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. 2011 Mar 15;14(6):957-71.
doi: 10.1089/ars.2010.3587. Epub 2011 Feb 2.

Sustained activation of nuclear erythroid 2-related factor 2/antioxidant response element signaling promotes reductive stress in the human mutant protein aggregation cardiomyopathy in mice

Namakkal Soorappan Rajasekaran  1 , Saradhadevi Varadharaj, Gayatri D Khanderao, Christopher J Davidson, Sankaranarayanan Kannan, Matthew A Firpo, Jay L Zweier, Ivor J Benjamin
Affiliations

Sustained activation of nuclear erythroid 2-related factor 2/antioxidant response element signaling promotes reductive stress in the human mutant protein aggregation cardiomyopathy in mice

Namakkal Soorappan Rajasekaran et al. Antioxid Redox Signal. .

Abstract

Inheritable missense mutations in small molecular weight heat-shock proteins (HSP) with chaperone-like properties promote self-oligomerization, protein aggregation, and pathologic states such as hypertrophic cardiomyopathy in humans. We recently described that human mutant αB-crystallin (hR120GCryAB) overexpression that caused protein aggregation cardiomyopathy (PAC) was genetically linked to dysregulation of the antioxidant system and reductive stress (RS) in mice. However, the molecular mechanism that induces RS remains only partially understood. Here we define a critical role for the regulatory nuclear erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein (Keap1) pathway--the master transcriptional controller of antioxidants, in the pathogenesis of PAC and RS. In myopathic mice, increased reactive oxygen species signaling during compensatory hypertrophy (i.e., 3 months) was associated with upregulation of key antioxidants in a manner consistent with Nrf2/antioxidant response element (ARE)-dependent transactivation. In transcription factor assays, we further demonstrate increased binding of Nrf2 to ARE during the development of cardiomyopathy. Of interest, we show that the negative regulator Keap1 was predominantly sequestrated in protein aggregates (at 6 months), suggesting that sustained nuclear translocation of activated Nrf2 may be a contributing mechanism for RS. Our findings implicate a novel pathway for therapeutic targeting and abrogating RS linked to experimental cardiomyopathy in humans. Antioxid.

