Modulation of Mitochondrial Respiration and Metabolic Signalling in Skeletal Muscle by Caveolin-3

Abstract

Caveolins (Cavs) represent the principal constituent of caveolae, and since their discovery have long been associated with the plasma membrane as regulators of cholesterol homeostasis and scaffolds for signalling molecules and receptors such as the insulin receptor. Recent work however, has indicated that Cavs can also localise to the membranes of intracellular organelles such as the mitochondria and lysosome, although what role they may play at these membranes remains poorly understood. The primary aims of my PhD were to determine the contribution that the muscle specific Cav3 isoform may make to mitochondrial respiratory capacity and to determine whether Cav3 has a role to play in the nutrient-dependent activation of mTORC1, an event that has been shown to be closely associated with the lysosome. Herein, I establish that Cav3 localises to the mitochondria in L6 skeletal muscle cells and in mouse gastrocnemius muscle. The loss of this Cav isoform using CRISPR/Cas9 gene editing, induces a significant change in mitochondrial protein profile in vitro that is broadly consistent with changes that are also seen in vivo in skeletal muscle of Cav3-deficient models and in cultured muscle cells expressing a loss of function disease associated Cav3 mutant. The change in mitochondrial protein profile associated with Cav3 loss is also linked with a significant increase in mitochondrial fragmentation, a reduction in mitochondrial respiratory capacity and dysregulation of cholesterol and mitochondrial cardiolipin content, all of which can be mitigated upon cellular re-expression of Cav3. I also demonstrate, for the first time, that Cav3 localises to lysosomal membranes in L6 skeletal muscle cells. Importantly, I show that cellular loss of Cav3 in L6 muscle cells is associated with a significant decrease in amino-acid induced mTORC1 activation as demonstrated by reduced phosphorylation of downstream physiological targets of mTORC1 that include S6K1, 4EBP1 and ULK-1. The reduced activation of the mTORC1/S6K1 axis is also associated with a marked decline in myocellular protein synthesis and a reduction in cellular proliferation. The results presented in this thesis indicate that Cav3 is a novel regulator of mitochondrial form and function and of lysosomal associated amino-acid induced mTORC1 activation in muscle cells.

Date of Award	2020
Original language	English
Awarding Institution	University of Dundee
Sponsors	Diabetes UK
Supervisor	Hari Hundal (Supervisor)