The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins

Abstract

The p97/VCP ATPase complex facilitates the extraction and degradation of ubiquitinated proteins from larger structures. We therefore studied if p97 participates to the rapid degradation of myofibrillar proteins during muscle atrophy. Electroporation of a dominant negative p97 (DNp97), but not the WT, into mouse muscle reduced fibre atrophy caused by denervation and food deprivation. DNp97 (acting as a substrate-trap) became associated with specific myofibrillar proteins and its cofactors, Ufd1 and p47, and caused accumulation of ubiquitinated components of thin and thick filaments, which suggests a role for p97 in extracting ubiquitinated proteins from myofibrils. DNp97 expression in myotubes reduced overall proteolysis by proteasomes and lysosomes and blocked the accelerated proteolysis induced by FoxO3, which is essential for atrophy. Expression of p97, Ufd1 and p47 increases following denervation, at times when myofibrils are rapidly degraded. Surprisingly, p97 inhibition, though toxic to most cells, caused rapid growth of myotubes (without enhancing protein synthesis) and hypertrophy of adult muscles. Thus, p97 restrains post-natal muscle growth, and during atrophy, is essential for the accelerated degradation of most muscle proteins.

Figure 1

Electroporation of DNp97 in TA blocks both denervation and starvation-induced atrophy. (A) Frequency histograms showing the distribution of cross-sectional areas of muscle fibres of TA either innervated or 9 days denervated and transfected or not with DNp97GFP. Muscles were electroporated with DNp97GFP plasmids at the same time as section of the sciatic nerve. According to the Kruskal–Wallis Test, differences (P<0.001) were found between innervated versus denervated and innervated versus denervated+DNp97. (B) Frequency histograms showing the distribution of cross-sectional areas of muscle fibres of TA either from fed or 2 days fasted mice and transfected or not with DNp97GFP. Muscles were electroporated with DNp97GFP plasmids and after 4 days mice were deprived of food for 2 days. According to the Kruskal–Wallis Test, differences (P<0.0001) were found between fed versus fasted, fed versus fasted+DNp97 and fasted versus fasted+DNp97. (C) A representative field of a transverse section of fibres expressing DNp97GFP from food-deprived mice. Scale bar represents 50 μm.

Figure 2

Electroporation of DNp97 or depletion of p97 using shRNA in TA increases fibre size in 7 days. (A–D) Frequency histograms showing the distribution of cross-sectional areas of TA muscle fibres either untransfected (control) or transfected for 7 days with WTp97 (A), DNp97 (B), a non-silencing shRNA (C) or a shRNA against p97 (D). According to the Mann–Whitney Test, the differences between DNp97 versus control and p97shRNA versus control were highly significant (P<0.0001). (E) Sixty micrograms of protein lysates from TA muscles electroporated with WTp97GFP (right leg) or DNp97GFP (left leg) were loaded. The anti-GFP antibody detection shows that equal amounts of WTp97GFP or DNp97GFP were expressed per muscle. The anti-p97 antibody detection reveals two bands: the endogenous (lower band) and the exogenous (upper band) p97. Rpl26 was used as loading control. (F) Seven days after electroporation with a plasmid for a non-silencing shRNA (right leg) or a shRNA against p97 (left leg), the contralateral TA muscles were frozen in isopentane cooled-liquid nitrogen from the same animal. Scale bar represents 1 cm. (G) The average weights of TA muscles 7 days after electroporation are plotted. Error bars indicate s.e.m. Paired t-test: *P<0.05 and ***P<0.003, n=5.

Figure 3

DNp97 increases cell diameter of myotubes. (A) Brightfield images of myotubes expressing GFP or DNp97 for 72 h are shown. Scale bar represents 50 μm. (B) The average diameter of myotubes expressing GFP or DNp97 for 72 h is plotted. ***P<0.0005, n=100. Error bars indicate s.e.m.

