bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of Human Thrombospondin-1 to the Pathogenesis of Gram-Positive Bacteria
Authors: Binsker Ulrike , Kohler Thomas P. , Hammerschmidt Sven
Published: February 2019

A successful colonization of different compartments of the human host requires multifactorial contacts between bacterial surface proteins and host factors. Extracellular matrix proteins and matricellular proteins such as thrombospondin-1 play a pivotal role as adhesive substrates to ensure a strong interaction with pathobionts like the Gram-positive Streptococcus pneumoniae and Staphylococcus aureus. The human glycoprotein thrombospondin-1 is a component of the extracellular matrix and is highly abundant in the bloodstream during bacteremia. Human platelets secrete thrombospondin-1, which is then acquired by invading pathogens to facilitate colonization and immune evasion. Gram-positive bacteria express a broad spectrum of surface-exposed proteins, some of which also recognize thrombospondin-1. This review highlights the importance of thrombospondin-1 as an adhesion substrate to facilitate colonization, and we summarize the variety of thrombospondin-1-binding proteins of S. pneumoniae and S. aureus.
Central Venous Stenosis after Hemodialysis: Case Reports and Relationships to Catheters and Cardiac Implantable Devices
Authors: Pacilio Mario , Borrelli Silvio , Conte Giuseppe , Minutolo Roberto , Musumeci Antonino , Brunori Giuliano , Veniero Patrizia , De Falco Vincenzo , Provenzano Michele , De Nicola Luca , Garofalo Carlo
Published: February 2019

The appropriate vascular access for hemodialysis in patients with cardiac implantable electronic devices (CIED) is undefined. We describe two cases of end-stage renal disease patients with CIED and tunneled central venous catheter (CVC) who developed venous cava stenosis: (1) a 70-year-old man with sinus node disease and pacemaker in 2013, CVC, and a Brescia-Cimino forearm fistula in 2015; (2) a 75-year-old woman with previous ventricular arrhythmia with implanted defibrillator in 2014 and CVC in 2016. In either case, after about 1 year from CVC insertion, patients developed superior vena cava (SVC) syndrome due to stenosis diagnosed by axial computerized tomography. In case 1, the patient was not treated by angioplasty of SVC and removed CVC with partial resolving of symptoms. In case 2, a percutaneous transluminal angioplasty with placement of a new CVC was required. To analyze these reports in the context of available literature, we systematically reviewed studies that have analyzed the presence of central venous stenosis associated with the simultaneous presence of CIED and CVC. Five studies were found; two indicated an increased incidence of central venous stenosis, while three did not find any association. While more studies are definitely needed, we suggest that these patients may benefit from epicardial cardiac devices and the insertion of devices directly into the ventriculus. If the new devices are unavailable or contraindicated, peritoneal dialysis or intensive conservative treatment in older patients may be proposed as alternative options.
The Immunological Association between Alopecia Areata and Respiratory Diseases: A Systematic Review
Authors: Sung Calvin T. , Choi Franchesca D. , Juhász Margit , Mesinkovska Natasha Atanaskova
Published: February 2019

Background: While alopecia areata (AA) has been associated with atopy, the immunological relationship is unclear, with the association of specific atopic and systemic respiratory diseases not established. The relationship between T-helper (Th)1-mediated AA and Th2-mediated atopy challenges the conventional Th1/Th2 paradigm of autoimmune disease categorization. Objectives: To determine the association between AA and atopic respiratory diseases in adults and children, and respiratory diseases in general. Method: All primary literature, excluding case reports, were identified within PubMed/MEDLINE, CINAHL, and Web of Science in May 2018 using the following search terms: “(alopecia OR hair loss) AND (respiratory OR pulmonary OR lungs OR asthma OR rhinitis OR bronchitis OR COPD OR atopy OR atopic).” Information from 32 articles meeting the inclusion and exclusion criteria was reviewed. Results: Among the 32 articles identified for inclusion, the prevalence of AA was more strongly associated with allergic rhinitis compared to asthma among pediatric and adult populations. While a significant association was identified between AA, allergic rhinitis, and a late age of onset, the association of AA and asthma remains controversial despite asthma’s prevalence among AA patients. No significant difference was identified with regard to the association of AA and non-atopic respiratory diseases between adult and pediatric patients. Conclusions: Adult and pediatric patients with AA warrant further workup for atopic respiratory diseases such as allergic rhinitis. AA may have an underlying Th2-mediated immunological component, which supports its association with atopic respiratory diseases and provides a new avenue for targeted therapies in select cases.
Developing Robust Standardised Analytical Procedures for Cannabinoid Quantification: Laying the Foundations for an Emerging Cannabis-Based Pharmaceutical Industry
Authors: Welling Matthew T. , Liu Lei , Hazekamp Arno , Dowell Ashley , King Graham J.
Published: February 2019

The plant genus Cannabis is a prolific producer of unique pharmaceutically relevant metabolites, commonly referred to as cannabinoids. Robust and standardised methods for the quantification of cannabinoids within botanical and drug forms is a critical step forward for an emerging Cannabis-based pharmaceutical industry, which is poised for rapid expansion. Despite a growing body of analytical methods for the quantification of cannabinoids, few have been validated using internationally accredited guidelines. Moreover, standardised methods have yet to be developed for application at various stages of manufacture as well as for different levels of processing and refinement. Validation parameters for establishing robust standardised methods for cannabinoid quantification within Cannabis-based drug forms are critically discussed. Determining an appropriate level of specificity (discrimination) among heterogeneous botanical matrices as well as evaluating accuracy (recovery) and inter-laboratory precision (reproducibility) within strict and volatile regulatory environments are potential obstacles to the establishment of robust analytical procedures. We argue that while some of these challenges remain unique to Cannabis, others are common to botanical-based drug development and manufacture. In order to address potential barriers to analytical method standardisation, a collaborative research initiative inclusive of academic and commercial stakeholders is proposed.
Abstract 107: The Mevalonate Pathway Controls Cardiomyocyte Proliferation
Authors: Hudson James E
Published: February 2019

