bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 212: Card9-mediated Signaling is Critical to Cytokine Production in Mice With PM Exposure
Authors: Liu Xuanyou , Cui Yuqi , Xiao Yichao , Zhu Qingyi , Jiang Meng , Duan Yunyan , Hao Hong , Abraham Joseph , Jiang Bimei , Shen Rulong , Sun Qinghua , LIU Zhenguo
Published: July 2021

Oxidative stress and inflammation are considered an important mechanism for the development of cardiovascular diseases. Cytokines including interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) play an important role in oxidative stress and inflammation. It is known that ambient fine particulate matter (
PM
) exposure is closely associated with cardiovascular diseases and oxidative stress. Caspase-recruitment domain 9 (
Card9
) signaling is critically involvement in the function of macrophages, neutrophils and monocytes that are important for oxidative stress and inflammation. The present study was designed to evaluate the role of CARD9-mediated signaling in cytokines production in mice with PM exposure. Both male wild-type (WT) C57BL/6 mice (8-10 weeks) and age-matched CARD9 knockout (KO) mice (with C57BL/6 background) were exposed to PM2.5 for 6 weeks via intranasal approach with PBS as the control. Serum concentrations of the cytokines including IL-6, IL-1β, and TNF-α were measured with ELISA in the mice before and after PM exposure. There was no difference in the serum levels of IL-6, IL-1β, or TNF-α between WT mice and CARD9 KO mice exposed to PBS. As expected, PM exposure substantially increased the serum levels of IL-6, IL-1β, and TNF-α in the WT mice (by up to 6 times). However, no significant increase in the serum concentrations for IL-6, IL-1β, and TNF-α was observed in CARD9 KO mice exposed to PM. Increased inflammatory infiltrations in the lungs were observed in the WT mice as compared to the CARD9 KO mice with PM exposure. In conclusion, the present study demonstrated that increased cytokines were produced in WT mice, but not in CARD9 KO mice with PM exposure. The data suggested that CARD9 signaling played a critical role in the production of inflammatory cytokines in the mice in response to PM exposure, and might contribute to the development of cardiovascular diseases related to PM exposure.
Abstract 213: Interleukins 17 a and 22: Are There Differences in Blood Concentrations of Patients With or Without Atherosclerosis ?
Authors: Oliveira Dinaldo C , Brasileiro Augusto , Oliveira Danielle , Heide Elayne , Gurgel Myrtson , Medeiros Orlando , Amorim Jessica
Published: July 2021

Introduction:
The role of the immune and inflammatory pathways in patients with atherosclerosis is important but not complete understood. The objective of this study was to evaluate if patients with coronary atherosclerosis have higher concentrations of interleukins 17 A and 22 when compared to patients without carotid atherosclerosis.

Hypothesis:
There are higher concentrations of interleukins 17 A and 22 in patients with coronary artery disease than in patients without carotid disease

Methods:
This is a cross-sectional, prospective, analytical study, conducted from August to December 2015, that enrolled 60 patients. We included 30 patients with stable CAD with coronary stenosis ≥ 50% according to current coronary angiography and 30 patients with chronic infectious parasitic disease without carotid atherosclerosis according to intimal medial thickness. Interleukins (IL) were evaluated in serum of patients. IL concentrations were expressed in pg / ml and the detectable minimum values of interleukins were: 17A = 15.62 pg/ml and 22 = 7.81 pg/ml. Descriptive and analytical statistical analyzes were performed. The Shapiro-Wilk normality test was applied to verify the normality of the data. Statistical tests were used to compare the variables and p-value < 0.05 was significant

Results:
There were 18 men and 12 women in the group of patients with coronary disease and 14 men and 16 patients without carotid disease. The main CAD risk factors (in the group of patients with coronary atherosclerosis) were: Hypertension 63%, Diabetes Mellitus 40%, dyslipidemia 33%, smoking 23%. The serum concentrations of interleukins 17A showed: patients with coronary atherosclerosis = 15.62 pg/ml vs patients without carotid atherosclerosis = 15.62 pg/ml. Serum concentrations of interleukins 22 showed: patients with coronary atherosclerosis = 7.81 pg/ml vs patients without carotid atherosclerosis = 7.81 pg/ml.

Conclusions:
Interleukin 17 and 22 concentrations were low in both groups of patients and there were no differences between patients with coronary atherosclerosis and no carotid atherosclerosis. Therefore, it is possible that these interleukins measured may not identify who has coronary atherosclerosis and who does not have carotid atherosclerosis.
Abstract 215: Allogeneic MSCs Improve Endothelial Function in Patients with Dilated Cardiomyopathy via an SDF-1α-mediated Mechanism and the Suppression of Pathologic Cytokines
Authors: Premer Courtney , Schulman Ivonne H , Balkan Wayne , Porras Valeria , Bellio Michael A , Hare Joshua M
Published: July 2021

