MitoQ regulates redox-related noncoding RNAs to preserve mitochondrial network integrity in pressure-overload heart failure

Abstract

Evidence suggests that mitochondrial network integrity is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial remodeling, the effect of mitochondrial-targeted antioxidants, such as mitoquinone (MitoQ), on the mitochondrial network in a model of HF (e.g., pressure overload) has not been demonstrated. Furthermore, the mechanism of this regulation is not completely understood with an emerging role for posttranscriptional regulation via long noncoding RNAs (lncRNAs). We hypothesized that MitoQ preserves mitochondrial fusion proteins (i.e., mitofusin), likely through redox-sensitive lncRNAs, leading to improved mitochondrial network integrity in failing hearts. To test this hypothesis, 8-wk-old C57BL/6J mice were subjected to ascending aortic constriction (AAC), which caused substantial left ventricular (LV) chamber remodeling and remarkable contractile dysfunction in 1 wk. Transmission electron microscopy and immunostaining revealed defective intermitochondrial and mitochondrial-sarcoplasmic reticulum ultrastructure in AAC mice compared with sham-operated animals, which was accompanied by elevated oxidative stress and suppressed mitofusin (i.e., Mfn1 and Mfn2) expression. MitoQ (1.36 mg·day^-1·mouse^-1, 7 consecutive days) significantly ameliorated LV dysfunction, attenuated Mfn2 downregulation, improved interorganellar contact, and increased metabolism-related gene expression. Moreover, our data revealed that MitoQ alleviated the dysregulation of an Mfn2-associated lncRNA (i.e., Plscr4). In summary, the present study supports a unique mechanism by which MitoQ improves myocardial intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) ultrastructural remodeling in HF by maintaining Mfn2 expression via regulation by an lncRNA. These findings underscore the important role of lncRNAs in the pathogenesis of HF and the potential of targeting them for effective HF treatment.NEW & NOTEWORTHY We have shown that MitoQ improves cardiac mitochondrial network integrity and mitochondrial-SR alignment in a pressure-overload mouse heart-failure model. This may be occurring partly through preventing the dysregulation of a redox-sensitive lncRNA-microRNA pair (i.e., Plscr4-miR-214) that results in an increase in mitofusin-2 expression.

Keywords: heart failure; long noncoding RNA; mitochondrial network; mitofusin; oxidative stress.

Fig. 1.

Mitoquinone (MitoQ) improves mitochondrial network integrity in pressure-overload mouse hearts. A: representative confocal image showing polarized and homogeneously-distributed mitochondrial membrane potential (ΔΨ_m) in sham cardiomyocytes. B: representative confocal images showing fully depolarized (cell 1) and partial, heterogeneously distributed (cell 2) ΔΨ_m in ascending aortic constriction (AAC) cardiomyocytes. C: representative confocal image showing improved ΔΨ_m in AAC+MitoQ cardiomyocytes. Insets, a–c: magnified views of ΔΨ_m distribution in areas denoting by squares in A–C. D: quantification and summary of ΔΨ_m. *P < 0.05 vs. sham; #P < 0.05 vs. AAC, n = 4–6 each group. AU, arbitrary units. E: representative transmission electron microscopy images showing the effects of AAC and MitoQ on cardiac mitochondrial networks in mice; n = 3–5 animals examined in each group.

Fig. 2.

Mitoquinone (MitoQ) increases the expression of cardiac mitochondrial fusion proteins in pressure-overload mice. A: mRNA expression of mitofusin (i.e., Mfn1 and Mfn2) and Opa1 (mitochondrial dynamin like GTPase 1). B: representative Western blotting and quantification of MFN1 protein expression. C: representative Western blotting and quantification of MFN2 protein expression. D: representative Western blotting and quantification of long (L; 100 kDa) and short (S; 83 kDa) isoform Opa1 protein expression. E: representative immunohistochemistry analysis of myocardial MFN2 protein expression. The protein or mRNA expression was normalized to GAPDH. *P < 0.05 vs. sham; #P < 0.05 vs. ascending aortic constriction (AAC); n = 6 in each group.

