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Autophagic-Lysosomal Inhibition Compromises Ubiquitin-Proteasome System Performance in a p62 Dependent Manner in Cardiomyocytes

Tian, Zongwen, Wang, Changhua, Hu, Chengjun, Tian, Yihao, Liu, Jinbao, Wang, Xuejun·2014·PLoS ONE
DOI 10.1371/journal.pone.0100715PMC PMC4069113
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Figure 1 Lysosomal inhibition accumulates both autophagic and proteasomal substrates in mice: hearts and kidneys. Male GFPdgn transgenic mice at 10 weeks of age were treated with bortezomib (BZM, 1 mg/kg, i.p.), bafilomycin A1 (BFA, 2.5 mg/kg/12 hrs, i.p.), or vehicle control (CTL). The mice were sacrificed at either 3 or 24 hours after the first injection and myocardial and kidney tissues were collected for total protein and RNA extraction and subsequent analysis. A and B, Representative images (A) and pooled of densitometry data (B) of western blot analyses for myocardial GFPdgn and other indicated proteins. C, Representative images (upper) and pooled densitometry data (lower) of western blot analyses for renal GFPdgn protein levels. D and E, Representative images (upper) and pooled densitometry data of reverse transcription (RT) PCR analyses of GFPdgn mRNA levels in heart and kidney tissues. Total RNA was used for the RT to synthesize the first strand cDNA which was subsequently used for GFPdgn and GAPDH duplex RT-PCR. GAPDH was probed as loading control. N = 4 mice/group. *p<0.05, **p<0.01 vs. CTL.
Figure 1 Lysosomal inhibition accumulates both autophagic and proteasomal substrates in mice: hearts and kidneys. Male GFPdgn transgenic mice at 10 weeks of age were treated with bortezomib (BZM, 1 mg/kg, i.p.), bafilomycin A1 (BFA, 2.5 mg/kg/12 hrs, i.p.), or vehicle control (CTL). The mice were sacrificed at either 3 or 24 hours after the first injection and myocardial and kidney tissues were collected for total protein and RNA extraction and subsequent analysis. A and B, Representative images (A) and pooled of densitometry data (B) of western blot analyses for myocardial GFPdgn and other indicated proteins. C, Representative images (upper) and pooled densitometry data (lower) of western blot analyses for renal GFPdgn protein levels. D and E, Representative images (upper) and pooled densitometry data of reverse transcription (RT) PCR analyses of GFPdgn mRNA levels in heart and kidney tissues. Total RNA was used for the RT to synthesize the first strand cDNA which was subsequently used for GFPdgn and GAPDH duplex RT-PCR. GAPDH was probed as loading control. N = 4 mice/group. *p<0.05, **p<0.01 vs. CTL.CC BY · PubMed Central
Figure 2 Representative confocal micrographs of mouse myocardial direct green fluorescence. Adult GFPdgn non-transgenic (A) and transgenic (B ∼ D) mice at 10 weeks of age were treated with bortezomib (1 mg/kg, i.p.; C), bafilomycin A1 (BFA, 2.5 mg/kg/12 hrs, i.p.; D), or vehicle control (DMSO, A and B). At 24 hours after the first BFA injection, ventricular myocardial tissues were collected and immediately immersion-fixed with 3.8% paraformaldehyde and processed for cryo-sectioning. The cryosections were mounted and imaged for direct fluorescence via confocal microscopy. Scale bar = 100 µm.
