Proteasome Inhibition Ameliorates Ang-Ⅱ-induced Cardiomyocyte Hypertrophy Through GSK-3β Pathway

BackgroundUbiquitin-proteasome system (UPS) is known as the most common protein degradation pathway in eukaryocytes. It has been reported to be involved in several biological processes, including cell cycle, cell proliferation and oxidative stress. The eukaryotic 26S proteasome is a large, multicatalytic protein complex composed of the 19S regulatory particle, which recognizes, deubiquitinates and unfolds target proteins, and the 20S core, which subsequently degrades target proteins via three proteolytic activities (chymotrypsinlike, trypsin-like and caspase-like). Proteolytic activities can be inhibited either reversibly by peptide aldehydes (e.g. MG132) and peptide boronates (e.g. MG262, bortezomib) or irreversibly byβ-lactone derivates (e.g. lactacystin) and epoxyketones (e.g. epoxomicin). Proteasome inhibitor could reduce the myocardium remodeling caused by pressure overload by inhibiting the activity of NF-κB, resulting in reverse of cardiomyocyte hypertrophy. Moreover, partial inhibition of proteasome activity could prohibit the cardiomyocyte hypertrophy in primary neonatal rat cardiomyocytes (NRCs).Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates cell functions including metabolism, transcription, translation, cell growth and apoptosis. According to the previous reports, GSK-3βwas a negative regulatory factor (NRF) for endogenous cardiomyocyte hypertrophy. Activation of GSK-3βcould deactivated the cardiomyocyte hypertrophy releasing factors, such as eukaryotic initiation factor (eIF) 2Bε, nuclear factor of activated T cells (NFAT), GATA4 and myocardin by phosphorylation, resulted in inhibition of cardiomyocyte hypertrophy. Our previous study indicated remarkable increase of GSK-3 activity after the inhibition of proteasome in human embryonic kidney (HEK) 293 cells. In this study, primary NRCs were used to investigate the effect of proteasome inhibition on cardiomyocyte hypertrophy and GSK-3 activity, as well as the underlying mechanism of proteasome inhibition on cardiomyocyte hypertrophy. All these could provide new approach for the mechanism of cardiomyocyte hypertrophy.Part oneEffect of proteasome inhibition on cardiomyocyte hypertrophy and GSK-3βObjective1. To investigate the effect of proteasome inhibition on cardiomyocyte hypertrophy2. To investigate the effect of proteasome inhibition on GSK-3βMethodsPrimary NRCs were used in this study. Cardiomyocyte hypertrophy were induced by AngⅡ(100nM), and lactacystin (1μM, a proteasome inhibitor) was given simultaneously, and incubated for 24 h. Chymotrypsin-like activity was detected with fluorescent peptide substrate. In addition, cardiomyocyte hypertrophy indexes including ANF mRNA expression, synthesis rate of protein and the cardiomyocyte surface area were detected. GSK-3 and phosphorylated GSK-3 (p-GSK-3-Ser-9) in myocardial cells were also detected by Western-blot.Results1. Chymotrypsin-like activity showed remarkable reduction after incubation with lactacystin for 24h (P<0.05).2. ANF mRNA expression, synthesis rate of protein and cardiomyocyte surface area increased significantly after incubation with lactacystin (P<0.05). 3. p-GSK-3β(ser9), the inactivated GSK-3, significantly decreased at 24 h after Ang-Ⅱand lactacystin treatment compared with cardiomyocyte hypertrophy group, indicating that GSK-3βactivity increased after lactacystin treatment .Brief summaryProteasome inhibition suppressed the cardiomyocyte hypertrophy and increased GSK-3βactivity induced by Ang-Ⅱ.Part twoThe potential effects of proteasome inhibition on cardiomyocyte hypertrophy. ObjectiveTo investigate the potential effects of proteasome inhibition on cardiomyocyte hypertrophy.MethodsLiC1(10μM), the inhibitor of GSK-3, was incubated with Ang-Ⅱ(100nM) and lactacystin (1μM) for 24h to down-regulate the activity of GSK-3. Chymotrypsin-like activity was detected with fluorescent peptide substrate. Moreover, cardiomyocyte hypertrophy indexes including ANF mRNA expression, synthesis rate of protein and the cardiomyocyte surface area were detected. Western-blot was applied to analyze the GSK-3 and p-GSK-3 in myocardial cells.Results1. Chymotrypsin-like activity showed remarkable reduction after incubation with lactacystin for 24h (P<0.05). Chymotrypsin-like activity showed remarkable reduction in LiC1 group (P<0.05).2. ANF mRNA expression, synthesis rate of protein and cardiomyocyte surface area showed reversed changes after incubation with LiC1.3. LiC1 could compromise the inhibiting effect of lactacystin on P-GSK-3βexpression induced by Ang-Ⅱ.Brief summaryProteasome inhibition suppressed cardiomyocyte hypertrophy through GSK-3 pathway. Conclusion1. Proteasome inhibiton suppressed cardiomyocyte hypertrophy induced by Ang-Ⅱ.2. Proteasome inbibition suppressed cardiomyocyte hypertrophy through GSK-3 pathway.