Spatio-temporal oscillations of individual mitochondria in cardiac myocytes reveal modulation of synchronized mitochondrial clusters

Abstract

Mitochondrial networks in cardiac myocytes under oxidative stress show collective (cluster) behavior through synchronization of their inner membrane potentials (DeltaPsi(m)). However, it is unclear whether the oscillation frequency and coupling strength between individual mitochondria affect the size of the cluster and vice versa. We used the wavelet transform and developed advanced signal processing tools that allowed us to capture individual mitochondrial DeltaPsi(m) oscillations in cardiac myocytes and examine their dynamic spatio-temporal properties. Heterogeneous frequency behavior prompted us to sort mitochondria according to their frequencies. Signal analysis of the mitochondrial network showed an inverse relationship between cluster size and cluster frequency as well as between cluster amplitude and cluster size. High cross-correlation coefficients between neighboring mitochondria clustered longitudinally along the myocyte striations, indicated anisotropic communication between mitochondria. Isochronal mapping of the onset of myocyte-wide DeltaPsi(m) depolarization further exemplified heterogeneous DeltaPsi(m) among mitochondria. Taken together, the results suggest that frequency and amplitude modulation of clusters of synchronized mitochondria arises by means of strong changes in local coupling between neighboring mitochondria.

Fig. 1.

Wavelet analysis of single mitochondrial signals. (A) Single mitochondria of a cardiomyocyte are identified and labeled within a hand-drawn grid of an averaged stack of frames in time. TMRE signal for a single oscillating mitochondrion (B). (C) The absolute squared wavelet transform over frequency and time. The major frequency component in a cluster of mitochondria (see whole myocyte mean signal in SI Text), dropped from about 15 to 5 mHz during the recording.

Fig. 2.

Mitochondrial frequency distribution in clusters for different myocytes (n = 9). (A) Frequency histogram for a specific frame obtained from wavelet analysis. There are three apparent major clusters. The amplitude distribution cutoff () is marked with a horizontal dashed line and the relevant cluster peak (in red) extends to frequencies defined by and (green lines). Because the correlation of the other two clusters peaks (in blue) with the major cluster peak is lower than 95%, the major cluster peak does not include these smaller clusters. (B) Mean cluster radius as a function of frequency. (C) Cluster area normalized by the full myocyte area as a function of frequency. (D) Cluster mitochondria count normalized by the total number of mitochondria for the major cluster as a function of frequency. SE bars are in red and the mean curve is in black. (E) Distribution of mitochondrial frequencies for all cluster mitochondria across all myocytes.

Fig. 3.

Coherence of mitochondria belonging to the major oscillating cluster, estimated at the mean cluster frequency (n = 9). To allow the statistical comparison among myocytes with unequal recording durations, the duration of the oscillations of each recording was normalized.

Fig. 4.

(A) Method for obtaining the amplitude of mitochondrial oscillations. After identifying the peaks of the mean TMRE signal of a major cluster of mitochondria, we determined the mean cluster area and frequency between consecutive peaks and determined the amplitude as the difference in their TMRE intensity. (B) The normalized cluster area is plotted against the oscillation amplitude values that have been normalized by the maximum amplitude value (n = 8). (C) Normalized mitochondrial cluster amplitude vs. normalized cluster area follows a linear pattern when plotted against the mean cluster frequencies (n = 6).

Fig. 5.

Isochronal map of a canine cardiac myocyte at the onset of flash-triggered oscillations. The red square signifies the area of the flash. About 25 s after the flash, an initial critical mass of mitochondria in the left bottom area of the myocyte triggers mitochondrial depolarizations by exciting a critical number of mitochondria. The excitation penetrates the whole myocyte within ≈10–12 s, leading to a conduction velocity of about 32 μm/s.