Fig. 3.

Mitoquinone (MitoQ) does not increase the expression of cardiac mitochondrial fission proteins in pressure-overload mice. A: mRNA expression of dynamin-related protein 1 (Drp1) and mitochondrial fission 1 (Fis1). AAC, ascending aortic constriction. B: representative Western blotting and quantification of DRP1 protein expression. C: representative Western blotting and quantification of the FIS1 protein expression. The protein or mRNA expression was normalized to GAPDH. *P < 0.05 vs. sham; n = 6 in each group.

Fig. 4.

Mitoquinone (MitoQ) improves cardiac mitochondrial-sarcoplasmic reticulum contact in ascending aortic constriction (AAC) mice. A–C: representative confocal images showing coimmunostaining of ryanodine receptor 2 (RyR2; red) and mitofusin 1 (MFN1; green) in sham (A), AAC (B) and AAC+MitoQ (C) myocardium. Line scanning (denoted by yellow lines in the overlay panels) shows relative position of RyR2 and MFN1 proteins in each group. D–F: quantification of MFN1 and RyR2 protein colocalization measured by Mander’s coefficient (D), %MFN1 overlapped with RyR2 (E), and %RyR2 overlapped with MFN1 (F). *P < 0.05 vs. sham; #P < 0.05 vs. AAC; n = 3 to 4 animals examined in each group.

Fig. 5.

Mitoquinone (MitoQ) attenuates oxidative stress and dysregulation of mitofusin 2 (Mfn2)-related noncoding RNAs. A: level of myocardial Plscr4 in nuclear factor erythroid-derived 2-related factor 2 (Nfe2l2)-knockout (KO) and the age-matched wild-type (WT) mice. B: myocardial oxidative stress measured by malondialdehyde (MDA) level. C: effects of ascending aortic constriction (AAC) and MitoQ on the level of Plscr4 in mouse hearts. D: effects of AAC and MitoQ on the level of miR-214 in mouse hearts. E: effect of MitoQ on mitochondrial reactive oxygen species (ROS) and membrane potential (measured by MitoSox and MitoView, respectively) in phenylephrine (PE)-treated adult mouse cardiomyocytes. F: effect of MitoQ on the level of Plscr4 in PE-treated adult mouse cardiomyocytes. *P < 0.05 vs. sham or control; #P < 0.05 vs. AAC or PE; n = 6 for A–D, and n = 4 to 5 for E and F in each group.

Fig. 6.

Mitoquinone (MitoQ) ameliorates dysregulation of cardiac metabolism-associated genes in ascending aortic constriction (AAC) mice. A: mRNA expression of mitochondrial pyruvate carriers 1 and 2 (Mpc1 and Mpc2), and carnitine palmitoyltransferases 1 and 2 (Cpt1 and Cpt2). B: mRNA expression of peroxisome proliferator-activated receptor-α (Ppar-α). C: mRNA expression of E2F transcription factor 1 (E2f1). D: mRNA expression of uncoupling proteins 1, 2, and 3 (Ucp1, Ucp2, and Ucp3). E: mRNA expression of cardiac mitofusin 1 and 2 (Mfn1 and Mfn2), Mpc2, Cpt1b, and Cpt2 in wild-type and Mfn2-knockout (KO) mice. *P < 0.05 vs. sham; #P < 0.05 vs. AAC; n = 5 to 6 in each group for A–D, and n = 4 in each group for E.

Fig. 7.

Schematics of the mechanisms through which mitoquinone (MitoQ) potentially regulates cardiac mitochondrial network integrity and contractile function in heart failure. Using an ascending aortic constriction (AAC)-induced pressure-overload mouse model, we demonstrate that MitoQ preserves mitofusin 2 (Mfn2) expression, which leads to increased intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) network integrity and improved cardiac contractile function in failing hearts. The mechanism is likely through suppressing oxidative stress and the dysregulation of a redox-sensitive, Mfn2-associated long noncoding RNA-microRNA (lncRNA)-microRNA (miRNA) (Plscr4-miR-214) pair. ROS, reactive oxygen species.