1. The Role of Mitochondrial Ubiquitin Ligase Activator of
NF-kB (Mulan) in Cardiomyocytes
David H. Xiang
under the direction of
Dr. Yanfei Yang
Dr. Ronglih Liao
Cardiac Muscle Research Laboratory
Brigham and Women’s Hospital
Harvard Medical School
Research Science Institute
July 28, 2015

2. Abstract
Motor neuron degeneration (mnd2) is a mouse disorder that has a similar phenotype to
Parkinson’s disease and causes death by 40 days of age. It is caused by a missense mutation
in the protease domain of the nuclear-encoded mitochondrial serine protease Omi. mnd2 was
first conjectured to be a neurodegenerative disorder, but previous experiments using rescued
mnd2 mice and Omi knockout mice showed that fully rescuing only the mitochondria in
the neurons of mnd2 mice does not fully cure the mnd2 mice. In fact, the rescued mnd2
mice showed abnormal heart conditions, such as premature cardiac aging and cardiac hy-
pertrophy. Eventually, these mice die from heart failure due to cardiac muscle dysfunction,
suggesting that Omi’s specific substrate in the mitochondria, the mitochondrial ubiquitin
ligase activator of Nf-kB (Mulan), also plays an important role in cardiomyocytes, or car-
diac muscle cells. This is because Mulan undergoes a process of ubiquitination in which
Mulan forms a complex with its substrate and degrades other regulatory proteins in the
mitochondria or cytosol. Previous studies have shown that a decrease in Omi activity causes
Mulan expression to rise significantly, leading to mitochondrial dysfunction and cell death in
the striatal neurons. We performed several immunocytochemistry stainings to show that for
the first time, overexpression of Mulan not only induces mitochondrial dysfunction and cell
death in cardiomyocytes, but also causes mitochondria to have a punctuated morphology in
cardiac muscle cells. This indicates that Mulan may also play a role in regulating proteins
that control mitochondrial dynamics, or the homeostatic equilibrium of fusion and fission of
mitochondria.
Summary
A spontaneous mutation in mice, motor neuron degeneration (mnd2), has been shown to
be controlled by the deficiency of serine protease Omi activity. mnd2 has almost identical
symptoms to Parkinson’s disease, a neurodegenerative disease that impairs motor move-
ment. One of the abnormalities of mnd2 is that rescued mnd2 mice still exhibit abnormal
heart muscle enlargement and cardiac tissue aging, and the mice eventually die from heart
failure. This is unexpected, as mnd2 was first characterized as a neurodegenerative disease.
To understand Omi’s role in the heart, we have to understand its specific substrate in the
mitochondria, which is the mitochondrial ubiquitin ligase activator of NF-kB (Mulan). Omi
closely controls Mulan, and under normal conditions, Omi plays a role in suppressing Mulan
overexpression and maintaining mitochondrial quality. Therefore, in mnd2 mice, when Omi
activity is supressed, Mulan expression levels increase dramatically. Our study shows, for
the first time, that Mulan overexpression in cardiac muscle cells induces mitochondrial dys-
function, morphological changes, and eventual cell death through many immunostainings,
demonstrating the importance of Mulan in cardiomyocytes.

3. 1 Introduction
1.1 The nuclear-encoded mitochondrial serine protease Omi
The nuclear-encoded mitochondrial serine protease Omi, which is localized in the intermem-
brane space (IMS) of the mitochondria as a member of the HtrA protease-chaperone family,
has been implicated in mitochondrial quality control and apoptosis [1]. Previous studies have
shown that motor neuron degeneration 2 (mnd2) is caused by a mutation in the protease
domain of Omi which leads to catastrophic symptoms in mice [2].
mnd2 was first identified in 1990 as a spontaneous, recessively inherited mutation that
induced abnormal weight gain, akinesis, loss of striatal neurons in a posteriomedial portion
of the basal ganglia, and death after 40 days [2]. After three weeks, most mnd2 mice will also
experience significant damage to neurons in the central nervous system, the brain stem, and
the spinal cord [2]. Additionally, mnd2 has a phenotype similar to features of a Parkinsonian
syndrome: decreased mobility, bended posture, and tremors [3].
A previous study proved that the missense mutation Ser276Cys in the protease domain of
Omi caused the mnd2 mutation. This missense mutation substitutes one nucleotide in exon 3
of the nuclear-encoded mitochondrial serine protease Omi and thus changes amino acid serine
276 to cysteine. The impact of this mutation is that the Ser276Cys mutation interferes with
the interface between the PDZ domain and protease domains, resulting in the loss of access to
the active site pocket. This causes mnd2 because the loss of protease activity of Omi increases
sensitivity to stress-induced cell death and causes loss of striatal neurons [2]. Additionally,
the loss of Omi protease activity leads to accumulation of misfolded and damaged proteins
in the mitochondria, which leads to progressive mitochondrial dysfunction, an important
mechanism in the early stages of many neurodegenerative disorders such as Parkinson’s
[2]. Moreover, the mutation reduces mitochondrial density and the mitochondria are more
susceptible to cellular stresses such as oxidative stress, in which excess reactive oxygen species
1

