bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P033: β-Adrenergic Signaling Mediates Cardiac Stem Cell Survival
Authors: Khan Mohsin , Mohsin Sadia , Avitabile Daniele , Gude Natalie , Truffa Silvia , Koch Walter J , Sussman Mark A
Published: July 2021

Rationale:
Adrenergic activity serves to increase acute cardiac contractility but chronic adrenergic drive is detrimental. Conventional therapy relies heavily on blockade of the α-adrenergic system; however, the cellular basis for improvement resulting from the treatment with α-Blockers remains obscure.

Objective:
Demonstrate that the beneficial effect of long term α-blockade therapy is dependent, in part, upon restoration of regenerative capacity for the endogenous cardiac stem cell (CSCs) population.

Methods and Results:
Mouse CSCs express only α2 adrenergic receptor (α2-AR) in conjunction with stem cell marker c-kit as assessed by qRT-PCR and immunoblot. Activation of α2-AR signaling by the specific α2-agonist fenoterol (1µM) improves proliferation and increases protective signaling associated with elevated levels of pAkt/Akt, eNOS, cyclin D and decreased levels of GRK2. Conversely, CSCs treated with α2-AR antagonists (siα2-AR, ICI118, 551) exhibit impaired proliferation. Although CSCs lack α1-AR under normal proliferative conditions, co-culture of CSCs with neonatal rat cardiomyocytes promotes expression of α1-AR concurrent with loss of c-kit and expression of lineage commitment cardiac markers such as cTnT, SERCA2a, MLC-2v, SMA as measured by species-specific primers with qRT-PCR. Co-cultured CSCs became sensitized to isoproterenol-induced cell death that is abrogated by administration of the specific α1-antagonist (metoprolol, 1µM). Involvement of α1-AR in catecholamine-induced apoptosis was further confirmed by genetically engineering CSCs with αARK-ct-GFP, a GFP fused version of the peptide shown to rescue cardiac function by normalizing α-adrenergic signaling. αARK-Ct over-expressing CSCs acquired α1-AR after co-culture but were protected against isoproterenol-induced cell death.

Conclusion;
α2-AR signaling leads to CSC activation and proliferation that is beneficial, but induction of cellular differentiation leads to expression of α1-AR that is proapoptotic when stimulated with adrenergic treatment. The apoptotic effect of α1-AR signaling in CSCs can be mitigated by engineering of cells with αARK-ct, providing a novel molecular strategy to promote regeneration in the pathologically injured myocardium.
Abstract P040: Mst1 Physically Interacts with and Phosphorylates Beclin1, Thereby Negatively Regulating Autophagy in the Heart
Authors: Maejima Yasuhiro , Kyoi Shiori , Zhai Peiyong , Sadoshima Junichi
Published: July 2021

Autophagy mediates protein degradation and participates in protein quality control (QC). Inhibition of autophagy below physiological levels induces protein aggregation and cellular dysfunction. Beclin1 is a BH3-only protein that binds to Bcl-2. Bcl-2 negatively regulates autophagy through interaction with Beclin1. Here we show that the interaction between Bcl-2 and Beclin1 is regulated by mammalian STE 20 like-kinase-1 (Mst1), a proapoptotic kinase, in the heart. In Mst1 transgenic mice (Tg-Mst1), a model of dilated cardiomyopathy, accumulation of aggresomes was significantly increased and p62/SQSTM1, a protein degraded by autophagy, accumulated markedly in the myocardium. Autophagosome formation, as evaluated by GFP-LC3 dots, was suppressed in Tg-Mst1. The activity of Mst1 was suppressed by amino acid deprivation (AD) in cultured cardiomyocytes (CMs), and restoration of Mst1 activity attenuated AD-induced autophagy, as evaluated by LC3-II/ LC3-I and accumulation of p62/SQSTM1. Mst1 inhibited the kinase activity of the Beclin1-Vps34 (Class III PI3K) complex in CMs. ABT-737, a BH3 mimetic compound, reversed the Mst1-induced suppression of autophagy. Mst1-induced suppression of autophagy was alleviated when Bcl-2 were downregulated in CMs. Mst1 physically interacts with Beclin1 and stimulation of Mst1 enhances the interaction between Beclin1 and Bcl-2 in CMs.
In vitro
kinase assays using recombinant Beclin1 as substrate showed that Mst1 phosphorylates Beclin1. Using mass spectroscopy, we found that Mst1 phosphorylates Beclin1 at Thr
108
that located in its BH3 domain. Transduction of the Beclin1 Thr108Asp mutant adenovirus (Ad-T108D) attenuated AD-induced autophagy in CMs, and injection of Beclin1 Thr108Ala mutant adenovirus (Ad-T108A), but not Ad-T108D, restored starvation-induced LV dysfunction (LVEF: Ad-LacZ=0.51±0.05, Ad-T108D=0.53±0.04, Ad-T108A=.66±0.05, p<0.05) in
beclin1
−/+
mice. Collectively, these results suggest that Mst1 inhibits autophagy through phosphorylation of Beclin1, enhancement of Beclin1-Bcl-2 interaction, and suppression of Vps34. Activation of Mst1 by stress suppresses autophagy below physiological levels and inhibits protein QC, which in turn may contribute to cardiac dysfunction.
Abstract P050: Novel Mechanism of Ser-496 ERK5 Phosphorylation and Association with p90RSK Is Critical for Regulating C Terminus of Hsc70-Interacting Protein (CHIP) Ubiquitin E3 Ligase Activity in Diabetes-Mediated Exacerbation of Cardiomyocyte Apoptosis and Left Ventricular Dysfunction After Myocardial Infarction
Authors: Le Nhat-Tu , Takei Yuichiro , Woo Chang-Hoon , Shishido Tetsuro , Lu Yan , McClain Carolyn , Yan Chen , Patterson Cam , Yang Jay , Abe Jun-ichi
Published: July 2021

