bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 310: Role of Cardiac Fibroblast in Cardioprotective Effects of Prior Transient ACE Inhibition
Authors: Hale Taben M , Biwer Lauren A , D’Souza Karen M
Published: July 2021

Prior treatment with the ACE inhibitor enalapril followed by washout protects against nitric oxide synthase inhibitor (L-NAME) induced fibrosis, cellular proliferation, and cardiac dysfunction. The present study investigated i) whether in vivo L-NAME administration induces a change in cardiac fibroblast phenotype that persists in vitro, ii) whether prior ACE inhibition protects against L-NAME induced changes in cardiac fibroblasts. SHR were divided into 3 groups: Control, L-NAME (C+L: 7d), enalapril+L-NAME (E+L: 14d enalapril + 14d washout + 7d L-NAME). MAP was measured by radiotelemetry (n=5-9), injury assessed by histology (n=6-10), and heart weight to body weight (HW/BW) was determined after 0 or 7 days of L-NAME in C+L and E+L (n=6-10). In separate rats cardiac fibroblasts were isolated after 7 days of L-NAME (C+L, E+L) or placebo (Con) and cultured to passage 1 (n=10-12). Gene expression was measured by quantitative real-time PCR. L-NAME increased MAP in C+L (22±4.1%) and E+L (21±3.6%) rats. Prior enalapril induced a persistent 13% reduction in HW/BW. L-NAME increased heart mass in E+L (7%) but not C+L; however, HW/BW remained 8% lower than C+L at sacrifice. L-NAME induced infarct in 70% of C+L and 40% of E+L hearts. Cardiac fibroblasts demonstrated a significant increase in proliferation rate in C+L, but not E+L, relative to control (C+L: 1.75-fold vs. con; E+L 1.09-fold vs. con). Fibroblasts from C+L hearts tended to have increased Collagen I and III gene expression. Despite hypertension, cardiac injury, and increased HW/BW; fibroblasts isolated from E+L proliferated at the same rate as those from control. In contrast, those isolated from C+L were hyperproliferative with a tendency toward increased capacity for collagen production. It may be that the fibroblast phenotype from E+L hearts would protect against infarct expansion and account, in part, for the previously reported cardioprotection in these rats.
Abstract 313: STIM1 Silencing Reverses Pressure-overload Induced Cardiac Hypertrophy In Mice
Authors: Bénard Ludovic O , Matasic Daniel S , Keck Mathilde , Lompré Anne-Marie , Hajjar Roger J , Hulot Jean-Sébastien
Published: July 2021

STromal Interaction Molecule 1 (STIM1), a membrane protein of the sarcoplasmic reticulum, has recently been proposed as a positive regulator of cardiomyocyte growth by promoting Ca2+ entry through the plasma membrane and the activation of Ca2+-mediated signaling pathways.

We demonstrated that STIM1 silencing prevented the development of left ventricular hypertrophy (LVH) in rats after abdominal aortic banding.

Our aim was to study the role of STIM1 during the transition from LVH to heart failure (HF). For experimental timeline, see figure.

Transverse Aortic Constriction (TAC) was performed in C57Bl/6 mice. In vivo gene silencing was performed using recombinant Associated AdenoVirus 9 (AAV9). Mice were injected with saline or with AAV9 expressing shRNA control or against STIM1 (shSTIM1) (dose: 1e+11 viral genome), which decreased STIM1 cardiac expression by 70% compared to control.

While cardiac parameters were similar between the TAC groups at weeks 3 and 6, shSTIM1 animals displayed a progressive and total reversion of LVH with LV walls thickness returning to values observed in sham mice at week 8. This reversion was associated with the development of significant LV dilation and severe contractile dysfunction, as assessed by echography. Hemodynamic analysis confirmed the altered contractile function and dilation of shSTIM1 animals. Immunohistochemistry showed a trend to more fibrosis. Despite hypertrophic stimuli, there was a significant reduction in cardiac myocytes cross-section area in shSTIM1-treated animals as compared to other TAC mice.

This study showed that STIM1 is essential to maintain compensatory LVH and that its silencing accelerates the transition to HF.
Abstract 315: Mice With Cardiac-restricted Overexpression Of Myozap Are Sensitized To Biomechanical Stress And Develop A Protein-aggregate Associated Cardiomyopathy
Authors: Frank Derk , Poyanmehr Reza , Rangrez Ashraf , Kuhn Christian , Grund Christine , Franke Werner W , Frey Norbert
Published: July 2021

The intercalated disc (ID) is a major component of the cell-cell contact structures of cardiomyocytes ensuring their electro-mechanical coupling. In previous work, we could identify and characterize Myozap, a novel cardiac-enriched ID protein, which interacts with several other ID proteins.

We could now show in cardiomyocytes that overexpression of Myozap activates the Rho-dependent SRF pathway. Consistently, RNAi-mediated Myozap knockdown blocked Rho-mediated SRF activation linking the ID to gene regulation and actin dynamics.

For further analyses of Myozap’s functions in vivo, we generated a mouse model with cardiac overexpression of Myozap cDNA (αMHC promoter). 1 year old mice developed cardiomyopathy with significant hypertrophy (heart weight/tibia length +43%, p<0.01, n=8-10) as well as LV dilation (LVEDD +20%, p<0.01) and a trend towards LV dysfunction (fractional shortening 50% vs. 42%, n=10, p=0.07). Consistently, these mice displayed upregulation of the hypertrophy-associated gene program (nppa 4.3-fold, p<0.001, and nppb 2.2-fold, p<0.01, n=7-9).

On the ultrastructural level, we could detect bulky aggregates containing Myozap, desmoplakin and other ID proteins. This aggregate-associated pathology closely resembled the changes in the hearts of patients suffering from desminopathies. Interestingly, desmin was not detectable in the aggregates in MyozapTG mice, but appeared to be displaced from the ID. Further molecular analyses in the aged mice also revealed dysregulation of typical members of the unfolded protein response (UPR) associated with upregulation of UPR-related apoptosis (cleaved caspase 12).