4. (ROS) production can lead to several other fatal diseases like cancer, Alzheimer’s disease,
and atherosclerosis [3].
However, Omi has also been shown to have a proapoptotic role by binding with inhibitor
of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif in the cytosol,
relieving the IAPs of their inhibitory function and resulting in increased apoptosis [3]. In
spite of this, the phenotype of Omi knockout mice is almost identical to that of mnd2 mice,
indicating that the inhibition of Omi activity induces mnd2 symptoms, not Omi release
in the cytosol [2]. Moreover, when second mitochondria-derived activator of caspase/direct
inhibitor of apoptosis-binding protein (Smac/DIABLO), another mammalian mitochondrial
protein that can interact and antagonize IAPs was knocked out, apoptosis did not decrease
and the phenotype of the knockout Omi mice was not changed. These results, proved in
2003, show that the missense mutation increases the mice’s susceptibility to cell death, not
Omi’s interactions with IAPs [2].
Recently, a study attempted to treat mnd2 mice by expressing Omi in the central nervous
system. The study managed to prevent premature death, but the mice developed accelerated
aging phenotypes, such as premature weight loss, heart enlargement, and eventual death
through cardiac muscle dysfunction by 12-17 months of age [1]. While the mice were rescued
from neurodegeneration, they still died very early, indicating that the Ser276Cys mutation
may play a more global role and is not simply isolated to the brain and that the substrate
of Omi may play a role in cardiomyocytes [4].
1.2 Ubiquitination
Ubiquitination is a process in which ubiquitin, a regulatory protein attaching to a substrate
protein, starts a signaling pathway that can signal protein degradation via the proteasome,
affect substrate protein activity, and prevent protein interactions. Ubiquitination is carried
out in three main steps: activation, conjugation, and ligation. It is performed by ubiquitin-
2

5. activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s).
First, Ubiquitin-like modifier activating enzyme 1 (UBA1) or another E1 enzyme marks
cellular proteins for degradation [5]. Then, E2 ubiquitin conjugating enzymes provide the
substrate specificity which brings it in proximity for direct transfer of ubiquitin to form
complexes with the E3 ligases. Mulan, one of these E3 ligases, will bind and ubiquitinate
specific substrates in the cytosol. One of these substrates is Omi, which has a proapoptotic
function when it is released to the cytosol through postmitochondrial mechanisms [6]. Omi
is translocated from the mitochondria to the cytosol after myocardial ischemia and has
been proven to promote cardiomyocyte apoptosis via a protease activity-dependent, caspase-
mediated pathway [7]. However, when in the IMS of the mitochondria, Omi has a unique
pro-survival function by regulating Mulan expression [8].
Figure 1: Ubiquitination pathway with Mulan representing the E3 ligase. Ubiquitination is
a system of activation, conjugation, and ligation by E1, E2, and E3 ligases. Figure modified
from [9].
1.3 Mitochondrial Ubiquitin Ligase Activator of NF-kB (Mulan)
Mitochondrial dysfunction underlies various human pathologies, including cancer, aging, and
cardiovascular disease. It can be caused by genetic mutation or external influences that affect
3

