| BackgroundHeart failure is the consequence of sustained, abnormal neurohormonal and mechanical stress and remains a leading cause of death worldwide. Pathological stress, such as hypertension, results in cardiac hypertrophy, myocardial apoptosis andfibrosis, altered microvascular structure, and chamber dilation, culminating in cardiac dysfunction and heart failure. However, the underlying mechanisms are far from clear.Notably, a causative role of oxidative stress in the pathogenesis of cardiovascular disease has been established. However, the antioxidant approaches of non-selective reactive oxygen species (ROS) scavenging for the treatment of cardiovascular disease are ineffective or even harmful. Accordingly, an effective therapy may require more specific targeting of either the source of oxidative stress or the endogenous antioxidant defense system. In this context, we have demonstrated that Nrf2, a master transcription factor in controlling the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes, is a negative regulator of cardiac pathological remodeling and dysfunction in diverse pathological settings. While it has been documented that Nrf2plays a mediator role in hydrogen sulfide-mediated cardioprotection, we and others have demonstrated that Nrf2might be a drug target for the treatment of cardiomyocyte injury and cardiac dysfunction. Despite the prominent contribution of Nrf2in cardiac protection, a direct link between cardiac specific activation of Nrf2 and cardiac protection in vivo remains to be established.Recently, emerging evidence has indicated that reductive stress due to an increase in reduced glutathione may causally contribute to cardiomyopathy induced by protein aggregation. In the setting of protein aggregation cardiomyopathy, there is a concern that the chronic activation of Nrf2secondary to oxidative stress is a contributing mechanism for the reductive stress-mediated heart failure. Although the’dark’side of Nrf2has not yet been demonstrated, Nrf2appears to be involved in the regulation of protein aggregation in autophagic substrate selection as well as the macroautophagy (commonly known asautophagy) per se. Autophagy is an evolutionarily conserved process that mediates the lysosome-dependent turnover of macromolecules and entire organelles. Importantly, autophagy and the ubiquitin proteasome system (UPS) are the major routes for the complete degradation/clearance of abnormal protein products in cells.UPS is usually effective in clearing soluble misfolded or damaged proteins via ubiquitination of the target proteins whereas autophagy is generally efficient in clearing less soluble or insoluble ubiquitinated protein aggregates. Upon a functional impairment of UPS, the ubiquitinated soluble abnormal proteins accumulate and aggregate into insoluble and toxic protein aggregates, and then autophagy is activated to be a major clearance route of the ubiquitinated proteins by default. Moreover, myocardial protein aggregation is often associated with cardiac proteasome insufficiency in various cardiomyopathies and heart failure while the activation of autophagy may result in either protective or detrimental consequences in the heart. These results raise an intriguing question whether Nrf2plays a role in the control of protein quality via regulating autophagy in the heart thereby contributing to cardiac remodeling and heart failure.Thus, we sought to investigate the impact of cardiac specific activation of Nrf2on cardiac maladaptive remodeling and dysfunction and explore the possibility of a novel role for Nrf2in regulating autophagy-mediated clearance of protein aggregates and preventing cardiac dysfunction. The results herein indicate that constitutive activation of Nrf2facilitates autophagic clearance of protein aggregates in the heart thereby protecting against cardiac dysfunction. In addition, Nrf2primarily suppresses necrosis rather than apoptosis in cardiomyocytes in a setting of toxic unbiquitinated protein overload via facilitating autophagic clearance of the toxic protein aggregates. Objectives 1. To investigate the impact of cardiac specific activation of Nrf2on cardiac maladaptive remodeling and dysfunction2. To explore the possibility of a novel role for Nrf2in regulating autophagymediated clearance of protein aggregates and preventing cardiac dysfunction.Materials and methods1. Generate transgenic mice with cardiomyocyte-specific overexpression of Nrf2(Nrf2ctg) Transgenic mice with cardiomyocyte-specific overexpression of Nrf2(Nrf2ctg) were generated in a FVB/NJ background using the transgene cassette containing a murine5.5kb alpha myosin heavy chain (α-MHC) promoter (a gift from Dr. Jeffrey Robbins).2. Transverse aortic arch constrictionMale C57B16/J mice at8weeks of age (BM30±2g) were subjected to sham or TAC operations. Mice were anesthetized by intraperitoneal injection of ketamine (80mg/kg) and xylazine (5mg/kg). The use of a horizontal incision at the level of the suprasternal notch allows direct visualization of the transverse aorta without entering the pleural space and thus obviates the need for mechanical ventilation. The transverse aorta was constricted between the right innominate and left carotid arteries to the diameter of a27-gauge needle using a7-0silk suture. Sham operations on sex-and age-matched mice were identical with the exception of omitting the actual aortic banding and served as controls for all experimental groups.3. Isolation of adult murine cardiac myocytesThe mouse is injected with heparin to prevent coagulation of blood in the coronary arteries. The mouse is then anesthetized, the chest opened and the heart rapidly removed and cannulated.The heartis perfused with a collagenase solution to digest the extracellular matrix. The heart is removed from the cannula and gently teased apart. A buffer containing serum is added at this point to stop the enzyme digestion and prevent overdigestion. The myocytes are incubated in buffer with increasing concentrations of calcium, finally achieving a concentration of1.2mM Ca2+as in our culture medium. The myocytes are plated on laminin-coated dishes and allowed to attach. After attachment, the nonattached cells are washed away. 4. Cell death assayCell death was measured using a cytotoxicity detection kit (630117, Clontech). Cells were treated with various stimuli as indicated for16h unless specified in figure legends. Cell viability was calculated as follows: cell viability=(LDH activity of cell lysate in Exp.)/(LDH activity of cell lysate in Con.)×100%. Alternatively, cell death rate=(LDH activity of supernatant in Exp)/(LDH activities of supernatant and cell lysate in Exp). Exp. and Con indicates experimental and control groups, respectively.5. Statistical analysisResults1. Generation of Nrf2cardiomyocyte-specific transgenic (Nrf2ctg) mice2. Cardiomyocyte-specific overexpression of Nrf2suppresses cardiac maladaptive remodeling and dysfunction in response to chronic pressure overload3. Nrf2enhances autophagosome formation and autophagicflux4. Nrf2does not inhibit apoptosis but suppresses the proteotoxic necroptosis in cardiomyocytes via facilitating the clearance of ubiquitinated toxic proteins.Conclusions1. Cardiomyocyte-specific activation of Nrf2suppressed myocardial oxidative stress as well as cardiac apoptosis,fibrosis, hypertrophy, and dysfunction in a setting of sustained pressure overload induced by transverse aortic arch constriction (TAC) in mice.2. The constitutive activation of Nrf2increased the steady level of autophagosomes while decreasing the ubiquitinated protein aggregates in the heart after TAC3. Nrf2gene gain-and loss-of-function approaches revealed that Nrf2enhances autophagosome formation and autophagicflux in cardiomyocytes4. While Nrf2minimally regulated apoptosis,it suppressed significantly the proteotoxic necrosis in cardiomyocytes5. Nrf2attenuated the proteocytotoxicity presumably via enhancing autophagy-mediated clearance of ubiquitinated protein aggregates in cardiomyocytes... |
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