EMRE is essential for mitochondrial calcium uniporter activity in a mouse model
Julia C. Liu, Nicole C. Syder, Nima S. Ghorashi, Thomas B. Willingham, Randi J. Parks, Junhui Sun, Maria M. Fergusson, Jie Liu, Kira M. Holmström, Sara Menazza, Danielle A. Springer, Chengyu Liu, Brian Glancy, Toren Finkel, Elizabeth Murphy
Julia C. Liu, Nicole C. Syder, Nima S. Ghorashi, Thomas B. Willingham, Randi J. Parks, Junhui Sun, Maria M. Fergusson, Jie Liu, Kira M. Holmström, Sara Menazza, Danielle A. Springer, Chengyu Liu, Brian Glancy, Toren Finkel, Elizabeth Murphy
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Research Article Cardiology Muscle biology

EMRE is essential for mitochondrial calcium uniporter activity in a mouse model

Abstract

The mitochondrial calcium uniporter is widely accepted as the primary route of rapid calcium entry into mitochondria, where increases in matrix calcium contribute to bioenergetics but also mitochondrial permeability and cell death. Hence, regulation of uniporter activity is critical to mitochondrial homeostasis. The uniporter subunit EMRE is known to be an essential regulator of the channel-forming protein MCU in cell culture, but EMRE’s impact on organismal physiology is less understood. Here we characterize a mouse model of EMRE deletion and show that EMRE is indeed required for mitochondrial calcium uniporter function in vivo. EMRE–/– mice are born less frequently; however, the mice that are born are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. Finally, to investigate the role of EMRE in disease processes, we examine the effects of EMRE deletion in a muscular dystrophy model associated with mitochondrial calcium overload.

Authors

Julia C. Liu, Nicole C. Syder, Nima S. Ghorashi, Thomas B. Willingham, Randi J. Parks, Junhui Sun, Maria M. Fergusson, Jie Liu, Kira M. Holmström, Sara Menazza, Danielle A. Springer, Chengyu Liu, Brian Glancy, Toren Finkel, Elizabeth Murphy

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Figure 1

EMRE is required for rapid mitochondrial calcium uptake.

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EMRE is required for rapid mitochondrial calcium uptake. (A) Western blo...
(A) Western blot analysis of MICU1, MCU, and EMRE protein expression in isolated mitochondria from WT and EMRE–/– hearts (n = 4 biological replicates per group). COX4 serves as a mitochondrial loading control. Molecular weights from the protein ladder are indicated on the left. (B) BN-PAGE analysis of isolated mitochondria from WT and EMRE–/– hearts, immunoblotted with MCU antibody (n = 3 technical replicates per group). Data are representative of at least n = 3 experiments on biological replicates. Molecular weights from the protein ladder are indicated on the left. (C) Extramitochondrial calcium traces with isolated mitochondria from WT and EMRE–/– hearts using the calcium indicator dye Calcium Green-5N. Arrows represent calcium addition of the concentration last indicated. Data are representative of at least n = 3 experiments on biological replicates. (D) Mitochondrial swelling assay measured by absorbance of isolated mitochondria from WT and EMRE–/– hearts in response to a 250 μM addition of calcium. Data are representative of at least n = 3 experiments on biological replicates. (E) Western blot analysis of MICU1, MCU, and EMRE protein expression in MEFs isolated from WT and EMRE–/– embryos that were stably transduced with either an empty vector or with a vector encoding a V5 epitope–tagged EMRE construct. Tubulin serves as a loading control. Molecular weights from the protein ladder are indicated on the left. (F) Representative traces of cytosolic calcium measurements in permeabilized MEFs using Calcium Green-5N. Arrows represent calcium addition of the concentration last indicated. Shown are WT MEFs, EMRE–/– MEFs, or EMRE–/– MEFs reconstituted with V5 epitope–tagged EMRE transgene. Data are representative of at least n = 5 experiments on independent cell cultures.