6. physiological conditions [10]. This dysfunction will lead to mitophagy, in which cells eat their
own mitochondria either through the autophagy-lysosome system or a highly selective process
that targets dysfunctional mitochondria [11].
Mitochondrial ubiquitin ligase activator of Nf-kB, or Mulan, is a E3 ligase located on the
outer mitochondrial membrane (OMM). Previous studies have also called it mitochondrial
ubiquitin ligase 1 (Mul1), mitochondrial anchored protein ligase (MAPL), among other vari-
ations [12, 13]. Mulan has a RING finger domain facing the cytoplasm and a large domain
in the IMS, giving it a direct link of communicating mitochondrial signals to the cytoplasm
and mitochondria.
It was proven in a recent study that Mulan is the specific substrate of Omi [4]. mnd2
mice exhibit a severe deficiency of Omi protease activity and have very similar phenotypes to
knockout Omi mice [1]. Omi closely regulates Mulan expression, as under normal conditions,
Omi plays a role in mitochondrial quality control. However, when Omi activity is suppressed,
Mulan expression increases dramatically in cardiomyocytes [4]. This may be why rescuing the
neuro-specific mitochondria in mnd2 mice only partially rescued the mice, as they eventually
died from heart failure through premature cardiac aging and cardiac hypertrophy [1].
1.4 Mitochondrial Dynamics
Mitochondria are normally assembled through biogenesis, naturally decline, and repaired or
destroyed through the system of mitochondrial dynamics. The term mitochondrial dynamics
refers to organelle fission, fusion, and subcellular translocation. It is crucial that the dam-
aged mitochondria be removed, because once damaged, the mitochondria start producing
Reactive Oxygen Species (ROS). Substantial production of ROS will lead to mitochondrial
DNA (mtDNA) damage and eventually cell death, as it causes oxidative stress. Mitochon-
drial fission and fusion are essential in maintaining mtDNA stability, respiratory function,
and preventing programmed cell death in a homeostatic balance. Fission is regulated by
4

7. dynamin-related protein 1 (Drp1) and has implications in managing stress response and
apoptosis. Fusion is regulated by mitofusin protein 1 (Mfn1) and mitofusin protein 2 (Mfn2)
on the OMM and optic atrophy protein 1 (Opa1) in the inner mitochondrial membrane
(IMM) (Figure 2). Excessive fusion leads to mitochondrial elongation. Mfn1 and Mfn2 me-
diate fusion between mitochondrial outer membranes and can dramatically affect the mor-
phology of affected mitochondria when overexpressed. The precarious balance between fusion
and fission is dictated by the up-and-down regulation of mitofusins and Drp1. While Mfn1,
Mfn2, Opa1, and Drp1 are all highly expressed in cardiomyocytes, ablation of these proteins
provokes severe cardiac dysfunction [11]. However, mitochondrial dynamism also regulates
mitochondrial quality control. Asymmetric fission, in which Drp1 splits the healthy and
impaired parts of the mitochondria apart, is directly integrated with mitophagy as the mi-
tophagosome will only engulf the damaged mitochondria fragment [11]. As a result, fusion
must be selective in maintaining mitochondria quality as it should only redistribute and fuse
the healthy mitochondria.
Figure 2: System of Mitochondrial Dynamics. The fusion proteins have a blue box outlined
around them and the fission protein Drp1 has a red box outlined around it. Figure modified
from [11].
5

8. 1.5 The Role of Mulan in Mitochondrial Dynamics
The various effects of Drp1 and Mfn2 might be regulated by Mulan overexpression through
Omi protease inactivity, which might lead to subsequent mitophagy. In cardiomyocytes,
problems with mitochondrial dynamics would pose catastrophic damage to health, as mito-
chondria in cardiomyocytes often generate the most ATP (about 5 kg a day). Moreover, all of
this ATP is used each and every day, creating a system in which there can be no aberrations
in mitochondrial health and function. Mulan, with a perfect location on the OMM, might
be the E3 ligase that leads to degradation of fusion and fission proteins.
Figure 3: Visualization of the locations of Mulan (on the OMM), Omi (in the IMS), and the
proteins responsible for regulating mitochondrial dynamics. Figure modified from [4].
1.6 Summary
mnd2 is caused by an Omi mutation that suppresses Omi protease activity. While mnd2
was first thought of as a neurodegenerative and neuromuscular disease, attempts to treat
mnd2 mice through targeted expression of Omi in the brain have resulted in heart compli-
cations and eventual heart failure. Since Omi is suppressed, it cannot carry out its main
function inside the mitochondria, which is regulating Mulan. As a result, Mulan expression
increases dramatically. Our project aim focused on exploring the effects and impact of Mulan
overexpression in cardiomyocytes.
6

