The Role of Troponin C in the Heart DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sean Carl Little Graduate Program in Integrated Biomedical Science Program The Ohio State University 2012 Dissertation Committee: Dr. Jonathan P. Davis, PhD (Advisor), Dr. Philip F. Binkley, MD Dr. Paul M.L. Janssen, PhD Dr. Mark T. Ziolo, PhD Copyright by Sean Carl Little 2012 Abstract Heart disease is a broad category composed of coronary artery disease, heart failure, arrhythmias and many other miscellaneous heart problems. A common characteristic for many of these diseases is the impairment of regulatory mechanisms believed to control the rate and extent of relaxation in the heart. An important component of myocardial relaxation is the inactivation of the myofilament, for which the rate- limiting steps are not completely understood. Depending on how fast the myofilament inactivates and how long it remains inactivated can influence the relaxation of the heart. Thus, the faster the myofilament is able to inactivate the more time the heart muscle is able relax to allow the heart to fill with blood. If the rate of myofilament inactivation is decreased, the heart relaxes slower and the heart is not able to relax to the same extent as before, resulting in inadequate filling of blood. The work described within focused on studying the molecular mechanisms of myofilament inactivation to contribute to the overall knowledge of heart relaxation. The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca2+ dissociation from Troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. To study these inactivation events we used a fluorescent TnC to measure both the rate of Ca2+ dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive ii to both Ca2+ dissociation and cross-bridge detachment, which allowed for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca2+ dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At sub-physiological temperatures (< 20oC), the rate of Ca2+ dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross- bridge detachment rate-limits relaxation. However, Ca2+ dissociation from TnC was not as temperature sensitive as cross-bridge detachment. At a near physiological temperature (35oC) and [ADP], the rate of cross-bridge detachment may actually be faster than the rate of Ca2+ dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation and TnC may play a vital role in rate- limiting relaxation. One way to further study the role of TnC in muscle physiology is to alter the function of the protein to determine its affect on Ca2+ binding. Cardiac TnCs that had altered Ca2+ binding properties in the Tn and reconstituted thin filament systems, were incorporated into ventricular myofibrils. These specifically designed TnCs had increased or decreased Ca2+ sensitivities, as well as modified Ca2+ dissociation rates when measured from the ventricular myofibrils. In addition, Ca2+ sensitized fluorescent TnCs were able to report the rate of Ca2+ dissociation from the neighboring, unlabeled, endogenous TnC. The modified TnC exposed the cross-talk that occurs between neighboring Tn’s in the myofilament that had different Ca2+ sensitivities. In addition, a Ca2+ desensitized TnC increased the rate of cross-bridge detachment in the presence of ADP, providing evidence that TnC may be able to influence the rate of thick iii filament inactivation in the dynamic myofilament. Thus, the TnCs with altered Ca2+ binding provided evidence that the rate of Ca2+ dissociation can be slowed and brought to light interactions that may occur in the dynamic and highly interactive myofilament. The specifically designed TnCs allowed us to test whether a direct increase or decrease in the Ca2+ sensitivity of the myofilament leads to a diseased phenotype. Upon incorporation of engineered TnCs into an animal model, the Ca2+ desensitized TnC, D73N, was able to recapitulate the diseased cardiac phenotype of a dilated cardiomyopathy as measured by echocardiography. This was accomplished by intra- peritoneal injection of adeno-associated virus serotype 9 (AAV-9) containing the D73N TnC into neonatal mice 1-2 days after birth. On the other hand, AAV-9 containing the Ca2+ sensitized, L48Q TnC, was unable to recapitulate the restrictive or hypertrophic cardiomyopathy phenotypes commonly associated with an increased myofilament Ca2+ sensitivity. AAV-9 containing GFP was used to verify that the AAV-9 was able to target the heart, as well as skeletal muscle, due to the use of a cyctomegalovirus promoter. This was a proof of principle study designed to test the feasibility of using AAV-9 to transduce the cardiomyocytes with modified TnCs. The work described within examined the effects that the modified TnCs had on the in vivo contractile properties of the heart. In addition, the results showed that an alteration in the Ca2+ sensitivity of the myofilament does not always lead to a diseased heart. In fact these engineered TnCs may be used as a treatment strategy against various cardiac diseases. iv Dedication DEDICATED TO THE MEMORIES OF JANE MARKOVICH AND CAROL MUIR v Acknowledgments I would like to thank all of those that have helped me throughout the years with everything from collaborations, insightful discussions, keeping me mentally focused during stressing times, and the many opportunities and interactions that I have had. To begin I would like to thank my advisor, friend, and colleague, Dr. Jonathan Davis. Jon has taught me how to critically think and to question everything in regards to experimental design and scientific thinking. In addition to scientific discussions, there were also many heated games of racquetball played throughout the years in which Jon gave me lessons in how not to control ones temper. I would like to thank my dissertation advisory committee: Drs. Philip Binkly, Paul Janssen, and Mark Ziolo for the invaluable insight into experimental design, collaboration, interpretation of the data, and guidance during my journey. I would like to thank Dr. Brandon Biesiadecki for his help in teaching me the ins and outs of gel chromatography in addition to the numerous spirited conversations. I would like to thank fellow graduate student Steve Roof for his tireless effort in the collection of the isolated cardiomyocyte data. I would like to thank Dr Jianchao Zhang, also known as Remington, for the production of the different viral constructs that were vital to the completion of my project. Lastly, I would like to thank my family who has always supported me in whatever endeavor it was that I chose and I am truly blessed to have them in my life. My loving wife, Jenna, has always supported me and always finds ways to pick me up when I stumbled. Without everyone’s help I would not be the scientist, and more importantly, the man I have become. vi Vita May 29th, 1984 ...............................................Born, Trumbull Hospital, Warren, Ohio 2002-2006 ......................................................B.A. Chemistry, Hiram College 2007-2012 ......................................................Graduate Research Associate, Department of Physiology and Cell Biology, The Ohio State University 2009-2011 .....................................................Graduate Research Fellow, Department of Physiology and Cell Biology, The Ohio State University Publications 1. Little SC, Biesiadecki BJ, Kilic A, Higgins RS, Janssen PM, Davis JP, The rates of Ca2+ dissociation and cross-bridge detachment from ventricular myofibrils as reported by a fluorescent cardiac troponin C. J Biol Chem 2012 Jun 20. [Epub ahead of print] 2. Nixon BR, Thawornkaiwong A, Jin J, Brundage EA, Little SC, Davis JP, Solaro RJ, Biesiadecki BJ, AMP activated protein kinase phosphorylates cardiac troponin I at Ser-150 to increase myofilament calcium sensitivity and blunt PKA dependent function. J Biol Chem 2012 Apr 6. [Epub ahead of print] vii 3. Little SC, Tikunova SB, Norman C, Swartz DR, Davis JP, Measurement of calcium dissociation rates from troponin C in rigor skeletal myofibrils. Front Physiology 2011 Oct 11; 2:70 4. Tikunova SB, Liu B, Swindle N, Little SC, Gomes AV, Swartz DR, Davis JP, Effect of calcium-sensitizing mutations on calcium binding and exchange with troponin C in increasingly complex biochemical systems. Biochemistry 2010 Mar 9; 49(9):1975-84 Fields of Study Major Field: Integrated Biomedical Science Program 1. Emphasis: Cardiac Muscle Physiology 2. Emphasis: Translational Research viii Table of Contents The Role of Troponin C in the Heart .................................................................................. 1 DISSERTATION ................................................................................................................ 1 Abstract ............................................................................................................................... ii Dedication ..........................................................................................................................