Functionally, pressure overload via transverse aortic constriction (TAC) caused exaggerated cardiac hypertrophy (heart/body weight 7.9 mg/g in Myozap TG-TAC vs. 5.6 mg/g in WT-TAC, n=11 each group, p=0.002) and loss of contractility (fractional shortening of 15.3% in Myozap TG-TAC vs. 33.0% in WT-TAC, n=11 each group, p<0.001). Similarly, a physiological stimulus (voluntary running) led to a less dramatic but significant premature LV dysfunction.

In conclusion, Myozap overexpression offers new insights in the pathogenesis of protein aggregate-associated cardiomyopathies.
Abstract 316: A Potential Mechanism Involving mTOR For The Expression Of CTGF In Agonist-stimulated Adult Cardiomyocytes
Authors: Sundararaj Kamala P , Balasubramanian Sundaravadivel , Pleasant Dorea , Kuppuswamy Dhandapani
Published: July 2021

Cardiac hypertrophy ensues as a response to multiple stimuli, such as mechanical stress, neurohumoral activation, growth factors and cytokines. Connective Tissue Growth Factor (CTGF), a potent fibrogenic cytokine, regulates a wide range of biological functions including ECM deposition, wound repair, angiogenesis, migration, differentiation, survival and proliferation. While CTGF overexpression in fibroblasts has been shown to be responsible for fibrosis in various organs, controversy exists about the source of CTGF. Since interstitial fibrosis contributes to ventricular wall stiffness and impairs diastolic function, understating how key factors such as CTGF are expressed and released for the genesis of fibrosis in the hypertrophying heart is important to develop new treatment options. To this end, we explored the signaling pathway(s) involved in the phenylephrine (PE), a hypertrophic agonist, induced expression of CTGF by cardiomyocytes (CMs). Since mammalian target of rapamycin (mTOR) is reported to regulate PE-induced hypertrophic signaling, we hypothesize that mTOR plays a role in PE induced CTGF expression in CMs.

To test if CMs produce CTGF, we treated adult feline CMs with phenylephrine. PE stimulated CTGF mRNA expression in a dose and time dependent manner. mTOR forms two distinct complexes, mTORC1 and mTORC2. Whereas both complexes are sensitive to a pharmacological inhibitor Torin1, only mTORC1 is sensitive to Rapamycin inhibition. Our results indicate that PE stimulated CTGF expression could be substantially enhanced by torin1 pretreatment of CMs. Moreover, shRNA mediated silencing of Rictor in CMs, one of the components of mTORC2, significantly augmented the PE induced CTGF mRNA expression. But mTORC1 inhibition using Rapamycin or activation of its downstream target S6K1 using Rapamycin resistant S6K1 adenovirus had no impact in PE -stimulated CTGF expression. The same trend was also observed in the level of secreted CTGF. In conclusion, these results strongly indicate that mTORC2 plays a repressive role in CTGF mRNA expression in adult CMs, and that the loss of such repression in PO myocardium might be a potential mechanism for the onset of cardiac fibrosis in hypertrophying myocardium.
Abstract 318: Dynamic Mass Redistribution Analysis Of Endogenous β-adrenergic Receptor Signaling In Cardiac Cells
Authors: Tilley Douglas G , Jacobson Marlene A , Carter Rhonda L , Yu Justine , Gristanti Laurel A
Published: July 2021

β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function, and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored the biological responses to βAR stimulation. We used label-free dynamic mass redistribution (DMR) to elucidate the biological response to stimulation of endogenous βAR in primary rat neonatal cardiac fibroblasts, and to begin to explore the pathways responsible for these effects. Isoproterenol (ISO, non-selective for β1/β2), salbutamol (Sal, β2-selective) and dobutamine (Dob, β1-selective) concentration-dependently induced increases in DMR response, with the Dob-induced response significantly less than either ISO- or Sal-induced DMR, consistent with the reduced expression of β1AR vs β2AR in cardiac fibroblasts. Addition of propranolol (Prop) competitively blocked the DMR effect induced by all 3 agonists, while the addition of cholera toxin (CTX) non-competitively blocked the effects of each agonist. Overall, the CTX-sensitive DMR effect accounted for approximately 75% of the response, while 25% of the biological response was CTX-insensitive. To confirm that the CTX-sensitive DMR response was mediated by downstream Gαs protein-dependent signaling, cAMP generation was assessed via FRET where ISO produced a similar concentration-effect curve and EC50 value as attained with DMR. All of the cAMP generation response was ablated by both Prop and CTX, indicating that the CTX-sensitive acute DMR effect to βAR stimulation is directly related to cAMP generation. Since ~25% of the DMR effect was not CTX-sensitive, other Gαs protein-independent signaling pathways are presumably activated in response to βAR stimulation in cardiac fibroblasts. To assess this possibility, EGFR internalization was quantitated as an indicator of βAR-mediated EGFR transactivation. EGFR internalization increased in response to ISO stimulation in a concentration-dependent manner, with a similar EC50 value as attained in the DMR assay. Defining the relative contributions of different βAR pathways in cardiac fibroblasts will help establish the importance of these pathways as potential therapeutic targets in the heart.
Abstract 320: Roles of Estrogen, AMPK and Micro RNAs in the Progression of Cardiac Hypertrophy
Authors: Lipovka Yulia , Konhilas John P
Published: July 2021

