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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
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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.
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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.