bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P138: PTEN Mediates Post--Myocardial Infarction Remodeling
Authors: Parajuli Nirmal , Yuan Yuan , Bedja Djahida , Cai Zheqing P
Published: July 2021

Background-
Following myocardial infarction (MI), innate immunity is activated, leading to cytokine production and initiation of inflammation. Interleukin (IL)-10 has been reported to attenuate post-MI remodeling by inhibiting expression of tumor necrosis factor (TNF)-α. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) plays an important role in myocardial ischemia-reperfusion injury. It is not known whether PTEN regulates cytokine production and cardiac remodeling. Here, we have investigated the hypothesis that PTEN mediates post-MI remodeling by inhibiting IL-10.

Methods and Results-
MI was induced in wildtype (WT) mice and
Pten
heterozygous (HET) mice. In a separate experiment,
Pten
adenoviruses or empty viruses were injected into the peri-infarct area of WT mice. Left ventricular (LV) anatomical and functional changes were assessed by echocardiography. Mononuclear cell infiltration was measured by histology. TNF-α, IL-10, matrix metalloproteinase (MMP)-2 and MMP-9 protein levels were analyzed in the peri-infarct area. At the end of the experiments, LV end-diastolic diameter (LVEDD) was decreased and fractional shortening (FS) was increased in HET mice compared with WT mice (LVEDD: 4.13±0.22 vs. 5.42±0.26 mm, p<0.01; FS: 21.9±2.6 vs. 11.5±1.1 %, p<0.001). Moreover, heart weight and infarct size were decreased in HET mice compared with WT mice. However, FS was decreased and LV rupture (LVR) was increased in
Pten
adenovirus-treated mice compared with empty vector-treated mice [FS: 8.2±1.1 vs. 21.5±3.2 %, p < 0.01; LVR: 100% (16/16) vs. 33% (3/9)]. Mononuclear cell infiltration was attenuated in HET mice and worsened in
Pten
adenovirus-treated mice. IL-10 protein levels were upregulated and TNF-α, MMP-2, and MMP-9 protein levels were downregulated in HET mice. The opposite effects were found in
Pten
over-expressing hearts.

Conclusions-
PTEN downregulation increases IL-10 production and inhibits inflammatory responses and attenuates cardiac remodeling; conversely, PTEN over-expression inhibits IL-10 expression and increases cardiac injury. Therefore, our studies demonstrate that PTEN mediates post-MI remodeling, at least in part, by regulating IL-10 production.
Abstract P141: Gender Differences in the Development of Severe Pulmonary Hypertension and Right Ventricular Dysfunction in Apolipoprotein E--Deficient Mice Are Eliminated with Increasing Age
Authors: Partow-Navid Rod , Umar Soban , Matori Humann , Iorga Andrea , Fogelman Alan M , Navab Mohamad , Eghbali Mansoureh
Published: July 2021

Apolipoprotein E (ApoE) is a multifunctional protein and its deficiency leads to the development of atherosclerosis in mice. Patients with pulmonary hypertension (PH) have reduced expression of ApoE in lung tissue. ApoE is known to inhibit endothelial and smooth muscle cell proliferation and has anti-inflammatory and anti-platelet aggregation properties. Young ApoE deficient mice have been shown to develop high fat diet-induced PH in a gender specific manner. Estrous cyclicity peaks at 7–8 months and declines by 9 months of age in mice. Here we investigated the effects of monocrotaline (MCT) on young and middle-aged ApoE deficient mice.Middle-Aged (MA) (11–12 month old) male (n=4) and female (n=4) and young (7–8 month old) male (n=5) and female (n=5) ApoE deficient mice were injected with a single intraperitoneal dose of MCT (60 mg/kg). Mice were closely monitored for ∼4 weeks with serial echocardiography for cardiopulmonary hemodynamic assessment. Direct cardiac catheterisation was performed terminally to record peak systolic right ventricular pressure (RVP). RV, LV, IVS and lung tissue was dissected and weighed. Trichrome staining and histochemical analyses were performed. At ∼4 weeks after MCT, MA male and female and young male mice developed severe PH (RVP: MA male=64±5 mmHg, MA female=71±4 mmHg, young male=60±5 mmHg, p=n.s between all the groups) whereas young females developed significantly less severe PH (RVP: 37±5 mmHg, P<0.05 vs. MA male and female, and young male). MA male and female and young male mice developed severe RV dysfunction (RV ejection fraction (RVEF): MA male=31±2%, MA female=28±4%, young male=36±1%, p=n.s between all the groups) whereas young females showed significantly better RV function (RVEF: 43±2%, P<0.05 vs. MA male and female, and young male). MA male and female mice also developed more severe RV hypertrophy (RV/LV+Septum, MA male=0.49, MA female=0.53, young female=0.39). MA male and female mice also manifested increased peripheral pulmonary artery muscularization and pulmonary fibrosis. Interestingly, the gender differences witnessed between young ApoE deficient male and female mice in the development of severe PH and RV dysfunction are abolished as the mice increase in age.
Abstract P162: Granulocyte Colony-Stimulating Factor Administration Reduced the Inducibility of Ventricular Tachyarrhythmias in Cardiac Hypertrophied Rats Through Phosphorylated Connexin-43
Authors: Shimojo Nobutake , Machino Takeshi , Xu Donzhu , Kimura Taizo , Koshitsuka Rumi , Kameda Yuri , Sakai Satoshi , Aonuma Kazutaka
Published: July 2021

Introduction:
Cardiac hypertrophy is an independent risk factor for sudden cardiac death from ventricular tachyarrhythmias (VT). Granulocyte colony-stimulating factor (G-CSF) has recently been reported to suppress VT after myocardial infarction by modulating the function of gap junctions between cardiomyocytes via maintaining Connexin-43 (Cx43). We hypothesized that the G-CSF could also regress an enhanced vulnerability to VT in the cardiac hypertrophy without ischemic fibrosis through regulating Cx43.