In industrialized countries, the prevalence of congestive heart failure (CHF) is increasing steadily and has become one of the leading causes of hospitalization. In addition, the risk of cardiovascular disease increases in post-menopausal women. Yet, the association between estrogen and the risk of CHF has not been adequately studied. Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. Our on-going studies indicate differential AMPK regulation through two miR species (miR195 and miR451) in a mouse model harboring a missense mutation (R403Q) in alpha-myosin heavy chain (αMHC) causing hypertrophic cardiomyopathy (HCM). Using bioinformatic algorithms (TargetScanMouse, 5.2), we were able to predict miR candidates that potentially target the AMPK axis. In addition, Altered expression of miRs that target AMPK axis was found in phenylephrine induced hypertrophic neonatal rat cardiomyocytes (NRCM). However, Estradiol treatment of NRCM blocked the hypertrophic changes induced by phenylephrine treatment. It was known that the activation of AMPK pathway inhibits cardiomyocyte hypertrophy. Our data showed that AMPK pathway was activated by Estradiol treatment, which can be blocked by estrogen receptor (ER) β antagonist. Therefore, estradiol increase AMPK pathway activation which in turn attenuate phenylephrine induced increase in cardiomyocyte cell size. Further studies are need to further explore the role of estrogen in the regulation of miR expression in hypertrophic cardiomyocytes, and the role of the expression changes of miRs regulated by estrogen in the development of hypertrophic phenotype.
Abstract 325: COX2 Expression is Increased in Human and Mouse Calcific Aortic Valve Disease and COX2 Inhibition Blocks the Induction of Osteogenic Gene Expression in Valve Interstitial Cells
Authors: Wirrig Elaine E , Hinton Robert B , Yutzey Katherine E
Published: July 2021

Calcific aortic valve disease (CAVD) is considered a disease of aging and is characterized by calcific nodule formation, stenosis of the valve, and induction of osteogenic gene expression. The klotho deficient mouse model of premature aging exhibits CAVD evident in calcific lesions in the aortic valve annulus. Klotho deficient mice develop calcification by 6 weeks of age, with minimal immune cell infiltration and increased osteogenic gene expression. Microarray analyses performed on aortic valve tissues of wild type and klotho deficient mice demonstrate that cyclooxygenase 2 (COX2) expression is increased in klotho deficient mice. We hypothesize that increased COX2 expression contributes to aortic valve calcification and that COX2 inhibition prevents osteogenic gene expression in aortic valve interstitial cells (aVICs). In klotho deficient mice, COX2 protein expression is increased throughout regions of valve calcification relative to wild type controls. Prior to the formation of calcified valve lesions, increased COX2 expression is localized to areas at risk of developing calcification in klotho deficient mice. Thus increased COX2 expression precedes valve calcification. In human explanted calcific aortic valves, COX2 expression also is increased in mineralized regions compared to controls. In bone, COX2 deficiency inhibits osteogenic gene induction. The ability of COX2 inhibition to block osteogenic gene expression was examined using porcine aVICs. Treatment of aVICs with osteogenic media induces bone markers, including osteocalcin, in addition to increasing COX2 gene expression. Furthermore, inhibition of COX2 activity in cells cultured under osteogenic conditions results in significantly decreased expression of osteogenic genes compared to controls. In vivo studies in progress provide initial evidence that loss of COX2 activity reduces aortic valve calcification in klotho deficient mice. In conclusion, COX2 expression is increased in regions of aortic valve calcification in both human and mouse CAVD tissues, and COX2 inhibition suppresses osteogenic gene expression in cultured aVICs. Targeting COX2 enzyme activity may be a potential therapeutic option for preventing or reducing aortic valve calcification in the future.
Abstract 326: Nucleostemin Induced by Pim-1 Kinase Antagonizes Senescence of Cardiac Progenitor Cells
Authors: Hariharan Nirmala , Joyo Anya Y , Avitabile Daniele , Samse Kaitlen M , Bailey Brandi , Quijada Pearl , McGregor Michael , Mohsin Sadia , Siddiqi Sailay , Sussman Mark A
Published: July 2021

Decline in stem cell functionality and regenerative capability is a crucial contributor to aging, upon which the c-kit+ cardiac progenitor cell (CPC) pool with enhanced regenerative potential decreases and CPCs with limited proliferative and differentiation potential progressively accumulate. With cardiac stem cells currently being used in the clinic, especially in the elderly population, there is a dire need to understand the phenotypic, molecular and functional characteristics of senescent stem cells to effectively manipulate them for therapy. Nucleostemin (NS), a nucleolar stress sensor protein associated with stem cell pluripotency and regeneration declines upon cardiac aging. We hypothesize that NS is a critical antagonizer of senescence in CPCs. NS expression decreases significantly (-59%, p<0.05), while the % of senescent cells increases in CPCs isolated from old mice (13 month, OCPC) relative to CPCs isolated from young mice (3 month, YCPC). OCPCs are morphologically different, characterized by flat, round cells (p<0.05) with increased bi- & poly-nucleation (p<0.05), slower proliferation (-80%, p<0.01) and altered cell cycle profiles, relative to YCPCs. Decreased NS level is causal for the phenotype observed in OCPCs, as silencing NS in YCPCs induces flattening of cells, increases bi- & poly-nucleation, decreases expression of stem cell marker c-Kit (-55%, p<0.05), upregulates cell cycle inhibitors p53 and p16 (4.2, 3.8 fold, p<0.01) and decreases proliferation (p<0.05). NS-mediated regulation of CPC senescence is p53 dependent, as silencing p53 rescues the phenotype induced by NS-silencing. Interestingly, NS is induced by Pim-1 kinase-mediated stabilization of transcription factor c-Myc, as NS protein expression decreases in CPCs upon knockdown of Pim-1 (p<0.05) or c-Myc (p<0.01); and in the absence of c-Myc, Pim-1 does not upregulate NS. Engineering OCPCs with NS is an effective strategy to antagonize senescence, as observed by decreased % senescent cells, increased proliferation and restored morphology. In conclusion, NS which is induced downstream of Pim-1 kinase, maintains multipotency and inhibits senescence in CPCs, consistent with cumulative evidence that Pim-1 induced cardiac regeneration is mediated in part by NS.
Abstract 339: Vasonatrin Peptide Attenuates Myocardial Ischemia/Reperfusion Injury in Diabetic Rats through PKG-Mediated Inhibition of Endoplasmic Reticulum Stress
Authors: Zhang Haifeng , Yu Liming , Shi Zhenwei , Lv Weifeng , Tie Ru , Liang Xiangyan , Yu Jun , Tao Ling
Published: July 2021

