bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 250: Pericardial Grafting Of Cardiac Progenitor Cells In The Three-dimensional Thick Scaffold Improves Cardiac Function After Myocardial Infarction In Mice.
Authors: Kondo Naomichi , Nagai Toshio , Liu Mei-Lan , Takahashi Toshinao , Kanda Masato , Matsuura Katsuhisa , Komuro Issei , Kobayashi Yoshio
Published: July 2021

Cardiac progenitor cell (CPC) therapy for heart disease has been examined enthusiastically. However, optimal scaffolds which maintain the transplanted cells are still elusive. We used clonally expanded stem cell antigen 1-positive CPCs from adult mice and produced a three-dimensional thick scaffold (CPC-scaffold), in which CPCs were cultivated up to 2 month with self-assembling peptide RADA16-I. Addition of designer self-assembling peptide containing the active motifs of 2-unit RGD binding sequence and IGF-1 promoted three-dimensional spreading and viability of CPCs. After making myocardial infarction (MI) with left coronary artery ligation in mice, we transplanted CPC-scaffold on the surface of infarction area and confined it inside of the pericardial space by closing parietal pericardium. Four weeks after transplantation, echocardiography showed that FS of treatment group (16±10%, n=17) was higher than that of control (MI only) group (10±6.8%, n=19) (P<0.05) and that LVDd of treatment group (5.4±1.0mm, n=17) was smaller than that of control group (6.2±1.1mm, n=19) (P<0.05). Infarction area was significantly decreased in treatment group (46±21%, n=17), compared to control group (59±19%, n=18) (P<0.05). Immunohistochemical staining for von-Willebrand factor (vWF) showed that the number of vWF-positive capillaries per mm2 in treatment group (16.8±3.2, n=5) was higher than that of control group(8.9±3.4, n=5) (P<0.05). There were many of CD31-positive capillaries with or without α-smooth muscle cell actin-expressing perivascular cells in the graft area. By using fluorescent-conjugated avidin, biotin-labeled scaffold was globally detected in the graft area 1 week after transplantation, but sparsely 4weeks after, suggesting that the transplanted scaffold was biodegradable. To examine whether transplanted CPCs remain in the scaffold, we labeled CPCs with red fluorescence protein (RFP). RFP+CPCs were observed in the graft area 4 weeks after transplantation of RFP+CPC-scaffold. FISH analysis showed that sex-mismatched CPCs were globally detected in the graft area on the surface of the heart. Therefore pericardial grafting of well-vascularized and cellularized CPC-scaffold was a useful method to improve cardiac function after MI.
Abstract 261: Robust Cardioprotection Afforded by a Previously Unrecognized Link between the Unfolded Protein Response and the Hexosamine Biosynthetic Pathway
Authors: Wang Zhao V , Deng Yingfeng , Gao Ningguo , Pedrozo Zully , Li Dan , Morales Cyndo , Criollo Alfredo , Luo Xiang , Tan Wei , Jiang Nan , Lehrman Mark , Rothermel Beverly , Lee Ann-Hwee , Lavandero Sergio , Mammen Pradeep , Ferdous Anwarul , Gillette Thomas , Scherer Philipp , Hill Joseph
Published: July 2021

Background:
The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in glucose metabolism and multiple diseases, regulation of the HBP remains largely undefined.

Methods & Results:
Here, we show that spliced Xbp1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers activation of the HBP by means of Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions known to promote O-GlcNAc modification. We go on to demonstrate that Xbp1s, acutely stimulated by ischemia/reperfusion (I/R) in heart, confers robust cardioprotection against I/R injury. We also show that HBP induction is required for this cardioprotective response. Mechanistically, HBP may mediate the adaptive branch of the UPR by activating autophagy and ER-associated degradation.

Conclusion:
These studies reveal that Xbp1s couples the UPR to the HBP, promoting robust cardioprotection during I/R.
Abstract 263: Enhance Cd34+ Cell Therapy For Limb Ischemia Using Novel Bioactive Peptide Amphiphiles Nanofibers
Authors: Yu Yang , Sleep Eduard , Zhou Junlan , Losordo Douglas W , Stupp Samuel I , Qin Gangjian
Published: July 2021

Background:
CD34+ cells have shown unprecedented promise for ischemic tissue repair in recent clinical trials; however, their recruitment, retention, and survival in the injured tissue remain one of the major barriers to the success of this novel therapeutic strategy.

Methods and Results:
In this study, we have developed a self-assembling bioactive peptide amphiphile (PA) nanofiber that displays the integrin-recognized cell adhesion epitope RGDS and tested this novel scaffold material for therapeutic delivery, retention, and functional support of human CD34+ cells in a mouse model of critical hind-limb ischemia (CLI). In culture, PA nanofibers, but not non-bioactive nanostructures or nanostructures with scrambled DGRS epitope, significantly enhanced the adhesion and survival of human CD34+ cells under a number of cellular stresses. In CLI nude mice, intramuscular injection of human CD34+ cells contained in the RGDS nanostructures led to a significantly better blood perfusion, motor ability, limb salvage, and treadmill endurance when compared with those of CD34+ cells contained in DGRS nanostructures, cells alone or RGDS alone. Histology analyses revealed a better viability and retention of CD34+ cells in the limb tissue of the RGDS nanostructures/CD34+ cells group than in the RGDS nanostructures alone or cells alone group. In consistent with the improved functional recovery, the density of capillaries in the ischemic limb of RGDS nanostructures/CD34+ cells group was significantly greater than in other groups.

