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Sarcolipin a Novel Regulator of the Cardiac Sarcoplasmic

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Sarcolipin a Novel Regulator of the Cardiac Sarcoplasmic SARCOLIPIN A NOVEL REGULATOR OF THE CARDIAC SARCOPLASMIC RETICULUM CALCIUM ATPase Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Poornima Bhupathy, M.S. ***** The Ohio State University 2008 Dissertation Committee: Approved by Dr. Muthu Periasamy, Advisor Dr. Cynthia A. Carnes ________________________________ Dr. Paul ML. Janssen Advisor Graduate Program in Molecular, Dr. Jill Rafael-Fortney Cellular and Developmental Biology ABSTRACT Cardiac contraction and relaxation are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+-uptake activity not only determines the rate of Ca2+ removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. It has been well documented over the years that altered expression and activity of SERCA2a can lead to systolic and diastolic dysfunction. The activity of SERCA2a is closely regulated by two structurally similar proteins, phospholamban (PLB) and sarcolipin (SLN). Although, the relevance of PLB has been extensively studied over the years, the role SLN in cardiac physiology is an emerging field of study. Therefore, the purpose of this study was to investigate the physiological significance of Sarcolipin, a novel 31 amino acid protein in the heart. Our hypothesis was that SLN interacts directly with cardiac SERCA2a and inhibits its function and this inhibitory effect can be modulated by SLN phosphorylation-dephosphorylation. One of the goals of this study was to perform detailed analyses of SLN protein expression during muscle development and in the diseased myocardium. Our findings indicate that (i) SLN co-localizes with both SERCA2a and PLB in the cardiac SR membrane. Further, using co-immunoprecipitation we showed that SLN interacts with both SERCA2a and PLB in cardiac myocytes. (ii) in small mammals, SLN expression is predominant in the atria but low in the ventricle and in skeletal muscle tissues, whereas in large mammals, SLN is quite abundant in skeletal muscle tissues than the atria (iii) SLN and SERCA2a are co-expressed in all striated muscle tissues studied except ventricle and co-ordinately regulated during muscle development and (iv) SLN protein levels are ~3 fold upregulated in ii the atria of heart failure dogs and ~30% decreased in the atria of hearts prone to myocardial ischemia. In addition, we found that in the atria, loss of phospholamban is compensated by the upregulation of SLN and overexpression of SLN leads to a decrease in PLB levels. These results taken together suggest that SLN is an important regulator of SERCA2a and its expression is modulated both during muscle development and cardiac pathology. The present study critically evaluated the relevance of SLN in cardiac physiology by generating a transgenic (TG) mouse model in which the SLN to SERCA2a ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium- handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. Protein and mRNA data indicate that sarcolipin is predominantly expressed in the atria. The role of SLN in atrial physiology however is unknown. Therefore in this study, we investigated the physiological significance of sarcolipin in the atria by generating a mouse model deficient for sarcolipin. The sarcolipin null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+, iii and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin null mice showed a blunted response to isoproterenol stimulation, implicating SLN as a mediator of β-adrenergic responses in atria. Our study documented for the first time that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulator of SERCA pump in the atria and ventricle. Data thus far indicate that SLN is a reversible inhibitor of SERCA2a. Therefore to dissect the mechanism of regulation of the inhibitory effect of SLN on SERCA2a we made use of adenoviral gene transfer and site directed mutagenesis. This study provides evidence for the first time that the highly conserved threonine 5 residue plays an important role in the regulation of SLN action on SERCA pump. We also provide evidence that threonine 5 can be phosphorylated by CaMKII in vitro. Future studies should be directed towards understanding the role of SLN as a target for CaMKII in mediating β -adrenergic response in the atria. In conclusion our data has identified SLN as a novel regulator of SERCA2a and as a mediator of β-adrenergic response in the atria. iv DEDICATION Dedicated to my parents v ACKNOWLEDGMENTS First and foremost I would like to express my sincere gratitude to my advisor Dr. Muthu Periasamy. I thank him for including me as part of his outstanding research group. His dedication for science and expertise in the field of cardiovascular physiology has instilled a deep sense of passion for science in me as well. Dr. Periasamy’s long discussions with me over the years helped me a great deal in coming up with novel ideas and testing them. He taught me how to become an independent thinker and scientist, and always encouraged me to write grants and papers which helped me develop my scientific writing skills. He also helped me improve my presentation and discussion skills. Without his constant guidance and support none of this thesis work would have been possible. I will be eternally grateful to Dr.Gopal J Babu for his invaluable help and support during my entire PhD journey. He has been both a friend and a guide. I particularly thank him for believing enough in my abilities to entrust me with this exciting and at times difficult Sarcolipin project. I thank Babu for teaching me all the techniques over the years and being patient with me when I made mistakes. He has been a great source of inspiration to me for his dedication and hardwork and all that he has achieved. I would also like to thank all the other past and present members of the Periasamy Lab. vi I thank Dr. David Bisaro for his constant help and counsel. My special thanks to Jan Zinaich for always lending a listening ear and for her sincere words of advice and encouragement over these years. I want to express my thanks to the members of my thesis committee Drs. Cynthia Carnes, Paul Janssen and Jill Rafael-Fortney for their invaluable advice and suggestions. I thank Dr. Christian Dumitrescu, Debra Wheeler and Bob Kelley in helping me set up the cardiac myocyte culture system. I would like to thank Dr. Loren Wold for his help with the Ion Optix set up. I also like to thank the faculty in the Department of Physiology and Cell Biology, especially Dr. Paul Janssen, Dr. Mark Ziolo, Dr. Cynthia Carnes, Dr. George Billman, Dr. Peter Reiser for their collaborations in this project. Finally, and most importantly my heart-felt thanks to each and everyone in my family and my friends, who make everything worthwhile. Special thanks to my parents to whom I dedicate my thesis. My mom for making me the person I am and my dad for his unconditional love and support and for believing in me and encouraging me to pursue my dreams. It is believed that rodents have saved more lives than 911! I therefore, cannot end this acknowledgment without thanking the rats and mice which have been an integral part of my research. This research was supported by grants from the National Institute of Health to Dr. Muthu Periasamy (NIH grant RO1-HL64140) and an American Heart Association predoctoral fellowship to Poornima Bhupathy (AHA Award Number: 0415170B). vii VITA April 3 1977 Born – Secunderabad, India 1995-1998 Bachelor of Science- Genetics, Zoology & Chemistry, Osmania University College for Women, Hyderabad, India. 1998-2000 Master of Science- Animal Sciences. Hyderbad Central University, Hyderabad, India 2002 Graduate Teaching Assistant. The Ohio State University, Columbus, OH 2001 – Present Graduate Student, The Ohio State University, Columbus, OH FELLOWSHIP July 2004- June 2006 American Heart Association Pre-doctoral Fellowship viii PUBLICATIONS 1) Muthu Periasamy, Poornima Bhupathy, Gopal J Babu Regulation of sarcoplasmic reticulum Ca2+ ATPase pump expression and its relevance to cardiac muscle physiology and pathology. Cardiovas .Res. 2008 Feb 1; 77(2):265-73. 2) Gopal J Babu, Poornima Bhupathy, Valeriy Timofeyev, Natalia N Petrashevskaya, Peter J. Reiser, Nipavan Chiamvimonvat, and Muthu Periasamy. Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility PNAS. 2007 Nov,104 (45);17867– 72 3) M.A. Hassan Talukder Anuradha Kalyanasundaram, Xue Zhao, Li Zuo, Poornima Bhupathy, Gopal J.
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