Regulation of acetylation restores proteolytic function of diseased myocardium in mouse and human

Abstract

Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.

Fig. 1.

HDAC inhibition in vitro enhances proteolytic activity of proteasomes in acute I/R injury and in end-stage ischemic heart failure. A, left-hand panel: murine cytosolic 20S proteasomal activity of the sham group (n = 5) was up-regulated by sodium valproate in a dose-dependent fashion (open circles, *p < 0.05 versus without sodium valproate); a significant increase in activity was reached at 0.04 mm, and the augmentation was maintained through 1 mm of sodium valproate. I/R injury significantly reduced 20S proteolytic activity (solid circles; $ denotes p < 0.05 versus sham). Sodium valproate at 0.04 mm restored the compromised 20S activity in the injured myocardium (solid circles; # denotes p < 0.05 versus without sodium valproate). This regulatory capacity was consistent with elevated doses of sodium valproate; an adverse effect was observed when 5 mm of sodium valproate was given. Right-hand panel: HDAC inhibition by SAHA up-regulated cytosolic 20S proteasomal activities in the healthy and injured myocardium. The maximal augmentation was achieved by 0.4 μm of SAHA, and the effect was consistent through 50 μm of SAHA. SAHA restored 20S activities in the I/R injured myocardium. p < 0.05 represents statistic significance; error bar represents standard error. B, left-hand panel: HDAC inhibition by sodium valproate significantly improved the proteolytic activity in diseased myocardium from end-stage heart failure patients (open circles, n = 5, *p < 0.05 versus without sodium valproate). A range of sodium valproate doses from 0.04 mm to 5 mm consistently achieved this regulatory capacity. Similar therapeutic effects on 20S proteolytic activities were also observed with HDAC inhibitor SAHA (open circles, n = 5, *p < 0.05 versus without SAHA). p < 0.05 represents statistic significance; error bar represents standard error.

Fig. 2.

Murine cardiac 20S proteasomes are targets of lysine acetylation. A, the mass spectrum of the N-terminal peptide from the α1 subunit of 20S proteasomes in its acetylated form. Digestion by the enzyme Glu-C was used to generate this N-terminal peptide. A cross-correlation score (Xcorr) of 3.83, determined by the Sequest algorithm, demonstrated a confident identification. B, the mass spectrum of peptide containing lysine-203 residue from the α5 subunit of 20S proteasomes. This lysine-203 residue was regulated by acetylation under physiological conditions. An Xcorr score of 3.71 demonstrated a confident identification.

Fig. 3.

Global in vivo HDAC inhibition enhances the proteolytic capacity and redistribution of 20S proteasomes and induces lysine acetylation at specific sites. A, the impact of HDAC inhibition on the expression of assembled cytosolic 20S complexes. Following 6 h of HDAC inhibition in vivo, the reduced cytosolic expression level of assembled 20S complexes was illustrated via immunoblotting with antibodies against subunit β2 (n = 5, *p < 0.05 versus without HDAC inhibition). B, HDAC inhibition in vivo significantly increased cytosolic proteasomal activities in the murine heart (n = 5, *p < 0.05 versus without HDAC inhibition). C, purified myocardial 20S from mice treated with HDAC inhibitors exhibited significant elevation of proteolytic activities (n = 5 technical replicates, *p < 0.05 versus without HDAC inhibition). Error bar represents standard error (A–C). D, HDAC inhibition in vivo significantly elevated acetylation of purified 20S proteasomal subunits as demonstrated in immunoblots against acetylated lysine residues. Immunoblottings with antibodies against subunits β2 and β7 served as loading controls. E, the mass spectrum of peptide containing lysine-201 residue from the β7 subunit of 20S proteasome. This novel site of acetylation was only detected following HDAC inhibition in vivo. An Xcorr score of 4.92 confirmed a confident identification.