Conclusions:
Our data demonstrate that integrin-specific binding epitope RGDS nanostructure enhances viability, adhesion, and angiogenic capability of human CD34+ cells. This pre-clinical study provides a rationale for further investigation of combining RGDS PAs with human CD34+ cells to enhance the effectiveness of cell therapy for ischemic limb disease.
Abstract 264: TNFR2-Bmx Signaling in Cardiac Stem Cells and Cardiac Repair
Authors: Zhou Huanjiao J , Huang Qunhua , Min Wang
Published: July 2021

While cytokine TNF via TNFR1 induces inflammation and apoptosis, it through its second receptor TNFR2 induces cell survival and migration by activating bone marrow non-receptor tyrosine kinase Bmx. Since Bmx has been implicated in self-renewal of stem cells, we hypothesize that TNF via TNFR2 activates Bmx in cardiac stem cells (CSCs) to mediate cardiac repair. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+) /CD45(-) /VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA (references: Stem Cells 2013;31:1881-1892). In the present study, we detect the c-kit(+)Lin28(+) CSCs populations in a mouse coronary artery ligation ischemic model. Furthermore, the c-kit(+) CSCs are reduced in TNFR2-KO and Bmx-KO mice. Mechanistically, we show a crosstalk between the TNFR2-Bmx and the c-Kit signaling pathways to mediate CSC proliferation, survival and migration. These observations suggest that TNFR2-Bmx signaling in c-kit(+) CSCs induces cardiac repair, providing a potential strategy to stimulate cardiac regeneration by TNFR2-specific agonists.
Abstract 269: Duo-impairment of the Ubiquitin-Proteasome System and Autophagy by Ablation of COP9 Signalosome Subunit 8 Activates a Programmed Necrosis Pathway Mediated by RIP1-RIP3 Kinases but not Cyclophilin D-regulated Mitochondrial Membrane Permeability
Authors: Wang Changhua , Jahn Andrea , Su Huabo , Wang Xuejun
Published: July 2021

Programmed cell death includes apoptosis and programmed necrosis (AKA, necroptosis). A well-recognized feature of necrosis is the loss of membrane integrity of the dying cell. Cells undergoing apoptosis, however, maintain their membrane integrity. We previously reported massive cardiomyocyte (CM) necrosis, before increased apoptosis is discernible, in mouse hearts with cardiomyocyte-restricted knockout of the COP9 signalosome subunit 8 (CR-Csn8KO). This is associated with defective autophagosome maturation and ubiquitin-proteasome system (UPS) dysfunction. Consequently, mice with perinatal CR-Csn8KO develop rapidly dilated cardiomyopathy (DCM) and die prematurely. The present study sought to search for the mechanisms underlying the CM necrosis. Here our immunoprecipitation revealed significant increases of RIP1-interacted RIP3 in CR-Csn8KO myocardium, indicative of activation of the RIP1-RIP3 pathway. The RIP1-RIP3 pathway is known to mediate necroptosis. To further test whether RIP1 activation plays an essential role in CM necrosis in CR-Csn8KO mice, we treated the mice with a RIP1 kinase specific inhibitor necrostatin-1 (Nec-1) or vehicle control via osmotic mini-pump implanted in the peritoneal cavity at 2 weeks of age. Nec-1 significantly suppressed CM necrosis as measured by the positivity of Evan’s blue dye (EBD) uptake (p<0.00001), prevented left ventricle dilatation (p<0.05) at 3 weeks, and delayed premature death of the CR-Csn8KO mice (p=0.0072). These results demonstrate that CM necrosis in CR-Csn8KO mice is necroptosis in which the RIP1-RIP3 kinases-mediated pathway plays a major pathogenic role, and that CM necroptosis is the primary cause of DCM and mouse premature death. Increased mitochondrial membrane permeability, which can be suppressed by inhibition of cyclophilin D, has been shown to play a critical mediating role in myocytes necrosis. Hence, we cross-bred CR-Csn8KO mice with cyclophilin D knockout mice and obtained the Kaplan-Meier survival curve. Unexpectedly, cyclophilin D deficiency exacerbated the premature death of CR-Csn8KO mice (p=0.007), indicating that cyclophilin D regulated mitochondrial membrane permeability does not play an important role in CM necrosis in CR-Csn8KO mouse hearts.
Abstract 271: Role of Arterial AT1 Receptor on the Regulation of GRK4 in Hypertension
Authors: Chen Ken , Liu Li , Chen Caiyu , Jose Pedro A , Zeng Chunyu
Published: July 2021

G protein-coupled receptor kinase 4 (GRK4) gene variants or increased GRK4 expression, via impairment of renal dopamine receptor and enhancement of renin angiotensin system function, impairs renal sodium excretion, resulting in sodium retention and an increase in blood pressure. Increased aortic stiffness, a risk factor in cardiovascular disease, may be related to increased activity of the renin-angiotensin system Whether or not GRK4 and the angiotensin type 1 receptor (AT1R) interact in the aorta is not known. We now report that GRK4 is expressed in vascular smooth muscle cells (VSMCs) of the aorta. Exogenous expression of the GRK4 variant 142V in aortic A10 cells increased AT1R protein expression and AT1R-mediated increase in intracellular calcium concentration. The increased AT1R expression was caused by an increase in AT1R mRNA expression via NF-kappa B, because blockade of NF-kappa B abolished those effects of GRK4 A142V. As compared with control (vector-transfected) cells, cells expressing 142V had higher NF-kappa B activity and more NF-kappa B bound to AT1R promoter. The increased AT1R expression in cells expressing GRK4 142V was associated with decreased AT1R degradation, which was ascribed to the lower AT1R phosphorylation. There was direct interaction between GRK4 and AT1R in A10 cells which was decreased by GRK4 that could have caused the lower AT1R phosphorylation and degradation. The regulation of GRK4 of AT1R expression was confirmed in GRK4142V transgenic mice, the AT1R expression was higher, while AT1R phosphorylation was lower in aorta in GRK4 142V than control mice. Angiotensin II-mediated vasoconstriction was higher in A142V mice. This study provides a mechanism that GRK4, via regulation of arterial AT1R expression and function, engaged into the pathogenesis of hypertension.
Abstract 282: Functional Analysis Of Micrornas In Vascular Disease
Authors: Wang Shusheng , Zhou Qinbo , Anderson Chastain
Published: July 2021

