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A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
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MMP-2 is localized to the mitochondria-associated membrane of the heart

Bryan G Hughes1, Xiaohu Fan, Woo Jung Cho

  • 1Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada;

American Journal of Physiology. Heart and Circulatory Physiology
|December 31, 2013
PubMed

Insights

Matrix metalloproteinase-2 (MMP-2) is primarily located in the mitochondria-associated membrane (MAM), not mitochondria. This MAM-localized MMP-2 may affect mitochondrial function by altering calcium signaling through calreticulin proteolysis.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Mitochondrial Research

Background:

  • Matrix metalloproteinase-2 (MMP-2) is known for extracellular matrix remodeling but also has intracellular roles.
  • Previous studies reported MMP-2 in mitochondria, a site of oxidative stress, without considering mitochondria-associated membranes (MAMs).
  • MAMs are specialized regions connecting the endoplasmic reticulum (ER) and mitochondria.

Purpose of the Study:

  • To investigate the precise subcellular localization of MMP-2, specifically differentiating between mitochondria and MAM.
  • To determine if MMP-2 associated with MAM impacts ER-mitochondrial communication.

Main Methods:

  • Immunogold electron microscopy on mouse heart sections to visualize MMP-2.
  • Immunofluorescence analysis of MMP-2:HaloTag fusion protein in cardiomyocytes, co-localizing with ER and mitochondrial markers.
  • Biochemical fractionation of crude mitochondrial preparations into purified mitochondria and MAM to assess MMP-2 distribution and activity.
  • In vitro proteolysis assays using MMP-2 on calreticulin.

Main Results:

  • Immunogold microscopy showed MMP-2 in mouse heart mitochondria.
  • Immunofluorescence revealed an ER-like distribution for expressed MMP-2, with higher colocalization with ER markers than mitochondrial markers.
  • Purified MAM fractions contained the majority of MMP-2 protein and activity, with minimal amounts in purified mitochondria.
  • MMP-2 was shown to proteolyze calreticulin, an ER/MAM protein, in vitro.

Conclusions:

  • The majority of MMP-2 previously identified in mitochondria is actually located in the mitochondria-associated membrane (MAM).
  • MAM-localized MMP-2 has the potential to influence mitochondrial function by proteolyzing calreticulin, thereby affecting ER-mitochondrial calcium signaling.
Keywords:
calreticulinmatrix metalloproteinase 2mitochondriamitochondria-associated membrane

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