Induction of cardiomyocyte proliferation is emerging as a promising strategy for heart regeneration. Using a recently developed human cardiac organoid system, we have recently discovered a small molecule (Compound 6.28) that inhibits both GSK3 and MST1 and drives robust proliferation (Mills et al., PNAS, 2017). However, chronic stimulation with this compound also causes a decline in contractile function, which would prevent its use as a therapeutic. We have now performed quantitative proteomics of 50 individual organoids under different conditions to de-convolute the effects of GSK3 and MST1 on the pro-proliferative response. GSK3 inhibition was found to be responsible for a cell cycle network whereas MST1 inhibition activated the mevalonate pathway; the detrimental functional effects were specifically associated with inhibition of GSK3. An additional screen of 105 compounds was performed to identify new compounds that could activate proliferation in human cardiac organoids without detrimental effects on functionality. We identified a compound targeting p38, which activated a similar cell cycle network to GSK3, and another compound targeting TGFBR/ACVR/BMPR, which activated the mevalonate pathway similar to MST1 inhibition. A strong synergistic pro-proliferative response was observed when both of these compounds were combined, without any detrimental effects on cardiac function. The pro-proliferative effect could be blocked by inhibition of β-catenin using ICRT14 or inhibition of the mevalonate pathway using a statin. In addition, we provide RNA-seq data revealing large scale transcriptional changes in the mevalonate pathway during heart maturation in vivo and demonstrate that blocking the mevalonate pathway in immature cardiomyocytes reduces proliferation. By re-supplementing different metabolites in the mevalonate pathway, a specific function for the prenylation branch of this metabolic pathway for proliferation was identified and direct inhibition of a downstream kinase activated by Rho prenylation, ROCK, also lead to a reduction in proliferation. Our results reveal a novel role for the mevalonate pathway in cardiomyocyte proliferation and suggest that inhibition of this pathway occurs during postnatal heart maturation.
Abstract 108: Molecular and Electrophysiologic Characterization of Obesity Mediated Atrial Fibrillation in
Mc4r-KO
Mice
Authors: Menon Ambili , McCauley Mark , Hong Liang , Sridhar Arvind , Perike Srikanth , Zhang Meihong , Lambers Erin , Darbar Dawood
Published: February 2019

Background:
Although a causal relationship between obesity and atrial fibrillation (AF) has recently been established, the underlying pathophysiological mechanisms remain unclear. Emerging evidence supports reduced cardiac Na
+
channel expression and current (I
Na
) as one potential contributing mechanism. Mutations in the melanocortin-4 receptor (
MC4R
) gene are the most common monogenic form of obesity in humans.

Objective:
We used a genetic
Mc4r-knockout, KO
mouse model of obesity to test the hypothesis that obesity increases susceptibility to AF by modulating the cardiac Na
+
channel.

Method:
Invasive electrophysiology studies and patch clamping of membrane potentials were performed in mice between 4-6 months of age to evaluate the molecular and whole-animal electrophysiologic phenotype.

Results:
Mc4r-KO mice were significantly heavier than wild-type lean controls. Pacing induced AF in 100% of Mc4r-KO mice, and 25% in controls; AF burden was more in Mc4r-KO mice than control mice. We showed that the expression of the cardiac Na
+
channel, I
Na
, and atrial action potential duration was reduced in Mc4r-KO mice. Furthermore, the oxidative stress biomarker F2-isoprostanes levels were elevated in MC4r-KO when compared to controls and these levels along with AF burden were reduced after 2 weeks of mitochondrial antioxidant (Mito-TEMPO) administration.

Conclusion:
We showed that mice with genetic susceptibility to obesity are not only more prone to develop AF but that this risk is mediated in part by modulation of the cardiac Na
+
channel and increased oxidative stress. These findings may have important implications for the management of obesity-mediated AF in humans.
Abstract 109: G Protein-coupled Receptor Kinase 2 Impairs Fatty Acid Metabolism in the Failing Heart Through Novel Mechanisms
Authors: Pfleger Jessica M , Gross Polina , Johnson Jaslyn , Gao Erhe , Houser Steven R , Koch Walter J
Published: February 2019

