bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P632: Nlrp3/inflammasome Activation Contributes To Aldosterone-induced Vascular Dysfunction In Type 2 Diabetes.
Authors: Ferreira Nathanne S , Bruder-Nascimento Thiago , Pereira Camila A , Zanotto Camila Z , Prado Douglas S , Alves-Filho José C , Carlos Daniela , Tostes Rita C
Published: June 2021

Diabetic patients and animal models of type 2 diabetes (DM2) display increased plasma aldosterone (aldo) levels. Aldo induces vascular inflammation and endothelial dysfunction. NOD-like receptors, which are pattern recognition receptors involved in a variety of host innate immune responses, promote vascular inflammation. We hypothesized that aldo via mineralocorticoid receptors (MR) activates the inflammasome platform in the vasculature of DM2 mice. Control (db/+) and diabetic (db/db) mice were treated with vehicle or spironolactone (spiro - MR antagonist, 50 mg/Kg/day). Mesenteric resistance arteries (MA) from db/db mice exhibited reduced acetylcholine (ACh) dilation, which was reversed by spiro [Emax (% of relaxation): db/+: 78.5±4.1; db/db: 40.5±6.4; db/+spiro: 77.0±3.8; db/db+spiro: 62.8±5.9 n=3-6 p<0.05]. Spiro treatment reduced caspase-1 and mature IL-1β content in MA from db/db mice. Spiro also reduced caspase-1 activity in macrophages from peritoneal lavage of db/db mice [% of activity: db/+: 33.9±2.5; db/db: 51.8±7.4; db/+spiro: 31.1±1.9; db/db+spiro: 34.8±3.8 n=4-7, p<0.05].
In vitro,
aldo increased mature IL-1β in vascular smooth muscle cells (VSMC) (cont: 0.9±0.01 ; LPS+Nigericine: 6.1±2.1 ; Aldo 4h: 9.7±2.6; LPS+Aldo 4h: 12.8±1.9 n=3-5, p<0.05). To determine whether aldo
in vivo
directly activates NLRP3/inflammasome in the vasculature and whether NLRP3 activation contributes to aldo-induced vascular injury, aldo was infused (600 ug/Kg/day for 14 days) in wild type (WT) and NLRP3 knockout mice (
NLRP3-/-
) after bone marrow transplantation from WT donor. The groups were constituted: WT->WT, WT->WT+aldo and WT->
NLRP3
-/-+aldo.
NLRP3 -/-
mice were protected against aldo-induced endothelial dysfunction [Emax: WT: 89.3±2.9; WT+aldo: 39.8±1.8;
NLRP3-/-
+aldo: 87.7±4.2, p<0.05]. Aldo treatment leaded to endothelial dysfunction in WT ->WT mice, but WT->
NLRP3
-/- mice were protected from aldo-induced endothelial dysfunction [Emax: WT->WT: 95.1±3.1; WT->WT+aldo: 57.1±4.7; WT->NLRP3-/-+aldo: 85.3±3.1 p<0.05]. These results suggest that NLRP3/inflammasome in the vasculature plays a crucial role on aldo/MR-induced vascular damage and on DM2-associated vascular dysfunction. Financial Support: FAPESP, CAPES, CNPq.
Abstract P636: Insulin Resistance in Obesity Results in Postprandial Salt and Water Loss
Authors: Irsik Debra L , Washington Ashley R , Alaisami Rabei , Brands Michael W
Published: June 2021

Obesity and insulin resistance contribute to the development of metabolic syndrome, a growing epidemic in our country. The obese Zucker rat is an experimental model of this disease. Previously, using Sprague Dawley rats, we have shown that the normal postprandial rise in insulin acts physiologically to prevent renal salt and water wasting after meals. This study tested whether the effects of postprandial insulin would be attenuated in insulin resistant rats and result in excess salt and water loss. Chronic artery and vein catheters were implanted in male lean and obese Zucker rats for infusion and blood sampling. Rats were housed in metabolic cages and their catheters were connected to dual-channel Instech swivels for access. Over a 24-hr period of ad libitum eating, blood glucose was not different between obese and lean rats (127±7 vs. 120±3 mg/dl) but obese rats were hyperinsulinemic (14.86 vs. 0.98 ng/ml). Obese rats had significantly greater urine volume than lean controls (22.5±1.2 vs. 14.7±0.9 ml) despite similar water intakes. Obese rats tended to excrete more Na+ than lean controls (3.46±0.15 vs. 2.97±0.35 mEq) with equal amounts of Na+ intake. To evaluate the response to a single meal while controlling for blood glucose, fasted rats were administered a glucose bolus (as 50% dextrose) that yielded peak levels of blood glucose that were not different in the two groups (589±11 vs. 596 ±3 mg/dl at t=5 min.). Plasma insulin increased from fasting in both groups to 26.35 and 9.34 ng/ml in obese and lean controls, respectively. Over the 4-hour period following the glucose administration, obese rats had significantly greater urine volume (8.6±1.3 vs. 2.2 ±0.6 ml) and Na+ excretion (0.53±0.11 vs. 0.25±0.09 mEq) than lean controls. This suggests that insulin resistance of obesity may impair the ability of postprandial insulin to participate in maintenance of Na+ and water homeostasis, but the potential role of insulin resistance specifically within the kidney requires further study.
Abstract P643: Potassium Differential Stress Response with Angiotensin II Blockade in Blacks
Authors: Stewart Deborah L , Harshfield Gregory A , Hanevold Coral , Mathur Sunil
Published: June 2021

Background:
In youths, we demonstrated stress increases systolic blood pressure (SBP) with decreased urinary sodium excretion (UNaV) and no change in urinary potassium excretion (UKV) in retainers (retain sodium during stress) but increased UNaV and UKV in excreters (excrete sodium during stress). Angiotensin receptor blockers (ARBs) are known to increase UNaV, yet are not commonly used in blacks.