microRNAs (miRNA, miR) are emerging as pivotal modulators of vascular development and disease. Research is my lab is focused on elucidating the functional mechanism and exploring therapeutic potential of microRNAs in vascular diseases. Our recent studies have shown that miR-23 and miR-27 in miR-23~27~24 family are required for proper angiogenesis and neovascularization in a laser-induced vascular injury model. Here we extend our study and provide evidence that miR-24 regulates actin dynamics in ECs through targeting multiple members downstream of Rho signaling, including Pak4, Limk2 and Diaph1 proteins. Consistent with the critical role for actin cytoskeleton in cell motility and proliferation, overexpression of miR-24 in ECs blocks stress fiber and lamellipodia formation, represses EC migration, proliferation and tube formation in vitro, as well as angiogenesis in an ex vivo aortic ring assay. Overexpression of miR-24 in transgenic mice represses postnatal retinal vascular development. Moreover, subretinal delivery of miR-24 mimics represses laser-induced CNV in vivo. Mechanistically, knockdown of miR-24 target protein LIMK2 or PAK4 inhibits stress fiber formation and tube formation in vitro, mimicking miR-24 overexpression phenotype in angiogenesis. Taken together, these findings demonstrate that miR-24 represses angiogenesis by simultaneously regulating multiple components in the actin cytoskeleton pathways, suggesting distinct function of miR-23~27~24 family members in angiogenesis. Manipulation of actin cytoskeleton pathways by miR-24 may represent an attractive therapeutic solution for numerous vascular diseases.
Abstract 290: Proliferation of Cardiac Fibroblasts Defines Early Stages of Genetic Dilated Cardiomyopathy and Precedes Myocardial Metabolic Derangement
Authors: Burke Michael A , Chang Stephen , Christodoulou Danos C , Gorham Joshua M , Wakimoto Hiroko , Seidman Christine E , Seidman Jonathan G
Published: July 2021

The complex molecular networks underpinning DCM remain poorly understood. To study distinct pathways and networks in the longitudinal development of DCM we performed RNAseq on LV tissue from mice carrying a human DCM mutation in phospholamban (PLN
R9C/+
) before phenotype onset (pre-DCM), with DCM, and during overt heart failure (HF), and also on isolated myocytes and non-myocytes from DCM hearts. PLN
R9C/+
mice show progressive fibrosis (20% vs. 1% control, p=6x10
−33
; n=3) associated with proliferation of cardiac non-myocytes (33% increase over control, p=6x10
−4
; n=3). Consistent with this, cardiac non-myocytes have upregulated gene expression and pathways, while these are generally downregulated in myocytes. Non-myocytes were enriched in fibrosis, inflammation, and cell remodeling pathways, from pre-DCM to HF. In contrast, myocytes were enriched for metabolic pathways only with overt DCM and HF. Myocytes showed profound derangement of oxidative phosphorylation with DCM (p=2.5x10
−41
; 44% (53/120) of pathway genes downregulated), suggesting mitochondrial dysfunction. Additionally, we detected probable inhibition of peroxisome proliferator-activated receptor (PPAR) signaling by diminished expression of pathway genes (Figure). DCM and hypertrophic remodeling was compared using RNAseq of a mouse model of HCM; similar patterns of fibrosis with myocyte metabolic dysregulation were evident despite unique differential gene expression patterns between models. DCM caused by PLN
R9C/+
is associated with early non-myocyte proliferation and later myocyte metabolic derangement possibly governed by altered PPAR signaling, and is common to DCM and HCM.
Abstract 298: Low-dose Interleukin-18 is Necessary to Adapt to Pressure Overload in Mice
Authors: Toshihiro Iwasaku , Shinichi Hirotani , Akiyo Eguchi , Naoki Suminatni , Yoshitaka Okuhara , Daisuke Morisawa , Hisashi Sawada , Yoshiro Naito , Tohru Masuyama
Published: July 2021

Background:
We previously reported that circulatory Interleukin-18 (IL-18) level elevates in heart failure (HF) patients. However, the roles of circulatory IL-18 in HF have not been fully elucidated. Continuous infusion of IL-18 induces cardiomyocyte hypertrophy and fibrosis, whereas IL-18 null mice were fragile to pressure overload. We hypothesized that systemic (circulatory) and local (cardiac) IL-18 have distinct effects on HF and cardiac remodeling.

Purpose:
The purpose of this study was to elucidate the role of circulatory IL-18 in adaptation to pressure overload in IL-18 null mice.

Methods:
Wild type (WT) mice and IL-18 null (IL-18-/-) were subjected to transaortic constriction (TAC). Sequential serum IL-18 levels myocardial IL-18 mRNA expressions were determined by ELISA and qRT-PCR in WT mice. After two week of TAC, IL-18 null mice were administered either (IP) saline or recombinant IL-18 intraperitoneally (10ng/20g every other day). Cardiac function was assessed by transthoracic echocardiography. Two weeks after TAC, myocardial samples were obtained. Haematoxylin and eosin stained sections and Masson’s trichrome staining sections were prepared.