Endothelial dysfunction is central to the pathophysiology of heart failure, including dilated cardiomyopathy (DCM). Current drug therapies are unable to halt the progression of DCM, compelling the emergence of novel stem cell therapy approaches. Mesenchymal stem cells (MSCs) are pro-angiogenic, immunomodulatory, antifibrotic, and stimulate endogenous endothelial progenitor (EPC) proliferation and function, thus having the potential to ameliorate endothelial dysfunction. We demonstrated that patients with DCM who received allogeneic MSCs had a significant improvement in endothelial function 3-months post treatment, whereas patients who received autologous MSCs had no improvement. Therefore, we hypothesized that allogeneic MSCs preferentially improve endothelial function via a mechanism involving the suppression of pathologic levels of vascular endothelial growth factor (VEGF), stromal derived factor-1 alpha (SDF-1α), and tumor necrosis factor alpha (TNFα). Accordingly, patient serum VEGF and TNFα were measured at baseline and 3 months post MSC treatment. In vitro, MSC secretion of SDF-1α and TNFα was also measured. Our results show that patients with DCM had elevated levels of VEGF (n=21, 581.2±812.2 pg/mL) and TNFα (n=15, 22±9.4 pg/mL) at baseline, and that only allogeneic MSCs were able to restore these levels toward normal (VEGF: n=10, Δ-267.1±252.1, P=0.01; TNFα: n=8, Δ-7.1±3.1 pg/mL, P=0.0005). While there was no difference in TNFα secretion by autologous or allogeneic MSCs (0.01±0.14 vs. 0.4±0.6 pg/mL), autologous MSCs secreted significantly higher levels of SDF-1α compared to allogeneic MSCs (n=12, 79.3±16.7 vs. 14.2±9.4 pg/mL, P=0.0001). In vitro secreted SDF-1α and serum VEGF and TNFα levels correlated with EPC bioactivity (ΔSDF-1α to ΔEPC-CFUs, R=-0.9, P<0.0001; ΔVEGF to ΔEPC-CFUs, R=-0.7, P=0.001; ΔTNFα to ΔEPC-CFUs, R=-0.6, P=0.01). These findings reveal a novel mechanism by which allogeneic MSCs secrete physiologic levels of SDF-1α resulting in physiologic levels of VEGF signaling, reduced TNFα, increased EPC bioactivity, and improved endothelial function. These findings have important clinical and biological implications for the use of MSCs in patients with DCM.
Abstract 216: Causal Role of Oxidized Lipids in Pulmonary Hypertension Development
Authors: Ruffenach Gregoire , Umar Soban , Vaillancourt Mylene , Grijalva Victor , O’Connor Ellen I , Moazeni Shayan , Cunningham Christine , Ardehali Abbas , Mahajan Aman , Reddy Srininivasa T , Eghbali Mansoureh
Published: July 2021

Pulmonary arterial hypertension (PAH) is a deadly disease characterized by increased pulmonary arterial pressure and pulmonary vascular occlusion. Recently, we and others demonstrated a robust increase in oxidized lipids, including 15-hydroxyeicosatetraenoic acids (15-HETE), in the lungs and plasma of PAH patients and animal models of pulmonary hypertension (PH). We hypothesized that diets rich in 15-HETE are sufficient to cause PH in wild type mice. We also examined whether 15-HETE or its metabolites are required to cause PH by comparing the effect of 15-HETE with 15-HETE methyl ester, which is a stable form of 15HETE that is not easily metabolized. C57BL/6 male mice were fed for 3 weeks with 15-HETE diet (5μg/day), 15-HETE methyl ester (15-HETE-ME, 5μg/day), or regular chow diet (n=8-21 mice/group). PH development was followed
via
weekly serial echocardiography. Right ventricular systolic pressure (RVSP) was measured via direct heart catheterization. RV hypertrophy index (RV/[IVS+LV]) was measured. Lung morphology and lipid accumulation were assessed using H&E and Oil red O staining. Echocardiography revealed the first sign of PH in mice on 15HETE diet as early as one week and a significant decrease in the pulmonary arterial acceleration time after 2 weeks of treatment (16.6±1.9 vs. 21.2±1.4 msec, p<0.05). Mice on 15HETE diet also had significantly higher RVSP (31.3±1.1 vs. 38.4±2.3 mmHg, p<0.05). Increase in RVSP was concomitant with significantly higher RV hypertrophy index (0.26 ± 0.02 vs. 0.33 ±0.02, p<0.05). Pulmonary arteriolar thickness was also significantly increased in mice on 15-HETE diet compared to regular diet (35.1±0.8 vs 53.4±1, p<0.05). Our new model of PH is not a model of atherosclerosis as there was no detectable plaque in aorta of the mice on 15-HETE diet. Finally, mice on 15-HETE-ME diet also developed PH as RVSP was significantly higher compared to control (31.3±1.1 vs. 39±3 mmHg, p<0.05). The severity of PH was similar in 15HETE-ME and 15HETE, confirming 15HETE itself and not its metabolites is sufficient to cause PH in wild type mice. We have developed a new and physiologically relevant animal model to study PH as a consequence of oxidized lipids overload as it occurs in humans with PAH.
Abstract 220: Plasma Extracellular RNAs In Lv Remodeling Post MI
Authors: Yeri Ashish S , Danielson Kirsty , Liu Xiaojun , Shah Ravi , Camacho Fernando , Tanriverdi Kahraman , Xiao Chunyang , Tschabrunn Cory , Anter Elad , Freedman Jane , Kwong Raymond , Rosenzweig Anthony , Wang Yaoyu , Das Saumya
Published: July 2021

Despite significant declines in early mortality after acute myocardial infarction (MI), heart failure (HF) remains a significant chronic complication following MI. Small non-coding RNAs are short (<200 nt) RNA transcripts that regulate networks of gene expression in cardiovascular disease (CVD), including fibrosis, atherosclerosis and arrhythmia. We hypothesized that plasma circulating extracellular RNAs (ex-RNAs) would be associated with cardiac remodeling by cardiac magnetic resonance in humans post-MI and dysregulated in animal models of ischemia. First, we found that plasma circulating ex-RNA profiles by RNA-seq are distinct in individuals with prior MI relative to healthy individuals without cardiovascular disease with several novel ex-RNAs detected (e.g., y-RNAs;
Figure 1
). Principal components of candidate RNAs selected from differential expression n RNA-seq were associated with LV phenotypes post-MI, specifically LV mass and fibrosis at 4-6 weeks post-MI and change in LV mass at 6 months. In animal and cellular models of ischemia, we found that (1) candidate miRNAs found in humans were expressed in a temporally and cell-specific fashion in the myocardium and (2) candidate miRNA expression increased in cardiomyocyte culture after hypoxia/reoxygenation. Collectively, these data add to a burgeoning literature implicating plasma circulating ex-RNAs as functional markers of acute cardiovascular disease involved in cardiac remodeling en route to HF.
Abstract 23: Exosome Inhibition Improved Blood Perfusion in Ischemic Hindlimb of db/db Diabetic Mice
Authors: Cheng Zhongjian , Garikipati Venkata Naga Srikanth , Cimini Maria , Wang Chunlin , Trungcao May , Tang Yan , Ye Yujia , Benedict Cindy , Goukassian David , Verma Suresh K , Kishore Raj
Published: July 2021

