Abstract 1845: Adult Cardiac Progenitor Cells Promote Angiogenesis and Cardioprotection through Their Secreted Soluble Vcam-1

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Katsuhisa Matsuura ◽  
Atsushi Honda ◽  
Toshio Nagai ◽  
Noritoshi Fukushima ◽  
Tatsuya Shimizu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Injury ◽  
Cell Sheet ◽  
Fold Increase ◽  
Neonatal Rat ◽  
Antibody Array ◽  
Cardiac Progenitor Cells ◽  
Protective Effects ◽  
Rat Cardiomyocytes ◽  
Promising Source

Although cardiac progenitor cells have been thought to be the promising source of cell therapy, the precise mechanisms of their paracrine action have not been fully elucidated. Since we observed that the transplantation of clonal expanded Sca-1 positive cardiac progenitor cells (cSca-1 cells) derived from adult murine heart by using cell sheet technique improved cardiac function of infarcted heart compared to adipose tissue derived mesenchymal cells (ATMC), we explored the secreted factors highly expressed in cSca-1 cells and identified that soluble VCAM-1 (sVCAM-1) was much abundant in cSca-1 cells compared to ATMC by using cytokine antibody array. cSca-1 cells-derived conditioned medium (CM) significantly enhanced endothelial migration and matrigel tube formation and these effects were abolished by knock down of VCAM-1 (Fold increase: control, 1.0; CM, 2.97 ± 0.21; siVCAM-1, 1.98 ± 0.09; siControl, 2.76 ± 0.05, p<0.01), suggesting that cSca-1 cells promote angiogenesis via their secreted sVCAM-1. We next examined whether sVCAM-1 conferred direct protective effects on cardiomyocytes. We exposed cardiomyocytes to 0.2 mM H 2 O 2 in the absence or presence of sVCAM-1 or CM and examined cardiomyocyte viability by MTT assay. The exposure of cardiomyocytes to H 2 O 2 significantly induced the cell injury. Interestingly when pretreated with sVCAM-1 or CM, the cell damages of cardiomyocytes by H 2 O 2 were significantly reduced. However when pretreated with anti-VLA4 antibody, a principal coreceptor of sVCAM-1, CM mediated cell protected effect was completely inhibited (Fold increase: control, 1.0; anti-VLA4, 0.89 ± 0.33; sVCAM-1, 1.69 ± 0.27; CM, 2.08 ± 0.28; CM+anti-VLA4, 1.07 ± 0.07, p<0.01), suggesting that a crucial role of the VLA4 in inducing survival of cardiomyocytes by CM. sVCAM-1 and CM induced phosphorylation of FAK, Akt, Erk and p38 MAPK in neonatal rat cardiomyocytes. When pretreated with wortmannin, SB203580 and PD98059, the cardioprotective effects of sVCAM-1 and CM significantly inhibited, suggesting that sVCAM-1 might protect cardiomyocytes from oxidative stress via integrated upregulation of Akt, Erk and p38MAPK. These findings suggest cardiac progenitor cells promote angiogenesis and cardioprotection through their secreted sVCAM-1.

Abstract 209: Exosomes Derived From c-kit+ Cardiac Progenitor Cells are Essential for Myocardial Repair After Acute Myocardial Function

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Sudhish Sharma ◽  
Grace E Bigham ◽  
Rachana Mishra ◽  
Flaviu Gruia ◽  
Philip Z Brohawn ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Progenitor Cells ◽  
Left Ventricular ◽  
Biologically Active ◽  
Neonatal Rat ◽  
Right Atrial ◽  
Cardiac Progenitor Cells ◽  
Myocardial Repair ◽  
Paracrine Factors ◽  
Rat Cardiomyocytes

