Lai 2022 Abstract Bioblast

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Lai Nicola
Lai Nicola, Kummitha CM, Hoppel CL(2022) Effect of isolation protocol of skeletal muscle mitochondrial subpopulations on bioenergetic function. Bioblast 2022: BEC Inaugural Conference.

Link: Bioblast 2022: BEC Inaugural Conference

Lai Nicola, Kummitha Chinna, Hoppel Charles L (2022)

Event: Bioblast 2022

Introduction The cardiac and skeletal muscle subsarcolemmal (SSmt) and interfibrillar (IFmt) mitochondria have different biochemical and structural properties affecting energy metabolism in health and disease states. In both muscles [1, 2], the method to isolate mitochondria affects the quality and quantity of the SSmt and IFmt separated by subcellular fractionation techniques. An isolation protocol for skeletal muscle SSmt and IFmt was proposed by our group [2] in which the mitochondrial yield was increased with a recovery close to 80 % of the mitochondria present in the original skeletal muscle sample; SSmt oxidative capacity was 10 % lower than that of IFmt; minor damage of the mitochondrial inner and outer membranes. A human study on skeletal muscle ultrastructure and bioenergetics showed a reduced mitochondrial oxidative capacity in patients with type 1 diabetes (T1D) [3]. Nevertheless, it was not investigated the effect of the disease on the bioenergetics of the two subpopulations of mitochondria. In this work, we compare the bioenergetic characteristics of SSmt and IFmt with those of the whole mitochondrial population. This comparison was obtained for both control (C) and T1D rats.

Methods The T1D was obtained from Lewis rats treated with streptozotocin. We used our protocol [2] to isolate skeletal muscle SSmt and IFmt of C and T1D rats. The same protocol [2] was modified to isolate the whole population (Wmt) of skeletal muscle mitochondria of C and T1D rats. The oxidative phosphorylation rate was measured with a digitized polarographic system [4] with substrates of the electron transfer system: palmitoyl carnitine, palmitoyl-CoA, glutamate, and succinate.

Results The yields of SSmt and IFmt of T1D rats (1.5±0.4; 3.5±1 mg/g) from rat skeletal muscle were lower than those of the control group (2±0.5; 5.5±0.5 mg/g). In contrast, the yield of the whole population of mitochondria was similar in both groups of rats (C 7±0.9; 6.8±0.5 mg/g). OXPHOS capacity P was measured in presence of glutamate as substrate with kinetically saturating concentration (2 mM) of ADP and of the uncoupler dinitrophenol (0.2 mM, DNP). The oxidative phosphorylation assay showed that in the C group the OXPHOS capacity was lower in SSmt (Wmt 4200±250; *SSmt 3200±200; IFmt 3800±150 pmol·s-1·mg-1, P<0.01) and a similar adenylate acceptor control ratio P/L (Wmt 18±4; SSmt 21±3; IFmt 30±4); in T1D group the OXPHOS capacity was lower in SSmt and IFmt (Wmt 4050±260; *SSmt 2000±230; *,#IFmt 2600±250 pmol·s-1·mg-1, *P<0.01 different from Wmt group, # different from SSmt) and a similar P/L (Wmt 14±2; SSmt 14±3; IFmt 16±6). The electron transfer capacity upon collapsing of mitochondrial potential with the uncoupler DNP was for C rats (Wmt 5040±220; *SSmt 3950±190; IFmt 4600±180 pmol·s-1·mg-1, P<0.01) and for T1D rats (Wmt 5200±330; *SSmt 2400±300; *,#IFmt 3000±400 pmol·s-1·mg-1, P<0.01 different from Wmt group, # different from SSmt). For both C and T1D groups of rats, respiration rate difference between SSmt and IFmt were also observed in presence of palmitoyl carnitine and palmitoyl-CoA substrates.

Conclusion The results of this study provide evidence of bioenergetic differences between the whole population and subpopulations of mitochondria. This work underlines that skeletal muscle bioenergetic differences may not be properly detected if both mitochondrial subpopulations are not isolated and biochemically characterized.


O2k-Network Lab: US OH Cleveland Hoppel CL


Affiliations

Lai N(1,2), Kummitha CM(2), Hoppel CL(3,4)
  1. Department of Mechanical, Chemical and Materials Engineering, University of Cagliari Italy
  2. Department of Biomedical Engineering, School of Medicine, Case Western Reserve University Cleveland USA
  3. Department of Pharmacology, School of Medicine, Case Western Reserve University Cleveland USA
  4. Department of Medicine, School of Medicine, Case Western Reserve University Cleveland USA. - nicola.lai@unica.it


References

  1. Palmer JW, Tandler B, Hoppel CL (1977) Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252:8731–9.
  2. Lai N, Kummitha C, Rosca MG, Fujioka H, Tandler B, Hoppel C (2019) Isolation of mitochondrial subpopulations from skeletal muscle: optimizing recovery and preserving integrity. Acta Physiol (Oxf):e13182.
  3. Monaco CMF, Hughes MC, Ramos SV, Varah NE, Lamberz C, Rahman FA, McGlory C, Tarnopolsky MA, Krause MP, Laham R, Hawke TJ, Perry CGR (2019) Altered mitochondrial bioenergetics and ultrastructure in the skeletal muscle of young adults with type 1 diabetes. Diabetologia 61:1411-23.
  4. Potter L, Krusienski D, Kennedy J, Hoppel CL, Lai N (2020) Integrated approach for data acquisition, visualization and processing of analog polarographic systems for bioenergetics studies. Anal Biochem 590:113515.

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