL-1 resulted in atypical mitochondrial morphology and function. This suggests that besides its anti-apoptotic role, MCL-1 may have broader functions in regulating mitochondrial dynamics and function. MCL-1 localizes to two different mitochondrial locations in myocytes. One form exists on the outer mitochondrial membrane (MCL-1
OM
) and a shorter cleaved form resides in the mitochondrial matrix (MCL-1
Matrix
). We found that overexpression of MCL-1
WT
or MCL-1
OM
, but not MCL-1
Matrix
, induces fragmentation and perinuclear aggregation of the mitochondria in a Drp1-dependent manner. Mutating MCL-1’s BH3 domain (G198E D199A), which is required for MCL-1’s anti-apoptotic function, completely abrogates its ability to induce perinuclear aggregation. Interestingly, a MCL-1-BCL-2 chimera, in which MCL-1’s BH domains are replaced with those of BCL-2, is still able to induce perinuclear aggregation. This suggests that the presence of a functional BH3 domain is sufficient for induction of perinuclear aggregation. We confirmed that there is increased interaction between endogenous MCL-1 and Drp1 in response to a variety of fission-promoting stimuli, including glucose deprivation, hypoxia, and treatment with rotenone or FCCP. MCL-1 overexpression also protects against cell death in response to these stimuli, but this protection is abrogated when Drp1 is knocked down using siRNA. Additionally, Drp1 levels are significantly increased at the mitochondria in the hearts of MCL-1
OM
transgenic mice, and this increase corresponds to an increased interaction between MCL-1 and Drp1 in these transgenic mice. Consistent with these findings, many of the mitochondria in these transgenic mice appear to be smaller than those from the WT mice, indicative of enhanced mitochondrial fission. Thus, our data suggests that MCL-1 functions as a positive regulator of Drp1-mediated mitochondrial fission.
Abstract 116: Inhibition of Autosis Attenuates Ischemia/reperfusion Injury in the Heart
Authors: NAH Gihoon , Fernandez Alvaro , Zhai Peiyong , Levine Beth , Sadoshima Junichi
Published: October 2019

Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. Recent studies provided evidence that excessive autophagy induces cell death with characteristic morphological and biochemical features, termed autosis. However, whether autophagy contributes to death in cardiomyocytes (CMs) is controversial. We here show that inhibition of autosis by targeting Rubicon attenuates ischemia/reperfusion (I/R) injury in the heart. Treatment with TAT-Beclin1 (TB1), a peptide that mobilizes endogenous Beclin 1, increased autophagic flux and induced cell death in CMs, which was inhibited by inhibitors of autophagy but not apoptosis or necrosis. CMs treated with TB1 at high doses showed typical morphological and biochemical features of autosis, namely an increased number of autophagic vacuoles with ballooning of perinuclear spaces and rescue of cell death by downregulation of Na
+
,K
+
-ATPase. To examine whether autosis is activated by I/R in the heart
in vivo
, mice were subjected to 30 minutes of ischemia followed by 24 hours of reperfusion, and autophagic flux and morphological features of CMs were examined at several time points. Autophagic flux was increased during ischemia and the early phase of reperfusion; however, more autophagosomes started to accumulate 6 hours after reperfusion due to suppression of autophagosome maturation, which was caused by increased Rubicon expression. CMs exhibited typical morphological features of autosis and Na
+
,K
+
-ATPase was significantly upregulated starting 6 hours after reperfusion. Injection of ouabain into cardiac glycoside- sensitive knock-in mice exhibited protection against I/R injury with decreased features of autosis. To normalize autophagic flux during I/R, we generated cardiac-specific Rubicon knockout (
rubi
-cKO) mice.
rubi
-cKO prevented the marked accumulation of autophagosomes and significantly reduced the size of infarct following I/R, with prominent suppression of autosis. These results indicate that autosis is triggered by dysregulated autophagic flux due to upregulation of Rubicon during the late phase of I/R in the heart and that inhibition of autosis protects the heart against I/R injury.
Abstract 118: Reduced Cardiac Transmembrane Protein 65 Resulted in Dilated Cardiomyopathy and Progressive Cardiac Fibrosis
in vivo
Authors: Teng Allen C , Gu Liyang , Di Paola Michelle , Fatah Meena , Chatterjee Diptendu , Kislinger Thomas , Hamilton Robert , Gramolini Anthony O
Published: October 2019

