The COP9 signalosome is required for autophagy, proteasome-mediated proteolysis, and cardiomyocyte survival in adult mice

Abstract

Background: The COP9 signalosome (CSN) is an evolutionarily conserved protein complex composed of 8 unique protein subunits (CSN1 through CSN8). We have recently discovered in perinatal mouse hearts that CSN regulates not only proteasome-mediated proteolysis but also macroautophagy. However, the physiological significance of CSN in a post-mitotic organ of adult vertebrates has not been determined. We sought to study the physiological role of CSN8/CSN in adult mouse hearts.

Methods and results: Csn8 was conditionally ablated in the cardiomyocytes of adult mice (CSN8(CKO)) using a temporally controlled Cre-LoxP system. Loss of CSN8 accumulated the neddylated forms of cullins and noncullin proteins, increased ubiquitinated proteins, and stabilized a surrogate substrate of the proteasome in the heart. Autophagic flux was significantly decreased, whereas autophagosomes were markedly increased in CSN8(CKO) hearts, indicative of impaired autophagosome removal. Furthermore, we observed increased oxidized proteins, massive necrotic cardiomyocytes, and morphological and functional changes characteristic of dilated cardiomyopathy in CSN8(CKO) mice.

Conclusions: CSN deneddylates substrates more than cullins and is indispensable to cardiomyocyte survival in not only perinatal hearts but also adult hearts. CSN8/CSN regulates both proteasome-mediated proteolysis and the autophagic-lysosomal pathway, critical to the removal of oxidized proteins in the heart.

Keywords: COP9 signalosome; NEDD8; autophagy; heart; proteasome endopeptidase complex.

Figure 1. Cardiomyocyte-restricted Csn8 knockout initiated in adult mice impairs deneddylation activities in the heart

At 5 days after the first tamoxifen injection, ventricular myocardium was sampled from Csn8^flox/flox (CTL) or Csn8^flox/flox::MerCreMer^tg (CSN8^CKO) littermate mice for western blot analyses. Representative images are shown with each lane representing a mouse. α-Tubulin or GAPDH was probed for loading control. (A) Changes in CSN8, CSN1 and CSN5. AU: arbitrary unit. (B) Increases in the neddylated form (arrow) and decreases in the native form (*) of cullin1 (Cul1), Cul 2, Cul3, and Cul4A in CSN8^CKO hearts. To verify the identity of the neddylated cullins, cultured neonatal rat cardiomyocytes were treated with a specific NEDD8 E1 inhibitor MLN4924 (MLN, 1µM) (Active Biochem, Maplewood, NJ) or vehicle (−) for 24 hours. MLN4924, which inhibits neddylation, abolished the slow-migrating band of all cullins, confirming their identity as neddylated cullins. (C) Increased Nedd8 conjugates in CSN8^CKO hearts.

Figure 2. Proteasome functional insufficiency in CSN8^CKO hearts

(A~C) CSN8^CKO hearts accumulate GFPdgn, a surrogate substrate of the UPS. GFPdgn was introduced into the CTL and CSN8^CKO mice via cross-breeding. At 3 days after the first injection of tamoxifen, ventricular myocardium of littermate mice was collected for western blot analyses (A) and immunofluorescence/confocal microscopy (B) for GFPdgn or RNA dot blot analyses (C). L.C., loading control; Scale bar = 40µm. (D) Proteasomal peptidase activity assays of ventricular myocardium at 3 days after the first tamoxifen injection, in the presence (+) or absence (−) of ATP.