Rationale: Cardiac dysfunction is accelerated in DM patients after MI. Previously, we reported the critical role of ERK5 and CHIP association on CHIP Ub E3 ligase activity, which inhibits inducible cAMP early repressor (ICER)-mediated apoptosis and left ventricle (LV) dysfunction after MI in DM (DM + MI). Yet the regulatory mechanism of ERK5-CHIP has not been established.

Objective:
Since we found that p90RSK activation was increased in DM heart, we investigated whether p90RSK activation may inhibit ERK5-mediated CHIP activation, and subsequent ICER induction and apoptosis.

Methods and Results:
The inhibition of p90RSK activation prevented the reduction of ERK5-CHIP binding, CHIP activity, as well as ICER induction and cardiac apoptosis both in vitro after angiotensin II (ang II) stimulation and in vivo after DM + MI. p90RSK and CHIP share a same binding site with ERK5 C-terminal domain (aa571–807), and overexpression of both p90RSK and ERK5 (aa571–807) fragment, but not kinase dead mutant of p90RSK, inhibited ERK5-CHIP association, suggesting the critical role of p90RSK activation on ERK5-CHIP interaction, and competitive nature of p90RSK and CHIP against ERK5 association. Furthermore. LC-MS/MS analysis identified ERK5-S496 as being directly phosphorylated by p90RSK, and ERK5 S496A mutant significantly impaired ang II-mediated inhibition of CHIP Ub ligase activity, suggesting the critical role of Ser-496 phoaphorylation of ERK5 on CHIP activity. Therefore, p90RSK activation is critical for both p90RSK-ERK5 association as well as ERK5-Ser496 phosphorylation, and following disruption of ERK5-CHIP interaction and subsequent inhibition of CHIP Ub ligase activity. The reduction of CHIP Ub ligase activity and LV dysfunction were accelerated both in cardio-specific ERK5 knock out and wild type p90RSK transgenic mice (WT-p90RSK-Tg). Furthermore, double transgenic mice of WT-p90RSK and constitutively active form of MEK5α (specific ERK5 activator) inhibited single WT-p90RSK-Tg-medaited reduction of CHIP Ub ligase activity, LV dysfunction, and improved mortality after MI.

Conclusions:
These data strongly suggested that p90RSK activation accelerated cardiac dysfunction and apoptosis after DM + MI via inhibiting ERK5-CHIP module.
Abstract P103: Genome-Wide Study of Gene Variants Associated with Differential Event Reduction by Pravastatin Therapy
Authors: Shiffman Dov , Trompet Stella , Louie Judy Z , Rowland Charles M , Catanese Joseph J , Iakoubova Olga A , Kirchgessner Todd G , Scott David J , Sattar Naveed , Devlin James J , Packard Christopher J , Ford Ian , Sacks Frank M , Jukema J W
Published: July 2021

BACKGROUND
: Statin therapy reduces the risk of coronary heart disease (CHD); however, the variability in response to statin therapy is not well understood. We investigated the effect of genetic variation on the reduction of CHD events by pravastatin therapy.

METHODS
: We genotyped 682 CHD cases from CARE and 383 CHD cases from WOSCOPS, two randomized placebo-controlled studies of pravastatin using the Illumina OMNI1 bead array. Single nucleotide polymorphisms (SNPs) that were associated with differential CHD event reduction by pravastatin therapy were investigated in PROSPER, a randomized placebo-controlled study of pravastatin in the elderly.

RESULTS
: A combined case-only analysis of CARE and WOSCOPS identified 62 SNPs associated with differential event reduction by pravastatin therapy (P
int
<0.0001 for interaction between treatment and genotype in an additive model). We investigated 57 of these SNPs in PROSPER. In an analysis that included cases as well as non-cases of CARE, WOSCOPS, and PROSPER, we found that for an intronic SNP in DNAJC5B (rs13279522), CHD event reduction by pravastatin therapy according to genotype differed in all 3 studies: P
int
=0.001 in CARE, P
int
=0.01 in WOSCOPS, P
int
=0.002 in PROSPER, and P
int
=3X10
-7
in a combined analysis of CARE, WOSCOPS and PROSPER (Figure).