Intercalated discs (ICDs) are unique and functionally indispensible to the heart, but its structural organization remains less understood. Previously, we showed that an ICD-bound transmembrane protein 65 (Tmem65) was required for Connexin 43 (Cx43) localization in cultured mouse neonatal cardiomyocytes, and that reduced Tmem65 was associated with a decrease and internalization of Cx43, and impaired electrical conduction between neighboring cardiomyocytes. Here, we investigated the role of Tmem65
in vivo
by injecting CD1 mice with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 (or scrambled) shRNA. Quantitative polymerase chain reactions and immunoblots confirmed greater than 90% reduction in Tmem65 expression in mouse ventricles compared to control samples. Immunoblots and immunofluorescence showed reduced and internalized Cx43 in Tmem65 knockdown (KD) hearts compared to controls, respectively. Kaplain-Meier survival plot showed that all Tmem65 KD mice died within 7 weeks (> 50% death 3 weeks post viral injection), whereas no death was seen in control mice. Tmem65 KD mice developed eccentric hypertrophic cardiomyopathy in 3 weeks and dilated cardiomyopathy with severe cardiac fibrosis in 7 weeks as confirmed by H&E and Masson’s Trichrome staining. Echocardiography also confirmed ventricular dilatation and showed a 60% reduction in cardiac output (19.27±1.46 mL/min in control vs. 6.63±0.52 mL/min in Tmem65 KD mice, p<0.01, n = 6 per group) at 7 weeks. Transmission electron microscopy showed an altered ICD pattern and disorganized myofibril structure in Tmem65 KD ventricles while control tissues had smooth ICDs and organized myofibers. Together, these findings suggest a critical role of Tmem65 in maintaining cardiac ICD and myofibers and loss of Tmem65 leads to cardiomyopathy
in vivo
.
Abstract 131: Cardiomyocyte Renewal and Cardiac Outcomes Following Injury in Young Swine
Authors: Agnew Emma J , Velayutham Nivedhitha , Moore Victoria , Alsaied Tarek , Baker Richard Scott , Riggs Kyle W , Zafar Farhan , Yutzey Katherine E
Published: October 2019

Objective:
Infants with severe congenital heart defects (CHD) typically require lifesaving cardiac surgery. Performing this surgery during a time when the myocardium can proliferate and repair could improve successful outcomes. The mouse heart can regenerate in the first week post-partum, but the mitotic activity of cardiomyocytes (CM) is extended to 1-2-months post-partum in the pig. Thus, the regulation of CM cell cycling ability and renewal in the hearts of young large mammals after injury was examined.

Methods & Results:
Pigs at postnatal day 30 (n=6) were subjected to cardiac ischemia (1-hour) by temporary occlusion of the left anterior descending (LAD) artery followed by reperfusion (IR), or to sham operation (n=6, no LAD occlusion). LAD occlusion, below the second diagonal branch, provided an effective injury as indicated by increased circulating cardiac troponin-I 2-hours after injury. In addition, ejection fraction (EF) decreased by 46% (57% to 31%) at 2 hours post-ischemia, which was then maintained to the 4-week study end point. Pigs were sacrificed 4 weeks after surgery and histology demonstrated evidence of scar formation in the area of injury. However, no change in number of proliferating CM or cell death (via pHH3 or TUNEL immunohistochemistry respectively) was detected in IR pigs vs sham, or between regions of the left ventricle.

Conclusions:
Here we report a successful ischemic injury method, at an age previously not reported in swine. Pigs did not continue to decline towards heart failure following an IR injury at 1 month of life, with preservation of EF up to 4-weeks post-injury. This is also without a change in CM cell cycling activity at 2 months of age. This study highlights that even in the presence of a scar, young, large mammals can adapt to cardiac injury to maintain cardiac function. The pathways regulating scar formation and CM cell cycling are being further investigated by RNA-seq studies. If similar mechanisms operate in humans, it may be beneficial to perform CHD surgeries at a younger age when the heart is able to better tolerate injury, to ultimately improve long term outcomes.
Abstract 132: Imaging-Based Assay for Screening of Cell Cycle Modifying Substances in Postnatal Cardiomyocytes
Authors: Becker Cora , Carillo Garcia Carmen , Freitag Patricia , Schade Dennis , Fleischmann Bernd K , Hesse Michael
Published: October 2019