Background:
Critical limb ischemia (CLI), a life-threatening condition characterized by pain at rest and tissue loss with ulcer and gangrene, imposes a major public healthy burden, resulting in high mortality and disability. The occurrence of CLI in patients with diabetes mellitus is very frequent. However, the effective therapy for CLI in diabetic patients is absent. Recent studies demonstrated that exosome from diabetic animals/cells has detrimental effects on the post-injury cardiovascular repair. Here, we tested the hypotheses that exosome inhibition
in vivo
improves blood flow recovery and protects skeletal muscle in ischemic hindlimbs of diabetic db/db mice following surgical ischemia.

Methods and Results:
Exosomes were isolated from bone-marrow derived progenitor cells or plasma in non-diabetic db/+ and diabetic db/db mice by ultracentrifugation. Diabetic exosome (5 ug/ml) inhibited tube formation of human cardiac microvascular endothelial cells. Unilateral hindlimb ischemia surgery was conducted by ligation of left femoral artery in 12-week old, male db/+ and db/db mice. Exosome inhibitor GW4869 (2 μg/g body weight) was given by intraperitoneal injection every other day for 4 weeks starting from one week before the HLI surgery. HLI mice injected with vehicle served as controls. Mice were divided into four groups: 1) db/+ + vehicle; 2) db/db+ vehicle; 3) db/+ GW4869; 4) db/db + GW4869. GW4869 decreased necrosis and loss of toe/toenail, improved blood flow, enhanced capillary/arterial density, skeletal muscle architecture and cell survival in ischemic hindlimb of diabetic db/db mice 21 days post-ligation.

Conclusions:
Although preliminary, our experiments suggest that therapeutic targeting of dysfunctional exosome secretion could represent a new avenue for the prevention and treatment of ischemic injury in diabetic patients.
Abstract 237: PRMT1-p53-Slug Axis Regulates Epicardial EMT and Ventricular Development
Authors: Jackson-Weaver Olan , Wu Jian , Gou Yongchao , Chen Yibu , Li Meng , Sucov Henry , Xu Jian
Published: July 2021

Rationale:
Epicardial epithelial-to-mesenchymal trasition (EMT) is a vital process in embryonic heart development. During EMT, epicardial cells acquire migratory and invasive properties, and differentiate into new cell types, including cardiac fibroblasts and coronary smooth muscle cells. Non-histone protein methylation is an emerging modulator of cell signaling. We have recently established a role for protein arginine methyltransferase-1 (PRMT1) in TGF-β-induced EMT in cultured cells.

Objective:
To determine the role of PRMT1 in epicardial EMT.

Methods and Results:
We investigated the role of PRMT1 in epicardial EMT in mouse epicardial cells. Embryonic day 9.5 (E9.5) tamoxifen administration of WT1-Cre
ERT
;PRMT1
fl/fl
;ROSA-YFP
fl/fl
mouse embryos was used to delete PRMT1 in the epicardium. Epicardial PRMT1 deletion led to reduced epicardial migration into the myocardium, a thinner compact myocardial layer, and dilated coronary blood vessels at E15.5. Using the epicardial cell line MEC1, we found that PRMT1 siRNA prevented the increase in mesenchymal proteins Slug and Fibronectin and the decrease in epithelial protein E-Cadherin during TGF-β treatment-induced EMT. PRMT1 siRNA also reduced the migration and invasion of MEC1 cells. We further identified that PRMT1 siRNA also increased the expression of p53, a key regulator of the Slug degradation pathway. PRMT1 siRNA increases p53 expression by decreasing p53 degradation, and shifted p53 localization to the cytoplasm.
In vitro
methylation assays further demonstrated that PRMT1 methylates p53. Knockdown of p53 increased Slug levels and enhanced EMT, establishing p53 as a regulator of epicardial EMT through controlling Slug expression. Furthermore, RNAseq experiments in MEC1 cells demonstrated that 40% (545/1,351) of TGF-β-induced transcriptional changes were prevented by PRMT1 siRNA. Furthermore, when p53 and PRMT1 were simultaneously knocked down, TGF-β induced transcriptional control of 37% (201/545) of these PRMT1-dependent genes was restored.

Conclusions:
The PRMT1-p53-Slug pathway is necessary for epicardial EMT in cultured MEC1 cells as well as in the epicardium
in vivo
. Epicardial PRMT1 is required for the development of compact myocardium and coronary blood vessels.
Abstract 240: Asb2-dependent Proteolysis of the Cytoskeleton Directs Cardiac Development and Disease
Authors: Yamak Abir , Hu Dongjian , Domian Ibrahim J
Published: July 2021

