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High Glucose Attenuates Anesthetic Cardioprotection in Stem-Cell–Derived Cardiomyocytes: The Role of Reactive Oxygen Species and Mitochondrial Fission

Canfield, Scott G. PhD; Zaja, Ivan MD; Godshaw, Brian BA; Twaroski, Danielle PhD; Bai, Xiaowen MD, PhD; Bosnjak, Zeljko J. PhD

doi: 10.1213/ANE.0000000000001254
Cardiovascular Anesthesiology: Research Report

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.

From the *Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin; and Departments of Physiology and Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin.

Accepted for publication February 2, 2016.

Funding: This work was supported in part by P01GM066730 and R01HL034708 from the National Institutes of Health, Bethesda, MD (to Dr. Bosnjak), and by grant R01GM112696 from the National Institutes of Health (to Dr. Bai).

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

Drs. Canfield and Zaja contributed equally to this work.

Reprints will not be available from the authors.

Address correspondence to Xiaowen Bai, MD, PhD, Departments of Physiology and Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53225. Address e-mail to

Diabetes mellitus contributes to >240,000 deaths a year with approximately 70% of those deaths attributable to cardiovascular disease and another 17% attributable to stroke.1,2 Volatile anesthetics are cardioprotective in the setting of a wide variety of stressors,3–9 but diabetes mellitus is a notable exception, a stressor in which volatile anesthetics are not cardioprotective. The cardioprotective effects of volatile anesthetics are attenuated in the presence of diabetes mellitus via mechanisms that are largely unknown.3,10–12

A substantial amount of laboratory data from various animal models demonstrates that high glucose concentrations induce an increase in mitochondrial reactive oxygen species (ROS) production contributing to attenuation of anesthetic cardioprotection.10,13–17 Other data suggest that administration of antioxidants may restore the cardioprotective effects of volatile anesthetics in hyperglycemic animals.14 Also, hyperglycemia-induced ROS generation and dynamin-related protein 1 (Drp1)–mediated mitochondrial fission have been shown to open the mitochondrial permeability transition pore (mPTP), resulting in cell death in rat cardiomyocytes.17

Most studies examining a lack of anesthetic-mediated cardioprotection in diabetes mellitus/hyperglycemia have been conducted in preclinical models.10,13,18–20 Although these studies are valuable, it is important to study human cardiomyocytes because there may be significant species differences. On the basis of recent progress in reprogramming to an induced pluripotent stem cell (iPSC) state from somatic cells, human cardiomyocytes can be differentiated using a variety of published protocols.21–24 These iPSC-derived cardiomyocytes (iPSC-CMs) resemble terminally differentiated human cardiomyocytes in many aspects23,25–27 and provide an in vitro human experimental model. We previously showed cardioprotection with isoflurane in human embryonic stem-cell–derived and iPSC-derived cardiomyocytes to a similar level as that observed in human myocardium.25,26,28 In addition, high glucose concentrations attenuated the cardioprotective effects conferred by isoflurane in iPSC-CMs.25 However, the underlying mechanisms remained unknown.

Thus, the aim of this study was to investigate the role of hyperglycemia in the attenuation of isoflurane-induced cardioprotection in a relevant human model. We hypothesized that high glucose concentration-induced ROS generation and mitochondrial fission contribute to the attenuation of cardioprotection in iPSC-CMs. In addition, we hypothesized that scavenging ROS or inhibiting mitochondrial fission in the presence of high glucose would restore isoflurane protection of iPSC-CMs.