Methods:
Dahl salt-sensitive rats were maintained for a 6 week-period on a high-salt diet as left ventricular hypertrophy models (LVHs) and a low-salt diet as controls (CONs). After 3-time subcutaneous injection (50 μ g/kg) of G-CSF and vehicle, the inducibility of VT was evaluated by rapid ventricular burst pacing. The electrical pulses for the induction of VT were square waves with 6 ms width at 6 V and delivered at 25 Hz for 30 sec. White blood cell was counted to confirm a response to the G-CSF treatment. Expression levels of phosphorylated and total Cx43 in the rat ventricles were analyzed by immunoblotting.

Results:
The LVHs showed apparent cardiac hypertrophy without pathological fibrotic changes. The G-CSF reduced the inducibility of VT compared to the vehicle in the LVHs (11 % vs. 63 %, p=0.04). Furthermore, the G-CSF eliminated the inducibility of VT in the CONs, although a difference in the inducibility of VT between with and without the G-CSF treatment did not reach statistical significance in the CONs (0 % vs. 33 %, p=0.60). White blood cell count in one microliter of blood was elevated by the G-CSF treatment in both LVHs (15083±4397 vs. 6976±1308, p<0.01) and CONs (19370±1174 vs. 7700±2335, p<0.01). The G-CSF increased phosphorylated Cx43 levels compared to the vehicle in both LVHs (1.4-fold vs. 1.2-fold, p<0.01) and CONs (1.4-fold vs. 1.0-fold, p=0.04), whereas the G-CSF did not affect total Cx43 levels in all groups.

Conclusion:
We demonstrated that the G-CSF administration ameliorated the electrophysiological stability in the rat model of cardiac hypertrophy by modulating the function of gap junctions through accelerating phosphorylation of Cx43.
Abstract P171: Low Concentrations of Aspirin Activate Cytoprotective Signaling Pathway Genes in Human Endothelial Cells: A Gene Expression Profiling Study
Authors: Kartha Reena V , Cheung Belinda W , Subramanian Subbaya , Schröder Henning
Published: July 2021

Low-dose aspirin is recommended for individuals with risk for heart disease and stroke in order to prevent thrombotic occlusive events. In addition to its well-established platelet inhibitory effects, increased expression of the antioxidant heme oxygenase-1 is considered to contribute to cardiovascular protection by aspirin. However little is known about the genome-wide changes in gene expression mediated by aspirin. In order to identify other molecular pathways and targets affected by aspirin, we investigated the global effects of aspirin treatment (3 μM and 100 μM)
in vitro
in human endothelial cells (Ea.hy 926) using HT-12 bead arrays (Illumina). The filtered genes were further analyzed using Ingenuity Pathway Analysis (IPA) software. We determined the gene expression profiles of endothelial cells treated with aspirin (3 μM and 100 μM) along with vehicle controls. Gene microarray analysis revealed significant upregulation of 134 genes (p <0.005 and >2 fold change) and downregulation of over 100 genes (p <0.005 and ≤0.5 fold change). Interestingly, the pattern of gene expression was similar between the two analyzed concentrations of 3 μM and 100 μM. The majority of upregulated genes were involved in gene expression, transcriptional regulation, cell-to-cell signalling and interaction and encoded transcription factors such as MAP kinases, phosphoinositide-3-kinases and cell cycle regulators (p<0.005). Several components of heterotrimeric G-proteins and G-protein-coupled receptors were also found to be upregulated which can potentially trigger phospholipase C signaling, suggesting an increase in overall intracellular second messenger signaling. In summary, our gene expression analysis suggests that aspirin at low and therapeutically relevant concentrations (3 μM) is effective in triggering cytoprotective signaling pathways some of which play a role in the regulation of heme oxygenase-1. Moreover, our study shows that low-dose aspirin results in altered expression profiles of a variety of genes. Future studies will have to address the biological and clinical relevance of these novel aspirin targets.
Abstract P174: Dynamics of the Renin-Angiotensin-Aldosterone System: Implications of Angiotensin-Peptide Fingerprinting on the Clinical Use and Development of Antihypertensive Drugs
Authors: Poglitsch Marko , Schuster Manfred , Loibner Hans
Published: July 2021

BACKGROUND:
The Renin-Angiotensin-Aldosterone-System (RAAS) is an extensively studied peptide hormone system which is critically involved in the regulation of blood pressure and other important physiologic functions. Angiotensin-(1-10) which is liberated from angiotensinogen by kidney secreted Renin, is sequentially metabolized to different N- and C-terminal truncated fragments with partially described biologic activities. Beside Angiotensin II (Ang-(1-8)), various other angiotensin peptides including Ang-(1-7), Ang-(1-9), Ang-(2-8) and Ang-(3-8) are known to possess physiologic activity while Ang-(1-7) and more recently Ang-(1-9) were described to antagonize pathologic effects of Ang-(1-8) via vasodilation and antifibrotic action respectively. The role of Ang-(1-8) in hypertension converts enzymes participating in Ang-(1-8) generation to favorable targets for antihypertensive drugs.

METHODS:
Human blood was collected from healthy volunteers followed by
ex-vivo
incubation at 37°C in the presence of different inhibitors of angiotensin metabolism. Angiotensin concentrations were determined employing a novel sensitive LC-MS/MS based method, which allows the simultaneous quantification of a broad panel of angiotensin peptides in human plasma.

RESULTS:
The investigation of different angiotensin metabolism modifiers revealed agent specific patterns of angiotensin peptide levels reflecting the specificity of these inhibitors. Spiking experiments allowed the determination of turnover rates for distinct angiotensin conversion reactions surrounded by their physiological matrix.

