In their recent clinical paper, Hara and co-authors describe their initial steps in untangling the pathogenesis of cardiac failure due to abnormal triglyceride deposition. They accomplished this by cellular proteomic profiling of skin fibroblasts harvested from two patients with the disease, comparing the fibroblasts with those obtained from three healthy volunteers.1
Caused by an enzyme mutation in the triglyceride (TG) hydrolysis pathway, triglyceride deposit cardiomyovasculopathy (TGCV) leads to skeletal and cardiac muscle damage due to lipid accumulation within cells. The disease is rare and usually leads to premature death unless heart transplantation is available. Apart from the defects in the TG degradation pathway, little is known about the disease pathogenesis. The mechanisms by which cardiac muscle and the coronary artery walls are specifically targeted for lipid accumulation have not yet been elucidated.
Hara et al. chose to examine differential protein expression in fibroblasts harvested from two patients with TGCV in the hope of discovering molecules involved in pathogenesis. Although cardiac tissue would seem more appropriate, fibroblasts are easy to harvest and culture in vitro; they also retain the TG pathway dysfunction leading to abnormal lipid accumulation. The researchers confirmed this latter observation using Oil Red O lipid staining to visualize the excess TGs accumulating in the cells in culture. Using microscopy at each stage of culture, the researchers were satisfied that their in vitro culture conditions maintained the TGCV phenotype of the patients’ fibroblasts under experimental conditions.
On the assumption that most pathways are controlled by proteins, the researchers used stable isotope labeling by amino acids in culture (SILAC) to investigate proteome profiles in the fibroblasts from the two TGCV patients. They compared the protein levels with those obtained from the volunteer subjects’ normal skin fibroblasts.
Once labeled, Hara et al. extracted cellular proteins, then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) before in-gel trypsin digestion. The researchers analyzed the digests by nano LC-MS/MS using an LTQ Orbitrap Velos hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific). They examined the spectral data using MaxQuant software v1.2.2.5, referring to the IPI Human Protein database for identification.
The researchers identified 4,033 proteins in total, of which 53 were differentially expressed in the patients’ fibroblast cultures. The researchers then used the Ingenuity Pathway Analysis (IPA) a tool for significant network discovery to identify key pathways affected by the differential expression. Unsurprisingly, “lipid metabolism” was the top ranked pathway in IPA network discovery for proteins both over- and under-expressed in the TGCV cells. Within this functional set, “lipid synthesis” ranked most highly as a repressed function (P = 5.76E-06, activation Z-score = -2.02), with “concentration of lipid” appearing as second ranked (P = 9.56E-05, activation Z-score 0.117).
Hara and co-authors chose 20 of the differentially expressed proteins to examine further by selective reaction monitoring/multiple reaction monitoring (SRM/MRM). They optimized conditions with an LTQ Orbitrap XL hybrid ion trap-Orbitrap mass spectrometer for preliminary ion fragmentation identification, then ran the assay on a TSQ Vantage triple stage quadrupole mass spectrometer (both Thermo Scientific) to quantify the peptides. Initial SRM/MRM data analysis was performed using Pinpoint software revision 1 (Thermo Scientific). Results confirmed the initial LC-MS/MS data for 14 of the proteins assayed.
As a further confirmation step, Hara and co-authors used microarray-based transcription analysis to examine gene expression. Real-time reverse transcription polymerase chain reaction (PCR) showed that RNA expression matched the differential proteomic profiles. Using quantitative PCR, the researchers selected 10 differentially expressed proteins for further gene analysis, demonstrating consistent results that supported prior experimental findings. The researchers also examined expression of two proteins known to be associated with lipid pathways and identified by results in earlier steps of their experiments, PLIN2 and filaggrin, in greater detail.
In summary, the researchers are confident that their proteome profiling and functional network analysis has, in combination with molecular characterization, uncovered mechanisms that may be important in the pathogenesis of TGCV. They suggest that the identification of proteins involved in disease development shows potential for developing new strategies for treatment and management of this rare, but fatal, condition.
Reference
1. Hara, Y., et al. (2013, December) “Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy,” Orphanet Journal of Rare Diseases, 8 (p. 197), doi: 10.1186/1750-1172-8-197.
Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer.
A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.
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