bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 131: PDE1 Inhibition Improves Cardiac Protein Quality Control
Authors: Zhang Hanming , Wang Xuejun "XJ"
Published: June 2021

Protein quality control (PQC) functions to minimize the level and toxicity of misfolded proteins in the cell. PQC relies on molecular chaperones and the targeted degradation of misfolded proteins. The latter is currently known to require the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). Virtually all cardiovascular diseases end up heart failure (HF), the leading cause of death of our society. UPS function insufficiency is implicated in the genesis of a large subset of HF, making cardiac PQC enhancement via promoting UPS and ALP function a promising therapeutic strategy to treat HF. Previously, we have demonstrated that stimulating protein kinase G (PKG) genetically or via inhibition of the type 5 phosphodiesterase (PDE5) improves UPS performance, facilitates the removal of misfolded proteins in cardiomyocytes and slows down the progression of cardiac proteinopathy in a transgenic mouse model (CryAB
R120G
). PKA has also been shown to enhance proteasomal function. Our preliminary studies reveal that myocardial protein levels of PDE1A, which suppresses both PKG and PKA, are remarkably elevated in the CryAB
R120G
mice. Hence we hypothesize that PDE1 inhibition (PDE1I) stimulates cardiac proteasomes via PKG and PKA activation and thereby protects against cardiac proteotoxicity. To test our hypothesis, we took advantage of a proven surrogate UPS substrate (GFPu or GFPdgn) as well as a
bona fide
misfolded protein (CryAB
R120G
) that is known to induce cardiac proteinopathy in human and mice. In cultured cardiomyocytes, PDE1 inhibitor LSN2790158 dose- and time-dependently decreased GFPu. Cycloheximide (CHX) chase assays further confirmed that PDE1I shortened the half-life of GFPu, indicative of improved UPS performance. Furthermore, PDE1I promoted the degradation of CryAB
R120G
. Our in vivo findings revealed that GFPdgn mice treated with LSN2790158 (3mg/kg, i.p.) displayed a significant reduction of myocardial GFPdgn protein but not mRNA levels. Taken together, our data strongly indicate that PDE1I improves cardiac UPS performance and PDE1 represents a potential target to treat cardiac diseases with elevated proteotoxicity.
Abstract 133: A Systems Genetics Approach to Identify Genetic Pathways and Key Drivers of Isoproterenol-induced Cardiac Hypertrophy and Cardiomyopathy in Mice
Authors: Rau Christoph D , Romay Milagros , Wang Jessica , Lusis Aldons J , Wang Yibin
Published: June 2021

Heart Failure (HF) is a complex disease involving numerous environmental and genetic factors. We previously reported a genetic analysis of HF traits in a population of inbred mouse strains treated with isoproterenol, a β-adrenergic agonist used to mimic catecholamine-driven cardiac hypertrophy. We now present a systems genetics analysis in which we have used left ventricular transcript levels from these mice to perform co-expression network modeling. We constructed gene networks composed of 8,126 genes and 20 modules using the wMICA algorithm. In the wMICA network generated from treated hearts, we identified a module with significant correlations to several HF-related phenotypic traits. Further analysis of this module showed significant over-representation of genes known to contribute to the development of HF. Using the causal modeling algorithm NEO, we identified the gene
Adamts2
as a putative master regulator of the module. We then validated the role of this gene through siRNA-mediated knockdown in neonatal rat ventricular myocytes (NRVM). Consistent with our model,
Adamts2
silencing was able to regulate the expression of the genes residing within the module as well as impairing isoproterenol-induced cell size changes
.
Our results provide a view of higher order interactions in heart failure with potential to facilitate diagnostic and therapeutic approaches.
Abstract 134: Increased Ca2+ Influx Through Cav1.2 in Vascular Smooth Muscle Cells Causes Hypertension Due to Impairment of Acetylcholine-dependent Vascular Relaxation
Authors: Zhang xiaoying , Cheng Zhongjian , Ai Xiaojie , Tang Mingxin , Hua Xiang , Houser Steven R , Navedo Manuel F , Santana Luis F , Wang Hong , Chen Xiongwen
Published: June 2021

Calcium channel plays an important role in smooth muscle contraction and relaxation and L-type calcium channel (Cav1.2) antagonist is widely used for treating hypertension. However, whether increasing Ca2+ influx into smooth muscle cells results in hypertension (through increasing smooth muscle cell contractility) or hypotension (through activating BKCa channels) is unclear. Aiming to answer this question, we established a transgenic (TG) mouse model with smooth muscle cell specific (SM22-α promoter driven) overexpression of a splicing variant of Cavβ2 subunit, Cavβ2a, tagged with GFP.

Methods and Results:
Immunostaining showed that Cavβ2a-GFP expressed in smooth muscle cells specifically and L-type Ca2+ current in smooth muscle cells from the 4
th
branch of mesenteric arteries of transgenic mice was significantly increased (5.1±0.7pA/pF in TG, n=12 vs. 2.2±0.5pA/pF in control, n=10). Telemetric blood pressure measurement showed that 24h-averaged systolic pressure was significantly increased (135.2±9.8mmHg, n=25) compared to that of that of control c57bl/6 mice (110.2±7.8mmHg, n=16). The diastolic pressure was also significantly elevated in TG mice than in control mice. Maximum contractility of the 4
th
branch mesenteric arteries measured with 40mM KCl was not different between control and TG groups. Sodium nitroprusside relaxed the phenylephrine-precontracted mesenteric artery in the same dose-dependent manner However, acetylcholine induced mesenteric artery relaxation was signficantly impaired in TG mesenteric arteries.

