Abstract
Multi-oscillatory behavior of mitochondrial inner membrane potential ΔΨm in self-organized cardiac mitochondrial networks can be triggered by metabolic or oxidative stress. Spatio-temporal analyses of cardiac mitochondrial networks have shown that mitochondria are heterogeneously organized in synchronously oscillating clusters in which the mean cluster frequency and size are inversely correlated, thus suggesting a modulation of cluster frequency through local inter-mitochondrial coupling. In this study, we propose a method to examine the mitochondrial network's topology through quantification of its dynamic local clustering coefficients. Individual mitochondrial ΔΨm oscillation signals were identified for each cardiac myocyte and cross-correlated with all network mitochondria using previously described methods (Kurz et al., 2010a). Time-varying inter-mitochondrial connectivity, defined for mitochondria in the whole network whose signals are at least 90% correlated at any given time point, allowed considering functional local clustering coefficients. It is shown that mitochondrial clustering in isolated cardiac myocytes changes dynamically and is significantly higher than for random mitochondrial networks that are constructed using the Erdös–Rényi model based on the same sets of vertices. The network's time-averaged clustering coefficient for cardiac myocytes was found to be 0.500 ± 0.051 (N = 9) vs. 0.061 ± 0.020 for random networks, respectively. Our results demonstrate that cardiac mitochondria constitute a network with dynamically connected constituents whose topological organization is prone to clustering. Cluster partitioning in networks of coupled oscillators has been observed in scale-free and chaotic systems and is therefore in good agreement with previous models of cardiac mitochondrial networks.
Highlights
The mitochondrial network in cardiac myocytes consists of highly organized and densely packed mitochondria whose inner membrane potential m can be triggered to enter several cycles of de-and re-polarizations by numerous stressors such as oxidative or metabolic stress
Mitochondrial inner membrane potential m was monitored with the cationic potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRE) and images were recorded with a two-photon laser-scanning microscope (MRC-1024MP, Bio-Rad) with excitation at 740 nm (Tsunami Ti:Sa laser, Spectra-Physics) and red emission of TMRE was collected at 605 nm using a band pass filter 578–630 nm (Aon et al, 2003)
SELECTION AND PROCESSING OF INDIVIDUAL MITOCHONDRIAL TMRE SIGNALS As detailed before (Kurz et al, 2010a,b), TMRE signals of individual mitochondria were extracted from planar images of isolated cardiac myocytes recorded at a rate of dt by manually applying a grid template on a pixel-by-pixel basis to the averaged image of n subsequent images in time starting at the onset of TMRE oscillations
Summary
The mitochondrial network in cardiac myocytes consists of highly organized and densely packed mitochondria whose inner membrane potential m can be triggered to enter several cycles of de-and re-polarizations by numerous stressors such as oxidative or metabolic stress (see Aon et al, 2008a for a review) These oscillations can be strictly localized in the form of transient single mitochondrial depolarizations (Nivala et al, 2011), individual or clustered mitochondrial m oscillations (Romashko et al, 1998; Kurz et al, 2010a) with clusters that can span the whole myocyte (Aon et al, 2004). The clustering coefficient can be used as a measure of the network’s robustness toward the functional deletion of single mitochondria or www.frontiersin.org
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