Purpose:
This study sought to determine if angiotensin II plays a role in the Na/K imbalance in retainers. The tested hypothesis was angiotensin II (Ang II) blockade stabilizes the urinary sodium/potassium (UNa/KV) excretion ratio during stress.

Methods:
Impact of Ang II on changes in SBP, UNaV, UKV and UNa/KV ratio in response to stress was tested in blacks after three day sodium/potassium controlled diet in a double-blind, placebo-controlled crossover design using irbesartan.

Results:
Excreters (n=84) and retainers (n=25) were identified and compared on placebo versus ARB. In response to Ang II blockade, excreters reduced their UNa/KV ratio and increased UNaV. Conversely, retainers did not change UNaV while sustaining their UNa/KV ratio. However, both groups increased SBP and UKV with a greater effect in excreters.

Conclusion:
These data suggest Ang II plays a significant role in the UNa/KV ratio homeostasis, through its impact on UNaV. Specifically, retainers are able to maintain their UNa/KV ratio during stress with Ang II blockade, whereas excreters are unable to maintain their UNa/KV ratio under the same treatment. Identification of this stress response pattern may be a critical need in providing personalized and effective treatment.
Abstract 101: Mitochondrial and Cardiac Protection Evoked by a Novel Mitochondria-targeting Peptide-idebenone Conjugate
Authors: Perry Justin B , Alleman Rick J , Tsang Alvin M , Winfrey William B , Allen Mitchell E , Green Tom D , McClung Joseph M , Brown David A
Published: June 2021

Dysfunction along the electron transport system contributes to cardiomyopathy in a number of mitochondrial diseases. Several pharmacological approaches are currently being tested for their ability to improve mitochondrial function, yet almost none of the leading candidates specifically target to mitochondria. In this study we determined the biological efficacy of a novel conjugate of idebenone and mitochondria-targeting peptide, SBT-61. Using a vertically-integrated approach we tested the ability of SBT-61 to improve functional recovery and bioenergetics in: perfused rat hearts subjected to ischemia-reperfusion, cultured cells exposed to hypoxia-reoxygenation injury, and isolated heart mitochondria where complexes I and/or III were inhibited. SBT-61 treatment preserved post-ischemic cardiac function as assessed by left ventricular developed pressure (40 ± 5 vs 24 ± 3 mmHg, P<0.0001), rate of contraction (1853 ± 236 vs. 1069 ± 109 mmHg/s, P<0.0001), rate of relaxation (-1349 ± 231 vs. -792 ± 52 mmHg/s, P<0.0001), and a 30% reduction in infarct size. In cells expressing an shRNA knockdown of glutathione reductase, treatment with SBT-61 normalized maximal mitochondrial respiration after hypoxia-reoxygenation. In isolated mitochondria with complexes I or III inhibited, SBT-61 stimulated respiration and was shown to directly reduce complex III and cytochrome c, indicating that SBT-61 can bypass defects in the electron transport system. These data highlight the cardioprotective potential of a mitochondria-targeted idebenone conjugate to stabilize mitochondrial bioenergetics.
Abstract 106: G Protein-coupled Receptor Kinase 2 Associates With Mitochondrial Proteins and Negatively Alters Mitochondrial Respiration After Myocardial Infarction
Authors: Pfleger Jessica , Sato Priscila , Xiao Kunhong , Koch Walter J.
Published: June 2021

During cardiac injury or stress, G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression levels and activity are increased, leading to a desensitization of myocardial β-adrenergic receptors (βARs) and contributing to the loss of contractile reserve. Up-regulated GRK2 has been shown to be pathogenic in the post-injured heart and is involved in the promotion of heart failure (HF). Consequently, inhibition of GRK2 has been shown to rescue several animal models of HF, as demonstrated by cardiac expression of the βARKct, a peptide inhibitor of GRK2. There is new evidence that GRK2 has other, non-GPCR dependent pathological functions within cardiomyocytes. For example, during conditions that increase oxidative stress, GRK2 has been shown to localize to the mitochondria, where it is a pro-death kinase. βARKct has also been shown to inhibit the mitochondrial translocation of GRK2 due to inhibition of Hsp90 binding, a chaperone found to bind and localize GRK2 to mitochondria. Our objective is to identify the functions and targets of GRK2 in the mitochondria and its role in the development of HF. We hypothesize that GRK2 has mitochondrial targets that impair mitochondrial function in vivo, contributing to the pathogenesis of HF. Our results show that ADP-stimulated and maximal respiration are significantly decreased in mitochondria isolated from mouse hearts 2 weeks post-myocardial infarction (MI). Cardiac-specific expression of the βARKct rescued the reduction in mitochondrial respiration post-MI, in the presence of pyruvate or palmitoylcarnitine. To identify the mitochondrial targets of GRK2, we performed interactome and phospho-interactome analyses on GRK2 immunoprecipitates from neonatal rat cardiomyocytes subjected to oxidative stress. These results revealed that GRK2 interacts with several mitochondrial proteins, including those involved in metabolism, oxidative stress, calcium handling and cell death. Thus we propose that during ischemia, an increase in mitochondrial GRK2 results in its association with proteins that compromise respiration and contribute to myocardial damage and HF. This demonstrates that understanding both the canonical and non-canonical roles of GRK2 in HF is essential for more targeted intervention for therapy.
Abstract 123: Adenoviral RhoA Regulation of Dynamin-related Protein 1 and Cardiac Mitochondrial Fission
Authors: Brand Cameron S , Tan-Sah Valerie P , Heller Brown Joan , Miyamoto Shigeki
Published: June 2021