Results:
IL-18 concentration in serum and IL-18 expression in myocardial tissue increased gradually after TAC in WT mice. Forty-seven % (7/15) of TAC-operated IL-18-/- mice and 12% (2/17) of TAC-operated WT mice died of heart failure by 14days. TAC-operated IL-18-/- mice exhibited more severe left ventricular (LV) remodeling, characterized by cardiomyocyte hypertrophy, extensive interstitial fibrosis and elevation of fetal gene expressions compared with TAC-WT mice. Recombinant IL-18 given intraperitoneally improved the survival rate to 100% (10/10) following TAC operation in IL-18-/- mice. Furthermore, exogenous IL-18 administration suppressed ventricular ANP mRNA expression and myocardial cross-sectional area enlargement to non-TAC-operated level, whereas LV enlargement and contractile dysfunction were only partially suppressed in IL-18-/- mice following TAC.

Conclusions:
Circulatory IL-18 exerts opposing effects on cardiac hypertrophy under pressure-overload. IL-18 produced in the heart may have an effect such as preserving contractile function.
Abstract 311: Inhibition Of Matrix Metalloproteinase-13 Dependent Protease-activated Receptor-1 Activation Attenuates Fibrotic Signaling
Authors: Dixon Allison E , Jaffre Fabrice , Mackman Nigel , Blaxall Burns C
Published: July 2021

Heart failure (HF) is the leading cause of morbidity and mortality in the United States and is characterized by progressive myocardial fibrosis, pathologic remodeling and deteriorating cardiac function. Cardiac fibroblasts (CF) are largely responsible for the secretion of ECM proteins as well as cytokines and growth factors in the heart. Upon injury or pathologic stimulation, CF transition to a myofibroblast phenotype, leading to excess production of ECM proteins and pro-inflammatory cytokines. Previous studies in our lab have indicated a role for protease-activated receptor-1 (PAR-1), the most highly expressed GPCR on CF, in pathologic cardiac remodeling. In particular, we reported the novel cleavage of PAR-1 via matrix metalloproteinase-13 (MMP-13) and cardioprotective effects of MMP-13 inhibition in an acute model of HF. Therefore, we hypothesize that MMP-13 plays an important role in cardiac remodeling through activation of PAR-1, particularly in the pathologic transition of CF to myofibroblasts.

To investigate this hypothesis, RNA was collected from hearts of mice infused with isoproterenol (ISO) for 7 days and concurrently treated with a specific MMP-13 inhibitor, WAY170523, or vehicle. To evaluate the effect of WAY170523, we used qRT-PCR to measure changes in the expression of fibrotic markers, COL1a1, COL3a1, and TGF-β. Inhibition of MMP-13 with WAY170523 attenuated expression of these genes compared to vehicle treated animals.

We previously reported that stimulation of CF and cardiomyocytes with MMP-13 induces phosphorylation of ERK1/2, a member of the MAPK family known to play a role in cardiac hypertrophy, and treatment with a direct PAR-1 antagonist decreased the activation of ERK1/2. We have found that ERK1/2 phosphorylation is directly attenuated following inhibition of MMP-13 with WAY170523.

Overall, these data suggest a role for MMP-13 dependent PAR-1 activation in pathologic myofibroblast transition and a potential therapeutic role for MMP-13 inhibition, possibly through its inhibition of ERK1/2 phosphorylation. Treatment with WAY170523 also attenuates markers of fibrosis in vivo, indicating a potential salutary role for MMP-13 inhibition in the treatment of HF.
Abstract 312: Regulation of Fibrotic Signaling Pathways by Desmosomal Armadillo Proteins in Cardiac Tissue
Authors: Dubash Adi D , Green Kathleen J
Published: July 2021

The process of fibrosis, described as accumulation of myofibroblasts and excessive deposition of extracellular matrix components, is a key development in the progression of multiple different types of cardiac disease. Nevertheless, little is known about the molecular mechanisms which cause the onset of fibrosis in cardiac disease. Fibrosis is a significant component of arrhythmogenic cardiomyopathy (AC), a genetic disorder characterized by replacement of healthy cardiomyocytes (CMs) with fibrous tissue, leading to arrhythmia and in certain cases, sudden death. AC is often characterized as a “disease of the desmosome”, as mutations for all obligate desmosome proteins have been found in cases of AC, including the desmosome armadillo proteins Plakophilin-2 (PKP2) and Plakoglobin (PG). PKP2 and PG are multi-functional proteins involved in both mechanical stabilization of the cardiac area composita, as well as mediation of desmosome-related signaling pathways. We have determined that loss of PKP2 or PG in neonatal CMs causes an aberrant increase in gene expression of pro-fibrotic stimuli such as transforming growth factor beta 1 (TGF-beta1) and Interleukin-6 (IL-6). In addition, p38 MAPK, a known mediator of inflammatory fibrosis, is activated upon loss of PKP2/PG. We hypothesize that mutation or loss of PKP2 or PG cause the recruitment and activation of cardiac fibroblasts via pro-fibrotic TGF-beta and p38MAPK signaling, resulting in pathological fibrosis characteristic of AC. Indeed, conditioned media from PKP2-silenced CMs causes an increase in fibronectin gene expression by freshly isolated cardiac fibroblasts. Our future experiments will investigate whether inhibition of TGF-beta or p38MAPK signaling can alleviate fibrotic gene production. By highlighting a novel link between desmosome armadillo proteins and pro-fibrotic signaling in cardiac tissue, this study provides mechanistic insights into the pathogenesis of AC, as well as advances our knowledge of potential therapeutic targets for combating fibrosis in multiple different types of heart disease or injury.
Abstract 314: Loss of Sarcospan has a Deleterious Effect on Cardiac Function in Aged Mice
Authors: Parvatiyar Michelle S , Marshall Jamie L , Jordan Maria C , Nguyen Reginald T , Roos Kenneth P , Crosbie-Watson Rachelle H
Published: July 2021

