Nrf2-Mediated Cardiac Maladaptive Remodeling and Dysfunction in a Setting of Autophagy Insufficiency

Abstract

Nuclear factor erythroid-2-related factor 2 (Nrf2) appears to exert either a protective or detrimental effect on the heart; however, the underlying mechanism remains poorly understood. Herein, we uncovered a novel mechanism for turning off the Nrf2-mediated cardioprotection and switching on Nrf2-mediated cardiac dysfunction. In a murine model of pressure overload-induced cardiac remodeling and dysfunction via transverse aortic arch constriction, knockout of Nrf2 enhanced myocardial necrosis and death rate during an initial stage of cardiac adaptation when myocardial autophagy function is intact. However, knockout of Nrf2 turned out to be cardioprotective throughout the later stage of cardiac maladaptive remodeling when myocardial autophagy function became insufficient. Transverse aortic arch constriction -induced activation of Nrf2 was dramatically enhanced in the heart with impaired autophagy, which is induced by cardiomyocyte-specific knockout of autophagy-related gene (Atg)5. Notably, Nrf2 activation coincided with the upregulation of angiotensinogen (Agt) only in the autophagy-impaired heart after transverse aortic arch constriction. Agt5 and Nrf2 gene loss-of-function approaches in combination with Jak2 and Fyn kinase inhibitors revealed that suppression of autophagy inactivated Jak2 and Fyn and nuclear translocation of Fyn, while enhancing nuclear translocation of Nrf2 and Nrf2-driven Agt expression in cardiomyocytes. Taken together, these results indicate that the pathophysiological consequences of Nrf2 activation are closely linked with the functional integrity of myocardial autophagy during cardiac remodeling. When autophagy is intact, Nrf2 is required for cardiac adaptive responses; however, autophagy impairment most likely turns off Fyn-operated Nrf2 nuclear export thus activating Nrf2-driven Agt transcription, which exacerbates cardiac maladaptation leading to dysfunction.

Keywords: NF-E2-related factor 2; angiotensinogen; autophagy; myocardial infarction; ventricular remodeling.

Figure 1

The effect of TAC on death, myocardial necrosis and pathological hypetrophy in WT and Nrf2^-/- mice. A, Survival rate. Nine wk old male littermates of WT and Nrf2^-/- mice were subjected to sham or TAC operation. The number of dead mice over an 8 wk period was counted daily. *p<0.05 vs. Nrf2^-/- group. B, The effect of Nrf2 knockout on myocardial necrosis after 3 days of TAC. Nine wk old male littermates of WT and Nrf2^-/- mice were subjected to sham or TAC operation. Green indicates the cardiomyocyte membrane. Red indicates the necrotic cells. *p<0.05 vs. sham (-) in the same group. C, Representative images of hearts in WT and Nrf2^-/- mice 4 wks after TAC surgery. D, Left panel: Representative echocardiographic images of WT and Nrf2^-/- mice 4 wks after TAC surgery. Right panel: LVPW;d of WT and Nrf2^-/- mice. *p<0.05 vs. sham (-) in the same group.

Figure 2

The effect of TAC on myocardial autophagy in mice. Male WT C57BL/6J mice at 9-10 wks of age were subjected to sham or TAC operation as indicated. A, Western blot analysis of myocardial LC3-I, LC3-II, and p62 expression. Left panel: representative immunoblots from 4 separate experiments. Right panel: densitometric analysis of LC3-II and p62 protein levels. n=4, * or # p<0.05 vs. control (0). There are no differences of LC3-II or p62 expression between control (0) and sham groups at each time point as indicated. B, Myocardial autophagic flux after TAC. Upper panel: representative immunoblots of LC3-I and LC3-II from 4 separate experiments. middle panel: densitometric analysis of LC3-II protein levels; bottom panel: quantified autophagic flux. n=4. ns, non-significant; CQ, chloroquine. There are no differences of LC3-II expression between control (0) and sham groups at each time point as indicated.

Figure 3

The effect of cardiomyocyte-restricted knockout of Atg5 on TAC-induced activation of Nrf2 in the murine heart. Male 6 wk-old mice with different genotypes as indicated were i.p. injected with tamoxifen (20 mg/kg/d) for 3 wks. After 2 wks for washing out tamoxifen from the body, these mice were subjected to sham or TAC operation for 4 wks. A, The efficacy of tamoxifen-induced knockout of Atg5 in the heart. Representative immunoblots of myocardial Atg5, p62, LC3-I and LC3-II from 8 separate experiments. B, qPCR analysis of myocardial Nrf2 and NQO1 mRNAs in the heart 4 wks after sham (-) and TAC. *p<0.05 vs. sham (-) in the same group. C, Western blot analysis of myocardial Nrf2 and NQO1 proteins in the heart 4 wks after sham and TAC. n=3 for each sham group and n=4∼5 for each TAC group. *p<0.05 vs. sham (-) in the same group.

