Nrf2-Mediated Cardiac Maladaptive Remodeling And Dysfunction In A Setting Of Autophagy Insufficiency

Background:Transcription factor erythroid-2 related factor 2 (Nrf2) is members of the basic leucine zipper transcription factor Cap ’n’ Coall (CNC) family, Nrf2 binds to a cis-acing enhancer sequences, we call this the antioxidant response element (ARE), the core of this nucleotide sequence is’5-RTGACNNNGC-3’, combined with this enhancer sequences can activate the expression of more than 200 kinds genes in different areas, including antioxidant gene, phase Ⅱ detoxifying enzymes and transcription factors, transporters, scavenger receptors as well as molecular chaperone, etc. Therefore, Nrf2 has extensive and complex biological functions, from classic antioxidant defense, to cell cycle regulation and protein quality control.The protect function of Nrf2 on the heart has been confirmed in many animal models, including the transverse aortic arch constriction (TAC) model, in which the increased pressure overload induce decompensated cardiac remodeling and cardiac dysfunction. Analysis at the molecular level, transcription factor Nrf2 drives antioxidant and detoxifying protein expression, to fight oxidative stress-mediated heart injury and dysfunction. At the same time, the study found that Nrf2 can promote the function of autophagy to remove toxic ubiquitin protein aggregation, so as to protect the heart from the toxic protein in the cell. But, in contrast to previous conclusions, a recent study found that in a protein aggregation induced cardiomyopathy animal models in mice, an ageing human missense (R120G) mutant of alpha B-crystallin (hCryABR120G) transgenic mouse, knockout Nrf2 gene can resist the reduction pressure of the heart, reduce the aggregation of ubiquitin protein in the heart, attenuate cardiac pathological hypertrophy, protect heart from heart failure. Therefore, the sustainability of Nrf2 activation maybe cause reductive stress and lead to hCryABR120G-induced cardiomyopathy. However, there are studies have found that the myocardial specificity knockout Nrf2 gene resistant to pressure-load induced decompensated cardiac remodeling and dysfunction, this conflict with the theory of Nrf2 mediated reduction pressure. On the other hand, previous studies have confirmed that, under the conditions of autophagy dysfunction, the activation of Nrf2 leads to liver damage. In addition, autophagy dysfunction in mice CryABR120G (mCryABR120G) gene plays an important role in the induction of cardiomyopathy. Given that hCryABR120G and mCryABR120G genes have almost the same nucleotide sequence, and aging itself is associated with autophagy insufficiency or damage, it is likely that in the aged hCryABR120G mouse heart Nrf2 mediated the negative role probably associated with autophagy insufficiency or impairment. To sum up, in different pathological conditions, the role of Nrf2 on the heart may be protective or detrimental. However, the exact mechanism for the dual effects of Nrf2 is not yet clear.In this study, we found that the pathophysiological consequence of Nrf2 activation in heart closely related to the integrity of the function of autophagy. When the autophagy function is completed the activation of Nrf2 play an important role on the adaptive response of the heart, however, when autophagy is impaired, the activation of Nrf2 becomes a mediator of cardiac maladaptive remodeling and dysfunction. In our study, we found that autophagy impairment probably blocks the nuclear Fyn-operated Nrf2 export, which leads to the Nrf2 accumulation in the nucleus, starting the original expression of angiotensinogen and deteriorating cardiac maladaptation, aggravating heart failure.Aims:1、To investigate the function of Nrf2 in the progress of cardiac maladaptive remodeling and dysfunction in response to pressure overload;2、To verify the relationship between the pathophysiological consequence of Nrf2 activation and the functional integrity of autophagy in the heart;3、To figure out the precise mechanism for Nrf2 playing different effects on the heart under different autophagy functional states.Part1:1、Nrf2 knockout and wild type mouse were subjected to transverse aortic arch constriction operation to construct heart failure model. Test the myocardial necrosis by EBD during the initial stage of cardiac adaptation. Detect the survival rate after the operation. Assess the level of cardiac maladaptive remodeling and dysfunction by echocardiography, qPCR, WGA, Masson and immunohistochemistry. Analyse the function of Nrf2 at different pathophysiological stage of heart failure;2、Examine autophagy functional states in the heart after TAC overtime by autophagy flux assay;3、We perform TAC operation on cardiomyocyte-restricted Atg5 knockout (Atg5-/-) mice to determine the impact of myocardial specific autophagy impairment on Nrf2 expression and activity in the heart after TAC.Part 2:1、Detect the expression of a heart failure related gene Agt in the autophagy insufficient heart by qPCR and western blot;2、We determine the effect of autophagy insufficient on TAC-induced myocardial activation of different cell signaling pathways, compare to the effect of Nrf2 knockout, and verify the correlation between two of them;3、We perform TAC operation on Nrf2/Atg5 double knockout mice to more specifically investigate the relationship between pathophysiological consequence of Nrf2 activation and the functional integrity of autophagy in the pressure overload heart by western blot and immunohistochemistry.Results:Part1:1、Knockout of Nrf2 impairs acute cardiac adaptation, however, ameliorates the progression of cardiac maladaptive remodeling leading to heart failure after pressure overload;2、Pathophysiological consequences of Nrf2 activation are linked to the functional integrity of autophagy in the heart after pressure overload;3、Nrf2 expression and activity are enhanced in the failing heart with autophagy impairment after pressure overload.Part 2:1、Nrf2 drives Agt expression in the pressure overloaded heart with functional insufficiency of autophagy;2、Autophagy impairment suppresses TAC-induced activation of Jak2/Fyn pathway which operates Nrf2 nuclear export for degradation in the pressure overloaded heart.Conclusions1.Nrf2 activation is cardioprotective when myocardial autophagy function is sufficient; however, it exacerbates cardiac maladaptive remodeling and dysfunction when myocardial autophagy function is insufficient;2. Nrf2 is capable of inhibiting myocardial necrosis in the heart with a sufficient function of autophagy, whereas it could enhance angiotensin Ⅱ signaling in the heart with an insufficient function of autophagy;3. Autophagy impairment results in Nrf2 nuclear accumulation and the subsequent activation of Nrf2-driven transcription of Agt, most likely due to the inactivation of Jak2/Fyn signaling for Nrf2 export and degradation.