Sarcospan (SSPN) has been shown to have an important role in stabilizing sarcolemmal dystrophin- and utrophin-glycoprotein adhesion complexes. Loss of sarcolemmal integrity leads to immune cell infiltration and inappropriate exchange of cellular contents with the extracellular milieu. Our laboratory has shown SSPN loss destabilizes skeletal muscle adhesion and reduces sarcolemmal dystrophin localization, whereas its complete loss due to mutation underlies development of Duchenne muscular dystrophy (DMD). Loss of dystrophin leads to cardiac dysfunction and early mortality in DMD patients. The role of SSPN in the heart is unknown. We present immunofluorescence data revealing reduction of dystrophin and the sarcoglycans with a coordinate increase of β1D integrin levels at the SSPN-null cardiac sarcolemma relative to WT. Also, SSPN loss decreases cardiac P-Akt levels, disrupting signaling promoting compensatory physiological hypertrophy. These studies suggest a fundamental role for SSPN in cardiac maintenance and function, since left ventricular mass increases with age and upon isoproterenol administration (0.8 mg/day for two wks). Aged SSPN-null mice developed hypertrophy, evidenced as increased heart/body weight ratio and left ventricular wall dimension. The SSPN-null mice lacked the characteristic initial rise in cardiac output, left ventricular ejection fraction (LvEF %), induced by chronic β-adrenergic stimulation. Functionally, aged SSPN-null hearts had an increased E/A ratio indicating restrictive ventricular filling and decreased fractional shortening F/S (%) upon isoproterenol administration. Aged untreated SSPN-null hearts had increased fibrosis compared to aged WT controls, however isoproterenol treated SSPN-null hearts displayed exacerbated fibrotic response compared to WT. To assess whether SSPN-null hearts have altered gene expression profiles during progression of pathogenesis, qRT-PCR will be utilized to measure differences in expression of fetal gene and calcium-handling proteins. In summary, we have found that SSPN has an important role in maintaining cardiac function, its loss exacerbates the hypertrophic response and localization of stabilizing adhesion complexes at the cardiac muscle sarcolemma.
Abstract 316: Cell-Matrix Contacts Regulate Age-Associated Cardiac Function
Authors: Sessions Ayla O , Kaushik Gaurav , Cammarato Anthony , Engler Adam
Published: July 2021

An increased deposition of ECM is observed in all advanced age heart failure patients. Therefore, it is necessary to investigate the effect of extracellular remodeling on mechanical function in genetically tractable, rapidly aging, and simple model organisms such as
Drosophila melanogaster
. The bilayered design of the Drosophila heart-tube makes it an easier model in which to study the interplay between ECM and cardiomyocytes as they regulate contraction. Here we present data from two common wildtype strains of
Drosophila
exhibiting different aging profiles in terms of cytoskeletal and ECM regulation and remodeling. Using a recently developed nanoindentation method to measure cardiomyocyte stiffness of intact
Drosophila
hearts, we have found that while
yellow-white
(
yw
) flies show midline stiffening at the intercalated discs (ICD) presenting a clear diastolic dysfunction with age, the
white-1118
(
w1118
) flies exhibit no ICD stiffening, but show an increase in thickness of the ECM layer between the ventral muscle (VM) and cardiomyocytes (CM). Paired with increased expression of ECM proteins, the
w1118
Drosophila
line may provide a good model for exploring the effect of cell-ECM contacts on regulating cardiac function with age. Knock-down of integral ECM genes LamininA and Viking (Collagen IV) result in no effect on cardiac performance in juvenile flies but causes a decrease in underlying cardiomyocyte stiffness and an increase in the contractile irregularity of heart beats. This suggests that the cell-ECM contacts in the basement membrane are intimately tied to coupling of the cardiomyocytes of the
Drosophila
heart-tube, which may have larger implications for elderly patients suffering from myocardial fibrosis and experiencing cardiomyocyte decoupling and resultant arrhythmias.
Abstract 323: Dronedarone Impairs Cardiac Mitochondrial Oxidative Phosphorylation in Dose-Dependent Manner
Authors: Emelyanova Larisa , Gudlawar Sirisha , Rizvi Farhan , Holmuhamedov Ekhson , Thakur Monika , Mirza Mahek , Jahangir Arshad
Published: July 2021

Introduction:
Dronedarone (DR), a new antiarrhythmic drug, was recently shown to worsen heart failure (HF) and mortality in patients with atrial fibrillation and left ventricular dysfunction. However, the mechanism underlying the adverse effect is not known. Since, myocardium depends on mitochondrial oxidative phosphorylation (OXPHOS), we hypothesized that DR impairs mitochondrial function, which could further compropmise energetic reserves predisposing to worsening of HF and death in patients with HF.

Methods:
Mitochondria isolated from rat heart (2 month old, SD) were treated with DR (1, 5, 10, 20, 50 μM), and the effect on oxygen consumption rate (OCR) in State 3 (St 3, ADP stimulated), State 4 (St 4o, oligomycin) and following FCCP addition were determined using Seahorse XF24 Analyzer in the presence of glutamate/malate (complex I substrates) and succinate/rotenone (complex II substrate).