Compromised contractility and energetics are hallmarks of the failing heart. Increased G protein-coupled receptor kinase (GRK)2 is central to the pathogenesis of heart failure (HF), via desensitization of beta-adrenergic receptors and, therefore, loss of contractile reserve. Additionally, we, and others, have reported that GRK2 has non-canonical activity that contributes to its pathological functionality in HF. For example, GRK2 can compromise fatty acid (FA) metabolism in cardiomyocytes, the mechanism of which remains unknown. Thus, our aim is to investigate the role of GRK2 in FA metabolism and bioenergetics in the heart. To do this, we measured FA uptake, cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination, and FA-driven bioenergetics in mice with cardiac-specific overexpression of GRK2, to levels observed in the human failing myocardium, or a GRK2 inhibitory peptide (TgGRK2 and TgβARKct, respectively) or in global heterozygous GRK2 knockout (GRK2
+/-
) mice. Additionally, we determined CD36 expression and phosphorylation following cardiac dysfunction induced by transverse aortic constriction (TAC). The results show a significant reduction in FA uptake rates (0.3-fold) and CD36 protein (0.8-fold), which was associated with an increase in its phospho-serine/threonine (0.7-fold increase) and ubiquitin-lysine (0.7-fold) content in the TgGRK2 mice. In contrast, these parameters were unchanged in the TgβARKct or GRK2
+/-
mice. A reduction in CD36 mRNA (0.7-fold) and protein levels (0.2-fold) were detected in post-TAC, failing hearts, which was associated with an increase is its phosphorylation (0.5-fold). Notably, normalization of CD36 protein levels is observed in GRK2
+/-
mice post-TAC. Maximal respiratory capacity was also enhanced in TgβARKct and GRK2
+/-
cardiomyocytes (0.6- and 0.8-fold, respectively). Together, our results show that up-regulation of GRK2 induces CD36 phosphorylation, ubiquitination, and downregulation, which is associated with reduced FA uptake. Thus, we propose that the increase in GRK2 observed during HF is, at least partly, responsible for reduced FA uptake and oxidation and may be an important nodal switch in substrate utilization during HF pathogenesis.
Abstract 110: Numb Family Proteins Modulate Cardiac Morphogenesis by Regulating N-cadherin Trafficking to Plasma Membrane
Authors: Wu Mingfu
Published: February 2019

Numb, an endocytic adaptor protein, is essential for multiple biologic processes. We previously reported that the deletion of Numb and its homologue Numblike (collectively known as, Numb Family Proteins or NFPs) specifically in heart causes a variety of morphogenetic defects, however, the key downstream target or signaling pathway that is disrupted and is responsible for each defect in the knockout was not determined. In this study, we investigated the cellular and molecular mechanisms of the trabeculation defect in the NFPs knockout. We found that during trabecular initiation, the cellular and mitotic spindle orientations of cardiomyocytes in the knockouts were significantly different from the control, which was further confirmed by the sparse cell-labeling / confetti clonal analysis. We then determined that N-Cadherin, which is required for trabecular formation in previous studies, is significantly reduced in the NFPs knockout. Genetic and biochemical tools were applied to demonstrate that Numb functions upstream of N-Cadherin and modulates its protein level to regulate trabeculation. Using chicken N-Cadherin driven by the aMHC promoter in transgenic mice, we found that cardiomyocytes that express chicken N-Cadherin in the NFPs knockout hearts preferentially localized to trabeculae compared to cardiomyocytes that do not express chicken N-Cadherin, thus partially rescuing the trabeculation defect of the knockout. This suggests that NFPs regulate trabeculation via maintaining the N-Cadherin protein level. To determine the mechanism of how NFPs regulate N-Cadherin protein, we generated a mouse line in which the endocytic motifs of Numb are deleted, and found the N-Cadherin protein level was not changed, indicating that Numb does not regulate N-Cadherin through endocytosis. Instead, we found that Numb localizes to the recycling endosome and that less N-Cadherin was localized to the membrane in the knockout, suggesting that NFPs are required for N-Cadherin recycling to the membrane. In summary, NFPs regulate N-Cadherin recycling to the plasma membrane during trabecular morphogenesis, and NFP deletion reduces N-Cadherin protein level, resulting in the trabeculation defect.
Abstract 115: Chromatin Microenvironments With Distinct Functionality During Cardiac Stress
Authors: Rosa-Garrido Manuel , J. Chapsky Douglas , Cabaj Maximilian , Karbassi Elaheh , Vondriska Thomas
Published: February 2019

Loss of the chromatin structural protein CTCF induces heart failure. Chromatin capture experiments (Hi-C), which detect genomic interactions, revealed global remodeling of chromatin structure after cardiac-specific depletion of CTCF or pressure overload (TAC). In the present study, we investigated epigenomic features (histone marks and DNA methylation) contributing to distinct behaviors of chromatin microenvironments. CTCF-KO or TAC induced gene expression changes mediated by a shift in compartmentalization between active and inactive chromatin (~4% of the genome). At the transcriptome level, genes significantly dysregulated in disease were involved in cardiac function (
e.g.
,
Mybpc3, Lmna
,
Hand2
), chromatin structure (
e.g.
,
Hmgb2
,
Rad21
), and histone modification (
e.g.
,
Hdac4
,
Mapk14, Foxo1
). Our Hi-C data allowed us to correlate changes in gene expression with localization within the nucleus, showing efficient segregation of differentially expressed genes into specific neighborhoods with similar expression behavior. Analyses of histone marks in these neighborhoods reveal enrichment of activating and inhibiting marks, H3K9ac and H3K9me3, respectively, in the specialized chromatin environments associated with gene expression or silencing. DNA methylation studies showed that transcriptionally active neighborhoods tended to have unmethylated CpGs, while repressive environments were methylated. Our Hi-C data also showed how active environments positively influence the expression of the genes that physically interact with them. For example, 351 unique genes with interacting partner genes had increased H3K9ac after TAC, and their 3D interacting partners included 200 upregulated genes and 104 downregulated genes, demonstrating similar transcriptional behavior in areas with similar epigenomic features and anatomical distribution in the nucleus. Heart failure involves conserved structural reprogramming of these chromatin microenvironments.
Abstract 116:
Mybphl
is a Novel Myofilament Component Implicated in Arrhythmia and Dilated Cardiomyopathy
Authors: Barefield David Y , Sell Jordan J , Puckelwartz Megan J , Wilsbacher Lisa D , Fishman Glenn I , McNally Elizabeth M
Published: February 2019