Congenital heart diseases (CHDs) account for 25% of birth defects and are major risk factors for adult cardiovascular problems. Partial disease penetrance is seen even in autosomal dominant disorders and genotype/phenotype correlations remain a clinical challenge; thus, the need to understand different regulators of cardiac formation. The Ubiquitin-Proteasome System (UPS) is important in controlling protein turnover during organ development but its role in the mammalian heart remains ambiguous. We have identified a specificity subunit of ubiquitin-mediated proteolysis (Asb2) as being specific for the cardiac myogenic lineage. Asb2 was previously shown to regulate hematopoietic and skeletal muscle cell differentiation through targeting filamin proteins (FlnA, B and C), actin-binding proteins important for cytoskeleton stabilization. In our present study, we show that Asb2 is markedly enriched in myocardial progenitor cells and cardiomyocytes. To investigate the role of Asb2 and UPS dependent proteolysis in heart development, we generated two cardiac-specific murine knockouts (KOs): Nkx
Cre
.Asb2
-/-
and Mef2c
Cre
.Asb2
-/-
(deleting Asb2 in early cardiomyocyte progenitors and anterior heart field progenitors, respectively). Both KOs are embryonic lethal with pericardial edema. We used tissue clarifying and confocal microscopy to define the morphological defects of the Asb2 null heart. Moreover, we found that FlnA is overexpressed in the hearts of these mice and its deletion therein partially rescues their lethality. In addition, using transcriptomic analysis on Asb2-null e9.5 hearts, we identified novel potential Asb2 targets in the heart. Finally, to understand the role of Asb2 in the differentiation and function of human cardiomyocytes, we used CRISPR/Cas9 genome editing technique to generate Asb2-null human induced pluripotent stem cells.

Collectively, our study provides a novel mechanistic understanding of the role of the UPS proteasome in cardiac development, myocardial function, and disease pathogenesis. Given recent interests in both the UPS and the cytoskeleton as therapeutic targets, our study provides an innovative platform for the development of pharmacotherapy for cardiac disease.
Abstract 25: P2Y14 Purinergic Receptor Overexpression: Letting Blind Cardiac Progenitor Cells ’See’ Again
Authors: Khalafalla Farid G , Kayani Waqas , Kassab Arwa , Ilves Kelli , Alvarez Roberto J , Chavarria Monica , Norman Benjamin , Sussman Mark A
Published: July 2021

Heart failure (HF) is a leading cause of death due to limited regenerative capacity of adult mammalian heart following injury. Autologous stem cell therapy holds promise for promoting cardiac regeneration. However, stem cells derived from aged/diseased organs exhibit poor growth and survival capabilities. Empowering cardiac progenitor cells (CPC) with prosurvival genes has been attempted. Nonetheless, the molecular mechanisms by which stem cells initially detect stress signals to stimulate appropriate regenerative responses are poorly understood. This study aims to explore the physiological responses mediated by purinergic receptors, which represent a major detector for extracellular nucleotides released during injury/stress, with a focus on P2Y
14
nucleotide receptor (P2Y
14
R) activated by extracellular UDP-conjugated sugars. P2Y
14
R mediates proliferation of keratinocytes and chemotaxis of neutrophils and hematopoietic stem cells (HSCs). In addition, P2Y
14
R enhances HSC resistance to stress-induced senescence and maintains regenerative capacity after injury. However, the physiological roles of P2Y
14
R in CPCs are largely unknown. Preliminary data show striking correlations between P2Y
14
R expression in human CPCs derived from HF patients (hCPCs) and patients’ ejection fraction (EF), where low EF corresponds to low P2Y
14
R expression hCPCs. Moreover, hCPCs with relatively slower growth kinetics and enhanced senescence exhibit dramatic decreases in P2Y
14
R expression compared to fast-growing hCPCs. P2Y
14
R overexpression improves hCPC proliferation, migration, survival under stress stimuli and reverses senescent-associated phenotypes. Furthermore, P2Y
14
R-overexpressing hCPCs show remarkable upregulation in the expression of paracrine factors critical for cardiac repair. Preliminary studies will be extended
in vivo
to assess whether P2Y
14
R overexpression in hCPCs improves their reparative potential for injured mouse myocardium. Overall, this study introduces a novel interventional molecular approach to improve the therapeutic outcome of hCPCs by enhancing their capability to detect stress-induced extracellular nucleotides and initiate proper regenerative responses through augmenting P2Y
14
R expression.
Abstract 271: Deletion of Delta-like 1 Homolog Accelerates Fibroblast-myofibroblast Differentiation and Induces Myocardial Fibrosis
Authors: Troncone Luca , Rodriguez Patricia , Sassi Yassine , Benard Ludovic , Ishikawa Kiyotake , Gordon Ronald E , Lamas Santiago , Laborda Jorge , Hajjar Roger J , Lebeche Djamel
Published: July 2021

Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. Here we uncover that Delta-like homologue 1 (Dlk1), a paternally imprinted gene encoding a transmembrane protein belonging to the Epidermal Growth Factor (EGF)-like family, orchestrates the process of cardiac fibroblast to myofibroblast differentiation and controls myocardial fibrosis. We first show that cardiomyocytes and cardiac fibroblasts express different Dlk1 mRNA spliced variants and its absence accelerates fibroblast differentiation into myofibroblasts in vitro. Overexpression of Dlk1 in cardiac fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process appears to be regulated by TGFβ-1 signaling, since fibroblasts lacking Dlk1 exhibited a higher activation of the TGFβ-1/Smad-3 pathway at baseline, leading to an earlier acquisition of the myofibroblast phenotype. Dlk1-null mice myocardium displayed increased TGFβ-1/Smad3 profibrotic activity, resulting in infiltration/accumulation of myofibroblasts, and induction and deposition of the extracellular matrix fibronectin extra domain A isoform and collagen, supporting a role for Dlk1 in cardiac fibrosis. Furthermore, these profibrotic events were associated with reduced myofibril integrity, myocyte hypertrophy and cardiac dysfunction. Interestingly, Dlk1 expression was downregulated in ischemic heart tissue from human patients and in the border and scar-zones of infarcted pigs’ hearts. This phenotype was paralleled by increased expression of the profibrotic markers, collagen I, lysyl oxidase and α-smooth muscle actin. Mechanistically, the inhibitory action of Dlk1 on cardiac fibroblast-myofibroblast differentiation is mediated by miR-370 direct targeting of TGFβ-R2/Smad-3 signaling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signaling leads to chronic fibrosis.
Abstract 274: Defective Branched Chain Amino Acid Catabolism Impairs Exercise Endurance
Authors: Abell Lauren , Shi Rufeng , Shao Dan , Kolwicz Stephen , Tian Rong
Published: July 2021