Results:
DR dose dependently reduced St 3 respiration both in the presence of complex I (Fig). In the presence of glutamate/malate, DR inhibited OCR by 16%, 20%, 25%, 39% and 100% at 1, 5, 10, 20, 50 μM, respectively, when compared to untreated control. At 20 μM, DR uncoupled mitochondria and increased St 4o respiration. DR at 50 μM was toxic with complete inhibition of OCR and loss of membrane potential. Similar results were observed when succinate/rotenone were used to assess complex II activity.

Conclusion:
DR has dose-dependent inhibitory effect on mitochondrial respiration, inhibiting OXPHOS at low concentration (1-10 μM), uncoupling at higher (20 μM) and toxic effect at 50 μM. Impairment of mitochondrial energetics could explain DR results reported in HF patients in clinical trials.
Abstract 325: Dissociation of Oxidative Stress from High Fatty Acid-Induced Cardiac Myocyte Death
Authors: Gutierrez Cortes Nicolas , Zhang Huiliang , Xu Shangcheng , Gong Guohua , Wang Wang
Published: July 2021

Metabolic syndrome, featured by obesity and diabetes, is an independent risk factor for cardiovascular disease. The high availability of free fatty acids characteristic of obesity has been proposed to increase fatty acid oxidation, impair mitochondrial function, stimulate reactive oxygen species (ROS) production and eventually lead to oxidative stress, which then would cause myocyte damage and heart dysfunction. However, the role of oxidative stress as the initial cause of obesity-induced heart failure is controversial.

We evaluated the time-dependent effect of fatty acids in vitro in adult rat cardiac myocytes (treated with palmitate and oleate, the major saturated and unsaturated fatty acids in the plasma, respectively), and in vivo in mice fed a high-fat diet (fat calories = 60%).

Palmitate and oleate dose-dependently stimulated mitochondrial respiration and superoxide flash activity, a respiration-coupled bursting ROS production event in single mitochondria. The flash frequency increased 2-fold (0.1 mM, the physiological level in plasma) and 3-fold (0.3 mM, the plasma level found in obese subjects) after only 2 hr incubation and up to 24 hr, increase that could be blocked by the CPT1 inhibitor etomoxir (100uM). Surprisingly, there was no obvious mitochondrial oxidative stress, although the high level of fatty acids caused mPTP opening and cell death at 48 hr. Further, aconitase activity was normal and NAD/NADH ratio was decreased, indicating a more reduced status rather than oxidative stress. Intriguingly, mitochondrial membrane potential was slightly lowered after only 2 hr of treatment. Finally, the high-fat diet-fed mice developed heart dysfunction after 12 weeks of treatment, but oxidative stress only appeared after 18 weeks.

In the light of these results, we hypothesize that high fatty acid supply induced multiple adaptive and mal-adaptive responses in cardiac myocytes. The increased mitochondrial respiration and bursting ROS production is offset by a mild uncoupling and increased reducing equivalents. Therefore, oxidative stress is unlikely an initial cause of myocyte death and heart dysfunction in obesity, but rather a consequence of mitochondrial dysfunction, although the exact cause of this dysfunction remains to be elucidated.
Abstract 335: Exercise Protects the Heart by Preserving Mitochondrial Membrane Potential During Early Reperfusion
Authors: Alleman Rick J , Patel Hetal D , Moukdar Fatiha , Brown David A
Published: July 2021

Exercise evokes adaptations intrinsic to the myocardium that protect against ventricular arrhythmia, yet the underlying mechanisms are not completely understood. We have previously shown that the transition to arrhythmia occurs concomitant with a collapse in mitochondrial membrane potential (ΔΨm). As our previous studies indicated that exercise preserves intracellular redox homeostasis, which directly influences mitochondrial energetics, we hypothesized that rats exposed to exercise (Ex, 10 d of treadmill running) would be protected against reperfusion arrhythmia via better maintenance of ΔΨm. To fully understand the temporal relationship between ΔΨm and cardiac electrical activity, two-photon microscopy images (using the fluorescent probe TMRM) and volume-conducted electrocardiogram were simultaneously recorded. Langendorff-perfused hearts underwent 40/30 min of ischemia/reperfusion. Exercise lowered the incidence of arrhythmia, with 3 of 8 Ex hearts experiencing tachycardia or fibrillation compared to 7 of 8 sedentary (Sed) hearts. Ex prevented the collapse of ΔΨm during the first 10 min of reperfusion (74±6.4% v 57±1.5% of baseline fluorescence intensity; P<0.05). To gain a more comprehensive understanding of energetics throughout the heterogeneous mitochondrial population, we then measured mean TMRM fluorescence intensity in isolated ventricular mitochondria harvested after reperfusion using flow cytometry (n=100,000 events per group). Interestingly, mean fluorescence intensity for ΔΨm was similar in Ex and Sed mitochondria (278±33 v 309 ±44 AU, respectively). Mitochondrial respiratory control ratios were also similar in Ex and Sed (9.03±0.70 v 9.00±0.92, respectively). Taken together, the isolated mitochondrial assessment did not reflect what was observed in vivo. This suggests that either intracellular factors influenced in vivo mitochondrial energetics, or our isolated mitochondria may have been enriched with predominantly healthy mitochondria. Our studies demonstrate for the first time that exercise prevents electrical dysfunction following an ischemic insult through better preservation of mitochondrial energetics, and that this preservation is only observed in the intact organ.
Abstract 338: p55γ Suppresses VSMC Proliferation and Neointimal Formation
Authors: Cao Chun-Mei , Xie Ning , Yao Yuan , Zhang Yan , Xiao Rui-Ping
Published: July 2021

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to various vascular diseases, but the factors that maintain VSMCs in a quiescent state remain poor understood. Phosphatidylinositol 3 kinases (PI3Ks) are important protein kinases that regulate vascular cell proliferation, but the biological and pathological functions of p55γ, a regulatory subunit of PI3K, and its regulation in the cardiovascular system are completely unknown. We aimed to determine the relationship between p55γ and vascular proliferation and neointimal formation.

