Isoflurane Preconditioning Elicits Competent Endogenous Mechanisms of Protection from Oxidative Stress in Cardiomyocytes Derived from Human Embryonic Stem Cells

Abstract

Background Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients, 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 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 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.

Advances in Regenerative Medicine

“…Although the mechanisms underlying the protective effect of anaesthetic-induced preconditioning have been shown to resemble those responsible for IPC (Zaugg et al, 2003), Mullenheim et al showed that administration of sevoflurane combined with IPC can synergistically protect rabbit hearts against myocardial infarction indicating the existence of parallel protective mechanisms (Mullenheim et al, 2003). In ESC-derived cardiomyocytes, brief exposure to the commonly used volatile anaesthetic, isoflurane, significantly attenuated cell death against oxidative stress through opening of mitoKATP channels, production of signalling ROS and inhibition of mPTP opening (Sepac et al, 2010). Other benefits include enhanced cell growth capacity and increased expression of pro-angiogenic VEGF as demonstrated in human EPCs preconditioned with sevoflurane (Lucchinetti et al, 2009).…”

“…During embryonic development, hepatocytes are differentiated from the definitive endoderm and the definitive endoderm is generated from the mesendoderm, which is induced under the influence of Nodal, a transforming growth factor beta (TGF-beta) family gene product (Zaret, 2000;. The hepatocyte differentiation from the definitive endoderm or mesendoderm is induced by FGF signalling from the cardiac mesoderm and BMP-4 signalling from the septum transversum mesenchyme (Duncan, 2003;Si-Tayeb et al, 2010;Zaret, 2001). Thus, the procedure for induction of hepatocyte differentiation from pluripotent stem cells consists of two steps: the first is definitive endoderm induction by activin, which acts in a similar way to Nodal, the second is hepatocyte induction by FGF and BMP-4.…”

Cardiac Tissue Engineering

“…In addition, we found that the cardiac muscle phenotype of the P19-derived cardiomyocytes was maintained when growing on collagen, a commonly used natural material in tissue engineering A similar cardiac muscle cell enrichment strategy has been employed using human and mouse ESCs, in which a MHC promoter drove antibiotic resistance or GFP expression [39][40][41][42][43][44][45][46][47]. A previous publication reported an 99% purity for human ESC-derived cardiomyocytes used the α-MHC promoter driving neomycin resistance approach [44].…”

“…Cardiomyocytes are dependent on their cell-cell and extracellular matrix interactions for survival, and thus are also sensitive to disruption, as reviewed in Robey et al, 2008). Strategies improving cardiomyocyte survival have been reported, and include reducing cardiomyocyte loss during transplantation by using a mixture of anti-apoptotic factors and prosurvival factors , treating dissociated cardiomyocyte cultures with a ROCK inhibitor , protecting cardiomyocytes from oxidative stress using isoflurane anesthetic preconditioning (Sepac et al, 2010), co-culturing dissociated cardiomyocytes with embryonic fibroblasts to improve attachment to substrates and EB formation , re-aggregating the cells following dissociation and cell sorting (Hattori et al, 2009;, and heat shock treatment conditioning (Zhang et al, 2001). …”

Cytoprotection and Preconditioning for Stem Cell Therapy

“…Although the mechanisms underlying the protective effect of anaesthetic-induced preconditioning have been shown to resemble those responsible for IPC (Zaugg et al, 2003), Mullenheim et al showed that administration of sevoflurane combined with IPC can synergistically protect rabbit hearts against myocardial infarction indicating the existence of parallel protective mechanisms (Mullenheim et al, 2003). In ESC-derived cardiomyocytes, brief exposure to the commonly used volatile anaesthetic, isoflurane, significantly attenuated cell death against oxidative stress through opening of mitoK ATP channels, production of signalling ROS and inhibition of mPTP opening (Sepac et al, 2010). Other benefits include enhanced cell growth capacity and increased expression of pro-angiogenic VEGF as demonstrated in human EPCs preconditioned with sevoflurane (Lucchinetti et al, 2009).…”