Diabetes mellitus (DM) increases morbidity/mortality of ischemic heart disease (IHD). Although atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) reduce the myocardial ischemia/reperfusion (MI/R) damage in non-diabetic rats, whether vasonatrin peptide (VNP), the artificial synthetic chimera of ANP and CNP, confers cardioprotective effect against acute MI/R injury, especially in diabetic patients, is still unclear. This study aimed to investigate the effects of VNP on MI/R injury in diabetic rats and the involved mechanisms. The high-fat diet-fed streptozotocin (HFD-STZ) induced diabetic rats were subjected to MI/R (30 min/4 h). VNP treatment (100 µg/kg, i.v., 10 min before R) significantly improved ± LV d
P
/dt
max
[(3242 ± 103) and -(2731 ± 79) mm Hg/s vs. (2936 ± 90) and -(2422 ± 83) mm Hg/s in DM group] and LVSP and reduced LVEDP, and reduced infarct size [(43.3 ± 3.6) % vs (53.5 ± 2.8) %], apoptosis index [(36.0 ± 2.1) % vs. (45.7 ± 3.5) %], caspase-3 activity, serum CK and LDH levels (n=8,
P
<0.05). Moreover, VNP inhibited endoplasmic reticulum (ER) stress by suppressing GRP78 and CHOP (n=3,
P
<0.05), and consequently increased the antiapoptotic protein Akt and ERK1/2 expression and phosphorylation levels (
P
<0.05). These effects were mimicked by 8-Br-cGMP (1 mg/kg, i.p., 20 min before R), a cGMP analogue, whereas inhibited by KT-5823 (0.5 mg/kg, i.p.), the selective inhibitor of PKG (both
P
<0.05). In addition, pretreated DM rats with TUDCA (50 mg/kg, i.p.), a specific inhibitor of ER stress, couldn’t further promote the VNP’s cardioprotective effect (
P
>0.05).
In vitro
study was performed using H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R, 3 h/6 h) and incubated with or without VNP (10
-8
mol/L). Gene knockdown of PKG1α with siRNA blunted VNP’s inhibition of ER stress and apoptosis (n=6,
P
<0.05), while overexpression of PKG1α resulted in significant decreased ER stress and apoptosis (n=6,
P
<0.01). In conclusion, VNP protects diabetic heart against MI/R injury by inhibiting ER stress via cGMP-PKG signaling pathway. These results suggest that VNP may have potential therapeutic value for the diabetic patients with ischemic heart disease.
Abstract 340: IL-10 And HSCRP Were Predictors For The Attenuation Of Heart Functions In Myocardial Ischemia Swine
Authors: Guo Shuzhen , Chuo Wenjing , Wang Yong , Chen Jianxin , Wang Juan , Feng Xuanchao , Lian Hongjian , Xie Hua , Yu Xue , Luo Liangtao , Wang Wei
Published: July 2021

Background and Objective:
Inflammation was one of the most important pathophysiological processes during myocardial ischemia. However, it is still poorly understood about the possibilities of these pro- or anti-inflammation factors be used as prognostic parameters for subsequent attenuation of heart functions.

Methods and Results:
Chinese experimental mini swines (25±4 kg) were instrumented with a size-matched Ameroid constrictor on the left anterior descending branch (LAD) under general anesthesia in sterile conditions (N=15). And a sham group (N=6) was included. 4 weeks after thoracotomy, more than 90% occlusion of the LAD was proved by coronary angiography. Early stage of ischemia was found by echocardiography which showed focal ischemia in the anterior wall of left ventricular (from apex to papillary muscle level) and unchanged ejection fraction. Meanwhile, Enzyme-linked immunosorbent assay of serum showed that IL-8 (86.83 ±10.96 vs. 130.3 ±10.94-pg/ml,
P
=0.0322) and IL-10 (0.9619 ±0.1421 vs. 1.660 ±0.1607-pg/ml,
P
=0.0111) were dramatically decreased compared to sham group, while no significant changes were found in IL-1 (0.3075 ±0.03558 vs. 0.3045 ±0.057371 -ng/ml,
P
= 0.9643), IL-6 (307.7 ±27.81 vs. 315.8 ±23.07-pg/ml,
P
=0.8637), high sensitivity C reactive protein (hsCRP) (12.94 ±1.324 vs. 9.943 ±1.442-ng/ml,
P
=0.2087) and TNF-a(1.769 ±0.1467 vs. 1.260 ±0.3209-ng/ml,
P
=0.1121). Furthermore, 8 weeks after thoracotomy, ejection fraction(EF) of myocardial ischemia animals were dramatically attenuated (44.69 ±4.494 vs. 60.60 ±3.420-%,
P
=0.0428). Subsequent EF (8 weeks postoperation) showed a good linear regression with the IL-10 and hsCRP concentration in the early stage of myocardial ischemia (4 weeks postoperation) (EF= 0.525+ 0.132* IL-10- 0.015* hsCRP,
P
=0.001) when stepwise regression was performed with all inflammation related factors above as dependent variables.