The branched chain amino acids (BCAA), leucine, isoleucine and valine, are essential for mammalians, and they play a positive role in exercise capacity, muscle development, and a lean body phenotype. BCAA supplementation is commonly paired with exercise in order to promote muscle growth, increase resistance to fatigue and reduce muscle soreness. On the other hand, elevated serum BCAA is strongly and positively correlated with the development of insulin resistance, coronary heart disease, and type II diabetes, and is predictive of patient response to therapeutics and intervention outcomes. We have previously shown that defective BCAA catabolism in mice impaired glucose metabolism in the heart and increased susceptibility to stress-induced cardiac damage. In this study we sought to determine the effects of elevated BCAA levels on skeletal muscle performance and response to exercise training using mouse models with systemically elevated BCAA levels. Supplementation of BCAA (1.5mg/g bodyweight/day, ratio of Leu:Ile:Val = 1.5:0.8:1) was administered to mice with impairment of BCAA catabolism due to the deletion of mitochondrial-localized protein phosphatase 2C (KO), a key enzyme in activating BCAA catabolism, and their littermate controls (CON). Mice were subjected to one week of daily exercise training via forced treadmill running and an exercise capacity test was performed at the beginning and end of training. Baseline maximum running time was decreased in the KO compared to CON (mean 73.4 and 82.5 min, respectively). One week of training resulted in increased exercise capacity in CON with an attenuated increase in KO mice (mean 136 and 112 min, respectively). BCAA supplementation did not further improve exercise capacity in CON (mean 131 min) and abrogated the response to training in KO (mean 70.1 min). Reduced exercise capacity positively correlated with elevated serum BCAA levels. Additionally, KO supplemented with BCAAs demonstrated elevated serum succinate, alanine and glutamate levels, which are metabolic markers of physiological stress. We conclude that short term supplementation of BCAA has no benefit for exercise capacity and accumulation of BCAAs has a negative effect on endurance exercise capacity.
Abstract 291: Platelet Function Monitoring After Percutaneous Coronary Intervention: Updated Meta-analysis of Randomized Trials.
Authors: Akinjero Akintunde M , Adegbala Oluwole , Edo-Osagie Esosa , Akinjero Nike , Akinyemiju Tomi
Published: July 2021

Background:
The need to balance bleeding and clotting risks after percutaneous coronary intervention (PCI) has led to interest in platelet function monitoring as a strategy to improve post-PCI outcomes. The prognostic value of platelet function testing in monitoring response to antiplatelet therapy after PCI remains unclear. Prior studies have been inconclusive. We sought to conduct an updated meta-analysis to address this gap in knowledge.

Methods:
We conducted a systematic search of EMBASE, PUBMED and the Cochrane libraries for studies since inception to December 2016 on platelet function monitoring. Our search yielded 203 studies, out of which 83 were extracted for full-text review. Only 3 studies met inclusion criteria. We pooled odds ratios using random-effects statistics, Mantel-Haenszel method. I2 and Chi- squared statistic was used to evaluate for heterogeneity. Publication bias was assessed using the funnel plot. Primary outcome was major adverse cardiovascular events (MACE). This was defined in the studies as a composite of cardiovascular death, myocardial infarction, stroke, stent thrombosis, and bleeding complications.

Results:
The 3 randomized controlled trials that were analyzed involved 3701 patients. There were 550 MACE (29.76%) in the platelet function monitored group compared with 514 (27.74%) in the control. MACE (Figure 1) was not significantly higher for the platelet function monitored group during follow-up compared with control (pooled Odds Ratio:1.11 [95% CI: 0.96-1.28], p = 0.15). Tests for heterogeneity were not significant, with I2 of 0%, Chi2 = 1.52 (p = 0.47); and small study bias was absent on visual inspection of the funnel plot.

Conclusions:
Platelet function monitoring continues to be used in practice. Results from this meta-analysis show no benefit of platelet function monitoring compared with conventional strategy with regards to MACE after PCI. Future research is needed to further evaluate this finding.
Abstract 294: Mitochondrial Inner Membrane Protein Plays a Critical Role in the Ischemic Myocardial Dysfunction and Determines the Outcome of Cardiac Recovery
Authors: Bopassa Jean C
Published: July 2021

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction and stroke, is the leading cause of death in the US. Indeed, reoxygenation of cardiomyocytes after an ischemic insult leads to mitochondrial Ca
2+
overload and increase in ROS generation that triggers the opening of mitochondrial permeability transition pore (mPTP), making this event crucial in the mechanism of cell death after I/R. Several proteins have been proposed contributing to formation and function of mPTP, but its exact molecular identity and mechanism still need to be elucidated. Cyclophylin D (CypD) deleted mice displayed a marked desensitization of the mPTP to Ca
2+
overload and oxidative stress establishing it as a key component of mPTP. However, CypD null mitochondria still exhibit mPTP activity. Using 2D-DIGE and mass spectrometry, we identified mitofilin, which plays an important role of maintaining mitochondrial cristae morphology, as a protein that the expression is reduced after I/R versus sham. We thus investigated the impact of mitofilin regulation in the mPTP formation. We found that:
i)
mitofilin
-/-
mice subjected to I/R exhibited an increase in myocardial infarct size, a reduced cardiac functional recovery, a reduced mitochondrial Ca
2+
retention capacity required to induce the mPTP opening and an increase in mitophagy compared to WT;
ii)
Knockdown of mitofilin in H9c2 myoblasts with siRNA led to: increase in apoptosis via activation of AIF-PARP1 pathway that is associated with S phase arrest of the cell cycle and nuclear fragmentation, increase in mitochondrial cristae disorganization, in ROS production and Calpain activity, as well as decrease in intracellular ATP production and mitochondrial membrane potential versus scramble siRNA;
iii)
mitofilin structurally bound to CypD and this interaction is abridged after the mPTP opening triggered by Ca
2+
overload after I/R. These results indicate that mitofilin plays an essential role in the mechanism of mPTP formation and cardiomyocyte death.
Abstract 297: GJA1-20k Protects the Heart From Ischemic Injury by Inducing Mitochondrial Metabolic Quiescence
Authors: Fu Ying , Basheer Wassim , Sun Baiming , Agvanian Sosse , Xiao Shaohua , Hong TingTing , Shaw Robin M
Published: July 2021

