title: High Glucose Attenuates Anesthetic Cardioprotection in Stem-Cell-Derived Cardiomyocytes: The Role of Reactive Oxygen Species and Mitochondrial Fission.
abstract: BACKGROUND
  Hyperglycemia can blunt the cardioprotective effects of isoflurane in the setting of ischemia-reperfusion injury. Previous studies suggest that reactive oxygen species (ROS) and increased mitochondrial fission play a role in cardiomyocyte death during ischemia-reperfusion injury. To investigate the role of glucose concentration in ROS production and mitochondrial fission during ischemia-reperfusion (with and without anesthetic protection), we used the novel platform of human-induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs).
  
  
  METHODS
  Cardiomyocyte differentiation from iPSC was characterized by the expression of CM-specific markers using immunohistochemistry and by measuring contractility. iPSC-CMs were exposed to varying glucose conditions (5, 11, and 25 mM) for 24 hours. Mitochondrial permeability transition pore opening, cell viability, and ROS generation endpoints were used to assess the effects of various treatment conditions. Mitochondrial fission was monitored by the visualization of fragmented mitochondria using confocal microscopy. Expression of activated dynamin-related protein 1, a key protein responsible for mitochondrial fission, was assessed by Western blot.
  
  
  RESULTS
  Cardiomyocytes were successfully differentiated from iPSC. Elevated glucose conditions (11 and 25 mM) significantly increased ROS generation, whereas only the 25-mM high glucose condition induced mitochondrial fission and increased the expression of activated dynamin-related protein 1 in iPSC-CMs. Isoflurane delayed mitochondrial permeability transition pore opening and protected iPSC-CMs from oxidative stress in 5- and 11-mM glucose conditions to a similar level as previously observed in various isolated animal cardiomyocytes. Scavenging ROS with Trolox or inhibiting mitochondrial fission with mdivi-1 restored the anesthetic cardioprotective effects in iPSC-CMs in 25-mM glucose conditions.
  
  
  CONCLUSIONS
  Human iPSC-CM is a useful, relevant model for studying isoflurane cardioprotection and can be manipulated to recapitulate complex clinical perturbations. We demonstrate that the cardioprotective effects of isoflurane in elevated glucose conditions can be restored by scavenging ROS or inhibiting mitochondrial fission. These findings may contribute to further understanding and guidance for restoring pharmacological cardioprotection in hyperglycemic patients.
authors.affiliation: From the *Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin; and †Departments of Physiology and Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin.
authors.forename: Scott G
authors.initials: SG
authors.lastname: Canfield
authors.forename: Ivan
authors.initials: I
authors.lastname: Zaja
authors.forename: Brian
authors.initials: B
authors.lastname: Godshaw
authors.forename: Danielle
authors.initials: D
authors.lastname: Twaroski
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Bai
authors.forename: Zeljko J
authors.initials: ZJ
authors.lastname: Bosnjak
mesh_terms: D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005947:Glucose / Q000633:toxicity*; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism / Q000473:pathology; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D007530:Isoflurane / Q000494:pharmacology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D063154:Mitochondrial Dynamics / Q000187:drug effects*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins / Q000378:metabolism; D009200:Myocardial Contraction / Q000187:drug effects; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D018685:Anesthetics, Inhalation; D000975:Antioxidants; D015415:Biomarkers; D008869:Microtubule-Associated Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; D007530:Isoflurane; D020558:GTP Phosphohydrolases; C110768:DNM1L protein, human; D034281:Dynamins; D005947:Glucose