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. 2014 Jun 24;9(6):e100715.
doi: 10.1371/journal.pone.0100715. eCollection 2014.

Autophagic-lysosomal inhibition compromises ubiquitin-proteasome system performance in a p62 dependent manner in cardiomyocytes

Zongwen Tian  1 , Changhua Wang  1 , Chengjun Hu  1 , Yihao Tian  1 , Jinbao Liu  2 , Xuejun Wang  1
Affiliations

Autophagic-lysosomal inhibition compromises ubiquitin-proteasome system performance in a p62 dependent manner in cardiomyocytes

Zongwen Tian et al. PLoS One. .

Abstract

Intracellular protein degradation is primarily performed by the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). The interplay between these two pathways has been rarely examined in intact animals and the mechanism underlying the interplay remains unclear. Hence, we sought to test in vivo and in vitro the impact of inhibition of the ALP on UPS proteolytic performance in cardiomyocytes and to explore the underlying mechanism. Transgenic mice ubiquitously expressing a surrogate UPS substrate (GFPdgn) were treated with bafilomycin-A1 (BFA) to inhibit the ALP. Myocardial and renal GFPdgn protein levels but not mRNA levels were increased at 24 hours but not 3 hours after the first injection of BFA. Myocardial protein abundance of key proteasome subunits and the activities of proteasomal peptidases were not discernibly altered by the treatment. In cultured neonatal rat ventricular myocytes (NRVMs), the surrogate UPS substrate GFPu and a control red fluorescence protein (RFP) were co-expressed to probe UPS performance. At 12 hours or 24 hours after ALP inhibition by 3-methyladenine (3-MA) or BFA, GFPu/RFP protein ratios and the protein half-life of GFPu were significantly increased, which is accompanied by increases in p62 proteins. Similar findings were obtained when ALP was inhibited genetically via silencing Atg7 or Rab7. ALP inhibition-induced increases in GFPu and p62 are co-localized in NRVMs. siRNA-mediated p62 knockdown prevented ALP inhibition from inducing GFPu accumulation in NRVMs. We conclude that in a p62-dependent fashion, ALP inhibition impairs cardiac UPS proteolytic performance in cardiomyocytes in vitro and in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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 2
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 3
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 4
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 5
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 6
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 7
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.
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