Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

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.

细胞膜胆固醇水平是调控细胞功能的重要因素。肌细胞膜胆固醇富集于脂筏（lipid rafts）与小窝（caveolae）——一类由细胞膜形成的烧瓶状内陷结构——之中。脚手架蛋白窖蛋白（caveolin）可促进小窝内胆固醇的富集，且窖蛋白与多种蛋白的相互作用能够调控这些蛋白的功能。本研究旨在探究心肌细胞胆固醇水平急性降低是否会改变亚细胞水平的蛋白激酶激活状态、细胞内钙离子浓度与心肌收缩功能。

方法：从成年斯普拉格-道利（Sprague Dawley）大鼠体内分离得到心室肌细胞，使用胆固醇降低剂甲基-β-环糊精（methyl-β-cyclodextrin，MβCD，5 mM，室温处理1小时）进行干预。随后检测细胞总胆固醇水平、窖蛋白-3（caveolin-3）的定位、亚细胞水平的细胞外调节蛋白激酶（ERK）与p38丝裂原活化蛋白激酶（mitogen activated protein kinase，MAPK）信号通路、心肌收缩功能以及细胞内钙离子浓度（[Ca²⁺]i）。

结果：MβCD处理可使细胞胆固醇水平降低约45%，并将窖蛋白-3从细胞骨架及曲拉通不溶性组分转移至曲拉通可溶性组分；同时可提升细胞骨架、胞质、曲拉通可溶性及曲拉通不溶性膜组分中ERK亚型的磷酸化水平，但未改变其亚细胞分布。与之相反，MβCD处理对p38的主要影响体现在p38α的亚细胞分布上，对p38的磷酸化水平影响微弱。胆固醇耗竭可提高心肌细胞的收缩幅度以及收缩与舒张速率，同时伴随舒张期与收缩期细胞内钙离子浓度的升高。

结论：本研究结果表明，细胞膜胆固醇水平急性降低可差异化调控心肌细胞基础状态下的亚细胞MAPK信号通路，同时提升细胞内钙离子浓度并增强心肌收缩功能。