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.

Gastrulating chick embryo as a model for evaluating teratogenicity: a comparison of three approaches.

BACKGROUND: Teratology studies must be carefully designed to minimize potential secondary effects of vehicle and delivery routes. A systematic method to evaluate chick models of early embryogenesis is lacking. METHODS: We investigated 3 experimental approaches that are popular for studies of early avian development, in terms of their utility for teratogen assessment starting at gastrulation. These included in vitro embryo culture, egg windowing followed by direct application of a carrier vehicle to the embryo, and injection of a carrier vehicle into the egg yolk. We also developed a morphologically based scoring system to assess development of the early embryo. RESULTS: The in vitro culture and egg windowing approaches both caused an unacceptably high incidence of central nervous system and cardiac abnormalities in vehicle-treated embryos, which made it difficult to identify teratogen-specific defects. In contrast, exposing chick embryos to vehicle via direct egg yolk injection did not induce developmental anomalies. CONCLUSIONS: Optimization of the exposure route of potential toxicants to the embryo is critical because control treatments can cause developmental anomalies. In ovo yolk injection minimizes perturbation of young embryos and may be appropriate for teratogen delivery.

Transient expression of TIP60 protein during early chick heart development.

Screening of an embryonic chick cDNA library revealed a gene product termed chick TIP60 (cTIP60) due to its homology with human TIP60, a founding member of the "MYST" family of proteins that possess functional motifs, including chromo, zinc finger, and histone acetyltransferase domains. cTIP60 expression was assessed during early chick embryogenesis, at the RNA level by using reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level by using Western blotting and immunohistochemistry. RT-PCR indicated that cTIP60 transcripts in whole embryos are present as early as Hamburger-Hamilton (HH) stage 5, diminishing after HH10. Western blotting of total embryonic protein revealed that cTIP60 was present in uniform quantities between HH3 and HH25. By contrast, Western blotting of protein from isolated hearts revealed that cTIP60 protein was strongly expressed at the earliest stages of heart development (HH11-13), diminishing thereafter. This finding was corroborated by immunohistochemistry, which revealed that cTIP60 protein was selectively expressed at high levels in the myocardium between HH 10-14. Considered in the context of its functional domains, these findings suggest that cTIP60 modulates transcriptional processes which regulate terminal cell differentiation, proliferation, or both, during early myocardial development.