Conclusion:
These results demonstrate that lower IL-10 and higher hsCRP in serum may be predictors for the attenuation of heart function caused by myocardial ischemia. Further Clinical trials may help to prove the efficiency of these novel insights of prognosis for ischemia heart diseases.
Abstract 349: Regulation Of Key Metabolic Enzymes By Tristetraprolin
Authors: Bayeva Marina , Sawicki Konrad , Ardehali Hossein
Published: July 2021

Introduction:
Heart disease and stroke are the primary causes of death in adults with type-2 diabetes thus strict metabolic control is essential in these patients. Here, we show that tristetraprolin (TTP), a protein involved in regulation of inflammation and iron homeostasis via mRNA degradation, may alleviate diabetic phenotype through modulation of key enzymes involved in gluconeogenesis and fatty acid (FA) metabolism in insulin-sensitive tissues.

Results:
TTP protein content was significantly reduced in livers of diabetic mice fed high-fat diet compared to control, suggesting that mRNA targets of TTP may be stabilized in diabetes. We then assessed the effects of TTP knockdown using siRNA in HepG2 liver cells on the expression of enzymes in four major metabolic pathways: glycolysis, gluconeogenesis, FA oxidation and synthesis. Consistent with reduced TTP, we found increased expression of a key gluconeogenic regulator - pyruvate dehydrogenase kinase 4 (PDK4). In silico analysis of the 3’ untranslated region (UTR) of PDK4 revealed five putative TTP binding sites. Importantly, elevated PDK4 levels were previously reported in diabetic patients, and are thought to exacerbate the disease by increasing hepatic glucose output. Assessment of FA metabolism revealed increased levels of PPARα and two of its targets, carnitine palmitoyltransferase I (CPT1) and fatty acid translocase (FAT). Consistent with increased FA flux, triglyceride levels were elevated with TTP siRNA in hepatocytes. In silico analysis revealed multiple well-conserved putative TTP-binding sites in the 3’UTR of PPARα, consistent with regulation by TTP. We also observed significant upregulation of PPARα and its targets in HL-1 cardiac cell line, indicating an overlapping function for TTP in insulin-sensitive tissues. Notably, cardiac-specific overexpression of PPARα was previously shown to cause a phenotype resembling diabetic cardiomyopathy, further suggesting that reduction of TTP in diabetes may exacerbate this disease.

Conclusions:
Our studies show that TTP is downregulated in diabetic mouse livers, and knockdown of this protein in hepatic and cardiac cells increases the levels of key metabolic enzymes, PDK4 and PPARα, both of which are implicated in diabetes and heart disease.
Abstract 358: Transient Receptor Potential Vanilloid 2 (TRPV2) contributes to myocardial contractility and hypertrophy via sarcoplasmic reticulum calcium handling
Authors: Rubinstein Jack , Singh Vivek P , Lasko Valerie M , Koch Sheryl E , Kranias Evangelia , Jones W. Keith , Lorenz John N
Published: July 2021

Background:
TRPV2 is a Ca2+ channel that we have recently discovered in cardiomyocytes. The absence of this channel negatively impacts baseline contractility while stimulation results in a positive inotropic response. What remains to be established is the mechanism of this receptor and its role, if any, in the development of hypertrophy.

Methods and Results:
We obtained isolated cardiomyocytes from wild type (WT) and TRPV2-/- (KO) mice and found that the sarcoplasmic reticulum Ca2+ content and Ca2+ transients were reduced along with fractional shortening in the KO cardiomyocytes (figure, panels A, B, C). In vivo echocardiography confirmed a decrease in ejection fraction in KO mice in comparison to the WT counterparts (panel D). The relevance of these findings was examined in 6 WT and 5 KO mice subjected to transverse aortic constriction (TAC). These mice were followed by echocardiography weekly for a total of 8 weeks post TAC. At the conclusion, the hearts were obtained for histological and molecular analyses. We demonstrated that the KO mice developed less LV hypertrophy in comparison to WT (via echocardiography and by heart weight/body weight ratios) (figure, panels E and F). Importantly, there was a 5 fold increase in TRPV2 expression assessed by PCR in TAC WT hearts, compared to WT not subjected to TAC (0.72±0.10 vs. 0.13±0.04; p<0.01). This suggests a role for TRPV2 not only in contractility, but also in the development of hypertrophy.

Conclusions:
We have discovered a novel cardiac channel that alters Ca2+ cycling and is capable of modulating cardiomyocyte contractility and hypertrophy, which could lead to novel therapeutic options in heart failure and hypertrophy.
Abstract 108: Interleukins 17 A and 22 in diabetic patients with stable coronary artery disease
Authors: Oliveira Dinaldo C , Heide Elayne , Rego Moacyr , Gomes Viviane R , Oliveira Danielle G , Pita Maira G
Published: July 2021

Introduction:
The role of the immune and inflammatory pathways in diabetic patients with coronary artery disease (CAD) is important but not complete understood. The aim of this study was to evaluate concentrations of the interleukins 17 A and 22 in diabetic patients with stable CAD.

Hypothesis:
Interleukins 17 A and 22 are not increased in diabetic patients with stable CAD.

Methods:
This is a cross-sectional, prospective, analytical study, conducted from August to December 2012. We included 15 diabetic patients (P) with stable CAD, CCS III or IV, ischemic myocardial scintigraphy, who had not been subjected to any kind of myocardial revascularization and with coronary stenosis ≥ 50% according to current coronary angiography. There were 20 healthy volunteers (C), to take up comparison of concentrations of interleukins (IL). Interleukins were evaluated in serum of patients and after 48 hours of cells in culture with and without stimulus. IL concentrations were expressed in pg / ml. Statistical analysis was performed using the Mann-Whitney or Student t test. P ≤ 0,05 was considered statistically significant.