G protein coupled receptors can signal downstream through various pathways, including activation of the small G protein RhoA. In cardiomyocytes, RhoA signaling is protective against ischemia/reperfusion injury. We have previously shown that this is mediated through downstream activation of Protein Kinase D (PKD), increased phosphorylation of cofilin, and diminished translocation of pro-apoptotic proteins to the mitochondria (Xiang et al, Sci. Signaling 2013). Mitophagy, a process that removes damaged mitochondria and limits mitochondrial death signaling, has also been suggested to be a cardioprotective response to oxidative stress. A step considered to be preliminary to clearance of damaged mitochondria via mitophagy is mitochondrial fission, and we hypothesized that RhoA signaling increases mitochondrial fission in cardiomyocytes. Constitutively active RhoA expressed in neonatal rat ventricular myocytes (NRVMs) was found to accumulate at the mitochondria. This was associated with an increase in small, fragmented mitochondria as observed by fluorescent confocal microscopy and electron microscopy, indicative of increased mitochondrial fission. The main protein involved in mitochondrial fission, dynamin-related protein 1 (Drp1), translocates from the cytosol to the mitochondria when activated. We used a tagged adenoviral Drp1 construct to determine whether expression of active RhoA changes Drp1 levels at the mitochondria. Mitochondrial Drp1 increased within 12 hours of adenoviral expression of active RhoA. Adenoviral RhoA expression also increased phosphorylation of Drp1 at serine-616 in NRVMs. In summary, we show that in cardiomyocytes, RhoA associates with mitochondria, can increase Drp1 phosphorylation and Drp1 mitochondrial localization, and can induce mitochondrial fission. The relationship between these mitochondrial signaling events and the protein kinases that are involved are currently under investigation. We suggest that G protein coupled receptors that stimulate RhoA can induce Drp1 accumulation and mitochondrial fission, which contributes to their cardioprotective effect.
Abstract 126: Triptonide is an Effective Inhibitor of Wnt Signaling
Authors: Hao Jijun , Chinison Jessica , Aguilar Jose S , Avalos Alan , Huang Ying , Chow Moses , Cameron Joshua
Published: June 2021

Canonical Wnt/β-catenin signaling plays important roles during vertebrate heart development and inhibition of this pathway has been shown to improve injured heart repair by promoting cardiac progenitor cells’ differentiation. Despite extensive research, to date, no approved Wnt inhibitors are available in clinic. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named
Tripterygium wilfordii
Hook F., and its cellular target has not been identified. Here we showed that triptonide effectively inhibited canonical Wnt/β-catenin signaling by targeting a very downstream nuclear component associated with c-termini-transaction domain of β-catenin. Tripterygium wilfordii Hook F., which contains triptonide, has been traditionally used in clinic in China, suggesting that triptonide may have ideal drug properties. Our discovery of triptonide as an effective Wnt inhibitor may represent an important step in identification of bioactive small molecule drugs to stimulate endogenous cardiac progenitor cells for cardiac repair.
Abstract 206: Stem Cell-derived Exosomes Induce Cardiac Repair via mRNA Modification
Authors: Mathiyalagan Prabhu , Liang Yaxuan , Martins Adriano S , Losordo Douglas W , Hajjar Roger J , Sahoo Susmita
Published: June 2021

Exosomes are cell-derived nanovesicles that carry and shuttle microRNAs (miRNAs) to mediate cell-cell communication. Vast majority of cell types including cardiac myocytes and progenitors actively secrete exosomes, whose miRNA contents are altered after physiological or pathological changes such as myocardial ischemia (MI). In this new study, we have discovered that chemical modification to mRNAs is a novel regulator of ischemia-induced gene expression changes in the heart. We hypothesized that the benefits of human CD34
+
stem cell-derived exosomes (CD34exo) are mediated by mRNA modifications in the target cells via miRNA delivery. MiRNA profiling and bioinformatic analysis identified that CD34exo is selectively enriched with a number of miRNAs that directly target genes implicated in regulation of mRNA modifications. Interestingly, under myocardial ischemia, there was a significant increase in mRNA modifications in the mouse heart, which was decreased by about 70% with CD34exo-treatment. In line with the in vivo MI data, in vitro hypoxic stimulation in neonatal / adult rodent myocytes and non-myocytes increased mRNA modifications and controls known regulators of those mRNA modifications. Loss-of-function studies for regulators of mRNA modifications attenuated hypoxia-induced changes to epitranscriptome indicating important roles for these molecules under stress conditions. Finally, using gain-of-function and loss-of-function studies, we demonstrate that miR-126, one of the most enriched miRNAs in CD34exo, plays a critical role in regulating the mRNA modifications. We conclude that miRNAs enriched in CD34exo mediate their cardioprotective effect at least in part, by regulating the mRNA epitranscriptome of the target cell. Our new data suggests hypoxia as a novel regulator of the mRNA epitranscriptome and provides novel insights to post-transcriptional gene regulation in the heart.
Abstract 21: Effect of Hypoxic Preconditioning on Cortical Bone Stem Cells (CBSCs)
Authors: Borghetti Giulia , Trappanese Danielle M , Mohsin Sadia , Troupes Constantine , Gross Polina , Houser Steven R
Published: June 2021

