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. 2017 Feb;113(2):160-170.
doi: 10.1093/cvr/cvw212. Epub 2016 Oct 29.

A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration

Huiliang Zhang  1 , Pei Wang  1 , Sara Bisetto  2 , Yisang Yoon  3 , Quan Chen  4 , Shey-Shing Sheu  2 , Wang Wang  5
Affiliations

A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration

Huiliang Zhang et al. Cardiovasc Res. 2017 Feb.

Abstract

Aims: Mitochondria in adult cardiomyocytes exhibit static morphology and infrequent dynamic changes, despite the high abundance of fission and fusion regulatory proteins in the heart. Previous reports have indicated that fusion proteins may bear functions beyond morphology regulation. Here, we investigated the role of fission protein, dynamin-related protein 1 (DRP1), on mitochondrial respiration regulation in adult cardiomyocytes.

Methods and results: By using genetic or pharmacological approaches, we manipulated the activity or protein level of fission and fusion proteins and found they mildly influenced mitochondrial morphology in adult rodent cardiomyocytes, which is in contrast to their significant effect in H9C2 cardiac myoblasts. Intriguingly, inhibiting endogenous DRP1 by dominant-negative DRP1 mutation (K38A), shRNA, or Mdivi-1 suppressed maximal respiration and respiratory control ratio in isolated mitochondria from adult mouse heart or in adult cardiomyocytes from rat. Meanwhile, basal respiration was increased due to increased proton leak. Facilitating mitofusin-mediated fusion by S3 compound, however, failed to inhibit mitochondrial respiration in adult cardiomyocytes. Mechanistically, DRP1 inhibition did not affect the maximal activity of individual respiratory chain complexes or the assembly of supercomplexes. Knocking out cyclophilin D, a regulator of mitochondrial permeability transition pore (mPTP), abolished the effect of DRP1 inhibition on respiration. Finally, DRP1 inhibition decreased transient mPTP-mediated mitochondrial flashes, delayed laser-induced mPTP opening and suppressed mitochondrial reactive oxygen species (ROS).

Conclusion: These results uncover a novel non-canonical function of the fission protein, DRP1 in maintaining or positively stimulating mitochondrial respiration, bioenergetics and ROS signalling in adult cardiomyocyte, which is likely independent of morphological changes.

Keywords: Adult cardiomyocyte; Dynamin related protein 1; Mitochondrial morphology; Mitochondrial permeability transition pore; Mitochondrial respiration.

