Pathogenesis of Heart and Liver Diseases in Acquired and Genetic Iron-overload Disorders ...

265 Pages·2016·22.56 MB·English

Pathogenesis of Heart and Liver Diseases in Acquired and
Genetic Iron-overload Disorders
Resveratrol as potential therapy

by

Subhash Kumar Das

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Experimental Medicine

Department of Medicine
University of Alberta

© Subhash Kumar Das, 2016
Abstract
Abnormal iron metabolism leads to cardiac and hepatic iron-overload disorders in an
epidemic proportion. Irregular iron absorption results in iron deposition in different
organs of the body including heart and liver. Iron-overload heart and liver diseases are
commonly observed in patients with genetic hemochromatosis and secondary iron-
overload, which are a common cause of end organ failure and mortality worldwide
basis. An excess amount of iron associated with iron induced oxidative stress that leads
to iron-overload cardiomyopathy and liver dysfunction respectively. We developed
murine models of iron-overload with cardiomyopathy and liver disease to understand
new insights into the pathogenesis and novel therapeutic effects of resveratrol.
Cardiac and hepatic iron-overload pathogenesis showed dose-dependent iron-
overload, oxidative stress, lipid peroxidation and fibrosis; however reduction in
SERCA2a protein with defective calcium cycling without any inflammatory response is
noticed in iron-overload hearts, which are key factors for the development of diastolic
and systolic dysfunction. In contrast, hepatomegaly, hepatic inflammation, hepatic cell
death and hepatic steatosis are well noticed in hepatic iron-overload. Resveratrol
therapy improved cardiac function by decreasing iron-induced oxidative stress,
myocardial lipid peroxidation, cardiac fibrosis, and hypertrophy; normalize calcium
cycling defects by improving SERCA2a. In contrast resveratrol, therapy also improved
liver function by reducing hepatic oxidative stress, lipid peroxidation, hepatic
inflammation, hepatic cell death and hepatic steatosis. Consistent with the role of iron-
induced myocardial oxidative stress in the development of heart disease, iron-overload
female mice showed normal cardiac function with better survival rate without iron
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induced oxidative stress, highlighting the antioxidant properties of the female sex
hormone estrogen. These results indicate that iron-induced oxidative stress is the key
driver in the development of heart and liver diseases. There is no effective therapy
available to prevent the global clinical burden of iron-overload, however, the mainstay
therapies for iron-overload are phlebotomy and chelation therapy and these therapies
have several limitations. Therefore, given the high degree of iron induced oxidative
damage, we proposed that the pleiotropic effects of resveratrol represent a potential
therapy to treat heart and liver diseases in iron-overloaded conditions.

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Preface:
Non-failing human hearts were collected as part of the Human Organ Procurement
and Exchange (HOPE) program for research at the Mazankowski Alberta Heart
Institute. Human cardiomyocytes were used to improve the translational impact of our
research. All experiments were performed in accordance with the institutional
guidelines and were approved by Institutional Ethics Committee. Informed consent
was obtained from all study subjects. The research projects in the current thesis are
mainly performed in Dr. Gavin Oudit lab.

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This thesis is dedicated
To my beloved parents for their endless love, support and encouragement, who
always reminded me as follow…….
“Karmanye vadhikaraste Ma Phaleshu Kadachana,”
“Ma Karmaphalaheturbhurma Te Sangostvakarmani”
“Keep on performing your duties without expecting any reward in return. You get
something or you don’t get anything, you should not worry about it. You should lead a
selfless life”
(Verses from Bhagwat Gita)

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ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr. Gavin Y. Oudit for the support that he has
provided throughout my Ph.D. graduate studies. Without his intellectual guidance, the
content of this thesis would not have been possible to accomplish.

I would also like to thank the committee members, Dr. Jason R.B. Dyck and Dr.
Michael Sean McMurtry, for their guidance, advice, invaluable suggestions and
constant support throughout the course of my Ph.D. program.

I sincerely would like to thank all of my collaborators, especially Dr. Jason R.B. Dyck
and Dr. Zamaneh Kassiri for allowing me to work with various ongoing projects,
independent from this thesis. I would also like to sincerely thank my other
collaborators Dr. Nancy C. Andrews and Dr. Roger J. Hajjar, for kindly providing us
the hemojuvelin knock-out mice and adenoviral SERCA2a, respectively.

My sincere gratitude is given to all my present and past lab members, for their
continued peer support and friendship, a wonderful experience in life during my time
in the lab.

This research would not have been possible without the financial assistance of
Alberta Innovative Health solutions (AIHS) studentship and Alberta HEART
STEADI-HF studentship as well as Heart and Stroke grant from Dr. Oudit.
Last but not least, I would like to thank my wife Sushreeta Sai for her continued and
unrelenting support. I would also like to thank my mother Susheela Das and my
father Bhaskar Das for letting me pursue my ambition.

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CONTENTS

Chapter 1: Literature Review……………………………………………………...1-46
1.1 General Introduction
1.2 Iron metabolism
1.2.1 Cellular iron metabolism
1.2.2 Systemic iron metabolism
1.3 Pathological conditions associated with iron-overload
1.3.1 Primary hemochromatosis
1.3.2 Secondary Iron-overload
1.4 Iron-overload cardiomyopathy
1.4.1 Clinical description of iron-overload cardiomyopathy
1.4.2 Pathophysiology of iron-overload cardiomyopathy
1.4.3 Role of gender in the pathogenesis of iron-overload cardiomyopathy

1.5 Hepatic Iron-overload disease
1.6 Iron induced oxidative stress
1.7 Current therapies
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1.8 Proposed therapies
1.8.1 Antioxidant therapy
1.8.2 Resveratrol in clinical trials
1.8.3 Amlodipine: L-Type calcium channel blocker
1.9 Hypothesis

Chapter 2: Overview of Research Protocol……………………………………...47-56

2.1 Iron-overload animal models
2.2 Experimental Animal Protocols
2.3 Sample size, Power of study and choice of statistical method

Chapter 3: Iron-overload injury and cardiomyopathy in acquired and genetic models
is attenuated by resveratrol therapy………………………………………………57-106

3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.4 Results
3.5 Discussion
3.6 Conclusion
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Chapter 4: Iron-overload cardiomyopathy in aged Juvenile hemochromatosis
genetic murine model and prevented by resveratrol…………………………107-131
4.1 Abstract
4.2 Introduction
4.3 Materials and Methods
4.4 Results
4.5 Discussion
4.6 Conclusion

Chapter 5: Resveratrol mediates therapeutic hepatic effects in acquired and
genetic murine models of iron-overload………………………………………132-157

5.1 Abstract
5.2 Introduction
5.3 Materials and Methods
5.4 Results
5.5 Discussion
5.6 Conclusion

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Chapter 6: Females are protected from iron-overload cardiomyopathy
independent of iron metabolism: key role of oxidative stress……………….158-189

6.1 Abstract
6.2 Introduction
6.3 Materials and Methods
6.4 Results
6.5 Discussion
6.6 Conclusion

Chapter 7: Discussion and future directions…………………………………190-203

7.1 Discussion
7.2 Future directions

References…………………………………………………………………….204-241

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