Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy

Abstract

BackgroundTriglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis.MethodsTo identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells.ResultsUsing SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search.ConclusionsWe performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future.

Imaging Modalities for Triglyceride Deposit Cardiomyovasculopathy

“…In TGCV, abnormal intracellular metabolism renders cells incapable of using long-chain fatty acids (LCFAs) (3,4) (Fig. 1), which play major roles in producing ATP in normal heart.…”

Tricaprin Rescues Myocardial Abnormality in a Mouse Model of Triglyceride Deposit Cardiomyovasculopathy

“…Hence, the development of specific treatment for TGCV is an urgent requirement. Our previous in vitro experiments on fibroblasts derived from patients with TGCV showed that LCFAs, the major energy source for the normal heart, accumulate as TG in cytoplasmic lipid droplets owing to ATGL deficiency 10,11 . To resolve the above-mentioned toxicity of LCFAs, we explored the development of nutritional therapeutic agents from medium-chain fatty acids MCFAs for the following reasons: 1 MCFAs have a long and safe history as they have been used clinically for 50 years in patients with metabolic and neurological disorders 12 14 ; 2…”

Diagnostic Criteria and Severity Score for Triglyceride Deposit Cardiomyovasculopathy

“…2, a lipid droplet maintenance protein, was a possible biomarker candidate in the context of lipid metabolism using fibroblasts from TGCV patients (17). Therefore, we can detect small lipid droplets as a PLIN 2 protein mass in cardiomyocytes ( Fig.…”