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Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells.

ABSTRACT: Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning.H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using a lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain-2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters.Differentiation yielded a high percentage (?85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling those of mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening.APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.

SUBMITTER: Sepac A

PROVIDER: S-EPMC2945423 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells.

Sepac Ana A Sedlic Filip F Si-Tayeb Karim K Lough John J Duncan Stephen A SA Bienengraeber Martin M Park Frank F Kim Jinhee J Bosnjak Zeljko J ZJ

Anesthesiology 20101001 4

<h4>Background</h4>Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning.<h4>Methods</h4>H1 hE ...[more]

PMID: 20823757

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Project description:BackgroundRepetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects from stroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance are unknown, though one exposure to hypoxia significantly increased peripheral B cell representation. For this study, we sought to determine if RHP specifically recruited B cells into the protected ischemic hemisphere, and whether RHP could phenotypically alter B cells prior to stroke onset.MethodsAdult, male SW/ND4 mice received RHP (nine exposures over 2 weeks; 8 to 11 % O2; 2 to 4 hours) or identical exposures to 21 % O2 as control. Two weeks following RHP, a 60-minute transient middle cerebral artery occlusion was induced. Standard techniques quantified CXCL13 mRNA and protein expression. Two days after stroke, leukocytes were isolated from brain tissue (70:30 discontinuous Percoll gradient) and profiled on a BD-FACS Aria flow cytometer. In a separate cohort without stroke, sorted splenic CD19+ B cells were isolated 2 weeks after RHP and analyzed on an Illumina MouseWG-6 V2 Bead Chip. Final gene pathways were determined using Ingenuity Pathway Analysis. Student's t-test or one-way analysis of variance determined significance (P < 0.05).ResultsCXCL13, a B cell-specific chemokine, was upregulated in post-stroke cortical vessels of both groups. In the ischemic hemisphere, RHP increased B cell representation by attenuating the diapedesis of monocyte, macrophage, neutrophil and T cells, to quantities indistinguishable from the uninjured, contralateral hemisphere. Pre-stroke splenic B cells isolated from RHP-treated mice had >1,900 genes differentially expressed by microarray analysis. Genes related to B-T cell interactions, including antigen presentation, B cell differentiation and antibody production, were profoundly downregulated. Maturation and activation were arrested in a cohort of B cells from pre-stroke RHP-treated mice while regulatory B cells, a subset implicated in neurovascular protection from stroke, were upregulated.ConclusionsCollectively, our data characterize an endogenous neuroprotective phenotype that utilizes adaptive immune mechanisms pre-stroke to protect the brain from injury post-stroke. Future studies to validate the role of B cells in minimizing injury and promoting central nervous system recovery, and to determine whether B cells mediate an adaptive immunity to systemic hypoxia that protects from subsequent stroke, are needed.

Dataset Information

Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells.

Publications

Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells.

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