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Human iPSC Cell Culture and Differentiation

All human cell experiments were approved by the institutional review board at the Medical College of Wisconsin. Human foreskin fibroblasts were reprogrammed as previously reported.29,30 iPSCs were a generous gift from Dr. Stephen Duncan, PhD (Medical College of Wisconsin, Milwaukee, WI). iPSCs were cultured as previously described.26 Before initiating cardiac differentiation, iPSCs were detached with Accutase (Innovative Cell Technologies, San Diego, CA) and seeded on Matrigel-coated (BD Biosciences, San Jose, CA) plates in mTeSR1 medium (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 10 μM ROCK inhibitor Y-27632 (EMD Millipore, Billerica, MA). Medium was changed daily for 4 days, and cells were incubated in 4% O2 and 5% CO2. At approximately 90% confluence, the medium was changed to mTeSR1 with the addition of Matrigel for 24 hours (matrix sandwich method), and cells were incubated in 20% O2 and 5% CO2. Then on day 0 of differentiation, the medium was changed to Roswell Park Memorial Institute (RPMI) medium supplemented with B27 supplement (RPMI/B27) without insulin (Life Technologies, Carlsbad, CA) with added Matrigel and activin A (100 ng/mL) (R&D Systems, Minneapolis, MN) for 24 hours. At day 1, the medium was changed to RPMI/B27 without insulin but with basic fibroblast growth factor (5 ng/mL) (Life Technologies) and bone morphogenetic protein-4 (5 ng/mL) (R&D Systems). Cells were cultured in 20% O2 and a 5% CO2 incubator for 4 days. On day 5, medium was changed to RPMI/B27 with insulin and changed every 2 days. Cells typically started contracting around day 12 to 14.21

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Microdissection and Single-Cell Dissociation

One month into differentiation, contracting cardiomyocytes were mechanically dissociated using a 27-gauge needle under a dissecting microscope (SMZ1000; Nikon, Tokyo, Japan). Dissected clusters were enzymatically dispersed in 0.05% trypsin-EDTA (Invitrogen, Waltham, MA) for 5 minutes. Trypsin was inactivated by Dulbecco Modified Eagle Medium containing 20% fetal bovine serum, and individual cells were plated onto Matrigel-coated glass coverslips (25,000 cells/cm2).

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Cardiomyocyte Differentiation Markers by Immunocytochemistry

Cells cultured on Matrigel-coated glass coverslips were fixed with 1% paraformaldehyde for 30 minutes and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and incubated with 10% donkey serum for 30 minutes to reduce nonspecific staining. Cells were incubated with primary antibodies for sarcomeric α-actinin (1:100 dilution; Sigma-Aldrich) and anti–cardiac-specific troponin T (1:100; Thermo Fisher Scientific, Waltham, MA) for 1 hour at 37°C. After 3 washes with phosphate-buffered saline, cells were incubated with fluorescently labeled secondary antibody Alexa Fluor 594 (1:1000; Invitrogen) for 1 hour at room temperature. Nuclei were counterstained with TOPRO-3 (1:1000; Invitrogen). Coverslips were then mounted onto slides and images acquired with a laser-scanning confocal microscope (Nikon Eclipse TE2000-U; Nikon).

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Genetic Marking of iPSC-CMs with a Lentiviral Vector

Genetic marking of iPSC-CMs was conducted similar to that in our previous study.26 The sodium-calcium exchanger 1 (NCX-1)-driven enhanced green fluorescent protein (eGFP) cassette was subcloned into lentiviral transfer plasmid pHR(+)c.Ub.MCSoligo.R(−)W(+). The NCX-1 promoter is specific for ventricular cardiomyocytes.31 Lentiviral vector assembly and titering were performed as previously described.32,33 One day after differentiation, cardiomyocytes were transduced with a lentiviral vector encoding human NCX-1–driven eGFP (multiplicity of infection, 1.8 × 104) (pNCX-1/eGFP) (provided by Dr. Eduardo Marbán, MD, PhD, Cedars-Sinai Hospital, Los Angeles, CA).

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Experimental Design

Twenty-four hours after lentiviral transduction, 11 mM glucose RPMI/B27 medium was replaced with RPMI/B27 medium containing 5 mM glucose. Cells were cultured for 4 days with daily medium changes. In 12-well plates, the cells were divided into 3 groups containing 5 mM glucose with 20 mM mannitol, 11 mM glucose with 14 mM mannitol, or 25 mM of glucose to normalize osmotic balance. iPSC-CMs were cultured in each condition for 24 hours.

