Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification

J Pharmacol Toxicol Methods. May-Jun 2018;91:50-58. doi: 10.1016/j.vascn.2018.01.004. Epub 2018 Jan 31.

Abstract

Introduction: In the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins.

Methods: SSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot.

Results: MFN2 and Cx43 were found predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM.

Discussion: Whereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation.

Keywords: Connexin-43; Heart; IFM; Methods; Mitochondria isolation; Mitofusin; Nagarse; SSM; Subtilisin BPN'.

MeSH terms

  • Animals
  • Cell Fractionation / methods
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mitochondria / metabolism*
  • Mitochondrial Proteins / analysis*
  • Mitochondrial Proteins / metabolism
  • Models, Animal
  • Myocardium / cytology
  • Oxygen Consumption / drug effects
  • Phenylmethylsulfonyl Fluoride / pharmacology
  • Pilot Projects
  • Proteomics / methods
  • Rats
  • Rats, Long-Evans
  • Rats, Wistar
  • Subtilisins / metabolism*

Substances

  • Mitochondrial Proteins
  • Phenylmethylsulfonyl Fluoride
  • Subtilisins