Dilated cardiomyopathy is under significant genetic influence. Using whole-genome sequencing, we found a premature stop variant (R255X) in the
MYBPHL
gene in a family with DCM and conduction system disease. A
Mybphl
null mouse model revealed systolic dysfunction with atrial and ventricular conduction system (VCS) abnormalities. We found MyBP-HL protein highly expressed in both human and mouse atria and in a subset of ventricular cardiomyocytes. We hypothesize that MyBP-HL regulates VCS function and loss of MyBP-HL causes ventricular arrhythmia and cardiac dysfunction. We now examined hearts and isolated VCMs from
Mybphl
WT, het, and null mice using immunofluorescence microscopy (IFM). Clusters of MyBP-HL+ ventricular cardiomyocytes were evident in WT hearts (~1/200,000), with only 10% as many MyBP-HL+ cardiomyocytes in het samples. In WT hearts, MyBP-HL+ cells localized in the right ventricular free wall, with significantly fewer MyBP-HL+ cells in the right ventricular free wall in het hearts. The VCS marker contactin-2 (Cntn2) was used to assess MyBP-HL co-localization in the VCS. MyBP-HL co-localized with Cntn2+ cardiomyocytes in the transition zone between the atria and ventricle near the AV node, as well as in a subset of Cntn2+ Purkinje fibers. MyBP-HL+ ventricular cardiomyocytes were also found outside the VCS, suggesting other roles for those cells in regulating ventricular function. To discern MyBP-HL myofilament localization, we used super-resolution structured illumination microscopy on atrial cardiomyocytes. MyBP-HL co-localized with cMyBP-C in the C-zone of the A-band but also occupied space in the inner portion of the A-band near the M-line. This localization was not altered in het mice; however, the ratio of MyBP-HL in the inner portion of the A-band was diminished in het mice compared to WT. This suggests that MyBP-HL may bind the light meromyosin coiled-coil tail that originates from the M-line. Following the discovery that loss of MyBP-HL is associated with DCM and arrhythmia, we identified that MyBP-HL associates with the VCS and reduced MyBP-HL levels reduce the amount of MyBP-HL positive VCMs. These data suggest that loss of MyBP-HL may impair the development and function of the VCS, resulting in arrhythmias.
Abstract 119: Aberrant Caesin Kinase 2 Signalling Predisposes the Heart to an Apoptotic Phenotype in Type 2 Diabetes
Authors: Smith Lauren E , Li Desmond K , Cordwell Stuart J , White Melanie Y
Published: February 2019

The clinical effects of myocardial ischemia/reperfusion injury (I/R) are typically significantly more adverse in patients with Type 2 Diabetes Mellitus (T2DM). We hypothesize that in T2DM hearts there are underlying molecular adaptations that, even in the absence of reduced functional output, can impact recovery if exposed to I/R. Given the likely rapid molecular response to I/R, we examined how T2DM alters basal myocardial protein phosphorylation to identify pathways associated with such adaptation. We employed a rat model that combined peripheral insulin resistance from energetic overload (high fat diet) and pancreatic insufficiency (streptozotocin), producing elevated blood glucose, serum triglycerides and reduced insulin sensitivity, consistent with a T2DM phenotype. Hearts were subjected to 60 mins
ex vivo
perfusion followed by protein extraction, trypsin digest, phosphopeptide enrichment and quantitative mass spectrometry. We identified abundance changes in >5400 phosphopeptides despite no evidence of functional perturbation in LV pressure or heart rate between control and T2DM hearts. Differentially abundant phosphosites were mapped to signal pathways. This showed Akt contributed to 36% of pathways with reduced phosphorylation in T2DM hearts. Reduced phosphorylation of Akt at S124/S129 was consistent with the global pathway data (log2 ratio: -1.10). Given that S129 is a casein kinase 2 (CK2) target, we next examined CK2 and CK2 signaling. We observed significantly reduced CK2 abundance (CK2α: 0.41±0.08 and CK2β: 0.54±0.09;
n
-fold±SEM) and reduced phosphorylation of CK2 targets (CK2 substrate pS/pT-D/E: 0.84±0.01;
n
-fold±SEM) by Western blot. This was further confirmed by the over-representation of CK2 target sequences (pS/pT-D/E) in the negatively regulated phosphopeptide set (D, 10.45-fold; E 6.82-fold). Reduced phosphorylation at CK2 regulated sites on catenin β1, caspase 9 and eukaryotic translation initiation factor 3, suggests T2DM elicits a pro-apoptotic environment. Increased cleavage of caspases 3 and 9 (2.15±0.28 and 1.32±0.08;
n
fold±SEM, respectively) confirmed these data. In summary, changes in CK2 signaling may ‘prime’ a pro-apoptotic environment in T2DM hearts that subsequently limits recovery in I/R.
Abstract 209: Downregulation of KLF2 in the Endothelium Contributes to the Pathogenesis in LMNA-related Dilated Cardiomyopathy
Authors: Sayed Nazish , Liu Chun , Himmati Farhan , Zhang Joe , Khanamiri Saereh , Chen Haodong , Moonen Jan-Ranier , Wnorowski Alexa , Matsa Elena , Cheng Linling , Sallam Karim , Rabinovitch Marlene , Wu Joseph C
Published: February 2019

Background:
Mutations in the gene that encodes the nuclear envelope proteins lamin A and C (LMNA) accounts for 6% of all cases of Dilated Cardiomyopathy (DCM). However, the molecular mechanisms that underlie “cardiolaminopathy” remain elusive, and it is unknown why mutations in this ubiquitously expressed gene have such a disproportionate effect on the heart.

