Perturbation of cullin deneddylation via conditional Csn8 ablation impairs the ubiquitin-proteasome system and causes cardiomyocyte necrosis and dilated cardiomyopathy in mice

Abstract

Rationale: Ubiquitin-proteasome system (UPS) dysfunction has been implicated in cardiac pathogenesis. Understanding how cardiac UPS function is regulated will facilitate delineating the pathophysiological significance of UPS dysfunction and developing new therapeutic strategies. The COP9 (constitutive photomorphogenesis mutant 9) signalosome (CSN) may regulate the UPS, but this has not been tested in a critical vertebrate organ. Moreover, the role of CSN in a postmitotic organ and the impact of cardiomyocyte-restricted UPS dysfunction on the heart have not been reported.

Objective: We sought to determine the role of CSN-mediated deneddylation in UPS function and postnatal cardiac development and function.

Methods and results: Cardiomyocyte-restricted Csn8 gene knockout (CR-Csn8KO) in mice was achieved using a Cre-LoxP system. CR-Csn8KO impaired CSN holocomplex formation and cullin deneddylation and resulted in decreases in F-box proteins. Probing with a surrogate misfolded protein revealed severe impairment of UPS function in CR-Csn8KO hearts. Consequently, CR-Csn8KO mice developed cardiac hypertrophy, which rapidly progressed to heart failure and premature death. Massive cardiomyocyte necrosis rather than apoptosis appears to be the primary cause of the heart failure. This is because (1) massive necrotic cell death and increased infiltration of leukocytes were observed before increased apoptosis; (2) increased apoptosis was not detectable until overt heart failure was observed; and (3) cardiac overexpression of Bcl2 failed to ameliorate CR-Csn8KO mouse premature death.

Conclusions: Csn8/CSN plays an essential role in cullin deneddylation, UPS-mediated degradation of a subset of proteins, and the survival of cardiomyocytes and, therefore, is indispensable in postnatal development and function of the heart. Cardiomyocyte-restricted UPS malfunction can cause heart failure.

Figure 1

Cardiomyocyte-restricted ablation of the Csn8 gene (CR-Csn8KO). A, The breeding scheme used to obtain CR-Csn8KO and littermate control (CTL) mice. The exons 4 through 6 were floxed in the Csn8-floxed allele (Csn8^flox). The deletor strain, αMHC-Cre⁺, expresses transgenic Cre under the control of the mouse myosin heavy chain-6 promoter (αMHC). The percent distribution of each genotype among 258 mice at birth is shown. B, Western blot analyses of Csn8 in myocardial extracts at the indicated age. C, Fluorescence confocal micrographs of myocardial cryosections immunostained for Csn8 (green). F-actin was stained with rhodamine conjugated Phalloidin. Arrows, cardiomyocytes; arrowheads, non-cardiomyocytes. Scale bar = 10 μm. D, Western blot analysis of Csn8 in the heart and the indicated non-cardiac organs at 3 weeks (wks). For all organs, the left lane is from CTL and the right from CR-Csn8KO mice.

Figure 2

Effects of Csn8 ablation on the protein abundance of other subunits, the complex distribution, the function of CSN in the heart. A, Western blot analysis of other CSN subunits in the heart at 2 weeks of age. B, Gel filtration followed by western blot analyses of CSN complex distribution. The results from probing Csn1, Csn2, and Csn6 consistently show significant reduction of CSN holocomplex in the CR-Csn8KO heart (KO). C, Western blot analysis of Nedd8 conjugates in total ventricular myocardium. D and E, Representative images (D) and a summary of densitometry data (E) of western blot analyses of the native and neddylated (marked by arrows) forms of cullin1 (Cul1), Cul2, Cul3, and Cul4A in ventricular myocardium at 2 weeks of age. Mean±SD, n=4; *:p<0.05 vs. CTL, Student's t-test.

Figure 3

CR-Csn8KO mice develop cardiac hypertrophy. A, The time course of changes in the body weight of CR-Csn8KO mice compared with their littermate CTLs. #: p<0.01 vs. CTL, Student's t-test. B, Representative images of whole hearts from 2-week old mice. Scale Bar=1mm. C, Comparison of the heart weight (Hw) to tibial length (TL) ratio and the ventricular weight to TL ratio between CTL and CR-Csn8KO mice at indicated ages. n = 6 ~ 8 for each group. *: p<0.05 vs. CTL; the same for other panels. D and E, Morphometric analysis of cardiomyocyte cross-sectional area (CSA) in 2-week-old mice. Representative images of H&E staining of myocardial sections (D) and a bar graph to summarize changes in CSA (E) are shown. Sections of 3 areas of each mouse heart and 3 mice per group were analyzed. Scale bar=20μm. F and G, RNA dot blot analyses of the steady state transcript levels of atrial natriuretic peptide (ANF) and skeletal α-actin (Sk. actin) in the ventricle of 2-week old mice. Total RNA (2 μg) isolated from ventricular myocardium was loaded on and cross-linked to nitrocellulose membrane and hybridized to p³²-labelled transcript-specific oligonucleotide probes. The bound probes were exposed to a phosphor-screen and imaged using a BioRad Personal Phosphoimager (F) and quantified using the Quantity-One software. GAPDH was probed as loading control. The relative changes are shown in panel (G).

