Abstract
Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation–contraction (E–C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E–C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.
Highlights
In skeletal muscle, action potentials propagate into the fiber interior through the transverse tubule (T-tubule) system, an intracellular membrane network composed of narrow tubules around 40–85 nm in diameter that originate from deep invaginations of the surface plasma membrane (Melzer et al, 1995; Jayasinghe and Launikonis, 2013)
We show that partial cholesterol extraction from single skeletal fibers isolated from mice adult skeletal muscle eliminated or significantly reduced electrically evoked Ca2+ transients, without affecting membrane permeability or causing sarcoplasmic reticulum (SR) calcium depletion
We propose that partial cholesterol removal impairs E–C coupling by modifying the interactions of the cholesterol-associated protein caveolin-3 with Type-1 ryanodine receptor (RyR1) and Cav1.1
Summary
Action potentials propagate into the fiber interior through the transverse tubule (T-tubule) system, an intracellular membrane network composed of narrow tubules around 40–85 nm in diameter that originate from deep invaginations of the surface plasma membrane (Melzer et al, 1995; Jayasinghe and Launikonis, 2013). The T-tubule network contains Cav1.1 voltage-dependent Ca2+ channels (Anderson et al, 1994)— known as dihydropyridine receptors (DHPR)—which play a crucial role in skeletal muscle excitation–contraction (E–C) coupling. Treatment of fetal skeletal muscle cells from mice with the cholesterol-lowering agent MβCD decreases surfaceconnected tubular elements and disorganizes the T-tubule system (Pouvreau et al, 2004). Previous reports indicate that cholesterol removal impairs E–C coupling in amphibian (Launikonis and Stephenson, 2001) and skeletal muscles from fetal mice (Pouvreau et al, 2004). To test the hypothesis that T-tubule cholesterol content is important for E–C coupling in adult muscle, the first aim of the present work was to examine in single skeletal muscle
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