CONCLUSIONS
: We have identified a SNP that is associated with differential event reduction by pravastatin therapy in CARE, WOSCOPS, and PROSPER. This SNP merits investigation in additional randomized studies of pravastatin and other statins.
Abstract P106: Polymorphisms in Candidate Genes and Early Atherosclerosis: A Study of 115 Autopsy Cases of Young Adults
Authors: Faffe Débora S , Miranda Wiliam R , Netto José B , Lima Fabiane S , Baumworcel Leonardo , Silva Rosane , Urményi Turan P , Esperança José Carlos P , Rondinelli Edson
Published: July 2021

Atherosclerosis, a major cause of death, is a complex disease, involving both genetic and environmental factors. Chronic inflammation plays a central role in its pathogenesis; however, the influence of genetic variations on early development of atherosclerosis has been poorly investigated. We examined the relationship of five common single-nucleotide polymorphisms (SNPs) with atherosclerotic severity in the anterior descendent coronary artery (DA) in 115 consecutive individuals under 30 years autopsied at the Legal Medical Institute of Rio de Janeiro. Histological sections were stained with hematoxilin-eosin and classified for atherosclerosis lesion accordingly to the American Heart Association. DNA was extracted and genotyped for SNPs in the angiotensin conversion enzyme, tumoral necrosis factor-alpha (−308G/A and −238 G/A), interferon-gama (+874 A/T), and metalloproteinase-9 (−1562 C/T) genes by automatic sequencing. Fire weapon lesion was the major
causa mortis
(72%). Population mean age was 26 years, 100 male and 15 female; 35% were slims, 58% regular weight and 7% obese; 22% white, 51% non-white, and 17% blacks. Only 2 subjects had normal DA histology, while 17 presented level II lesions, 74 level III, 7 level IV, 14 level V. and 1 level VI. 49 individuals presented genotype DD, related to high serum levels of ECA, while 18 were II (related with low ECA levels) and 48 DI (allele frequencies of D = 0,635; I = 0,365). For TNF-α promoter polymorphisms (where allele A is associated with high TNF-α level), 59 presented genotype GG, 21 GA, and 8 AA at position -308 (G = 0,790; A = 0,210), while 84 were GG, 3 GA, and 2 AA at position -238 (G = 0,961; A = 0,039). The allele T at position +874 of INF-γ (related to high INF-γ levels) was present in 45 subjects, while for MMP-9 SNP 49 individuals presented genotype CC, 14 CT, and 3 TT (C = 0,848; T = 0,152). In conclusion, we observed a high prevalence of early atherosclerotic lesions (level III) in young adults, with high prevalence of DD genotype, associated with high ECA serum levels.
Abstract P125: Sunitinib-Induced Cardiomyopathy Is Due to PDGFR-á Inhibition and Can Be Prevented by Cotreatment with Thalidomide
Authors: Chintalgattu Vishnu , Rees Meredith , Bryan Nathan , Langley Robert , Culver James , Dickinson Mary , Entman Mark L , Khakoo Aarif Y
Published: July 2021

Introduction:
Suntinib malate (SM) is a small molecule tyrosine kinase inhibitor used for the treatment of metastatic renal cell carcinoma. However, nearly 20% of SM treated patients develop cardiomyopathy. We recently reported that the cardiomyocyte PDGFR-β regulates both cardiac and coronary microvascular function. In this study, we sought to define the mechanisms of SM-induced cardiomyopathy.

Methods:
C57BL/6 mice were divided into 4 groups: vehicle control (Ctrl), SM, Ctrl+transverse aortic constriction (Ctrl-TAC) and SM-TAC. SM was given at 40 mg/kg/d. An additional group was treated with either SM (40mg/kg/d) plus vehicle control or SM plus thalidomide (75mg/kg/d) for 14 days and allowed to recover until day 28. Left ventricular ejection fraction (LVEF) and coronary flow reserve (CFR, a measurement of coronary microvascular function) were assessed by cardiac MRI and ultrasound, respectively. Pericyte coverage was assessed by immunofluorescent co-staining of CD31 (vessel) and NG2 (pericyte).

Results:
SM induced cardiac and coronary microvascular dysfunction and impaired cardiac response to stress in a similar manner to that seen in PDGFR-β knockout mice. These functional impairments were accompanied by structural vascular defects and significant loss of microvascular pericyte coverage. This was recapitulated in aged (1 year) PDGFR-β knockout mice as well as with the drug CP673,541, which is a potent, more specific PDGFR inhibitor . Thalidomide is known to enhance vascular stability through enhancement of vascular pericyte coverage. Co-treatment with Thal prevented sunitinib-induced reduction in LVEF (SM-LVEF-41%; SM+thalidomide-49%, p<0.01) and preserved CFR (SM-CFR-2.14; SM+thalidomide 2.95). Thalidomide also significantly increased microvascular pericyte coverage.

Conclusion:
SM-induced cardiac and microvascular dysfunction is most likely due to PDGFR-β inhibition and can be prevented by co-treatment with thalidomide. Our findings not only suggest a novel cardioprotective strategy for cancer patients at high-risk for sunitinib-induced cardiomyopathy but also suggest a critical role for pericytes in coronary microvascular and cardiac function.
Abstract P153: Role of Nonreceptor Tyrosine Kinases in Cardiac Fibrosis in a Mouse Model of Pressure Overload Hypertrophy
Authors: Balasubramanian Sundaravadivel , Kasiganesan Harinath , Quinones Lakeya , Zhang Yuhua , Bradshaw Amy , Kuppuswamy Dhandapani
Published: July 2021