Background: Human cardiac progenitor cells (hCPCs), identified by ckit + /CD45 - , provide a promising therapeutic option following myocardial infarction (MI) as their clinical relevance has been validated in the S tem C ell I nfusion in P atients with I schemic Cardi o myopathy (SCIPIO) Phase I clinical trial. The mechanism for their functional recovery of the injured myocardium is unknown. Hypothesis: We hypothesized whether CPCs secrete biologically active exosomes and if these exosomes could provide cardioprotection after myocardial infarction (MI). Methods and results: Exosomes were isolated from cultured CPCs, generated from the biopsies of right atrial appendage (RAA) from neonatal (nCPCs) and adult (aCPCs) patients with normal functioning myocardium. TEM showed that both CPCs secrete microvesicles, which fall within the same size range as exosomes (80-170nM, diameter). FACS performed for canonical exosomal surface markers CD63, ALIX and CD9 confirmed the presence of exosomes in the secretome of CPCs. Quantification of exosomes by Nanosight NS300 showed that nCPCs produce more than twice the amount of exosomes as compared to aCPCs in 48 hours. Exosomes were internalized by cardiomyocytes, endothelial cells and fibroblasts, within the myocardium. CPCs derived exosomes enhanced angiogenesis as analyzed by HUVEC tube assay formation and proliferation of neonatal rat cardiomyocytes while inhibiting their apoptosis in the presence of oxidative stress and inflammation. Intra-myocardial injection of exosomes into rat myocardium after MI restored ejection fraction (CPCs 63.74±3.68% vs CPCs-exosomes 62 ± 2.97%), attenuated adverse left ventricular remodeling and reduced infarct size which were comparable to CSC therapy at 28 days post MI. CPC exosomes also contain distinctive cargo of miRs and proteins. Immunoblot analysis shows that CPC exosomes are enriched in the paracrine factors VEGFA, ANG1, SCF1 and HGF1, with cardioprotective roles. Conclusion: Our findings identify exosomes as the smallest functional unit and potential biomarkers of CPC therapy. CPCs derived exosomes can be utilized as an off the shelf cell-free therapy which eliminates several shortcomings of cell therapy, including cell retention, cell rejection and arrhythmia.

scholarly journals Responses of hypertrophied myocytes to reactive species: implications for glycolysis and electrophile metabolism

2011 ◽  
Vol 435 (2) ◽  
pp. 519-528 ◽  
Author(s):  
Brian E. Sansbury ◽  
Daniel W. Riggs ◽  
Robert E. Brainard ◽  
Joshua K. Salabei ◽  
Steven P. Jones ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxygen Consumption ◽  
Energy Demand ◽  
Lactate Production ◽  
Fold Increase ◽  
Cellular Protein ◽  
Reactive Species ◽  
Neonatal Rat ◽  
Glycolytic Flux ◽  
Rat Cardiomyocytes

During cardiac remodelling, the heart generates higher levels of reactive species; yet an intermediate ‘compensatory’ stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischaemia or in the failing heart. For this, we measured energetics in control and PE (phenylephrine)-treated NRCMs (neonatal rat cardiomyocytes) under basal conditions and when stressed with HNE (4-hydroxynonenal). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a 2-fold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homoeostasis under conditions of increased oxidative stress.

Abstract 1582: Adenosine A3 Receptor Deficiency Exerts Unanticipated Protective Effects on Pressure Overloaded-Induced Left Ventricular Hypertrophy and Dysfunction in Mice

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Zhongbing Lu ◽  
John Fassett ◽  
Xin Xu ◽  
Xinli Hu ◽  
Guangshuo Zhu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ventricular Hypertrophy ◽  
Pressure Overload ◽  
Heart Association ◽  
Left Ventricular ◽  
Neonatal Rat ◽  
Protective Effects ◽  
Rat Cardiomyocytes ◽  
Extracellular Adenosine ◽  
Myocardial Oxidative Stress