Appropriate volumes of isoflurane stock solution (Baxter, Deerfield, IL) were sonicated into culture media to achieve 0.5 mM (approximately 1 minimum alveolar concentration) concentration like in previous studies.26,34 Media without isoflurane (control) was simultaneously sonicated. At the end of each experiment, isoflurane concentration was analyzed by gas chromatography. iPSC-CMs in each glucose group were then subjected to either exposure to 0.5 mM isoflurane in media or control media (without isoflurane) for 30 minutes. After control and isoflurane exposure, the medium was exchanged for 30 minutes with culture medium containing 5, 11, or 25 mM of glucose, but without isoflurane, to remove any remaining isoflurane.

iPSC-CMs were then subjected to oxidative stress conditions for 2 hours using 100 μM hydrogen peroxide (H2O2) (Calbiochem, La Jolla, CA) and 10 mM 2-deoxy-glucose (Sigma-Aldrich), an inhibitor of glycolysis. Control cultures did not receive these treatments.

Figure 1

Figure 1

Trolox (250 μM; Sigma-Aldrich), a scavenger of ROS, or mdivi-1 (50 μM; Sigma-Aldrich), a Drp1 inhibitor (mitochondrial division inhibitor-1), was added immediately after the 24 hours oxidative stress. iPSC-CMs were then returned to their original glucose groups for 4 hours. Then cell viability, ROS generation, and mitochondrial fission assays were all conducted for all groups. Experimental protocols are summarized in Figure 1.

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Lactate Dehydrogenase Assay

Lactate dehydrogenase (LDH) activity was used as an assay of iPSC-CM cytotoxicity. iPSC-CMs were plated at 1 × 104 cells per well in 96-well plates and cultured in 100 μL of RPMI/B27 medium supplemented with the appropriate glucose concentrations as previously discussed. LDH was measured via a colorimetric cytotoxicity assay kit according to the manufacturer’s directions (Roche Diagnostics Corporation, Indianapolis, IN).

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Laser-Scanning Confocal Microscopy for Mitochondria Membrane Potential and mPTP Opening Assays

Four days after the lentiviral vector transduction, confocal imaging was used to identify living iPSC-CMs by NCX-1–driven eGFP expression.26 Tetramethylrhodamine ethyl ester (TMRE; 30 nM; Invitrogen) was used to evaluate mitochondrial membrane potential (ΔΨm). The opening of the mPTP was assessed as described previously in our laboratory,35 a method based on mPTP induction by photoexcitation-generated oxidative stress.36–39 The mPTP opening was detected by rapid dissipation of ΔΨm, observed as a loss of TMRE fluorescence, which is sensitive to inhibition of mPTP opening.35 The data were analyzed with Image J software (National Institutes of Health, Bethesda, MD).

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Western Blotting

Cultured cells were washed with phosphate-buffered saline and lysed in ice-cold lysis buffer (20 mM Tris-HCI, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were then sonicated and centrifuged for 20 minutes at 14,000 g. Supernatant was collected and protein concentration was determined using Bradford assay (Bio-Rad, Hercules, CA) according to the protocol provided by the company. Proteins (30 μg/lane) were separated on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat milk or 5% bovine serum albumin for 1 hour at room temperature, then probed overnight with primary antibodies against phosphoSer616 Drp1 (pSer616 Drp1) (Cell Signaling, Danvers, MA), Drp1 (BD Transduction Laboratories, Lexington, KY), β-actin (Abcam, Boston, MA) followed by appropriate secondary antibodies conjugated to horseradish peroxidase. Bands were visualized using enhanced chemiluminescence (Pierce, Rockford, IL), and intensities were analyzed using ImageJ 1.41. Data are shown in arbitrary units.

