Role of the ubiquitin-proteasome system in cardiac dysfunction of adipose triglyceride lipase-deficient mice.
Bottom Line: Dysfunction of the UPS was accompanied by activation of NF-κB signaling.Chronic treatment of ATGL-deficient mice with the PPARα agonist Wy14,643 improved proteasomal function, prevented NF-κB activation and decreased oxidative stress.In summary, our data point to a hitherto unrecognized link between proteasomal function, PPARα signaling and cardiovascular disease.
Affiliation: Department of Pharmacology and Toxicology, University of Graz, Universitätsplatz 2, A-8010 Graz, Austria. Electronic address: email@example.com.Show MeSH
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Mentions: Fig. 1A shows a representative Western blot of ATGL expression in cardiac homogenates of WT, AKO, WT/cTg, and AKO/cTg mice. Two different exposure times were chosen to better illustrate the massive ATGL overexpression in transgene animals (WT/cTg and AKO/cTg). To examine the involvement of UPS in the pathogenesis of cardiac dysfunction observed in AKO mice, total levels of ubiquitinated proteins as well as protein expression of the ubiquitin-activating enzyme E1 (UBE1a) were measured in cardiac homogenates of WT, AKO, WT/cTg, and AKO/cTg mice (Fig. 1B). As shown in Fig. 1C, homogenates of AKO hearts showed markedly increased levels of ubiquitinated proteins, whereas only minor accumulation was observed in WT/cTg and AKO/cTg mice compared to WT controls (Fig. 1C). Protein expression of UBE1a, which catalyzes the initial step of the UPS cascade (i.e. activation of ubiquitin moieties through thioester formation), was more than 2-fold increased in AKO hearts (Fig. 1D). Expression of UBE1a was normalized to WT levels upon cardiomyocyte-directed overexpression of ATGL in AKO mice. To investigate whether increased protein ubiquitination was a consequence of 26S proteasomal dysfunction, chymotrypsin-like activity was measured in cardiac cytosols of WT and AKO animals. Peptidase activity was monitored over a range of ATP concentrations (0–4 mM) to ensure maximal activation of the 26S proteasome. ATP bidirectionally modulated proteasomal activity with maximal activation observed at 10–30 μM ATP (Fig. 1E). Normalizing proteasomal activity to basal values (measured in the absence of exogenous ATP) resulted in a similar activation profile of the UPS in cardiac homogenates of WT and AKO mice (Fig. 1F). The selective proteasome inhibitor MG132 (1 μM) decreased chymotrypsin-like activity to a similar extent (~ 90%) in WT and AKO animals (Fig. 1E). Protein expression of the 19S regulatory cap was not affected by ATGL deficiency (Fig. 1G). Western blot analysis of the mitochondrial marker citrate synthetase revealed that protein expression was significantly decreased in AKO animals by 41 ± 8%. Similar results were obtained for prohibitin (53 ± 18%), however, results failed to reach statistical significance (Fig. 1H).
Affiliation: Department of Pharmacology and Toxicology, University of Graz, Universitätsplatz 2, A-8010 Graz, Austria. Electronic address: firstname.lastname@example.org.