SUMMARY The mitochondrion is the primary source of reactive oxygen species (ROS) in eukaryotic cells. With the aid of a novel mitochondrial matrix-targeted superoxide indicator, here we show that individual mitochondria undergo spontaneous bursts of superoxide generation, termed “superoxide flashes”. Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and reflect elementary events of superoxide production within single mitochondria across a wide diversity of cells. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore (mPTP) and are fueled by electron transfer complexes-dependent superoxide production. While decreased during cardiac hypoxia/anoxia, a flurry of superoxide flash activity contributes to the destructive rebound ROS burst observed during early reoxygenation after anoxia. The discovery of superoxide flashes reveals a novel mechanism for quantal ROS production by individual mitochondria and substantiates the central role of mPTP in oxidative stress related pathology in addition to its well-known role in apoptosis.

Calcium (Ca 2+ ) and reactive oxygen species (ROS) constitute the most important intracellular signaling molecules participating in the regulation and integration of diverse cellular functions. Here we briefly review cross-talk between the two prominent signaling systems that finely tune the homeostasis and integrate functionality of Ca 2+ and ROS signaling systems can be both stimulatory and inhibitory, depending on the type of target proteins, the ROS species, the dose, duration of exposure, and the cell contexts. Such extensive and complex cross-talk might enhance signaling coordination and integration, whereas abnormalities in either system might propagate into the other system and undermine the stability of both systems.

The mitochondrion is essential for energy metabolism and production of reactive oxygen species (ROS). In intact cells, respiratory mitochondria exhibit spontaneous "superoxide flashes", the quantal ROS-producing events consequential to transient mitochondrial permeability transition (tMPT). Here we perform the first in vivo imaging of mitochondrial superoxide flashes and tMPT activity in living mice expressing the superoxide biosensor mt-cpYFP, and demonstrate their coupling to whole-body glucose metabolism. Robust tMPT/superoxide flash activity occurred in skeletal muscle and sciatic nerve of anesthetized transgenic mice. In skeletal muscle, imaging tMPT/superoxide flashes revealed labyrinthine three-dimensional networks of mitochondria that operate synchronously. The tMPT/ superoxide flash activity surged in response to systemic glucose challenge or insulin stimulation, in an apparently frequency-modulated manner and involving also a shift in the gating mode of tMPT. Thus, in vivo imaging of tMPTdependent mitochondrial ROS signals and the discovery of the metabolism-tMPT-superoxide flash coupling mark important technological and conceptual advances for the study of mitochondrial function and ROS signaling in health and disease.

Mitochondria in many types of cells are dynamically interconnected through constant fusion and fission, allowing for exchange of mitochondrial contents and repair of damaged mitochondria. However, constrained by the myofibril lattice, the ∼6,000 mitochondria in the adult mammalian cardiomyocyte display little motility, and it is unclear how, if at all, they communicate with each other. By means of target-expressing photoactivatable green fluorescent protein (PAGFP) in the mitochondrial matrix or on the outer mitochondrial membrane, we demonstrated that the local PAGFP signal propagated over the entire population of mitochondria in cardiomyocytes on a time scale of ∼10 h. Two elemental steps of intermitochondrial communications were manifested as either a sudden PAGFP transfer between a pair of adjacent mitochondria (i.e., "kissing") or a dynamic nanotubular tunnel (i.e., "nanotunneling") between nonadjacent mitochondria. The average content transfer index (fractional exchange) was around 0.5; the rate of kissing was 1‰ s −1 per mitochondrial pair, and that of nanotunneling was about 14 times smaller. Electron microscopy revealed extensive intimate contacts between adjacent mitochondria and elongated nanotubular protrusions, providing a structural basis for the kissing and nanotunneling, respectively. We propose that, through kissing and nanotunneling, the otherwise static mitochondria in a cardiomyocyte form one dynamically continuous network to share content and transfer signals.mitochondrial dynamics | nanotubule I n most cells, mitochondria are highly dynamic organelles that constantly undergo shape changes through fusion and fission. This results in a form of communication that allows for the exchange and distribution of soluble and membranous components, including metabolic intermediates, signaling messengers, proteins, lipids, and mitochondrial DNAs, and provides a mechanism for repairing damaged mitochondria and maintaining a healthy mitochondrial population (1-3). Disorders of mitochondrial fusion and fission have been associated with developmental defects, neurodegenerative diseases, and cardiovascular diseases (4-7).In adult mammalian cardiomyocytes, mitochondria occupy ∼40% of the cell volume and are rigidly organized between bundles of myofilaments (interfibrillar mitochondria), under the sarcolemma (subsarcolemmal mitochondria), and around the nucleus (perinuclear mitochondria). This arrangement and the apparent lack of motility, a prerequisite for mitochondrial fusion in other types of cells, raise the question of whether mitochondria in adult cardiomyocyte communicate with each other dynamically. Mitochondrial dynamics in cardiac cell lines or neonatal cardiomyocytes (8) do not provide a direct answer to this question because mitochondria in these cells are not constrained strictly. Limited studies with adult cardiomyocytes have been inconclusive and even controversial. Whereas low-amplitude and high-frequency mitochondrial fluctuations have been visualized and quantified in living adult cardiomyo...