Results:
There were 6 men and 9 women in the group of the diabetic patients and 12 men and 8 women in the controls. The age was similar between the groups (61.6 ± 6.7 years vs 57.9 ± 9.4, p = ns). The main CAD risk factors: hypertension 73%, smoking 60%, dyslipidemia 40%, prior myocardial infarction 33%. The comparison between the groups showed: IL 17 A: Serum: P = 3.91 (3.91 -- 3.91 ) vs C = 3.91 (3.91 -- 3.91 ), p = 0.82; culture 48 hours without stimulus: P = 3.91 (3.91 -- 3.91) vs C = 3.91 (3.91 -- 3.91), p = 0.06; culture 48 hours with stimulus: P = 199 (3.91 -- 520) vs C = 154 (3.91 -- 574), p = 0.90. IL 22: Serum: P = 15.63 (15.63 -- 41.09) vs C = 15.63 (15.63 -- 41.09), p = 0.34; culture 48 hours without stimulus: P = 7.81 (7.81 -- 7.81) vs C = 7.81 (7.81 -- 7.81), p = 0.09; culture 48 hours with stimulus: P = 113.79 (7.81 - 248.63) vs C = 322.87 (7.81 - 628.49), p = 0.14.

Conclusions:
There were no differences in concentrations of IL 17 A and 22, does not matter in serum or cell in culture. In conclusion in diabetic patients with stable CAD the concentrations of interleukins 17 A and 22 were not increased.
Abstract 121: Combinatorial Tailored Polymers Enhanced Maturation of Human iPSC-CMs
Authors: Chun Young Wook , Feaster Tromondae K , Boire Timothy , Sheng Calvin C , Sung Hak-Joon , Hong Charles C
Published: July 2021

There is a tremendous interest in human cardiomyocytes generated from patient-derived induced pluripotent stem cells (iPSC-CMs) for the study and possible treatment of human heart diseases. Despite their vast potential, a significant impediment to a broader application of iPSC-CMs to study human myocyte biology is the structural and functional immaturity of iPSC-CMs. Growing evidence indicates that synthetic polymers utilized as extracellular substrates can exert significant effects on in vitro tissue generation, although the underlying mechanisms remain largely unknown. Based on the profound impact of the extracellular matrix of developing embryos on in vivo organogenesis, we hypothesize that engineered polymer substrates will likewise influence in vitro maturation of iPSC-CMs. A subset of combinatorial polymers was synthesized by polymerizing poly(ε-caprolacton) (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL), abbreviated as x%PEG-y%PCL-z%cPCL (x, y, and z: molar %). We investigated effects of the polymer composition on maturation of iPSC-CMs with respect to the beating behavior, mitochondrial function and molecular profiles after 30 days in culture on polymer scaffolds. Results showed the 4%PEG-96%PCL scaffold promoted the most active beating in iPSC-CMs at 30 days and further, that the mitochondrial function, as assessed by tetramethyl rhodamine methylester (TMRM) was significantly increased in the iPSC-CMs cultured on 4%PEG-96%PCL over other polymers. Molecular profiling analysis indicates 4%PEG-96%PCL scaffolds enhanced the expression of MYL2 (a commonly accepted marker of mature ventricular myocytes) as well as of components of the intermediate filaments linking the plasma membrane to the myofilament. In summary, although the polymers we used here exhibit similar physicochemical properties, they have divergent effects on iPSC-CM differentiation. Thus, specific chemical compositions of synthetic substrates can exert profound influence on in vitro maturation of hiPSC-CMs. Our work exploring the effects of synthetic biomaterials on human stem cell differentiation could pave the way for a successful translation of ongoing advances in tissue engineering to new treatments for human heart diseases.
Abstract 129: Embryonic Stem Cell Derived Exosomes Revive Endogenous Repair Mechanisms In Failing Heart
Authors: Khan Mohsin , Verma Suresh K , Mackie Alexander R , Vaughan Erin , Garikipati Srikanth , Lambers Erin , Ramirez Veronica , Ellingson Kenneth , Abramova Tatiana , Ito Aiko , Krishnamurthy Prasanna , Misener Sol , Qin Gangjian , Koch Walter J , Kishore Raj
Published: July 2021

Rationale:
Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to ethical concerns, lack of autologous donors and teratoma formation. Recently, it has been observed that beneficial effects of stem cells are mediated by exosomes secreted out under various physiological conditions. ESCs have the ability to produce exosomes however their effect in the context of the heart is unknown.

Objective:
Determine the effect of ESC derived exosomes for cardiac repair and modulation of CPCs functions in the heart following myocardial infarction.

Methods and Results:
Exosomes were isolated from murine ESCs (mES Ex) or embryonic fibroblasts (MEFs) by ultracentrifugation and verified by Flotillin-1 immunoblot analysis. Induction of pluripotent markers, survival and in vitro tube formation was enhanced in target cells receiving ESC exosomes indicating therapeutic potential of mES Ex. mES Ex administration resulted in enhanced neovascularization, cardiomyocyte survival and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex mediated considerable enhancement of cardiac progenitor cell (CPC) survival, proliferation and cardiac commitment concurrent with increased c-kit+ CPCs in vivo 4 weeks after mES Ex transfer. miRNA Array analysis of ESC and MEF exosomes revealed significantly high expression of miR290-295 cluster in the ESC exosomes compared to MEF exosomes. The underlying beneficial effect of mES Ex was tied to delivery of ESC miR-294 to the heart and in particular CPCs thereby promoting CPC survival and proliferation as analyzed by FACS based cell death analysis and CyQuant assay respectively. Interestingly, enhanced G1/S transition was observed in CPCs treated with miR-294 in conjunction with significant reduction of G1 phase.

Conclusion:
In conclusion, mES Ex provide a novel cell free system for cardiac regeneration with the ability to modulate both cardiomyocyte and CPC based repair programs in the heart thereby avoiding the risk of teratoma formation associated with ESCs.
Abstract 131: Age dependent regenrative ability of ckit+ cells Derived from Neonatal and adult Hearts
Authors: Mishra Rachana , Mishra Rachana , Sharma Sudhish , Desmukh Savitha , Balachandaran Keerti , Chen Ling , Kaushal Sunjay
Published: July 2021

Background:
Human adult c-kit
+
cardiac stem cells (CSCs) alleviates post-myocardial infarction left ventricle dysfunction in animal models and a Phase I clinical study. The regenerative capacity of CSCs in the very young patients with non-ischemic congenital heart defects has not been explored, even in the most surgically challenging Hypoplastic Left Heart Syndrome (HLHS) patients. We hypothesized that isolated neonatal-derived ckit
+
CSCs have higher regenerative abilities than adult-derived CSCs and might address the anatomical deficiency of HLHS myocardium.