Rationale:
Cortical Bone Stem Cells (CBSCs) improve cardiac performance when delivered in an infarcted heart, in part, through secretion of paracrine factors. However transplanted cells can die due to hypoxia or other hostile fators within the infarcted heart. Hypoxia is known to also influence several biological process including migration, proliferation and secretory activity.

Objective:
the aim of this study is to define the effects of hypoxia on biological properties of CBSCs.

Methods and Results:
CBSCs were isolated from C57Bl/6 mouse, expanded in vitro and characterized. CBSCs were subjected to hypoxia (1% O
2
) or normoxia (21% O
2
) condition and then several biological properties were analysed. Hypoxia resulted in upregulation of CAIX and GLUT1 expression, two HIF1α downstream targets (3.2 and 5.1 fold increase of GLUT1; 6.7 and 65 fold increase of CAIX after 6 and 24 hours respectively). This indicates an effective hypoxic response activation in our experimental conditions. Cell proliferation was assessed by CyQuant assay: no significant difference between normoxic and hypoxic cells were found after 1 and 2 days. Growth curves were determined to evaluate hypoxia effects at longer time points. Hypoxic cells had the same growing rate as cells in normoxia in the first days, but their number increased at longer time points, indicating different kinetics with respect to normoxic when cells were grown in a challenging condition like an exhausted media. CBSCs migration and chemotaxis decreased when cells were cultured at 1%O
2
. Hypoxia preconditioning increased the paracrine activity of CBSCs. VEGF expression was increased after 6 hours (3 fold), and this effect remain stable after 24 hours and 3 days of 1% O
2
. Hypoxia resulted in 2.2 and 1.9 fold increase of SDF1 after 24 hours and 3 days respectively and a 1.8 fold increase of IGF1 after 1 day. The expression of bFGF, Angiopoietin and SCF was not affected.

Conclusion:
CBSCs can survive hypoxia and their paracrine activity increases without adversely affecting their proliferative capacity. In vivo experiments are needed to investigate if hypoxic preconditioning will enhance the ability of CBSCs to improve the structure and function of the injured heart.
Abstract 212: Aggregation of Child Cardiac Progenitor Cells into 3D Spheres Improves Endothelial Lineage Commitment
Authors: Trac David Q , Xu Chunhui , Davis Michael E.
Published: June 2021

Congenital heart disease is rarely cured by surgery and can lead to life-threatening, intractable right ventricular heart failure (HF). In particular, children with hypoplastic left heart syndrome have a 10 year transplant-free survival rate of 50-75% despite palliative surgical repair. Currently, no effective stem-cell based treatments are available for pediatric HF. Recent stem-cell based clinical trials have been limited by poor differentiation rates and low cell retention. Additionally, we have shown that human cardiac progenitor cells (hCPCs) have reduced regenerative potential as they age, starting as early as 1 year old. We propose the aggregation of CPCs into scaffold-free spheres to improve the differentiation of child CPCs into mature cardiac phenotypes by enhancing intercellular Notch signaling. Notch signaling activity has been implicated in the regulation of CPC fate decisions and prior research in our lab has shown that intramyocardial delivery of Notch-ligand containing hydrogels improves cardiac function. Child CPC spheres were produced at a size of 1500 cells per sphere using a microwell array and cultured in suspension. Using immunohistochemistry, we showed that aggregation of CPCs increased Notch1 expression compared to parallel monolayer cultures. This effect is not limited to CPCs and was recapitulated in spheres of Chinese hamster ovarian cells transfected with Notch1-YFP. Additionally, Notch signaling pathway gene array data showed increased expression of the Notch-cleaving metalloprotease ADAM10 (3.6-fold) and Notch ligand DLL1 (25.0-fold) in CPC spheres by 3 days in culture compared to monolayer cultures. By 14 days in culture, we showed that aggregation of CPCs robustly increases the expression of the GATA4, a cardiac transcription factor associated with angiogenesis, and VEGFR1, an early marker of endothelial lineage commitment. Based on our results, we hypothesize that aggregation of CPCs into spheroids increases endothelial differentiation via a Notch-dependent mechanism. Transplantation of CPC spheres may improve cardiac function
in vivo
compared to transplantation of single CPCs. The results from our project will facilitate the development of autologous stem-cell based therapies for pediatric HF.
Abstract 213: Evolving Challenges in Promoting Stem Cell Based Cardiovascular Repair and Regeneration
Authors: Valarmathi Mani T , Li Jiang
Published: June 2021

Introduction:
Use of adult stem cells in the stimulation of mammalian cardiac muscle regeneration is in its infancy, and to date, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone-marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific cells, and also exhibit functional synchronization. As a result, this necessitates the development of an appropriate in vitro three-dimensional (3-D) model of cardiomyogenesis and prompts the development of a 3-D cardiac muscle construct for tissue engineering purposes, especially using the adult stem cells.

