Regulation of the mitochondrial integrated stress response and ATF4 by acute contractile activity in mouse muscle

Abstract

In the context of energetically demanding conditions, such as exercise, skeletal muscle relies on mitochondria to support its metabolic requirements. Maintenance of mitochondria and their bioenergetic availability is thus critical to sustain the metabolic flexibility of muscle. The quality of the organelle network is highly regulated by the coordination of mitochondrial quality control (MQC) processes. One MQC pathway that has garnered recent interest is the integrated stress response (ISR), which is activated in response to a variety of stimuli. The transcription factor ATF4, denoted as the main effector of this response pathway, serves to ameliorate cellular stress by upregulating a plethora of cytoprotective genes, such as CHOP and ATF5. Current literature has shown that the ISR is activated upon mitochondrial stress/perturbations to restore optimal organelle function and health. However, the precise mechanism that constitutes the activation of ISR and thus the response of ATF4 following acute exercise-induced stress is unknown. Therefore, our objective is to determine the extent of ISR/ATF4 induction in vivo and to test whether this pathway is required for the maintenance of muscle function. To investigate this, a mouse in situ hindlimb protocol was utilized to acutely stimulate muscles at 0.25-1 tetani/per second for 9 mins, followed by a 1-hour recovery period. The kinases, CAMKIIα and JNK2, were robustly phosphorylated/activated 6-fold immediately following the protocol. The initial stage of ISR activation, reflected in the ratio of phosphorylated to total-eIF2α protein levels, was also increased in response to acute contractile activity, evident in the recovery phase. Downstream of ISR activation, the total protein expression of ATF4 remained unchanged, however, contractile activity induced an increase in the localization of ATF4 to the nucleus. Robust 2-fold increases in the mRNA expression of ATF4 and CHOP were also observed and enhanced following the recovery period. Changes in ATF4 mRNA appeared to be independent of transcriptional activation, as assessed using an ATF4 promoter-reporter plasmid. An in vitro cell free mRNA decay assay revealed an increase in ATF4 mRNA stability post-stimulation, possibly as a result of cellular compartmental changes in the RNA binding proteins, HuR and CUGBP1. RNA sequencing analyses also confirmed the observed increase in related ISR genes following an exhaustive bout of exercise. These data suggest that acute contractile activity is indeed suffcient to induce mitochondrial stress and activate the ISR, corresponding to the induction of ATF4. Future work will reveal the underlying mitochondrial signal responsible for activating this pathway, as well as the downstream consequences for the regulation of MQC processes in maintaining mitochondrial homeostasis. This work was supported by funds from the Canadian Institutes for Health Research (CIHR). Victoria C. Sanfrancesco is the recipient of the CIHR Frederick Banting and Charles Best Canada Graduate Scholarship-Master's (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.