Methods and Results:
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
+
cells using immune-activated magnetic bead selection, regardless of the initial weight or age. More than 85% ckit+ cells were isolated from both groups and these cells were negative for tryptase, collagen, CD45, CD34 and CD31. Single cell proliferation assay showed neonatal ckit+ cells are significantly more proliferative compare to adult ckit
+
cells. Neonatal c-kit
+
cells showed significantly higher telomere length (p=0.0286) compare to Adult. The neonatal-derived c-kit
+
cells secreted higher levels of VEGF-A, ANG, and SDF-1α when compared to adult-derived c-kit
+
cells. When transplanted into infarcted myocardium, neonatal-derived c-kit
+
cells had a significantly higher ability to preserve myocardial function, prevent adverse remodeling, and enhance blood vessel preservation when compared to adult-derived c-kit
+
CSCs. . Treatment with Neonatal-derived ckit
+
cells also augmented the preservation/formation of neovessels (isolectin B4) and arterioles (α-SMA) compared to adult-derived ckit
+
cells

Conclusions:
Neonatal-derived c-kit
+
cells have a strong regenerative ability when compared with adult-derived c-kit
+
cells that may depend on angiogenic cytokines. This has important implications in the potential use of CSCs in future HLHS clinical trials.
Abstract 141: Influence of the Collagen Processing Heat Shock Protein 47 on Cardiomyocyte Homeostasis and Maturation
Authors: Sur Sumon , Christalla Peter , Roa Angelica , Zimmermann Wolfram H
Published: July 2021

PURPOSE:
Heat shock protein 47 (Hsp47) is a collagen-specific molecular chaperone required for maturation of collagen type 1. Little is known about the role of Hsp47 in the heart. This study aims to investigate whether Hsp47 is important for cardiomyocyte maturation and assembly into functional syncytia.

METHODS:
We made use of recently developed 2D and 3D culture platforms to scrutinize the role of Hsp47 for cardiomyocyte maturation and function, i.e., (1) 2D model: culture of embryonic stem cell-derived cardiomyocytes (ESC-CM) on ECMs derived from both mouse embryonic wild type (WT)- and Hsp47 (-/-) fibroblasts and (2) 3D model: construction of engineered heart muscle (EHM) consisting of purified ESC-CMs with WT- or Hsp47 (-/-) fibroblasts. Cultures were investigated using confocal microscopy and isometric force measurements.

RESULTS:
ESC-CMs showed mainly immature polygonal shaped morphology and disorganized sarcomeric structures when cultured on ECM secreted by Hsp47 (-/-) fibroblasts; this morphology was in clear contrast to the mostly anisotropic cardiomyocyte appearance in control conditions. EHM with Hsp47 (-/-) fibroblasts did not develop measurable contractile forces. In contrast, WT fibroblast-supplemented EHMs contracted regularly (WT: 0.14±0.1 mN at 2.4 mM Ca2+; n=8/group). Histological analysis of Hsp47 (-/-) EHMs showed mainly rounded and underdeveloped cardiomyocytes comparable to the cardiomyocyte phenotype observed in EHMs containing only ESC-CMs, without fibroblasts.

CONCLUSIONS:
Hsp47 influences the deposition of ECM-collagen and affects cardiomyocyte morphology and functionality in our 2D and 3D culture models. Our data collectively suggest that Hsp47 may be an attractive target for the regulation of cardiac tissue homeostasis.
Abstract 147: δ-opioid Receptor Activation Increases Nrf2 Nuclear Translocation And Stabilizes Mitochondrial Membrane Potential Of Hypoxic Cells
Authors: Cao Shan , Chen Tao , Chao Dongman , Wen Guoqiang , Balboni Gianfranco , Xia Ying
Published: July 2021

Conditions that limit O2 supply for a prolonged period lead to cardiac injury. However, current strategies against hypoxic cardiac injury are limited in clinical settings. It is of utmost importance to find novel clues for protecting cells from hypoxic injury. Our work has recently shown that δ-opioid receptor (DOR) activation strongly regulates microRNA expression in the heart exposed to prolonged hypoxia, suggesting a role of DOR in cardiac adaptation to hypoxic stress. To further determine DOR's action on cell survival under hypoxia and the underlying mechanisms, we exposed HEK293t cells to hypoxia for a prolonged period and then investigated cellular viability and changes in mitochondrial membrane potential and the nuclear factor erythroid 2-related factor 2 (Nrf2), which are thought to be involved in the regulation of cell survival under hypoxia. Our data show that prolonged hypoxia (0.5% of O2 for 16-48 hours) caused serious cellular injury. DOR activation with UFP-512, a potent and specific DOR agonist, prevented the collapse of mitochondrial membrane potential and markedly attenuated cell injury in the hypoxic condition. The DOR-mediated protection was largely reversed by DOR antagonism with naltrindole. Furthermore, we observed that DOR activation increased Nrf2 translocation from cytoplasm to nucleus. The DOR-mediated cytoprotection was largely abolished by the "knock-down" of Nrf2. Our results suggest that DOR activation increases Nrf2 nuclear translocation and stabilizes mitochondrial membrane potential during the process of cellular protection against hypoxic stress.

SC and TC are equal contributors.