Hypothesis:
Despite the fact that LMNA is abundantly expressed in endothelial cells (ECs) and mutations in LMNA are known to induce EC dysfunction, little is known about the EC-specific phenotype of LMNA-related DCM. As EC dysfunction has been known to contribute to DCM, we hypothesize that EC dysfunction due to LMNA mutation has a significant impact on the pathogenesis and disease progression of DCM.

Results:
Intriguingly, our preliminary data showed that iPSC-ECs derived from patients (n=5) harboring the LMNA-mutation exhibit decrease functionality as seen by impaired angiogenesis and decreased NO production (control iPSC-ECs vs LMNA iPSC-ECs; p<0.05). Similarly, genome editing of isogenic iPSC lines enabled us to recapitulate the EC disease phenotype further allowing us to dissect the effects of LMNA mutations on EC function. LMNA corrected iPSC-ECs (via use of CRISPR/Cas9 genome editing tool to correct the single mutated copy in heterozygous patient’s iPSCs) showed restoration of EC function. Whole genome RNA-sequencing identified Krüppel-like Factor 2 (KLF2) as a potential transcript responsible for EC dysfunction in LMNA-mutated patients. Importantly, treatment of LMNA-mutated ECs with KLF2 agonists showed rescue of EC dysfunction. Furthermore, iPSC-derived cardiomyocytes (iPSC-CMs) from LMNA-mutated patients that exhibited DCM phenotype, showed improvement in CM physiology when co-cultured with iPSC-ECs treated with KLF2 agonists.

Conclusion:
This study is a first step towards understanding the molecular mechanisms of cardiolaminopathy by modeling endothelial dysfunction using patient-specific iPSCs. Moreover, our results suggest that improving EC function in cardiolaminopathy patients could have a significant impact on the pathogenesis of DCM. Results from this work could potentially lead to new strategies that could improve the management of DCM patients.
Abstract 211: Modified Endothelial Progenitor Cell (EPC) Transplantation Improves Diabetic Kidney Disease
Authors: Sen Sabyasachi , Kundu Nabanita , Asico Laureano , Domingues Cleyton , Jose Pedro
Published: February 2019

DKD is a major vascular complication of diabetes, which is associated with glomerulosclerosis and poor perfusion. Therefore, improving the renal perfusion may help to treat the sclerotic kidney disease. EPCs have been shown to improve tissue perfusion. Here, we investigated whether transplanting apoptosis resistant genetically modified mouse EPCs (p53 gene silenced, transiently, using Adenovirus ex-vivo), could improve angiogenesis and renal perfusion, in a hyperglycemic type 1 diabetes milieu. Mesenchymal stromal cells (MSC) transplantation has also been used in DKD however concern for epithelial to mesenchymal transformation remains.

Methods:
We compared the efficacy of p53shEPC with MSC, Null-EPC and saline in STZ-induced type 1 diabetic C57BL/6J mouse model. 8 weeks after STZ delivery stable hyperglycemia, polyuria and proteinuria was confirmed followed by transplantation of 0.3 million EPC/ MSC bilaterally, under each kidney capsule. We compared our results with non-STZ normal C57Bl6J mouse. Urine was collected weekly for volume, protein and creatinine estimation. Blood creatine and renal blood flow was measured by laser Doppler, at sacrifice. qRT-PCR on harvested kidneys were performed focusing on genes associated with angiogenesis.

Results:
Polyuria was reduced in p53shEPC transplanted animals with urine volume close to non DM animals and half the volume compared to saline and MSC group at week 4 and 6. Protenuria levels were the lowest in p53shEPC group. Plasma creatinine was lowest in p53shEPC group (3-fold lower) compared to Null EPC group Enhanced blood flow by laser doppler was also noted with delivery of p53sh-EPCs vs controls, such as null EPC (1.2 fold), saline (3 folds) at wk4&6. Markers for angiogenesis, such as eNOS mRNA (4.5 fold, p=0.002) and VEGF-A mRNA (1.5 fold, p=0.03) upregulated significantly post p53 silenced EPC transplantation compared to null EPC group. Capillary density staining is pending.

Conclusion:
Apoptosis resistant EPCs help to improve proteinuria, plasma creatinine, polyuria and renal perfusion most likely by helping to increase angiogenesis and may have a prominent therapeutic role in DKD. MSC transplantation results were consistently less efficacious compared to p53silenced EPC transplantation.
Abstract 223: Activation of CDK2 Mediates Doxorubicin-induced Cardiotoxicity
Authors: Xia Peng , Liu Yuening , Liu David , Cheng Zhaokang
Published: February 2019