Hypothesis:
Functioning vascularized cardiac tissue can be generated by the interaction of human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human multipotent mesenchymal stem cells (hMSCs) on a 3-D prevascularized collagen cell carrier (CCC) scaffold.

Methods and Results:
In order to achieve the above aim, we have developed an in vitro 3-D functioning vascularized cardiac muscle construct using hiPSC-ECMs and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured on 3-D CCCs for 7 days under vasculogenic culture conditions, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of micro vessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed dramatic neo-angiogenesis and neo-cardiomyogenesis.

Conclusions:
Thus, our unique 3-D co-culture system provided us the apt in vitro functioning prevascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty.
Abstract 22: Identification of the Nature of Cardiac Resident c-kit
+
Cells
Authors: Cai Chen-Leng , Sultana Nishat , Zhang Lu , Yan Jianyun , Chen Jiqiu , Cai Weibin , Razzaque Shegufta , Jeong Dongtak , Sheng Wei , Bu Lei , Xu Mingjiang , Huang Guo-Ying , Hajjar Roger , Zhou Bin , Moon Anne
Published: June 2021

Identifying a
bona fide
population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. For more than a decade, c-kit, a receptor tyrosine kinase expressed in certain types of hematopoietic stem cells, has been recognized as a marker of resident CSCs in mammals. It was shown that c-kit
+
cells are multipotent, with differentiation potential to become cardiomyocytes, endothelial, and smooth muscle cells
in vitro
and after cardiac injury. Here, we provide new insights into the nature of cardiac resident c-kit
+
cells. By targeting the
c-kit
locus with several reporter genes in mice, we unexpectedly found that c-kit
+
cells rarely co-localizes with cardiac progenitor marker Nkx2.5 or myocardial marker cTnT. Instead, c-kit labels an endocardial population from embryonic stage to adulthood. After acute cardiac injury, the c-kit
+
cells still retain their endothelial identity and do not become cardiomyocytes. Our study supports the notion that cardiac c-kit
+
cells are in fact endothelial cells and not CSCs. This finding suggests an urgent need to re-evaluate the mechanisms by which c-kit
+
cells contribute to heart repair or regeneration given their endothelial identity.
Abstract 223: Parkin is Critical for the Clearance of Damaged Mitochondria via an Autophagy-independent Pathway
Authors: Hammerling Babette C , Cortez Melissa Q , Najor Rita H , Gustafsson Åsa B
Published: June 2021

Functional mitochondria are essential for highly metabolic organs such as the heart. When mitochondria are damaged they can release pro-death factors and reactive oxygen species which in turn can result in cell death. The E3 ubiquitin ligase Parkin plays an important role in clearing damaged mitochondria via the autophagy pathway to protect cells against unnecessary cell death. Interestingly, we have found that Parkin can mediate clearance of damaged mitochondria via an autophagy-independent pathway. In fact, Parkin promotes clearance of depolarized mitochondria at the same rate in both wild-type (WT) and autophagy deficient Atg5-/- mouse embryonic fibroblasts (MEFs) in response to the mitochondrial uncoupler FCCP. We also found that Parkin-mediated ubiquitination is critical for this process as disease associated mutants of Parkin were incapable of inducing mitochondrial clearance in Atg5-/- MEFs. Upon further investigation, we observed a significant increase in the number of Rab5+ early- and Rab7+ late endosomes in both WT and Atg5-/- MEFs after depolarization of mitochondria with FCCP or valinomycin, indicating activation of the endosomal-lysosomal degradation pathway. We did not observe activation of the endosomal pathway after exposure to actinomycin D, an inhibitor RNA synthesis and activator of apoptosis, confirming that mitochondrial damage specifically activates the endosomal degradation pathway. We also observed activation of the endosomal pathway in neonatal myocytes in response to FCCP treatment or after exposure to simulated ischemia/reperfusion (sI/R). Overexpression of the dominant negative Rab5S34N significantly enhanced sI/R-mediated cell death, suggesting that this is a protective pathway activated by cells in response to stress. Moreover, Beclin1 is well known to regulate activation of autophagy. Here, we found that knockdown of Beclin1 inhibited both the number of Rab5+ early endosomes and their colocalization with mitochondria in response to either FCCP or sI/R in myocytes, suggesting that Beclin1 is a critical upstream regulator of the endosomal degradation pathway. Thus, our data suggest that Parkin mediates clearance of damaged mitochondria via both the autophagy and endosomal pathways in cells.
Abstract 228: HYQVIA in Postural Orthostatic Tachycardia Syndrome(POTS)
Authors: Ahsan Muhammad M , Thompson Tara , Ashangari Chandralekha , Suleman Amer
Published: June 2021

