Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals

Abstract

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.

A reaction to mitochondria in action

“…Mitochondria in skeletal muscle cells exhibited spontaneous SO flashes that occurred throughout the mitochondrial network of individual cells. SO flashes occurred asynchronously in individual mitochondria distributed along axons of the sciatic nerve and, interestingly, SO flashes occurred only in mitochondria that were stationary and not in mitochondria that were being npg actively transported along the axons [9]. Additional experiments in the new study showed that, as was the case in cell culture, the SO flashes are associated with and require the opening of mPTP.…”

“…Fourth, how is the frequency of the SO flashes modified by physiological signaling and in pathological settings? Fang et al [9] found that when mice were administered either glucose or insulin the frequency of the mitochondrial SO flashes increased. Glucose might increase SO flash frequency by providing more substrate for oxidative phosphorylation, and insulin might act by increasing glucose transport.…”

“…There is considerable evidence that mitochondrial alterations and oxidative stress play important roles in the development of insulin resistance in muscle cells and damage to pancreatic β-cells in diabetes [4]. Because the impact of elevated blood glucose levels and insulin on the production of SO by individual mitochondria was previously unknown, Fang et al [9] imaged mt-cpYFP fluorescence in skeletal muscle cells during the administration of glucose or insulin to the mice. There was a marked increase in the SO flash frequency of mitochondria in response to systemic administration of either glucose or insulin.…”

“…The Many new questions arise from the data obtained using the novel mt-cpYFP probe [8,9]. First, what is the physiological function(s) of mitochondrial SO flashes?…”

“…Recently, Fang et al [9] provide an important advance towards understanding the behaviors and functions of mitochondria in vivo. Previous attempts to study mitochondrial SO production and mPTP opening in vivo had involved measurements made using isolated mitochondria, or relied on indirect indicators of SO levels (hydroethidine fluorescence) and mPTP opening (cytochrome c release into the cytosol), and did not reveal the existence of mitochondrial SO flashes.…”