Cardiovascular diseases are the main cause of death in the industrial world. Especially large myocardial infarctions and the potentially resulting left ventricular dysfunction are chronical diseases with a poor prognosis. Due to limited endogenous cardiac repair capacity, we are investigating the regulation of the cell cycle in cardiomyocytes (CMs) as a potential treatment strategy. We hypothesize that controlled induction of cardiomyocyte proliferation will lead to an increase in muscle mass and improvement of cardiac function. We are using CMs from double transgenic αMHC-H2B-mCherry/CAG-eGFP-anillin mice for the analysis of postnatal CM development. This double transgenic system enables us to unequivocally identify CM nuclei as well as cell cycle variations like acytokinetic mitosis (mitosis without cytokinesis), resulting in binucleated CMs, and endoreplication (mitosis without cytokinesis and karyokinesis), leading to polyploid CMs. With the goal of inducing authentic cell division, we are screening for pro-proliferative substances in P1 and P6 postnatal CMs of αMHC-H2B-mCherry/CAG-eGFP-anillin mice. Here we will present the detailed analysis of several screening hits demonstrating an increase in cell cycle activity leading mainly to binucleation but only to limited cell division in CMs.
Abstract 138: A Novel Cellular and Genetic Approach to Investigate the Cardioprotective Role Played by Endothelial Nitric Oxide Synthase in Myocardial Infarction
Authors: Gentile Carmine , Kesteven Scott , Wu Jianxin , Bursill Christina , Davies Michael J , Figtree Gemma
Published: October 2019

The loss of regenerative properties in adult cardiomyocytes (CMs) is directly linked to their inability to proliferate. Following an extensive ischaemic event in an aged heart, fibrotic scar formation is the only repair process and eventually heart failure develops. However, molecular and cellular cues in the neonatal heart support that cardiac regeneration is possible in presence of proliferating CMs. Based on previous studies demonstrating that endothelial nitric oxide synthase (eNOS) regulates proliferation in both endothelial cells (ECs) and CMs, we hypothesized that eNOS signaling could play a cardioprotective role. To test our hypothesis, we injected different combinations of co-cultured ECs and CMs in the LV muscle wall of MI mice (permanent LAD ligation). First, injected cells were isolated from either WT or KO eNOS neonatal mice and then co-cultured to form 3D vascularized cardiac spheroids (VCSs), which were eventually transplanted in adult MI mice on the day of the procedure. Control infarcted animals received media-only (vehicle). Other mice received a suspension of co-cultured VCSs in media as follows:
i
) WT CMs and ECs;
ii
) WT CMs and KO ECs;
iii
) KO CMs and WT ECs. Following 28 days, injection of WT cells increased the ejection fraction (EF%) by 20% compared with control animals (61%±4% and 41%±11%, respectively). When eNOS was absent in either CMs or ECs, the EF% was 40%±5% and 46%±2%, respectively, suggesting that the eNOS-mediated protection is dependent on its presence in both cells. Histological analyses confirmed the presence of WT VCSs in MI mice, contributing to a thicker wall thickness compared to vehicle MI mice. No VCSs were observed in the LV wall when KO cells were injected. Therefore, our results strongly suggest that eNOS may play a major role via both an autocrine (CMs) and paracrine (ECs) mechanism. Current studies are focusing on further evaluating the mechanism(s) for this eNOS-mediated protective role. To our knowledge, this is the first study combining cellular and genetic approaches to evaluate the cardioprotective role of eNOS in the heart. A better understanding of this mechanisms may have significant impact for the development of improved molecular and cell therapeutics (including stem cells) for heart failure patients.
Abstract 144: Ultrasound Mediated Transfection of SERCA2a and Cx43 Genes Assisted Bone Marrow Stem Cells Transplantation to Improve Heart Failure and Ventricular Arrhythmia After Myocardial Infarction
Authors: Mu Yuming , Wang Wei , Tayier Baihetiya , Guan Lina
Published: October 2019

Objective:
We aimed to investigate the possibility of combining BMSCs transplantation (BMSCs T) with sarcoplasmic reticulum Ca2+ATase (SERCA2a) and connexins 43 (Cx43) genes transfection mediated by ultrasound-targeted microbubble destruction (UTMD) to treat heart failure (HF) and ventricular arrhythmia (VA) after MI.

