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.

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Altered metabolic states due to obesity and diabetes have a direct impact on renal structure and function. Obesity and diabetes leads to several key morphological, subcellular fibrotic remodeling, nephropathy and renal failure. However the clear signaling mechanism is not known. Leptin receptor deficiency is associated with obesity and hypercholesterolemia. We showed that acute cholesterol depletion increased cellular function and phosphorylation of p38, ERK and AKT kinases. Hence we hypothesized that obesity enhances phosphorylation of p38, ERK1/2 and AKT kinases in the kidney. To test our hypothesis we used kidneys from 25 weeks old four leptin receptor deficient db/db obese mice and their age matched four c57BL6 (wild type) mice. Transverse slices were made from similar position of all kidneys. Kidney slices were chopped into very small pieces and homogenized under the ice cold lysis buffer with protease and phospahtase inhibitors. The low spin pellets were discarded and the supernatants were collected. Kidney lysates were run through 7–12% poly acrylamide gel electrophoresis. Phosphorylations of p38, ERK1/2 and AKT were measured using western blot and cell signaling antibodies. The results were normalized to β‐actin as a control for protein loading using NIH image J software. Data of phospho proteins expressed in arbitrary units for p38 (14477 ±1703 vs. 22488±2773, p<0.03), ERK1/2 (12408 ±1965 vs. 21807±1467, p<0.01) and AKTThr308 (10173 ±462 vs. 18250±1198, p<0.01) were more in obese db/db mice compared to wild type mice. Total p38, ERK1/2 and AKT were not different between db/db and wild type mice. We conclude that obesity is associated with enhanced phosphorylation of p38, ERK and AKTThr308 which could be associated with altered renal structure and function.Support or Funding InformationHamad Medical Research Center #16304This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.