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

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

Indicators of increased ER stress and UPR in aged D2-mdx and human dystrophic skeletal muscles

Duchenne muscular dystrophy (DMD) is a progressive muscle disease that results in muscle wasting, wheelchair dependence, and eventual death due to cardiac and respiratory complications. In addition to muscle fragility, dystrophin deficiency also results in multiple secondary dysfunctions, which may lead to the accumulation of unfolded proteins causing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The purpose of this investigation was to understand how ER stress and the UPR are modified in muscle from D2-mdx mice, an emerging DMD model, and from humans with DMD. We hypothesized that markers of ER stress and the UPR are upregulated in D2-mdx and human dystrophic muscles compared to their healthy counterparts. Immunoblotting in diaphragms from 11-month-old D2-mdx and DBA mice indicated increased ER stress and UPR in dystrophic diaphragms compared to healthy, including increased relative abundance of ER stress chaperone CHOP, canonical ER stress transducers ATF6 and pIRE1α S724, and transcription factors that regulate the UPR such as ATF4, XBP1s, and peIF2α S51. The publicly available Affymetrix dataset (GSE38417) was used to analyze the expression of ER stress and UPR-related transcripts and processes. Fifty-eight upregulated genes related to ER stress and the UPR in human dystrophic muscles suggest pathway activation. Further, based on analyses using iRegulon, putative transcription factors that regulate this upregulation profile were identified, including ATF6, XBP1, ATF4, CREB3L2, and EIF2AK3. This study adds to and extends the emerging knowledge of ER stress and the UPR in dystrophin deficiency and identifies transcriptional regulators that may be responsible for these changes and be of therapeutic interest

Soleus and EDL muscle weights and function.

<p>* indicates significantly different from C57; # indicates significantly different from mdx. C57 (n = 6–7) mdx (n = 6) mdxQ (n = 5–6).</p

Dystrophin deficiency increased fibronectin in soleus muscles compared to healthy muscles.

<p>A-C) Representative 10x immunohistochemical images for fibronectin (red) and DAPI (blue). All images have been uniformly brightened to make the fibronectin signal easier to see. D) The percent positive pixels were quantified. * indicates significantly different from C57. Width of white bar represents 100 microns. C57 (n = 7) mdx (n = 6) mdxQ (n = 6).</p

Dystrophin deficiency causes histological injury that is not corrected by quercetin.

<p>A-C) Representative images from H&E-stained, reconstructed soleus muscle cross sections. E) Extra cellular nuclei, F) centralized nuclei, and G) total contractile area were calculated. * indicates significantly different from C57. Width of black bar represents 250 microns. C57 (n = 7) mdx (n = 6) mdxQ (n = 6).</p

Animal activity was increased by dietary quercetin enrichment.

<p>At the conclusion of the investigation animal behavior was quantified using an ethological approach where a 10 minute observation period was divided into 15 second blocks. A) The number of time blocks spent sitting or exhibiting sedentary behavior was quantified. B) We also quantified the number of active behaviors. * indicates significantly different from C57; # indicates significantly different from mdx. C57 (n = 7) mdx (n = 7) mdxQ (n = 6).</p

Transcript expression in the soleus.

<p>Data are shown as fold change relative to C57. * indicates significantly different from C57; # indicates significantly different from mdx. C57 (n = 7) mdx (n = 6) mdxQ (n = 6).</p