Molecular Biology
oROS-HT variants were all cloned based on the pC1 plasmid backbone from pC1-HyPer-Red (Addgene ID: 48249). Primers for point mutations or fragment assembly required to generate the oROS-HT screening variants were designed for In Vitro Assembly cloning (IVA) technique81, and they were ordered from Integrated DNA Technologies (IDT). All gene fragment amplifications were done using Superfi-II polymerase (Invitrogen; 12368010). Amplification of the DNA fragment was verified with agarose gel electrophoresis. 30 minutes of DpnI enzyme treatment were done on every PCR product to remove the plasmid template from PCR samples. Circulaization or assembly of the PCR products was achieved with the IVA technique, while the linear DNA products were transformed into competent E.Coli cells (DH5ɑ or TOP10) and grown on agar plates that contain kanamycin selection antibiotic (50 µg/mL). Upon colony formation, single colonies were picked and grown in 5mL cultures containing LB Broth (Fisher BioReagents; BP9723-2) and selection antibiotic (/kanamycin; 50 µg/mL) overnight (37°C, 230 RPM). DNA was isolated using Machery Nagel DNA prep kits (Machery Nagel; 740490.250). Sanger sequencing (Genewiz; Seattle, WA) or Whole plasmid nanopore sequencing (Plasmidsarus; Eugene, OR) of the isolated plasmid DNA was used to confirm the presence of the intended mutation. Genes encoding the final variants were cloned into a CAG-driven backbone, pCAG-Archon1-KGC-EGFP-ER2-WPRE (Addgene; #108423), using the methods above. All subsequences were verified with Sanger sequencing (Genewiz; Seattle, WA) or Whole plasmid nanopore sequencing (Plasmidsarus; Eugene, OR).
Protein structure prediction and analysis
Protein structure analysis and plotting were performed using Chimera-X-1.2.1. Oxidized [PDB:1I6A] and reduced [PDB:1I69] crystal structures of ecOxyR were imported from the Protein Data Bank (PDB). Pairwise residue distance between reduced and oxidized ecOxyR structure was achieved by aligning both structures using a matchmaker algorithm that superimposes protein structures by creating a pairwise sequence alignment and then fitting the aligned residue pairs to derive pairwise residue distances. The structure of Variant 213-214 was predicted using ColabFold24. (msa_method= mmseqs2, homooligomer= 2, pair_msa= False, max_msa= 512:1024, subsample_msa= True, num_relax= None, use_turbo= True, use_ptm= True, rank_by= pLDDT, num_models= 3, num_samples= 1, num_ensemble= 1, max_recycles= 24, tol= 0, is_training= False, use_templates= False). The putative position of JF635 was incorporated into the ColabFold prediction of Variant 213-214 to report JF635 bound cpHaloTag structure (PDB:6U2M) with the matchmaker algorithm.
Chemicals
Halotag ligand of Janelia Fluor (JF-HTLs) 635, 585 described in this paper were generously provided by Janelia Materials. Stock solutions of JF-HTLs were prepared in 100% DMSO at 200µM. Cells described in this study were incubated in 200nM JF-HTL for 1 hour prior to imaging unless specified. H2O2 working solutions were freshly prepared before every experiment from H2O2 solution 30 % (w/w) in H2O (Sigma-Aldrich, H1009). A stock solution of Menadione (Sigma-Aldrich, M9429) was prepared in 100% DMSO at 50mM. A stock solution of Auranofin (Tocris Bioscience, 46-005-0) was prepared in 100% DMSO at 50mM.
HEK Cell culture and transfection.
Human Embryonic Kidney (HEK293; ATCC Ref: CRL-1573) cells were cultured in Dulbecco’s Modified Eagle Medium + GlutaMAX (Gibco; 10569-010) supplemented with 10% fetal bovine serum (Biowest; S1620). When cultures reached 85% confluency, the cultures were seeded at 150,000/75,000 cells per well in 24/48-well plates, respectively. 24 hours after cell seeding, the cells were transfected using Lipofectamine3000 (Invitrogen; L3000015) at 1000/500 ng of DNA per well of a 24/48-well plate, according to the manufacturer’s instructions.