Endogenous adenosine can protect the overloaded heart against the development of hypertrophy and heart failure, but the contribution of A 1 receptors (A 1 R) and A 3 receptors(A 3 R) is not known. To test the hypothesis A 1 R and A 3 R can protect the heart against systolic overload, we exposed A 3 R gene deficient (A 3 R KO) mice and A 1 R KO mice to transverse aortic constriction (TAC). Contrary to our hypothesis, A 3 R KO attenuated 5 weeks TAC-induced left ventricular (LV) hypertrophy (ratio of ventricular mass/body weight increased to 7.6 ±0.3 mg/g in wild type (Wt) mice as compared with 6.3±0.4 mg/g in KO), fibrosis and dysfunction (LV ejection fraction decreased to 43±2.5% and 55±4.2% in Wt and KO mice, respectively). A 3 R KO also attenuated the TAC-induced increases of myocardial ANP and the oxidative stress markers 3-nitrotyrosine(3-NT ) and 4-hydroxynonenal. In addition, A 3 R KO significantly attenuated TAC-induced activation of multiple MAP kinase pathways, and the activation of Akt-GSK signaling pathway. In contrast, A 1 R-KO increased TAC-induced mortality, but did not alter ventricular hypertrophy or dysfunction compared to Wt mice. In mice in which extracellular adenosine production was impaired by CD73 KO, TAC caused greater hypertrophy and dysfunction, and increased myocardial 3-NT, indicates that extracellular adenosine protects heart against TAC-induced ventricular oxidative stress and hypertrophy. In neonatal rat cardiomyocytes induced to hypertrophy with phenylephrine, the adenosine analogue 2-chloroadenosine (CADO) reduced cell area, protein synthesis, ANP and 3-NT. Antagonism of A3R significantly potentiated the anti-hypertrophic effects of CADO. Our data demonstrated that extracellular adenosine exerts protective effects on the overloaded heart, but A 3 R act counter to the protective effect of adenosine. The data suggest that selective attenuation of A 3 R activity might be a novel approach to attenuate pressure overload-induced myocardial oxidative stress, LV hypertrophy and dysfunction. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

Abstract 81: End-Stage Human Failing Heart Increases Functional Cardiac Progenitor Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Rachana Mishra ◽  
David Simpson ◽  
Sudhish Sharma ◽  
Evan Colletti ◽  
Sunjay Kaushal
Keyword(s):  
Progenitor Cells ◽  
Fold Increase ◽  
Myosin Heavy Chain Isoforms ◽  
Functional Improvement ◽  
Cardiac Progenitor Cells ◽  
Failing Heart ◽  
Normal Human ◽  
Fetal Reprogramming ◽  
The Right ◽  
End Stage

Background: Human cases of end-stage heart failure provide the rare opportunity to test whether the cells within these hearts exhibit different phenotypic characteristics than those in healthy hearts. We tested whether the failing heart, as it reverses to a well-known fetal reprogramming state, also compensates by increasing the number of functional cardiac progenitor cells. Methods and Results: We examined samples from end-stage human failing hearts and also normal human hearts to quantitate the expression of various cardiac progenitor markers, by immunofluorescence, flow cytometry and RT-PCR. We further tested whether the cardiac progenitor cells were functional in a rodent model of myocardial infarction. All the failing end-stage hearts (N=13) reversed to a fetal state by switching their myosin heavy chain isoforms from beta to alpha. Additionally, atrial natriuretic factor was increased. Compared to normal congenital myocardium, failing end-stage hearts had a 2 to 5 fold increase in the number of C-kit+ and ISL-1+ cardiac progenitor cells (P<0.5). The numbers of cardiac progenitor cells was highest in the right atria as compared to other chambers of the end-stage heart. Cardiac progenitor cells isolated from failing hearts expressed several stemness markers that were upregulated compared to normal human hearts. FACS and IF analysis demonstrated significantly (P<0.5) higher c-kit expression in CDCs derived from end stage patients compare to normal congenital myocardium. Also, there was a tendency for increased FLK1 and Sca-1 expression in CM patients respectively. Transplanted cardiac progenitor cells from end-stage hearts promoted greater myocardial regeneration and functional improvement in the infarcted rat myocardium than transplanted cardiac progenitor cells derived from normal congenital patients (EF=57+3 vs. 41.5±3, P<0.05). Conclusion: Our results show an increase of progenitor cells within the end-stage heart that have the functional potential to regenerate the myocardium. Stimulating the differentiation and increasing the population of cardiac progenitor cells may provide a novel therapeutic strategy for these end-stage hearts.