Figure 4

CR-Csn8KO mice develop dilated cardiomyopathy and die prematurely. A and B, Representative images (A) and key parameters (B) of the M-mode echocardiography from 3-week-old mice. LVEDD, left ventricle (LV) end-diastolic dimension; LVESD, LV end-systolic dimension; PWTd, posterior wall thickness at the end of diastole; FS, fractional shortening. C, Changes in the Lung weight to TL ratio and the kidney weight to TL ratio during the first 4 weeks (wks) after birth. n = 6 ~ 8 for each group. *: p<0.05 vs. CTL; the same for the other panels. D, A Kaplan-Meier survival curve. p<0.0001, the log rank test.

Figure 5

CSN8 deficiency in cardiomyocytes impairs UPS proteolytic function. A and B, GFPdgn transgene was introduced into CR-Csn8KO and littermate control mice through cross-breeding. Ventricular myocardium was collected at 3 weeks of age for analyses reported here. A, Western blot analysis of myocardial GFPdgn. Representative images are shown in the upper panel and the densitometric data (n=6 for each group) are shown in the graph at the bottom. A non-specific band serves as a loading control (L.C.). *: p < 0.01 vs. CTL. Representative fluorescence confocal micrographs of ventricular myocardial sections from CTL/GFPdgn and CR-Csn8KO/GFPdgn mice were shown in panel B. Csn8 and GFPdgn were immunostained red and green, respectively. C and D, Littermate CR-Csn8KO and CTL mice at 3 weeks of age were used for western blot analysis (C) and confocal microscopy of immunofluorescence staining (green, D) for total ubiquitinated proteins (Ub.) in ventricular myocardium. In panel D, F-actin was stained using Rhodamine-conjugated Phalloidin (red) and the nuclei were stained blue with DAPI. Insets are the enlarged images of the arrowhead-pointed areas. Scale bar = 20 μm.

Figure 6

Effects of Csn8 deficiency on proteasomal abundance and peptidase activities in the heart. A, Changes in proteasomal peptidase activities at 3 weeks. Specific synthetic fluorogenic peptide substrates were used to measure the activity of the indicated peptidases in the crude myocardial protein extracts from the CTL (n=4) and the CR-Csn8KO (n=6) mouse ventricles. *: p < 0.05 vs. CTL. B, Representative images of western blot analyses for the indicated proteasome subunits. C, Gel filtration and subsequent western blot analyses for the size distribution of representative proteasome subunits. D, Western blot analyses of the indicated representative E3 ligases. GAPDH and β-tubulin were probed for loading control.

Figure 7

Effects of Csn8 deficiency on cardiomyocyte apoptosis. A, Representative confocal micrographs of TUNEL-stained (green) myocardial sections from 3-week old mice. The sections were counterstained for F-actin (red) with rhodamine phalloidin and for the nucleus (blue) with TO-RPO 3. To challenge the hearts, mice were treated with isoproterenol (ISO, i.p., 15 mg/kg) 24 hours before being sacrificed. Scale bar=20 μm. B, Quantification of TUNEL-positive cardiomyocyte nuclei. N = 3 mice/group. *: p<0.05, #: p<0.01. C, Representative images of western blot analysis of full length (FL) and cleaved caspase (Casp.) 3 and Bcl2 in ventricular myocardium from 3-week-old mice. D, Kaplan-Meier survival analysis of the impact of cardiac overexpression of Bcl-2 on CR-Csn8KO mice.

Figure 8

CR-Csn8KO induces myocytes necrosis. A and B, Representative confocal micrographs of Evans blue dye (EBD) incorporation (red in A) and immunostained CD45⁺ cells (green in B) in myocardium at 3 weeks. EBD was intraperitoneally injected 18 hours before the mice were sacrificed. The cell membrane was stained green in panel A with FITC-conjugated wheat germ agglutinin. Cardiomyocytes were stained red in panel B with rhodamine phalloidin. C and D, Quantification of EBD infiltrated cells (C) and CD45⁺ cells (D). n = 3 mice/group. Bar=50 μm; *: p<0.05, #: p < 0.01 vs. CTL.