Figure 2 Representative confocal micrographs of mouse myocardial direct green fluorescence. Adult GFPdgn non-transgenic (A) and transgenic (B ∼ D) mice at 10 weeks of age were treated with bortezomib (1 mg/kg, i.p.; C), bafilomycin A1 (BFA, 2.5 mg/kg/12 hrs, i.p.; D), or vehicle control (DMSO, A and B). At 24 hours after the first BFA injection, ventricular myocardial tissues were collected and immediately immersion-fixed with 3.8% paraformaldehyde and processed for cryo-sectioning. The cryosections were mounted and imaged for direct fluorescence via confocal microscopy. Scale bar = 100 µm.CC BY · PubMed Central
Figure 3 Lysosomal inhibition does not alter proteasome abundance and proteasome peptidase activities in mouse hearts. Mice were treated as described in Figure 1. A, Representative images of western blot analyses for the representative subunits of the 19S proteasome (Rpt6) and the 20S proteasome (α3, α4, β2, and β5) in the total protein extracts from ventricular myocardium. B, Pooled densitometry data of β2 and β5 subunits as analyzed in A. C, Proteasome peptidase activity assays. Synthetic fluorogenic substrates specific for chyomtrypin-like, caspase-like, and trypin-like peptidases were used to measure the proteasomal peptidase activities in the crude protein extracts from ventricular myocardium collected from mice treated with BFA or vehicle control (CTL) for 24 hours. The assay was performed in the absence (-) or presence (+) of ATP to detect the activity of the 20S and the 26S proteasome, respectively. Proteasome inhibitor-suppressible activities were attributed to the proteasome. N = 4 mice/group. **p<0.01 vs. the respective group without ATP.
Figure 3 Lysosomal inhibition does not alter proteasome abundance and proteasome peptidase activities in mouse hearts. Mice were treated as described in Figure 1. A, Representative images of western blot analyses for the representative subunits of the 19S proteasome (Rpt6) and the 20S proteasome (α3, α4, β2, and β5) in the total protein extracts from ventricular myocardium. B, Pooled densitometry data of β2 and β5 subunits as analyzed in A. C, Proteasome peptidase activity assays. Synthetic fluorogenic substrates specific for chyomtrypin-like, caspase-like, and trypin-like peptidases were used to measure the proteasomal peptidase activities in the crude protein extracts from ventricular myocardium collected from mice treated with BFA or vehicle control (CTL) for 24 hours. The assay was performed in the absence (-) or presence (+) of ATP to detect the activity of the 20S and the 26S proteasome, respectively. Proteasome inhibitor-suppressible activities were attributed to the proteasome. N = 4 mice/group. **p<0.01 vs. the respective group without ATP.CC BY · PubMed Central
Figure 4 Dynamic effects of ALP inhibition via 3-MA or BFA on p62 protein levels and a surrogate UPS substrate in cultured neonatal rat ventricular myocytes (NRVMs). Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu, Ad-RFP) 48 hr before 3-methyladenine (3-MA) or bafilomycin A1 (BFA) treatment. A ∼ C, The time course of protein level changes in the GFPu/RFP ratio and the p62/β-tubulin ratio in NRVMs treated with 3-MA (3 mM) or vehicle control. Representative images of western blots (A) and quantitative data from 3 repeats (B, C) are shown. Paired t-test, *p<0.01. D and E, 3-MA and BFA concentration-dependently increases GFPu/RFP ratio at 24 hours. At 24 hr after the drug treatment, the cells were harvested for total protein extraction and western blot analyses of the indicated proteins. Representative images (D) and pooled densitometry data (E) are shown. Two-way ANOVA followed by the Scheffé's test; *p<0.05, **p<0.01 vs. CTL; n = 3 repeats/group.
Figure 4 Dynamic effects of ALP inhibition via 3-MA or BFA on p62 protein levels and a surrogate UPS substrate in cultured neonatal rat ventricular myocytes (NRVMs). Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu, Ad-RFP) 48 hr before 3-methyladenine (3-MA) or bafilomycin A1 (BFA) treatment. A ∼ C, The time course of protein level changes in the GFPu/RFP ratio and the p62/β-tubulin ratio in NRVMs treated with 3-MA (3 mM) or vehicle control. Representative images of western blots (A) and quantitative data from 3 repeats (B, C) are shown. Paired t-test, *p<0.01. D and E, 3-MA and BFA concentration-dependently increases GFPu/RFP ratio at 24 hours. At 24 hr after the drug treatment, the cells were harvested for total protein extraction and western blot analyses of the indicated proteins. Representative images (D) and pooled densitometry data (E) are shown. Two-way ANOVA followed by the Scheffé's test; *p<0.05, **p<0.01 vs. CTL; n = 3 repeats/group.CC BY · PubMed Central
Figure 5 Inhibition of the ALP stabilizes a UPS substrate. NRVMs were cultured and infected with Ad-GFPu/Ad-RFP viral mixture as described in Figure 4 . Forty-eight hours later, the cells were treated with 3-MA (2 mM, A and B) or BFA (10 nM, C and D), or respective vehicle control (CTL) for 12 hrs; then, the treatment of cycloheximide (CHX, 50 µM) was initiated. Immediately before (0 min) or 5, 10, 15, 30, 60, and 120 min after CHX administration, the cells were harvested for western blot analyses for GFPu and RFP. Representative western blot analysis images (A, C) and densitometry data pooled from 3 biological repeats (B, D) are shown. *p<0.05, **p<0.01 vs. CTL.