CONCLUSION:
The measurement of blood concentrations of angiotensin peptides provides important information about the substrate specificities and activities of enzymes involved in their metabolism. This allows the identification of potential causes for pathologic conditions, facilitating a patient specific therapeutic modulation of the RAAS. The overall assessment of angiotensin peptide concentrations (Angiotensin-Peptide-Fingerprinting) represents a powerful tool to gain a more detailed understanding of blood pressure regulation, enabling the pharmacologic characterization of RAAS modifying agents in use and development.
Abstract P183: Cardiac Deletion of CaMKII Delta and Gamma Protects Against Heart Failure Despite Activation of Calcineurin Signaling
Authors: Kreusser Michael M , Lehmann Lorenz H , Keranov Stanislav , Gröne Josef-Hermann , Katus Hugo A , Olson Eric N , Backs Johannes
Published: July 2021

CaMKII delta and gamma are the major CaMKII genes expressed in the heart, and both are up-regulated in response to pressure overload. Recently, we have demonstrated that CaMKII delta single knockout mice are protected against cardiac hypertrophy and remodeling. However, the role of CaMKII gamma and potential redundant functions of CaMKII delta and gamma are still elusive. The aim of the present study was to evaluate the function of CaMKII delta and gamma by a cardiomyocyte-specific double knockout mouse model(delta/gamma-CKO). Strikingly, whereas delta and gamma single knockout mice displayed only slightly reduced levels of cardiac phospholamban (PLN) phosphorylation at the CaMKII phosphorylation site threonin 17, in delta/gamma-CKO mice there was almost no residual PLN-threonin-17 phosphorylation detectable. Surprisingly and in contrast to delta and gamma single knockout mice, delta/gamma-CKO mice did develop cardiac hypertrophy after transverse aortic constriction (TAC). Despite cardiac hypertrophy we observed markedly reduced cardiac fibrosis and apoptosis. Microarray analysis revealed a distinct different gene expression profile pointing to an activation of calcineurin in delta/gamma-CKO mice after TAC. Phosphorylation of calcineurin at serine 197, which leads to an inactivation of its enzymatic activity, was almost abolished in delta/gamma-CKO mice. To test the therapeutical implications of a complete myocardial CaMKII knockout, an tamoxifen-inducible knockout system was established. Knockout of CaMKII delta and gamma was induced by administration of tamoxifen three weeks after TAC surgery. Whereas control mice did develop overt heart failure and cardiac remodeling 16 weeks after TAC, delta/gamma-iCKO mice recovered from cardiac dysfunction. Taken together, our mouse genetic studies demonstrate that CaMKII delta and gamma are promising drug targets to restore cardiac function after pathological stress. These data also unmask a cross talk of CaMKII to endogenous calcineurin signaling, which results in adaptive cardiac hypertrophy and not pathological remodeling.
Abstract P189: RhoA Functions as an Antihypertrophic Switch in the Mouse Heart
Authors: Vanhoutte Davy , Van Berlo Jop , York Allen J , Zheng Yi , Molkentin Jeffery D
Published: July 2021

Background.
Small GTPase RhoA has been previously implicated as an important signaling effector within the cardiomyocyte. However, recent studies have challenged the hypothesized role of RhoA as an effector of cardiac hypertrophy. Therefore, this study examined the
in vivo
role of RhoA in the development of pathological cardiac hypertrophy.

Methods and results
. Endogenous RhoA protein expression and activity levels (GTP-bound) in wild-type hearts were significantly increased after pressure overload induced by transverse aortic constriction (TAC). To investigate the necessity of RhoA within the adult heart, RhoA-LoxP-targeted (RhoA
flx/flx
) mice were crossed with transgenic mice expressing Cre recombinase under the control of the endogenous cardiomyocyte-specific β-myosin heavy chain (β-MHC) promoter to generate RhoA
βMHC-cre
mice. Deletion of RhoA with β-MHC-Cre produced viable adults with > 85% loss of RhoA protein in the heart, without altering the basic architecture and function of the heart compared to control hearts, at both 2 and 8 months of age. However, subjecting RhoA
βMHC-cre
hearts to 2 weeks of TAC resulted in marked increase in cardiac hypertrophy (HW/BW (mg/g): 9.5 ± 0.3 for RhoA
βMHC-cre
versus 7.7 ± 0.4 for RhoA
flx/flx
; and cardiomyocyte size (mm
2
): 407 ± 21 for RhoA
βMHC-cre
versus 262 ± 8 for RhoA
flx/flx
; n ≥ 8 per group; p<0.01) and a significantly increased fibrotic response. Moreover, RhoA
βMHC-cre
hearts transitioned more quickly into heart failure whereas control mice maintained proper cardiac function (fractional shortening (%): 23.3 ± 1.2 for RhoA
βMHC-cre
versus 29.3 ± 1.2 for RhoA
flx/flx
; n ≥ 8 per group; p<0.01; 12 weeks after TAC). The latter was further associated with a significant increase in lung weight normalized to body weight and re-expression of the cardiac fetal gene program. In addition, these mice also displayed greater cardiac hypertrophy in response to 2 weeks of angiotensinII/phenylephrine infusion.