Primary rat neuron isolation
Primary cortical neurons were prepared as previously described82,83. Briefly, 24-well tissue culture plates were coated with matrigel (mixed 1:20 in cold-PBS, Corning; 356231) solution and incubated at 4°C overnight before use. Sterile dissection tools were used to isolate cortical brain tissue from P0 rat pups (male and female). Tissue was minced until 1mm pieces remained, then lysed in equilibrated (37°C, 5% CO2) enzyme (20 U/mL Papain (Worthington Biochemical Corp; LK003176) in 5mL of EBSS (Sigma; E3024)) solution for 30 minutes at 37°C, 5% CO2 humidified incubator. Lysed cells were centrifuged at 200xg for 5 minutes at room temperature, and the supernatant was removed before cells were resuspended in 3 mLs of EBSS (Sigma; E3024). Cells were triturated 24x with a pulled Pasteur pipette in EBSS until homogenous. EBSS was added until the sample volume reached 10 mLs before spinning at 0.7 rcf for 5 minutes at room temperature. The supernatant was removed, and enzymatic dissociation was stopped by resuspending cells in 5 mLs EBSS (Sigma; E3024) + final concentration of 10 mM HEPES Buffer (Fisher; BP299-100) + trypsin inhibitor soybean (1 mg/ml in EBSS at a final concentration of 0.2%; Sigma, T9253) + 60 µl of fetal bovine serum (Biowest; S1620) + 30 µl 100 U/mL DNase1 (Sigma;11284932001). Cells were washed 2x by spinning at 0.7 rcf for 5 minutes at room temperature and removing supernatant + resuspending in 10 mLs of Neuronal Basal Media (Invitrogen; 10888022) supplemented with B27 (Invitrogen; 17504044) and glutamine (Invitrogen; 35050061) (NBA++). After final wash spin and supernatant removal, cells were resuspended in 10 mLs of NBA++ before counting. Just before neurons were plated, matrigel was aspirated from the wells. Neurons were plated on the prepared culture plates at the desired seeding density. Twenty-four hours after plating, 1µM AraC (Sigma; C6645) was added to the NBA++ growth media to prevent the growth of glial cells.Plates were incubated at 37°C and 5% CO2 and maintained by exchanging half of the media volume for each well with fresh, warmed Neuronal Basal Media (Invitrogen; 10888022) supplemented with B27 (Invitrogen; 17504044) and glutamine (Invitrogen; 35050061) every three days.
Brain slice imaging
Organotypic whole hemisphere (OWH) rat brain slice preparation: Male rats on postnatal day (P)10 were administered an overdose intraperitoneal injection of pentobarbital (120–150 mg/kg). Animals were then quickly decapitated and whole brains were extracted, cut into hemispheres, and placed into ice-cold dissecting media consisting of 0.64% w/v glucose, 100% Hank's Balanced Salt Solution (HBSS), 1% penicillin–streptomycin. Whole-hemisphere live slices of 300 μm were obtained using a tissue chopper as previously described.84 Slices were then transferred to 35 mm, 0.4 μm-pore membrane inserts in six-well plates and cultured in 1 ml of 5% heat-inactivated horse-serum slice culture media (SCM) consisting of 50% Minimum Essential Media (MEM), 45% HBSS, 1% GlutaMAX, and 1% penicillin–streptomycin. Slices were cultured in a sterile incubator at constant temperature (37°C), humidity, and CO2 level (5%).
AAV transduction and confocal imaging: After 1 day in vitro (DIV), crude AAV9-CAG-oROS-HT prep was added to the slices to be expressed. At the end of the 3-day incubation, 1 μM JF635-HTL was added to the slices for an additional 48 hours. OWH brain slices were transferred to 35mm confocal dishes (VWR, 75856-742). Confocal images were acquired with 10x (Nikon Plan Apo 10x Objective, 0.45 numerical aperture) and 20x (Nikon Plan Apo 10x Objective, 0.75 numerical aperture) magnifications (Nikon Corporation, Minato City, Tokyo, Japan). Brain slice tile scans were obtained with the Cy5 channel before multiple representative images were taken from both the cortex and striatum of each slice. Image acquisition settings were kept consistent before and after the 300µM H2O2 stimulation.
