MiP2005: Session 5

Mitochondrial Physiology Network 10.9: 59-60 (2005) - download pdf


Depression of oxidative phosphorylation under normoxia during invertebrate diapause and strategies for mitochondrial stabilization.

J Reynolds1, X-H Liu2, A Aksan2, M Menze1, G Elliott2, M Toner2, Steve Hand1 

1Division of Cellular, Developmental and Integrative Biology, Department of Biological Sciences, Louisiana State University, Baton Rouge, USA; 2Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, USA. – shand@LSU.edu

    In response to cues signifying the approach of winter, adult brine shrimp (Artemia franciscana) produce encysted embryos that enter diapause.  Diapause is a specific type of dormancy that is genetically programmed into the life cycle, is controlled by endogenous factors, and is characterized by developmental and metabolic arrest.  Diapause affords profound tolerance to severe environmental stresses.  We show that respiration rates of diapause embryos collected from the field (Great Salt Lake, Utah, USA) are reduced up to 92 % compared with non-diapause embryos when measured under conditions of normoxia and full hydration [1]. However, mitochondria isolated from diapause embryos exhibit rates of state 3 and state 4 respiration on pyruvate that are equivalent to those for non-diapause embryos with active metabolism; a reduction in state 3 and state 4 respiration (15 % – 27 %) was measured with succinate for two of three collection years. Respiratory control ratios for diapause mitochondria are comparable to or higher than those for non-diapause embryos. The P:O flux ratios are statistically identical.  Our calculations suggest that respiration of intact, non-diapause embryos is operating close to the state 3 oxygen fluxes measured for isolated mitochondria. For one collection year, cytochrome c oxidase (COX) activity was 53 % lower in diapause mitochondria; the minimal impact of this COX reduction on mitochondrial respiration appears to be due to the 31 % excess COX capacity in A. franciscana mitochondria. Transmission electron micrographs of embryos reveal mitochondria that are well differentiated and structurally similar in both states. As inferred from the similar amounts of mitochondrial protein extractable, tissue contents of mitochondria in diapause and postdiapause embryos are equivalent. Taken together, metabolic depression during diapause cannot be fully explained by lower mitochondrial densities in vivo or by the properties measured for isolated mitochondria. Rather, downregulation may be due to reversible inhibition of oxidative phosphorylation in vivo that is not maintained in isolated mitochondria (e.g., via phosphorylation of cytochrome c oxidase subunit 1 [2], a diffusible inhibitor, or substrate limitation).

    In nature, diapause embryos and their mitochondria are commonly exposed to prolonged periods of anoxia and severe desiccation [cf., 3]. During these periods of restricted energy flow, structure and functional capacity of mitochondria must be retained and opening of the mitochondrial permeability transition pore (MPTP) avoided if embryos are to survive and recover [4]. Trehalose content in A. franciscana embryos is approximately 400 mM. This sugar is well known for its ability to stabilize macromolecules during drying and has been used extensively to improve the desiccation tolerance of mammalian cells.  We show evidence that trehalose is present in the matrix of mitochondria from A. franciscana embryos. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the MPTP.  Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography shows that the sugar permeated rapidly into the matrix upon opening the MPTP.  The concentration of intra-matrix trehalose reaches 0.29 mmol/mg protein (~190 mM) in 5 min.  Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After rehydration, the inner membrane integrity was assessed by measurement of membrane potential with the fluorescent probe JC-1. Results show that following drying to similar water contents, the mitochondria loaded with trehalose have significantly higher inner membrane integrity than those without trehalose. These findings suggest the presence of trehalose in the matrix improves desiccation tolerance of isolated mitochondria.

   Supported by NIH grant 1-RO1-GM071345-01 and DARPA contract N00173-01-1-G011.

1.  Reynolds J, Hand S (2004) Differences in isolated mitochondria are insufficient to account for respiratory depression during diapause in Artemia franciscana embryos.  Physiol. Biochem. Zool. 77: 366-377.

2.  Lee I, Salomon A, Ficarro S, Mathes I, Lottspeich F, Grossman L, Hüttemann M (2005) cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity.  J. Biol. Chem. 280: 6094–6100.

3.  Covi J, Treleaven W, Hand S (2005) V-ATPase inhibition prevents recovery from anoxia in Artemia franciscana embryos: Quiescence signaling through dissipation of proton gradients.  J. Exp. Biol. 208 (in press).

4.  Menze M, Hutchinson K, Laborde S, Hand S (2005) Mitochondrial permeability transition in the crustacean Artemia franciscana: Absence of a Ca2+-regulated pore in the face of profound calcium storage.  Amer. J. Physiol. 289: R68-R76.

5.  Liu X-H, Aksan A, Menze M, Hand S, Toner M (2005) Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria. Biochim. Biophys. Acta (accepted with revision).

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