Abstract
Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility.
Highlights
Cholesterol is a key lipid component of cell and organelle membranes that regulates membrane fluidity
[1] Caveolae, specialized forms of lipid rafts that contain the scaffolding protein caveolin, are flask-shaped invaginations in the plasma membrane yielding membrane microdomains that serve to compartmentalize signal transduction. [2],[3],[4] In addition to being enriched in cholesterol lipid rafts and caveolae, are characterized by their resistance to detergent (Triton X-100) solubilization. [5],[6],[7] Caveolin, which has a high affinity for cholesterol, binds to numerous proteins in various cell types. [2],[3],[4] Evidence that the function of these proteins is directly modulated by their localization in caveolae or association with caveolin is based on the results of an increasing number of studies utilizing cholesterol depletion and/or caveolin knockout
Alpha-actinin, a sarcomeric protein enriched in the Z lines, exhibited significant expression in the pellet generated by the low-speed spin (designated as cytoskeletal (CySk)) and the tritoninsoluble membrane fraction, with much less in the triton-soluble membranes
Summary
Cholesterol is a key lipid component of cell and organelle membranes that regulates membrane fluidity. The reduction of membrane free cholesterol levels, with agents such as methyl-β-cyclodextrin (MβCD), disrupts the structure of lipid rafts and caveolae leading to altered cell signaling and function. [18],[19] the effects of reductions in cholesterol on adult cardiomyocyte signaling have not been reported. [20],[21],[22],[23] Reports nearly 25 years ago indicated that in vitro function of various cardiac ion pumps, such as the sarcolemmal Na+Ca2+ exchanger and the Na+-K+ ATPase, could be modulated by changes in cholesterol levels, [24,25] and cholesterol depletion alters L-type calcium current in cardiomyocytes. The purpose of this study was to determine the effects of acute cholesterol depletion with methyl-β-cyclodextrin (MβCD) on cardiomyocyte subcellular signaling and function
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