Isola 2022 Abstract Bioblast 2nd

Isola Raffaella, Lai Y, Hoppel CL, Noli R (2022) Mitochondrial morphology and bioenergetics in C2C12 myotubes. Bioblast 2022: BEC Inaugural Conference.

Link: Bioblast 2022: BEC Inaugural Conference

Isola Raffaella, Lai Y, Hoppel CL, Noli R (2022)

Event: Bioblast 2022

Mitochondrial cristae are dynamic compartments, capable of taking various shapes under different physiological conditions. Previous studies used two-dimensional models to observe cristae morphology and/or assessed mitochondria bioenergetics [1,2]. However, no study investigated on the relationship between the three-dimensional structure of the cristae and the bioenergetic activity of the cells on a relevant number of mitochondria.

This preliminary study compares the three-dimensional structure of the cristae, by high resolution scanning electron microscopy (HRSEM), with mitochondrial bioenergetic activity. Being preliminary, we tested the efficacy of two permeabilizing agents, digitonin and α-chaconine, on oxidative phosphorylation (OXPHOS) detection in whole cells.

To this end, we used mouse myoblasts cell cultures (C2C12) after myogenic differentiation by switching their culture medium with a low-serum medium. Mitochondrial OXPHOS has been assessed by a Clark-type electrode on digitonin- or α-chaconine- permeabilized myotubes [3]. Myotubes bioenergetics have been investigated with complex I (malate plus pyruvate), II (rotenone plus succinate), III (rotenone plus DHQ, durohydroquinone) and IV (rotenone plus ascorbate plus tetramethyl-p-phenylenediamine, TMPD) substrates, PalCoA as lipid substrate and DNP (dinitrophenol) as uncoupler.

For morphological examination, myoblasts and myotubes were treated with or without permeabilizing agents and underwent osmium maceration technique [4], which allows three-dimensional observation of cristae by HRSEM.

We titrated both permeabilizing agents to find the optimal concentration for promoting substrate entry in myotubes without compromising the integrity of the outer mitochondrial membrane. The resulted best concentrations were: 0.008 % for digitonin and 30 µM for α-chaconine. Higher concentrations were detrimental to myotubes.

The basic mitochondrial morphology revealed pleomorphic mitochondria, that were thin and elongated, or short and roundish. The shape of mitochondrial cristae in thin mitochondria was mainly lamellar, while in roundish mitochondria was more various and often coexistence of lamellar and tubular cristae was observed. In general, a manifest change of cristae appearance was not evident after adding the permeabilizing agent, regardless of digitonin or α-chaconine administration. Untreated myoblasts displayed similar morphologies.

Bioenergetic data were consistent in digitonin and α-chaconine treated cells, and for complex I were 10.91±1.68 and 9.5±3.15 nmol/min/10*6 cells, respectively.

In conclusion, the osmic maceration technique allowed us to state that apparently no significant differences in cristae morphology were detected in myotubes after either digitonin or α-chaconine cell membrane permeabilization.

Digitonin and α-chaconine seem to have a comparable effect in obtaining a reliable bioenergetic activity of myotubes, as a consequence of permeabilization, with a slight additional efficacy for digitonin.

Acknowledgements. This work was supported by the Research Cooperation Agreement with the Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA.

1. Mannella CA, Lederer WJ, Jafri MS (2013) The connection between inner membrane topology and mitochondrial function. J Mol Cell Cardiol, 62: 51-7. https://doi.org/10.1016/j.yjmcc.2013.05.001
2. Ding C, Wu Z, Huang L, Wang Y, Xue J, Chen S, Deng Z, Wang L, Song Z, Chen S (2015) Mitofilin and CHCHD6 physically interact with Sam50 to sustain cristae structure. Sci Rep, 5, 16064. https://doi.org/10.1038/srep16064
3. Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc.3: 965-76. https://doi.org/10.1038/nprot.2008.61.
4. Riva A, Loy F, Isola R, Isola M, Conti G, Perra A, Solinas P, Testa Riva F (2007) New findings on 3-D microanatomy of cellular structures in human tissues and organs. An HRSEM study. PMID: 17703594

• Keywords: Mitochondria morphology; C2C12 cells; OXPHOS; scanning electron microscopy; cristae ultrastructure

Affiliations

Isola R¹, Lai Y¹, Hoppel CH², Noli R¹

1. Dept Biomedical Sciences, Univ. of Cagliari, Italy
2. Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA - [email protected]

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