Conclusion.
These data identify RhoA as an antihypertrophic molecular switch in the mouse heart.
Abstract P193: Nuclear Localization of the α1B-Adrenergic Receptor Subtype Is Required for Hypertrophic Signaling in Cardiac Myocytes
Authors: Wu Steven C , Cypher Andrew L , Healy Chastity L , Wright Casey D , Huang Yuan , O'Connell Timothy D
Published: July 2021

We previously demonstrated that α1-adrenergic receptors (α1-ARs) in the heart are required for physiologic hypertrophy during development and prevent a maladaptive response to pathologic stress. We have also shown that the major subtypes, α1A and α1B, both localize to the nucleus in adult cardiac myocytes. Importantly, we have defined a nuclear α1A-ERK survival and an α1A-PKCδ-cTnI inotropic signaling pathway that both originate at the nucleus and are transduced to cytosolic targets, suggesting that the α1A is required for cardiac myocyte survival and contractility. However, less is known about the molecular function of the α1B. In the current study, we examined the role of the α1B-subtype in hypertrophic signaling. First, we identified a bi-partite nuclear localization sequence (NLS) in the carboxy-terminal tail of the receptor. Mutation of the NLS (α1B-NLSmut) disrupted its localization to the nucleus when expressed in adult cardiac myocytes. We then compared hypertrophic signaling of the wild-type α1B- to the mutated receptor by reconstitution in cardiac myocytes lacking endogenous α1B (α1BKO) receptors. Activation of the wild-type receptor by the α1-agonist phenylephrine in α1BKO myocytes restored hypertrophic signaling, as we observed increased phosphorylation of protein kinase C (PKC) isoforms δ and ε, and histone deacetylases (HDAC) 4 and 5. We also observed increased expression of the hypertrophic gene marker, atrial natriuretic factor (ANF). Expression of the α1B-NLSmut failed to activate hypertrophic signaling in α1BKO cardiac myocytes despite phenylephrine stimulation. Together, our data show that nuclear localization of the α1B-subtype is required for hypertrophic signaling and overall further suggest that α1-adrenergic receptors are functional only at the nucleus in cardiac myocytes.
Abstract P196: The Impact of MicroRNA195 on the Lkb1/AMPK Signaling Axis and Hypertrophic Cardiomyopathy
Authors: Chen Hao , Mckee Laurel , Untiveros Gustavo , Perez Jessica , Konhilas John
Published: July 2021

Objectives:
MicroRNAs (miRs) have been identified as chief post-transcriptional regulators of cardiac disease progression. In addition, a critical role of the adenosine monophosphate-activated kinase (AMPK) pathway in the development of myocardial hypertrophy has been revealed. Yet, regulation of the AMPK pathway by miRs in the heart has not been addressed. We hypothesized that components of the AMPK pathway are targeted by miRs and alter AMPK signaling in a mouse model of hypertrophic cardiomyopathy (HCM).

Methods and results:
Using real-time PCR, a candidate miR screen that included 22 miRs implicated in pathological cardiac disease and/or metabolic dysregulation was performed on hearts from 60-, 120-, and 240-day-old transgenic HCM male mice harboring an R403Q mutation in the myosin heavy gene. Among early (60 day) elevated miRs were miR-195 and -451. Both miR-195 and -451 have conserved target sites in the 3′ UTR of CAB39 (MO25), a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK and its subsequent activation. We further confirmed the elevation of miR-195 and -451 by Northern blotting. Next, we demonstrated specific expression and a similar distribution pattern of miR-195 and -451 in cardiomyocytes of R403Q HCM hearts by in situ hybridization. To determine whether the conserved sites in MO25 3′ UTR acted as functional targets, either the miR-195 or miR-451 target sequence was cloned into a luciferase expression vector. MiR-195 but not miR-451 suppressed luciferase activity compared to the missense sequence control vector in C2C12 cells. In addition, over-expression of miR195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase). Finally, parallel changes were measured in 60 day R403Q HCM male hearts that included reduced MO25 expression and lowered phosphorlation of AMPK and Acetyl CoA carboxylase.

Conclusion:
Our findings indicate that miR-195 targets the LKB1/AMPK signaling axis and suggest a functional role for miR-195 elevation in R403Q HCM disease progression.
Abstract P217: Nuclear Protein Import as the Missing Link in Phenylephrine-Induced Cardiomyocyte Hypertrophy
Authors: Chahine Mirna N , Mioulane Maxime , Földes Gabor , Lyon Alexander , Harding Sian E
Published: July 2021

During cardiac hypertrophy, cardiomyocytes (CM) present alterations in gene expression and increased contractile protein content. Nuclear protein import (NPI) is critical in regulating gene expression, transcription, and subsequently cell hypertrophy. However, it is unknown how the nuclear transport machinery (transport receptors and nuclear pore complex (NPC)) functions to sustain increased demands for nucleocytoplasmic trafficking. The aim of this study was to determine if exposure of adult CM to phenylephrine (PE) affects hypertrophy by altering NPI and NPC density. Comparisons were made to adult failing rat and human CM. Rat myocytes were enzymatically isolated from adult hearts, and used for immunocytochemistry, qPCR and western immunoblotting. Failing CM were obtained from explanted human hearts at the time of transplant and from a rat model of myocardial infarction-induced hypertrophy and failure. Rat adult CM exposed for 48h to PE were injected with a protein import substrate (Alexa488-BSA-NLS) to visually monitor nuclear import with the confocal microscope. The effects of P38 MAPK inhibitor, HDAC inhibitor, Exportin-1 (CRM-1) inhibitor, and GSK-3 β inhibitor were investigated. Cell and nuclear sizes were increased in PE treated-adult rat CM and in the adult failing rat and human CM compared to normal CM. In contrast, PE depressed the rate and maximal NPI (by 65 +/- 3.4 % (3.55 from 5.46), p<0.05) as well as nucleoporin p62 mRNA and protein expression levels in adult rat CM compared to non-treated CM. Nucleoporin p62, cytoplasmic Ranbp1, and nuclear translocation of importins (Imp.α and β) relative densities were also decreased in PE treated-adult rat CM and in adult failing rat CM and human heart tissue compared to normal controls. On the contrary, CRM-1 nuclear export relative density was increased during the same pathological conditions. Thus NPI downregulation is linked to an increased nuclear export required by CM to generate the hypertrophic phenotype. All these effects were P38MAPK, HDAC and CRM-1 dependent but GSK-3Beta independent in rat CM. Our results show that alterations in NPI and NPC density occur in failing CM as well as in CM under hypertrophic stimuli. NPI may represent a critical therapeutic target in hypertrophic conditions.
Abstract P249: Cardiac Troponin I Pro82Ser Variant Impairs Myofilament Cooperativity, Induces Diastolic Dysfunction, and Blunts β-Adrenergic Response
Authors: Frazier Aisha , Ramirez-Correa Genaro A , Bedja Djahida , Shen Xiaoxu , Gao Wei Dong , Zhu Guangshuo , Cingolani Oscar H , Takimoto Eiki , Murphy Anne M
Published: July 2021

