A Critical Role of Nrf2 In Protecting Cardiomyocytes Against Oxidative Stress and Ischemic Injury

Persistent Link:
http://hdl.handle.net/10150/333336
Title:
A Critical Role of Nrf2 In Protecting Cardiomyocytes Against Oxidative Stress and Ischemic Injury
Author:
Strom, Joshua
Issue Date:
2014
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Release 13-Aug-2015
Abstract:
Coronary heart disease (CHD) remains the single leading cause of natural death worldwide. Despite significant advances in the diagnosis and treatment, CHD accounts for 1 out of every 6 deaths in the United States. Myocardial infarct (MI) as a result of CHD causes irreversible damage to the heart through the loss of viable myocardial tissue. Patients surviving the initial MI are at risk of developing heart failure due to lost contractile function and adverse cardiac remodeling. Improvement in the survival rates for MI have led to an increase in the incidence of heart failure, affecting approximately 5 million people in the United States. Although treatment of heart failure has improved, the mortality rates of heart failure remain high with 1 in 5 dying within the first year of diagnosis and 50% dying within 5 years. The cost of caring for heart failure patients ranks number one in Medicare. Oxidative stress plays an important role in the etiology and pathophysiology of CHD and heart failure. The transcription factor Nrf2 is a master regulator of cellular antioxidant defense mechanisms, controlling the expression of numerous antioxidant and detoxification genes through the Antioxidant Response Element (ARE) in the promoter regions. The cytoprotective effects of Nrf2 have been demonstrated in a variety of organs and disease states; however, the role of Nrf2 in the heart and heart disease has not been defined. The work presented here defines roles of Nrf2 in limiting cardiac injury and the progression to heart failure (Chapter II), protecting cardiac myocytes from oxidative stress through the preservation of mitochondria (Chapter III), and mediating the infarct reducing effects of statins, one of the most prescribed pharmacological agent (Chapter IV). In order to investigate a role of Nrf2 in the pathology of ischemic injury in the heart, a mouse model of ischemia and myocardial infarct by occlusion of the left anterior descending coronary artery was used. Nrf2 knockout mice subjected to ischemia/reperfusion injury experienced a larger infarct size than wild-type mice. Furthermore, mice lacking Nrf2 experienced a higher mortality rate and an accelerated progression to heart failure, indicated by severely compromised contractile function and reduced cardiac output, within 10 days following an MI. Morphological examination revealed maladaptive remodeling, including myocyte hypertrophy, heart enlargement, and dilated left ventricle, in Nrf2 KO mice that was absent in WT mice. Analysis of cardiac function by echocardiogram revealed increased left ventricular mass, increased systolic volume, decreased fraction shortening, reduced ejection fraction, and decreased cardiac output in Nrf2 KO mice. Nrf2 KO mice also demonstrated expression of biomarkers of heart failure, such as expression of fetal gene program, with elevated levels of β-MHC, ANF, and BNP mRNA in the myocardium. Interestingly, a lack of immune cell infiltrate and myofibroblasts as well as a deficiency in collagen deposition were observed in the infarcted region of hearts from Nrf2 KO mice. These data indicate that Nrf2 plays an important role in protecting the myocardium from ischemic injury and the progression to heart failure. Lack of Nrf2 response results in deficiency of wound healing and instead initiation of maladaptive remodeling, leading to heart failure. Mitochondria are key sources of reactive oxygen species (ROS) generation, as well as important targets for ROS-induced cell injury. Cardiac myocytes have the highest content of mitochondria among all cell types and can be particularly susceptible to mitochondrial dysfunction due to the high metabolic demand associated with the contractile function of the heart. With cardiomyocytes (CMCs) isolated from neonatal rats and kept under tissue culture conditions, mitochondria exist in elaborated networks. Such networks were replaced by predominately individual punctate mitochondria 24 hours after exposure to a sublethal dose of H₂O₂. Mitochondrial morphology was altered with membrane swelling and disorganization of inner cristae with areas of condensation. Disrupted mitochondrial morphology was associated with a loss of membrane potential and decreased expression of mitochondrial proteins involved in the electron transport chain, such as cytochrome b and cytochrome c. Nrf2 overexpression prevented H₂O₂ from inducing morphological changes in mitochondria and the reduction of cytochrome b and cytochrome c expresssion. Although Nrf2 is known as a transcription factor regulating antioxidant and detoxification genes, Nrf2 overexpression did not significantly reduce the level of protein oxidation as measured by carbonyl formation. Instead, we found that Nrf2 localizes to the outer mitochondrial membrane, suggesting a direct role of Nrf2 in mitochondrial protection. As further evidence of a direct role in mitochondrial protection, a cell-free system of mitochondria isolated from the myocardium of Nrf2 knockout mice were more sensitive to permeability transition, an indicator of mitochondrial dysfunction. Combined, these data suggest that Nrf2 protects mitochondria from oxidant injury likely through direct interaction with mitochondria. In the clinic, statins are now commonly administered for patients experiencing MI or