Differentiation of stem cell-derived cardiomyocytes and neurons
hiPSC culture and cardiomyocyte differentiation (diffusion study): Undifferentiated IMR90 (WiCell) hiPSCs were maintained on Matrigel (Corning) coated tissue culture plates in mTeSR1 (Stemcell Technologies). Cardiomyocyte-directed differentiation was performed using a modified small molecule Wnt-modulating protocol using Chiron 99021 and IWP-4 as previously described.85,86. Lactate enrichment was performed following differentiation to purify hiPSC-CMs.87
hiPSC culture and cardiomyocyte differentiation (Auranofin study):
Undifferentiated human induced pluripotent stem cells (hiPSCs) (WTC11, Male) were maintained on Matrigel (Corning) coated tissue culture plates in mTeSR1 (Stemcell Technologies). Cardiomyocyte-directed differentiation was performed using the RBA-based modified method as previously described88. Spontaneous contraction was observed on day 8 post-induction. On day 12 post-induction, media was reduced to 1 mL in preparation for 45 minutes heat-shock at 42°C on day 13. After heat shock, the media was changed to 1 mL of fresh RMPI+B27+ins. On day 14, cells were dissociated with 0.05% Trypsin (Thermo-Fisher) and frozen in BAMBANKER for storage in LN2. These cardiomyocytes were thawed in 90% RPMI+B27+ins and 10% Knockout Serum (KSR) with 10μM ROCK inhibitor and plated on matrigel coated plates. 24 hours after thaw, media was replaced with fresh RPMI+B27+ins and changed every other day.
hiPSC culture and cortical neuron differentiation
Neurons were generated from the previously characterized wild type CV background human induced pluripotent stem cell line89–91. Neural progenitor cells (NPCs) from this cell line were differentiated from hiPSCs using dual-SMAD inhibition and NPCs were differentiated into neurons as previously described (Knupp et al., 2020; Shin et al., 2023). Briefly, for cortical neuron differentiation from NPCs, NPCs were expanded into 10 cm plates in Basal Neural Maintenance Media (BNMM) (1:1 DMEM/F12 (#11039047 Life Technologies) + glutamine media/neurobasal media (#21103049, GIBCO), 0.5% N2 supplement (# 17502-048; Thermo Fisher Scientific,) 1% B27 supplement (# 17504-044; Thermo Fisher Scientific), 0.5% GlutaMax (# 35050061; Thermo Fisher Scientific), 0.5% insulin-transferrin-selenium (#41400045; Thermo Fisher Scientific), 0.5% NEAA (# 11140050; Thermo Fisher Scientific), 0.2% β-mercaptoethanol (#21985023, Life Technologies) + 20 ng/mL FGF (R&D Systems, Minneapolis, MN). Once the NPCs reached 100% confluence, they were switched to Neural Differentiation Media (BNMM +0.2 mg/mL brain-derived neurotrophic factor (CC# 450–02; PeproTech) + 0.2 mg/mL glial-cell-derived neurotrophic factor (CC# 450–10; PeproTech) + 0.5 M dbcAMP (CC# D0260; Sigma Aldrich). Neural Differentiation Media was changed twice a week for 21 days, at which point the differentiation is considered finished. Neurons were replated at a density of 500,000/cm2.
Immunofluorescence staining
Immunofluorescence staining performed for Nrf2 translocation study were done using polyclonal Nrf2 antibody (PA5-27882, Invitrogen) and Donkey anti-Rabbit IgG Alexa Fluor 488 (A21206, Invitrogen). HEK293 cells for each condition were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in 0.2% Triton-x solution for 1 hour. After blocking the fixed cells for 1 hour with 0.5% Bovine Serum Albumin (BSA) blocking buffer in TBST, Cells were then incubated with primary antibodies diluted in the blocking buffer overnight at 4°C. The next day, cells were washed 3 times with PBS. They were then incubated in a secondary antibody solution containing secondary antibodies diluted in 0.5% BSA in PBS overnight at 4°C. Counterstaining was performed with Vectashield containing DAPI (Vector Labs).
Microscopy
Imaging experiments described in this study were performed as follows unless specifically noted. Epifluorescence imaging experiments were performed on a Leica DMI8 microscope (Semrock bandpass filter: GFPratio ex/em: FF01-391-23/FF01-520-35, GFP ex/em: FF01-474-27/FF01-520-35, RFP ex/em:FF01-554-23 or FF01-578-21/FF01-600-37, Far-red ex/em: FF01-635-18/FF01-680-42) controlled by MetaMorph Imaging software, using sCMOS camera (Photometrics Prime95B) and 20x magnification lens (Leica HCX PL FLUOTAR L 20x/0.40 NA CORR) or 10× objective (Leica HC PL FLUOTAR L 10x/0.32 NA) Confocal imaging experiments were performed on Leica SP8 confocal microscope from Imaging Core of Institute of Stem Cell and Regenerative Medicine. Cells were imaged in live cell imaging solution with 10mM glucose (LCIS+, Gibco, A14291DJ). Image analysis methods are described below.
