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 tMPT-dependent 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.

Figure 1

Pan-tissue expression of mt-cpYFP in transgenic mice. (A) A neonatal mt-cpYFP transgenic mouse under UV illumination. Top: plasmid construct for generating the transgenic mouse. The full-length mt-cpYFP DNA was cloned into pUC-CAGGS vector downstream of the chicken β-actin promoter. Arrows indicate the positions of forward and backward primers for genotyping. (B-D) Confocal images of mt-cpYFP fluorescence in myocardium (B), gastrocnemius muscle (C) and sciatic nerve trunk (D) at low and high magnifications. Note the cell type-specific pattern of intracellular mitochondrial distribution. (B-D) use the same scale bar in D.

Figure 2

Imaging tMPT/superoxide flashes in living mice. (A) In vivo imaging of a mt-cpYFP transgenic mouse under anesthesia. An upright confocal microscope was used to image hind limb skeletal muscle and sciatic nerve trunks exposed after skin incision. (B) Punctiform tMPT/superoxide flashes in mouse gastrocnemius. From top to bottom: xy view of mitochondria in doublet bands flanking the z-lines; time lapse, enlarged views of the flash in a boxed region of 1.57 μm × 1.76 μm; and time course plot of the superoxide flash. (C) Characteristics of skeletal muscle (Sk) and sciatic nerve (SN) superoxide flashes. ΔF/F₀, amplitude; T_p, time to peak; T₅₀, half decay time. (D) Time-lapse images and time course plot of a tMPT/superoxide flash in an axon (outlined in blue) of sciatic nerve trunk. The arrow marks the mitochondrion undergoing excitation. (E) Surface plots showing punctiform (left), linear (middle) and lamellar (right) tMPT/superoxide flashes in gastrocnemius. (F) Transient loss of ΔΨm (indexed by TMRM signal) during synchronized tMPT/superoxide flashes (the same as those in E). Similar results were obtained in four other fibers.

Figure 3

Synchrony and geometry of functional mitochondrial network in skeletal muscle. (A) Synchrony in a lamellar tMPT/superoxide flash. Data are shown as xy view (top) and space-time plots along the transverse (yt) and longitudinal (xt) directions of the flash. Line plots show the rising phase of the flash (normalized by respective amplitude) at locations marked by colored bars flanking the space-time plots. Dashed line marks the simultaneous take-off of the flash. (B) Synchronized tMPT/superoxide flash in xyz stack. Subset z-stacks corresponding to quiescent and excited states of a functional network. Arrowheads mark longitudinal elements on a z-plane of the network. (C) Three-dimensional views of the boundary of the network in B. See Materials and Methods for details of digital reconstruction and Supplementary information, Video S4 for animation.

Figure 4

Decoding metabolic status by the frequency of tMPT/superoxide flashes. (A) Representative examples showing tMPT/superoxide flash activities in basal conditions and after glucose or insulin challenges in different mt-cpYFP transgenic mice. Data are presented as overlay of xy view of a region of gastrocnemius muscle under study (bottom) and surface plot of all superoxide flashes combined from a 100-s data set. (B, C) tMPT/superoxide flash activity (column bars) during glucose (B, 2 g/kg body weight, n = 9 mice) or insulin challenge (C, 1 U/kg body weight, n = 6 mice). (D, E) Histogram analysis of unitary properties of superoxide flashes. F/F₀, amplitude; FDHM: full duration at half maximum. The traces represent nonlinear least square fittings to the FDHM histograms with multiple Gaussian functions and data from all three groups were constrained simultaneously, yielding μ₁ = 7.5, μ₂ = 14, σ₁ = 4.9 and σ₂ = 7.1. The individual component ratio was a₁:a₂ = 17.3:3.6, 9.6:9.1 or 10.5:8.3 for basal, glucose or insulin group, respectively. n = 68-150 events from 4-6 mice. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus basal.