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Mitochondrial Morphology Measurement

Cells (4 × 104) were plated on glass coverslips coated with Matrigel. To visualize mitochondrial morphology, cells were incubated in 30 nM TMRE.40,41 Images were captured with Nikon TE2000 inverted microscope (514-nm excitation/585-nm emission) using 60× oil objective (numerical aperture 1.4) and analyzed with ImageJ. Images were contrast-enhanced with 0.5% saturation level, convoluted with 5 × 5 Laplacian kernel, with nonzero diagonal terms, which defines structures based on abrupt changes in fluorescence. Bandpass filtering was applied to remove structures below optical resolution limit of 0.2 μm, and then images were segmented by thresholding and binarized. The morphology of each mitochondrion was described by 2 factors: aspect ratio (major/minor axis of ellipse) and form factor (4π × area/perimeter2).42 Both factors have a minimal value of 1, which represents perfect circle. Whereas aspect ratio (AR) is an indicator of length, form factor (FF) denotes length and branching. Hence, low values of AR and FF signify fragmented, unbranched mitochondria, and higher values mean more elongated, branched mitochondria.43,44 For each group, 50 random cells from 3 independent experiments were analyzed.

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ROS Measurements

The levels of ROS production in iPSC-CMs were evaluated using the fluorescent probe dihydroethidium (DHE) (Molecular Probes, Grand Island, NY). Cells were loaded with 5 μM DHE for 25 minutes at room temperature in RPMI. Images were acquired with a confocal microscope and analyzed using ImageJ software.

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Statistical Analysis

Data are presented as mean ± SD. Each experimental group consisted of iPSC-derived CMs from ≥3 separate differentiations. For the statistical analyses, SigmaStat 3.0 software (Systat Software, Inc., San Jose, CA) was used. Statistical comparisons were performed using 1-way analysis of variance (ANOVA) from the pooled data. Equal variance and Shapiro-Wilk normality assumptions were satisfied for the residuals of each ANOVA model with a P value of 0.05 to reject. Within each glucose condition, all pairwise comparisons were conducted within the ANOVA context using post hoc t tests with the pooled variance estimate followed by Holm-Sidak stepdown correction for multiple testing. Adjusted P values are displayed in the text, and all unadjusted and adjusted P values along with 99% confidence intervals can be found in the Supplemental Digital Content ( P value <0.05 was considered significant.

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Characterization of Cardiomyocytes Derived from iPSCs

Figure 2

Figure 2

iPSC-CM contractility was seen within 2 weeks of differentiation. Immunostaining verified the presence of the cardiac proteins: cardiac-specific troponin T and sarcomeric α-actinin (Fig. 2A). Dissociated cardiomyocytes were labeled with a lentiviral vector expressing eGFP (Fig. 2, B and C). Approximately 80% to 90% of dissociated cells expressed eGFP, indicating a high efficiency of differentiation (Fig. 2D). Living eGFP-expressing cells were used for subsequent confocal studies (Fig. 2C).

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Isoflurane Delayed mPTP Opening and Protected iPSC-CMs Against Oxidative Stress in 5 and 11 mM but Not in 25 mM Glucose

Isoflurane-induced delay in mPTP opening directly translates to a reduction in cell death after an ischemia–reperfusion injury.26,35,45 Isoflurane treatment delayed the mPTP opening time in iPSC-CMs in the 5- (P < 0.001) and 11-mM (P < 0.001) glucose groups compared with control without isoflurane treatment. However, isoflurane-induced delay in mPTP opening was not observed in iPSC-CMs in the 25-mM glucose group (P > 0.99) (Fig. 3A).

Figure 3

Figure 3

Oxidative stress was induced in the iPSC-CMs by exposure to 100 μM H2O2 with the addition of 10 mM 2-deoxy-D-glucose for 2 hours. iPSC-CMs that were pretreated with isoflurane before oxidative stress had less LDH release in 5-mM (P = 0.0047) and 11-mM (P = 0.0189) glucose groups compared with stress control groups. Isoflurane did not, however, reduce LDH release in iPSC-CMs in 25 mM glucose compared with the stress control group (P = 0.8924) (Fig. 3B).