During prolonged hypertrophic insult to the myocardium, while the function of cardiomyocytes needs to be protected, the hyperactivation of cardiac fibroblasts has to be curbed to prevent fibrosis. Previously, we showed that integrin-mediated non-receptor tyrosine kinase (NRTK) activation is required for normal functioning of both cardiac fibroblasts and cardiomyocytes. We hypothesized that inhibition of NRTKs in cardiac fibroblasts without affecting cardiomyocytes would be beneficial to the stressed myocardium. Our initial studies using kinase inactive forms of Src, Pyk2 and FAK expressed adenovirally in isolated primary cardiac fibroblasts showed that the pro-fibrotic signaling events as studied by fibronectin and collagen deposition are downregulated. Our in vivo studies in mouse transverse aortic constriction (TAC) model suggest that dasatinib, a multikinase NRTK inhibitor administration via a peritoneally implanted mini-osmotic pump is able to preserve ventricular geometry and function and reduce the accumulation of fibrotic extracellular matrix (ECM) proteins upon 4 wk pressure overload. Data obtained from cell culture experiments with kinase inactive NRTKs and dasatinib suggest that NRTK inhibition is able to reduce the proliferation, migration and mitogenic signaling in cardiac fibroblasts without affecting the cell survival pathways in cardiomyocytes. These data indicate that NRTKs play a significant pro-fibrotic role in cardiac fibroblasts and curbing the activity of NRTKs could be a potential therapeutic approach to treat fibrosis in hypertrophic heart diseases.
Abstract P159: Th17 Cells Are Indispensable for Cardiac Inflammation in Autoimmune Myocarditis
Authors: Yamashita Tomomi , Nakayama Hiroyuki , Fujio Yasushi
Published: July 2021

Background:
Chronic inflammation is closely associated with heart failure; however, the molecular and cellular mechanisms of inflammatory reactions remain to be fully elucidated. Interleukin(IL)-17 is a pro-inflammatory cytokine that is produced by Th17 cells. Th17 cells are induced by IL-6 and transforming growth factor(TGF-β), through up-regulation of its specific transcriptional factor, retinoic acid receptor-related orphan nuclear receptor(ROR)γt. Th17 cells are reported to participate in pathogenesis of chronic inflammatory disease. In this study, we examined the functional roles of Th17 signaling in cardiac inflammation using murine experimental autoimmune myocarditis(EAM) model.

Methods and Results:
EAM was induced in wild-type(WT) and/or RORγt-eGFP knock-in Balb/c mice by immunization twice with α-myosin heavy chain peptide. Three weeks after the first immunization, the mRNA expression of RORγt was up-regulated and correlated with the disease severity in heart. Additionally, in immunostaining and flow cytometry analysis, we observed a significant number of Th17 cells among inflammatory cells infiltrating myocardium from WT and RORγt-eGFP knock-in heterozygous mice. These data showed that Th17 cells were remarkably recruited in EAM heart. Moreover, depression of Th17 differentiation suppressed disease severity completely with inhibition of RORγt and IL-17 transcriptional activity, whereas IL-17 neutralizing antibody to lesser extent. Finally, RORγt-eGFP knock-in homozygous mice were protected from cardiac inflammation.

Conclusion:
Our results indicated that Th17 activation through RORγt pathway is a critical mechanism for the development of myocarditis. However, IL-17 is not crucial for induction of EAM, suggesting that other Th17 producing pro-inflammatory cytokines are involved. It could be proposed that Th17 cells are novel therapeutic target against chronic inflammation in heart.
Abstract P164: N-acetylglucosamine Conjugated to Nanoparticles Enhances Myocyte Uptake and Improves Delivery of a Small Molecule P38 Inhibitor for Postinfarct Healing
Authors: Gray Warren D , Che Pao Lin , Brown Milton , Ning Xinghai , Murthy Niren , Davis Michael E
Published: July 2021

An estimated 985,000 new myocardial infarctions (MI) will occur in the U.S. in 2011. While many will survive the initial insult, the early damage will eventually lead to heart failure for which the only definitive cure is transplantation. Cardiomyocyte (CM) apoptosis is a large contributor to cardiac dysfunction, and although potential therapeutic molecules exist to inhibit apoptotic pathways, drug delivery methods are lacking. This damage is largely regional and thus localized delivery of therapeutics holds great potential. However, CMs are relatively non-phagocytic, which precludes existing delivery schemes using polymeric particles that target phagocytic cells. Recently, the carbohydrate N-acetyl-glucosamine (GlcNAc) was discovered to be bound and internalized by CMs, providing a potential mechanism for drug delivery. Here we demonstrate efficacy of a drug delivery system comprising a drug-loaded biodegradable polyketal nanoparticle that is surface-decorated with GlcNAc. Inclusion of the sugar enhanced uptake by CMs in vitro as measured by intracellular activated fluorescence. When delivered in vivo following ischemia-reperfusion injury, GlcNAc-decorated particles loaded with the p38 inhibitor SB239063 reduced infarct size and improved acute cardiac function. This was in contrast to our published data demonstrating no acute effect of non-sugar decorated, p38 inhibitor-loaded particles. These data suggest a novel therapeutic option to enhance uptake of drug-loaded nanoparticles to CMs, and perhaps to reduce the large amount of CM cell death following myocardial injury.
Abstract P170: Nicorandil Inhibits Gáq-Induced Chronic Heart Failure in Transgenic Mice
Authors: Hirose Masamichi , Takeishi Yasuchika , Shimojo Hisashi , Kashihara Toshihide , Nakada Tsutomu , Mende Ulrike , Yamada Mitsuhiko
Published: July 2021

Introduction:
Beneficial effects of nicorandil on the treatment of hypertensive heart failure (HF) and ischemic heart disease have been suggested. However, whether nicorandil has inhibitory effects on HF and ventricular arrhythmias caused by the activation of G protein alpha q (Gαq) -coupled receptor (GPCR) signaling pathway still remains unknown. We examined effects of chronic and acute administration of nicorandil on the development of HF and ventricular action potential (VAP) in transgenic mice with transient cardiac expression of activated Gαq (Gαq-TG), respectively.