Figure 3. Marked increases of autophagic vacuoles in CSN8^CKO hearts

(A, B) Representative images of western blot analyses of LC3 and p62 (A) and a summary of LC3-II densitometry data (B) in CTL and CSN8^CKO hearts at 5 days after the first tamoxifen injection. (C, D) Probing autophagy in CSN8^CKO hearts using GFP-LC3. GFP-LC3 was introduced into the CTL and CSN8^CKO mice via cross-breeding. Perfusion-fixed ventricular myocardium from CTL::GFP-LC3 mice and CSN8KO::GFP-LC3 littermate mice at 5 days after the first injection of tamoxifen was subjected to GFP-LC3 direct fluorescence confocal microscopy. Images from a 2.1 µm-thick slide of tissue were projected (C, representative images) and analyzed for the GFP-LC3 puncta density (D). The inset in panel C shows the area indicated by the arrow in a higher magnification. Bar=10 µm. (E, F) Electron micrographs of myocardium from CTL (E) and CSN8^CKO (F) hearts. Abundant autophagosomes (Av) containing degenerating mitochondria and/or other cytoplasmic contents are evident in CSN8^CKO hearts.

Figure 4. Impaired autophagic flux in CSN8^CKO hearts

Three days after the first tamoxifen injection, CTL and CSN8^CKO littermate mice were treated with two doses of BFA (3µmol/kg, i.p.) or vehicles with 1 hour in between, and sacrificed 2 hour after the first dose. LC3-II protein levels in ventricular myocardium were quantified using western blot analysis. Representative western blot images (A) and densitometric quantification (B) of LC3 proteins are presented.

Figure 5. CSN8 deficiency in adult hearts accumulates abnormal proteins and increases cardiomyocyte necrosis

Ventricular myocardium from CTL and CSN8^CKO littermate mice at 5 days after the first tamoxifen injection were used for the following analyses. (A) Western blot analysis of myocardial total ubiquitinated proteins (Ub). (B) Immunofluorescence analysis of the subcellular localization of Ub (green) in cardiomyocytes. Sarcomeric α-actinin was counter-immunostained (red) to identify cardiomyocytes. Note increased ubiquitin-positive aggregates (arrowheads) in CSN8^CKO cardiomyocytes. (C) Western blot analysis of DNP-derivatized protein carbonyls. The carbonyls of oxidized proteins in the total myocardial protein extracts were first derivatized by DNP and then detected by immunoblot for DNP. The representative image is shown as the upper panel. Before the immunoblotting, the proteins on the same membrane were visualized by MemCode protein (Pro.) stains (Pierce) (bottom panel). (D, E) Increased Evans Blue Dye (EBD, red) incorporation into the cardiomyocytes of CSN8^CKO hearts. Perfusion-fixed ventricular myocardium was collected 18 hours after an intraperitoneal injection of EBD (100 mg/kg) and processed for cryosections. FITC-conjugated wheat germ agglutinin and rhodamine-conjugated phalloidin were used to stain cell membrane (green) and myofibrils (blue), respectively. The percentage of EBD positive cardiomyocytes in 2 or 3 random fields per heart and 3 hearts per group was measured and presented in panel E.

Figure 6. Gravimetric and cardiac fetal gene expression changes in CSN8^CKO mice

CTL and CSN8^CKO mice were used 5 days after the first injection of tamoxifen. (A) Gravimetric measurements. (B) Changes in the transcript levels of atrial natriuretic factor (ANF), skeletal (Sk.) α-actin, and sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) in ventricular myocardium. RNA dot blot analyses were performed using radioactively labeled transcript-specific oligonucleotide probes.

Figure 7. Changes in left ventricle (LV) geometry and function in CSN8^CKO mice

CTL and CSN8^CKO mice were used 5 days after the first injection of tamoxifen. (A) Echocardiographic assessments. LV end-diastolic dimension (LVEDD), end-systolic dimension (LVESD), posterior wall thickness in diastole (PWTd), and fraction shortening (FS) were determined using transthoracic M-mode echocardiography. (B) Hemodynamic assessments. LV systolic pressure (LVSP), LV end diastolic pressure (LVEDP), the maximum first derivative of LV pressure (dP/dt_max) and the minimum first derivative of LV pressure (dP/dt_min) were measured by LV catheterization via carotid artery.