Conclusions:
Increasing Ca2+ influx through the L-type Ca2+ channel in smooth muscle cells causing hypertension via impairment of acetylcholine-dependent relaxation.
Abstract 211: ATF6 and the Adaptive ER Stress Response Enhances Proliferation and Viability in Mouse Cardiac Stem Cells
Authors: Stauffer Winston T , Doroudgar Shirin , Stephens Haley N , Bailey Brandi , Glembotski Christopher C
Published: June 2021

Rationale:
Cardiac stem cells (CSCs) are beneficial when administered to infarcted mouse or rat hearts. Though the mechanism of these benefits is unknown, CSC vitality likely plays a major role. Thus, investigating the factors governing CSC survival in the ischemic heart may lead to more effective therapeutic strategies. Our previous studies showed that misfolded proteins accumulate in the sarco/endoplasmic reticulum (SR/ER) of the ischemic heart. The transcription factor, ATF6, is a key component of the adaptive ER stress response because it induces genes that reduce the accumulation of misfolded proteins, improving myocyte survival during ischemic stress. While our lab has shown that, in cardiac myocytes, ATF6 is cardioprotective in the ischemic heart, neither the ER stress response nor ATF6 have been examined in CSCs. We hypothesize that ATF6 and the adaptive ER stress response are critical for optimal survival of CSCs.

Objective/Methods:
To gauge the relevance of the ER stress response in CSCs, we used MTT assays to compare the viabilities of mouse CSCs to neonatal rat ventricular myocytes (NRVM) subjected to treatments that mimic ischemic ER stress in the heart. We also assessed the effect of inhibiting ATF6 on both the ER stress response and CSC viability by using chemical inhibition of ATF6 activation or siRNA-mediated ATF6 knock down.

Results:
We found that, compared to NRVM, CSCs exhibited lower levels of adaptive ER stress response gene expression and decreased viability in response to ER stress. Thus, relative to NRVM, the adaptive ER stress response is not fully developed in CSCs. We also found that either chemical inhibition of ATF6 activation or ATF6 knock down decreased adaptive ER stress response gene expression. Strikingly, ATF6 inhibition or knockdown decreased CSC viability and cell number by as much as 70%.

Conclusions:
Thus, compared to cardiac myocytes, CSCs exhibit a reduced adaptive ER stress response and are more sensitive to ER stress, suggesting that enhancement of the ATF6-mediated adaptive ER stress response in CSCs may be a viable therapeutic approach for enhancing stem cell-mediated myocardial repair.
Abstract 218: Cardiac Side Population Cells Give Rise to Cardiomyocytes in the Adult Heart
Authors: Yellamilli Amritha , Bender Ingrid , Ren Yi , van Berlo Jop H
Published: June 2021

Therapies that enhance cardiac regeneration have the potential to improve heart failure outcomes. One strategy to enhance cardiac regeneration is by activating endogenous cardiac progenitor cells. Cardiac side population cells (cSPCs) are proposed progenitor cells that reside in the heart; however, their putative progenitor cell properties are based on cell culture data and transplantation studies performed with isolated cSPCs. To determine the endogenous regenerative potential of cSPCs
in vivo
, we generated a mouse model that harbors an Abcg2-driven, tamoxifen-inducible Cre recombinase and crossed it to a GFP reporter mice. One month after tamoxifen treatment, we obtained efficient GFP-labeling of side population cells with 47.0 ± 11.05% of bone marrow side population cells and 75.8 ± 10.87% of cSPCs. Importantly, during a one-month chase period, we observed a three-fold increase in GFP-labeled cardiomyocytes when compared to a 1-week chase period. We quantified the extent of GFP-labeling of cardiomyocytes using adult cardiomyocyte isolation, where we measured 0.8% GFP labeled cardiomyocytes after a one-month chase period. We also observed labeling of many other cell types in the heart, such as endothelial cells, smooth muscle cells, and pericytes. We are currently testing to what extent cardiac injury enhances cSPC derived cardiomyocyte formation. Using our mouse model that efficiently labels side population cells
in vivo
, we demonstrated that
cSPCs
contribute cardiomyocytes in the adult heart
in vivo
.
Abstract 219: Induced Pluripotent Stem Cells as a Potential Cell Source for Tissue Engineered Heart Valves
Authors: Yonezawa Aline L , Singh Monalisa , Safranski David , Dupont Kenneth M , Xu Chunhui , Davis Michael E
Published: June 2021

Despite recent advances in tissue engineered heart valves (TEHV), one of the major challenges is finding a suitable cell source for seeding TEHV scaffolds. Native heart valves are durable because valve interstitial cells (VICs) maintain tissue homeostasis by synthesizing and remodeling the extracellular matrix. In this study, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived into induced mesenchymal stem cells (iMSCs) using our feeder-free protocol and then further differentiated into VICs using a 3D cell culture environment. The differentiation efficiency was quantified using flow cytometry, immunohistochemistry staining, RT-PCR, and trilineage differentiation. In addition, iMSCs were encapsulated in polyethylene (glycol) diacrylate (PEGDA) hydrogels of varying stiffness, grafted with adhesion peptide (RGDS), to promote cell proliferation, remodeling, and further differentiation into VIC-like cells. VICs phenotype was characterized by the expression of αSMA, vimentin, F-actin, and the ECM production after 7, 14, and 21 days. The results demonstrated that using our feeder-free differentiation protocol, iMSCs were differentiated from iPSCs. Our iMSCs had a 99.9% and 99.4% positive expression for MSC markers CD90 and CD44, respectively. As expected, there was 0.019% expression of CD45, which is a hematopoietic marker. In addition, iMSCs differentiated into adipogenic, chondrogenic, and osteogenic. When MSC derived cells were encapsulated in PEGDA hydrogels that mimic the leaflet modulus, we observed expression of αSMA and F-actin after 7 days. Thus, the results from this study suggest that iPSCs can be a suitable cell source for TEHV by using a feeder-free differentiation approach and 3D culture.
Abstract 226: δ-opioid Receptor Activation Protects the Brain From Hypoxic-ischemic Injury by Downregulating Inflammatory Cytokines and Upregulating Nrf-2
Authors: Qiu Jie , Chao Dongman , Ding Guanghong , Xia Ying
Published: June 2021

