Abstract
Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism.
Highlights
From the ‡Department of Neurobiology, Physiology, and Behavior, §Department of Physiology and Membrane Biology, University of California, Davis, California 95616; ¶Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202
This is the first report of proteomic and metabolomic comparisons between mutations associated with mild (R278C) and severe (I79N) phenotypes in any sarcomeric gene. An advantage of this investigation is that both mutations are well characterized and occur in the same gene (TnT), which reduces the potential for artifacts when interpreting the results. Another advantage of these studies is that the I79N and R278C mutations are not associated with hypertrophy in patients or in the mouse models [7, 13,14,15,16,17]
Familial hypertrophic cardiomyopathy (FHC)-causing troponin T (TnT) mutations are associated with a high risk of sudden cardiac death, and the mechanisms underlying the disease are not well understood
Summary
DTT, triethylphosphine, iodoethanol, and ammonium bicarbonate (NH4HCO3) were purchased from Sigma-Aldrich 20S Proteasome Activity—25 g protein sample was combined with 1ϫ Chemicon Buffer (25 mM HEPES, 0.5 mM EDTA, 0.05% Nonidet P-40, 0.001% SDS, pH 7.5), bort or an equal volume of DMSO, and water and incubated for 20 min at room temperature. Calpain Activity—Protein sample (50 g) was combined with calpain activation buffer (25 mM Tris, 0.5 mM EDTA, 5 mM CaCl2, 75 mM NaCl, 0.25 mM DTT), 25 M calpain inhibitor IV (Calbiochem San Diego, CA) or an equal volume of DMSO, and incubated for 20 min at room temperature. The membrane was incubated in peroxidase-conjugated anti-rabbit secondary antibody diluted 1:5000 in 5% milk/TBST for 1 h, washed three times in TBST for 5 min per wash, incubated with Clarity Western ECL Substrate, and imaged as described in the previous section. Results are presented as mean Ϯ standard deviation (S.D.), and a value of p Ͻ 0.05 was considered statistically significant
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