Methods:
HF rats received BMSCs T 4 weeks after AMI. Two days later, microbubbles carried SERCA2a and Cx43 genes were injected via tail vein, then ruptured by irradiation. 63 rats were divided into 7 groups : 1)SHAM; 2)HF; 3)UTMD; 4)BMSCs + UTMD; 5)BMSCs + UTMD-SERCA2a; 6)BMSCs + UTMD-Cx43; 7)BMSCs+ UTMD-SERCA2a/Cx43-1:1. Left ventricular ejection fraction (LVEF), cardiac tolerance to electrical stimulation were examined 28 days after BMSCs T.

Results:
The LVEF of gene transfection groups were improved than before: BMSCs + UTMD-SERCA2a (39.6±3.6% vs. 34.3±4.3%); BMSCs + UTMD-Cx43 (40.1±5.6% vs. 35.1±5.2%); BMSCs + UTMD-SERCA2a/Cx43-1:1 (41.7±6.8% vs. 35.1±5.2%) (P < 0.05). Stimulating with 7V voltage, the frequencies of inducing VA in BMSCs + UTMD-SERCA2a, BMSCs + UTMD-Cx43 and BMSCs + UTMD-SERCA2a/Cx43-1:1 were 13.07±4.77Hz, 13.94±1.29Hz and 13.97±1.91Hz, which were higher than HF and non-gene groups (P < 0.05). With 8V, the frequencies in BMSCs + UTMD-Cx43 and BMSCs+ UTMD-SERCA2a/Cx43-1:1 were also higher than other groups but SHAM (P < 0.05). The frequency of double-gene group was higher than single-gene groups with 8V (P < 0.05).

Conclusion:
Our results suggest that BMSCs T assisted by UTMD-mediated SERCA2a and Cx43 genes transfection can improve HF and VA after MI, and especially for the stability of cardiac electrical activity with double-gene.
Abstract 146: Investigating and Inhibiting Neutrophil Extracellular Trap Formation in the Heart
Authors: Sayegh Michael N , Wang Lanfang , Shin Eric Y , Han Woojin M , Brown Milton E , Davis Michael E , Garcia Andres J , Levit Rebecca D
Published: October 2019

Neutrophil extracellular traps (NETs) have been observed in multiple diseases of the cardiovascular system, such as myocardial infarction and atherosclerosis. We have recently shown that adenosine (ADO) inhibits NET formation and may be an important endogenous regulator. CD73 is expressed on many cell types and catalyzes the extracellular formation of ADO from AMP. Here, we first attempt to implicate NETs in cardiac injury, and we posit that human NETs negatively impact cardiac function by influencing native leukocyte recruitment and activation. Second, we prototype a CD73-functionalized hydrogel delivery vehicle for adenosine and test its function in vitro. We introduced NETs obtained from stimulated neutrophils into healthy hearts by intramyocardial injection in two-month-old rats. We used echo to assess cardiac function at days 0, 1 and 3 of injection. We measured the abundance of cardiac leukocyte subpopulations by flow cytometry at days 1 and 3 after injection, with immunostaining for CD45 (all leukocytes), myeloperoxidase (MPO, neutrophils), CD68 (macrophages), CD3 (T-cells), B220 (B-cells) and citrullinated histone 3 (citH3, NETs). To deliver adenosine as a NET antagonist, we designed a polyethylene (PEG) hydrogel composed of a 4-armed PEG incorporating a VPM protease-degradable crosslinker for cargo release, and an RGD peptide to enhance gel-tissue attachment. We tested gel polymerization by measuring storage and loss moduli at 4% and 6% PEG content (w/v) and 0, 1 uM and 1mM RGD concentrations. We functionalized the gel with CD73 to test its adenosine production capability. We found a significant decreased in cardiac function assessed by global longitudinal strain at day 1 in the NET group compared to saline. By flow cytometry, we found an increase in neutrophils, macrophages and NET formation at day 1, while B- and T-cells were increased at day 3. We found that increasing the PEG content of our hydrogel, but not RGD, increases gel stiffness. We also found that the CD73 functionalized hydrogel successfully catalyzes the formation of adenosine in vitro. In conclusion, we found that NETs negatively impact cardiac function, and successfully tested an adenosine hydrogel source in vitro. Next, we will study the effect of our gel on NETs in vivo.
Abstract 147: Screening for Developmental and Injury-Induced Genes That Facilitate Heart Regeneration
Authors: Shah Akansha M , Cui Miao , Wang Zhaoning , Tan Wei , Liu Ning , Bassel-Duby Rhonda , Olson Eric N
Published: October 2019