Hypoxia-ischemia (HI) insult induces inflammatory/oxidative injury and causes neurological disability and mortality. However, there are limited therapeutic options at present. Our recent studies have shown that the δ-opioid receptor (DOR) is neuroprotective against hypoxic-ischemic stress. In this work, we asked if DOR activation protects the brain against hypoxic-ischemic encephalopathy (HIE) by regulating inflammatory cytokines and Nrf-2, a basic leucine zipper protein that regulates the expression of antioxidant proteins. Our results showed that DOR activation with UFP-512 treatment significantly decreased TNF-α, IL-6, and ICAM-1 expression (P<0.05) in the left cortex of the rat with HI injury, while DOR inhibition with naltrindole significantly increased their expression (P<0.05). In contrast, DOR activation induced an opposite effect on IL-10 expression (P<0.05) and had no appreciable effect on IL-18 (P>0.05). In the control group, no significant difference was detected after the vehicle treatment. Furthermore, we investigated the role of Nrf-2 in the DOR neuroprotection since our recent data suggest that DOR signaling promotes the expression and relocation of Nrf2 and thus induces cytoprotection. HI significantly increased nuclear Nrf-2 protein, while DOR activation by pretreatment with UFP-512 before HI further elevated the level of nuclear Nrf-2 protein by 70% (P<0.05) in the cortex. On the other side, DOR inhibition by the pretreatment with naltrindole completely abolished the HI- and DOR-induced increase in nuclear Nrf-2 protein. In the control, the vehicle treatment did not have any significant effect on nuclear Nrf-2 protein. These first data suggest that DOR activation protects the brain from HIE by downregulating the expression of inflammatory cytokines and upregulating Nrf-2 activity.

This work was supported by NIH (AT-004422), Memorial Hermann’s Foundation and Vivian L Smith Neurologic Foundation. GD was supported by 973 Program (2012CB518502) and NSFC (81574053 & 81590953) of China.
Abstract 229: Increased Afterload Following Myocardial Infarction Promotes Conduction-dependent Arrhythmias That are Unmasked by Hypokalemia
Authors: Motloch Lukas J , Ishikawa Kiotake , Xie Chaoqin , Hu Jun , Aguero Jaume , Fish Kenneth R , Hajjar Roger J , Akar Fadi G
Published: June 2021

Background-and-Objectives:
Arterial hypertension worsens the prognosis of post-myocardial infarction (MI) patients. Its impact on electrophysiological function, however, is unknown. To address this, we developed a new porcine model of post-MI remodeling with chronically increased afterload (MI/iAL). We compared the electrophysiological substrate of this model to that of a standard model of MI without increased afterload and to naïve pigs (Sham).

Methods-and-Results:
Yorkshire pigs underwent aortic banding 1-month following MI, and were allowed to survive with increased afterload for another 2-months. MI/iAL but not MI or Sham animals exhibited widespread interstitial fibrosis and increased profibrotic gene expression with no major changes in Cx43 expression or localization. Arterially-perfused wedges from MI/iAL but not MI or Sham pigs were prone to pacing-induced VT/VF when challenged with hypokalemia. Investigation of the electrophysiological substrate revealed the dependence of these arrhythmias on impaired conduction and not repolarization. When excitability was reduced with fast pacing and hypokalemia, MI/iAL preparations exhibited conduction velocity (CV) slowing to critical levels never attained in sham or MI preparations before their eventual loss of capture. A steep negative slope of rate-dependent CV slowing was observed exclusively in MI/iAL preparations consistent with a higher safety factor for propagation of slow wavefronts before onset of VT/VF.

Conclusions:
MI/iAL animals exhibit widespread interstitial fibrosis, a profibrotic gene expression program and marked susceptibility to arrhythmias that are unmasked by hypokalemia. Structural remodeling in MI/iAL produces a substrate that promotes the successful propagation of critically slow wavefronts leading to reentrant arrhythmias.
Abstract 241: MDM2 E3 Ligase-mediated Ubiquitination and Degradation of HDAC1 in Vascular Calcification
Authors: Kook Hyun , Kwon Duk-Hwa , Eom Gwang Hyeon , Min Hyun-Ki , Yoon Somy , Kim Yong Sook , Ahn Youngkeun
Published: June 2021

Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.
Abstract 242: Priming for Action: Interventions That Promote IKs-Channel Opening Restore Defective cAMP-Dependent Upregulation in Long-QT1 Syndrome
Authors: Volders Paul G , Spätjens Roel L , Seyen Sandrine R , Menini Gabriele , Moreno Vadillo Cristina
Published: June 2021

Introduction:
Patients with the KCNQ1 mutation A341V show a high incidence of sympathetically-triggered ventricular tachyarrhythmias and sudden cardiac death. A341V decreases basal IKs and disrupts cAMP-dependent upregulation. The latter can be rescued by the phosphomimetic substitution KCNQ1 S27D. We hypothesize that A341V imposes a different conformational state of the IKs channel, which prevents full phosphorylation of KCNQ1 S27 and therefore loss of upregulation. We have now examined whether different strategies that promote channel opening can rescue cAMP-dependent upregulation.

