Dynamics of Mitochondrial Redox and Energy Networks: Insights from an Experimental–Computational Synergy - Systems Biology of Metabolic and Signaling Networks - page 120

Experiment Model

TMRE

GSB

31

1.0

12

10

0.9

30

8

0.8

6

29

0.7 4

2

0.6 28

0 100 200 300 400 500

0 100 200 300

Time (sec)

Time (s)

Fig. 5.7 Glutathione oscillations. (a) Experimental demonstration of GSH oscillations (70 s
period) recorded simultaneously with ΔΨ m . Oscillations were triggered after a localized laser
flash as described in Fig. 5.4 in freshly isolated cardiomyocytes loaded with 100 nM tetramethylr-
hodamine methyl ester (TMRM) and 50 μ M monochlorobimane, MCB (Cortassa et al. 2004 ).
Reproduced from Cortassa, Aon, Winslow, O'Rourke (2004) Biophys J 87, 2060-73

blocking the mitochondrial ATP synthase (Fig. 5.6c ). Mitochondrial oscillations
could be interrupted by acute inhibition of mitochondrial ROS production
(Fig. 5.6d ), or by blockage of mitochondrial IMAC (Aon et al. 2003 ); these were
later found to be activated under moderate oxidative stress (Aon et al. 2007b ).
In the respiratory chain, oscillations were suppressed when we inhibited electron
transfer complex I (rotenone), III (antimycin A/myxothiazol), or IV (cyanide), or
the phosphorylation machinery F 1 F 0 ATPase (oligomycin) or adenine nucleotide
translocator (ANT, bongkrekic acid). On the other hand, we reinforced the scav-
enging system of cardiac cells by adding a superoxide dismutase mimetic or
N-acetyl cysteine (Aon et al. 2008a ). Although we could suppress the mitochondrial
oscillations by preincubation in the presence of ROS scavengers, it took much
longer than respiratory inhibitors to see the effect (1-3 min vs. 1 h) (Aon
et al. 2003 ). The scavengers also slowed the initial

ΔΨ m depolarization wave

(Aon et al. 2004a ; Cortassa et al. 2004 ).
Model simulations showed the ability to reproduce the ~100 s oscillatory period
and the phase relationship between

ΔΨ m and NADH observed experimentally
(Fig. 5.5 ). The model was also able to simulate other major experimental findings,
including (1) the requirements of ROS to cross a threshold so as to trigger fast ΔΨ m

depolarization, (2) the suppressive effect of inhibitors of the electron transport
chain, adenine nucleotide translocator (ANT) and the F 1 F 0 ATPase on ROS pro-
duction and

ΔΨ m oscillation (Cortassa et al. 2004 ), (3) the effects of anion channel
inhibitors, and (4) the sensitivity of the oscillator to the level of ROS scavengers.
After validation, the model led to predictions that were tested in the experimental
system. The model anticipated oscillatory behavior of reduced glutathione (GSH),
and that was experimentally demonstrated as well as its phase relationship with
ΔΨ m (Fig. 5.7 ) (Cortassa et al. 2004 ).