Cardiac troponin I is a key regulatory protein for muscle contraction and necessary for an adequate β-adrenergic response. The Proline 82 Serine (P82S) sequence variant of troponin I gene (
TNNI3
) is associated with late onset hypertrophic cardiomyopathy (HCM). Our preliminary data has shown that cTnIP82S is heterozygous in 3% of African Americans and is associated with increased LV mass in hypertensive black men. We hypothesize that cTnIP82S variant could influence the hypertrophic response to hypertension and/or the malignancy of the phenotypic expression when combined with known HCM disease causing myofilament mutations. We created a transgenic cTnIP82S mouse model. Echocardiography at baseline in older Tg and NTg mice (64-65 wks) showed impaired ejection time and relaxation, Tg display longer isovolumetric relaxation time (IVRT) (Tg 29.7±0.26 vs NTg 23± 0.23 msec, n=8 vs n=7 p<0.05) and a significantly higher TEI index. In the young NTg and Tg (17 wks) there were no echocardiographic differences. However, preliminary data suggest that 1 wk after transverse aortic coarctation (TAC), diastolic dysfunction was evident in Tg, and 10 weeks after TAC LV mass was also increased compared to NTg. Skinned fibers studies showed in the Tg mice borderline lower maximal Force (F
max
) and myofilament calcium sensitivity, whereas
n
Hill showed a marked depression (Tg 1.72±0.17 vs NTg 3.09±0.44, n=7 vs n=5, p<0.05), this effect was not due to the global myofilament phosphorylation pattern. In addition, intact twitching cardiac muscle studies in Tg mice revealed an impaired dose-dependent β-adrenergic acceleration of relaxation, evidenced by a failure to increase relaxation ([-dF/dt min]/[+dF/dt max]) and accelerate calcium transients decay after isoproterenol. Pressure-volume (PV) loop studies confirm that Tg mice fail to increase: Δ change in dP/dt max (NTg 4,928.3±7.7 vs Tg 843.3±10.7, n=4 vs n=4 p<0.05) in response to isoproterenol. TnIP82S variant is near a region of TnI-TnT interaction; this could explain its dramatic effects on myofilament cooperativity. Overall these studies suggest that the expression of cTnIP82S variant in the mice heart induces diastolic dysfunction, impairs relaxation at baseline and after β-adrenergic stimulation.
Abstract P254: Protein Kinase C Binding Protein 1 Inhibits Hypoxia-Inducible Factor 1 Action in the Heart
Authors: Walton Chad B , Veal David , Mafnas Chrisy , MacCannell Keith A , Anderson Cynthia D , Shohet Ralph V
Published: July 2021

The response to hypoxia in tissues is regulated by the heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1). We have investigated the transcriptional effects of hypoxia-inducible factor 1 alpha (HIF-1α) in the heart by expressing an oxygen-stable form of HIF-1α in cardiac myocytes of transgenic mice. The result in most cases is regulation of an expected panoply of genes that restore homeostasis during hypoxia, with corresponding phenotypic changes including contractile dysfunction and increased capillary density. In mice that do not show this phenotype, the mRNA for protein kinase c binding protein 1 isoform 2 (PRKCBP1) was much more abundant, as was the protein, and chromatin immunoprecipitation shows a predominant binding of HIF to the promoter of this gene. Sequencing of the promoter region of the PRKCBP1 gene from the two phenotypes revealed an unexpected 480 bp insert in the HIF-resistant animals containing two canonical HIF binding sites. The protein co-immunoprecipitates with HIF and inhibits HIF transcriptional activity in cell culture. In FVB mice, that contain the promoter insert, PRKCBP is induced by ischemia and co-localizes with HIF in the infarct region. It may be responsible for the greater susceptibility of this strain to heart failure after infarction. We have confirmed with genetic, transcriptional, biochemical, and physiological data that Prkcbp1 inhibits HIF activity through direct interaction, in a mechanism mediated by transcriptional control.
Abstract P264: Interaction of Collecting Duct-Derived Prorenin and Soluble Prorenin Receptor Increases Intraluminal Renin Activity and Augments Intratubular Angiotensin II Formation in Ang II-Dependent Hypertensive Rats
Authors: Prieto Minolfa C , Liu Liu , Gonzalez Alexis A , Seth Dale M , Navar L Gabriel
Published: July 2021