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Figures

FIG. 1.
FIG. 1.
Tissue-specific transgenic (TG) overexpression of human mutant αB-crystallin (hR120GCryAB) induces hypertrophic cardiomyopathy in mice. (A) Heart-specific overexpression of mutant protein induces pathological ventricular hypertrophy and biatrial thrombosis at 6 months of age. No such phenotypical (hypertrophy) changes and cardiac dysfunction are evident in the 3-month-old mice. (B) Organ-to-body (heart/body) weight ratios on autopsy confirm the cardiac hypertrophy in the hR120CryAB-TG mouse at 6 months of age but not in the 3-month-old mice. n = 8 or more mice from each group (p < 0.01). (C, D) Redox markers (GSH and GSH/GSSG): Determined the concentrations of reduced and oxidized glutathione in nontransgenic (NTG) and TG mouse heart ventricles (n = 4) at 3 and 6 months. At 3 months, no statistically significant change in GSH levels was recorded among the experimental groups. Dramatic increase in GSH and GSH/GSSG redox ratio was evident in the TG mouse at 6 months, indicating highly enhanced reducing environment along with pathological hypertrophy. Values are mean + standard deviation for 4 or animals in each group (*p < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).
FIG. 2.
FIG. 2.
Total reactive oxygen species (ROS) formation is enriched in myocardium of the TG mouse heart at 3 months but not at 6 months. (A) Electron paramagnetic resonance (EPR) signals for 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) in the NTG and TG mouse heart tissues. Representative EPR signals for CMH showing prominent differences between the NTG and TG hearts with higher radical generation in the TG at 3 months but no significant difference after 6 months. (B) Graph of the radical formation in the ventricular tissue determined using CMH spin probe. Significantly increased radical formation is evident in the 3-month-old TG mouse hearts compared with the NTG (*p < 0.05). However, the ROS levels were not significantly different between the NTG and TG at 6 months. (C–E) Determination of ROS in the myocardial tissue sections using fluorescent (dihydro difluro diacetate [H2DCFDA]/dihydroethidium [DHE]) probes. (C) Staining with DCFDA/DHE: Determination of ROS using fluorescent probes (H2DCFDA/DHE) and microscopy. Frozen tissue sections were incubated with 10 (M of H2DCFDA/DHE for 30 min at 37°C. Sections were then washed with 1 × PBS, mounted and analyzed by Zeiss 510 Meta confocal microscopy. Significantly increased ROS levels were seen in the TG mouse heart compared with NTG at 3 months (p < 0.01), but these values were insignificant at 6 months of age, suggestive of a more highly reduced environment (D, E).
FIG. 3.
FIG. 3.
Nuclear translocation of nuclear erythroid 2-related factor 2 (Nrf2) is prominent in the TG mice overexpressed with mutant CryAB at 3 and 6 months of age. (A) Representative Western blots (WBs) of cytosolic and nuclear fractions obtained from 3-month-old NTG and mutant CryAB (TG) mice. Each lane indicates an individual mouse. Along with abundant expression of mutant protein (nonphosphorylated/phosphorylated forms), the nuclear translocation of Nrf2 is predominant in the TG mouse heart. (B) Densitometry analysis for CryAB and P-(S59)-CryAB reveals 3- and >4-fold increase, respectively, in the cytosol of TG mice compared to the NTG. A significant (∼50%) reduction of Nrf2 in the cytosol of TG mice is evident (*p < 0.01). (C) Obviously, the nuclear translocation of Nrf2 is highly significant (∼8-fold) in the TG compared to the NTG mice (C). Densitometry analysis was not performed for mutant CryAB (phosphorylated and nonphosphorylated forms) due to their exclusive presence in the nuclear fraction of TG mice. (D) Representative WBs of cytosolic and nuclear fractions obtained from 6-month-old NTG and mutant CryAB (TG) mice. Each lane indicates an individual mouse. Along with abundant expression of mutant protein (nonphosphorylated/phosphorylated forms) Nrf2 is translocated into the nucleus in the TG mouse heart. (E) Densitometry analysis for CryAB and P-(S59)-CryAB reveal ∼5- and >8-fold increase, respectively, in the cytosol of TG mice compared to the NTG at 6 months of age. A huge reduction (∼90%) of Nrf2 in the cytosol of TG mice is evident, whereas significant Nrf2 nuclear translocation is also evident in the NTG at 6 months, indicating age-associated activation. As expected, nuclear translocation of Nrf2 is prominent (∼6-fold when compared to NTG) in the TG mouse heart at 6 months, suggesting sustained nuclear translocation of Nrf2 during the pathogenesis of cardiomyopathy (F) (*p < 0.01).
FIG. 4.
FIG. 4.
