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. 2013 Oct 1;100(1):63-73.
doi: 10.1093/cvr/cvt150. Epub 2013 Jun 12.

Nrf2 deficiency prevents reductive stress-induced hypertrophic cardiomyopathy

Sankaranarayanan Kannan  1 , Vasanthi R Muthusamy, Kevin J Whitehead, Li Wang, Aldrin V Gomes, Sheldon E Litwin, Thomas W Kensler, E Dale Abel, John R Hoidal, Namakkal S Rajasekaran
Affiliations

Nrf2 deficiency prevents reductive stress-induced hypertrophic cardiomyopathy

Sankaranarayanan Kannan et al. Cardiovasc Res. .

Abstract

Aims: Mutant protein aggregation (PA) cardiomyopathy (MPAC) is characterized by reductive stress (RS), PA (of chaperones and cytoskeletal components), and ventricular dysfunction in transgenic mice expressing human mutant CryAB (hmCryAB). Sustained activation of nuclear erythroid-2 like factor-2 (Nrf2) causes RS, which contributes to proteotoxic cardiac disease. The goals of this pre-clinical study were to (i) investigate whether disrupting Nrf2-antioxidant signalling prevents RS and rescues redox homeostasis in hearts expressing the mutant chaperone and (ii) elucidate mechanisms that could delay proteotoxic cardiac disease.

Methods and results: Non-transgenic (NTG), transgenic (TG) with MPAC and MPAC-TG:Nrf2-deficient (Nrf2-def) mice were used in this study. The effects of Nrf2 diminution (Nrf2±) on RS mediated MPAC in TG mice were assessed at 6-7 and 10 months of age. The diminution of Nrf2 prevented RS and prolonged the survival of TG mice (∼50 weeks) by an additional 20-25 weeks. The TG:Nrf2-def mice did not exhibit cardiac hypertrophy at even 60 weeks, while the MPAC-TG mice developed pathological hypertrophy and heart failure starting at 24-28 weeks of age. Aggregation of cardiac proteins was significantly reduced in TG:Nrf2-def when compared with TG mice at 7 months. Preventing RS and maintaining redox homeostasis in the TG:Nrf2-def mice ameliorated PA, leading to decreased ubiquitination of proteins.

Conclusion: Nrf2 deficiency rescues redox homeostasis, which reduces aggregation of mutant proteins, thereby delaying the proteotoxic pathological cardiac remodelling caused by RS and toxic protein aggregates.

Keywords: Nrf2; antioxidants; cardiomyopathy; protein aggregation; reductive stress.