Methods:
Chinese hamster ovary cells were transfected with wild type (WT) and heterozygous KCNQ1 A341V, with KCNE1 and Yotiao. In the absence and presence of cAMP/okadaic acid (OA), we examined IKs responses to (1) the KCNQ1 I346-K358 short peptide (shown to unlock the closed state of IKs through its binding to the endogenous S4-S5L of KCNQ1), (2) the double mutant A341V/L353K (L353K was previously shown to promote constitutive current), and (3) the channel activator ML277 (1 μmol/L).

Results:
As previously shown, WT but not A341V IKs was upregulated by intrapipette cAMP/OA. The unlocking effect of the I346-K358 peptide and the constitutive conduction by the co-presence of L353K facilitated mutant IKs enhancement by cAMP/OA (+61% and +71%, respectively), along with a -10 mV shift of the IV curve. Similar results were found for the IKs activator ML277. In baseline conditions, ML277 increased both WT and A341V IKs by approximately 60%. The IKs response to ML277 in cells stimulated with cAMP/OA was amplified more than twofold in WT, but strikingly also in A341V channels. This larger ML277 effect is thus likely explained by an augmented cAMP-dependent regulation of A341V IKs.

Conclusions:
Pharmacological and molecular interventions that promote IKs-channel opening rescue (at least partly) from defective cAMP-dependent upregulation by the hot spot mutation A341V. Our results suggest novel therapeutic strategies to restore the beta-adrenergic control of repolarization in long-QT1 syndrome.
Abstract 263: A Decline in Kidney-derived Protein Klotho Contributes to Aging-related Heart Failure
Authors: Sun Zhongjie , Chen Kai
Published: June 2021

Background & Objective:
The heart function declines in the aged population. At age 80 years, LV contractile function is less than half of what it was at age 20 years. Aging is a recognized risk factor for heart diseases. For instance, the prevalence of heart disease increases with age. The mortality from heart diseases is higher in the aged than in the young population. Klotho is a recently discovered anti-aging gene that is primarily expressed in kidneys. The secreted Klotho is released into blood. The objective of this study is to investigate if a decline serum Klotho levels contributes to aging-related heart failure.

Method & Results:
The results showed that ejection fraction, stroke volume and cardiac output were decreased in aged mice (24 months)
vs.
the adult mice (10 months), indicating that aging impairs heart function. The serum level of Klotho was decreased significantly in aged mice. Interestingly, daily injection of recombinant Klotho protein rescued the aging-related decline in heart function. Mutation of Klotho gene (

KL
-/-

)
resulted in heart failure which was associated with a significant increase in serum phosphate levels (hyperphosphatemia). Low phosphate diet delayed but did not prevent the development of heart failure in male

KL
-/-

mice, suggesting that hyperphosphatemia partially contributes to Klotho deficiency-induced heart failure. Unfortunately, low phosphate diet failed to improve heart failure in female

KL
-/-

mice. Interestingly, administration of estrogen decreased hyperphosphatemia and delayed the development of heart failure in

KL
-/-

although it did not prevent Klotho deficiency-induced heart failure. Therefore,

Conclusion:
Klotho deficiency contributes to aging-related heart failure. Hyperphosphatemia accelerated the development of Klotho deficiency-induced heart failure. Administration of recombinant Klotho protein is an effective therapeutic strategy for heart aging. (supported by NIH R01 HL118558, AG049780, DK093403).
Abstract 269: Miofibroblast Tgf-beta Signaling in a Proteotoxic Mouse Model
Authors: Bhandary Bidur , Meng Qinghang , Osinska Hanna , Shay-Winkler Kritton , Gulick James , Bhuiyan Md. Shenuarin , Robbins Jeffrey
Published: June 2021

Introduction:
Transforming Growth Factor Beta (TGFβ) is an important cytokine in mediating the fibrogenic response and, in particular, cardiac fibrosis. Extensive fibrosis accompanies the cardiac remodeling that occurs during development of the protein conformation-based disease caused by cardiomyocyte-specific expression of a mutant, small, heat shock-like protein and chaperone, aB crystallin (CryABR120G). During the onset of fibrosis, fibroblasts are activated to the so-called “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that TGFβ signaling processes that result in significant cardiac fibrosis in a mouse model of proteotoxic heart disease are mediated by cardiac fibroblasts, rather than cardiomyocytes. Here, we have partially ablated TGFβ signaling only in cardiac myofibroblasts to observe if cardiac fibrosis is reduced.

Aims and Methods:
The objective of this study was to understand the contributions of fibroblast-derived TGFβ signaling to the development of cardiac fibrosis in a proteotoxic mouse model that results in significant cardiac fibrosis. To test the hypothesis we partially deleted the myofibroblast specific canonical and non-canonical signaling by crossing

CryAB
R120G

mice with
Tgfbr1
or
Tgfbr2
floxed mice. The double transgene containing mice were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter. Echocardiography, Masson’s Trichome staining, PCR arrays, IHC and western blots were performed to characterize the fibrotic progression in

CryAB
R120G

transgenic mice.

Results:
We observed that myofibroblast-targeted partial knockdown of
Tgf
βr1
signaling prolonged survival, modestly reducing fibrosis and improving cardiac function
.
Similarly,
Tgf
βr2
partial knockdown prolonged survival, modestly reducing fibrosis without improving cardiac function during fibrosis development in CryAB
R120G
mice.