9. 2 Materials and Methods
2.1 Isolation and Culture of Neonatal Rat Ventricular Myocytes
Primary cultures of ventricular myocytes were obtained from 1-day-old Wistar rats (cat#003,
Charles River Laboratories) and prepared using a Neonatal Heart Dissociation Kit (Miltenyi
Biotec Inc.) according to the manufacturer instructions. Briefly, after enzymatic digestion,
ventricles were subjected to mechanical dissociation using a gentleMACS Dissociator. Cell
suspensions were applied to a discontinuous Percoll gradient and myocyte layers were har-
vested and cultured.
2.2 Western Blotting
Neonatal Rat Ventricular Myocytes infected by LacZ and Mul1 adenovirus were lysed in
1x lysis buffer (Cell Signaling #9803), harvested for Western Blot using primary antibodies
a tag for Mulan, anti-Flag (1:500) (Sigma), anti-GAPDH (1:1000) (Trevigene), and anti-
CoxIV (1:1000) (Life Tech), and subcellularly fractionated for the cytosol and mitochondria.
20 μg of protein were loaded to PAGEr Gold Precast Gel (Lonza #58505), run at 80V for
30 minutes and 100V for 20 minutes, and transferred to Immobilon-FL transfer membrane
(EMD Millipore) in an electrophoresis machine overnight in 4◦
C at 30V. After transfer, the
membranes were blocked with 3% BSA in PBS for 1 hour at room temperature and then
incubated for primary antibody in 4◦
C overnight. The membranes were washed 3 times with
PBS-T (1x PBS + 0.1% Tween20). Secondary antibodies, IRDye 680RD donkey anti-rabbit
700 (Li-Cor) and IRDye 800CW donkey anti-mouse 800 (Li-Cor) were then added and spun
for 1 hour at room temperature. The membranes were then washed again 3 times with PBS-T
buffer. Finally, the membranes were scanned with the Li-Cor Odyssey CLx Infrared Imaging
System.
7

10. 2.3 Live Cell Imaging and Staining
Cardiomyocytes were kept in cell culture medium at 37◦
C. The medium was changed, and
5nmol/L of tetramethylrhodamine, ethyl ester, perchlorate (TMRE) (Life Technologies) and
100nmol/L of mitotracker green (Life Technologies) were added to the medium and incubated
for 30 minutes at 37◦
C. Afterwards, the cells were viewed under a LSM 700 (Zeiss) confocal
microscope using a 63X oil immersion objective. Fluorescence was excited with a 488 nm
laser (Mitotracker Green) and a 555 nm laser (TMRE). Selection of images was done in a
blind manner regardless of treatment.
2.4 Immunocytochemistry of Cardiomyocytes
Cardiomyocytes were fixed with PBS containing 4% paraformaldehyde, permeabilized with
0.3% Triton X-100 PBS, blocked with PBS containing 3% BSA and incubated with the de-
sired primary antibodies, including anti-Mulan (1:50, Zervos Lab), anti-Flag (1:50, Sigma),
and anti-Tom20 (1:50, Santa Cruz). The cell death staining was treated with terminal
deoxynucleotidyltransferase (TdT) dUTP Nick-End Labeling (TUNEL), Mitotracker, and
Hoechst. Alexa Fluor 488 and Alexa Fluor 555 Dye-conjugated secondary antibodies (Life
Technologies) were used for fluorescent confocal analysis (dilution of 1:100).
2.5 Colocalization Analysis
Using ZEN software, a random line segment was chosen on the merged picture of the car-
diomyocytes, and the colocalization of primary antibodies Flag and Tom20 was found, with
the x-axis symbolizing the distance of the line segment from beginning to end and the y-axis
signifying the intensity of the two primary antibodies.
8

