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1 Neuronal Nitric Oxide Synthase in Viral Heart Disease Sameelah Reed Biology, School of Arts and Sciences Clark Atlanta University, Atlanta, Georgia Medical Student Training Program (MSTP) SURF Summer Internship 2002 Dr. Kirk U. Knowlton Department of Medicine The University of California, San Diego, La Jolla, California Introduction: Cardiovascular disease is among leaders of mortality in America. Today, statistics show that one in every five Americans is affected by some form of cardiovascular disease and an estimated 2,500 Americans die from it daily. There are many causes of heart failure such as coronary artery disease, myocardial infarction and hypertension. Cardiomyopathy, a disease that damages heart muscles caused by infections, alcohol or drug abuse, is another disease that contributes to heart failure. Researchers are interested in investigating the pathway that leads to this disease. Dr. Knowlton’s laboratory is interested in the role of Coxsackievirus B3 (CVB3) in cardiomyopathy. CVB3 causes some of the acquired forms of cardiomyopathy; cardiomyopathy also has hereditary forms. CVB3 is part of the enterovirus genus and is a common cause of myocarditis, a disease characterized by inflammation and infection of the myocytes. Infection with CVB3 results in cardiomyopathy, which ultimately leads to heart failure, although the mechanism by which this occurs has yet to be elucidated. Previous work in the lab has shown that the CVB3 viral protease 2A cleaves dystrophin. Dystrophin is a large cytoskeletal protein that links the plasma membrane to actin. The function of dystrophin is partly to prevent heart muscle cells from sheer or contractile stress. Protease 2A cleaves dystrophin at its third hinge region, which leads to the disruption of the dystrophin-glycoprotein complex (DGC). Consequently, the sarcolemma or cell membrane of heart cells, become more susceptible to sheer/contractile stress. The DGC is important because it consists of dystrophin, α-syntrophin, β-syntrophin and neuronal nitric oxide synthase (nNOS). The association of nNOS through α-syntrophin to the DGC in skeletal muscle is supported by previous work. Although there is considerable information about nNOS in skeletal muscle, little is known about its localization in cardiac muscle. It is well known that nNOS produces nitric oxide (NO) and other superoxide radicals from arginine. The significance of NO is that it has been shown to inhibit the cleavage of dystrophin by protease 2A, which allows dystrophin to maintain its proper function at the sarcolemma. Therefore, it has been hypothesized that nNOS may protect dystrophin from cleavage by protease 2A. The main objective of this research project is to investigate the role of nNOS located in heart muscle cells. Methods: For this study, mice were used as the animal model system. There are two major techniques used to complete experiments concerning nNOS. First, immunofluorescent staining 2 was done to try to localize nNOS in the cell through the use of a primary antibody specific to nNOS and a fluorescent secondary antibody specific to the primary. The primary antibody was a polyclonal rabbit antibody to the C terminus nNOS and the secondary antibody was an FITC conjugated goat anti-rabbit. Tissue sections from CVB3 infected and uninfected wild-type mouse hearts were used. Fixations of the tissue sections included various solutions such as methanol, acetone or 4% Para formaldehyde. For each experiment there were positive and negative controls. The positive controls were skeletal muscle sections, while the negative controls received only the secondary. Tissues sections were stained and photographed. Secondly, tagged nNOS cDNA was cloned. The purpose of doing this is to facilitate localization of nNOS in cells since the immunofluorescent staining patterns were inconsistent. Cloning the nNOS cDNA involved several steps. Those steps include: Polymerase Chain Reaction (PCR), DNA extraction from gel, digestions of vectors and fragments, fragment purification, ligation, transformation, mini-preps and maxi-preps. Primers used in PCR were designed before starting cloning process. The two plasmids used were called pcDNA and Blue Script (pBlue). The restriction enzymes used were HindIII, BamHI, AatII, ECORI and XhoI. Briefly, RNA was isolated, cDNA was synthesized from brain and heart tissues, the cDNA was amplified, fragments and vectors were digested with restriction enzymes, and each fragment was inserted into the vectors separately. Detailed protocols can be referenced elsewhere. Results: In wild-type tissue not infected with CVB3, nNOS was shown to be at the sarcolemma. In wild-type tissue infected with CVB3, nNOS and dystrophin appear to be at the sarcolemma of some cells, however, absent in others. Evan’s Blue Dye (EBD) correlated with the absence of nNOS or dystrophin, which are the infected cells. EBD will only enter the cell if the membrane is permeable, which means that the CVB3 has disrupted the sarcolemma. Due to a limited amount of time, only two of the three fragments of the nNOS cDNA had been cloned. Hence, the cloning project is incomplete. Discussion: Thus far, the following conclusions have been made. Dystrophin is at the sarcolemma and has been well published. Based on the results of the nNOS stains, they do not appear as consistent as anticipated, which still makes the localization of nNOS unclear. Nonetheless, we worked on the best way to stain nNOS such as fixation, amount of antibody, and detergent (Tween 20 vs. Triton X). Thus far, methanol, a 1:100 dilution of antibody and Tween 20 yield the best results. Additionally, an experiment using Chinese Hamster Ovary (CHO) cells support the hypothesis concerning the role of nNOS and NO. These cells were transfected with dystrophin and had an added aspecific nNOS NOS inhibitor. This study showed that with a nNOS inhibitor, which blocks the production of NO, the rate of cell death increased because NO was not present and could not interfere with the CVB3 protease 2A cleavage of dystrophin. Once dystrophin has been cleaved the cell has essentially lost it protection because the dysfunctional DGC can not prevent sheer stress. Therefore nNOS is an essential protein in cardiac cells and may be a contributing factor to protecting dystrophin from CVB3. Future studies will be to continue the cloning of the tagged nNOS cDNA so that it could be used for other projects. 3 References: 1. Lee GH, Badorff C, Knowlton KU. Dissociation of sarcoglycans and the dystrophin carboxyl terminus from the sarcolemma in enteroviral cardiomyopathy. Circulation Research. 2000; 87:489-495. 2. Durbeej M, Campbell KP. Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models. Current Opinion in Genetics & Development. 2002; 12:349-361. 3. Badorff C, Berkely N, Mehrotra S, Talhouk JW, Rhoads RE, Knowlton KU. Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue. The Journal of Biological Chemistry. 2000; v275 15:11191-11197. 4. Knowlton KU, Chien KR. Inflammatory pathways and cardiac repair: the affliction of infarction. Nature Medicine. 1999; v5 10:1122-1123. 5. Badorff C, Lee GH, Lamphear BJ, Martone ME, Campbell KP, Rhoads RE, Knowlton KU. Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nature Medicine. 1999; v5 3:320-326.