Method and Results:
Nicorandil (6 mg/kg/day) or vehicle was chronically administered in Gαq-TG mice for 24 weeks from 8 weeks of age, and then ventricular function, and electrical and structural changes were investigated in the hearts. Chronic nicorandil administration improved the reduction of left ventricular fractional shortening (p < 0.001) in Gαq-TG hearts. During 10 min of electrocardiogram recording, premature ventricular contractions (more than 20 beats/min) were observed in 7 of 10 vehicle-treated Gαq-TG but in none of 10 nicorandil-treated Gαq-TG hearts (p < 0.01). QT interval was significantly shorter in nicorandil-treated Gαq-TG than in vehicle-treated Gαq-TG hearts (p < 0.05). Chronic nicorandil administration improved the increased ventricular interstitial fibrosis (p < 0.05) but not cardiac hypertrophy in Gαq-TG left ventricles. Real time RT-PCR revealed that mRNA expression levels of s sulfonylurea receptor 2B (SUR-2B) were decreased in vehicle-treatd Gαq-TG but not in nicorandil-treated Gαq-TG. In addition, chronic nicorandil increased endotherial nitric oxide syntheses gene expression in Gαq-TG hearts (p < 0.05). Acute nicorandil administration (1 microM) significantly shortened the prolonged VAP duration and reduced the number of PVCs in vehicle treated Gαq-TG hearts.

Conclusions:
These findings suggest that nicorandil inhibits ventricular electrical and structural remodeling and arrhythmias through the shortening of VAP duration and the increased expression of SUR-2B and eNOS in a mouse model of HF.
Abstract P172: Circulating Heat Shock Protein 70 and Antiheat Shock Protein 70 Antibodies in Atrial Fibrillation: Relation with Atrial Fibrillation Type and Response to Catheter Ablation
Authors: Kornej Jelena , Reinhardt Claudia , Kosiuk Jedrzej , Arya Arash , Hindricks Gerhard , Adams Volker , Husser Daniela , Bollmann Andreas
Published: July 2021

Background:
HSP and anti-HSP antibodies have been associated with AF development and progression. This study investigated the possible association between circulating heat shock protein 70 (HSP70) and anti-HSP70 antibodies as well their changes and rhythm outcome after atrial fibrillation (AF) catheter ablation.

Methods:
In 67 patients with AF (59±11 years, 66 % male, 66 % lone AF) undergoing catheter ablation, circulating HSP70 and anti-HSP70 antibodies levels were quantified before and 6 months after catheter ablation. Serial 7-day Holter ECGs were used to detect AF recurrences.

Results:
At baseline, HSP70 was detectable in 14 patients (21 %), but there was no correlation between clinical or echocardiographic variables and the presence or the level of HSP70. Patients with paroxysmal AF (n=39) showed lower anti-HSP70 antibodies (median 43, IQR 28 - 62 µg/ml) than patients with persistent AF (n=28; 53, 41 - 85 µg/ml, p=.035). Using multivariable regression analysis, AF type was the only variable associated with anti-HSP70 antibodies (Beta=.342, p=.008). At 6 months, HSP70 was present in 27 patients (41 %, p<.001 vs. baseline) with an overall increase (median 0, IQR 0 - 0 vs. 0, 0 - 0.09 ng/ml, p=.029). Similarly, there was an increase of anti-HSP70 antibodies (48, 36 - 72 vs. 57, 43 - 87 µg/ml, p<.001). AF recurrence rates were higher in patients with HSP70 increase >0.025 ng/ml (32 vs. 11 %, p=.038) or anti-HSP70 antibodies increase >2.5 µg/ml (26 vs. 4 %, p=.033).

Conclusion:
HSP70 and anti-HSP70 antibodies may be involved in the progression of AF and AF recurrence after catheter ablation.
Abstract P177: Traditional Chinese Medication Qiliqiangxin Inhibits Cardiac Hypertrophy, Remodeling, and Dysfunction During Chronic Pressure Overload in Mice
Authors: Zou Yunzeng , Gong Hui , Lin Li , Zhou Ning , Li Lei , Liang Yanyan , Wu Jian , Zhou Jingmin , Ge Junbo
Published: July 2021

Qiliqiangxin (QL), a traditional Chinese medicine, has been used in the treatment of chronic heart failure. However, whether QL can prevent cardiac hypertrophy and remodeling in the hypertensive is unknown. We here compared the effects of QL with Losartan on the development of cardiac hypertrophy in a mice model of pressure overload. Constriction of transverse aorta (TAC) or sham operation was imposed to C57B/L6 mice and QL (0.6mg/Kg/day), Losartan (13.4mg/Kg/day) or vehicle was then administrated to them. Cardiac hypertrophy, remodeling, functions and fibrosis were evaluated by echocardiography, catheterization, histology, and examination of specific gene expression and ERK phosphorylation. Local apoptosis, autophagy, TNF-α/IGF-1, angiotensin II type 1 receptor (AT1-R), and especially the proliferation of cardiomyocytes and phosphorylation of ErbB2 and ErbB4 were examined
in vivo
to elucidate the mechanisms. Two weeks later, TAC resulted in a significant cardiac hypertrophy in vehicle group, which was significantly suppressed in either QL or Losartan group. At the end of 4 weeks, QL treatment effectively abrogated TAC-induced the development of myocardial remodeling, dysfunction, fibrosis, and the increases in apoptosis, autophagy, TNF-α to IGF-1 ratio and AT1-R expression, which were comparable to Losartan treatment. However, QL, but not Losartan, enhanced proliferation of cardiomyocytes at 4 weeks after TAC, which was paralleled with dowregulation of
C/EBP
β
, upregulation of
CITED4
, and increases in ErbB2 and ErbB4 phosphorylation. Thus, QL inhibits myocardial inflammation and cardiomyocyte death, and promotes cardiomyocyte proliferation, leading to an ameliorated cardiac remodeling and function in a mice model of pressure overload. The possible mechanisms may involve inhibition of AT1-R and activation of ErbB receptors.
Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart
Authors: Baskin Kedryn K , Salazar Rebecca , Chen Wenhao , Taegtmeyer Heinrich
Published: July 2021