Conclusion:
These findings suggest that, in a model of proteotoxic heart disease, myofibroblast based TGFβ signaling in the heart may contribute to cardiac hypertrophy/dysfunction but cannot account entirely for the fibrotic response.
Abstract 27: Interplay of Prostaglandin E2 and Major Histocompatibility Comeplex-II Determines Immunoprivilege of Allogeneic Mesenchymal Stem Cells in the Ischemic Heart
Authors: Sareen Niketa , Sequiera Glen Lester , Abu Al-Rub Ejlal , Moudgil Meenal , Dhingra Sanjiv
Published: June 2021

The unique immunomodulatory properties of bone marrow derived allogeneic mesenchymal stem cells (MSCs) make them excellent candidate cell type for clinical applications. The outcome of preclinical studies and initial clinical trials suggested that after transplantation to the infarcted heart, allogeneic MSCs were able to improve cardiac function. However the long term fate of transplanted cells in these studies was not determined. We recently reported that allogeneic MSCs improved heart function after implantation in the infarcted heart. However, late after implantation, transplanted cells lost their immunoprivilege, and were rejected. Therefore we started extensive investigations to study the mechanisms of rejection. Our data demonstrate that MSC immunoprivilege was found to be mediated by prostaglandin E2 (PGE2), the levels of this soluble factor decreased in rat MSCs after exposure to hypoxia/ischemic conditions which was associated with loss of immunoprivilege and rejection of transplanted allogeneic MSCs. We observed increased cytotoxicity in hypoxic MSCs caused by allogeneic T cells in the
in vitro
co-culture. Furthermore, blocking PGE2 biosynthesis in normoxic MSCs increased the immunogenicity of MSCs. MSCs immunoprivilege is reported to be established by the absence of major histocompatibility complex class II (MHC-II) molecules. Our data suggest that MHC-II expression increased in MSCs after exposure to hypoxia. PGE2 treatment of hypoxic MSCs decreased MHC-II expression and preserved their immunoprivilege. In a rat myocardial infarction (MI) model, maintaining PGE2 levels with a biodegradable, temperature sensitive hydrogel in the infarct region prevented rejection of transplanted allogeneic MSCs and restored cardiac function after five weeks of cell transplantation. In conclusion, immunoprivilege of allogeneic MSCs was maintained by PGE2 mediated regulation of MHC-II levels, exposure to hypoxia/ischemia decreased PGE2 and increased MHC-II levels that was associated with loss of immunoprivilege and rejection of MSCs. Maintaining PGE2 levels preserved immunoprivilege and restored cardiac function after an MI.
Abstract 272: miRNA-7 Leads to Enhanced Fibrosis in the Heart
Authors: Gupta Manveen , Wang Xi , Martelli Elizabeth , Sathyamangla Prasad
Published: June 2021

MicroRNA-7 (miR-7) is known to target epidermal growth factor receptors (EGFRs) in oncogenic cells however, less is known about its role and function in the heart. Our studies have identified reciprocal expression pattern for ERBB2 (a member of EGFR family) in the context of miR-7 expression in human heart failure and in mice post-TAC (Transverse Aortic Constriction). To directly determine the role of miR-7 in the heart, we generated transgenic (Tg) mice with cardiomyocyte-specific overexpression of miR-7 (miR-7 Tg). miR-7 Tg mice are characterized by marked loss in ERBB2 expression compared to their wildtype littermate controls. Also, miR-7 Tg mice have age-dependent deterioration in cardiac dysfunction and is associated with dilation as measured by echocardiography (3 months - 60% FS, 6 month -52% FS and 12 months - 24%FS) and yet, they survive over 18 months as assessed by Kaplan-Meier curves. To investigate whether pathological stress would accelerate the deterioration in cardiac function, miR-7 Tg mice were subjected to TAC for two weeks. In contrast to the wild type littermates that showed significant hypertrophic response, miR-7 Tg mice have accelerated cardiac dysfunction and dilation following two weeks of TAC. Histological analysis shows increased collagen deposition in miR-7 Tg mice which is further accelerated following after TAC compared to their sham controls. These observations suggest that ERBB2 expression in the cardiomyocytes may play a critical role in delaying the pro-fibrotic response post-TAC by supporting an adaptive hypertrophic response in response to stress. Our study will discuss the mechanisms of increased fibrosis observed in miR-7 Tg mice subjected to TAC.
Abstract 273: T cell Sphingosine 1-Phosphate Receptor 1 Signaling Possesses Intrinsic Anti-fibrotic Effects in Murine Heart
Authors: Jin Zhuqiu , Abdullah Chowdhury S
Published: June 2021

T lymphocyte infiltration into myocardium has been observed in patients with myocardial infarction and diabetic cardiomyopathy. T cell trafficking from primary lymphoid organs into blood depends on S1P receptor 1 (S1P
1
) activation and S1P concentration gradients. However, effects of T cell S1P
1
signaling activation on cardiac fibrosis and function remain unknown. Mice with conditional knockout of T cell S1P
1
were generated by cross-bred of S1P
1
loxp-loxp
mice with
Lck-Cre
transgenic mice. T cell S1P
1
mutant mice (TS1P
1
KO) and wildtype littermate (WT) were utilized. To explore the effect of T cell S1P
1
on cardiac fibrosis and myocardial function, additional groups with reconstitution of T cells containing S1P
1
were conducted by injection of freshly prepared T cells from WT into TS1P
1
KO. Body weight, glucose levels, and behavior change were recorded twice a week. After 11 weeks, Langendorff heart perfusion was prepared. Cardiac contractile force was recorded. Trichrome staining was used to evaluate the extent of cardiac fibrosis. CD4, CD8, and Foxp3 regulatory T cells in the blood and spleen were quantified with flow cytometry. Expression of TGF-beta and CD34 in mouse myocardium was determined by using immunohistochemistry. Comparing with WT control, TS1P
1
KO mice exerted increased cardiac fibrosis and low cardiac contractile force (P<0.05). TGF-beta expression and CD34 stained cells were significantly enhanced in TS1P
1
KO mice. Both CD4 and CD8 T cells in the blood and spleen were reduced significantly in TS1P
1
KO. Reconstitution of T cells with S1P
1
into TS1P1KO attenuated cardiac fibrosis and myocardial dysfunction. In conclusion, T cell S1P
1
signaling plays a critical role in anti-fibrosis and cardioprotection.
Abstract 274: Expression and Phosphorylation States of Eukaryotic Elongation Factor 2 (EEF2) and EEF2 Kinase in Cardiomyocytes From Hypertrophy Models
Authors: Kameshima Satoshi , Okada Muneyoshi , Ikeda Shiro , Watanabe Yuki , Yamawaki Hideyuki
Published: June 2021