Abstract 18698: Identification of a Novel Cardiac Progenitor Population Expressing Pw1/peg3 in the Murine Heart

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Elisa Yaniz-Galende ◽  
Luigi Formicola ◽  
Nathalie Mougenot ◽  
Lise Legrand ◽  
Jiqiu Chen ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Fold Increase ◽  
Cardiac Regeneration ◽  
Cardiac Repair ◽  
Cardiac Progenitor Cells ◽  
Average Percentage ◽  
Pathological Conditions ◽  
Reporter Mice ◽  
Novel Target

The myocardium responds to injury by recruiting cardiac progenitor cells (CPCs) to the injured tissue to promote cardiac repair. Although different classes of CPCs have been identified, their contribution in physiological and pathological conditions remains unclear. PW1 gene has recently been proposed as a marker of resident adult stem and progenitor cell populations in several adult tissues. Our goal was to characterize and determine the role of PW1+ population in the heart. Here, we employ immunostaining and fluorescence-activated cell sorting (FACS) analysis in PW1-reporter mouse to perform qualitative and quantitative analyses of PW1+ population in the heart. We first found that PW1+ cells are mainly located in the epicardium and myocardial interstitium of normal hearts. The average percentage of PW1+ cells, as assessed by FACS, was 1.56±1.41%. A subset of PW1+ cells also co-express other CPC markers such as Sca-1 (52±22%) or PDGFR1α (43±14%). In contrast, a very small proportion of PW1+ cells co-express c-kit (6±5%). To investigate the contribution of PW1+ cells in pathological conditions, we then performed myocardial infarction (MI) by LAD ligation in PW1-reporter mice. We found that MI resulted in a 3-fold increase in the number of PW1+ cells in infarcted mice compared with sham-operated groups, at 1 week post-MI (1.16%±0.47% in sham versus 3.43%±0.82 in MI). This population preferentially localized in the injured myocardium and border area. PW1+ cells were isolated by FACS from the whole infarcted heart from PW1-reporter mice. In vitro differentiation assays reveal that purified PW1+ cells are multipotent and can spontaneously differentiate into smooth muscle cells, endothelial cells and cardiomyocyte-like cells. Taken together, our data identify a novel PW1+ cardiac progenitor population with the potential to undergo differentiation into multiple cardiac lineages, suggesting their involvement in cardiac repair in normal and pathological conditions. The discovery of a novel population of cardiac progenitor cells, augmented following MI and with cardiogenic potential, provides a novel target for therapeutic approaches aimed at improving cardiac regeneration.

scholarly journals Sophoricoside ameliorates cardiac hypertrophy by activating AMPK/mTORC1-mediated autophagy

2020 ◽  
Vol 40 (11) ◽  
Author(s):  
Maomao Gao ◽  
Fengjiao Hu ◽  
Manli Hu ◽  
Yufeng Hu ◽  
Hongjie Shi ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Hypertrophy ◽  
Cardiac Fibrosis ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Dependent Manner ◽  
Protective Effects ◽  
Rat Cardiomyocytes ◽  
Pathological Cardiac Hypertrophy ◽  
Compound C

Abstract Aim: The study aims to evaluate protective effects of sophoricoside (Sop) on cardiac hypertrophy. Meanwhile, the potential and significance of Sop should be broadened and it should be considered as an attractive drug for the treatment of pathological cardiac hypertrophy and heart failure. Methods: Using the phenylephrine (PE)-induced neonatal rat cardiomyocytes (NRCMs) enlargement model, the potent protection of Sop against cardiomyocytes enlargement was evaluated. The function of Sop was validated in mice received transverse aortic coarctation (TAC) or sham surgery. At 1 week after TAC surgery, mice were treated with Sop for the following 4 weeks, the hearts were harvested after echocardiography examination. Results: Our study revealed that Sop significantly mitigated TAC-induced heart dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis. Mechanistically, Sop treatment induced a remarkable activation of AMPK/mTORC1-autophagy cascade following sustained hypertrophic stimulation. Importantly, the protective effect of Sop was largely abolished by the AMPKα inhibitor Compound C, suggesting an AMPK activation-dependent manner of Sop function on suppressing pathological cardiac hypertrophy. Conclusion: Sop ameliorates cardiac hypertrophy by activating AMPK/mTORC1-mediated autophagy. Hence, Sop might be an attractive candidate for the treatment of pathological cardiac hypertrophy and heart failure.