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High Glucose Induces Mitochondrial Fission, Hyperpolarization of the Mitochondrial Membrane Potential, and Generation of ROS

Figure 4

Figure 4

Increased mitochondrial fragmentation was observed when iPSC-CMs were exposed to 25 mM glucose (Fig. 4A), indicating mitochondrial fission. Mitochondrial fragmentation was observed as a decrease in form factor and aspect ratio, indicating reduced mitochondrial branching and length, respectively (Fig. 4B). We also observed that 24 hours of exposure to 25 mM glucose dose-dependently hyperpolarized the mitochondrial membrane potential, observed as an increase in TMRE fluorescence (Fig. 4C). Furthermore, 25-mM glucose exposure led to hyperpolarization of the mitochondrial membrane potential compared with 5 mM glucose (P < 0.001). Both 11- and 25-mM glucose concentrations increased superoxide levels compared with 5 mM (P< 0.001, P < 0.001, respectively) in iPSC-CMs measured with DHE fluorescence. Furthermore, 25-mM glucose exposure increased superoxide levels compared with 11-mM glucose concentration (P < 0.001) (Fig. 4D).

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Elevated Glucose Conditions Increased the Abundance of Activated Drp1

Figure 5

Figure 5

Mitochondrial fission is primarily regulated by Drp1 phosphorylation. We observed that elevated glucose concentrations increased the abundance of pSer616 Drp1 protein (5 vs 11 mM, P < 0.001; 5 vs 25 mM, P < 0.001). In addition, compared with control, H2O2-induced oxidative stress increased pSer616 Drp1 (P < 0.001), whereas adding the ROS scavenger Trolox resulted in reduced levels of the protein (P < 0.001) compared with H2O2-induced oxidative stress, further confirming the strong correlation between ROS and mitochondrial fission (Fig. 5, A–D). Isoflurane preconditioning reduced H2O2-induced increase in pSer616 Drp1 only in 5-mM glucose conditions (5 mM: P = 0.0009; 11 mM: P = 3.9685; 25 mM: P = 2.6886) (Fig. 5, E and F).

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Anesthetic Cardioprotection via Reduced ROS Is Abrogated in High-Glucose Conditions

Figure 6

Figure 6

Oxidative stress measured with DHE fluorescence induced a similar level of superoxide generation in the 5-, 11-, and 25-mM glucose groups compared with control groups (Fig. 6, A–C). Isoflurane treatment reduced ROS levels in 5- and 11-mM glucose groups (P < 0.001, P < 0.001, respectively) but not in 25-mM glucose group (P = 0.1426). Mdivi-1 and Trolox both reduced ROS levels in all glucose conditions. We observed the greatest reduction in oxidative stress-induced ROS generation when iPSC-CMs were pretreated with isoflurane and treated with Trolox in all glucose groups.

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Inhibiting Mitochondrial Fission and Scavenging ROS Restored Cardioprotection in 25 mM Glucose

Figure 7

Figure 7

iPSC-CMs treated with isoflurane before the oxidative stress had lower LDH release in 5- and 11-mM glucose groups (P = 0.00014, P = 0.0001, respectively) but not in the 25-mM glucose group (P = 0.6167) (Fig. 7, A–C). In the 5- and 11-mM glucose groups, we observed that mdivi-1 and Trolox were cardioprotective individually; treatment with both Trolox and isoflurane conferred the greatest protection (Fig. 7, A and B). Isoflurane exposure did not lead to a reduced LDH release from oxidant stress in the 25-mM glucose group. However, LDH release was similar to controls when isoflurane was used in conjunction with mdivi-1 or Trolox in the 25-mM glucose group (P < 0.001, P < 0.001, respectively). mdivi-1 alone did not provide any level of cardioprotection (Fig. 7C).

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The main findings of this study in human iPSC-CMs were that high glucose concentrations attenuated isoflurane-induced cardiac protection, at least in part by increasing ROS generation and mitochondrial fission. Elevated glucose concentrations induced mitochondrial fission, mitochondrial membrane hyperpolarization, and increased ROS generation. Finally, scavenging of ROS or inhibiting mitochondrial fission restored the isoflurane cardioprotection, despite elevated glucose concentrations.