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K, also known as calmodulin (CaM)-dependent protein kinase III) is regulated by both CaM-dependent and -independent mechanisms. Activated eEF2K phosphorylates and inactivates a specific substrate, eEF2. eEF2 activation facilitates protein translation. It is recognized that increased protein synthesis is one of the primary factors for cardiomyocyte hypertrophy. In fact, angiotensin II, which induces cardiomyocyte hypertrophy, was reported to facilitate eEF2 dephosphorylation (activation) and protein synthesis in rat isolated cardiomyocytes. We have previously demonstrated that protein expression of eEF2K was increased specifically in left ventricles (LV) of spontaneously hypertensive rats (SHR). However, expression and phosphorylation states of eEF2K and eEF2 in LV of other cardiac hypertrophy models are unknown. The aim of this study was to explore it. Male C57BL/6NJcl mice and Wistar rats received transverse aortic constriction (TAC) and isoproterenol (5 mg/kg; ISO) injection, respectively, which induced cardiac hypertrophy. After 3 and 28 days from TAC operation and 7 days from ISO injection, LV were isolated and used for Western blotting (WB) and immunohistochemistry (IHC). Echocardiography was done in TAC mice before LV isolation. In TAC-induced hypertrophied LV (3 days), eEF2K expression was significantly increased (p<0.01 vs. SHAM) and its phosphorylation at Ser366 was significantly decreased (p<0.05 vs. SHAM). Consistently, eEF2 phosphorylation was significantly increased (p<0.01 vs. SHAM). In LV from ISO rats, eEF2K phosphorylation at Ser366 was significantly decreased as determined by WB (p<0.01 vs. control). In addition, eEF2K- and phosphorylated eEF2-positive cardiomyocytes were increased as determined by IHC. These changes were also confirmed in LV from SHR. At 28 days after TAC, fractional shortening was significantly decreased (from 56.6±1.6% to 44.4±2.3%, p<0.01). Interestingly, eEF2 phosphorylation in LV was significantly decreased (p<0.05 vs. SHAM). The present results suggest the potential role of eEF2K/eEF2 signals in the pathogenesis of cardiac hypertrophy/failure.
Abstract 287: Obesity Accelerates T Cell Senescence
Authors: Shirakawa Kohsuke , Yamamoto Tsunehisa , Enso Jin , Fukuda Keiichi , Sano Motoaki
Published: June 2021

Obesity is associated with accelerated biological aging and predisposes to the early onset of aging-related diseases. We investigate how obesity causes aging. Diet-induced adiposity was associated with an accumulation of programmed cell death-1 (PD-1)+ memory phenotype (MP) CD4+ T cells having features of cell senescence in visceral adipose tissue (VAT). The PD-1+ MP CD4+ T cells stimulated macrophage infiltration and promoted M1 macrophage polarization, while reducing regulatory T cells in VAT. High fat diet (HFD) induced conversion of naïve T cells to PD-1+ MP T cells in a macrophage- and B cell-dependent manner. Immunological depletion of PD-1+ T cells attenuated adipose inflammation and reversed insulin resistance in HFD-induced obese mice, while adoptive transfer of PD-1+ MP CD4+ T cells in VAT induced adipose inflammation and insulin resistance in non-obese mice. We propose that T cell senescence originating in VAT contributes to the mechanism of how obesity causes aging.
Abstract 292: The Role of the ATF6 Branch of the ER Stress Response in the Endocrine Heart
Authors: Blackwood Erik A , Glembotski Christopher C
Published: June 2021

Rationale:
Atrial natriuretic peptide (ANP) is stored in the heart in large dense core granules of atrial myocytes as a biologically inactive precursor, pro-ANP. Hemodynamic stress and atrial stretch stimulate coordinate secretion and proteolytic cleavage of pro-ANP to its bioactive form, ANP, which promotes renal salt excretion and vasodilation, which, together contribute to decreasing blood pressure. While the ATF6 branch of the ER stress response has been studied in ventricular tissue mouse models of myocardial ischemia and pathological hypertrophy, roles for ATF6 and ER stress on the endocrine function of atrial myocytes have not been studied.

Objective/Methods:
To address this gap in our knowledge, we knocked down ATF6 in primary cultured neonatal rat atrial myocytes (NRAMs) using a chemical inhibitor of the proteolytic cleavage site enabling ATF6 activation and siRNA and measured ANP expression and secretion basally and in response to alpha- adrenergic agonist stimulation using phenylephrine. We also compared the ANP secretion from wild- type mice and ATF6 knockout mice in an ex vivo Langendorff model of the isolated perfused heart.

