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. 2011 Jul 22;109(3):296-308.
doi: 10.1161/CIRCRESAHA.111.244707. Epub 2011 Jun 9.

Autophagy and p62 in cardiac proteinopathy

Affiliations

Autophagy and p62 in cardiac proteinopathy

Qingwen Zheng et al. Circ Res. .

Abstract

Rationale: Recent studies suggest an important role of autophagy in protection against αB-crystallin-based (CryAB(R120G)) desmin-related cardiomyopathies (DRC), but this has not been demonstrated in a different model of cardiac proteinopathy. Mechanisms underlying the response of cardiomyocytes to proteotoxic stress remain incompletely understood.

Objective: Our first objective was to determine whether and how the autophagic activity is changed in a mouse model of desminopathy. We also investigated the role of p62 in the protein quality control of cardiomyocytes.

Methods and results: Using an autophagosome reporter and determining changes in LC3-II protein levels in response to lysosomal inhibition, we found significantly increased autophagic flux in mouse hearts with transgenic overexpression of a DRC-linked mutant desmin. Similarly, autophagic flux was increased in cultured neonatal rat ventricular myocytes (NRVMs) expressing a mutant desmin. Suppression of autophagy by 3-methyladenine increased, whereas enhancement of autophagy by rapamycin reduced the ability of a comparable level of mutant desmin overexpression to accumulate ubiquitinated proteins in NRVMs. Furthermore, p62 mRNA and protein expression was significantly up-regulated in cardiomyocytes by transgenic overexpression of the mutant desmin or CryAB(R120G) both in intact mice and in vitro. The p62 depletion impaired aggresome and autophagosome formation, exacerbated cell injury, and decreased cell viability in cultured NRVMs expressing the misfolded proteins.

Conclusions: Autophagic flux is increased in desminopathic hearts, and as previously suggested in CryAB(R120G)-based DRC, this increased autophagic flux serves as an adaptive response to overexpression of misfolded proteins. The p62 is up-regulated in mouse proteinopathic hearts. The p62 promotes aggresome formation and autophagy activation and protects cardiomyocytes against proteotoxic stress.