Postural Orthostatic Tachycardia Syndrome (POTS) is a form of dysautonomia associated with variety of symptoms like Headache, Abdominal discomfort, Dizziness/presyncope, Nausea, Fatigue, Lightheadedness, Sweating Sleep disorder, Tremor, Anxiety, Palpitations, Exercise intolerance which is believed to be caused by an underlying infectious and/or autoimmune trigger. HYQVIA is an immune globulin with a recombinant human hyaluronidase indicated for the treatment of Primary Immunodeficiency (PI) in adults. This includes, but is not limited to, common variable immunodeficiency (CVID), X-linked agammaglobulinemia, congenital agammaglobulinemia, Wiskott-Aldrich syndrome, and severe combined immunodeficiencies. HYQVIA is a gammaglobulin and is a slow metabolizer given subcutaneously once a month.There is data that Intravenous immunoglobulin(IVIg) helps POTS patients. We present a case of 57 year old female diagnosed with autonomic failure,orthostatic intolerance and primary immune deficiency. She was given IVIg for primary immune deficiency. She was previously reported for severe autonomic failure. From then, she was doing extremely well, had more energy and she thinks more clearly. She also had much better attitude. She was getting albumin every 2 weeks or so for volume expansion. She felt IVIg had helped her. Her immunoglobulin was switched from Gammagard to HYQVIA 35 grams. After she did, her blood pressure had gone up. She took it with albumin and she felt palpitations and chest pain.She was stopped on albumin, as albumin and HYQVIA combined could cause more volume expansion and push her into fluid overload or even congestive heart failure. After she stopped albumin without HYQVIA, she did well. Similarly, she cut back on midodrine which is an α1 adrenergic agonist, and works by stimulating receptors that noradrenaline normally works on. After swallowing, midodrine is rapidly converted into another, more active drug that binds to noradrenaline receptors causing blood vessels to narrow, thereby increasing blood pressure andreduction in heart rate in POTS patients. She was only HYQVIA and had been feeling extremely well.
Abstract 238: Loss of AKAP150 Promotes Pathological Remodeling and Heart Failure Propensity by Disrupting Calcium Homeostasis and Contractile Reserve
Authors: Li Lei , Li Jing , Drum Benjamin , Chen Yi , Yin Haifeng , Guo Xiaoyun , Luckey Stephen , Gilbert Merle , McKnight G. S , Scott John , Santana Luis F , Liu Qinghang
Published: June 2021

Impaired Ca
2+
cycling and myocyte contractility are a hallmark of heart failure triggered by pathological stress such as hemodynamic overload. The A-Kinase anchoring protein AKAP150 has been shown to coordinate key aspects of adrenergic regulation of Ca
2+
cycling and excitation-contraction in cardiomyocytes. However, the role of the AKAP150 signaling complexes in the pathogenesis of heart failure is largely unknown. Here we investigate how AKAP150 signaling complexes impact Ca
2+
cycling, myocyte contractility, and heart failure susceptibility following pathological stress. We detected a significant reduction of AKAP150 expression in the failing mouse heart induced by pressure overload. Importantly, cardiac-specific AKAP150 knockout mice were predisposed to develop dilated cardiomyopathy with severe cardiac dysfunction and fibrosis after pressure overload. Loss of AKAP150 also promoted pathological remodeling and heart failure progression following myocardial infarction. However, ablation of AKAP150 did not appear to affect chronic activation of calcineurin-NFAT signaling in cardiomyocytes or pressure overload- or agonist- induced cardiac hypertrophy. Immunoprecipitation studies showed that AKAP150 was associated with SERCA2, phospholamban, and ryanodine receptor-2, providing a targeted control of sarcoplasmic reticulum Ca
2+
regulatory proteins. Mechanistically, loss of AKAP150 led to impaired Ca
2+
cycling and reduced myocyte contractility reserve following adrenergic stimulation or pressure overload. These findings define a critical role for AKAP150 in maintaining Ca
2+
homeostasis and myocardial ionotropy following pathological stress, suggesting the AKAP150 signaling pathway may serve as a novel therapeutic target for heart failure.
Abstract 244: Differential mRNA Expressions in Thoracic Aortic Aneurysm in Patients with Bicuspid and Tricuspid Aortic Valve
Authors: Zhu Yuanjia , Hanick Andrea , Wright Mariah , Barnard John , Roselli Eric E , Van Wagoner David R
Published: June 2021

Objective:
Bicuspid aortic valve (BAV), the most common congenital heart condition, is frequently associated with thoracic aortic aneurysm (TAA). Aortic mRNA expression patterns in BAV patients and expression differences between BAV and tricuspid aortic valve (TAV) patients are unclear. We sought to compare mRNA expressions in TAA in patients with BAV and TAV.

Methods and Results:
Ascending aorta tissue was obtained from 58 aortic aneurysm repair patients (31 BAV, 27 TAV) and from 8 heart transplant donors. Illumina Human HT-12v4 microarrays were used to assess mRNA expression. After standard QC and filtering, probes were analyzed using multivariable linear regression analysis, adjusting for age, gender, medication use (beta blockers, ACE-Is, ARBs), and 10 surrogate variables. Ingenuity Pathway Analysis (IPA) was used to identify pathways and top regulator effect networks that were differentially expressed in BAV vs. TAV and BAV vs. donors.

