Unravelling the Mechanisms of Energetics-Induced Fast-to-Slow Phenotypic Shift in Creatine-Deficient Mice

Skeletal muscle is the most predominant tissue in the human body and serves as the organ of movement. To sustain its function, a muscle specializes by expressing unique fiber compositions that define its phenotype. A slow phenotype consists primarily of type I and IIa fibers that are metabolically oxidative, exhibit lower force production, but demonstrate fatigue resistance. Conversely, a fast phenotype is predominantly composed of type IIx and IIb fibers that are metabolically glycolytic, generate greater force, but are more prone to fatigue. ATP hydrolysis is essential for muscle function, particularly contraction, and its concentration must remain stable.

Muscle fiber metabolic specialization matches the myosin heavy chain (MHC) isoform ATP consumption, ensuring adequate energy provision. When an energetic imbalance occurs, elevated AMP and ADP levels activate the energetic sensor AMP-activated protein kinase (AMPK). While acute AMPK activation restores energetic equilibrium by stimulating metabolism, chronic AMPK activity has been associated with atrophy and fast-to-slow phenotypic shift.

Muscle tissue possesses an ATP buffering system known as the creatine kinase (CK) system. The CK enzyme catalyzes ADP rephosphorylation to ATP using phosphocreatine (PCr) and the reverse reaction, phosphorylating creatine (Cr) to PCr using ATP. The high rate of CK activity and elevated PCr concentrations in muscle enable rapid compensation for sudden ATP depletion during contraction. Disruption of the CK system induces muscle atrophy and fast-to-slow phenotypic shifts associated with AMPK activation. However, the energetics and muscle-dependent consequences of mice lacking endogenous Cr biosynthesis remain poorly understood. Cr biosynthesis can be suppressed by knockout Guanidinoacetate N-methyltransferase (GAMT KO) or L-Arginine: Glycine amidinotransferase (AGAT KO).

Using Western blot and immunostaining techniques, we investigated the muscle phenotype, atrophy, and AMPK activation in fast muscle gastrocnemius (GAST) and slow muscle soleus (SOL) of both GAMT KO and AGAT KO mice. We also evaluate the basal activation of AMPK and its upstream kinases expression between fast and slow muscles. Additionally, we examined the effects of energetic deprivation on Ca²⁺ dynamics in stimulated C2C12 myotubes and measured Ca²⁺-related proteins in AGAT KO mice.

In this study, we provide evidence that basal AMPK activity is higher in slow compared to fast muscle. We suggest that AMPK activity may be compartmentalized, with activation being influenced by local rather than global energetics. Cr-deficient mice GAMT KO and AGAT KO exhibited radically different muscle phenotypes. GAMT KO mice showed subtle changes in fast muscle (GAST) mass and phenotype independently of AMPK activity. In contrast, AGAT KO showed a severe atrophy and fast-to-slow phenotypic shift in GAST associated with high AMPK activity. We speculated whether the use of GAA as a Cr substitute in GAMT KO may prevent AMPK activation and phenotypic adaptations. Based on our results and new findings by collaborators (Dr. Florian Britto, Institut Cochin, Paris, France), we discussed the potential involvement of AMPK in the phenotypic shift. Finally, we showed that energetic deprivation enhances the Ca2+ release by ryanodine receptors and theorized a new potential mechanism of energetics-induced fast-to-slow phenotypic shift.

SUPERVISORS

Supervisor: Rikke Birkedal
Co-supervisor: Marko Vendelin

OPPONENTS

Associate prof. Rasmus Kjøbsted, PhD; University of Copenhagen, Department of Nutrition, Exercise and Sports, Denmark.
Professor Allen Kaasik, PhD; University of Tartu, Department of Pharmacology, Estonia.

TIME OF DEFENCE

3 December 2025 at 14:00 at the Department of Chemistry and Biotechnology, room SCI109

THESIS

You can download PDF of the thesis https://doi.org/10.23658/taltech.87/2025