11. 2.6 Statistics
All values are expressed as mean±SEM. The standard error was found from the standard
deviation and the number of samples; the values were calculated and graphed using Microsoft
Excel.
3 Results
3.1 Exogenous Mulan is Localized to the Mitochondria in Car-
diomyocytes
Previous studies have shown that Mulan overexpression was localized in mitochondria when
using neuro-specific cells [4]. However, since we are exploring Mulan’s role in cardiomyocytes,
we first have to confirm that treatments of exogenous Mulan (foreign Mulan by adenovirus
injection) are still localized to the OMM in mitochondria in cardiomyocytes.
Neonatal rat ventricular myocytes (NRVMs) infected by LacZ (control protein) or Mulan
adenoviruses for 48 hours were harvested for subcellular fractionation to cytosol and mito-
chondrial fractions and for Western Blotting. Cells were also stained using anti-Flag and
anti-Tom20 antibodies and with Hoescht staining for the nucleus.
The Western Blot showed that exogenous Mulan was localized in the mitochondria (Fig-
ure 4). Anti-flag is the tag for foreign Mulan and has the highest expression in the Mulan
overexpression treatment, which confirms that exogenous Mulan is localized in the mitochon-
dria (Figure 4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein involved
in glycolysis, or the breakdown of glucose for the production of adenosine triphosphate
(ATP), and therefore is only present in the cytosol [14]. Therefore, the Western Blot con-
firms this statement, as the blot shows that GAPDH is only present in the cytosol and is not
affected by Mulan overexpression. Lastly, the Western Blot shows that cytochrome c oxidase
9

12. (CoxIV), which is located in the inner mitochondrial membrane and is involved in oxidative
phosphorylation, is only present in the mitochondria, again as expected [15].
Figure 4: Overexpression of foreign Mulan is localized to mitochondria in cardiomyocytes.
Flag is the tag for exogenous Mulan, GAPDH is the cytosolic control, and CoxIV is the inner
mitochondrial control.
To further confirm that exogenous Mulan is localized to the OMM of the mitochondria
in cardiomyocytes, we performed an immunostaining to show that foreign Mulan colocalized
with the OMM by using both anti-Flag staining for Mulan and anti-Trans-outer membrane
20 (anti-Tom20), which stains for the OMM (Figure 5). The merged picture immediately
showed a visual colocalization of exogenous Mulan and the OMM, indicating that Mulan
overexpression introduced via viral infection still remained in the OMM of the mitochondria
in cardiomyocytes. To further quantitatively confirm the visual representation, colocalization
analysis was performed on a randomly chosen segment of the merged staining picture (Figure
6). As expected, the graph showed that the intensity lines of Flag and Tom20 were essentially
superimposed, further confirming that overexpressed Mulan is localized to the mitochondria,
and more specifically, the OMM.
10

13. Figure 5: Immunocytochemistry of cardiomyocytes shows that exogenous Mulan colocalizes
on the OMM of the mitochondria. Tom20 and Flag primary antibodies are clearly colocalized
as the merged picture showed the mitochondria turning a yellow hue. The blue line on the
merged picture shows the area chosen for colocalization analysis.
Figure 6: Colocalization analysis of a randomly selected area on the merged picture. Flag and
Tom20 intensity essentially overlap each other, confirming that exogenous Mulan is localized
to the OMM of the mitochondria.
3.2 Mulan Overexpression Causes Mitochondrial Dysfunction Over
Time in Cardiomyocytes
Because mnd2 mice exhibit an almost complete lack of Omi protease activity, Omi can no
longer manage mitochondrial quality, leading to an increase in mitochondrial dysfunction.
After previously determining that overexpressed foreign Mulan will still localize to the mito-
11