Figure 4

Nrf2-mediated upregulation of Agt in the autophagy impaired heart after TAC. A, TAC-induced expression of Agt, Nrf2, and NQO-1 in the heart. Male WT mice at 9 wks of age were subjected to sham or TAC operation. Left ventricles of mice before and after 1, 2, and 4 wks post-surgery were subjected to qPCR analysis of Agt, Nrf2, and NQO-1 mRNA expression. *p<0.05 vs. Sham. Sample numbers for each group are indicated in the figure. B, The effect of cardiomyocyte-restricted knockout of Atg5 on Agt expression in the heart 4 wks after sham (-) or TAC (+). Male mice with different genotypes were treated as described in Figure 3. Upper panel: qPCR analysis of Agt mRNA expression in the heart. Middle panel: representative immunoblots, and Bottom panel: densitometric analysis of Agt protein in the heart. n=4, *p<0.05 vs. sham (-) in the same group. C, Effect of Nrf2 knockout on Agt expression in the heart 4 wks after sham (-) or TAC (+). Male 6 wk-old littermates of WT and Nrf2 received the same treatment as described in Figure 3. Upper panel: qPCR analysis of Agt mRNA expression in the heart. Middle panel: representative immunoblots, and Bottom panel: densitometric analysis of Agt protein in the heart. n=4, *p<0.05 vs. WT sham (-).

Figure 5

Jak2/Fyn pathway in the autophagy impaired heart after TAC. Male mice with different genotypes were treated as described in Fig. 3. Western blot analysis of p-Jak2, Jak2, p-Stat3, Stat3, p-Fyn, and Fyn in A) Agt5^fl/fl, MerCreMer⁺, and cardiomyocyte-restricted Atg5^-/- as well as B) WT and Nrf2^-/- mice 4 wks after sham (-) and TAC. Results are representative immunoblots from 4 separate experiments.

Figure 6

The effect of Nrf2 and Atg5 knockdown on Ang II-induced activation of the Jak2/Fyn/Agt signaling axis in cardiomyocytes. Rat neonatal cardiomyocytes infected with adenovirus of control scramble shRNA (Ad-shCtr) and Ad-shNrf2 were further transfected with control siRNA (siCtr) and siAgt5 prior to Ang II treatment as described in the Online Supplement. A, Representative immunoblots from 4 separate experiments. B, Densitometric analysis. n=4, *p<0.05 vs. the control (vehicle treated Ad-shCtr+siCtr cells). C, The efficacy of Nrf2 and Atg knockdown. Upper panel: qPCR analysis of Nrf2 mRNA expression and Western blot analysis of Nrf2 target gene NQO1 in rat neonatal cardiomyocytes infected with Ad-shCtr and Ad-shNrf2. n=4, *p<0.05 vs. Ad-shCtr infected cells. Lower panel: representative immunoblot of LC3 and Agt5 in the rat neonatal cardiomyocytes transfected with siCtr and siAtg5 from 4 separate experiments.

Figure 7

The effect of Fyn kinase inhibition on Ang II-induced Nrf2 activation and Agt expression in cardiomyocytes with a setting of autophagy impairment. A, A time-course study of Ang II-induced mRNA expression of NQO1 and Agt. Serum starved rat neonatal cardiomyocytes were treated with Ang II (1 M) in serum free DMEM as indicated. n=4, *p<0.05 vs. Ang II (0 h) in the same group. B, The effect of Jak2 inhibitor AG490 and Fyn kinase inhibitor PP2 on Ang II-induced mRNA expression of NQO1 and Agt. Serum starved rat neonatal cardiomyocytes were treated with Ang II (1 μM), AG490 (1 μM), and PP2 (1 μM) in serum free DMEM as indicated for 48 h. n=4, *p<0.05 vs. vehicle, PP2, AG490, or Ang II alone treated groups. ns; non-significant. C, The effect of PP2 on Ang II-induced mRNA expression of NQO1 and Agt in the settings of Nrf2 and/or Atg5 knockdown. Rat neonatal cardiomyocytes were treated as in Figure 6 and stimulated with Ang II and PP2 as in Figure 7B. n=4, *p<0.05 vs. Ang II-treated Ad-shCtr+siCtr group. ns; non-significant.

Figure 8

The role of Jak2/Fyn signaling in regulating Nrf2 nuclear accumulation in the pressure overloaded heart. A, Immunochemical staining of p-Fyn and Nrf2 in the heart at 4 wks after TAC. Left panel: representative images from 4 separate experiments. Right panel: % positive nuclei values for p-Fyn and Nrf2. B, The effect of Nrf2 deficiency on Agt expression in the heart with cardiomyocyte-specific knockout of Atg5 at 4 wks after TAC. Left panel, Western blot analysis of Agt protein expression. n=4, *p<0.05 vs. Sham (-) in the same group. Right panel, qPCR analyses of Agt mRNA expression. n=8, *p<0.05. vs. Sham(-) in the same group. Nrf2^+/+∷Atg5^-/-; cardiomyocyte-restricted Atg5 knockout mice. Nrf2^-/-∷Atg5^-/-; double Nrf2 and cardiomyocyte-restricted Atg5 knockout mice. C, Schematic depiction of the results and proposed hypothesis.