Results:
ATF6 knockdown in NRAMs significantly impaired basal and phenylephrine-stimulated ANP secretion. ATF6 knockout mice displayed lower levels of ANP in atrial tissue at baseline as well as after phenylephrine treatment. Similarly, in the ex vivo isolated perfused heart model, less ANP was detected in effluent of ATF6 knockout hearts compared to wild-type hearts.

Conclusions:
The ATF6 branch of the ER stress response is necessary for efficient co-secretional processing of pro-ANP to ANP and for agonist-stimulated ANP secretion from atrial myocytes. As ANP is secreted in a regulated manner in response to a stimulus and pro-ANP is synthesized and packaged through the classical secretory pathway, we posit that ATF6 is required for adequate expression, folding, trafficking, processing and secretion of biologically active ANP from the endocrine heart.
Abstract 293: MicroRNA-125b-5p Protect the Mouse Heart from Ischemic Injury by Repressing Pro-apoptotic Bak1 And Klf13 in Cardiomyocytes
Authors: Broskova Zuzana , Park Kyoung-mi , Wang Yongchao , Kim Il-man
Published: June 2021

Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs), small non-coding RNAs that post-transcriptionally regulate target genes. For example, miR-125a is up-regulated in patients with heart failure (HF), while miR-125b is down-regulated in patients with end-stage dilated cardiomyopathy (DCM) and ischemic DCM. Circulating levels of these two miRs have been recently proposed as potential biomarkers of HF. We previously showed that β1-adrenergic receptor-mediated cardioprotective signaling through β-arrestin1 stimulates the processing of miR-125a and miR-125b in mouse heart (Figure A-C). Here, we hypothesize that these two miRs might confer cardioprotection against ischemic injury. Using cultured cardiomyocyte (CM) and
in vivo
approaches, we show that these miRs are ischemic stress-responsive protectors against CM apoptosis. CMs lacking miR-125a or miR-125b have an increased sensitivity to stress-induced apoptosis, while CMs overexpressing miR-125a or miR-125b have increased phospho-AKT pro-survival signaling. Moreover, we demonstrate that loss-of-function of miR-125b in mouse heart causes abnormalities in cardiac structure and function after myocardial infarction. The cardioprotective roles of the two miRs during ischemic injury are in part attributed to direct repression of the pro-apoptotic genes
Bak1
and
Klf13
in CMs (Figure D). In conclusion, these findings reveal pivotal roles for miR-125a and miR-125b as important regulators of CM survival during cardiac injury.
Abstract 297: The Oracle™ Device: A New Snare for Removal of Thrombus
Authors: Heuser Richard R
Published: June 2021

Stroke is a leading cause of serious disability and death worldwide. Randomized trials have shown benefits of mechanical thrombectomy compared to usual cases with intravenous thombolysis. A simple intuitively obvious device for clot removal would be ideal.

To deal with thrombus removal, we introduce the ORACLE™ snare. This snare pictured has the ability to be placed through an .014 system to remove thrombus or other foreign material throughout the body (Figure 1). The Oracle Retrieval Device is composed of three primary parts: an outer sheath tube, a core wire, and 4 capture loops. The 180cm device has loop sizes that range from 2 - 6 millimeters in diameter (Figure 2). There is an ABS plastic operating handle on the proximal end which is used to direct the device as well as to lock the capture loops in the expanded and retracted positions.

Utilizing the ORACLE™ snare, we have successfully removed thrombus in a canine model (3 out of 3 removed successfully) and bovine model (2 out of 2 removed successfully) with minimal loss of endothelial cells (Figure 3).

With the emergence of dedicated thrombus removal devices for stroke care, more challenges have occurred in treating smaller vessels. Simple intuitively obvious devices available in all carotid and stroke centers as well as centers performing coronary interventions would be ideal. The ORACLE™ may be one approach to remove thrombus and can be placed through .014 compatible catheters.
Abstract 301: Initial Increase of
Klf5
and
Ppara
Expression After Myocardial Ischemia/Reperfusion in Mice Appears to be Critical for Survival
Authors: Pol Christine J , Valenti Mesele-Christina , Schumacher Sarah M , Yuan Ancai , Gao Erhe , Goldberg Ira J , Koch Walter J , Drosatos Konstantinos
Published: June 2021

Krüppel-like factors (KLF) have important roles in metabolism. We previously found that KLF5 is a positive transcriptional regulator of peroxisome proliferator-activated receptor α (
Ppara)
, a central regulator of cardiac fatty acid oxidation (FAO). Mice with cardiomyocyte-specific
Klf5
ablation
(
α

MHC-Klf5
-/-
)

had reduced cardiac
Ppara
expression and FAO. At age 6-12 months these mice develop distinct cardiac dysfunction. The role of PPARα activation in I/R injury is unclear as both beneficial and detrimental effects have been reported. We aimed to study if
Ppara
expression changes during I/R are driven by KLF5 and explore its protective or detrimental role. Wild type mice were subjected to
in vivo
I/R or sham surgery. I/R resulted in an initial increase in
Ppara
, and its target gene pyruvate dehydrogenase kinase 4 (
Pdk4)
mRNA after 2h reperfusion, followed by decreased expression after 24h reperfusion. The
Ppara
expression is associated with parallel changes in cardiac
Klf5
mRNA expression. Concurrent, there was a decrease of cardiac FAO-related genes carnitine palmitoyl-transferase 1β (
Cpt1b),
very long chain acyl-CoA dehydrogenase
(Vlcad),
and acyl-CoA oxidase (
Aox)
in mice with I/R. To define the cell type causing the temporal changes in
Klf5
and
Ppara
after I/R we isolated primary cardiomyocytes and fibroblasts. Our data suggest a similar effect in primary isolated cardiomyocytes only.
Klf5
mRNA expression is increased after 2 hour hypoxia and normalized after 4 hour re-oxygenation in cardiomyocytes, whereas there are no changes after hypoxia/normoxia in fibroblasts. To assess the importance of cardiomyocyte KLF5 in I/R we used
α

