Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation-contraction
Rubén J Lopez1, Barbara Mosca1, Susan Treves1
1*Departments of Anesthesia and of Biomedicine, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland.
Abstract:
The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.
Insights
Ablating raptor in skeletal muscle affects glycogen storage and calcium release. This impacts excitation-contraction coupling by altering ryanodine receptor function.
Area of Science:
- Biochemistry
- Molecular Biology
- Physiology
Background:
- Mammalian target of rapamycin (mTOR) is a kinase regulating cell growth.
- mTOR exists in two complexes, mTORC1 and mTORC2.
- Regulatory associated protein of mTOR (raptor) is crucial for mTORC1 function.
Purpose of the Study:
- Investigate the effects of raptor ablation in skeletal muscle on excitation-contraction (E-C) coupling.
- Examine changes in glycogen phosphorylase and calcium handling in raptor knockout (KO) muscle.
Main Methods:
- Generated muscle-specific raptor knockout (RamKO) mice.
- Analyzed glycogen phosphorylase association with sarcoplasmic reticulum (SR) membranes.
- Performed equilibrium binding assays for ryanodine and dihydropyridine receptors (DHPRs).
- Measured calcium transients and elementary calcium release events (ECREs) in flexor digitorum brevis (FDB) fibers.
Main Results:
- In RamKO muscle, glycogen phosphorylase predominantly associated with SR membranes in its inactive form.
- The ratio of ryanodine receptors to DHPRs increased in raptor KO skeletal muscle membranes.
- While global calcium transients were similar, hyper-osmotic shock induced more frequent and larger elementary calcium release events in raptor KO FDB fibers.
Conclusions:
- Skeletal muscle-specific raptor ablation alters the protein composition and function of the E-C coupling machinery.
- mTORC1 signaling influences glycogen metabolism and calcium release channel regulation in skeletal muscle.
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