Hypoxic oROS-HT sensor maturation in HEK293
2-day post-seeding of HEK293 cells in 24 well plates (150,000 cells/well), culture media was swapped from complete DMEM media (as mentioned above) to complete Fluorobrite DMEM (A1896701, Gibco) with 20mM HEPES. After 2-hour of acclimation, cells were transfected (Lipofectamine-based, as described above) with pC1-oROS-HT-C199S (Loss-of-function), with 100nM JF635-HTL. Immediately after the transfection, transfected cells were either incubated at 37°C in an atmospheric environment or under hypoxic conditions. For hypoxic conditions, culture plates were transferred into a sealable chamber. The chamber was flushed with N2 for 10 min at a flow rate of 10 L/min before being placed into the incubator. Approximately 18 hours after, epifluorescence imaging were performed as described earlier.
Multiplexed experiments
oROS-HT/SypHer3s: HEK293 cells were co-transfected with pC1-oROS-HT/pC1-SypHer3s or pC1-oROS-HT-C199S/pC1-SypHer3s as described above. 2 days post-transfection, both sensors expressed in HEK293s were imaged using epifluorescence microscope. pH change experiment for oROS-HT-C199S were performed with HEK293s in PBS (10010001, Gibco) prepared at pH of 6, 7.44, and 9. Fluorescence level for GFP and Far-red profile were captured every 1.5 seconds. Sequential pH-changes plus Menadione applications were performed with HEK293s in PBS (pH 7.44), which was changed to PBS (pH 6) followed by menadione stimulation prepared in PBS (pH 6). Fluorescence level for GFP and Far-red profile were captured every 2 seconds.
oROS-HT/Grx1-roGFP2: HEK293 cells were co-transfected with pC1-oROS-HT and pC1-Grx1-roGFP2 as described above. 2 days post-transfection, both sensors expressed in the cells with live cell imaging solution with 10mM glucose (LCIS+, Gibco, A14291DJ) were imaged using an epifluorescence microscope. For the sequential response of oROS-HT/Grx1-roGFP2 to 10µM H2O2, fluorescence level for GFP and Far-red profile were captured every second. For the response to Auranofin, fluorescence levels for GFPratio, GFP, and Far-red profiles were captured every minute.
oROS-HT/Fluo-4: hiPSC-CMs were transfected with pCAG-oROS-HT as described above. 2 days post-transfection, cells were incubated with Fluo-4 (Invitrogen, F14201) at 5µM and JF635-HTL in RPMI + B27+insulin for 1 hour prior to imaging. For the response to Auranofin, fluorescence level of GFP profile (10Hz) and Far-red (0.1Hz) profile were acquired every 10 seconds for hiPSC-CMs in HEPES-buffered RPMI + B27+insulin.
Analysis
Analysis of cell fluorescence imaging data was done by FUSE, a custom cloud-based semi-automated time series fluorescence data analysis platform written in Python. First, the cell segmentation quality of the selected Cellpose92 model was manually verified. For the segmentation of cells expressing cytosolic fluorescent indicators, model ‘cyto’ was selected as our base model. If the selected Cellpose model was low-performing, we further trained the Cellpose model using the Cellpose 2.0 human-in-the-loop system93. Using an “optimized” segmentation model, fluorescence time-series data is extracted for each region of interest. This allows for unbiased extraction of change in cellular fluorescence information for a complete set of experimental samples. Extracted fluorescence data is normalized as specified in the text using a custom Python script.
Computational Cell Scale Modeling
We used an existing model of iPSC-CM membrane kinetics53 with one modification. Based on experimental observations, the spontaneous beating of the iPSC-CMs was observed to be around 0.5 Hz. To reflect this observation in our computational simulations, we increased the maximal value of the inward rectifier potassium (IK1) by a factor of 1.71484375. This change resulted in a decrease in spontaneous beating rate from 1.1 Hz to 0.5 Hz. To simulate ROS effects on iPSC-CMs, we ran simulations in which we modified parameters corresponding to maximal efflux via the SR Ca2+ ATPase (SERCA2a), SR Leak amplitude, and maximal conductance of the L-type Ca2+ channel (gCaL). The perturbation factor for SERCA efflux varied from 0.1 to 1.0 in steps of 0.1. SR Leak amplitude and gCaL were both increased from the default level (1.0) to 2.0 in steps of 0.1. Simulations of bioelectrical activity were conducted using openCARP94, a cardiac electrophysiology modeling software that is freely available for non-commercial reuse (see: http://opencarp.org/). Stimulated Cai values were post-analyzed with custom-written python scripts. Scripts and files used to run all simulations can be found at the Github depository.