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 5

Characterization of the role of EMRE in a muscular dystrophy mouse model exhibiting mitochondrial calcium overload.

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Characterization of the role of EMRE in a muscular dystrophy mouse model...
(A) Western blot analysis of MCU and EMRE protein expression in quadriceps muscle from WT (n = 2 biological replicates) and LAMA2–/– (n = 3 biological replicates) mice. VDAC1 serves as a mitochondrial loading control. Molecular weights from the protein ladder are indicated on the left. (B and C) Quantification of MCU (B) and EMRE (C) protein expression in A, normalized to VDAC1. Data are represented as mean ± SD; results of unpaired t test with Welch’s correction. *P < 0.05. (D) Survival curve for control (n = 26), EMRESM-KO (n = 16), LAMA2–/– (n = 19), and EMRESM-KO LAMA2–/– (n = 17) mice (all biological replicates). (E) Body weights for 4-week-old male control (n = 14), EMRESM-KO (n = 10), LAMA2–/– (n = 13), and EMRESM-KO LAMA2–/– (n = 11) mice (all biological replicates). (F) Body weights for 4-week-old female control (n = 13), EMRESM-KO (n = 10), LAMA2–/– (n = 9), and EMRESM-KO LAMA2–/– (n = 15) mice (all biological replicates). (G) Assessment of muscle strength using an inverted grid test on 4-week-old control (n = 15), EMRESM-KO (n = 9), LAMA2–/– (n = 18), and EMRESM-KO LAMA2–/– (n = 17) mice (all biological replicates). (H) Representative image of an isolated flexor digitorum brevis (FDB) muscle fiber loaded with the fluorescent calcium-sensitive dye Rhod-2 AM (in red), the mitochondria mass stain MitoTracker Green (in green), and the ratio of Rhod-2 AM to MitoTracker Green (heatmap). (I) Relative levels of matrix calcium in FDBs isolated from control (n = 32 cells from n = 5 mice), EMRESM-KO (n = 25 cells from n = 4 mice), LAMA2–/– (n = 22 cells from n = 3 mice), and EMRESM-KO LAMA2–/– (n = 21 cells from n = 3 mice) mice (biological replicates) as measured by the ratio of Rhod-2 AM to MitoTracker Green. Data are represented as mean ± SD. Data were first compared by 1-way ANOVA followed by unpaired t test with Welch’s correction to determine significance for each pair of data sets. *P < 0.05; **P < 0.01.