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. 2022 Jul 26;11(8):1454.
doi: 10.3390/antiox11081454.

Noninvasive Diagnosis of the Mitochondrial Function of Doxorubicin-Induced Cardiomyopathy Using In Vivo Dynamic Nuclear Polarization-Magnetic Resonance Imaging

Yukie Mizuta  1   2 , Tomohiko Akahoshi  3 , Hinako Eto  4 , Fuminori Hyodo  5 , Masaharu Murata  4 , Kentaro Tokuda  6 , Masatoshi Eto  4 , Ken Yamaura  2
Affiliations

Noninvasive Diagnosis of the Mitochondrial Function of Doxorubicin-Induced Cardiomyopathy Using In Vivo Dynamic Nuclear Polarization-Magnetic Resonance Imaging

Yukie Mizuta et al. Antioxidants (Basel). .

Abstract

Doxorubicin (DOX) induces dose-dependent cardiotoxicity via oxidative stress and abnormal mitochondrial function in the myocardium. Therefore, a noninvasive in vivo imaging procedure for monitoring the redox status of the heart may aid in monitoring diseases and developing treatments. However, an appropriate technique has yet to be developed. In this study, we demonstrate a technique for detecting and visualizing the redox status of the heart using in vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) with 3-carbamoyl-PROXYL (CmP) as a molecular imaging probe. Male C57BL/6N mice were administered DOX (20 mg/kg) or saline. DNP-MRI clearly showed a slower DNP signal reduction in the DOX group than in the control group. Importantly, the difference in the DNP signal reduction rate between the two groups occurred earlier than that detected by physiological examination or clinical symptoms. In an in vitro experiment, KCN (an inhibitor of complex IV in the mitochondrial electron transport chain) and DOX inhibited the electron paramagnetic resonance change in H9c2 cardiomyocytes, suggesting that the redox metabolism of CmP in the myocardium is mitochondrion-dependent. Therefore, this molecular imaging technique has the potential to monitor the dynamics of redox metabolic changes in DOX-induced cardiomyopathy and facilitate an early diagnosis of this condition.

Keywords: dynamic nuclear polarization–magnetic resonance imaging; heart; mitochondria; nitroxyl radical; oxidation reduction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Redox imaging of the mouse heart after injection of nitroxyl radical. (A) Experimental set-up using the in vivo DNP–MRI system (Keller). (B) Chemical structure of the redox probe, 3-carbamoyl-PROXYL, and its nitroxyl reduction in the tissue, indicating the redox status. (C) Cardiac imaging by 1.5 T animal MRI. (D) In vivo DNP–MRI images of the heart 1 min after injecting 3-carbamoyl-PROXYL with DNP ON and OFF (i.e., with and without EPR irradiation, respectively).
Figure 2
Figure 2
Effects of DOX injection on survival, cardiac function, and histology. (A) Kaplan–Meier survival curve showing survival after DOX injection. n = 10. * p = 0.0392 vs. control group. (B,C) Echocardiography performed in mice before DOX injection at 2 and 6 days after DOX injection. (C) Representative images of 2D M-mode echocardiograms. Ejection fractions (EFs) were quantified. n = 6 in each group. ** p < 0.01. (D) Representative photomicrographs of heart sections with H&E staining 6 days after DOX injection. Scale bar, 50 μm. (E,F) Immunohistochemical detection of oxidative DNA damage with 8-OHdG. Representative photomicrographs of the 8-OHdG-positive nuclei. Scale bar, 50 μm. 8-OHdG-positive nuclei/total cells (%) were normalized to the control group. n = 5 in each group. ** p < 0.01.
Figure 3
Figure 3
Redox imaging of the hearts of DOX-treated mice using in vivo DNP–MRI. (A) Representative imaging of temporal changes in DNP–MRI after intravenous injection of CmP at days 2 and 6 after DOX injection. (B) Comparison of the reduction rate of DNP image intensity in control and DOX-treated mice. Reduction rates were calculated by the slope of image intensity in the region of interest corresponding to the enhancement by CmP. n = 5–6 in each group. * p < 0.05, ** p < 0.01. (C) Comparison of body weight decay rate in control and DOX-treated mice at days 2 and 6 post-DOX injection. n = 5–6 in each group. ** p < 0.01. (D) Analysis of the total CmP radical concentration (sum of oxidized and reduced forms) in the heart and blood. Total CmP concentration was obtained after reoxidation by treating the heart tissue and blood samples with 2 mM potassium ferricyanide via X-band EPR. n = 4 in each group.
Figure 4
Figure 4
Assessment of CmP dynamics in H9c2 cells. (A) A typical ESR signal attenuation of CmP in four-fold diluted H9c2 cells homogenate solution over 30 min. (B) Comparison of the reduction rate of CmP radical concentration over 30 min in control H9c2 cells vs. KCN-treated H9c2 cells and control H9c2 cells vs. DOX-treated H9c2 cells. CmP concentration was measured by X-band EPR. n = 3 in each group. * p < 0.05, ** p < 0.01. (C) Oxygen consumption rate (OCR) over 82 min calculated using relative fluorescence unit slopes in control H9c2 cells vs. DOX-treated H9c2 cells. n = 3 in each group. ** p < 0.01.
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