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Figures

Figure 1
Figure 1
Expression of a DRC-linked mutant desmin increases autophagic flux in mouse hearts and NRVMs. A and B, Confocal microscopic analysis of GFP-LC3 distribution in ventricular myocardium. D7-des tg mice were cross-bred with GFP-LC3 mice. The resultant GFP-LC3::D7-des double tg (DTG) mice and their littermate GFP-LC3 single tg mice were analyzed at 2 months of age. The representative images (A) and the quantitative analysis of the number of GFP-LC3 puncta (B) are presented. The GFP dot data (B) were quantified from the 3 randomly selected fields per section, 3 representative sections per heart, and 3 hearts per group. C and D, Increases in autophagic flux in the D7-des tg mouse heart. D7-des tg and NTG littermate mice at 2 months were intravenously injected with one dose of bafilomycin A1 (BFA, 6µmol/kg) or vehicle control (CTL) at 3 hours before the hearts were harvested for western blot analyses of LC3. Representative images (C) and a summary of LC3-II densitometry data (D) are presented. *p<0.05 vs. CTL. E and F, Autophagic flux was increased in NRVMs expressing DRC-linked mutant desmin (MT-Des). Cultured NRVMs were infected with Ad-MT-Des or Ad-β-gal (as control). Six days after infection, the cells were treated with BFA (100nM) or DMSO for 3 hours before being harvested for western blot analysis for the indicated proteins. Representative images (E) and a summary of densitometry data (F) are presented. *p<0.05 vs. CTL; #p<0.05 vs. Ad-β-gal/CTL; N=3/group in all cases. AU = arbitrary units.
Figure 2
Figure 2
Representative electron micrographs. Perfusion-fixed ventricular tissues from 2-month-old D7-des Ntg (A, C, E) and tg (B, D, F) mice at the baseline (A~D) or 1 hour after the second BFA injection (E, F; 2 intraperitoneal injections with 1 hour in between, 3µmol/kg/injection) were used for electron microscopy. G: protein aggregates. Arrows or open arrow point to autophagosomes. Bar=1µm.
Figure 3
Figure 3
Inhibition of autophagy by 3-methyladenine (3-MA) promotes the ability of MT-des expression to accumulate ubiquitinated proteins (Ub-proteins). A and B, NRVMs were infected with Ad-MT-des. Starting one day after infection, cells were treated daily with 3-MA (1.5mM) or DMSO for 4 days. The level of Ub-proteins, desmin (Des), and GAPDH were measured using western blot analyses. Ub-proteins and the high molecular weight Des (HMW-Des) in MT-des expressing cells were markedly increased by 3-MA. All Ub-proteins in the lane as illustrated in the representative image (A) are included in the densitometry. In each cohort, the densitometric reading of the Ad-MT-des lane was defined as 1AU and used to normalize other lanes on the same image. A summary of quantitative data from 3 repeats is presented (B). C and D, Representative images (C) and a summary of densitometry data (D) of semi-quantitative RT-PCR analyses for the mRNA levels of transgenic MT-des. GAPDH was analyzed for loading control. N.S., not significant; t-test, n = 3.
Figure 4
Figure 4
Enhancement of autophagy by rapamycin decreases the ability of MT-des expression to accumulate ubiquitinated proteins (Ub-proteins). A and B, NRVMs were infected with Ad-MT-des one day before the initiation of the daily rapamycin (Rapa, 200nM) or DMSO treatment. After 4 daily treatments, the cells were harvested for western blot analyses of the indicated proteins. The representative image (A) and a summary of quantitative data of 3 repeats (B) are presented. The quantification of Ub-proteins was performed as in Figure 3B. C and D, Cycloheximide (CHX) chase assay for c-myc-tagged MT-des. CHX (10µM) was used to block protein synthesis. MT-des protein levels at the indicated time points after CHX treatment were measured using western blot analyses for the c-myc tag. A representative image is shown in C. The relative levels of myc-MT-des at post-CHX time points to that of the 0 hour time point were summarized in D. *p < 0.05 vs. the DMSO group, t-test, n = 4.
Figure 5
Figure 5
The levels of the steady state p62 protein and mRNA are increased in the hearts of mouse models of cardiac proteinopathy. Ventricular myocardium was collected from 2-month-old D7-des, 3-month-old CryABR120G (R120G), and their NTG littermates for extraction of total proteins (A and B) or total RNA (C and D). A and B, Western blot analysis for p62 protein. C and D, Semi-quantitative RT-PCR analyses of p62 mRNA levels in D7-des (C) and R120G (D) mouse hearts. The representative images are presented on the left and a summary of densitometric data are shown on the right of each panel. GAPDH was included for loading controls. * p<0.05, **p<0.01, ***p<0.001 vs. NTG, t-test, n=3~4 mice/group.
Figure 6
Figure 6
p62 Knockdown decreases LC3-II in cardiomyocytes. NRVMs were infected with Ad-HA-CryABR120G (Ad-R120G), Ad-Myc-MT-des, or Ad-β-gal as indicated on day 0. The cells were also transfected with either luciferase siRNA (Luc-siRNA, as control) or p62 siRNA using the Lipofectamine reagent on day1 and day 4. On day 7, the cells were harvested for western blot analyses of the indicated proteins. p62 Knockdown significantly reduced LC3-II levels in both the control and the MT-des or CryABR120G (R120G) expressing cells. The representative image (A and C) and a summary of quantitative data from 4 repeats (B and D) are presented. * p<0.05 vs. β-gal/Luc-siRNA; # p<0.05; two-way ANOVA followed by the Scheffé's test.
Figure 7
Figure 7
p62 is required for efficient formation of aggresomes in NRVMs expressing CryABR120G. NRVMs were treated as described in Figure 6. On day 7 after Ad-HA-CryABR120G infection, cultured NRVMs were fixed in 2% paraformaldehyde and used for indirect double immunofluorescence labeling for the HA-tag of HA-CryABR120G (green) and for p62 (red, A) or SEC61α (red, B). Nuclei were stained blue with DAPI. HA-labeled aggregates, induced by HA-CryABR120G expression, co-localize with p62 (A) and SEC61α (B). To show the normal distribution of SEC61α, the images of the groups infected with Ad-β-gal were obtained by a longer exposure (1.5s) compared to the Ad-CryABR120G groups (400ms).
Figure 8
Figure 8
p62 depletion decreases the insoluble ubiquitinated (Ub−) proteins and the insoluble CryABR120G protein in NRVMs overexpressing CryABR120G (R120G) and exacerbates cell injury, and reduces cell survival in cultured NRVMs overexpressing CryABR120G or MT-des. NRVMs were treated as described in Figure 6. A ~ D, Cells were lysed with a lysis buffer containing 1% Triton X-100. Triton X-100 soluble and insoluble fractions of the cell lysates were used for western blot analysis for the abundance of ubiquitinated proteins or HA-tagged CryABR120G. GAPDH and α-actinin were probed as loading controls for proteins in the soluble and the insoluble fractions, respectively. Representative images (A) and a summary of quantitative data (B, C and D) are presented. E ~ G, The following assays were performed on day 7. E, LDH activities in the cultured medium were measured to assess cell injury. F, Cell viability was assessed by the MTT assay. Cell viability was deceased by expressing CryABR120G or MT-des, and was further reduced by p62 knockdown. G, Changes in the LDH/MTT ratio. * p<0.05, vs. β-gal/luc-siRNA; # p<0.05; n=4.

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