Simultaneous Measurement Of Force And Fluorescence In Single Guinea-pig Cardiac Myofibrils

Abstract

Hypercholesterolemia is a major risk factor for cardiovascular events. Among them, abnormal VSM contractions such as vasospasm are caused by Ca 2þ -sensitization of VSM contraction. However, the relationship between cholesterol (CHOL) and the VSM Ca 2þ -sensitization has not been clarified yet. Our recent studies showed that a sphingosylphosphorylcholine (SPC)/Src family tyrosine kinase (Src-TK) / Rho-kinase (ROK) pathway mediates the Ca 2þ -sensitization, and SPC indeed induces severe vasospasm in vivo. We found that serum CHOL potentiates the SPC/Src-TK/ROK pathway leading to Ca 2þ -sensitization in both human and rabbit: the extent of SPC-induced Ca 2þ -sensitization correlated well with total CHOL and LDL-CHOL, but inversely correlated with HDL-CHOL. Moreover, the depletion of CHOL by b-cyclodextrin destroyed CHOL-enriched membrane lipid rafts and abolished the SPC-induced ROK translocation and Ca 2þ -sensitization, suggesting the SPC-induced translocation of ROK to lipid rafts. Taken together, we suggested that not only CHOL, but also lipid rafts mediates the SPC-induced Ca 2þ -sensitization. Understanding the mechanism(s) by which lipid rafts promotes Ca 2þ -sensitization in human VSM requires the elucidation of lipid raft protein composition. As a first step, we succeeded for the first time in purifying lipid rafts from human VSM by sucrose density gradient ultracentrifugation, which were confirmed by western blot of raft marker proteins such as caveolin-1 and flotillin-1. Subsequently, using mass spectrometry (MALDI TOF-MS), proteomic analysis was performed to compare the protein compositions between lipid raft and non-lipid raft membrane fractions. The ongoing studies have identified so far previously unreported novel raft-localized proteins, in addition to the known proteins, including lipid-or GPI-anchored proteins and membrane proteins. We are attempting to accumulate functional data to suggest that some novel signaling molecules contribute to an SPC/Src-TK/ROK pathway leading to the VSM Ca 2þ -sensitization.