Figure 1.
(A) Fluorescent confocal images of a single isolated FDB muscle fiber loaded with MitoSox red (125 nM) and Mitotracker green (20 nM). (i)Fluorescent image of fiber loaded with mitotracker, (ii) Fluorescent image of fiber loaded with MitoSox obtained using excitation at 405 nm, (iii) Fluorescent image of fiber loaded with MitoSox obtained using excitation at 488 nm, (iv) merge of bright field and fluorescent images, (v) merged fluorescent images i–iii, (vi) bright field image. Scale bar = 25 µm. (B) Fluorescence values from MitoSox red loaded fibers (n = 3) with time, prior to -, and after exposure to 10 µM antimycin A (denoted by the black bar). Data from excitation at 405 nm is claimed to reflect 2-hydroxy MitoSox and excitation at 488 nm monitors non-specific oxidation products of MitoSox.
Figure 2.
(A) MitoSox red loaded single isolated skeletal muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) merged images i and ii, (iv) bright field image, scale bar = 25 µm. Figures B and D show the relative fluorescence with time from skeletal muscle fibers loaded with MitoSox red (data presented from 405 nm excitation). Fibers were either non-stimulated or subjected to two periods of electrically stimulated contractions during the time periods denoted by a black bar. No significant effect of stimulation over the whole time course was found in the absence of L-NAME (Fig. 2B) whereas in the presence of L-NAME (Fig. 2D) a significant effect of stimulation was found (repeated measures, F = 5.3, P = 0.04) compared with non stimulated fibers. Figures C and E show the rate of change in relative fluorescence (derived from figures B and D ) between the indicated time points with the stimulation periods denoted by black bars. Data shown in Figures B and C were from untreated fibers, data in Figures D and E were from fibers in the presence of 100 µM L-NAME. *P<0.05 compared with non-stimulated fibers at the same time point (n = 6–7 for all groups).
Figure 3.
(A) DHE loaded single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) merged images i and ii, (iv) bright field image, scale bar = 50 µm. Figures B and D show relative fluorescence with time from skeletal muscle fiber nuclei (data presented from 405 nm excitation only, n = 8). Fibers were either at rest or subjected to muscle contraction as denoted by the black bar. Figures C and E show the rate of change in relative fluorescence (derived from figures B and D respectively) between indicated time points. Data shown in Figures B and C were from untreated fibers, data in Figures D and E were from fibers in the presence of 100 µM L-NAME. *P<0.05 compared with non-stimulated fibers at the same time point.
Figure 4.
(A) DHE loaded single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) Fluorescent image obtained with 405 nm emission and 450/35 nm emission monitoring non-oxidised DHE, (iv) bright field image, scale bar = 50 µm, (v) merged figures i–iii. (B) Data from muscle fibers that were either stimulated (as denoted by black bar) or non-stimulated (n = 6 for all groups). Solid symbols show 2-HE fluorescence over time measured from nuclei showing the acute increases in fluorescence following contractions and open symbols show the “unoxidised” DHE fluorescence measured from the same muscle fibers. Cytosolic fluorescence from “unoxidised” DHE decreased by approximately 50% over sixty minutes in both experimental groups reflecting loss of DHE from the cell. (C) Rate of change in relative fluorescence with time for cytosolic DHE (derived from figure B). *P<0.05, compared with non stimulated fibers at the same time point.
Figure 5.
(A) Cyto-HyPer transfected single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) bright field image, (iv) merged images i and ii, scale = 50 µm. (B) Non-transfected single muscle fiber, (i) Fluorescent image obtained using excitation at 405 nm, (ii) Fluorescent image obtained using excitation at 488 nm, (iii) bright field image, (iv) merged images i and ii, scale = 50 µm. (C) Ratio of fluorescence values at excitations of 488/405 nm from fibers transfected with HyPer that were either stimulated (as denoted by black bars) or non- stimulated (n = 7 for both groups). (D) Rate of change in ratio of fluorescence at excitations of 488/405 nm from fibers transfected with HyPer. *P<0.05, compared with non stimulated fibers at the same time point.