MHC-Klf5
-/-

mice. Interestingly, despite reduced FAO, 7 month old
αMHC-Klf5
-/-
mice subjected to I/R had a marked increase in mortality; 4 of 7
αMHC-Klf5
-/-
mice died within the first 24h of reperfusion while no mortality was observed in age-matched wild type mice that underwent I/R. In conclusion, I/R is associated with an increase in
Klf5
and
Ppara
in the first hours of reperfusion followed by a decrease in
Klf5
and
Ppara
, likely accounted for by cardiomyocytes. Increased mortality for
α

MHC-Klf5
-/-

mice with I/R injury suggests that the initial increase may be an adaptive response that is critical for survival.
Abstract 308: Differential Regulation of Alpha1a-adrenergic Receptor and its Genetic Variant Signaling by Novel Spinophilin-RGS2 Signalosome in Human Cells
Authors: Babaeva Ekaterina , Gradinaru Irina , Schwinn Debra A , Oganesian Anush
Published: June 2021

α
1-
adrenergic receptors (α
1a
, α
1b
, α
1d
), members of G protein-coupled receptor (GPCR) family, regulate stress-facilitated changes in the cardiovascular (CV) system and human blood pressure (BP) via smooth muscle cell (SMC) proliferation and vasoconstriction. The α
1a
AR is the predominant subtype in human heart and resistance vessels. The α
1a
AR G247R (247R) genetic variant was discovered in a patient with severe hypertension and in a cohort of patients enriched for CV disorders. We reported a unique coupling of this genetic variant to βarrestin/MMP/EGFR transactivation pathway in rat fibroblasts, cardiomyoblasts and vascular SMCs leading to hyperproliferation and hypertrophy.

Here we present that differential interaction of 247R or α
1a
AR (WT) with scaffolding/regulatory proteins mediate unique signaling of 247R. Physiological levels of 247R but not WT upregulate endogenous levels of scaffolding protein spinophilin (SPL) in rat SMCs and cardiomyoblasts. SPL knockdown or overexpression of RGS2 (negative
R
egulators of
G
-protein
S
ignaling) inhibits 247R-mediated hyperproliferation. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that SPL binds RGS2 and prevents 247R-RGS2 interaction.

To determine if 247R hyperproliferation is in effect in human cells, we generated human iPSCs from primary fibroblasts and differentiated them to SMCs with characteristic morphology and expression of SMC markers (RT-PCR at various passages). Consistent with our findings in rat cells, expression of 247R but not WT in hiPSC-derived SMCs triggered ~70% increased proliferation. Agonist treatment resulted in increased hypertrophy in 247R cells compared with WT.

Co-immunoprecipitation from HEK293 cells expressing HA-α
1
ARs and full length Myc-SPL or Flag-RGS2 revealed that SPL exhibits ~2-fold stronger interaction with WT than with 247R. Consequently, SPL recruits RGS2 and inhibits receptor signaling. In contrast, weaker SPL-247R interaction results in diminished RGS2 inhibitory effect allowing hyperproliferation of 247R cells.

Thus, we identify novel SPL/RGS2 signalosome responsible for α
1a
AR signaling in human cells and identify SPL as a potential novel target for treatment of α
1
AR-mediated human CV disorders.
Abstract 309: Deletion of a Specific Type-I Transforming Growth Factor-beta Receptor Attenuates Thoracic Aortic Aneurysm Development
Authors: Stroud Robert E , Lieser Sarah L , Franklin Adam N , Nadeau Elizabeth K , Mukherjee Rupak , Ikonomidis John S , Jones Jeffrey A
Published: June 2021

Introduction:
Pathological remodeling with thoracic aortic aneurysms (TAA) occurs as a result of an imbalance between deposition and degradation of the extracellular matrix. TGF-β is a soluble peptide growth factor that signals through a heteromeric receptor complex involving two type-I receptors (either TGF-βR1 or ALK-1) to regulate matrix homeostasis. Alterations in type-1 TGF-β receptor abundance are coincident with TAA formation, suggesting matrix regulation shifts from a TGFβR1 to an ALK-1 mediated pathway. This study tested the hypothesis that ALK-1 signaling is required for TAA development.

Methods and Results:
TAAs were induced (periadventitial CaCl
2
: 0.5 M, 15 mins) in TGF-βR1- (TGF-βR1
+/-
), and ALK-1
+/-
deficient mice, and compared to wild-type littermate controls. Baseline aortic diameters (pre-procedural) were similar in TGF-βR1
+/-
(795±42 μm), ALK-1
+/-
(712±46 μm), and control animals (744±52 μm TGF-βR1
+/-
line; 735±22 ALK-1
+/-
line). Mice were randomized for terminal study (biochemistry, histology) at 2, 4, 8, or 16 weeks (7-10 mice/group, M/F) post-TAA induction. The change in aortic diameter was computed for each mouse as a percent increase from its own baseline measurement. Aortic dilatation was observed in all groups at 2 weeks post-TAA induction and continued to increase through 16-weeks. However, the change aortic dilation over time was attenuated in the ALK-1
+/-
deficient mice vs. littermate control (Figure).

Conclusions:
These results demonstrate that signaling through the ALK-1 receptor is necessary for TAA development, and suggest that therapeutic strategies aimed at ALK-1 inhibition may reduce or prevent the clinical progression of TAA.