Figure 4

In myotubes, DNp97, but not WTp97, decreases degradation of both short- and long-lived proteins. (A, B) Myotubes were infected with adenoviruses encoding GFP (dotted line), WTp97 (dashed line) or DNp97 (full line) for 36 h and then incubated with ³H-tyrosine for the last 20 min (A) or 20 h (B) to differentially label short- and long-lived proteins, respectively. Error bars indicate s.d. n=6. (C) Equal amounts of protein lysates from myotubes expressing WTp97 or DNp97 were loaded and blotted with antibodies against: RGSH₄, polyubiquitinated proteins (FK1) and GAPDH as loading control. Thirty-six hours after infection with DNp97-expressing virus, myotubes start to accumulate ubiquitinated proteins.

Figure 5

In myotubes, DNp97, unlike WTp97, completely blocks the stimulation of proteolysis by caFoxO3, without altering the content of some atrogenes. (A) Myotubes expressing GFP, WTp97 or DNp97 for 24 h were given fresh media containing a second adenovirus expressing GFP or caFoxO3 supplemented with ³H-tyrosine to label long-lived proteins. Error bars indicate s.d. ANOVA was performed followed by Tukey’s HSD procedure. *P<0.05 DN–GFP versus GFP–GFP, **P<0.05 GFP–caFoxO3 versus GFP–GFP, ***P<0.05 DN–caFoxO3 versus GFP–caFoxO3, n=4. The DNp97-induced decrease in proteolysis was calculated by subtracting the protein degradation rate in the DNp97 overproducing cells from that in the controls (GFP–GFP or GFP–caFoxO3 expressing cells). Error bars indicate s.d. *P<0.001. (B) Overexpression of caFoxO3 in myotubes does not change the endogenous level of p97 protein. Equal amounts of protein from myotubes expressing for 48 h GFP (first three lanes) or caFoxO3 (last three lanes) were loaded. Endogenous p97, FoxO3 and GAPDH were immunoblotted. (C) The content of some atrogenes (atrogin-1, MuRF1, LC3 and Gabarapl1) increases upon overexpression of caFoxO3 but does not change in myotubes expressing WTp97 or DNp97 for 48 h. A lysate of cells treated with Bortezomib for 2 h has been also loaded as additional control for accumulation of polyubiquitin conjugates. Bortz: Bortezomib.

Figure 6

In myotubes, DNp97 blocks the FoxO3-induced proteolysis by inhibiting both proteasomal and lysosomal degradation. (A) Myotubes treated as described in Figure 5A were given fresh DMEM (control) containing 2% HS with or without the proteasomal (Bortezomib=Bortz) or lysosomal (ammonium chloride=NH₄Cl) inhibitors. Rates of proteolysis were determined starting 2 h later. Error bars indicate s.d. n=4. (B) The amount of proteolysis sensitive to each inhibitor represented the amount of proteasome or lysosome-mediated degradation and was calculated from data in Figure 6A by subtracting the rates of proteolysis in cells treated with the inhibitors from those of untreated cells. Error bars indicate s.d. (C) The DNp97-induced decrease in proteolysis was calculated by subtracting the amount of lysosomal or proteasomal protein degradation rate (i.e., the inhibitor-sensitive component) in the DNp97 overproducing cells from that in the controls (GFP-expressing cells). ***P<0.005 and *P<0.05 versus GFP–GFP controls. Error bars indicate s.d.

Figure 7

The myosin-chaperone UNC45B, a substrate of p97, does not alter muscle fibre size as DNp97. (A) Equal amounts of protein from normal and denervated TA muscles or from TA muscles of fed or food-deprived mice were analysed at the indicated times. Band quantitation analysis showed that the content of UNC45B does not change during two types of atrophy. (B) Frequency histograms showing the distribution of cross-sectional areas of TA muscle fibres either untransfected (control) or expressing UNC45B for 7 days. According to the Mann–Whitney Test, the differences between UNC45B versus control were significant (P<0.0001). (C) The average weights of TA muscles expressing for 7 days GFP or UNC45B are plotted. Error bars indicate s.e.m. No differences were found. n=5. (D, E) Frequency histograms showing the distribution of cross-sectional areas of TA muscle fibres either untransfected (control) or expressing for 7 days a shRNA for UNC45B (D) or shRNA for UNC45B and a plasmid for DNp97 (E). According to the Mann–Whitney Test, the differences between shRNA for UNC45B versus control (P<0.005) and between shRNA for UNC45B+DNp97 versus control (P<0.0001) were significant.