Abstract 319: Mirna-125b Reduces Ischemia / Reperfusion (i/r)-induced Apoptosis Through Targeting Multiple Targets in Intrinsic and Extrinsic Apoptosis Pathways

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Peipei Wang ◽  
Qiying Chen ◽  
Yue Zhou ◽  
Arthur M Richards
Keyword(s):  
Cell Count ◽  
Cell Injury ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Protective Effects ◽  
Extrinsic Pathway ◽  
Multiple Genes ◽  
Induced Apoptosis

Apoptosis is mediated through extrinsic and intrinsic pathways, both play a role in ischemia/reperfusion (I/R) injury. Predicted targets for miRNA-125b include extrinsic pathway mediators Traf6 and Tnfrsf1b, and intrinsic mitochondria regulators Bcl-2 family pro-apoptotic effectors Bak1 and BH3-only facilitators Bim, Bmf, Puma. We hypothesized that miRNA-125b directly targets multiple genes to reduce I/R-induced apoptosis. Myoblast H9c2 cells underwent 16 hours 0.2% O 2 hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R) and were transfected with miRNA-125b mimic vs. scrambled mimic control (25 nmol, miR-125b-M vs. MC) and miR-125b inhibitor vs. inhibitor control (miR-125b-I vs. IC). Cell count/viability, WST assay, cell injury-induced LDH release and apoptotic marker Casp3/7 were measured. Cells were trypsinized for assessment of apoptosis (7-AAD and annexin V double staining) and lysed for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Bak1 plasmids were cloned and transfected into H9c2 for recovery studies. The effects were verified in neonatal rat ventricular myocytes (NRVM). miRNA-125b-M significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-125b-M vs. MC p<0.05). qPCR confirmed that (1) miR-125b expression was reduced in H-R; (2) RISC-loaded (immunoprecipitation pull-down Ago-2) miR-125b increased by ~35 fold and reduced to ~3% following mimic and inhibitor transfection respectively; (3) multiple apoptosis-related genes were reduced by miR-125b-M, Bak1, Bmf, Bim, Puma, Traf6 and Tnfrsf1b. All changes were confirmed by WB. Luciferase reporter assays indicated miR-125b bound to the 3’-UTR of all genes tested except Traf6. Total apoptotic cell numbers and Casp3/7 release were significantly reduced by miR-125b-M. The protective effect of miRNA-125b was partially abolished by Bak1 overexpression (pCMV-Myc-Bak1 and miR-125b co-transfection). Protective effects of miRNA-125b were further verified in NRVM. MiRNA-125b inhibitor reversed protective effects and target changes at mRNA and protein level. miR-125b is powerfully cardioprotective in I/R injury due to directly targeting multiple genes in the extrinsic and intrinsic apoptotic pathways.

Abstract P379: Inhibition Of O-glcnac Transferase Ogt Activates P38 Signaling In Neonatal Rat Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Kyriakos Papanicolaou ◽  
Natasha Zachara ◽  
Deepthi Ashok ◽  
Agnes Sidor ◽  
D B Foster ◽  
...  
Keyword(s):  
Heart Failure ◽  
Intracellular Signaling ◽  
Ventricular Myocytes ◽  
Fold Increase ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Creb Phosphorylation ◽  
Glcnac Transferase