Figure 5 Inhibition of the ALP stabilizes a UPS substrate. NRVMs were cultured and infected with Ad-GFPu/Ad-RFP viral mixture as described in Figure 4 . Forty-eight hours later, the cells were treated with 3-MA (2 mM, A and B) or BFA (10 nM, C and D), or respective vehicle control (CTL) for 12 hrs; then, the treatment of cycloheximide (CHX, 50 µM) was initiated. Immediately before (0 min) or 5, 10, 15, 30, 60, and 120 min after CHX administration, the cells were harvested for western blot analyses for GFPu and RFP. Representative western blot analysis images (A, C) and densitometry data pooled from 3 biological repeats (B, D) are shown. *p<0.05, **p<0.01 vs. CTL.CC BY · PubMed Central
Figure 6 Representative confocal fluorescence micrographs of NRVMs grown on cover glasses. Ad-GFPu infection and BFA (10 nM) treatment are as described in Figure 4 . At 24 hr after BFA treatment, the cells were fixed with 4% paraformaldehyde and subject to immunofluorescence staining for p62 (red). GFPu direct fluorescence (green) and p62 indirect immunofluorescence were visualized and captured using a confocal microscope. In the BFA treated cells, both p62 and GFPu are increased and the increased GFPu is co-localized with p62. Scale bar = 50 µm.
Figure 6 Representative confocal fluorescence micrographs of NRVMs grown on cover glasses. Ad-GFPu infection and BFA (10 nM) treatment are as described in Figure 4 . At 24 hr after BFA treatment, the cells were fixed with 4% paraformaldehyde and subject to immunofluorescence staining for p62 (red). GFPu direct fluorescence (green) and p62 indirect immunofluorescence were visualized and captured using a confocal microscope. In the BFA treated cells, both p62 and GFPu are increased and the increased GFPu is co-localized with p62. Scale bar = 50 µm.CC BY · PubMed Central
Figure 7 Autophagic inhibition accumulates GFPu in a p62 dependent manner. Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu/RFP) 24 hours before transfection with siRNA for Atg7 (siAtg7), Rab7 (siRab7) and/or for p62 (sip62). A luciferase-specific siRNA (siLuc) was used as control for siRNA infection and off-target effects. The cells were harvested at 48 h later for extracting total proteins. A, Shown are representative images out of 3 repeats. B, pooled densitometry data. *p<0.05 vs. CTL.
Figure 7 Autophagic inhibition accumulates GFPu in a p62 dependent manner. Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu/RFP) 24 hours before transfection with siRNA for Atg7 (siAtg7), Rab7 (siRab7) and/or for p62 (sip62). A luciferase-specific siRNA (siLuc) was used as control for siRNA infection and off-target effects. The cells were harvested at 48 h later for extracting total proteins. A, Shown are representative images out of 3 repeats. B, pooled densitometry data. *p<0.05 vs. CTL.CC BY · PubMed Central

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Metadata

Authors
Tian, Z., Wang, C., Hu, C., Tian, Y., Liu, J., & Wang, X.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-06-24
Volume
9
Issue
6
Pages
e100715
ISSN
1932-6203
DOI
10.1371/journal.pone.0100715
PMC
PMC4069113
Cited by
35
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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