Previous studies have shown that iPSC-CMs are a viable model to investigate the underlying mechanisms of anesthetic cardioprotection25 because they respond similarly to human atrial myocytes28 and human embryonic stem-cell–derived cardiomyocytes.26 The iPSC used in this study were from normal subjects. The use of iPSC from subjects with specific cardiovascular diseases significantly enriches the experimental platforms available to researchers. In addition, cultured human iPSC-CMs can be exposed to countless culture conditions that mimic various disease environment conditions. Although the iPSC-CMs generated in the short differentiation protocol were immature, they still proved valuable as a model system for studying stressed human cell responses to anesthetic-mediated protection. In this study, we used a small portion of that potential to investigate how elevated glucose concentrations attenuate volatile anesthetic cardioprotection.

Mitochondria are highly dynamic organelles. They continuously move, fuse, fragment, and divide. Normal balance between fusion and fission events of the mitochondrial outer membrane is required to maintain cellular homeostasis.46 Unbalanced fission–fusion can affect a variety of biological processes such as apoptosis and contribute to pathological processes such as heart failure.47–50 The most important mitochondrial fission protein is Drp1, which is mainly cytosolic, but on activation, it translocates to mitochondrial scission sites. When Drp1 is phosphorylated (at Ser616), mitochondrial fission occurs.51 In addition, an increase in ROS production during oxidative stress conditions has been shown to activate (phosphorylated) Drp1 in human umbilical vein endothelial cells, neurons, and cardiomyocytes.40,52–54 We found that high glucose concentrations alone increased mitochondrial fission, and the ratio of phosphorylated Drp1 to total Drp1 levels in iPSC-CMs is similar to that reported in earlier findings in rat neonatal cardiomyocytes.17

Mitochondrial dynamics have a key role in mitochondrial bioenergetics and function.55 Zanna et al.56 demonstrated the correlation between mitochondrial fusion and oxidative phosphorylation. Inhibition of mitochondrial fusion resulted in reduced oxygen consumption. Loss of mitochondrial membrane potential, reduced endogenous respiration, and capacity to increase respiration were observed in human dermal fibroblasts. Alterations in one of the mitochondrial membrane fusion proteins also affected mitochondrial metabolism in a similar manner. Other evidence suggests that alterations in mitochondrial fission proteins could affect mitochondrial metabolism. Knockdown of Drp1 with ribonucleic acid (RNA) interference in HeLa cells reduced the basal rate of oxygen consumption, decreased the coupled respiration, and lowered the rate of adenosine triphosphate synthesis.57 Similarly, high glucose concentration-induced mitochondrial fission and inhibition of mitochondrial fission by a dominant negative form of Drp1 markedly impaired mitochondrial ability to increase respiratory rate.16 ROS stress during deep laser scanning-induced cellular damage also mediates mitochondrial fission in neonatal rat ventricular cardiomyocytes, human lung adenocarcinoma cells, and kidney fibroblast cells.58

ROS generation contributes to the development and progression of diabetes mellitus.59,60 In addition, excessive ROS generation is 1 of the major causes of stress in cardiomyocytes after ischemia–reperfusion injury61–63 and plays a role in opening of the mPTP.64 The mitochondrial respiratory chain is the major site of ROS production within the cell. Superoxide is a byproduct of normal respiration through 1-electron reduction of dioxygen. Both in vivo and in vitro studies show increased superoxide production during high glucose exposure.16,65,66 The mitochondrial enzyme, manganese superoxide dismutase, converts superoxide to hydrogen peroxide, which, in the presence of ferrous or cuprous ions, forms the highly reactive hydroxyl radical, which damages all classes of biomolecules. Hydrogen peroxide, however, can create a hydroxyl radical (1 of the strongest oxidant agents) by interaction with ferrous ions in the catalytic Fenton reaction. There is some doubt that a similar reaction can be induced in mammalian cells, but studies involving different cells such as H9C2, endothelial cells, and neurons conclusively show that H2O2 can induce oxidative stress and cell death.67,68 The availability of free iron and copper within mitochondria is uncertain, although the reaction of superoxide with the iron sulfur center in aconitase releases ferrous iron. Excitation of fluorescence molecules using high-energy laser light is known to produce ROS resulting from the interaction of the long-lived triplet state of the excited dye molecule with molecular oxygen, which in turn leads to mPTP opening, depolarization of mitochondrial membrane, cytochrome c release, and cell death. We found that high glucose concentrations increased ROS generation and antioxidant Trolox used in conjunction with isoflurane-reduced oxidative stress-induced ROS in all groups. These findings confirm that ROS generation is a key mediator in the attenuation of anesthetic cardioprotection in the presence of high glucose concentrations. We also observed that isoflurane was successful in reducing oxidative stress-induced pSer616 Drp1 levels in lower glucose concentrations. These results taken together indicate a strong correlation between ROS generation and mitochondrial fission in iPSC-CMs.

