A Cell-Based Phenotypic Assay to Identify Cardioprotective Agents

Guo, Stephanie; Olm-Shipman, Adam J.; Walters, Andrew M.; Urciuoli, William R.; DeVito, Stefanie Renee; Nadtochiy, Sergiy M.; Wojtovich, Andrew P.; Brookes, Paul S.

doi:10.1161/circresaha.111.263715

Cited by 27 publications

(23 citation statements)

References 60 publications

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“…Cellular glycolysis was determined by measuring the ECAR in NRCMs using a Seahorse Bioscience XF24 extracellular flux analyzer (Billerica) as described previously (41). An increased oxygen consumption rate (OCR) immediately following the addition of palmitate substrate to the cells was used as an indicator for FAO in the XF24 Analyzer.…”

Section: The Journal Of Clinical Investigationmentioning

confidence: 99%

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy

Wu¹,

Chang²,

Khechaduri³

et al. 2014

J. Clin. Invest.

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Section: The Journal Of Clinical Investigationmentioning

confidence: 99%

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy

Wu¹,

Chang²,

Khechaduri³

et al. 2014

J. Clin. Invest.

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“…H9c2 myoblasts, derived from embryonic rat ventricle, were an excellent model system in which to directly test the effects of V 1A R signaling during in vitro stress, as they express cardiac and skeletal isoforms of L-type Ca 21 channels, sarcolemmal ATPase splice variants characteristic of a normal heart and endogenous V 1A R, which respond to AVP with typical Ga q protein-coupled receptor responses (Hescheler et al, 1991;Sipido and Marban, 1991;Hammes et al, 1994;Tran et al, 1995;Reilly et al, 1998;Chen and Chen, 1999;Guo et al, 2012). During the development of the H/R model, we initially found that H/R increased caspase 3/7 activity and decreased cell viability with enhanced robustness over normoxic conditions only under serum-free conditions, whereas the presence of fetal bovine serum protected the cells.…”

Section: Avp Protects H9c2mentioning

confidence: 99%

Arginine Vasopressin Enhances Cell Survival via a G Protein–Coupled Receptor Kinase 2/β-Arrestin1/Extracellular-Regulated Kinase 1/2–Dependent Pathway in H9c2 Cells

et al. 2013

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Circulating levels of arginine vasopressin (AVP) are elevated during hypovolemia and during cardiac stress. AVP activates arginine vasopressin type 1A (V 1A )/Ga q -coupled receptors in the heart and vasculature and V 2 /Ga s -coupled receptors in the kidney. However, little is known regarding the signaling pathways that influence the effects of V 1A receptor (V 1A R) activation during cellular injury. Using hypoxia-reoxygenation (H/R) as a cell injury model, we evaluated cell survival and caspase 3/7 activity in H9c2 myoblasts after treatment with AVP. Pretreatment of H9c2 cells with AVP significantly reduced H/R-induced cell death and caspase 3/7 activity, effects that were blocked via both selective V 1A R inhibition and mitogenactivated protein kinase (MEK1/2) inhibition. AVP increased extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation in a concentration-dependent manner that was sensitive to MEK1/2 inhibition and V 1A R inhibition, but not V 1B R or V 2 R inhibition. Discrete elements of the V 1A /Ga q -protein kinase C (PKC) and V 1A /G protein-coupled receptor kinase (GRK)/b-arrestin signaling cascades were inhibited to dissect the pathways responsible for the protective effects of V 1A R signaling: Ga q (overexpression of Gq-I-ires-green fluorescent protein), PKC (administration of Ro 31-82425; 2-[8-(aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide, HCl, bisindolylmaleimide X, HCl), GRK2 [C-terminal GRK2 peptide overexpression and small interfering RNA (siRNA) knockdown], GRK5 (siRNA knockdown), and b-arrestin1 (siRNA knockdown). These studies demonstrated that both Ga q /PKC-and GRK2/b-arrestin1-dependent V 1A R signaling were capable of inducing ERK1/2 phosphorylation in response to AVP stimulation. However, AVP-mediated protection against H/R was elicited only via GRK2-and b-arrestin1-dependent signaling. These results suggest that activation of the V 1A R in H9c2 cells mediates protective signaling via a GRK2/b2 arrestin1/ERK1/2-dependent mechanism that leads to decreased caspase 3/7 activity and enhanced survival under conditions of ischemic stress.

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“…air) with subsequent short-term exposure to different O 2 concentrations during various treatments (drugs, photodynamic irradiation, hypoxia-reperfusion, etc.) [4,5,7,[9][10][11][12]. The consequences of long-term exposure to hyperoxic or physioxic environments on the subsequent cell killing effects of cancer treatments (e.g.…”

Section: Introductionmentioning

confidence: 99%

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

Ferguson

Smerdon

Harries

et al. 2018

Free Radical Biology and Medicine

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In vivo, mammalian cells reside in an environment of 0.5-10% O 2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O 2 ) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

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A Cell-Based Phenotypic Assay to Identify Cardioprotective Agents

Cited by 27 publications

References 60 publications

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy

Arginine Vasopressin Enhances Cell Survival via a G Protein–Coupled Receptor Kinase 2/β-Arrestin1/Extracellular-Regulated Kinase 1/2–Dependent Pathway in H9c2 Cells

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

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