In BAV vs. TAV, MMP9, ESM1 and IL1B were downregulated 0.63, 0.74 and 0.77 fold, whereas COMP was upregulated 2.29 fold (p < 0.05 for all). In BAV vs. donors, IL1R2, MCEMP1, IL18RAP and ACAN were downregulated 0.66, 0.2, 0.2 and 0.05 fold, while ADAMTS8, CCL21 and CIDEA were upregulated 6.97, 6.24 and 4.65 fold (p < 0.0001). IPA analysis showed P53 as the top upstream regulator for both BAV vs. TAV (p = 1.9e-8) and BAV vs. donors (p = 2.3e-25). IRF7, which protects against angiotensin II-induced hypertrophy in cardiomyocytes, was also a significant upstream regulator for BAV vs. TAV (p = 3.6e-7). Additionally, miR-124-3p in BAV vs. TAV (p = 6.3e-7) and BAV vs. donors (p = 1.7e-22), as well as miR-96-5p in BAV vs. TAV (p = 4e-7) were predicted significant upstream regulators. Immune response was shown to be the top regulator effect network in BAV vs. TAV, whereas perivascular fibrosis was shown to be an important regulator effect network in BAV vs. donors. Cell stress pathways were also activated in BAV vs. donors.

Conclusions:
Distinct pathways and regulator effect networks exist in TAA with BAV or TAV. TAV associated TAA is more associated with inflammatory responses, while BAV aortopathy is more associated with extracellular matrix remodelling and cell stress. MiRs may also play an important role in BAV aortopathy.
Abstract 247: Rtf1 Controls Sarcomere Integrity and Cardiac Function
Authors: Lu Fei , Langenbacher Adam , Gao Chen , Ren Vincent , Wang Yibin , Chen Jau-Nian
Published: June 2021

Sarcomere integrity must be continuously maintained in cardiomyocytes to ensure contractile function; however, the genetic programs controlling this process are not completely understood. We have previously shown that the RNA polymerase II associated protein Rtf1 directs cardiac progenitor cell formation during early cardiogenesis. Here we report that in mature cardiomyocytes, Rtf1 controls gene programs essential for the maintenance of sarcomere structure and cardiac function. Cardiomyocyte-specific ablation of Rtf1 in mice results in dilated cardiomyopathy characterized by thinning of the myocardial walls, dilation of the ventricular cavity and impaired left ventricle contractile function. Rtf1 knockdown in neonatal rat ventricular myocytes model results in the breakdown of sarcomeres and downregulation of myofibrillar gene programs accompanied by reduced abundance of monoubiquitinated H2BK120 and epigenetic marks associated with actively transcribed genes. In zebrafish embryos, mutation in Rtf1’s histone modification domain arrests cardiomyocyte differentiation and heart tube formation. Together these findings demonstrate that Rtf1 controls cardiomyocyte maturation, myofibril integrity and cardiac function by modulating cardiac epigenetic landscapes.
Abstract 255: Role of Connexin 43 in Muscular Dystrophy Cardiomyopathy
Authors: Gonzalez J. Patrick , Ramachandran Jayalakshmi , Kang Chifei , Xie Lai-Hua , Shirokova Natalia , Contreras Jorge , Fraidenraich Diego
Published: June 2021

Duchenne muscular dystrophy (DMD) is caused by an X-linked mutation that leads to the absence of dystrophin, resulting in life-threatening arrhythmogenesis and associated heart failure. The downstream events that lead to cardiomyopathy are largely unexplored. We unveiled a novel role for the gap junction protein connexin43 (Cx43) in maintaining cardiac conduction and ultimately in preventing heart failure from occurring in DMD. In mild mdx and severe mdx:utr mouse models of DMD, and human DMD tissues, Cx43 was found to be aberrantly upregulated and mislocalized to lateral sides of cardiomyocytes, with an abnormal phosphorylation pattern. We found that a triplet of serine residues, which plays a key role in a variety of cardiac pathologies, was hypophosphorylated in mdx relative to wild-type hearts. Electrocardiography on isoproterenol (Iso) challenged mice showed that both DMD mouse models developed arrhythmias and died within 24 hours, while wild-type mice were free of pathology. Administering peptide mimetic inhibitors of lateralized Cx43 channels prior to Iso challenge protected mdx mice from arrhythmogenesis and death. Cx43 copy number reduction in mdx:Cx43(+/-) mice not only ameliorated acute but also chronic, long-term symptoms of cardiomyopathy in mdx mice. Overall, our findings suggest that Cx43 lateralization contributes significantly to DMD cardiomyopathy and that selective inhibition of misplaced, upregulated channels may provide substantial benefit.
Abstract 257: Determining the Potential Role of cTnT Isoform Switching in the Development of Early Childhood Tropomyosin-linked DCM
Authors: Lynn Melissa , Holeman Teryn , Tal-Grinspan Lauren , Jin J.P. , Tardiff Jil C
Published: June 2021