The adult mammalian heart has a limited capacity to regenerate upon injury, such as myocardial infarction (MI). MI in adult mouse hearts results in a reduced cardiac function, due to extensive loss of cardiomyocytes and formation of scar tissue by activated fibroblasts. In contrast, the newborn postnatal day 1 (P1) mouse heart is able to regenerate and restore its cardiac function following injury. However, this regenerative ability dramatically declines after P7. The molecular properties and mechanisms that facilitate neonatal heart regeneration remain unknown. To gain further insight into the realm of cardiac regeneration, our lab performed transcriptome analysis by bulk and single cell RNA-seq on P1 and P8 hearts at various time points following MI surgery. We also performed enhancer profiling by H3K27ac ChIP-seq. Coupling of both these approaches has uncovered two different gene sets that that may promote the regenerative ability of neonatal hearts: developmental genes and injury-induced genes. Amongst the neonatal-specific developmental genes, Igf2bp3, a RNA-binding protein, and Hic2, a transcriptional regulator, have been shown to promote neonatal rat ventricular cardiomyocyte (NRVM) proliferation and are currently being characterized for their involvement in neonatal heart regeneration by using gain-of-function and loss-of-function in vivo approaches. Amongst the injury-induced genes, we identified mitogens secreted from macrophages that promote NRVM proliferation. This is in line with the differential immune response elicited by P1 hearts upon MI-injury. We have also shown that an epicardial secreted factor in P1 hearts can enhance human umbilical vein endothelial cell tube formation, indicative of its role in angiogenesis. In summary, by using a multi-layered genomic analysis, we identified various genes that are involved in different cellular responses during neonatal heart regeneration. This comprehensive analysis of the neonatal regenerative response provides insights into the molecular basis of neonatal heart regeneration, and can potentially identify genes that can be modulated to facilitate adult heart regeneration.
Abstract 149: Endothelial-specific Overexpression of Metallothionein Prevents Diabetes Mellitus-induced Impairment in Ischemia Angiogenesis via Preservation of Hif-1á/sdf-1 in Endothelial Progenitor Cells
Authors: Wang Kai , Dai Xiaozhen , He Junhong , Yang Chengkui , Wintergerst Kupper , Epstein Paul , Cai Lu , Tan Yi
Published: October 2019

Diabetes mellitus associated dysfunction of endothelial progenitor cells (EPCs) may contribute to dysregulation of endothelial regeneration. Whether oxidative protection of EPCs by an antioxidant, such as the protein metallothionein (MT) can have positive angiogenic effects and by what mechanisms remain unclear. Endothelial-specific MT overexpression (JTMT) mice were made diabetic by high fat diet followed by streptozocin administration (HFD/STZ). Diabetic hind limb ischemia (HLI) was established by femoral artery ligation. Bone marrow mononuclear cells (MNCs) collected from JTMT mice were transplanted into db/db mice with HLI. Blood reperfusion was monitored. High glucose and hypoxia conditions in culture were adopted to mimic diabetic ischemia. Compared with wild-type (WT) littermates, JTMT mice were resistant to diabetes-induced impairment in ischemia angiogenesis and blood reperfusion in HFD/STZ diabetes. Similarly, transplantation of MT overexpressing MNCs showed better therapeutic effect on db/db mice with HLI than transplantation of WT MNCs. These changes were accompanied by increased mobilization and infiltration of EPCs in JTMT mice, and enhanced incorporation of EPCs into capillaries in JTMT MNCs transplanted db/db mice. Furthermore, EPCs from JTMT mice exhibited augmented cell survival, tube formation, and migration capacities under diabetic ischemia-like conditions. Mechanistically, the expression of hypoxia-inducible factor 1α (HIF-1α) and the secretion of stromal cell-derived factor (SDF-1) in blood and expression in ischemic tissues were upregulated in JTMT and JTMT-MNC transplanted db/db mice, and in cultured JTMT EPCs, which were accompanied by marked amelioration of oxidative stress. However, MT-mediated elevation of SDF-1 and improvements of function in EPCs were all abrogated by siRNA knockdown of HIF-1α expression without effect on the anti-oxidative capacity of MT. Endothelial-specific MT elevation is sufficient to protect against diabetes mellitus-induced impairment of ischemia angiogenesis by promoting EPC function. The benefits of MT are predominantly mediated by upregulation of the HIF-1α/SDF-1 pathway.
Abstract 160: Atg7-Dependent Activation of Mitochondrial Autophagy in Cardiomyocytes
Authors: Abdullah Chowdhury S , Aishwarya Richa , Alam Shafiul , Morshed Mahboob , Miriyala Sumitra , Panchatcharam Manikandan , Bhuiyan Md. Shenuarin
Published: October 2019

