Dynamics of Mitochondrial Redox and Energy Networks: Insights from an Experimental–Computational Synergy - Systems Biology of Metabolic and Signaling Networks - page 117

5.2.1 Experimental Studies: A Brief Phenomenological
Description of Mitochondrial Oscillations

The mitochondrial oscillator was first described experimentally under pathophysi-
ological conditions of metabolic stress, e.g., substrate deprivation (Romashko
et al. 1998 ), or oxidative stress (Aon et al. 2003 ). The mechanisms underlying the
synchronization and propagation of mitochondrial oscillations in intact
cardiomyocytes were explored in detail employing two-photon laser scanning
fluorescence microscopy (Aon et al. 2003 ).
Experimentally, oscillations were triggered in a reproducible manner, a key for
studying the underlying mechanisms under controlled conditions. Two-photon
microscopy gives a detailed spatial picture of the mitochondrial network as can
be seen in Fig. 5.4 , in which a freshly isolated cardiomyocyte loaded with mem-
brane potential and ROS sensors is shown. The fluorescence spatiotemporal dynam-
ics along a line drawn throughout the longitudinal axis of an individual cell can be
obtained. A time-line image of fluorescence intensity of TMRM or CM-DCF results
in 2D plots that contain the whole spatial and temporal information of the stack of
images. In these pseudo-color plots the blue bars correspond to mitochondrial
membrane potential ( ΔΨ m ,) depolarization, and the yellow zones in between to

ΔΨ m repolarization. These 2D plots clearly show that while the oscillations affect
the whole cell, the flashed zone remains depolarized with high ROS (Fig. 5.4 ), and
oxidized NADH (not shown) (Aon et al. 2003 ). After about 1 min, whole-cell
mitochondrial oscillations are triggered whereby both

ΔΨ m and the reduced state of

NADH are synchronized into phase; with each ΔΨ m depolarization an associated

burst in the rate of ROS production occurs.

5.2.2 Modeling Studies: A Brief Description
of the Mitochondrial Oscillator

A model describing mitochondrial energetics and Ca 2+ handling (Cortassa
et al. 2003 ) was extended to describe the key features of the proposed mechanism
of mitochondrial oscillations based on our experimental findings (Cortassa
et al. 2004 ). The addition to this model of a leak of electrons from the respiratory
chain to produce the free radical superoxide, O 2 . , as previously proposed for an
outwardly rectifying inner membrane anion channel (IMAC) modeled after the
centum pS channel in which conductance is O 2 . activated (Borecky et al. 1997 ),
and a cytoplasmic ROS scavenging system in the cytoplasm, was sufficient to
support limit-cycle oscillations within certain parametric domains of our model
(Cortassa et al. 2004 ). The normal anion permeability of IMAC would permit the
passage of O 2 . from the matrix to the cytoplasmic side of the inner membrane.
In addition, the IMAC opening probability was assumed to be increased by O 2 .
at an external site.