The mRNA of the
GJA1
gene that encodes gap junction protein connexin 43 (Cx43) undergoes alternative translation, producing N-terminal truncated smaller protein isoforms. Unlike the well-characterized full-length Cx43 (GJA1-43k), little is known about the function and regulation of the smaller isoforms. Here, we report that global ischemia/reperfusion injury in Langendorff-perfused mouse hearts upregulates endogenous GJA1-20k, the most abundant isoform of alternative translation. Biochemical fractionation indicates that the induced GJA1-20k is preferentially enriched in cardiomyocyte mitochondria. When introduced in vitro through adenovirus-medicated gene expression, exogenous GJA1-20k, but not full length protein GJA1-43k, localizes to mitochondria and improves the survival and viability of adult cardiomyocytes when subjected to oxidative stress. In vivo gene transfer of GJA1-20k through retro-orbital injection of AAV9 virus results in lower mitochondria-dependent basal oxygen consumption as well as maximal respiratory capacity in cardiomyocytes, unlike GFP or GJA1-43k controls. Thus GJA1-20k, but not GJA1-43k, protects the heart against ischemic injury induced by permanent LAD ligation. As compared to GFP control group, myocardial infarct size in GJA1-20k treated hearts is reduced by 30% at 72 hours post LAD ligation. These results indicate that endogenous GJA1-20k is induced upon stress and has strong tropism to mitochondria. Increased GJA1-20k induces mitochondrial metabolic quiescence and affords cardioprotection in vitro and in vivo. Alternatively translated GJA1-20k acts as a novel mitochondrial stress protein and demonstrates therapeutic potential against ischemic injury.
Abstract 3: Loss of Type 9 Adenylyl Cyclase Triggers Reduced Phosphorylation of Hsp20 and Diastolic Dysfunction
Authors: Baldwin Tanya A , Li Yong , Wang Yan , Brand Cameron S , Dessauer Carmen W
Published: July 2021

Adenylyl cyclase type 9 (AC9) is found tightly associated with the scaffolding protein Yotiao and the I
Ks
ion channel in heart. But apart from potential I
Ks
regulation, physiological roles for AC9 are unknown. Utilizing a gene-trap mouse line that disrupts expression of AC9, we show that loss of AC9 reduces less than 2% of total AC activity in heart but eliminates Yotiao-associated AC activity. AC9
-/-
mice exhibit no structural abnormalities but show a significant bradycardia. Global changes in PKA phosphorylation patters are not altered in AC9
-/-
heart, however basal phosphorylation of heat shock protein 20 (Hsp20) is significantly decreased. AC9 binds Hsp20 in a Yotiao-independent manner, while deletion of AC9 decreases Hsp20-associated AC activity in heart, consistent with and an AC9-Hsp20 complex.

Phosphorylation of Hsp20 occurs largely in ventricles and is vital for the cardioprotective effects of Hsp20. Decreased Hsp20 phosphorylation suggests a potential baseline ventricular defect for AC9
-/-
. Doppler echocardiography of AC9
-/-
mice displays a decrease in the early ventricular filling velocity and ventricular filling ratio (E/A), indicative of grade 1 diastolic dysfunction. Our findings unveil potential new roles for AC9 in cardiac function and emphasize the importance of local cAMP production in the context of macromolecular complexes.
Abstract 30: miR34a Affects the Functional Potential of Human Cardiac Progenitor Cells
Authors: Mishra Rachana , Sharma Sudhish , Cheng Wen C , Kingsbury Tami J , Saha Progyaparamita , Civin Curt I , Kaushal Sunjay
Published: July 2021

Background:
miR-34a expression is increased in adult hearts and promotes age- associated cardiomyocyte cell death. Here we explored the effect of miR-34a on the secretome of cardiac progenitor cells (CPCs) and their potential to recover the myocardial functional and tissue regeneration in response to injury.

Hypothesis:
We hypothesize that
miR-34a/HSF1 pathway
is directly involved in controlling the functional activity of CPCs during chronological aging.

Methods:
Human specimens were obtained during routine cardiac surgical procedures from right atrial appendage tissue discarded from 2 age groups: neonates and adults patients. We developed a reproducible isolation method that generated c-kit
+
Cd45
-
cells using immune-activated magnetic bead selection, regardless of the initial weight or age.

