Fig 1.
Schematic overview of the protocol showing sample preparation from either pools of larval zebrafish or dissected adult cardiac tissue.
Samples are processed using the DMPO spin trap or CMH spin probe by EPR spectroscopy. Figure icons created in BioRender.
Table 1.
Fitted parameters for DMPO radical adducts in zebrafish larval spectrum.
Provided g-factor and hyperfine splitting (HFS) for each species in the simulated spectrum.
Fig 2.
Representative experimental and simulated EPR spectra of DMPO (100 mM) in a pool/20 of zebrafish larvae.
The dashed line represents the experimental EPR spectrum, and the solid line shows the simulated spectrum.
Fig 3.
Stable radical adducts generated from the DMPO spin trap from a Fenton reaction and CMH spin probe reacting with .
(A) Representative experimental and simulated EPR spectra of DMPO (100 mM) in the Fenton reaction system. The dashed line represents an experimental EPR spectrum, while the solid line shows a simulated spectrum. (B) Representative experimental and simulated EPR spectra of CMH (1 mM) in zebrafish adult hearts. The dashed line represents an experimental EPR spectrum, while the solid line shows the simulated spectrum.
Fig 4.
EPR spectra and quantifications of detected from zebrafish larvae and adult heart tissue using CMH under rotenone and NAC-treated conditions.
(A-C) Analysis of levels in DMSO-treated (control) and rotenone-treated larvae. (D-F) Analysis of
levels in DMSO-treated (control) and rotenone-treated adult hearts. (G-I) Analysis of
levels in rotenone-treated (control) and NAC-treated adult hearts. Representative EPR spectra are shown for controls, DMSO-treated larvae (dashed line; A), DMSO-treated adult hearts (dashed line; D), and rotenone-treated adult hearts (dashed line; G). Representative EPR spectra are shown for rotenone-treated larvae (solid line; A), rotenone-treated adult hearts (solid line; D), and rotenone + NAC-treated adult hearts (solid line; G); quantification of normalized EPR signal amplitude (B, E, H), and normalized double integration (C, F, I). Rotenone treatment increases
levels in larvae (B, C) and adult hearts (E, F) compared to DMSO-treated larvae and adult hearts. NAC reduces
levels in adult hearts following rotenone treatment (H, I). A 1 mM CMH concentration was used for all conditions; n=6 replicates/pools of larvae and n = 4 replicates/adult hearts. NAC, N-acetyl cysteine; Rot, rotenone. Error bar corresponds to SEM.
Fig 5.
EPR spectra and quantification of detected from zebrafish adult heart tissue using CMH under rotenone and PEG-SOD treated conditions.
Representative EPR spectra are shown for rotenone-treated adult heart (dashed line; A), and rotenone + PEG-SOD treated heart (solid line; A). Quantification of normalized EPR signal amplitude (B). A 1 mM CMH concentration was used for all conditions; n=3 replicates/adult hearts. PEG-SOD, polyethylene glycol-conjugated superoxide dismutase; Rot, rotenone. Error bar corresponds to SEM.
Fig 6.
Rotenone-treated heart sections from wild-type adult zebrafish atrium and ventricle indicate an increase in compared to DMSO-treated samples using DHE.
(A) Maximum intensity projections of confocal images from DMSO-treated and rotenone-treated wild-type adult zebrafish atrium and ventricle frozen sections. (B) Quantification of fluorescence intensity from maximum intensity projections reveal a higher amount of in wild-type adult zebrafish atrium and ventricle sections following rotenone treatment. Scale bar, 10 μm. a.u., arbitrary units. Error bar corresponds to SEM.