Upregulation of collecting duct (CD)-derived renin (CD renin) in angiotensin II (Ang II)-dependent hypertension may provide a pathway for intratubular Ang II formation by acting on angiotensinogen (AGT) delivered from proximal tubule segments. Recently, a prorenin/renin receptor (PRR) has been cloned and shown to enhance renin and prorenin activation. The soluble form of the PRR (sPRR) is augmented in the renal inner medulla of chronic Ang II-infused rats. The present study was performed to determine if renin is secreted into the lumen by the CD cells in chronic Ang II-infused rats and to establish the functional contribution of sPRR to the enhanced renin activity in distal nephron segments. Accordingly, urinary levels of renin (
uRen
) and Ang II (
uAngII
) were measured by RIA in chronic Ang II-infused male Sprague-Dawley rats [80 ng/min, SC minipumps for 14 d, n=10] and sham-operated rats [n=10]. Systolic blood pressure increased in the Ang II rats by Day 5 and continued to increase throughout the study (Day 13; Ang II: 175±10 vs. sham: 116±2 mmHg;
p
<0.05). Although plasma renin activity (PRA) was suppressed in the Ang II-infused rats, renal medullary renin content was significantly augmented (12,605±1,343 vs. 7,956±765 ng Ang I/h/mg;
p
<0.05). The excretion of
uAngII
was also increased (3,813±431 vs. 2,080±361 fmol/day;
p
<0.05). In addition, renin and prorenin excretion rates increased progressively and were markedly augmented by Day 13 of Ang II infusion [renin (8.6±1.5 vs. 2.8±0.5x10
-6
Enzyme Units Excreted (EUE) /day; prorenin: 15.8 ± 2.8 vs. 2.6 ± 0.7x10
-3
EUE /day,
p
<0.05). Renin and prorenin protein levels examined by Western Blot in the urine were similarly increased. Importantly, the incubation of urine samples of Ang II-infused rats with recombinant human prorenin showed increased Ang I formation compared to sham-operated rats. In conclusion, in chronic Ang II-infused rats, the presence of sPRR in the urine reflects augmented enzymatic activity of prorenin secreted by the principal cells of the CD, which increase intratubular Ang II de novo formation in the distal nephron segments thus contributing to enhanced sodium reabsorption during Ang II-dependent hypertension.
Abstract P265: Adenosine 2A Receptor Activation Regulates Expression of Scavenger Receptors CD36 and LOX-1 in Human Macrophages and Aortic Endothelial Cells
Authors: Voloshyna Iryna , Littlefield Michael J , Carsons Steven , Reiss Allison B
Published: July 2021

Objectives of the study.
A variety of scavenger receptors (SR) have been linked to lipid accumulation in the vasculature. Among these, CD36 and LOX-1 are proposed to play a prominent role in atherosclerosis. Adenosine is a potent cellular signaling molecule with anti-atherogenic properties. Our previous work has shown that agonists specific for the adenosine A
2A
receptor (A
2A
R) enhance cholesterol efflux, preventing macrophage foam cell transformation. Here we report additional atheroprotective effects of A
2A
R activation through changes in SR expression.

Methods.
Primary human aortic endothelial cells (HAECs) and THP-1 human macrophages were incubated in the presence of oxLDL (20 μg/ml) and: a) growth media b) DMSO vehicle c) the A
2A
R agonist CGS-21680 (1 μM) d) the A
2A
R antagonist ZM-241385 (10 μM, 1h) + CGS-21680 (1 μM) (n=5). SR expression was evaluated by real-time PCR. PCR results were confirmed by immunofluorescence analysis with CD36 and LOX-1-specific primary antibodies, followed by incubation with FITC conjugated secondary antibodies.

Results.
CGS-21680 downregulated CD36 and LOX-1 expression vs. untreated HAEC (67.89±9.55% and 49.00±2.55%, respectively, n=6, P<0.01). This effect was abrogated by ZM-241385, resulting in upregulation of these SR to 175.9±22.67% and 172.10±39.6%, respectively, vs. control (n=6, P<0.001). The pattern of SR expression changes in THP-1 macrophages upon exposure to CGS-21680 and ZM-241385 was the same as in HAEC. PCR results were confirmed by quantization of fluorescence intensity with immunofluorescence confocal microscopy.

Summary.
A
2A
R activation is a promising therapeutic approach to prevent lipid overload in arterial vasculature. Our data support an anti-atherogenic role for the A
2A
R in both cholesterol influx and efflux pathways. Several new agents that target the A
2A
R are in development and they may hold promise as treatment for atherosclerotic cardiovascular disease.
Abstract P273: Enhancement of Proteasomal Function Protects Against Proteinopathy and Myocardial Ischemia-Reperfusion Injury in Mice
Authors: Li Jie , Horak Kathleen M , Su Huabo , Sanbe Atsushi , Robbins Jeffrey , Wang Xuejun (XJ)
Published: July 2021

The ubiquitin-proteasome system degrades most intracellular proteins, including misfolded proteins. Proteasome functional insufficiency (PFI) was observed in experimental proteinopathies and implicated in many human common diseases but its pathogenic role has not been established because a measure to enhance proteasome function in the cell has not been reported until very recently. We have recently discovered that overexpression of proteasome activator 28α (PA28α) enhances proteasome-mediated removal of abnormal proteins in the cell and protects against oxidative stress in cultured cardiomyocytes (
FASEB J 2011;
25(3):883-93
). Here we have extended the
in vitro
discoveries to intact animals. First, we created inducible transgenic mice with cardiomyocyte-restricted PA28α overexpression (CR-PA28αOE). CR-PA28αOE does not alter the homeostasis of normal proteins and cardiac function but increases the degradation of a surrogate misfolded protein in the heart. This marks the establishment of the first animal model of benign enhancement of proteasomal function. Second, by breeding CR-PA28αOE mice with a well-established mouse model of mutant αB-crystallin (CryAB
R120G
) based desmin-related cardiomyopathy, a
bona fide
cardiac proteinopathy, we demonstrate that CR-PA28αOE markedly reduced aberrant protein aggregation and cardiac hypertrophy and the lifespan of the animals significantly increased. Furthermore, PA28α knockdown promoted, while PA28αOE attenuated, CryAB
R120G
protein accumulation in cultured cardiomyocytes. Finally, we tested the effect of proteasome functional enhancement on myocardial ischemia/reperfusion (I/R) injury induced by ligation and subsequent release of the left anterior descending artery. We found that the infarct size was significantly reduced and cardiac function recovery during reperfusion was markedly facilitated in the CR-PA28αOE mice, compared with their littermate control mice. We conclude that (1) benign enhancement of cardiac proteasome function can be achieved by CR-PA28αOE; (2) PFI plays a major pathogenic role in proteinopathy and myocardial I/R injury; and (3) upregulating PA28α is potentially a novel therapeutic strategy for proteinopathies and disease alike.
Abstract P277: Cardioprotection Enabled by Proteomic Remodeling of the Mitochondrial Respiratory Chain
Authors: Lotz Christopher , Deng Ning , Zhang Jun , Wang Yueju , Zong Chenggong , Liem David , Youn Ji-Youn , Cai Hua , Wu Christine , Yates John , Weiss James , Ping Peipei
Published: July 2021

The mitochondrial respiratory chain is a collection of five multi-protein complexes, whose unobstructed functionality represents a pivotal element at the crossroads of cell death or survival. However, its molecular composition and stochiometric information remains elusive and the adaptive abilities of the chain remain largely unknown.