Introduction:
Autophagy-related protein 7 (Atg7) has been studied for its role in cellular macroautophagy induction and regulation in different cell systems. We have previously reported that Atg7 regulates cardiac macroautophagy. However, whether Atg7 plays any role in the initiation and regulation of mitochondrial autophagy (mitophagy) in cardiomyocytes remains elusive.

Objective:
We investigated subcellular localization and molecular function of Atg7 in the regulation of mitophagy through a series of
in vitro
and
in vivo
experiments.

Methods and Results:
We isolated mitochondria from mouse hearts by subcellular fractionation and conducted biochemical experiments which revealed localization of Atg7 on the mitochondrial membrane. Next, we co-stained for Atg7 in isolated heart mitochondria stained with Mitotracker dye and found Atg7 localizes to mitochondrial membrane. To elucidate molecular function of Atg7 on mitophagy, adenovirus-mediated Atg7 overexpressed cultured neonatal rat cardiomyocytes (NRCs) were treated with mitophagy inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP)
in vitro
and stained with organelle-specific mitochondria and lysosome dyes to count mitophagy fluorescent dots by confocal microscopy. Atg7 overexpressed NRCs exhibited an increase in mitophagy dots than β-gal treated NRCs following CCCP induction. Further, we have treated cardiac specific Atg7 overexpressed (Atg7 Tg) mice and littermate non-transgenic (Ntg) mice with CCCP to assess mitophagy induction
in vivo
. Immunoblotting confirmed enhanced accumulation of mature autophagolysosomal marker LC3-II protein in mitochondrial fraction from Atg7 Tg hearts.

Conclusions:
Our
in vitro
and
in vivo
findings indicate that Atg7 localizes to the mitochondrial membrane, regulates recruitment and initiation of mitophagy machinery, and facilitates mitophagy in cardiomyocytes.
Abstract 161: Il4 Increases Phagocytosis of Necrotic Cells in a Cd36-dependent Manner
Authors: Chen Mingzhuang
Published: October 2019

Macrophages are a population of immune cells with high heterogeneity, which can acquire distinctive phenotypes under different stimuli, and each subtype has specific function in immune surveillance and tissue homeostasis. Different with traditional activated macrophages, which are mainly primed by pathogen motifs or inflammatory stimuli and are involved in pathogen clearance and immune defence, alternatively activated macrophages by Th2 cytokine IL4 have been shown to be functioning in cell debridement, promising a crucial role in tissue repair and tissue homeostasis. However, the mechanisms underlying how IL4 signaling promotes cell debridement have not been fully understood. To address this question, we aim to investigate whether and how IL4 signaling promotes necrotic cell clearance, which is the primary source of cell debris and share similar biological properties. In our study, we demonstrated that IL4 enhances phagocytosis of necrotic cells, and the downstream IL4Rα-STAT6 axis signaling pathway is involved. We also found that CD36, a class B scavenger receptor, may be, at least to some extent, responsible for IL4-accelerated phagocytosis. Beside increasing engulfment of necrotic cells, IL4 treatment also enhances the processing of engulfed material, indicating multi-dimensional effects of IL4 on phagocytosis activity of macrophages. By uncovering the molecular mechanisms of IL4 promoting phagocytosis activity of macrophages, we are able to better understand the role of IL4 signaling in cell debridement, and this may help to develop new strategies in promoting tissue repair and regeneration in chronic diseases.
Abstract 171: The Effects of Adolescent Binge Alcohol Exposure to Cardiovascular Structure and Function
Authors: Ai Lizhuo , Perez Edith , Heroux Maxime , Zlobin Andrei , Asimes AnnaDorothea , Cao Quan , Chung Charles S , Pak Toni R , Kirk Jonathan A
Published: October 2019