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Figures

Figure 1
Figure 1
DRP1 inhibition altered mitochondrial morphology in adult cardiomyocytes. (A) Representative images of mitochondrial targeted JC1 showing the morphology of adult cardiomyocytes with or without the indicated treatments. (B–C) Quantitative analysis of mitochondrial size (B, area) and aspect ratio (C) with ImageJ software. N = 357–1774 mitochondria from 17–65 cells and 4–9 rats. *P < 0.05 vs. Control.
Figure 2
Figure 2
DRP1 inhibition caused mitochondrial fusion in H9C2 myoblast cells. (A) Representative images showing the morphology of mitochondria in H9C2 cells with manipulations of fission and fusion proteins. (B–E) Quantitative analysis of mitochondrial morphology showing mitochondrial size, aspect ratio, and form factor. N = 1689–7786 mitochondria from 13–36 cells and 3–4 independent experiments. *, #: P < 0.05 or 0.01 vs. Control, respectively.
Figure 3
Figure 3
Inhibiting endogenous DRP1 activity suppressed cardiac mitochondrial respiration. (A) Mdivi-1 dose-dependently inhibited respiratory control ratio (RCR, State 3/State 2 ratio) in isolated mitochondria from adult mouse heart. N = 3 independent experiments. (B) Mdivi-1 (50 µM) suppressed State 3 respiration and RCR in permeabilized adult cardiomyocytes. N = 6 from three rats. (C–E) Representative images and summarized data showing adenovirus mediated overexpression of the dominant negative mutation K38A (C), WT DRP1 (D), or DRP1 shRNA (shDRP1) in adult cardiomyocytes. N = 3. *P < 0.05 vs. Control (Scrambled shRNA). (F) Titration of the number of adult cardiomyocytes for OCR measurement by Seahorse system (XF96). N = 5 in each group. (G) Effects of manipulating DRP1 on RCR of intact adult cardiomyocytes measured by Seahorse system. Mdivi-1 (50 µM) added for 30 min and S3 compound (2 µM) for 24 h before measurement. N = 4–12. The RCR was calculated by the ratio between maximal OCR (induced by 1 µM FCCP) and basal OCR. *, #: P < 0.05 or 0.01 vs. Control.
Figure 4
Figure 4
DRP1 inhibition had no effect on ETC supercomplexes assembly and activity. (A) Blue native gel image showing Mdivi-1 did not cause significant changes in ETC supercomplexes assembly. N = 3 mice. (B) Effects of Mdivi-1 and K38A on the enzyme activity of Complexes I–V and citrate synthase in adult rat cardiomyocytes. N = 6–11 rats.
Figure 5
Figure 5
DRP1 stimulated mPTP and through which regulated mitochondrial respiration. (A) The effect of Mdivi-1 (50 μM) on respiration inhibition was abolished by mPTP inhibitor, cyclosporine A (CsA, 1 μM) in permeabilized adult cardiomyocyte from WT mice, and was missing in permeabilized adult cardiomyocytes from mice lacking the mPTP regulator, cyclophilin D (Ppif−/−). N = 3–4 mice in each group. *P < 0.05 vs. Control or WT. (B) Overexpression of K38A suppressed laser-induced mPTP openings (as indicated by loss of TMRM signal) in intact adult cardiomyocytes. N = 92–100 mitochondria from 7–8 cells and three rats. #P < 0.01 vs. Control. (C) Increased mitochondrial Ca2+ in K38A overexpressed myocytes. N = 11–31 cells from three rats. *P < 0.05 vs. Control. (D) Mitochondrial membrane potential in K38A overexpressed myocytes was maintained. N = 16–27 cells from three rats.
Figure 6
Figure 6
DRP1 modulated mitochondrial flash and ROS. (A, B), Representative images (A) and summarized data (B) showing that Mdivi-1 inhibited the frequency of mitochondrial flash in perfused beating heart from mt-cpYFP transgenic mouse. The white boxes in (A) indicate the location of flashes during a 100 s serial scanning. N = 3 mice. #P < 0.01 vs. Control. (C, D) Enlarged serial images (C) and traces (D) showing the fluorescence changes during a typical mitochondrial flash observed in the perfused heart. The intervals between each images in C is 2 s, the colour coding indicates relative fluorescence intensity. (E) Summarized data showing the frequency of mitochondrial flash in Midivi-1 treated adult cardiomyocytes from rat in the presence or absence of respiration substrate, pyruvate (1 mM). N = 16–29 cells from 3–5 rats. *, †: P < 0.05 or 0.001 vs. Control, respectively. (F) Summarized data showing K38A or shDRP1 inhibited mitochondrial flash in adult cardiomyocytes from rats. N = 26–29 cells from four rats. *, #: P < 0.05 or 0.01 vs. Control, respectively. (G) Mdivi-1 or DRP1-K38A decreased constitutive mitochondrial ROS in adult cardiomyocytes monitored by the slop of fluorescence increase of MitoSox. N = 11–16 cells from 3–4 rats. *P < 0.05 vs. Control.
Figure 7
Figure 7
Schematic diagram showing the canonical and non-canonical roles of endogenous DRP1 in adult cardiomyocytes. The recruitment of DRP1 to mitochondria (upper panel) can induce fission (lower right panel), which is the canonical function of DRP1. At the same time, DRP1 also positively regulates mitochondrial respiration in adult cardiomyocytes (lower left panel). This novel non-canonical function of DRP1 may involve transient mPTP and contribute to bioenergetics, Ca2+ and ROS signalling in adult cardiomyocytes. Whether the canonical and non-canonical functions are connected or independent to each other needs further study. ETC, electron transport chain.
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