We found that elevated glucose concentrations attenuated the cardioprotective effects of isoflurane. Interestingly, the cotreatment of Trolox or mdivi-1 and isoflurane had a significantly greater protection in iPSC-CMs than the treatment with mdivi-1 or Trolox alone. These results show that scavenging ROS and blocking mitochondrial fission can restore the ability of cardioprotection to protect iPSC-CMs from an oxidative stress event. Our studies indicated that both ROS and fission interact and influence one another with both contributing to the attenuation of anesthetic cardioprotection in elevated prolonged high glucose conditions.

One limitation of our study was the method of induced cellular injury. We generated oxidative stress with high H2O2 and 2-deoxyglucose. These exaggerated stress conditions were chosen when more physiologic hypoxia/reoxygenation conditions did not produce a damage phenotype (data not shown). Second, we used only 24 hours of exposure to high glucose concentrations in the attenuation of anesthetic cardioprotection in iPSC-CMs after finding that iPSC-CMs became adapted to high glucose concentrations after 48 hours and no longer responded with ROS generation and/or mitochondrial fission (data not shown).

The range of glucose concentrations used in this study was chosen based on animal studies. Of note, typical culture media for iPSC-CMs contains 11 mM glucose, which is physiologically hyperglycemic. Additional studies are needed to investigate the effects of these chronic high glucose concentrations on various iPSC-derived cell types to fully understand the mechanisms of anesthetic cardioprotection and how different disease states affect this cardioprotection. Finally, in this study, we focused on the effects of high glucose concentrations on the attenuation of the cardioprotection provided by isoflurane. It would be valuable to look at other effectors of diabetes mellitus, for example, fatty acids and their role on the attenuation of the cardioprotective effects of anesthetics.

In conclusion, our study shows that (1) iPSC-CMs are useful for studying the underlying mechanisms by which high glucose concentrations attenuate volatile anesthetic cardioprotection; and (2) inhibiting mitochondrial fission or scavenging ROS restores the cardioprotective properties of anesthetic cardioprotection in high glucose conditions in iPSC-CMs.

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Name: Scott G. Canfield, PhD.

Contribution: This author is an equal first author contributor. This author helped to design the study, conduct the study, collect and analyze data, and prepare the manuscript.

Attestation: Scott G. Canfield approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author.

Name: Ivan Zaja, MD.

Contribution: This author is an equal first author contributor. This author helped to design the study, conduct the study, collect and analyze the data, and prepare the manuscript.

Attestation: Ivan Zaja approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is an archival author.

Name: Brian Godshaw, BA.

Contribution: This author helped to conduct the study, collect the data, and analyze the data.

Attestation: Brian Godshaw approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Danielle Twaroski, PhD.

Contribution: This author helped conduct the study, collect the data, and analyze the data.

Attestation: Danielle Twaroski approved the final manuscript.

Name: Xiaowen Bai, MD, PhD.

Contribution: This author helped to design the study, conduct the study, and prepare the manuscript.

Attestation: Xiaowen Bai approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Zeljko J. Bosnjak, PhD.

Contribution: This author helped to design the study, conduct the study, and prepare the manuscript.

Attestation: Zejko J. Bosnjak approved the final manuscript.

This manuscript was handled by: Charles W. Hogue, MD.

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The authors thank Aniko Szabo, PhD (Associate Professor, Institute for Health and Society, Division of Biostatistics, Medical College of Wisconsin), for help with the statistical analysis.

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