With the rapid increase of cancer survivors due to improved diagnosis and therapy in the past decades, cancer treatment-related cardiotoxicity is becoming an urgent concern for modern society. The anthracycline doxorubicin (DOX), one of the most effective chemotherapeutic agents to date, is known to cause cardiomyopathy through induction of cardiomyocyte apoptosis. We previously showed that the cyclin-dependent kinase (CDK) inhibitor p21 promotes resistance against DOX-induced apoptosis. In this study, we demonstrated that CDK2 activity and expression were significantly increased following DOX exposure in mouse heart and cultured neonatal rat cardiomyocytes (NRCMs). DOX-induced apoptosis, as measured by cleavage of caspase 3 and PARP, TUNEL staining, and MTT assay, was significantly suppressed by inhibition of CDK2 activity using small-molecule inhibitors, or CDK2-specific siRNA. Conversely, overexpression of CDK2 augmented DOX-induced apoptosis. DOX injection-induced CDK2 activation in mouse heart was correlated with upregulation of the BH3-only protein Bim. Mechanistically, CDK2 mediated DOX-induced expression of Bim, which was essential in triggering Bax/Bak-dependent mitochondrial outer membrane permeabilization. Pharmacological inhibition of CDK2 robustly repressed DOX-induced mitochondrial depolarization. These findings identify CDK2 as a key determinant of DOX-induced apoptosis in cardiomyocytes. Activation of CDK2 is necessary for DOX-induced Bim expression and mitochondrial damage. Also, pharmacological inhibition of CDK2 might be an effective cardioprotective strategy against anthracycline cardiotoxicity.
Abstract 228: RNA Aptamer 9.14T79VRT7 Modifies Canine Ex vivo Platelet Reactivity
Authors: Talentino Spencer E , Joseph Matthew E , Byington Chase M , Bratton Camille F , Hatten Caitlin M , Shimmin Gabriel S , Anderson Cole P , Wilson Jenna L , Huttinger Allyson L , Wheeler Debra G , Nimjee Shahid M
Published: February 2019

Objective:
To compare the prophylactic and thrombolytic effects of RNA aptamer (9.14T79VRT7) on ex vivo canine platelet function.

Approach and Results:
We previously demonstrated that inhibition of von Willebrand Factor (VWF) by a targeted RNA aptamer prevents thrombosis and thrombolyses stabilized clots in a FeCl
3
-induced murine vascular injury model suggesting a pivotal role for VWF in the pro-thrombotic and anti-thrombotic milieu. We hypothesize that 9.14T79VRT7, which demonstrated no hemorrhagic complications and greater re-perfusion compared to rTPA in murine and canine thrombotic models, may affect additional agonist pathways to mitigate platelet activation, aggregation, and adhesion. Platelet aggregation before and after 9.14T79VRT7 addition was analyzed utilizing WB aggregometry based on impedance in five adult beagles. Agonist concentrations were selected from previous publications which had optimized platelet reactivity in canine WB and included: collagen (3.2 ug/ul), ADP (20 uM), arachidonic acid (0.5 mM), and botrocetin (1 ug/ul). The 9.14T79VRT7 concentration used (25 nM) was selected as the most effective as assessed by Total Thrombus Analysis System (TTAS). Botrocetin elicited the greatest response before 9.14T79VRT7 addition, followed by Collagen, ADP, and Arachidonic acid. Impedance resulting from prophylactic addition of 9.14T79VRT7 before Collagen, ADP, and Botrocetin resulted in a 50.00%, 34.83%, and 38.26% change in amplitude, respectively and a 43.70%, 14.28%, and 35.14% change in slope, respectively (p<.0001). There was no significant difference in response after Arachidonic acid. Impedance resulting from thrombolytic addition of 9.14T79VRT7 after agonist response to Collagen, ADP, and Botrocetin resulted in a 78.66%, 94.08%, and 84.58% change in amplitude, respectively and a 67.28%, 61.28%, and 63.73% change in slope, respectively (p<.0001). Although amplitude and slope decreased with 9.14T79VRT7 addition after Arachidonic acid, the change was not significant. Both control groups (9.14T79VRT7 and platelet buffer diluent) resulted in no significant changes.

Conclusion:
9.14T79VRT7 markedly changes platelet response both before and after agonist treatment in canine ex vivo impedance analysis.
Abstract 230: Repair of Aortic Coarctation in Neonates Less than Two Kilogram
Authors: Chen Qiang , Fleming Thomas , Caputo Massimo , Stoica Serban , Tometzki Andrew , Parry Andrew
Published: February 2019

Objective:
Though a significant number of low birth weight and premature neonates are born with aortic coarctation, waiting for the child to grow before intervening rarely permits significant useful weight gain and puts the child at risk of interim complications. Previous studies of early operation on these patients have shown a high hospital mortality up to 25% (25 out of 100) and 30% (30 out of 100) recurrence in one year. We reviewed our data to ascertain whether modern approaches allow better outcomes for these children.

Methods:
All patients weighing less than 2 kg with isolated coarctation between August 2008 and November 2015 were studied by retrospective chart review to ensure greater than 24 months follow-up. Eleven patients underwent extended end-to side surgical repair. In-hospital and medium term follow-up data were collected. Data are expressed as median (range).

Results:
Weight at time of surgery was 1.8kg (1.6-1.9). There were no deaths, in-hospital or during follow-up. One patient developed postoperative chylothorax. In-hospital stay was 18 days (4-47). At follow-up of 68 months (28-115) echocardiographic velocity across the repair was 1.6m/sec (1.4-4.5). One patient with William’s syndrome had evidence of recoarctation and required balloon coarctoplasty followed by stenting. This patient had grossly abnormal vessels at the time of initial surgery with aortic wall thickness greater than 3mm.