Decreased ubiquitination and increased activity of Nrf2 in the TG mouse. (A) TransAM-Nrf2 activity assay: Nuclear extracts from NTG and TG mouse (n = 6) at 6 months were incubated with the precoated antioxidant response element (ARE) oligonucleotides. Using ant-Nrf2-ab and HRP-conjugated secondary, specific activity for Nrf2 was measured in a plate reader. Results were expressed as A450 nm ± standard deviation for four mice in each group (*p < 0.05). (B) Ubiquitination of Nrf2: Representative immunoprecipitation (IP) (with poly-ubiquitin-ab) and WB (with anti-Nrf2-ab) analysis performed in NTG and TG cytosolic fractions indicating decreased ubiquitination of Nrf2, suggesting its enhanced nuclear translocation in the TG mouse at 3 and 6 months (**p < 0.01). (C) Nrf2 interaction with Kelch-like ECH-associated protein (Keap1): Representative WB analysis showing poor interactions between the Keap1 and Nrf2 in the TG mice compared with NTG and this observation is in line with decreased ubiquitination and enhanced nuclear translocation of Nrf2. Rb-IgG was used as control to perform IP with NTG/TG samples.
FIG. 5.
FIG. 5.
Sustained Nrf2 nuclear translocation in the TG hearts induces gene expression of ARE-dependent antioxidants. Real-time RT-PCR determinations of Nrf2 target genes in NTG and TG mouse at 3 (A–H) and 6 months (I–P) were performed using Qiagen-mouse primer sets (n ≥ 6). Data were first normalized to Arbp1 expression and then to the corresponding gene expression in the NTG group (Delta-delta-CT method). While there is an increasing trend in the messenger RNA (mRNA) expression for major targets of Nrf2, catalase (∼5-fold increase) and NADH-quinone oxidase (Nqo1) genes were significantly upregulated at 3 months (A). At 6 months, most of the major antioxidant genes (Nqo1, catalase, hemoxygenase-1 [Ho1], glutamyl cysteine ligase (modulatory) [Gclm], glutamyl cysteine ligase (catalytic) [Gclc], and glucose 6 phosphate dehydrogenase [G6pd]) were significantly upregulated (I–P) to facilitate the highly reduced environment (*p < 0.05 vs. NTG).
FIG. 6.
FIG. 6.
Increased transactivation of key antioxidant genes in the TG hearts induces their protein expression at 3 and 6 months. (A, D) Representative WB experiments of cytosol or nucleus from NTG and TG mice at 3 and 6 months, respectively. Protein blots were probed with anti-Ho1, Nqo1, super oxide dismutase-1 (Sod-1), catalase, and γ-Gcl. Each lane indicates an individual mouse. (B, C, E) Densitometry analysis were performed as relative intensity values calculated as mean arbitrary units obtained from the WB shown in (A) and (D), respectively (*p < 0.05, **p < 0.01, vs. NTG). Significant increases in the Ho1 and Nqo1 in the cytosol and Nqo1 in the nucleus of TG mice were evident at 3 months. At 6 months, Ho1 protein is decreased, whereas the same increasing trend exists with the Nqo1 protein expression in the cytosol and nucleus of TG mice in association with reductive stress.
FIG. 7.
FIG. 7.
Sequestration of Keap1 in to the mutant CryAB aggregates facilitate Nrf2 nuclear translocation. Immunofluorescence analysis was performed using the CryAB (Rb.ab, green) and Keap1 (SC-Goat ab, red) and merged with the nuclear staining (DRAQ1). CryAB and Keap1 were colocalized around the perinuclear area of neither Nrf2-KO nor NTG mouse hearts (A, B, D). Prominent colocalization of these proteins is evident in the R120G CryAB-Tg at 3 and 6 months of age (C, E). Robust interactions could be observed in 6-month-old TG mice, indicating that the Keap1 is sequestered into the aggregates, which favors sustained dissociation and stabilization of Nrf2. Inset arrows and boxes in each panel showing magnified view (100 μm) of the Keap1-CryAB colocalization with the aggregates (yellow on merged images).
FIG. 8.
FIG. 8.
Determination of Keap1 protein expression and its interactions by IP and WB. (A, B) WB probing Keap1 in the cytosol of NTG and TG hearts: Although there is no significant change in the protein levels of Keap1 among the NTG and TG hearts at 3 months, significant decrease (p < 0.05) of cytosolic Keap1 is evident in the TG mouse at 6 months, indicating its sequestration, leading to decreased Nrf2 binding. (C) Determination of Keap1 and mutant CryAB protein interactions: IP using anti-CryAB, Keap1 WBs showing robust interactions among these proteins in the TG mouse heart tissue at 6 months, indicating possible sequestration of Keap1 into the protein aggregates. Rb-IgG were used for IP with NTG/TG cytosol. WB images are representative of multiple analysis (n = 4 or more) and are statistically significant (*p < 0.01; **p < 0.05). Densitometry analysis confirmed significant increase of CryAB and Keap1 aggregation in cytosol and nucleus of TG mice.
FIG. 9.
FIG. 9.
Schematic proposal for Nrf2 activation and mechanism for reductive stress in the mutant-protein aggregation cardiomyopathy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).
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