In the present study, we have demonstrated that p55γ expression is markedly downregulated in primary cultured VSMCs in response to mitogenic stimulation and in carotid arteries after balloon injury, and that overexpression p55γ profoundly inhibits mitogenic stimuli and injury induced VSMC proliferation as well as neointimal formation. p55γ overexpression inhibited, whereas knockdown of p55γ promoted PDGF-BB- and serum-induced VSMC proliferation. Importantly,
in vivo
adenoviral gene transfer of p55γ into carotid arteries attenuated, while knockdown of p55γenhanced balloon injury-induced neointimal formation. Furthermore, p55γ sequentially upregulated p53 and p21, resulting in cell-cycle arrest in S phase; knockdown of either p53 or p21 blocked p55γ-induced VSMC growth arrest. Mechanistically, p55γ interacted with and stabilized p53 protein by blocking MDM2-mediated p53 ubiquitination and degradation, subsequently activating its target gene p21. Concurrently, p55γ upregulated Bcl-xl expression, which counterbalanced p53-mediated apoptosis. These findings mark p55γ as a novel upstream regulator of the p53-p21 signaling pathway which negatively regulates VSMC proliferation, suggesting that malfunction of p55γ may trigger vascular proliferative disorders.

* Correspondence to Chun-Mei Cao (caochunmei@pku.edu.cn) or Rui-Ping Xiao (xiaor@pku.edu.cn).
Abstract 34: A Human G109E Polymorphism in Inibitor-1 Compromises Cardiomyocyte Function and Induces Arrhythmias
Authors: Haghighi Kobra , Pritchard Tracy J , Singh Vivek P , Das Parthib , Vanderbilt Erica L , Rubinstein Jack , Kranias Evangelia G
Published: July 2021

Protein phosphatase 1 (PP1) has emerged as a nodal regulator of function and survival in the heart. Indeed, the increased activity of this enzyme in failing hearts contributes to depressed SR Ca-cycling and deteriorative remodeling. PP1 is negatively regulated by endogenous inhibitor-1, which is considered a promising therapeutic target. Increases in inhibitor-1 activity and decreases in PP1 protect against ischemia/reperfusion injury, chronic isoproterenol stimulation and heart failure progression. We recently identified a polymorphism (G109E) in the inhibitor-1 gene in heart failure patients with a frequency of 6%. Expression of G109E (called mutant) in rat cardiomyocytes resulted in ~20% decreases in contractile parameters, Ca-transients and sarcoplasmic reticulum Ca-load. This depressed function was rescued by isoproterenol. Interestingly, when subjected to stress conditions (2 Hz +/- Iso), the mutant cells were more susceptible to aftercontractions. Similar findings were obtained by expression of G109E in inhibitor-1 knockout cardiomyocytes in the absence of endogenous protein. The underlying mechanisms included reduced binding of mutant to PP1, increased PP1 activity and hyper-phosphorylation of S2814 in ryanodine receptor (RyR), promoting aberrant SR Ca-release. These findings were also reflected by in vivo cardiac overexpression of G109E. Contractile and Ca-kinetic parameters were depressed by ~30% in mutant cardiomyocytes, while isoproterenol relieved these inhibitory effects. Stress conditions were associated with induction of Ca waves and aftercontractions in G109E cells. Furthermore, serial caffeine/Iso injections in vivo, elicited higher incidence of ventricular ectopy (bigeminy, trigeminy and non-sustained ventricular tachycardia) in mutant mice, whereas WTs had normal rhythm. Our findings suggest that G109E and increased PP1 may dephosphorylate the RyR and promote Ca-leak, in agreement with previous reports. Subsequently, the increased Ca-levels activate CAMK, leading to hyperphosphorylation of RyR and further increases in Ca-leak. Thus, inhibitor-1 is critical in cardiac function and represents a key control in balancing phosphatase/kinase activities to stabilize Ca-cycling in the heart.
Abstract 359: Negative Regulation Of The Fgf-16 Gene Promoter By Doxorubicin Is Associated With A Decrease In Nkx2.5 Binding
Authors: Wang Jie , Jin Yan , Cattini Peter A
Published: July 2021