Figure 8

DNp97 becomes tightly associated to myofibrils in mouse TA (even more after denervation) and causes accumulation of ubiquitinated actin and MyLC2 in myotubes. (A) The purity of the myofibrils extracted by adult muscle is assayed by immunoblotting for proteins localized to nucleus (HDAC1) or to the mitochondrial membrane (VDAC1). Sol: soluble. Myo: myofibrils. (B) Myofibril isolation from paired mouse TA expressing WTp97GFP (left leg) or DNp97GFP (right leg) for 7 days. Fluorescence measurements of the soluble, the myofibrillar fraction and the washes from myofibrils with increasing salt concentration were plotted as a percentage of total fluorescence. The changes shown are typical of results obtained three times, although the absolute amounts of p97 in these fractions varied among experiments. (C) Gentle myofibril isolation from paired mouse muscles expressing DNp97GFP and denervated or not for 9 days (*P<0.005, n=3). Error bars indicate s.e.m. (D) Representative images by confocal microscopy of longitudinal sections of TA muscle expressing WTp97–MycHis or DNp97–MycHis (p97E578Q) for 7 days were stained with anti-Myc antibodies (green). While WTp97 shows a diffuse and nuclear distribution, the DN mutant clearly displays a striation-like pattern. Scale bar represents 5 μm. (E) Soluble and myofibrillar fractions (before and after 0.6 M KI extraction) from whole-muscle homogenate were stained for MyHC, MyLC2, actin, p97 and HDAC1. (F) In extracts of myotubes expressing GFP or DNp97 for the indicated times, the contractile proteins were assayed by immunoblotting. Both actin and MyLC2 (unlike MyHC and MyBP-C) were found in part as ubiquitinated species upon 72 h expression of DNp97. No appreciable difference of the MyHC/actin ratio of Coomassie Blue-stained myofibrils from muscles expressing WTp97 or DNp97 was found, suggesting that both thin and thick filaments are degraded by p97-dependent mechanisms (WTp97 muscles: 1.74±0.31; DNp97 muscles: 1.99±0.19, n=3, P=0.52). Figure source data can be found with the Supplementary data.

Figure 9

In myotubes, DNp97 binds actin (which also binds Ufd1) or MyLC2 or MyLC1 (which also binds p47) and results in accumulation of actin or MyLC2 as ubiquitinated species. (A–D) Immunoprecipitation of actin (A) or MyLC2 (B) or Ufd1 (C) or p47 (D) was performed from myotubes overexpressing GFP, WTp97–RGSH₄ (in the presence of DMSO or 1 μM Bortezomib for 3 h) or DNp97–RGSH₄ for 48–72 h. As control, immunoprecipitation of the same samples with appropriate IgG was carried out. Actin, RGSH₄, polyubiquitinated proteins (FK1), MyLC2 and 1,Ufd1, p47 and MyHC were immunoblotted. Figure source data can be found with the Supplementary data.

Figure 10

The content of p97 and two of its cofactors are induced together with proteasomal subunits and the shuttling factor mR23A in Gastrocnemius 10 days after denervation. (A, B) Levels of p97 and its cofactors Ufd1, p47 and Ufd2 (A) and of the proteasomal subunit of the 19S regulatory particle Rpt1, the 20S catalytic core and the shuttling factor mR23A (B) were measured by immunoblotting on extracts from mouse Gastrocnemius at different times after cutting the sciatic nerve. Equal amounts of protein were loaded. Densitometry data and statistical analysis of protein levels are plotted. Error bars indicate s.e.m. (A) *P<0.05, n=6. (B) **P<0.01 and ***P<0.005, n=7. (C) Gastrocnemius weight was decreased 10 days after denervation. *P<0.001, n=4.