14. chondria, NRVMs were infected by LacZ or Mulan adenoviruses at 100 moi (multiplicity of
infection) for a total of 48 hours and stained for mitotracker green (200 nmol/L) and TMRE
(5 nmol/L) dyes for 30 minutes. They were examined using live cell confocal imaging at a
period of 24 hours after infection and then 48 hours after infection. This allows us to see the
mitochondrial activity and thus determine whether or not they are functioning normally.
When mitochondria are active, they will exhibit a negative membrane potential inside
the mitochondria, as the mitochondria must have a proton gradient in order to efficiently
generate ATP. TMRE will be sequestered by active mitochondria as it is a negative membrane
potential sensitive dye. Mitotracker green will stain the entire mitochondria regardless of
activity.
At 24 hours, the mitochondria from cardiomyocytes treated with LacZ and Mulan had a
similar amount of active mitochondria when compared to the total number of mitochondria
present. However, after 48 hours, the amount of active mitochondria in the Mulan over-
expressed treatment dramatically dropped, indicating that Mulan overexpression leads to
gradual mitochondrial dysfunction as the mitochondria are no longer active, meaning they
cannot generate ATP efficiently anymore and might be subjugated to mitophagy (Figure
7). Furthermore, malfunctioning or damaged mitochondria might start producing increased
levels of ROS, which would lead to more catastrophic damage to the cardiomyocytes.
12

15. Figure 7: Approximately 200 NRVMs infected by LacZ or Mulan adenoviruses at 100 moi for
a total of 48 hours were stained for mitotracker green (200 nmol/L) and TMRE (5 nmol/L)
dyes. Then, they were analyzed with live cell confocal imaging using a Zeiss LSM700 con-
focal microscope at 37◦
C. (A) shows that when the mitochondria were imaged at 24 hours,
the LacZ and Mulan treated mitochondria showed no significant difference in mitochondria
activity. (B) shows staining results of mitochondria activity at 48 hours in the Mulan over-
expressed mitochondria, in which the activity dropped severely, indicating mitochondrial
dysfunction.
3.3 Mulan overexpression induces cell death in cardiomyocytes
A curious observation we made when we analyzed the immunohistochemistry stain for mito-
chondrial function was that the number of live cardiomyocytes in the Mulan treatment group
decreased dramatically. To confirm this hypothesis, we used terminal deoxynucleotidyltrans-
ferase (TdT) dUTP Nick-End Labeling (TUNEL) staining to detect apoptosis. Essentially,
TUNEL stains for nicked DNA fragments, which occurs in cells undergoing apoptosis. What
13

16. we found confirmed our earlier observation, as the Mulan overexpression treatment had
a much higher percentage of TUNEL positive cells when compared to the control LacZ
treatment, which indicates that Mulan overexpression also induces death in cardiomyocytes
(Figure 8).
Figure 8: Representative images of the cardiomyocytes were taken, and the TUNEL fluo-
rescence was excited at 555 nm and WGA at 488 nm to make sure there are no spectral
overlap issues. Cardiomyocytes were stained with TUNEL (cell death) exhibiting a red color,
Mitotracker showing a grey color, and Hoescht (nucleus) displaying a blue color. Over 200
cardiomyocytes were counted and used for analysis. Mulan treated cardiomyocytes exhibited
a higher ratio of TUNEL positive cardiomyocytes to total cardiomyocytes, indicating that
Mulan overexpression induces cell death.
3.4 Punctated mitochondrial morphology occurs through Mulan
overexpression in cardiomyocytes
Interestingly, we noticed that not only did the Mulan treated cardiomyocytes exhibit mito-
chondrial dysfunction, but also a different mitochondrial shape. The Mulan overexpressed
cardiomyocytes had much smaller circular fragments making up the mitochondrial area,
14

17. whereas the control group’s mitochondria were more elongated. This led us to create a
method for quantifying the degree of fragmentation in the hopes of using these results for
further analysis and research.
Our classification system is as follows, with the percentage representing the amount of
circular fragments divided by the total mitochondrial area: 0% to 20% indicates a filamental
morphology (not fragmented), 20% to 80% shows a mixed morphology (moderately frag-
mented), and 80% to 100% suggests a punctated morphology (fully fragmented) (Figure 9).
We manually count the number of fragmented circles, trace the area of the mitochondria, and
take the ratio. To determine these categories, we engaged in extensive discussion with collab-
orating laboratories (especially the Zervos lab at the University of Central Florida), leading
us to create what we believe is the best organizational system for classifying mitochondrial
fragmentation.
In the Mulan overexpression treatment, the percentage of fully fragmented cells was
the largest, indicating that Mulan overexpression leads to mitochondrial fragmentation in
cardiomyocytes, which might implicate Mulan as having a role in mitochondrial dynamics
(Figure 10).
Figure 9: Visualization of the three categories of mitochondrial morphology: Filamental (0-
20%), Mixed (20-80%), and Punctated (80-100%).
15