Supported by HD-034852, AT-004422, NSFC 81060096 and 81260204.
Abstract 153: IL-10 Accelerates Re-Endothelialization and Inhibits Post-injury Intimal Hyperplasia following Carotid Artery Denudation by Attenuating TNF-alpha-induced Endothelial Cell Dysfunction
Authors: Verma Suresh K , Krishnamurthy Prasanna , Mackie Alexander R , Vaughan Erin E , Khan Mohsin , Srikanth Garikipathy V , Ramirez Veronica , Abramova Tatiana , Misener Sol , Qin Gangjian , Kishore Raj
Published: July 2021

The association of inflammation with atherosclerosis and restenosis is now fairly well established. Restenosis, a persistent complication of percutaneous vascular interventions, is thought to be a complex response to injury, which includes early thrombus formation, neointimal growth and acute inflammation. Mononuclear phagocytes are likely participants in the host response to vascular injury, via the secretion of cytokines and chemokines, including TNF-alpha (TNF). Others and we have previously shown that IL-10 inhibits TNF and other inflammatory mediators produced in response to cardiovascular injuries. The specific effect of IL-10 on endothelial cell (EC) biology is not well elucidated. Here we report that in a mouse model of carotid denudation, IL-10 knock-out mice (IL10KO) displayed significantly delayed ReEndothelialization and enhanced neointimal growth compared to their WT counterparts. Exogenous treatment of recombinant IL-10 dramatically blunted the inflammatory cell infiltration and neointimal thickening while significantly accelerating the recovery of the injured endothelium both WT and IL10KO mice. In vitro, IL10 co-treatment reversed TNF-mediated growth arrest, EC cell cycle inhibition, EC-monocyte adhesion and EC apoptosis. At signaling level, IL-10 reduced TNF-induced activation of JNK MAP kinase while simultaneously activating PI3K/Akt pathway. Because IL-10 function and signaling are important components for control of inflammatory responses, these results may provide insights necessary to develop strategies for modulating vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.
Abstract 155: Cardiac Function And Calcium Handling in Obesity-Resistant rats
Authors: Leopoldo Andre , de Sá Felipe , Estevam Wagner , Jacobsen Bruno , Ferron Artur , da Cunha Márcia , Castardeli Edson , Campos Dijon , Cicogna Antonio , Lima Leopoldo Ana Paula
Published: July 2021

Experimental studies have demonstrated that obesity induced by different types of high-fat and/or high-energy diets also lead to myocardial dysfunction. Nevertheless, few studies have evaluated the myocardial function in obesity-resistant rats. Moreover, the mechanisms underlying the participation of calcium (Ca
2+
) handling on cardiac function in this model remain unknown. The aim of this study was to investigate in rodent model of obesity-resistant. In addition, as obesity-resistant group is fed a high-fat diet but did not become obese, this study tested whether obesity-resistant model develops cardiac abnormalities and impairment of calcium handling as obesity-prone. Male 30-day-old
Wistar
rats were fed standard (C) and alternately four palatable unsaturated high-fat diets (Ob) for 15 weeks. After experimental protocol, Ob rats consuming the unsaturated high-fat diets were ranked based on adiposity index. Rats on the unsaturated high-fat diets exhibiting the greatest adiposity index were referred to as OP, whereas those exhibiting the lowest adiposity index were referred to as OR. Obesity was determined by adiposity index and comorbidities were evaluated. Myocardial function was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic and lusitropic maneuvers. After 15 weeks, final body weight, total body fat, adiposity index, triglycerides levels and heart weight were significantly greater in OP rats than C and OR rats, however, there was no change in systolic blood pressure between groups. The C, OP and OR muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus was compromised in OP rats. In conclusion, obesity-resistant model by unsaturated high-fat diet, after 15 weeks, does not display nutritional and metabolic characteristics of obesity-prone. Furthermore, obesity-resistant does not promote cardiac abnormalities and impairment of calcium handling visualized in obesity-prone.
Abstract 163: Genetic Basis of Isoproterenol-Induced Cardiac Fibrosis
Authors: Rau Christoph D , Avetisyan Rozeta , Stein Dorit , Romay Milagros , Wang Jessica , Wang Yibin , Lusis Aldons J
Published: July 2021

Background:
Cardiac fibrosis is a common pathology in the diseased heart, which can cause a loss of elasticity and contractile dysfunction. Cardiac fibrosis is a complex process driven by many pathological triggers which involve numerous genes, pathways and cell types. Despite its importance, the genetic basis for the development of cardiac fibrosis has not been systematically explored.

Methods and Results:
We have developed a resource, the Hybrid Mouse Diversity Panel (HMDP) for high resolution GWAS and systems genetics study of quantitative traits in mice. Eight week old female mice from 80 unique inbred strains of the HMDP were given 30 ug/g/day of isoproterenol (ISO) for three weeks and cardiac fibrosis was assessed by Masson Trichrome staining which revealed a wide spectrum in the degree of fibrosis among the HMDP strains both before and after treatment. Using the Efficient Mixed Model Algorithm, we identified 13 significant or suggestive loci contributing to cardiac fibrosis, many containing numerous gene candidates. Within one of these loci, Abcc6, an orphan ABC transporter linked to the human disease pseudoxanthoma elasticum, was identified as a possible candidate for ISO-induced cardiac fibrosis. A splice-site mutation present in 19 strains of the HMDP was significantly linked to a higher degree of ISO-induced cardiac fibrosis(P=1E-4) but was not linked to increased fibrosis in untreated animals(P=0.25). Targeted genetic knockout of Abcc6 promoted ISO-induced cardiac fibrosis while reintroducing the wildtype Abcc6 allele to an genetic strain homozygous for the Abcc6 splice site mutation significantly alleviated ISO-induced cardiac fibrosis.

Conclusion:
A GWAS performed on levels of cardiac fibrosis observed in ISO treated animals using HMDP mice as model system uncovered significant genetic contributions to stress-induced cardiac fibrosis. Abcc6 is a novel gene contributing to ISO-induced cardiac fibrosis in the heart.