Results and Conclusion:
Here we are reporting a novel finding that knock down of miR-34a in adult-derived CPCs (aCPCs) significantly increases their cell survival, paracrine secretion that directly correlated with significantly improved LV function in the myocardial infarcted (MI) rodent model in comparison to aCPCs. Previously, we have showed that heat shock factor 1 (HSF1) is the key chaperone pathway that affects CPC’s growth potential and functional activity. Here we are showing that up-regulation of miR-34a in CPCs are attributed towards down regulation of HSF1 and their paracrine secretion. Data showed not only novel insights into the effects of miR34a/heat shock pathway on CPCs survival and controlling their paracrine behavior but it is also paramount to characterize and dissect the mechanism of cell therapy in order to generate the most optimally designed clinical trial in adult heart -failure patients.
Abstract 314: BRaf Plays a Major Role in Gene Expression in Cardiomyocytes in Mouse Hearts
in vivo
Authors: Hardyman Michelle A , Fuller Stephen J , Meijles Daniel N , Rostron Kerry A , Leonard Sam J , Sugden Peter H , Clerk Angela
Published: July 2021

Introduction:
Raf kinases lie upstream of ERK1/2 with BRaf being the most highly expressed and having the highest basal activity. V600E BRaf mutations constitutively activate ERK1/2 and are common in cancer. The role of BRaf in the adult heart is yet to be established. ERK1/2 regulate cardiomyocyte gene expression, promoting cardiac hypertrophy and cardioprotection, but effects of ERK1/2 may depend on signal strength.

Hypothesis:
Our hypotheses are that BRaf is critical in regulating ERK1/2 signaling in cardiomyocytes and, whilst moderate ERK1/2 activity is beneficial, excessive ERK1/2 activity is detrimental to the heart.

Methods:
We generated heterozygote mice for tamoxifen- (Tam-) inducible cardiomyocyte-specific knockin of V600E in the endogenous BRaf gene. Mice (12 wks) received 2 injections of Tam or vehicle on consecutive days (n=4-10 per group). Kinase activities and mRNA expression were assessed by immunoblotting and qPCR. Echocardiography was performed (Vevo2100). M-mode images (short axis view) were analyzed; data for each mouse were normalized to the mean of 2 baseline controls.

Results:
V600E knockin did not affect overall BRaf or cRaf levels in mouse hearts, but significantly increased ERK1/2 activities within 48 h (1.51±0.05 fold). Concurrently, mRNAs for hypertrophic gene markers including BNP and immediate early genes (IEGs) increased signficantly. At 72 h, expression of BNP, Fosl1, Myc, Ereg and CTGF increased further, other IEGs (Jun, Fos, Egr1, Atf3) declined, and ANF was upregulated. In contrast, expression of α and β myosin heavy chain mRNAs was substantially downregulated (0.46/0.41±0.05 relative to controls). Within 72 h, left ventricular (LV) mass and diastolic LV wall thickness had increased (1.23±0.05 relative to controls), but cardiac function was severely compromised with significant decreases in ejection fraction and cardiac output (0.53/0.68±0.09 relative to controls) associated with increased LV internal diameters and cardiac volumes.

Conclusions:
Endogenous cardiomyocyte BRaf is sufficient to activate ERK1/2 in mouse hearts and induce cardiac hypertrophy associated with dynamic temporal changes in gene expression. However, excessive activation of ERK1/2 in isolation is detrimental to cardiac function.
Abstract 32: The Role of Cyclin A2 in Adult Human Cardiomyocyte Plasticity
Authors: Ranjan Amaresh , Vadakke Madathil Sangeetha , Chaudhry Hina
Published: July 2021

The adult mammalian heart is known to have a very low abundance of progenitor cells which can take part in active cycling and regeneration after damage. Cardiomyocytes exit the cell cycle soon after birth coincident with the silencing of cyclin A2 (CCNA2). In our previous studies, we demonstrated that viral delivery of
Ccna2
induces cardiac regeneration in infarcted hearts of small and large animal models. However, the molecular mechanism whereby Ccna2 induces cardiac regeneration and increase in cardiac function deserves further study. To explore further, we isolated adult mouse cardiomyocytes and induced
Ccna2
expression by using adenovirus transfection and cultured them for 3 weeks. Co-expression of the mature cardiac marker troponin Tc with the immature cardiac marker non-muscle myosin IIB was observed. Additionally, expression of epithelial to mesenchymal transition markers (vimentin and FSP1) was observed. Also, decreased expression of mature cardiac markers
α-MHC
,
ckmt2
and
cTnT
was noted. To study the factors responsible for human cardiomyocyte plasticity and cell division, we have optimized a novel method for culturing adult human cardiomyocytes in our laboratory. We cultured cardiomyocytes isolated from heart tissue obtained from a 55 yr old male patient. After transfection with CCNA2 adenovirus made for human use (cTnT promoter driving human
CCNA2
cDNA), they were co-transfected with two more adenoviruses (1) cTnT-GFP to label cardiomyocytes (green) and (2) CMV-α-actinin-m-Cherry to label the sarcomere (red). Time lapse live epifluorescence microscopy was carried out for 70 hrs and time lapse movies were prepared (please refer the youtube link to see a representative time lapse movie
https://youtu.be/OBrJGCq7YCA
). Movies were analyzed to calculate the cytokinesis index in samples transfected with (Test) and without (Null) CCNA2 adenoviruses. We observed a significantly higher cytokinesis index in CCNA2 samples versus Null. We are further investigating the role of cyclin A2 in dedifferentiation of adult human cardiomyocytes to generate immature or progenitor cardiac cells and their contractile status, which could be utilized for regeneration and functional restoration of damaged adult heart tissue.
Abstract 33: Combination of Allogeneic Mesenchymal and Kidney-derived Stem Cells Promotes Kidney Repair in a Swine Model of Chronic Kidney Disease
Authors: Rieger Angela C , Tompkins Bryon A , Natsumeda Makoto , Florea Victoria , Rodriguez Jose , Rosado Marcus , Balkan Wayne , Hare Joshua M , Schulman Ivonne H
Published: July 2021

Background:
Chronic kidney disease (CKD) has a high prevalence (~14% of the population) and is associated with a significantly increased risk of cardiovascular disease and a 15-fold higher rate of mortality than the general population. Current therapies slow disease progression but do not repair organ damage, leading to end-stage renal disease. Stem cell therapy has the potential to promote repair via neovascularization and antifibrotic effects. We tested the renal reparative capacity of allogeneic mesenchymal stem cells (MSCs) and kidney ckit+ stem cells (c-kit) in an established swine model of CKD.