CHD. Statins have become mainstays in the treatment of hypercholesterolemia and atherosclerosis as inhibitors of the rate limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. In addition, statins have been shown to elicit pleiotropic effects, including plaque stabilization, maintenance of endothelial function, anti-inflammatory actions, and antioxidant capabilities, independent of effects on cholesterol synthesis. Recently, these pleiotropic effects have been implicated in providing acute protection against ischemia and reperfusion injury, which has led to the use of high dose statins clinically before revascularization of an ischemic event. I have found that administration of atorvastatin in mice induced Nrf2 protein levels in the heart, brain, lung, and liver. While atorvastatin reduced infarct size following an MI in wild-type mice, this protective effect was lost in mice lacking Nrf2. Failure of atorvastatin to protect against MI in Nrf2 knockout mice indicates that Nrf2 plays a critical role in mediating the protective effects of acute statin treatment. Nrf2 induction by statins is a novel discovery. In order to understand the mechanism of such statin effect, I used an in vitro cell system, in which a variety of statins, atorvastatin, simvastatin, lovastatin, and pravastatin, were found to elevate Nrf2 protein levels. Elevation of Nrf2 by statins was independent of increased protein stability or transcriptional regulation. Instead, statins increased Nrf2 mRNA association with ribosomal complexes and induced Nrf2 protein through a translational mechanism. Recruitment of Nrf2 mRNA to ribosomes and induction of Nrf2 protein was dependent on activation of PI3 kinase. These studies provide evidence that Nrf2 plays a critical role in protecting cardiac myocytes and the heart from oxidative stress and MI. In the absence of Nrf2, mice experienced worse cardiac injury following MI and quickly advanced to heart failure. Mechanistically, this work has identified a novel role of Nrf2 in preserving mitochondrial morphology and integrity during oxidative stress through a direct interaction with the outer mitochondrial membrane. Finally, a newly defined role of Nrf2 induction by statins in mediating protection against MI by acute statin therapy indicates that modulation of Nrf2 may represent a viable pharmacological target for cardiac protection in humans.
Type:
text; Electronic Dissertation
Keywords:
myocardial infarction; Nrf2; oxidative stress; statin; Medical Pharmacology; mitochondria
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Medical Pharmacology
Degree Grantor:
University of Arizona
Advisor:
Chen, Quin M.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleA Critical Role of Nrf2 In Protecting Cardiomyocytes Against Oxidative Stress and Ischemic Injuryen_US
dc.creatorStrom, Joshuaen_US
dc.contributor.authorStrom, Joshuaen_US
dc.date.issued2014-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.releaseRelease 13-Aug-2015en_US
dc.description.abstractCoronary heart disease (CHD) remains the single leading cause of natural death worldwide. Despite significant advances in the diagnosis and treatment, CHD accounts for 1 out of every 6 deaths in the United States. Myocardial infarct (MI) as a result of CHD causes irreversible damage to the heart through the loss of viable myocardial tissue. Patients surviving the initial MI are at risk of developing heart failure due to lost contractile function and adverse cardiac remodeling. Improvement in the survival rates for MI have led to an increase in the incidence of heart failure, affecting approximately 5 million people in the United States. Although treatment of heart failure has improved, the mortality rates of heart failure remain high with 1 in 5 dying within the first year of diagnosis and 50% dying within 5 years. The cost of caring for heart failure patients ranks number one in Medicare. Oxidative stress plays an important role in the etiology and pathophysiology of CHD and heart failure. The transcription factor Nrf2 is a master regulator of cellular antioxidant defense mechanisms, controlling the expression of numerous antioxidant and detoxification genes through the Antioxidant Response Element (ARE) in the promoter regions. The cytoprotective effects of Nrf2 have been demonstrated in a variety of organs and disease states; however, the role of Nrf2 in the heart and heart disease has not been defined. The work presented here defines roles of Nrf2 in limiting cardiac injury and the progression to heart failure (Chapter II), protecting cardiac myocytes from oxidative stress through the preservation of mitochondria (Chapter III), and mediating the infarct reducing effects of statins, one of the most prescribed pharmacological agent (Chapter IV). In order to investigate a role of Nrf2 in the pathology of ischemic injury in the heart, a mouse model of ischemia and myocardial infarct by occlusion of the left anterior descending coronary artery was used. Nrf2 knockout mice subjected to ischemia/reperfusion injury experienced a larger infarct size than wild-type mice. Furthermore, mice lacking Nrf2 experienced a higher mortality rate and an accelerated progression to heart failure, indicated by severely compromised contractile function and reduced cardiac output, within 10 days following an MI. Morphological examination revealed maladaptive remodeling, including myocyte hypertrophy, heart enlargement, and dilated left ventricle, in Nrf2 KO mice that was absent in WT mice. Analysis of cardiac function by echocardiogram revealed increased left ventricular mass, increased systolic volume, decreased fraction shortening, reduced ejection fraction, and