Current Grant
04/25 – 03/26
In this current grant period, we wish to expand our exploration of the functional role of the raised cholesterol content in BlaJ muscle fibres compared to wild-type (WT) from the mildly dystrophic tibialis anterior (TA) fibres to the strongly dystrophic psoas fibres; and further to look at the effect of aging. Firstly, we wish to determine whether our observation that decreased sarcoplasmic reticulum (SR) calcium (Ca2+) content following the depletion of tubular (t-) system membrane cholesterol content in BlaJ TA fibres is due to the subsequent loss of stabilization of the RyR by its inhibitory interaction with the t-system Dihydropyridene Receptor (DHPR). That is, we wish to know whether cholesterol stabilizes the DHPR in the absence of dysferlin in the t-system membrane? Secondly, we will determine the importance of cholesterol in the t-system membrane for maintaining SR Ca2+ content in the strongly dystrophic psoas muscle at a young age (8-weeks) and in TA and psoas at an older age (36-weeks). Additionally, we will explore changes in basal Ca2+ signalling in the BlaJ and WT muscle and the effects of statins on SR Ca2+ content in this renewal.
Project Results
Previous Grant Period
01/24 – 3/25
During this grant period, we explored the differences in cholesterol content and its functional implications between wild type (WT) and BlaJ mouse muscle. To do this we utilized two cholesterol-interacting agents, saponin and methyl-β-cyclodextrin (MβCD). Saponin is an agent that bind cholesterol in the lipid bilayer and when aggregated in sufficient number forms non-specific pores, while MβCD acts by removing cholesterol molecules from the bilayer allowing the effects of cholesterol depletion of the bilayer to be observed. We tracked the movements of fluorescent dyes across the plasma membrane of muscle while applying saponin as an assay of relative cholesterol content between the genotypes in tibialis anterior (TA) muscle fibres. Our assays suggested a greater cholesterol content in the outer sarcolemma of BlaJ fibre compared to WT but no difference in tubular (t-) system membrane could be determined. However, we note that local clustering of cholesterol in the bilayer is not excluded by these assays. Next, our functional assays explored the properties of the sarcoplasmic reticulum (SR) in retaining its endogenous Ca2+ content and the Ca2+ handling properties of the t-system membrane. By depleting the t-system membrane of cholesterol in WT and BlaJ TA fibres with MβCD, we found that the Ca2+ content of the SR decreased further in the BlaJ than WT fibres. We also found that the properties of the t-system membrane PMCA TA muscle fibres of TA BlaJ were more sensitive to cholesterol depletion than WT, but store-operated Ca2+ entry properties were unaffected by the cholesterol depletion. These results and our previous work on ryanodine receptors in WT and BlaJ muscle fibres (Meizoso-Huesca et al 2025) have allowed us to develop to a hypothesis that the raised cholesterol expression in BlaJ TA fibres (Haynes et al 2019) may provide a stabilizing factor to reduce RyR Ca2+ leak to maintain the mildly dystrophic status in some dysferlin-deficient muscle.
Haynes VR, Keenan SN, Bayliss J, Lloyd EM, Meikle PJ, Grounds MD, Watt MJ (2019). Dysferlin deficiency alters lipid metabolism and remodels the skeletal muscle lipidome in mice. J Lipid Res. 60, 1350-1364.
Meizoso-Huesca A, Lamboley CR, Krycer JR, Hodson MP, Hudson JE, Launikonis BS (2025). Muscle-specific Ryanodine receptor 1 properties underlie limb-girdle muscular dystrophy 2B/R2 progression. Nat Commun. 16, 3056.