We employed a quantitative proteomic approach to investigate the hypothesis that cardioprotection against ischemic injury is afforded by a salutary proteomic remodeling of the mitochondrial respiratory chain.

The respiratory chain of cardiac mitochondria isolated from wild type (WT) mice and from mice expressing a constitutively active protein kinase Cε (AE-PKCε) were characterized using 15N-stable isotope labeled murine models (SILAM), as well as a label-free method in conjunction with high resolution LC-MS/MS, respectively. Enzymatic function of electron transport chain and the ATP synthase were evaluated (n=7/group); and mitochondrial superoxide production was examined by ESR-spectroscopy (n=3/group).

Three novel and important observations are made: (i) five individual respiratory complexes exhibited a molar ratio of 1:1:1.4:1.2:4.5 in the WT-heart; (ii) subunits within the five complexes encoded by the mitochondrial genome were expressed at much lower abundance (p<0.05) than those encoded by the nuclear genome; and (iii) Genetic cardioprotection by AE-PKCε elicited a proteomic remodeling of complex I and III, mitochondria from AE-PKCε exhibited an increased expression of multiple subunits, including the catalytic complex III subunit Cyc1. This finding was accompanied by a preserved complex III activity (p<0.05), as well as tempered superoxide production (p<0.01) subsequent to Ca2+-induced damage.

This is the first study documenting a salutary proteomic remodeling of the mitochondrial respiratory machinery in cardioprotection. Quantitative proteomics technology enabled novel information and new insights into mitochondrial biology.
Abstract P278: Fibroblast Growth Factor 21 Mediated Protection of Ischemic Myocardium
Authors: Liu Shu Q , Tefft Brandon , Kharitonenkov Alexei , Ren Yupeng , Zhang Li-Qun , Wu Yu H
Published: July 2021

Myocardial ischemia is a prevalent disorder causing heart failure. As cardiomyocyte death is the principal cause of cardiac functional deficits, a recognized treatment is to minimize cardiomyocyte death post myocardial ischemia. While extensive investigations have been conducted, few effective cardioprotective agents have been developed for clinical applications. The goal of this investigation is to test and establish a cardioprotective agent based on fibroblast growth factor 21 (FGF21), a secretory protein participating in innate cardioprotective responses. In myocardial ischemia induced by LAD coronary artery ligation in the mouse, hepatocytes upregulated FGF21 mRNA by 11 folds at 12 hrs, followed by an increase in the FGF21 protein level in hepatocytes and the serum. Administration of recombinant FGF21 to mice immediately post MI (50 ng/gm, IV, twice/day for 3 days) resulted in a significant reduction in the fraction of myocardial infarction (MI) with reference to the LV wall volume below the LAD ligation (38+/−5 and 21+/−4%, n=7, vs. albumin administration 51+/−8 and 31+/−5%, n=6, at day 1 and 10, respectively, p<0.05 for both times) in association with improved LV dp/dt. In FGF21 overexpressing mice, the volume fraction of MI (36+/−6%, n=6) was significantly lower than that in wildtype mice (48+/−6%, n=5, p<0.05) at day 1 post MI. Furthermore, FGF21 administration to normal mice induced phosphorylation of FGFR1, PI3K, Akt, and BAD in cardiomyocytes within 10 – 30 min. These molecules were also phosphorylated in cardiomyocytes within 1 day post MI. Injection of siRNA specific to FGFR1, PI3K, or Akt to the LV anterior wall at 6 locations about 2 mm apart (diffusion range of FITC-siRNA tested by fluorescence microscopy) 3 days prior to MI resulted in a reduction in the protein level of FGFR1, PI3K, or Akt by 65+/−9, 71+/−11, or 68+/−12%, respectively (n=3), within the region of siRNA injection at 1 day post MI in association with a reduction in BAD phosphorylation and an increase in the fraction of MI by 9+/−3, 8+/−2, or 11+/−3%, respectively (n=3). These observations suggested that FGF21 contributed to myocardial protection possibly via the FGFR1-PI3K-Akt-BAD signaling mechanisms and recombinant FGF21 may be potentially used as a cardioprotective agent.
Abstract P288: ATP Signaling Stimulates Fibroblast Activation: The Role of Pannexin1 Channels in Fibrosis
Authors: Duffy Heather S , Dolmatova Elena , Baum Jennifer , Keith Kimberly , Boassa Daniella , Sosinsky Gina , Kontaridis Maria I
Published: July 2021

BACKGROUND-We have shown that Pannexin1 (Panx1), a novel ATP channel, is found in ventricular cardiac myocytes and upregulated on myocyte sarcolemmal membranes during acute ischemia. We hypothesize that the channel is part of a protein complex that plays a role in downstream cell signaling post myocardial infarction (MI).

METHODS-Using a canine model of MI, where the left anterior descending coronary artery is occluded (CO) we examined epicardial tissue from hearts subjected to 0–3 hrs of CO. Immunoprecipitation (IP) with subsequent silver stain studies revealed novel binding partners for Panx1. Co-immunoprecipitation studies (Co-IP) were performed to confirm Panx1 binding partners. In vitro dye uptake studies and ATP assays were used to determine the function of Panx1 channels under hypoxic conditions.

