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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Sepac
Ana
A
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
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
D018685:Anesthetics, Inhalation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D007530:Isoflurane; D054086:KATP Channels / Q000187:drug effects / Q000502:physiology; D016086:Lentivirus / Q000235:genetics; D008564:Membrane Potentials / Q000502:physiology; D042282:Microdissection; D018613:Microscopy, Confocal; D008929:Mitochondria, Heart / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D018384:Oxidative Stress / Q000502:physiology; D010101:Oxygen Consumption / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism
D018685:Anesthetics, Inhalation; D054086:KATP Channels; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D007530:Isoflurane
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