decreased cardiac output in Nrf2 KO mice. Nrf2 KO mice also demonstrated expression of biomarkers of heart failure, such as expression of fetal gene program, with elevated levels of β-MHC, ANF, and BNP mRNA in the myocardium. Interestingly, a lack of immune cell infiltrate and myofibroblasts as well as a deficiency in collagen deposition were observed in the infarcted region of hearts from Nrf2 KO mice. These data indicate that Nrf2 plays an important role in protecting the myocardium from ischemic injury and the progression to heart failure. Lack of Nrf2 response results in deficiency of wound healing and instead initiation of maladaptive remodeling, leading to heart failure. Mitochondria are key sources of reactive oxygen species (ROS) generation, as well as important targets for ROS-induced cell injury. Cardiac myocytes have the highest content of mitochondria among all cell types and can be particularly susceptible to mitochondrial dysfunction due to the high metabolic demand associated with the contractile function of the heart. With cardiomyocytes (CMCs) isolated from neonatal rats and kept under tissue culture conditions, mitochondria exist in elaborated networks. Such networks were replaced by predominately individual punctate mitochondria 24 hours after exposure to a sublethal dose of H₂O₂. Mitochondrial morphology was altered with membrane swelling and disorganization of inner cristae with areas of condensation. Disrupted mitochondrial morphology was associated with a loss of membrane potential and decreased expression of mitochondrial proteins involved in the electron transport chain, such as cytochrome b and cytochrome c. Nrf2 overexpression prevented H₂O₂ from inducing morphological changes in mitochondria and the reduction of cytochrome b and cytochrome c expresssion. Although Nrf2 is known as a transcription factor regulating antioxidant and detoxification genes, Nrf2 overexpression did not significantly reduce the level of protein oxidation as measured by carbonyl formation. Instead, we found that Nrf2 localizes to the outer mitochondrial membrane, suggesting a direct role of Nrf2 in mitochondrial protection. As further evidence of a direct role in mitochondrial protection, a cell-free system of mitochondria isolated from the myocardium of Nrf2 knockout mice were more sensitive to permeability transition, an indicator of mitochondrial dysfunction. Combined, these data suggest that Nrf2 protects mitochondria from oxidant injury likely through direct interaction with mitochondria. In the clinic, statins are now commonly administered for patients experiencing MI or CHD. Statins have become mainstays in the treatment of hypercholesterolemia and atherosclerosis as inhibitors of the rate limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. In addition, statins have been shown to elicit pleiotropic effects, including plaque stabilization, maintenance of endothelial function, anti-inflammatory actions, and antioxidant capabilities, independent of effects on cholesterol synthesis. Recently, these pleiotropic effects have been implicated in providing acute protection against ischemia and reperfusion injury, which has led to the use of high dose statins clinically before revascularization of an ischemic event. I have found that administration of atorvastatin in mice induced Nrf2 protein levels in the heart, brain, lung, and liver. While atorvastatin reduced infarct size following an MI in wild-type mice, this protective effect was lost in mice lacking Nrf2. Failure of atorvastatin to protect against MI in Nrf2 knockout mice indicates that Nrf2 plays a critical role in mediating the protective effects of acute statin treatment. Nrf2 induction by statins is a novel discovery. In order to understand the mechanism of such statin effect, I used an in vitro cell system, in which a variety of statins, atorvastatin, simvastatin, lovastatin, and pravastatin, were found to elevate Nrf2 protein levels. Elevation of Nrf2 by statins was independent of increased protein stability or transcriptional regulation. Instead, statins increased Nrf2 mRNA association with ribosomal complexes and induced Nrf2 protein through a translational mechanism. Recruitment of Nrf2 mRNA to ribosomes and induction of Nrf2 protein was dependent on activation of PI3 kinase. These studies provide evidence that Nrf2 plays a critical role in protecting cardiac myocytes and the heart from oxidative stress and MI. In the absence of Nrf2, mice experienced worse cardiac injury following MI and quickly advanced to heart failure. Mechanistically, this work has identified a novel role of Nrf2 in preserving mitochondrial morphology and integrity during oxidative stress through a direct interaction with the outer mitochondrial membrane. Finally, a newly defined role of Nrf2 induction by statins in mediating protection against MI by acute statin therapy indicates that modulation of Nrf2 may represent a viable pharmacological target for cardiac protection in humans.en_US
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectmyocardial infarctionen_US
dc.subjectNrf2en_US
dc.subjectoxidative stressen_US
dc.subjectstatinen_US
dc.subjectMedical Pharmacologyen_US
dc.subjectmitochondriaen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMedical Pharmacologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorChen, Quin M.en_US
dc.contributor.committeememberChen, Quin M.en_US
dc.contributor.committeememberLarson, Douglasen_US
dc.contributor.committeememberVanderah, Todden_US
dc.contributor.committeememberRonaldson, Patricken_US
dc.contributor.committeememberWondrak, Georgen_US
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