18. Figure 10: Mulan overexpression greatly increased the number of fully fragmented mito-
chondria when compared to the control treatment, indicating that Mulan overexpression can
induce mitochondrial fragmentation in cardiomyocytes. The graph quantifies the degree of
fragmentation in the control and Mulan treatment groups.
4 Future Research
Short term goals include understanding the mechanism and consequences of mitochondrial
fragmentation in cardiomyocytes due to Mulan overexpression. This fragmentation might be
the result of downregulation of proteins Drp1 and Mfn2, which are essential in mitochondrial
dynamics and maintaining the general health and efficiency of mitochondria. If Drp1 and
Mfn2 are specific substrates of Mulan, then that would mean that Mulan also has a role in
managing the homeostatic equilibrium of mitochondria in cardiomyocytes.
Long term goals include understanding the complete pathway in which neurodegenerative
diseases work. Understanding the causes and symptoms not experienced in the brain would
help in developing a more complete treatment. This would combine many different fields, from
neurobiology to cardiology, and indicates that some of the most fatal and complex diseases
affect numerous organs in the body. This new approach necessitates a lot of collaboration and
16

19. creative thinking but is also very exciting because it allows researchers of different disciplines
to unify their work.
5 Conclusion
Previous studies showed that attempts to rescue the neuro-specific Omi deficient cells in
mnd2 mice led to heart abnormalities and eventual failure and death in 12-17 months. We
have proved that Omi’s substrate, Mulan, which is overexpressed in mnd2 mice, plays a role in
cardiomyocytes in mitochondrial dysfunction, cell death, and mitochondrial fragmentation.
mnd2 was previously thought of as a neurodegenerative disease, but our study shows that
it also has roots in cardiomyocytes. This might prove to be the link to understanding the
pathology of Parkinson’s disease, which has a similar phenotype to mnd2.
When we first started, we were expecting Mulan overexpression to lead to some sort of
dysfunction in cardiomyocytes. However, we did not expect that Mulan overexpression could
lead to such a drastic change in mitochondrial function. Most surprisingly, we were amazed
when we noticed that Mulan overepression also led to changes in mitochondrial morphology,
which is definitely an interesting research quandary for future work.
6 Acknowledgments
I would first like to thank my mentor, postdoctoral fellow Dr. Yanfei Yang, for directly
teaching and advising me on my project. I would also like to thank principal investigator
Dr. Ronglih Liao for providing me the chance to gain invaluable skills and experiences at a
world class research facility. Additionally, I would like to thank everyone else at the Brigham
and Women’s Hospital/Harvard Medical School Cardiac Muscle Research Laboratory for
welcoming me into the lab and always being helpful. I would also like to thank Ber¸san ¨Ozcan,
17

20. my tutor, for helping me revise my paper and providing me with advice on professional
writing skills and lexicon. Furthermore, I would like to thank Dr. Frederick Chen and Mr.
Scott Berk for sponsoring me this summer and providing me the means to attend RSI. Lastly,
I would like to thank the Center for Excellence in Education, the Research Science Institute,
and MIT for arranging my mentorship.
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23. Appendix A Glossary
Mulan: Mitochondrial ubiquitin ligase activator of NF-kB
Omi: Nuclear-encoded mitochondrial serine protease
mnd2: Motor neuron degeneration 2
IAP: Inhibitors of apoptosis proteins
Smac/DIABLO: Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-
binding protein
OMM: Outer mitochondrial membrane
IMM: Inner mitochondrial membrane
IMS: Intermembrane space
Drp1: Dynamin-related protein 1
Mfn1: Mitofusin protein 1
Mfn2: Mitofusin protein 2
Opa1: Optic atrophy protein 1
mtDNA: mitochondrial DNA
ROS: Reactive Oxygen Species
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