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old,
n
=8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1
in vitro
exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.
Abstract P206: Automated Imaging Reveals a Switch Between Reversible and Irreversible Cardiac Myocyte Hypertrophy
Authors: Ryall Karen A , Saucerman Jeffrey J
Published: July 2021

Cardiac hypertrophy increases risk for heart failure, arrhythmia, and sudden death. Little is known about the specific signaling pathways that distinguish reversible forms of hypertrophy from irreversible forms which lead to heart failure. This project utilizes a novel high-throughput cell phenotype imaging and analysis protocol to study the reversibility of cardiac myocyte hypertrophy in a scalable cell culture model. Primary cardiac myocytes were transfected with GFP plasmid driven under a cardiac specific troponin T promoter. Five by five mosaic images (∼100 cells) within each well of a 96-well plate were recorded with an automated XYZ stage and focus. Post-processing algorithms automatically background corrected, segmented cell edges, quantified cell phenotypes, and tracked cells between measurements. This platform therefore has the ability to track changes in area and shape of hundreds of individual cells over a time period of about a week. Cell shape changes after washout of a dose response to the hypertrophic agonist phenylephrine (PE) showed that hypertrophy was reversible at low but not high levels of alpha-adrenergic signaling: a cellular “toggle switch”. Specialized alpha-adrenergic receptor (αAR) antagonists were used to study a potential mechanism for this biphasic response. Prazosin, an αAR antagonist that could act at the sarcolemma and also be transported inside the cell, reversed PE-induced hypertrophy. In contrast, CGP-12177a, an αAR antagonist that acts at the sarcolemma, did not reverse PE-induced hypertrophy. These experimental results and a computational model support the hypothesis that cellular uptake of PE and activity at nuclear alpha-adrenergic receptors may explain the biphasic response in the reversibility of PE-induced hypertrophy.
Abstract P210: Human Atrial Nonmyocytes Secrete Prohypertrophic, High-Molecular-Weight FGF-2, Which Is Upregulated by Angiotensin II via AT-1 and AT-2 Receptors, as Well as ERK and MMP Activation
Authors: Santiago Jon-Jon R , McNaughton Leslie J , Nickel Barbara E , Fandrich Robert R , Arora Rakesh C , Kardami Elissavet
Published: July 2021

Background:
Very little is known about the expression and role of fibroblast growth factor-2 (FGF-2) isoforms in the human heart. Using the rat model we have documented that high molecular weight Hi-FGF-2 rather than the commonly studied 18 kDa low molecular weight isoform Lo-FGF-2 is a potent inducer of cardiac hypertrophy
in vitro
and
in vivo
; and that Hi-FGF-2 is expressed and secreted predominantly by cardiac non-myocytes (fibroblasts). We have now examined (i) the expression of Hi-FGF-2 in adult human heart (atria) and heart-derived non-myocytes (HDNM), and; (ii) signals regulating Hi-FGF-2 expression in HDNM.

Methods/Results:
Atrial tissue, obtained from patients undergoing cardiac surgery, (blinded study), was used to obtain extracts, and to isolate migratory cells (fibroblastic, HDNM). All tissue extracts (n=30) contained Hi- as well as Lo-FGF-2, assessed by Western blotting. Amounts of total FGF-2 varied from 1.5 - 25.5 pg per µg of extracted protein. Immunohistochemistry of paraffin-embedded atrial tissue sections and immunofluorescence of HDNM illustrated that human Hi-FGF-2 is localized mainly in the nucleus but is also present in cytoplasm. As was the case with rat- (ventricle and/or atria) derived fibroblasts, HDNM expressed predominantly Hi-FGF-2 (90% of total). The expression/secretion of Hi-FGF-2 by HDNM, as well as by human embryonic heart-derived fibroblasts, was significantly up-regulated by angiotensin II (Ang II). Simultaneous inhibition of both AT-1 as well as AT-2 receptors (by losartan and PD123319, respectively) was required to fully prevent Ang II-induced Hi-FGF-2 up-regulation. In addition, both inhibition of ERK activation (by U0126), or MMP activity (by MMP-2 Inhibitor I) fully prevented Ang II-induced up-regulation of human Hi-FGF-2.