An oft noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. Recently two unrelated multigenerational families were identified with the tropomyosin (Tm) mutation Asp230Asn (D230N), exhibiting a striking “bimodal” distribution of severity. In these families, many children (<1 year) with D230N Tm presented with a severe form of DCM that led to sudden, often fatal CHF, while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function into young adulthood. To explain this improvement, despite the persistence of D230N Tm, we hypothesized that the phenotype is modified by other thin filament (TF) isoforms. Thus we propose the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). We have shown that D230N Tm leads to a more stable (rigid) filament primarily at the C-terminus of Tm near the Tm overlap, a crucial region for TF regulatory function that cTnT modulates. Myofilaments from D230N Tm mice exhibited a small decrease in calcium sensitivity of force development that was significantly reduced in the presence of cTnT1, supportive of a modulatory role. We assessed cardiac performance in our novel D230N Tm x cTnT1 double transgenic (DTg) mice, % FS was similarly reduced for D230N Tm and DTg mice at 2 months likely due to persistent endogenous expression of cTnT1. Divergent cardiac remodeling occurred at 4 months at which point DTg mice had significantly reduced % FS compared to D230N, indicating that additive exposure to cTnT1 is detrimental to the function of D230N hearts. Additionally,
In vitro
studies on non-failing and failing human heart tissue found that RNA levels of cTnT1 are significantly higher in failing hearts. Thus modulation by cTnT1 could be a more general mechanism for the progressive remodeling seen in heart failure.
Abstract 288: Novel Mechanisms of Non-coding Genomic Regulation Identified in Cardiac Disease-in-a-dish Models
Authors: Kumar Aditya , Thomas Stephanie , Wong Kirsten , Tenerelli Kevin , Lo Sardo Valentina , Ferguson William , Topol Eric , Baldwin Kristin , Engler Adam
Published: June 2021

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) at gene loci that affect cardiovascular function, and while mechanisms in protein-coding loci are obvious, those in non-coding loci are difficult to determine. 9p21 is a recently identified locus associated with increased risk of coronary artery disease (CAD) and myocardial infarction. Associations have implicated SNPs in altering smooth muscle and endothelial cell properties but have not identified adverse effects in cardiomyocytes (CMs) despite enhanced disease risk. Using induced pluripotent stem cell-derived CMs from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs and either CAD positive or negative, we assessed CM function when cultured on hydrogels capable of mimicking the fibrotic stiffening associated with disease post-heart attack, i.e. “heart attack-in-a-dish” stiffening from 11 kiloPascals (kPa) to 50 kPa. While all CMs independent of genotype and disease beat synchronously on soft matrices, R/R CMs cultured on dynamically stiffened hydrogels exhibited asynchronous contractions and had significantly lower correlation coefficients versus N/N CMs in the same conditions. Dynamic stiffening reduced connexin 43 expression and gap junction assembly in R/R CMs but not N/N CMs. To eliminate patient-to-patient variability, we created an isogenic line by deleting the 9p21 gene locus from a R/R patient using TALEN-mediated gene editing, i.e. R/R KO. Deletion of the 9p21 locus restored synchronous contractility and organized connexin 43 junctions. As a non-coding locus, 9p21 appears to repress connexin transcription, leading to the phenotypes we observe, but only when the niche is stiffened as in disease. These data are the first to demonstrate that disease-specific niche remodeling, e.g. a “heart attack-in-a-dish” model, can differentially affect CM function depending on SNPs within a non-coding locus.
Abstract 30: In vitro Fabrication of Human Cardiac Tissues With Porcine Perfusable Blood Vessels
Authors: Inui Akitoshi , Sekine Hidekazu , Sano Kazunori , Dobashi Izumi , Yoshida Azumi , Matsuura Katsuhisa , Kobayashi Eiji , Ono Minoru , Shimizu Tatsuya
Published: June 2021

The definitive treatment of severe heart failure is heart transplantation; however the number of heart transplantation procedures performed in Japan per year ranges from 30-40 due to donor shortage. Therefore, recently other treatments such as ventricular assist device or regenerative therapy by human cardiac tissue engineering have been developed and are considered as appropriate alternatives. We have developed an original technology, which was named cell-sheet based tissue engineering to fabricate functional three-dimensional tissue by layering cell sheets. The utilization of this technique allowed us to successfully engineer thick rat cardiac tissue with perfusable blood vessels in vitro. Here, we demonstrate a technique to engineer human cardiac tissue with perfusable blood vessels using cardiac cell sheets derived from human induced pluripotent stem cells, and porcine small intestine as a vascular bed for perfusion culture. The small intestine was harvested from with a branch of the superior mesenteric artery and vein and underwent mucosal resection after harvested tissue was cut open. To engineer cardiac tissue with perfusable blood vessels, cardiac cell sheets co-cultured with endothelial cells, were triple-layered and then was overlaid on the vascular bed in the bioreactor system. One day after perfusion culture, overlaid cardiac tissues pulsated spontaneously and were synchronized. The cardiac tissue construct was viable tissue without any observable necrosis. Furthermore we examined the possibility of transplantation of the in vitro engineered human cardiac tissue with the connectable host artery and vein. Engineered cardiac tissue was removed from the bioreactor system after 4-day perfusion, and transplanted to another pig heart. The branch of the superior mesenteric artery and vein of the graft were then reconnected to the host internal thoracic artery and vein. When the cardiac tissue reperfused, it began to beat spontaneously after a few minutes. We believe that this method is useful to fabricate functional cardiac tissue and may become an appropriate treatment for severe heart failure.
Abstract 304: Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-mediated Angiogenesis
Authors: Chen Hong
Published: June 2021

Background:
VEGF-induced binding of VEGFR2 to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2, however the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain if such determinants were critical for physiological angiogenesis
in vivo
.

Methods and Results:
Structural modeling uncovered two novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Further, we found that the VEGF-induced VEGFR2-epsin interaction promoted c-Cbl-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties
in vitro
, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing.

Conclusions:
Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.