The mitogen activated protein kinase (MAPK) p38 is important in cardiac hypertrophic responses and p38 inhibition has been tested as a potential therapeutic approach to heart failure. p38 is tightly regulated by upstream kinases and phosphatases. While p38 inhibitors suppress cardiac hypertrophy in vitro and in animal models, the partial efficacy of p38 inhibitors in clinical trials for heart failure illustrates the need for a deeper understanding of p38-regulatory mechanisms. O -linked N-Acetylglucosamine ( O -GlcNAc) on Ser/Thr residues is a ubiquitous intracellular modification ( O -GlcNAcylation) that participates in intracellular signaling, often occurring in counterpoint to phosphorylation. O -GlcNAcylation is catalyzed by O -GlcNAc Transferase (OGT) and removed by O -GlcNAc-Ase (OGA). Given the crucial regulation of p38 activity by phosphorylation, we hypothesized that O -GlcNAcylation regulates p38 phosphorylation during basal and hypertrophic cardiomyocyte signaling. Treating neonatal rat ventricular myocytes (NRVM) with OSMI-1 (inhibitor of OGT) significantly decreased O -GlcNAcylation (0.48 ± 0.02, P <0.001 vs. vehicle), whereas treatment with Thiamet-G (inhibitor of OGA) significantly increased O -GlcNAcylation (3.0-fold increase ± 0.35, P <0.05 vs. vehicle). OSMI1 treatment induced the phosphorylation of p38 at its activation site (3.9-fold increase ± 0.46, P <0.001 vs. vehicle) and promoted the phosphorylation of the downstream target, heat shock protein Hsp27 (8-fold increase ± 1.3, P <0.0001 vs. vehicle) and transcription factor Creb (3.3-fold increase ± 0.12, P <0.001 vs. vehicle). OSMI-1 had an additive effect in inducing p38 and Creb phosphorylation following hypertrophic stimulation by phenylephrine (3.1-fold and 1.4-fold increase vs. phenylephrine respectively, P <0.05). Treatment with the p38 inhibitor SB202190 abolished the phosphorylation of Hsp27 and Creb that was induced by OSMI-1. Canonical upstream activators of p38 include the MAP3Ks, TAK1 and ASK1. However, we found that treatment with ASK1 or TAK1 inhibitors (GS-444217 and Takinib, respectively) either alone, or in combination, did not negate the phosphorylation of p38 by OSMI-1. We conclude that regulation of p38 by OGT activity could occur at a level downstream of canonical MAP3Ks or through non-canonical pathways.

Abstract 546: S-palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of Gc-1 in Cardiomyocytes

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Christos Tzimas ◽  
Ilaria Russo ◽  
Wen Dun ◽  
Sevde Yilmaz ◽  
Sudarsan Rajan ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Redox Regulation ◽  
Soluble Guanylyl Cyclase ◽  
Fold Increase ◽  
Transcript Level ◽  
Neonatal Rat ◽  
Membrane Microdomains ◽  
Post Translational Modification ◽  
Cysteine Oxidation ◽  
Rat Cardiomyocytes

Objectives: The nitric oxide (NO) receptor soluble guanylyl cyclase (GC-1) can localize to cell membrane microdomains, including caveolae. In failing hearts, GC-1 relocalizes away from caveolae and becomes oxidized. How this occurs is unknown. We hypothesized that S-palmitoylation, a reversible, enzyme-catalyzed, post-translational modification that enhances membrane affinity of protein substrates, mediates caveolae-localization and modulates redox regulation of GC-1. Methods: Adult male C57BL/6 mice were subjected to transverse aortic constriction (TAC) versus thoracotomy (Sham). Modified acyl-biotin exchange (ABE) assays were performed to assess S-palmitoylation of GC-1 in failing TAC vs non-failing Sham hearts. Using bioinformatics, we identified 5 conserved cysteines in the GC-1α subunit as potential S-palmitoylation sites. We created cysteine-serine mutants of GC-1α at each and all 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). Mutants and WT GC-1α were expressed in neonatal rat cardiomyocytes (NRCM) and their palmitoylation status compared by ABE. We assessed localization and oxidation of GC-1α (CS* vs WT) by confocal microscopy and redox Western, respectively. Results: In TAC hearts, S-palmitoylation of GC-1α was half of that in Sham ( p =0.02). Likewise, palmitoyl-transferase ZDHHC16 was downregulated in TAC vs Sham by 31% ( p =0.01) at the transcript level and by 56% ( p =0.03) at the protein level. In NRCMs, S-palmitoylation of CS* was markedly diminished (6.9%, p <0.001), while that of C15S, C176S, and C497S were modestly diminished (23.8%, p=0.03; 23.2%, p=0.01; 22.8%, p=0.01), compared to WT (54.7%). Moreover, S-palmitoylation of WT but not CS* was decreased by palmitoylation inhibitor 2-BP (17.8%, p<0.0001). Confocal microscopy showed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed increased oxidation of CS* vs WT upon H 2 O 2 treatment (1.7-fold increase, p=0.008). Conclusions: Our data support that GC-1α is S-palmitoylated at C15, C176, and C497. S-palmitoylation of GC-1α at these 3 cysteines appears to protect GC-1α against oxidation and to mediate its membrane localization.

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