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Figures

Figure 1
Figure 1
Nrf2 deficiency rescues redox homeostasis, prevents sudden cardiac death, and extends survival of the MPAC-TG mice. (A) Genotyping for human mutant R120GCryAB (TG) and Nrf2 deficiency (nrf2±) using specific primers sets. (B) Q-PCR determination of myocardial Nrf2-mRNA levels in NTG, MPAC-TG, and TG:Nrf2-def mice (n = 4–6/gp). (C) Immunoblots for the Nrf2 protein in the nuclear extracts of hearts from the above mouse models exhibiting either abundant (in MPAC-TG) or reduced Nrf2 (in TG:Nrf2-def) expression. (D) Immunoblots showing CryAB expression in the total homogenates of mouse hearts at 2 months of age. No significant difference was observed between TG vs. TG:Nrf2-def mice (n = 3/group). (E) Kaplan–Meier Survival plot (n ≥ 10 mice/group). (F) Representative images of hearts showing the gross morphology and reduced hypertrophy in Nrf2-def mice at 6–7 months of age. Images were obtained at 4× using a light microscope. (G) Heart-to-body weight ratios at 6–7 months of age showing significant hypertrophy in TG mice. (H) Quantitative PCR analysis of cardiac hypertrophy markers (n = 4–6/group). *NTG vs. TG; $NTG vs. TG:Nrf2-def; P < 0.05. (I) Myocardial GSH state determined by enzyme-kinetic assays (n = 4–6/group). (J) Scheme representing the transcriptional role of Nrf2 on antioxidants and GSH metabolism. *NTG vs. TG; $NTG vs. TG:Nrf2-def; P < 0.05.
Figure 2
Figure 2
Deficiency of Nrf2 preserves the cardiac function in the MPAC-TG mice. (A) M-mode images from echocardiographic analysis (Vevo2100, VisualSonics) with high-resolution (40 MHz) ultrasound to evaluate the systolic function. The LV cavity (white) and wall thickness (grey) are displayed in diastole and systole for each genotype at 6–7 months of age (n = 5 mice/group). (B) Quantitative analysis of echocardiography for systolic function (FS, ejection fraction) and hypertrophy (the end-diastolic and end-systolic LV mass from 2D imaging, and diastolic dimensions of the interventricular septal, and posterior walls from M-mode imaging). *NTG vs. TG; P < 0.05.
Figure 3
Figure 3
(A) Disruption of Nrf2 down-regulates antioxidants transcript levels in the MPAC-TG mice. QPCR analysis showing the transcript levels of antioxidant enzymes at 6–7 months of age. The mRNA data were normalized to Arbp1 or Gapdh of the respective samples and fold changes were calculated by comparing with the NTG group, (n = 4–6/group). *NTG vs. TG; $NTG vs. TG:Nrf2-def; P < 0.05. (B–D) The effect of Nrf2 deficiency on antioxidant enzymes in the MPAC-TG mice. (B) Immunoblots of glutathione-S-reductase (GSR), γ-glutamyl cysteine synthetase (γ-GCS), glucose-6-phosphate dehydrogenase (G6PD), hemoxygenase-1 (HO1), NADH-quinone oxidase (NQO1), superoxide dismutase-1 (Cu/Zn-SOD), superoxide dismutase-2 (Mn-SOD), and catalase in the heart tissues from NTG, TG, and TG:Nrf2-def mice at the age of 6–7 months. (C) and (D) Densitometry analysis of the immunoblots were performed from three sets of experiments and expressed as mean average ± SD for 4–6 animals/group. *NTG vs. TG; $NTG vs. TG:Nrf2-def; P < 0.05.
Figure 4
Figure 4
Rescue of redox homeostasis prevents or delays PA in the MPAC-TG mice. (A) Representative western blots from soluble and insoluble fractions, and total homogenates of hearts (n = 4–6). Signals probed against anti-CryAB antibody showing soluble and insoluble aggregates in TG and TG:Nrf2-def mice. (B) Respective densitometry analysis from three independent experiments/animals. (C) Q-PCR analysis for human-mutant CryAB gene expression (n = 3–4 animals/group). (D) IF analysis for protein aggregates. Cryo-sections from heart tissues were fixed and processed with anti-CryAB or anti-Hsp25 antibodies and imaged by confocal microscopy. Green, protein aggregates; blue, DAPI nuclear staining; arrows indicate aggregates around the nucleus. Fluorescence was quantified by appropriate densitometry analysis using the Simple PCI 6 Imaging Software (Hamamatsu Corporation, Sewickley, PA). *NTG vs. TG; $NTG vs. TG:Nrf2-def; P < 0.05.
Figure 5
Figure 5
Rescue of redox homeostasis prevents ubiquitination and favours basal oxidation of proteins in MPAC-TG mice. Representative western blots for poly-ubiquitination from (A) soluble and (B) insoluble fractions of myocardial proteins. A 12% poly-acrylamide gel was used to separate proteins, which were then probed with mono- and poly-ubiquitin antibodies to detect ubiquitinated proteins. Densitometry analyses of immunoblots indicate increased protein ubiquitination in the insoluble fraction of TG mice. (C) Representative western blot from the total homogenate probed for anti-4-HNE antibody showing oxidized proteins. Densitometry indicates decreased protein oxidation in TG. Data are expressed as mean ± SE (n = 4–6) mice. (D) Effects of Nrf2 deficiency on proteasome and cathepsin L function. Cathepsin L activity is significantly increased in TG:Nrf2-def hearts (n = 3/group). *NTG vs. TG; $NTG vs. TG:Nrf2-def; P < 0.05.
Figure 6
Figure 6
Rescue of redox homeostasis ameliorates ER stress in the MPAC-TG mice. (A) Q-PCR analysis showing Grp94 RNA levels from NTG, TG, and TG:Nrf2-def mice. (B) Immunoblots for Grp94 and Grp78 indicating ER stress in TG. This was partially rescued by Nrf2 deficiency. (C) IP and Co-IP with αBC or Grp78 antibodies followed by immunoblotting showing sequestration of Grp78 in TG mice. Data are mean ± SD for 3–4 animals/group. *NTG vs. TG; $NTG vs. TG:Nrf2-def; P < 0.05.
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Comment in

  • Reducing damage through Nrf-2.
    Balligand JL. Balligand JL. Cardiovasc Res. 2013 Oct 1;100(1):1-3. doi: 10.1093/cvr/cvt204. Epub 2013 Aug 22. Cardiovasc Res. 2013. PMID: 23970486 No abstract available.

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