Proteomic Profiling of H-Ras-G12V Induced Hypertrophic Cardiomyopathy in Transgenic Mice Using Comparative LC-MS Analysis of Thin Fresh-Frozen Tissue Sections

Determination of disease-relevant proteomic profiles
from limited
tissue specimens, such as pathological biopsies and tissues from small
model organisms, remains an analytical challenge and a much needed
clinical goal. In this study, a transgenic mouse disease model of
cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided
a system to explore the potential of using mass spectrometry (MS)-based
proteomics to obtain a disease-relevant molecular profile from amount-limited
specimens that are routinely used in pathological diagnosis. Our method
employs a two-stage methanol-assisted solubilization to digest lysates
prepared from 8-μm-thick fresh-frozen histological tissue sections
of diseased/experimental and normal/control hearts. Coupling this
approach with a nanoflow reversed-phase liquid chromatography (LC)
and a hybrid linear ion trap/Fourier transform-ion cyclotron resonance
MS resulted in the identification of 704 and 752 proteins in hypertrophic
and wild-type (control) myocardium, respectively. The disease driving
H-Ras protein along with vimentin were unambiguously identified by
LC-MS in hypertrophic myocardium and cross-validated by immunohistochemistry
and western blotting. The pathway analysis involving proteins identified
by MS showed strong association of proteomic data with cardiovascular
disease. More importantly, the MS identification and subsequent cross-validation
of Wnt3a and β-catenin, in conjunction with IHC identification
of phosphorylated GSK-3β and nuclear localization of β-catenin,
provided evidence of Wnt/β-catenin canonical pathway activation
secondary to Ras activation in the course of pathogenic myocardial
hypertrophic transformation. Our method yields results indicating
that the described proteomic approach permits molecular discovery
and assessment of differentially expressed proteins regulating H-Ras
induced hypertrophic cardiomyopathy. Selected proteins and pathways
can be further investigated using immunohistochemical techniques applied
to serial tissue sections of similar or different origin.