Conclusions:
We have shown for the first time that human heart-derived fibroblastic cells express and secrete pro-hypertrophic Hi-FGF-2 in culture; and thus are likely to do so
in vivo
. Our data also suggest that the beneficial effects of drugs targeting Ang II signal transduction may be due, in part to their effects on Hi-FGF-2 accumulation.
Abstract P219: Role of Angiotensin Type I Receptor and β
1
Integrin in Stretch-Induced Activation of Akt and JNK in Cardiac Myocytes
Authors: Gerilechaogetu FNU , Feng Hao , Golden Honey B , Cristales Ricardo J , Dasuri Kumar , Nizamutdinov Damir , Foster Donald M , Tong Carl , Dostal David E
Published: July 2021

Cardiac hypertrophy is a common response to mechanical stimuli, such as increased hemodynamic load and exercise. However, the underlying mechanotransduction processes are poorly understood. Recent studies indicate that both β
1
integrin and angiotensin type I receptor (AT
1
R) can serve as mechanoreceptors in the intact myocardium and isolated cardiac myocytes. Mechanical stress induced activation of Akt and JNK has been associated with various aspects of hypertrophic growth. We used primary cultures of NRVM plated on deformable membranes to test whether static stretch-induced activation (phosphorylation) of Akt and JNK involves β
1
integrin and/or AT
1
R. In these studies, pretreatment with the AT
1
R antagonist Losartan (10 µM), significantly decreased mechanical stretch induced activation of Akt (Akt
473
, 47.1 ± 6.1%, p<0.01 and Akt
308
, 33.1 ± 3.2%, p<0.01, n=5), but not JNK after 15 min, compared to stretch alone. Although expression of dominant-negative β
1
D
-integrin (Tac-β
1
D
) alone had no effect on stretch-induced Akt and JNK activation, the addition of Losartan to the culture medium decreased stretch-induced activation of both Akt (Akt
473
, 74.9 ± 10.4%, p<0.01 and Akt
308
, 66.1 ± 8%, p<0.01, n=5) and JNK (60.1 ± 2.5%, p<0.01, n=5), compared to stretch in lacZ expressing cells. Because Rac1 is known to be activated by mechanical stretch in NRVM, we determined whether Rac1 is required for stretch-induced Akt and JNK activation. Adenovirus-mediated expression of dominant-negative Rac1 decreased mechanical stretch induced activation of Akt (Akt
473
, 36.3 ± 7.6%, p<0.05 and Akt
308
, 29.8 ± 6.2%, p<0.05, n=5), but not JNK. Whereas, in NRVM expressing dominant-negative Rac1, Losartan pretreatment further decreased stretch-induced activation of both Akt (Akt
473
, 64.9 ± 10.4%, p<0.05 and Akt
308
, 50.8 ± 7.8%, p<0.01, n=5) and JNK (66.7 ± 11%, p<0.05, n=5). In summary, Akt activation in acutely stretched NRVM was mediated through the AT
1
R, whereas JNK activation could be mediated through either AT
1
R or β
1
integrin. The regulation Akt and JNK was also found to be significantly dependent on Rac1 activation. In conclusion, this study suggests that both AT
1
R and β
1
integrin are important mechanosensors that activate pathways that lead to cardiac myocyte hypertrophy.
Abstract P237: Exercise Training Prevents Hypercholesterolemia-Induced Cardiac Mitochondrial Dysfunction
Authors: McGee Allison M , McCommis Kyle S , Laughlin M H , Bowles Douglas K , Baines Christopher P
Published: July 2021

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We hypothesized that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response, and that exercise training could reverse this phenotype. FH pigs were obtained from the University of Wisconsin. Control, normolipidemic farm pigs were maintained on standard pig chow. After 4 months on a high-fat diet, the FH pigs were switched to the standard pig chow, and randomized to sedentary or exercise groups. The exercise group underwent a progressive treadmill-based training program for 4 months. At the end of the training protocol the animals were sacrificed and the heart removed. MPT was assessed by mitochondrial swelling in response to Ca2+. Protein nitrotyrosylation, GSH levels, and antioxidant enzyme expression were also examined. FH pigs did show an increased MPT response despite no change in the expression of putative MPT pore components adenine nucleotide translocase (ANT), mitochondrial phosphate carrier (PiC), and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation and decreased GSH levels. This was associated with concomitant decreases in the expression of mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD) and thioredoxin-2 (Trx2). However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce oxidative stress, and increase MnSOD expression. We conclude that hypercholesterolemia causes increased oxidative stress and enhances the MPT response in the porcine myocardium, and that exercise training can correct for both the increased oxidative stress and MPT alterations observed with hypercholesterolemia.
Abstract P246: Inhibition of Endothelial Senescence Ameliorates Insulin Resistance of Obese Mice
Authors: Yokoyama Masataka , Minamino Tohru , Okada Sho , Tateno Kaoru , Moriya Junji , Shimizu Ippei , Nojima Aika , Ito Takashi , Ogasawara Yoko , Komuro Issei , Kobayashi Yoshio
Published: July 2021

