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, 10 (3), e0119687
eCollection

Overexpression of TFAM or Twinkle Increases mtDNA Copy Number and Facilitates Cardioprotection Associated With Limited Mitochondrial Oxidative Stress

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Overexpression of TFAM or Twinkle Increases mtDNA Copy Number and Facilitates Cardioprotection Associated With Limited Mitochondrial Oxidative Stress

Masataka Ikeda et al. PLoS One.

Abstract

Background: Mitochondrial DNA (mtDNA) copy number decreases in animal and human heart failure (HF), yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM) or Twinkle helicase in volume overload (VO)-induced HF.

Methods and results: Two strains of transgenic (TG) mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT) mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP) activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS) upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM) overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice.

Conclusions: The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Characterization of TFAM and Twinkle (TW) mice.
(A) Expression of human TFAM (hTFAM) in left ventricle (LV) and aorta in TFAM and wild-type (WT) control mice. (B) mtDNA copy number in myocardium from TFAM and TW mice by real-time PCR (n = 4). (C) Expression of endogenous murine TFAM (mTFAM) and mitochondrial complex proteins in LV of TFAM and TW mice. (D) Transcription of mtDNA-encoded genes in TFAM and TW mice (n = 6). Data are expressed as mean ± SEM. *P < 0.05 vs. WT, **P < 0.01 vs. WT, analyzed by Student’s t-test.
Fig 2
Fig 2. Analysis of TFAM and Twinkle (TW) mice 8 weeks after arteriovenous fistula creation.
(A) Heart weight/body weight (HW/BW) (n = 12) (B) M-mode echocardiogram (C) Lung weight/body weight (LW/BW) in TFAM and TW mice (n = 12). (D) Quantification of perivascular fibrosis standardized by vascular circumference (n = 6). (E) mtDNA copy number in left ventricle (LV) of TFAM (upper panel) and TW (lower panel) mice (n = 6). (F) Mitochondrial electron transport chain enzymatic activities in TFAM (upper panel) and TW (lower panel) mice (n = 4–5). Data are expressed as mean ± SEM. *P < 0.05 vs. WT + Sham, **P < 0.01 vs. WT + Sham, P < 0.01 vs. WT + VO, †† P < 0.01 vs. WT + VO, analyzed by one-way ANOVA followed by post hoc Tukey’s test.
Fig 3
Fig 3. Matrix-metalloproteinase (MMP) gelatinase activity and reactive oxygen species (ROS) production in the heart tissues of TFAM mice and Twinkle (TW) mice at 8weeks after creating arteriovenous fistula.
(A) Representative images and quantification of MMP gelatinase activities in LV from TFAM or TW mice at 8 weeks after creating AVF (n = 6), **P < 0.01 vs. WT+Sham, P < 0.01 vs. WT+VO, †† P < 0.01 vs. WT+VO, analyzed by one-way ANOVA followed by post hoc Tukey’s test. (B) Representative images and quantification of Dihydroethidium staining (DHE) staining of LV from TFAM or TW mice (n = 6), **P < 0.01 vs. WT+Sham, P < 0.01 vs. WT+VO, †† P < 0.01 vs. WT+VO, analyzed by one-way ANOVA followed by post hoc Tukey’s test. Scale bar, 100μm. All data are mean±SEM.
Fig 4
Fig 4. Volume overload (VO)-induced hemodynamic load and mRNA expression of matrix-metalloproteinase (MMP)-2, MMP-9 and other VO-upregulated molecules in TFAM and Twinkle (TW) mice at 24 hours after creating arteriovenous fistula.