Methods:
Yorkshires pigs (n=27) underwent 5/6 nephrectomy via renal artery embolization and 4-weeks later received either: MSC (10х10
6
), c-kit (10х10
6
), combination (MSC+c-kit; 1:1 ratio [5х10
6
each]), or placebo (each n=5). Allogeneic cell therapy was delivered via the patent renal artery of the remnant kidney. Kidney functional parameters and renal MRI were measured at baseline, and at 4- and 12-weeks (euthanasia) post-embolization.

Results:
The CKD model was validated from baseline to 4 weeks by an increased creatinine: (Δ1.1 ± 0.15 mg/dl; p<0.0001), BUN (Δ13.50 ± 2.99mg/dl; p=0.0003), and urine protein/creatinine ratio (Δ0.311mg/g; p=0.018), and decreased GFR (Δ49.82 ±6.41 ml/min; p=0.0002). Mean arterial pressure (MAP) was not different between groups from baseline to 4 weeks. After 12 weeks, there was a significant difference in MAP between groups (p=0.04), with an increase in the placebo group (19.97± 8.65 mmHg, p=0.08). BUN and creatinine levels improved in all of the groups from 4-12 weeks. GFR also improved in all the groups, but with the greatest effect in the combination group (76± 23.83ml/min; p= 0.03) from 4-12 weeks. Urine protein/creatinine ratio did not change in placebo but decreased in cell treated groups. There was no evidence of immune rejection as evaluated in a complete body necropsy.

Conclusion:
Allogeneic MSCs and kidney-derived stem cells are safe in a CKD swine model. The combination of stem cells was shown to be more efficacious in improving kidney function. These novel findings have important implications for the advancement of cell therapy for CKD.
Abstract 333: MED13-dependent Regulation of Cardiac Thyroid Hormone Signaling
Authors: Minerath Rachel , Martins Ines , Grueter Chad
Published: July 2021

Thyroid hormone (TH) is a key regulator of cardiac metabolism. While hypothyroidism is known to result in adverse cardiac effects, the molecular mechanisms that modulate TH signaling are not completely understood. Mediator is a multiprotein complex that coordinates signal dependent transcription factors with basal transcriptional machinery. Mediator complex protein, MED13, was previously demonstrated to repress numerous thyroid receptor (TR) response genes in the heart. Furthermore, we have previously demonstrated that mice overexpressing cardiac MED13 (MED13cTg) treated with propylthiouracil (PTU), an inhibitor of T3 biosynthesis, were resistant to PTU-dependent decreases in cardiac contractility. Here we demonstrate that MED13 expression is induced in the hearts of mice in response to a PTU diet. To elucidate the role of MED13 in transcriptional regulation of cardiac TH signaling, cardiac-specific Med13 knockout mice (MED13cKO) and control mice were placed on a PTU diet or normal chow diet for 4 weeks. An additional group of mice on PTU diet were treated acutely with thyroid hormone (T3). While heart weight to body weight ratios did not differ between genotypes, RNA sequencing was performed from hearts of these mice to understand the role of MED13 in TR-dependent transcription. Echocardiography was performed to assess cardiac function in these mice. In addition, histology was performed to evaluate cardiac structure and fibrosis. These studies demonstrate that MED13 is induced in response to hypothyroidism and further deciphers molecular mechanisms of MED13 regulation of TR-dependent transcription.
Abstract 34: Acetylation of GATA 4 Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes
Authors: Singh Vivek P , Mathison Megumi , Pinnamaneni Jaya P , Sanagasetti Deepthi , Belaguli Narasimhaswamy S , Somvanshi Sonal , Olive Jacqueline K , Yang Jianchang , Rosengart Todd K
Published: July 2021

Objective:
Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2c and Tbx5), has recently been demonstrated, suggesting a promising statregy for cardiac regeneration. However, the efficiency of direct reprogramming is usually relatively low and requires extensive epigenetic redesigning, although the underlying mechanism are largely unknown.

Methods:
In a recent study, we created a novel mutation in rat GATA 4 by replacing lysine residue with glutamine at position 299 i.e. (K299Q), to mimic constitutive acetylation and examined whether constitutive acetylation of GATA4, when compared with wild type GATA4, further enhance GMT-mediated direct reprogramming efficiency of induced cardiomyocytes
in vitro
and accordingly ventricular function after myocardial infarction in rat,
in vivo
.

Results:
We found that acetylated GATA 4 (K299Q), in the presence of Mef2c and Tbx5 upregulated cardiac-specific markers, suppressed fibroblast genes, in rat cardiac fibroblasts (RCFs) more efficiently when compared with Mef2c, Tbx5 plus wild type GATA4. FACS analyses revealed that G(K299Q) MT induced significantly more cardiomyocyte marker cardiac troponin T (cTnT) expression compared with GMT alone. Mechanistic studies demonstrated that the K299Q substitution, resulting in enriched p300 occupancy at the GATA 4 promoter, induced acetylation of Histine 3, decreased HDAC expression. In addition, substitution augmented the increase in an acetylated form of GATA-4 and its DNA binding and transcriptional activity, compared with wildtype GATA 4. In agreement with upregulated cTNT gene expression
in vitro
, echocardiographic analysis demonstrate that the acetylated G(K299Q) MT vectors have improved effect in enhancing ventricular function than GMT vectors from postinfarct baselines as compared to negative control [G(K299Q) MT, 15.6% ± 2.7%; G(WT)MT, 12.8% ± 1.7%; GFP, -2.3% ± 1.1%].

Conclusions:
Collectivily, these data indicate that acetylated GATA4 (K299Q) significantly increases reprogramming efficiency of induced cardiomyocytes (iCMs),
in vitro
and
in vivo,
and provide new insight into the molecular mechanism underlying cardiac regeneration.