Conclusions:
Neonates below 2kg can undergo coarctation repair safely with low incidence of recurrence. Waiting for growth in this cohort of patients cannot therefore be justified.
Abstract 241: In Vitro Disease Modeling and Discovery of Small Molecules for the Treatment of Duchenne Muscular Dystrophy Utilizing Patient iPSC-derived Cardiomyocytes
Authors: Sun Chicheng , Qadir Deena , Accorsi Anthony , Moore Natalie , Lin Ling , Cacace Angela , Graef John
Published: February 2019

Duchenne Muscular Dystrophy (DMD) is a fatal disease of skeletal and cardiac muscle characterized by progressive muscle weakness and atrophy. Current average life expectancy is in the mid-20s and mortality is often linked to end stage cardiomyopathy. DMD is a monogenic disease caused by loss-of-function mutations in the dystrophin gene. While dystrophin is critical for muscle function by connecting the cytoskeleton to the sarcolemma and extracellular matrix, its absence can be largely compensated for by up-regulation of utrophin (UTRN), the fetal orthologue of dystrophin. Because of the importance of UTRN expression in the heart in both DMD patients and preclinical models, we utilized DMD patient iPSC-derived cardiomyocytes as a relevant cellular model to perform in vitro pharmacology as well as functional assessment. Human iPSC-derived cardiomyocytes recapitulate key properties of bona fide cardiomyocytes, such as expression of cardiac genes, spontaneous contractility, and response to chronotropic drugs (eg. isoproterenol and hERG channel blockers). We have created an isogenic pair of healthy control and DMD iPSC lines with exon 51 deletion using CRISPR/Cas9 technology. Functional measurements of cardiomyocyte electrophysiology were performed by multielectrode array (MEA) and fluorometric imaging plate reader (FLIPR) platforms. High-throughput assays were developed to measure UTRN expression in cardiomyocyte culture in 384-well plates. Importantly, we have identified small molecules that increased UTRN expression in both patient and isogenic DMD iPSC-derived cardiomyocytes. Our study represents a valuable strategy for small molecule drug discovery for the treatment of DMD cardiomyopathy based on iPSC technology.
Abstract 243: Modeling of Diastolic Dysfunction in Induced Pluripotent Stem Cell-derived Cardiomyocytes From Hypertrophic Cardiomyopathy Patients
Authors: Wu Haodi , Yang Huaxiao , Rhee June-Wha , Zhang Joe , Lam Chi Keung , Sallam Karim , Chang Alex C , Ma Ning , Blau Helen , Bers Donald , Wu Joseph
Published: February 2019

Aims:
Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. Yet its cellular mechanisms are not fully understood, and presently, there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD and as a platform for drug discovery.

Methods and results:
In the present study, beating iPSC-CMs were generated from healthy recruits and HCM patients with evident DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, retarded relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca
2+
imaging indicated elevated diastolic [Ca
2+
]
i
and abnormal Ca
2+
handling in HCM iPSC-CMs, which were exacerbated by β-adrenergic challenge. Combining Ca
2+
imaging and traction force microscopy (TFM), we observed enhanced myofilament Ca
2+
sensitivity (measured as dF/Δ[Ca
2+
]
i
) in HCM iPSC-CMs. These results indicated that cytosolic diastolic Ca
2+
overload, slowed [Ca
2+
]
i
decline and increased myofilament Ca
2+
sensitivity, collectively impair the relaxation of HCM cells. Treating HCM iPSC-CMs with partial blockade of Ca
2+
or late Na
+

current

reset diastolic Ca
2+
homeostasis, restored diastolic function and improved long-term survival, suggesting disturbed Ca
2+
signaling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type calcium channel (LTCC) and transient receptor potential channels (TRPCs) in HCM iPSC-CMs, which likely contribute to diastolic [Ca
2+
]
i
overload.

Conclusion:
In summary, this study recapitulated DD at the single cell level, and revealed novel mechanisms and potential therapeutic targets of DD using HCM iPSC-CMs.
Abstract 249: Cortical-bone Derived Stem Cells Improve Cardiac Outcomes After Myocardial Infarction by Modulating the Inflammatory Response
Authors: Hobby Alexander R , Sharp Thomas E , Feldsott Eric , Mohsin Sadia , Kubo Hajime , Berretta Remus , Houser Steven R
Published: February 2019

Rationale:
Cortical-bone derived stem cells (CBSCs) improve cardiac pump function and reduce scar size after myocardial infarction (MI) compared to Cardiac-Derived and Mesenchymal Stem Cells. However, the mechanism for these improved outcomes is not known. Recent data in a swine model of MI has demonstrated acute changes to the immune response elicited by MI in CBSC treated animals compared to vehicle controls, suggesting that CBSCs may improve post-MI remodeling by modulation of the immune response.

Objective:
Identify the changes in the immune response of CBSC-treated animals and determine the role of these changes in cardiac healing and wound repair.

Methods and Results:
Using multi-color flow cytometry, we found that murine bone marrow-derived macrophages (BMDMs) cultured and exposed to CBSC conditioned culture medium
in vitro
express higher levels of the mannose receptor CD206 than controls, indicating alternative “M2” activation. In addition, CD86 expression on BMDMs exposed to the classical (M1) activators interferon-γ (INF-γ) and lipopolysaccharide (LPS) in conditioned CBSC media was significantly lower than cells treated only with INF-γ and LPS. Blocking the IL-4 receptor on the BMDMs with a monoclonal antibody in conditioned media decreased CD206 expression, increased CD86 expression, and failed to mitigate the activation elicited by INF-γ and LPS. Exposing the BMDMs to conditioned media also increased the amount of the anti-inflammatory cytokine IL-1 receptor antagonist (IL1-RA) released compared to controls, matching previous data from a swine-model of MI that showed increased IL-1RA blood levels in CBSC-treated swine 72 hours post-MI. Together these data implicate IL-4 as a possible mechanism through which CBSCs influence the inflammatory response raised to MI.