While the effects of alcohol consumption on the adult heart have been well studied, the impact to the adolescent heart is almost entirely unknown. Adolescents primarily consume alcohol in a binge pattern, which elevates blood alcohol content (BAC) to 0.08 g/dL within 2 hours. During adolescence the body grows rapidly, and the heart must also grow (through cellular hypertrophy) to meet this increasing demand. Our goal was to determine the impact of adolescent binge alcohol exposure on the heart, using an outbred rat model.

On postnatal day (PND) 37 (adolescence), rats were gavaged 3 g/kg EtOH for 3 days, H2O for 2 days, and EtOH for 3 more days, then sacrificed following the last dose (Binge). The control group received only H2O (Water). Both groups had normal food and water intake and weight gain. BAC was 0.08 g/dL in the Binge group and 0 in the Water group. Water rats experienced a ~40% increase in LV end-diastolic volume (LVEDV) due to normal growth along with a parallel increase in end-systolic volume such that ejection fraction (EF) was preserved. Conversely, Binge rats only had half that increase in LVEDV, and a decrease in systolic diameter resulting in increased EF. We hypothesize alcohol impaired normal cardiac growth, and systolic function increased to maintain cardiac output. Increased systolic function suggested beta-adrenergic activation, which was supported by increased troponin I phosphorylation at the PKA sites in the Binge group.

Doppler indicated the E/A ratio increased with age in the Water group, but in the Binge group the E/A ratio did not change with age. Further, the Binge group displayed increased single cell passive stiffness level (sarcomere length: 1.7-2.4 μm) compared to Water, likely due to altered titin phosphorylation. Lastly, we performed RNA-seq and identified 58 down-regulated and 10 up-regulated genes in the Binge group. Many of these genes suggest a switch in substrate utilization from fatty acid to glucose metabolism in the Binge rats.

These data reveal a previously unappreciated pathological impact of adolescent binge alcohol exposure. The young heart can compensate for these consequences at first and appear healthy. However, the long term impact of these effects may be significant and whose underlying cause was previously unknown.
Abstract 173: Development of an Auto-Segmentation Technique Using a Convolutional Neural Network for the Segmentation of the Ventricular Cavity in Zebrafish
Authors: Teranikar Tanveer Ashwini , Ma Xin , Bailey Zachary , Lim Jessica , Gaire Bijan , Hirsch Martin , Kim Won Hwa , Lee Juhyun
Published: October 2019

Light sheet fluorescence microscopy (LSFM) with a synchronization algorithm is a unique imaging technique that can be used to image the beating zebrafish heart in 4-dimensions (4-D). However, natural arrhythmic diastolic and systolic contraction and relaxation of the heart can cause aberrations in 4-D image reconstruction. These aberrations are observed as blurred lines, spikes, or holes in the myocardium of the heart, which can lead to complications when analyzing biomechanics by computational fluid dynamics (CFD). In this study, we imaged a beating zebrafish heart at 4 days post fertilization (dpf) using a 10x objective lens with our single-sided LSFM system scanning with a 2 μm step size. The obtained images were analyzed in 3-D to ensure that there were limited aberrations in the wall of the myocardium. Then, the ventricular cavity of these 3-D images was hand segmented at various time points that were spaced within two cardiac cycles. These hand segmented images were used to train a convolutional neural network (CNN) which would have the capability of performing segmentation of the ventricular chamber automatically. This auto-segmentation tool was then used to segment the ventricular cavity of ten samples contained within one cardiac cycle that had not previously been used for training purposes. The ventricular volume of these ten images calculated after auto-segmentation was found to be comparable to the images when hand segmented. Then, a Gaussian wavelet filter was used after auto-segmentation to remove aberrations and minimize errors in the CFD simulation. Both the auto-segmented images and the hand-segmented images were analyzed using a CFD solver to simulate the hemodynamic shear stress through the cardiac cycle. The results from these simulations showed no significant difference between shear stress when the images were hand-segmented or auto-segmented, and there was no leakage observed between the two sets. The results using the auto-segmentation code are promising for future applications in cardiac research where low-quality images obtained under fast acquisition times can be enhanced without the traditional costly deconvolution techniques. This can lead to faster and more accurate analysis of the ventricular cavity for related diseases.
Abstract 175: Evaluation of Sepsis-Related Cytokines on Endothelial Cell Permeability
Authors: Campbell Carmen E , Boisvert William A
Published: October 2019

