Grx2 Regulates Skeletal Muscle Mitochondrial Structure and Autophagy

Glutathione is an important antioxidant that regulates cellular redox status and is disordered in many disease states. Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that plays a pivotal role in redox control by catalyzing reversible protein deglutathionylation. As oxidized glutathione (GSSG) can stimulate mitochondrial fusion, we hypothesized that Grx2 may contribute to the maintenance of mitochondrial dynamics and ultrastructure. Here, we demonstrate that Grx2 deletion results in decreased GSH:GSSG, with a marked increase of GSSG in primary muscle cells isolated from C57BL/6 Grx2−/− mice. The altered glutathione redox was accompanied by increased mitochondrial length, consistent with a more fused mitochondrial reticulum. Electron microscopy of Grx2−/− skeletal muscle fibers revealed decreased mitochondrial surface area, profoundly disordered ultrastructure, and the appearance of multi-lamellar structures. Immunoblot analysis revealed that autophagic flux was augmented in Grx2−/− muscle as demonstrated by an increase in the ratio of LC3II/I expression. These molecular changes resulted in impaired complex I respiration and complex IV activity, a smaller diameter of tibialis anterior muscle, and decreased body weight in Grx2 deficient mice. Together, these are the first results to show that Grx2 regulates skeletal muscle mitochondrial structure, and autophagy.

Ultrastructure of hyphal cells of the dermatophyte Trichophyton tonsurans

Background and Purpose: Trichophyton tonsurans is a widely distributed anthropophilic dermatophyte causing different diseases of skin. In the literature limited data are available about the morphogenesis of vegetative mycelium of T. tonsurans and related anthropophilic dermatophytes. The aim of present study was to describe ultrastructural patterns of development, cellular organellography and septal pore apparatus structure of in vitro growing vegetative mycelium of T. tonsurans. Materials and Methods: Trichophyton tonsurans strain RCPFF 214/898 was grown on solid Czapek’s Agar (CzA) at 28ºС. For investigation of colonies morphology we used methods of light-, scanning and transmission electron microscopy (SEM and TEM). Results: Differences in morphogenesis of aerial and substrate hyphae were revealed. Mitochondrial reticulum and fibrosinous bodies were shown in T. tonsurans for the first time. The septal pore apparatus in hyphal cells of was comprised Woronin bodies and septal pore plugs. Woronin bodies (0.18 μm), located with 1‒4 near the pore, were spherical, membrane-bound, and had a homogeneous, electron-dense content. The cells of aerial and submerged hyphal cells of T. tonsurans contain two nuclei. Conclusion: Mature cells of substrate hyphae appeared more active than comparable cells in the aerial mycelium. During the maturation process, the differences in number and morphology of mitochondria, number of vacuoles, and in the synthesis of different types of storage substances were revealed. Presence of “mitochondrial reticulum” and variable types of storage substances in submerged hyphal cells suggested higher levels of metabolic activity compared to aerial mycelium.

PO-033 Mitochondria-associated Ampk mediates mitochondrial quality control in skeletal muscle

Objective Mitochondria exist as a complex, interconnected reticulum with the degree of complexity at homeostasis varying by cell type. Declines in mitochondrial quality is a hallmark of numerous diseases and is partly maintained through the targeted removal and degradation of damaged/dysfunctional regions of the reticulum known as mitophagy. We have demonstrated that acute exercise causes targeted degradation of a subset (<1%) of the mitochondrial reticulum through mitophagy in an AMPK-dependent manner. However, how such spatial regulation is achieved is unclear. Given that AMPK is a well-known bioenergetics sensor critical for maintenance of energetic homoeostasis, and numerous studies have linked AMPK-signaling to mitochondrial remodeling and functional adaptations under physiological and pathological conditions, we hypothesized that AMPK regulates mitophagy by being physically associated with the mitochondria. Methods C57BL/6 mice (3 or 20 months of age) were kept in standard conditions (12:12 light, dark cycle) on normal chow. Enriched mitochondrial fractions from skeletal muscle were obtained via differential centrifugation and percoll gradient isolation. Skeletal muscle energetic stress was induced via acute treadmill running, direct electrical stimulation, or hindlimb ischemia-reperfusion. Somatic gene transfer into skeletal muscle was done via electraporation. Confocal microscopy of whole mount skeletal muscle was performed at least 10 days post-electraporation to assess MitoTimer fluorescence Red:Green Ratio and Mitophagy (i.e. presence of pure red puncta). Results We have discovered that AMPK is enriched in isolated mitochondria from both mouse and human skeletal muscle (mitoAMPK) and that, in mice, mitoAMPK consists exclusively of ⍺1/β2/γ1 isoforms. Furthermore, skeletal muscle mitoAMPK Thr172 phosphorylation, indicative of activation, is increased following electrical stimulation, ischemia-reperfusion, and acute treadmill running. By co-transfecting mouse FDB muscle with pMitoTimer and a mitochondrially-targeted AMPK inhibitor peptide (mitoAIP), we show that mitoAMPK activity is required to maintain mitochondrial quality (as evidenced by increased MitoTimer Red:Green ratio) and exercise-induced mitophagy (MitoTimer pure red puncta). Interestingly, association of mitoAMPK with mitochondria declines by ~50% in skeletal muscle in old mice and coincides with poor mitochondrial quality (increased MitoTimer Red:Green ratio), which may explain the loss of mitochondrial quality and metabolic flexibility with aging. Conclusions Our current working hypothesis is that activation of mitoAMPK by exercise plays an instrumental role in promoting mitochondrial remodeling, hence contractile and metabolic adaptations, by spatially recognizing damaged regions of the mitochondrial reticulum. Future research on mitoAMPK will significantly improve the mechanistic understanding of exercise training-induced adaptation in skeletal muscle and AMPK biology.

Lactate is oxidized outside of the mitochondrial matrix in rodent brain

The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD+, suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD+-dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD+-dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.