Various stimuli can induce irreversible cell growth arrest, termed cellular senescence. This response is controlled by negative regulators of the cell cycle such as p53. Accumulating evidence suggests a potential relationship between cellular senescence and age-associated diseases including type 2 diabetes. Here we show a crucial role for endothelial p53 in the regulation of insulin resistance. We found that treatment of endothelial cells with high glucose and palmitate synergistically increased p53 expression. Consistent with the in vitro results, endothelial expression of p53 was markedly up-regulated when the mice were fed a high-calorie diet, suggesting that excessive calorie intake promotes endothelial senescence. To investigate the role of endothelial p53 in type 2 diabetes, we analyzed metabolic parameters in endothelial cell-specific p53 conditional knockout (ECp53CKO) mice on a high-calorie diet. In spite of no difference in dietary intake, ECp53CKO mice had a significantly smaller weight and less fat accumulation than control mice. Moreover, ECp53CKO mice showed better insulin sensitivity and glucose tolerance than control littermates. ECp53CKO demonstrated a significant increase in oxygen consumption and had a higher core body temperature compared with control mice. Next we considered some assumed mechanisms of relationship of endothelial cell p53 expression and metabolic disorders. As a result, we found that ECp53CKO mice had higher glucose uptake in skeletal muscles than control. These results indicate that inhibition of endothelial senescence ameliorates insulin resistance by increasing energy consumption via glucose uptake and suggest that endothelial p53 will be a novel therapeutic target for type 2 diabetes.
Abstract P251: Mechanical Stretch Induces Phosphorylation of Cardiac Myosin Binding Protein-C
Authors: Liu Yang , Feng Hao , Gerilechaogetu FNU , Sadayappan Sakthivel , Dostal David E , Tong Carl W
Published: July 2021

Normal hearts increase contractile force in response to mechanical stretch caused by increasing volume. Although this phenomenon has been extensively studied from myofilaments’ spacing perspective, possible coupling of mechanical-sensing signaling to modulation of myofilament function remains unknown. Cardiac myosin binding protein-C (MyBPC3) is a component of heart muscle thick filament. Phosphorylation of MyBPC3 releases its inhibition on cross-bridge cycling to increase cardiac contractility. Thus, we postulate that mechanical stretch of the myocardium causes phosphorylation of MyBPC3 to increase contractility. We tested this hypothesis by performing static stretch of 20% from baseline on cultured neonatal rat cardiac myocytes (NRCM) at durations of 2, 5, 15, 30, and 60 minutes. NRCM culture provides the advantage of cells living in an environment free of adrenergic stimulation to avoid catecholamine stimulation mediated phosphorylation of MyBPC3. Site-specific phospho-serine antibodies were used to detect phosphorylation of rat equivalent of S282 and S302 of mouse MyBPC3. We used MyBPC3 antibody made from a different species than site-specific phospho-serine antibodies to account for loading. S282P transiently peaked after 5 minutes of stretch, whereas S302P continued to increase with time through 60 minutes (see
figure
). Consequently, our data show that mechanical stretch alone can cause phosphorylation of MyBPC3 as a mechanism that couples different signaling pathways to myofilament function.
Abstract P283: Parkin Mediates Mitophagy in Cardioprotection
Authors: Andres Allen M , Huang Chengqun , Ratliff Eric P , Hernandez Genaro , Lee Pamela , Gottlieb Roberta A
Published: July 2021

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.
Abstract P312: Tracheal Instillation of Diesel Exhaust Particles Exacerbates Myocardial Ischaemia and Reperfusion Injury in Rats
Authors: Robertson Sarah , Thomson Ashleigh L , Shaw Catherine A , Miller Mark R , Newby David E , Hadoke Patrick W , Gray Gillian A
Published: July 2021

Episodes of increased air pollution are associated with higher cardiovascular mortality. The adverse effects of air pollution have been attributed to particulate matter, especially ultrafine particles. This study addresses the hypothesis that ultrafine diesel exhaust particles (DEP) exacerbate myocardial ischaemia reperfusion (I/R) injury secondary to induction of a systemic inflammatory response. Wistar rats (n=5–6/group) received DEP (0.5 mg) or saline vehicle by intratracheal instillation. 6h later I/R was induced either
in vivo
or
ex vivo
in isolated buffer perfused hearts. Lung inflammation was confirmed 6h after DEP instillation by increased levels of neutrophils, total protein and IL-6 in bronchoalveolar lavage fluid. However, there was no evidence for systemic inflammation as assessed by plasma cytokine levels (IL-6, TNF-α, and CRP) or by neutrophil priming (CD11b expression) or activation (CD62L expression).
In vivo
, systolic blood pressure was significantly higher in DEP-instilled rats (129 ± 7 mmHg) than in saline controls (92 ± 3 mmHg, P<0.01), consistent with increased autonomic activation. Arrhythmias occurred intermittently after induction of ischaemia (in total 8.2±1.2 s in the saline group) and were more prevalent in DEP-instilled rats (32.9±5.0s, P<0.001). Fatal arrhythmias occurred in 60% of rats receiving DEP but not at all in saline controls. Following reperfusion, infarct size (extent of triphenyltetrazolium chloride staining) was significantly increased after DEP (34.7 ± 1.2% left ventricle) vs saline-instilled (10.3 ± 1.2%, P<0.001). Infarct size was similarly potentiated in hearts isolated from DEP instilled rats and perfused
ex-vivo
. Histological examination confirmed the absence of inflammatory infiltrate in hearts prior to I/R. Prior exposure to pollution
in vivo
thus renders the heart more vulnerable to I/R injury, either
in situ
in the body or
ex vivo
when the heart is isolated from systemic mediators and cells. Systemic inflammation does not appear to be necessary for this ‘priming’ effect of DEP. The role of autonomic activation in promoting cardiac arrythmia
in vivo
after DEP instillation and in determining the ability of the heart to withstand subsequent I/R injury merits further investigation.