Fig 1.
Characterization of subcellular fractions in control and MβCD treated myocytes.
(1A) A representative blot demonstrating the subcellular distribution of α-actinin and cytochrome c oxidase subunit IV (cyt c ox) in time control myocytes (lanes 1 and 2) and myocytes treated with MβCD (lanes 3 and 4). (1B) Western blot demonstrating the effect of MβCD (lanes 3–4) on the subcellular distribution of caveolin-3 (cav-3); time control myocytes are in lanes 1–2. (1C) Summarized results showing the distribution of cav-3 in control and MβCD treated myocytes. Cardiomyocytes were treated with the cholesterol-depleting agent MβCD (5 mM) for 1 hour at room temperature. Subcellular fractions are: cytoskeletal (CySk), triton-insoluble (Insol), triton-soluble (Sol) and cytosol (Cyto). In the cytosol fraction caveolin-3 was not different and negligible compared to other fractions and therefore not given in the bar graph. *p < 0.05 vs. time control, n = 3–4/group.
Fig 2.
Subcellular analysis of the effect of MβCD on p44/p42 ERK in rat cardiomyocytes.
(2A) A representative blot of dually-phosphorylated and total p44 and p42 ERK in control and MβCD treated cells. (2B) The effects of MβCD on the subcellular distribution of phosphorylated and (2C) total p44 ERK. Phosphorylated p44 values shown in (2B) are normalized to total p44 ERK in each fraction. Normalized phosphorylated and total p42 ERK are shown in Figs (2D) and (2E), respectively. *p < 0.05 vs. time control, n = 5/group. Fig (2F) is a representative western blot showing the effect of enhancing cardiomyocyte cholesterol with MβCD + cholesterol on ERK phosphorylation.
Fig 3.
Effect of cholesterol depletion with MβCD on subcellular p38 MAPK.
(3A) Representative western blot showing expression of dually-phosphorylated and total p38α MAPK in cytoskeletal (Cytsk), cytosolic (Cyto), triton-soluble (Sol) and triton-insoluble (Insol) fractions. Summarized blot results for dually phosphorylated p38 normalized to total p38α and total p38α are shown in Figs (3B) and (3C), respectively. *p < 0.05 vs. control, n = 4/group. Fig (3D) shows a pp38 western blot in myocytes treated with MβCD + cholesterol.
Fig 4.
Effects of cholesterol depletion with MβCD and cholesterol enrichment with MβCD + cholesterol on cardiomyocyte contractility.
(4A) Contractility of percentage shortening, (4B) maximal rate of shortening, and (4C) maximal rate of relaxation. Contractility parameters were calculated as described in the text. * p<0.05 vs. control, n = 20–30/group.
Fig 5.
Effects of MβCD and MβCD + cholesterol on intracellular calcium.
(5A) Diastolic and systolic Fluo-4 fluorescence values. (5B) Maximal rate of Fluo-4 increase during systole, and (5C) maximal rate of Fluo-4 decrease during diastole. * p<0.05 vs. control, n = 20–30/group.