The endothelium plays a critical role in the progression from sepsis to organ failure because endothelium lines all blood vessels and organs in the body to provide a selective barrier. The endothelium is responsible for dynamically regulating the passage of small molecules, fluid, and cells between the bloodstream and tissues. In sepsis, many factors play a role in compromising the endothelium. Dysfunctional endothelium results in development of capillary leak, tissue hypoperfusion, hypoxia, edema, and shock. This study evaluates the individual contributions immunomodulatory cytokines as well as a potential synergistic effect of cytokine combinations on vascular endothelial permeability. Major components of intercellular junctions including tight junction and associated cell adhesion molecules are also assessed microscopically. Specifically, endothelial cell monolayer response to an array of sepsis-related factors including TNF-alpha, IL-1b, IL-6, IL- 8, IL-10, IL-12, IFN-g, and TGF-b by a novel approach using the in-vitro Electric Cell-substrate Impedance Sensing (ECIS) biosensor model is presented.
Abstract 180: Blood Outgrowth Endothelial Cell-derived Exosomes Mediate Therapeutic Neovascularization
Authors: Janssens Stefan P , Wibowo Arief , Gillijns Hilde , Caluwe Ellen , Veltman Denise , Delrue Leen , Bartunek Jozef
Published: October 2019

Background:
Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models. We hypothesized that BOECs promote angiogenesis via secretion of exosomes, extracellular vesicles with an important role in cell-to-cell communication.

Methods:
We derived exosomes from BOECs of patients with ischemic heart failure (HF with LVEF <35%) and age-matched controls (CON) in normoxia and 1% O
2
hypoxia by differential ultracentrifugation and used nano tracking (NTA) and immunoblot analysis to identify size, number, and surface markers. We characterized protein and miRNA content by proteomic LC-MS-Orbitrap analysis of trypsin-digested exosomes and validated selected peptides using ELISA. We measured expression of pro-angiogenic miRNAs and tested in vitro angiogenic potential of HUVECs in the presence or absence of exosomes using matrigel 2D-tube formation and 3D-spheroid sprouting assays.

We compared in vivo angiogenesis using a wound healing model in nude mice.

Results:
NTA showed higher exosome concentrations in hypoxia compared to normoxia (14.38±0.23x10E8 vs 12.05±0.23x10E8 particles/ml, n=7, P<0.001) with robust expression of exosome markers TSG101 and Flotilin-1. Compared to serum stimulation (positive control), BOEC-derived exosomes similarly enhanced 2D vascular network growth and 3D spheroid sprouting angiogenesis whereas absence of serum or exosomes did not (P<0.001). Bioinformatic proteome analysis identified 249 shared proteins in HF and CON, and 72 proteins exclusively present in HF with abundant expression of matricellular proteins and angiogenic growth factors. Exosomes derived in hypoxia contained significantly higher levels of pro-angiogenic miR-210-3p, miR-210-5p and miR-21-3p (P<0.05 vs normoxia, n=6). Closure of the skin wound after 4 and 5 days was significantly better in animals treated with BOEC-derived exosomes and accompanied with greater density of CD31-positive neovessels.

Conclusion:
BOEC-derived exosomes effectively induce vascular network formation and sprouting angiogenesis and stimulate wound healing in vivo. The identified protein and miRNA content in BOEC-derived exosomes may constitute a promising allogenic approach for therapeutic neovascularization.