Proteasome inhibition decreases cardiac remodeling after initiation of pressure overload

Abstract

We tested the possibility that proteasome inhibition may reverse preexisting cardiac hypertrophy and improve remodeling upon pressure overload. Mice were submitted to aortic banding and followed up for 3 wk. The proteasome inhibitor epoxomicin (0.5 mg/kg) or the vehicle was injected daily, starting 2 wk after banding. At the end of the third week, vehicle-treated banded animals showed significant (P<0.05) increase in proteasome activity (PA), left ventricle-to-tibial length ratio (LV/TL), myocyte cross-sectional area (MCA), and myocyte apoptosis compared with sham-operated animals and developed signs of heart failure, including increased lung weight-to-TL ratio and decreased ejection fraction. When compared with that group, banded mice treated with epoxomicin showed no increase in PA, a lower LV/TL and MCA, reduced apoptosis, stabilized ejection fraction, and no signs of heart failure. Because overload-mediated cardiac remodeling largely depends on the activation of the proteasome-regulated transcription factor NF-kappaB, we tested whether epoxomicin would prevent this activation. NF-kappaB activity increased significantly upon overload, which was suppressed by epoxomicin. The expression of NF-kappaB-dependent transcripts, encoding collagen types I and III and the matrix metalloprotease-2, increased (P<0.05) after banding, which was abolished by epoxomicin. The accumulation of collagen after overload, as measured by histology, was 75% lower (P<0.05) with epoxomicin compared with vehicle. Myocyte apoptosis increased by fourfold in hearts submitted to aortic banding compared with sham-operated hearts, which was reduced by half upon epoxomicin treatment. Therefore, we propose that proteasome inhibition after the onset of pressure overload rescues ventricular remodeling by stabilizing cardiac function, suppressing further progression of hypertrophy, repressing collagen accumulation, and reducing myocyte apoptosis.

Fig. 1.

Proteasome activation by cardiac hypertrophy. Measurement after 1 wk banding. A: ATP dependence of proteasome activity in sham-operated vs. banded hearts (n = 4/group). B: increased proteasome activity in adult mouse cardiomyocytes isolated from banded hearts vs. sham-operated hearts (n = 3/group). *P < 0.01 vs. sham.

Fig. 2.

Proteasome inhibition regresses preexisting hypertrophy. Measurement of proteasome activity, left ventricular (LV) weight-to-tibial length and lung weight-to-tibial length ratios, and ejection fraction in hearts from mice submitted to 1, 2, or 3 wk banding and treated daily with vehicle (○) or epoxomicin (•) during the third week (n = 6/group for all panels). #P < 0.05 vs. 3 wk vehicle; *P < 0.01 vs. sham.

Fig. 3.

Prevention of collagen accumulation by proteasome inhibitors during pressure overload. A: Masson's trichrome staining of transversal sections of hearts submitted to 3 wk banding and treated daily with epoxomicin (E) or vehicle (V) during the last week compared with sham-operated hearts. B: accumulation of extracellular matrix (measured as a percentage of the surface of the LV tissue section) after 3 wk banding in presence of epoxomicin (•) or vehicle (○) compared with that of sham-operated animals (n = 4/group). *P < 0.05 vs. corresponding sham; #P < 0.05 vs. corresponding vehicle. C: picric acid Sirius red staining after 3 wk banding (Bg) and treatment with epoxomicin or vehicle compared with shams. D: quantification of collagen (measured as a percentage of the surface of the LV tissue section) in the same groups (n = 4/group). #P < 0.01 vs. corresponding vehicle; *P < 0.01 vs. corresponding sham.

Fig. 4.

Regulation of collagen transcription by proteasome inhibitors. A: quantitative PCR (qPCR) for collagen type I and III transcripts in sham-operated animals vs. 3-wk banded (Banding) mice treated with vehicle or with epoxomicin during the last week. Data are normalized per cyclophilin transcript. B: NF-κB activity measured by ELISA. C: qPCR for matrix metalloprotease-2 (MMP-2) transcript in sham-operated animals vs. 3-wk banded mice treated with vehicle or with epoxomicin during the last week. Data are normalized per cyclophilin transcript. D: protein expression of the NF-κB subunit p65 and the NF-κB inhibitor IκBα. Data are normalized per expression of α-actin. #P < 0.05 vs. corresponding vehicle; *P < 0.05 vs. sham (A–C: n = 5/group; D: n = 6/group).

Fig. 5.

Sequential effects of proteasome inhibitors in the overloaded heart. Proteasome activity, NF-κB activity, collagen type I transcript, LV-to-tibial length ratio, and ejection fraction in banded mice treated with vehicle or epoxomicin for 1 day, starting 2 wk after banding, and analyzed 24 h later. #P < 0.05 vs. corresponding vehicle; *P < 0.05 vs. sham (Top and bottom: n = 4/group).

Fig. 6.

Epoxomicin does not affect myocyte apoptosis. A: measurement of apoptosis by transferase-mediated dUTP nick-end labeling (TUNEL) and cardiac cell size by myocyte cross-sectional area in sham-operated animals vs. 3-wk banded mice treated with vehicle or epoxomicin during the last week. #P < 0.05 vs. corresponding vehicle; *P < 0.05 vs. sham (n = 5/group). B: daily treatment of sham-operated mice with vehicle (Veh) or escalating doses of epoxomicin for 1 wk followed by the measurement of apoptosis by TUNEL and chymotryptic-like and tryptic-like proteasome activities. *P < 0.05 vs. vehicle (n = 3/group). C: treatment of isolated cardiac myocytes with vehicle, different concentrations of epoxomicin, or with 100 μM H₂O₂ for 24 h and measurement of polyubiquitylation, proteasome activity, and myocyte apoptosis by caspase-3 activity. *P < 0.05 vs. vehicle (n = 3/group).

Fig. 7.

Proposed mechanism for the action of proteasome inhibitors in the overloaded heart. Upon stress, in this case pressure overload, the transcription factor NF-κB is activated upon removal of its inhibitory subunit IκB, which is degraded by the proteasome. NF-κB activation stimulates the expression of collagen isoforms, which leads to cardiac remodeling, further alteration in cardiac structure, and increased stress. Inhibition of the proteasome by epoxomicin prevents IκBα degradation, thereby restraining NF-κB activity, which prevents the increase in expression of collagen.