Doxorubicin (DOX) is a widely used and effective anti-cancer drug, but it is also cardiotoxic, which can lead to heart failure, and so strategies are needed to protect the heart. Fibroblast growth factor 16 (FGF-16) is preferentially expressed and released from cardiomyocytes after birth. Evidence suggests that FGF-16 decreases the risk of heart damage and limits the negative effects of heart remodeling (hypertrophy and fibrosis) after injury in vivo. Exogenous addition of FGF-16 also increased resistance to the loss of contractility in an isolated heart model of acute DOX-induced injury. Thus, how endogenous FGF-16 production and by extension function is affected by DOX treatment is of interest. The FGF-16 gene is highly conserved between human and murine species. Alignment of sequences indicates a conserved Nkx2.5 binding site in the proximal promoter region that is associated with a previously characterized TATA box. Nkx2.5 is an important factor in vertebrate heart development and congenital disease. Furthermore, Nkx2.5 RNA levels are decreased with DOX treatment. Thus, the possibility that DOX negatively affects FGF-16, perhaps through an effect on Nkx2.5 levels or binding, was investigated. Neonatal rat cardiomyocytes were treated with DOX, and FGF-16 RNA levels decreased 75% within 6 hours as assessed by qPCR. Inhibition of transcription with actinomycinD had no effect on the DOX-induced decrease in FGF-16 RNA levels. Further support for an effect of DOX on FGF-16 transcription was obtained by transfection of cardiomyocytes with a hybrid 747 bp mouse FGF-16 promoter/luciferase reporter gene that was treated with DOX; a significant decrease in luciferase activity was observed. Electrophoretic mobility shift and chromatin immunoprecipitation assays suggest reduced Nkx2.5 protein-DNA interaction with this site after DOX treatment in vitro and in situ, respectively. These data indicate that DOX decreases FGF-16 RNA expression and this correlates with a decrease in Nkx2.5 levels and association with the proximal promoter region. A direct effect of Nkx2.5 on FGF-16 promoter activity awaits further testing. Thus, a decrease or loss of FGF-16 synthesis might contribute to the DOX-induced damage and/or response to injury.
Abstract 361: Inactivation of Cardiac Foxo1 by Insulin Signaling Is Required for Cardiac Function and Suppression of β-Myosin Heavy Chain Gene Expression
Authors: Qi Yajuan , Zhu Qinglei , Zhang Kebin , Thomas Candice , Kumar Rajesh , Baker Kenneth M. , Guo Shaodong
Published: July 2021

Heart failure is a leading cause of morbidity and mortality in the USA and is closely associated with diabetes mellitus. The molecular link between diabetes and heart failure is incompletely understood. We recently demonstrated that insulin receptor substrate 1, 2 (IRS1, 2) are key components of insulin signaling and their dysfunction mediates insulin resistance, resulting in metabolic dysregulation and heart failure. Loss of IRS1 and IRS2 is associated with downstream Akt inactivation and in turn activation of the forkhead transcription factor Foxo1. To determine the role of Foxo1 in control of heart failure in insulin resistance and diabetes, we generated mice lacking Foxo1 gene specifically in the heart. Mice lacking both IRS1 and IRS2 in adult hearts exhibited severe heart failure, loss of mitochondria, and a remarkable increase in the β-isoform of myosin heavy chain (β-MHC) gene expression, while deletion of cardiac Foxo1 gene largely prevented the heart failure and the loss of mitochondria, and resulted in a decrease in β-MHC expression. The effect of Foxo1 deficiency on rescuing cardiac dysfunction was also observed in db/db mice and high-fat diet (HFD) mice. Using cultures of primary ventricular cardiomyocytes, we found that Foxo1 interacts with the promoter region of β-MHC and stimulates gene expression, mediating an effect of insulin that suppresses β-MHC expression. Taken together, our study suggests that Foxo1 has important roles in promoting diabetic cardiomyopathy and controls β-MHC expression in development of cardiac dysfunction. Targeting Foxo1 and its regulation will provide novel strategies in preventing metabolic and myocardial dysfunction and influencing MHC plasticity in diabetes mellitus.
Abstract 363: Class I HDAC Inhibition Causes Selective Activation of Pro-Survival Kinases at Reperfusion in Post-Ischemic Hearts
Authors: Aune Sverre E , Herr Daniel J , Menick Donald R
Published: July 2021

Background:
Histone deacetylase (HDAC) enzymes inhibit gene expression through chromatin compaction and exacerbate myocyte death following ischemia-reperfusion (IR) in the heart. In our prior study, disinhibition of antioxidant gene and protein expression was achieved through 24 hour pretreatment with entinostat, a class I HDAC inhibitor, and was correlated with improved heart contractile recovery from IR. Additionally, ERK1/2 was phosphorylated, indicating activation of the reperfusion injury survival kinase (RISK) pathway. HDAC inhibitors also enhance posttranslational acetylation of nonhistone proteins through unmasking of acetyltransferase activity, which may influence heart survival from injury.

Hypothesis:
We hypothesized that inhibition of class I HDACs during reperfusion in postischemic hearts would activate the RISK pathway and improve contractile recovery.

Methods:
Hearts from male Sprague-Dawley rats (n=4/group) were isolated and perfused in Langendorff mode. Global ischemia was induced by cessation of perfusion for 30 min, followed by 60 min of reperfusion, during which hearts were perfused with vehicle or 10, 100 or 1000 nM entinostat. Results are reported vs vehicle as mean±SE.

Results:
At the end of reperfusion, rate pressure product (mm hg/min) was improved with 10 nM entinostat (17226±1187, p<0.001) vs vehicle (11024±1810), as were +dP/dtmax, -dP/dtmax, and developed pressure. The improvement in pressure development with 10 nM entinostat was due primarily to prevention of end diastolic dysfunction in reperfusion. LV function was not significantly preserved in hearts treated with 100 or 1000 nM entinostat. Hearts reperfused with 10 nM entinostat showed reduced infarct area (35%) vs vehicle (64%), along with increased shuttling of 14-3-3 protein, Akt and phospho-ERK from cytoplasm to nucleus. These effects were reduced with higher concentrations of inhibitor. Notably, histone acetylation was increased with higher concentrations of entinostat, but not with 10 nM.

Conclusions:
Inhibition of class I HDACs during reperfusion resulted in specific activation of RISK members without acetylation of histone, indicating that acetyltransferase enzymes may mediate activation of pro-survival kinases in the IR heart.