(A) Left ventricular end diastolic pressure (LVEDP) in C57BL/6J mice after 24 h of volume overload (VO) (n = 4). (B) VO-induced mRNA expression of MMP-2, MMP-9, connective tissue growth factor (CTGF), tissue inhibitor of metalloprotease (TIMP)-1,TIMP-2, TIMP-3, and TIMP-4 in the hearts of C57BL/6J mice, as measured by real-time PCR (n = 4) at 24 h of VO (n = 4). (C) Comparison of LVEDP in WT and transgenic (TG) mice at 24 h after VO creation (n = 6–12). (D) mRNA expression of MMP-2, MMP-9, CTGF, TIMP-1, TIMP-3, and TIMP-4 in the hearts of WT and TG mice under VO, as measured by real-time PCR (n = 6–12). Data are expressed as mean ± SEM. *P < 0.05 vs. Sham, **P < 0.01 vs. Sham, P < 0.01 vs. WT + VO, †† P < 0.01 vs. WT + VO, analyzed by Student’s t-test.
Fig 5
Fig 5. Effect of TFAM overexpression on mitochondrial DNA (mtDNA), mitoROS and mRNA expressions of matrix-metalloproteinase (MMP)-2 and MMP-9 in rat neonatal myocytes.
(A) Human TFAM expression at 72 h in neonatal rat ventricular myocytes infected by LacZ (control)-adenovirus or TFAM-adenovirus (multiplicity of infection [MOI] = 0.3, 1, and 3). (B) mtDNA copy number in TFAM-adenovirus infected myocytes at 72 h after infection (1 MOI) (n = 4–5). (C) Rotenone (Rot) or DMSO (Vehicle [Veh])-induced mitoROS detected by dihydroethidium (DHE) staining in LacZ- or TFAM-adenovirus (1 MOI) infected myocytes. Scale bar, 100 μm (left panel). Quantification of fluorescence intensity in LacZ- or TFAM-adenovirus (1MOI) infected myocytes (right panel; n = 3). (D) Rotenone-induced mRNA expressions of matrix-metalloproteinase (MMP)-2 (left panel) and MMP-9 (right panel) in LacZ- or TFAM-adenovirus (1 MOI) infected myocytes (n = 3), as measured by real-time PCR. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, analyzed by one-way ANOVA followed by post hoc Tukey’s test.
Fig 6
Fig 6. Mitochondrial reactive oxygen species (mitoROS) assay using H9c2 overexpressing human TFAM (hTFAM) or rat Twinkle-Flag (rTwinkle) and mitochondria from TFAM mice and Twinkle (TW) mice.
(A) Protein expression of hTFAM in H9c2 overexpressing hTFAM (left panel) and rTwinkle-Flag in H9c2 overexpressing rTwinkle (right panel). (B) Quantification of mtDNA copy number in H9c2 overexpressing hTFAM and rTwinkle by real-time PCR, (C) Antimycin A-induced mitoROS using MitoSOX probe in H9c2 overexpressing hTFAM and rTwinkle, Scale bar, 50 μm (upper panel). Quantification of fluorescence-intensity in the cytoplasm of H9c2 cells overexpressing hTFAM or rTwinkle vs. controls (lower panel; n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, analyzed by one-way ANOVA followed by post hoc Tukey’s test.
Fig 7
Fig 7. Nitration of mitochondrial protein and oxidized mtDNA extracted from mitochondria of TFAM mice and Twinkle (TW) mice at 8 weeks after creating arteriovenous fistula.
(A) Fluorescence intensity rate obtained by an in vitro assay using mitochondria isolated from WT and both transgenic mice together with NBD-Me-TPP (n = 6). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, analyzed by one-way ANOVA followed by post hoc Tukey’s test. (B) Western blots of mitochondrial proteins from wild type (WT) and both transgenic (TG) mice using anti-NO2-tyrosine antibody and anti-complex II antibody. Arrows a, b, and c identify reduced tyrosine nitration in both TG mice. Arrow d shows an additional nitrated protein (probably nitration of the overexpressed human TFAM protein).

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Grant support

This work was supported by JSPS KAKENHI Grant Number 23220013, 23591084, 50163043, and the Strategic Funds for the Promotion of Science and Technology from Ministry of Culture, Sports, Sciences and Technology. This work was also supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labour, and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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