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. 2021 Sep 14;118(37):e2025932118.
doi: 10.1073/pnas.2025932118.

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy

Joshua C Drake  1   2 , Rebecca J Wilson  3   4 , Rhianna C Laker  3 , Yuntian Guan  3   5 , Hannah R Spaulding  3 , Anna S Nichenko  2 , Wenqing Shen  3 , Huayu Shang  3 , Maya V Dorn  3 , Kian Huang  3 , Mei Zhang  3   6 , Aloka B Bandara  2 , Matthew H Brisendine  2 , Jennifer A Kashatus  7 , Poonam R Sharma  8 , Alexander Young  3 , Jitendra Gautam  9 , Ruofan Cao  10 , Horst Wallrabe  10   11 , Paul A Chang  12 , Michael Wong  12 , Eric M Desjardins  13   14 , Simon A Hawley  15 , George J Christ  8   16 , David F Kashatus  7 , Clint L Miller  4   8   17   18 , Matthew J Wolf  3   6 , Ammasi Periasamy  10   11 , Gregory R Steinberg  13   19 , D Grahame Hardie  15 , Zhen Yan  1   5   6   20
Affiliations

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy

Joshua C Drake et al. Proc Natl Acad Sci U S A. .

Abstract

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5' AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Keywords: AMPK; exercise; mitochondria; mitophagy; skeletal muscle.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Identification of enymatically active AMPK on OMM in vivo. (A) Whole-cell lysates (WC), postnuclear lysates (PNL), and the corresponding enriched mitochondrial fractions of Percoll gradient isolation from mouse GA and Heart were probed for pan-AMPKα with Vdac, Catalase, and α-tubulin as loading and purity controls (n = 3). CS denotes mixed whole-tissue lysate comprised of mouse skeletal muscle, heart, and liver. (B) Immunofluorescence confocal microscopy of longitudinal sections of C57BL/6 mouse plantaris muscle probed by pan-AMPKα (red) and Cox4 (green) antibodies and DAPI for nuclear DNA (blue). Representative image of n = 3. (Scale bar, 20 µm.) (C) Enriched mitochondrial and cytosolic fractions isolated via differential centrifugation from mouse GA muscle and probed for each AMPK subunit isoform. n = 3. (D) Enriched mitochondrial fractions from frozen GA muscle of AMPKβ1/β2 knockout (KO) and wild-type littermate mice (WT). n = 3 per group. (E) Enriched mitochondrial fractions from mouse GA were treated with/without trypsin and probed for AMPKα1/α2/β2/γ1 (n = 2 per condition). An illustration of the physical association of AMPK with OMM is presented below. (F) Enriched mitochondrial fractions from human skeletal muscle biopsies (n = 2) and left ventricle biopsies (n = 3) were probed for AMPKα1/α2/β2/γ1. (G) AMPK activity in WC, cytosolic (Cyto), and enriched mitochondrial (Mito) factions with (+) and without (-) AMP (n = 3). All data presented as mean ± SEM. ***P < 0.001 by two-way ANOVA.
Fig. 2.
Fig. 2.
mitoAMPK is activated by mitochondrial energetic stress with spatial specificity. (A) Single FDB muscle fibers from C57BL/6 mice transfected with pmitoABKAR were cultured on phenol-red–free Matrigel coated glass plates, and FLIM/FRET efficiency (E%) was measured at rest and following 20 min of electrical stimulation-induced contractions and illustrated as representative heat map images (Left) and histogram (Right). (B) Calculated mean E%. n = 18 fibers across four independent experiments. (C) Representative heat map image of C2C12 myoblasts transfected with pmitoABKAR and imaged via confocal microscopy prior to 15 and 60 min following administration of 2.5 µM Oligomycin. (D) Data presented as normalized FRET ratio (FRET/cerulean). n = 21 cells per timepoint across two independent experiments. (E) Enriched mitochondrial fractions from GA muscles of sedentary (Sed) or immediately after 90 min of gradient treadmill running (Ex) in dnAMPKα2 and WT littermate mice were probed for p-AMPKα1/2 (T172) and pan-AMPKα. WT-sed (n = 13), WT-Ex (n = 13), dnTG-Sed (n = 7), and dnTG-Ex (n = 8). (F) Quantitative data of phosphorylated AMPK relative to total AMPK and total AMPK relative to Vdac. (G) Enriched mitochondrial fractions and corresponding cytosolic fractions from GA muscles of sedentary mice following 3 d metformin treatment (250 mg/kg via I.P.) or saline were probed for p-AMPKα1/2 (T172) and pan-AMPKα. For both groups, n = 5. (H) Quantitative data of phosphorylated AMPK relative to total AMPK in both Mito and Cyto fractions as well as total AMPK relative to Vdac. (I) Oxygen consumption rates of permeabilized TA muscle fibers in the presence of glutamate (10 mM) and malate (1 mM) were added to determine complex I leak respiration in the presence of physiological free ADP levels (20 µM) followed by titration of Metformin into the chamber (n = 3, run in triplicate). (J) Representative trace of complex I leak respiration during metformin titration. All data are presented as mean ± SEM. Results of the paired Student’s t test (B), one-way ANOVA (D), two-way ANOVA (F), unpaired Student’s t test (H), and repeated measures ANOVA (I) are *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3.
Fig. 3.
mitoAMPK activity regulates mitochondrial quality control. (A) Live confocal imaging of C2C12 myoblasts transfected with pCIneo, pmitoAIP, and pmitoAIP(TA) carrying mCherry (red) and stained with MitoTracker Deep Red (gray) with nontransfected cells (NT) as control. (B) Quantification of mitochondria occupied area as fold change relative to pCI-neo transfected cells. pCIneo (n = 73), NT (n = 78), pmitoAIP (n = 29), and pmitoAIP(TA) (n = 30) between three independent experiments. (C) Representative images of C57BL/6J mouse (10 to 12 wk) FDB fibers cotransfected with either pMitoTimer and pCIneo or pMitoTimer and pmitoAIP(-mCherry). Images are merged red and green channels. (Scale bar, 20 µm.) (D) Quantification of MitoTimer Red:Green fluorescence intensity and pure red puncta. n = 10 per group. Data presented as mean ± SEM. Results of one-way (B) or two-way ANOVA (D) are *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 4.
Fig. 4.
A working model of the regulation and function of mitoAMPK in mitochondrial remodeling. Mitochondrial energetic stress under the conditions of ischemia, muscle contraction, and/or pharmacological inhibition of mitochondrial respiratory chain will lead to subcellular increase of AMP and/or ADP in the vicinity of damaged/dysfunctional mitochondria (indicated by mitochondria of red color), which binds to and activates mitoAMPK (increased phosphorylation by upstream kinases). Activation of mitoAMPK promotes mitochondrial biogenesis, fission, and mitophagy through phosphorylating PGC-1a, MFF1, and ULK1, respectively. Cooperation of PGC-1a and NRF1/2 action in the nuclear genome with TFAM action in the mitochondrial genome drives mitochondrial biogenesis to add new, functional mitochondria (indicated by mitochondria of green color). Activated MFF1 interacts with DRP1 in executing mitochondrial fission for the physical separation the damaged/dysfunctional portion of mitochondria from the mitochondrial reticulum. Activated ULK1 promotes formation and targeting of autophagosome to damaged/dysfunctional mitochondria, which fuses with lysosome for degradation in autolysosome. This working model points to the central role of mitoAMPK in sensing mitochondrial energetic stress and regulating mitochondrial remodeling with subcellular specificity.
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