Fiber Type Shift and Cell Size in Skeletal Muscle-Specific ATF3 Knockout Mice and Myotube ATF3 Overexpression

Activating Transcription Factor 3 (ATF3) is a stress-responsive gene implicated in the regulation of adaptations to challenges in cellular homeostasis. ATF3 mRNA is transiently and rapidly elevated in skeletal muscle following injury or after an acute bout of resistance or endurance exercise. Knockout of ATF3 in satellite cells enhances their activation following injury in the short term but exhausts the satellite cell pool in the long term. As a result, whole-body knockout of ATF3 impairs skeletal muscle adaptations to exercise training. However, the role of myocyte-specific ATF3 in regulating skeletal muscle cell phenotype remains unclear. In this study, we aimed to investigate whether ATF3 expression is required to maintain normal muscle fiber morphology and if it is sufficient to promote myotube hypertrophy. We induced a skeletal muscle cell-specific knockout of ATF3 using systemic delivery of MyoAAV-HSA-Cre (ATF3-KO; n = 20) or MyoAAV-HSA-EV (EV; n = 15) and harvested the diaphragm for fiber type and cross-sectional area measurements. Knockout of ATF3 was confirmed by genotyping 12–16 weeks post-injection. For the overexpression studies, C2C12 myoblasts were transfected with either a mouse ATF3 expression plasmid (ATF3-OE) or a control plasmid (EV), differentiated into myotubes, and their cell morphology was examined, using myosin heavy chain staining, 5 days post-differentiation. Our analysis showed a significant decrease in the percentage of type IIa fibers (EV: 64 ± 14%, ATF3-KO: 52 ± 18%; p = 0.047) and a corresponding increase in IIb/x fibers (EV: 26 ± 10%, ATF3-KO: 39 ± 21%; p = 0.039), with no significant change in type I fibers (EV: 10 ± 7.3%, ATF3-KO: 9.0 ± 6.8%; p = 0.65). However, there were no significant differences between groups in Feret’s diameter of fibers type I (EV: 37.0 ± 7.1 µm, ATF3-KO: 39.7 ± 7.2 µm; p = 0.30), IIa (EV: 40.3 ± 1.4 µm, ATF3-KO: 39.2 ± 3.3 µm; p = 0.22), and IIb/x (EV: 35.0 ± 5.3 µm, ATF3-KO: 34.3 ± 5.1 µm; p = 0.70) or the cross-sectional area of fibers type I (EV: 1520 ± 428 µm 2 , ATF3-KO: 1763 ± 502 µm 2; p = 0.16), IIa (ATF3-KO: 1700 ± 218 µm 2 , EV:1751 ± 103 µm 2 ; p = 0.41), and IIb/x (EV:1382 ± 340 µm 2 ; ATF3-KO: 1359 ± 373 µm 2 p = 0.85). In vitro transfection of C2C12 cells led to a 3- to 4-fold increase in myotube ATF3 mRNA, but no significant changes were observed in myotube width (EV: 36.8 ± 12.0 µm, ATF3-OE: 39.5 ± 8.8 µm; p = 0.67). Overall, our study suggests that in adult mice myocyte-specific ATF3 regulates type II muscle fiber (myosin heavy chain) composition but not fiber size in the absence of physiological stimulus or stress (e.g., exercise). Our experiments with C2C12 cells suggest that ATF3 overexpression alone is insufficient to cause myocyte hypertrophy. NIH R01 HL130318 to LFF. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.