Conclusion:
Soluble factors secreted by CBSCs change the phenotype of immune cells
in vitro
and
in vivo
. A possible mechanism for the beneficial effects of CBSCs on cardiac wound healing after MI may come from their ability to modulate the monocyte/macrophage response to preserve cardiac structure and function.
Abstract 255: Mechanical Stretch is Not a Major Cause of Ectopic Activation During Early Stage Regional Ischemia in an Isolated Left-Ventricular Working Heart Model
Authors: Zhang Hanyu , Yu Han , Walcott Gregory P , Rogers Jack M
Published: February 2019

Background:
Acute regional ischemia is a leading cause of death. Ectopic beats may initiate ventricular arrhythmias in the first hour after coronary occlusion; however, the origin of these beats remains poorly understood. Some studies suggest that abnormal mechanical stretch at the ischemic border zone may contribute to the initiation of ectopy. If mechanical stretch plays a major role in triggering ectopic beats, we therefore expect:
I
, left ventricular working (LVW) hearts will have higher frequency of post-occlusion ectopic activity than non-beating (NB) hearts;
II
, in LVW hearts, ectopic beats will arise from ventricular sites undergoing mechanical stretch.

Methods and Results:
Isolated pig hearts were assigned to NB or LVW groups (8 per group) and prepared for Langendorff or LVW perfusion, respectively. Contraction of NB hearts was suppressed with 10 μM blebbistatin. Acute regional ischemia was induced by LAD occlusion. Using a newly developed optical mapping system, electrical activity and mechanical deformation were simultaneously recorded from 0 to 60 minutes after LAD occlusion. Mechanical stretch (positive strain in 2 principal directions) was observed in the ischemic and border zones of LVW but not NB hearts (Figure, Panel A). There was no significant difference in the number of ectopic events in LVW hearts compared to NB hearts (58±35 vs. 32±36, p=0.17). Of the 45 ectopic events that originated from the LV mapping region in LVW hearts, <20% (6) were from sites that underwent mechanical stretch in the prior cardiac cycle (Figure, Panel B).

Conclusion:
Mechanical stretch does not play a major role in triggering ectopic beats in early acute regional ischemia in this model.
Abstract 256: Precision Medicine in Cardiac Channelopathy: Integrating Genome Editing and Induced Pluripotent Stem Cells to Decipher Variant of Unknown Significance
Authors: Garg Priyanka
Published: February 2019

Background:
The long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsade de pointes (TdP) and sudden cardiac death (SCD). Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of unknown significance (VUS), for which there is insufficient evidence of pathogenicity.

Objectives:
To demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy.

Methods and Results:
Peripheral blood mononuclear cells (PBMCs) were isolated from a carrier with a novel missense variant (T983I) in the
KCNH2
(LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (iPSC-CMs). Whole-cell patch clamp recordings revealed significant prolongation of the action-potential duration (APD) and reduced rapidly activating delayed rectifier K
+
current (
I
Kr
) density in VUS iPSC-CMs compared to healthy control iPSC-CMs. ICA-105574, a potent
I
Kr
activator, enhanced
I
Kr
magnitude and restored normal APD in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder.

Conclusions:
Our results suggest that the KCNH2
T983I
VUS may be classified as potentially pathogenic.
Abstract 261: Activation of the Thromboxane/Prostanoid Receptor Contributes to Elevated End-Diastolic Calcium in Cardiomyocytes and Cardiac Fibrosis Following Right Ventricular Pressure Overload
Authors: Carrier Erica J , Kim Kyungsoo , Noll Natalie A , Macias-Perez Ines , Merryman W. David , Knollmann Bjorn C , West James D
Published: February 2019

Like its systemic counterpart, pulmonary arterial hypertension (PAH) results in remodeling and fibrosis of the right ventricle as it attempts to adapt to the increased pressure overload. This eventually leads to contractile dysfunction, and RV failure is the primary cause of death in PAH patients. The G protein-coupled thromboxane/prostanoid (TP) receptor is expressed in vascular smooth muscle and immune cells, and is upregulated in cardiomyocytes following PAH. Activation of the cardiac TP receptor increases cardiomyocyte intracellular calcium and can lead to arrhythmias. We previously reported that oral treatment with the TP receptor antagonist ifetroban prevents RV fibrosis in a mouse pressure overload model of PAH. Here, we investigate the effects of TP receptor activation on calcium handling in RV cardiomyocytes and explore treatment of established RV remodeling with ifetroban, compared with prevention. Fixed pressure overload of the RV via pulmonary arterial banding (PAB) caused an increase in contractility and resting (end-diastolic) intracellular calcium in individual cardiomyocytes after 3 weeks; this occurred in conjunction with RV dilation, fibrosis, and stiffness. Surprisingly, total calcium content of the sarcoplasmic reticulum was increased following PAB. Antagonism with ifetroban decreased formation of fibrosis in a time-dependent manner. However, treatment with antagonist following establishment of RV fibrosis still prevented the cardiomyocyte increase in end-diastolic calcium. This suggests a multi-factorial contribution of the TP receptor in the RV response to PAH. Further studies continue to analyze changes in calcium-dependent signaling, as well as the contribution of the cardiomyocyte TP receptor to both fibrosis and calcium handling.