RESULTS-IP followed by silver staining revealed multiple potential binding partners for Panx1 in the heart. Following 3 hrs CO some interactions increased while others decreased. Co-immunoprecipitation identified one of these partners for Panx1 as the MAGUK scaffolding protein SAP97, an interaction that is significantly increased following 3 hr CO (n=3, p<0.05). Immunostaining showed that CO increased trafficking and stabilization of Panx1 channels at the cell membranes. In vitro dye uptake studies show that cells increase dye uptake following hypoxia, an event that is blocked by the Panx1 channel blocker, Probenecid. Hypoxia also causes increased ATP release likely due to opening of Panx1 channels. ATP release activated fibroblasts and stimulated early fibrosis signaling.

CONCLUSIONS-Our data show the presence of a Panx1 protein complex in cardiac myocytes that is scaffolded at the cell membranes by SAP97. Under cellular stress Panx1 channels release ATP to induce early fibrotic events. We We conclude that Panx1 plays a key functional role in ATP signaling and formation of fibrosis in the post-MI heart.
Abstract P296: Intralipid Protects the Heart Against Ischemia/Reperfusion Injury More Efficiently than Cyclosporine-A
Authors: Li Jingyuan , Youn Ji-Youn , Cai Hua , Eghbali Mansoureh
Published: July 2021

It has been shown that intralipid protects the heart against ischemia reperfusion (I/R) injury. Here we compared the cardioprotective effect of Intralipid (ILP) with cyclosporine-A (CsA), a potent inhibitor of the mitochondria permeability transition pore (mPTP) opening. Isolated hearts (Langendorff) from male mice were subjected to 20 minutes of global ischemia followed by reperfusion with Krebs Henseleit buffer (CTRL), additional 1% ILP (ILP group) or 1.5µM CsA (CsA group). The reperfusion duration was 40 min for measurement of cardiac function and infarct size, 10 min for measurement of mitochondrial calcium retention capacity and 5 min for superoxide production measurements using electron spin resonance. Post-ischemic administration of ILP improved the heart functional recovery even better than CsA as Rate pressure product (RPP) at the end of 40 min was significantly higher in ILP group compared to CsA group (13676±611 mmHg*beats/min (n=6) in ILP group vs. 7426±1158 mmHg*beats/min (n=3) in CsA, P<0.01). Consistent with the heart function, the infarct size was also significantly smaller in ILP compared to CsA group (18.3±2.4% (n=9) in ILP vs. 29.9±2.0% (n=3) in CsA, P<0.01). The heart functional recoveries were significantly higher and the infarct sizes were significantly smaller in both groups compared to CTRL (RPP=2999±863 mmHg*beats/min (n=6), the infarct size=54.8±2.9% (n=10)). ILP was as efficient as CSA in inhibiting mPTP opening as calcium retention capacity (CRC) was not significantly different between the mitochondria isolated from the heart which were reperfused with ILP or CSA (274.3±8.4 nM/mg-mitochondria protein (n=6) in ILP vs. 260.3±2.9 nM/mg-mitochondria protein (n=6) in CsA). The CRC in both groups were higher than CTRL (168.6±9.6 nM/mg mitochondria protein (n=7)). ILP prevented superoxide production in mitochondria similarly as CSA (0.344±0.1104 (n=4) in ILP vs. 0.5744±0.1445 (n=4) in CsA) which were significantly lower than CTRL (1±0.1197 (n=4), normalized to CTRL). In conclusion, although ILP inhibits the opening of the mPTP as efficiently as CsA, ILP is more effective than CsA in reducing the infarct size and improving the heart functional recovery.
Abstract P314: Grainyhead-like 3 Plays an Essential Role in Endothelial Cell Function in a NO-Dependent Manner
Authors: Lukosz Margarete , Mlynek Arne , Altschmied Joachim , Haendeler Judith
Published: July 2021

Aging of human endothelial cells (EC) is associated with reduced nitric oxide (NO) bioavailability, decreased migratory capacity and an increase in Src kinase activation and apoptosis sensitivity. We recently identified the transcription factor grainyhead-like 3 (GRHL3) as a pro-migratory transcription factor in EC. A role for GRHL3 in aging processes was suggested by reduced expression in brains from old mice. Therefore, we wanted to investigate the regulation of GRHL3 by NO and Src kinase and GRHL3 effects on NO-bioavailabilty, apoptosis and migration. We treated EC either with physiological concentrations of NO or the Src kinase inhibitor PP2. In both cases GRHL3 expression was increased (3.75 fold and 4.50 fold, respectively). In addition, both treatments induced migration and inhibited apoptosis. Interestingly, overexpression of GRHL3 activated endothelial nitric oxide synthase (eNOS), its upstream regulator Akt and subsequently increased the S-NO content of EC. This demonstrates that GRHL3 enhances NO-bioavailability in EC, which is inseparably tied to apoptosis protection and migration. Along this line, GRHL3 overexpression reduced apoptosis of EC (1.89 fold reduction of basal apoptosis vs. empty vector transfected cells). Interestingly, this anti-apoptotic effect was dependent on NO synthesis by eNOS, since the eNOS inhibitor L-NMMA completely abrogated the protective effect of GRHL3. Having demonstrated a pro-migratory effect of GRHL3, we wanted to know whether this effect is mediated by induction of vascular endothelial growth factor (VEGF) expression. Surprisingly, GRHL3 overexpression did not change VEGF protein levels. To exclude a bystander effect of GRHL3 in EC migration, we knocked down expression with shRNA. Reduction of GRHL3 mRNA levels decreased basal and NO-induced EC migration (scr: 73 +/− 16 migrated cells; shGRHL3: 26 +/− 12 migrated cells; scr+NO: 149 +/− 19 migrated cells; shGRHL3+NO: 36 +/− 10 migrated cells) demonstrating an essential role in this process. Taken together, these data suggest that GRHL3 is essential for EC functions compromised during aging.