University of Southern California Dissertations and Theses

TNNI3K expression affects nucleation of cardiomyocytes and skeletal myoblasts

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TNNI3K expression affects nucleation of cardiomyocytes and skeletal myoblasts

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Content TNNI3K Expression Affects Nucleation of Cardiomyocytes
and Skeletal Myoblasts

by

Kimberly Lim

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2018

Copyright 2018 Kimberly Lim

ii
Acknowledgements

I would like to thank my mentor, Dr. Henry Sucov, for his guidance and encouragement
during my time at USC. Under his mentorship, I had pushed myself to execute critical thinking,
perform experiments with poise and exercise independence as a scientist. I am especially grateful
for his patience and understanding when I was faced with difficult challenges. I would also like
to thank my committee members, Dr. Jian Xu and Dr. Ellen Liu, for their time and support in
helping me prepare for my thesis. Also, I would like to extend my gratitude towards the
members of the Sucov Lab: Michaela Patterson, Susana Cavallero, Hua Shen, Peiheng Gan, and
Alexa Velasquez. Thank you all for your unwavering support whenever I needed a helping hand.
In addition, I would like to thank Kim Staats; for all the late nights we spent together on my
thesis, for the reminders to hold myself up and be confident, and for checking up on me
throughout the process, thank you for being a great friend. Lastly, I would like to thank everyone
who has been there for me throughout this journey, especially my family and friends. Thank you
for your steadfast support.

Ad majorem Dei gloriam.

iii
Table of Contents

ACKNOWLEDGEMENTS II

LIST OF FIGURES V

ABSTRACT VI
CHAPTER I: INTRODUCTION 1
1.1 HEART DEVELOPMENT AND DISEASE 1
1.2 HEART REGENERATION 2
1.3 MNDCM POPULATION VARIES ACROSS INBRED MOUSE STRAINS AND PREDICTS 3
REGENERATIVE CAPACITY
1.4 CARDIAC TROPONIN I-INTERACTING KINASE 4
1.5 TNNI3K CONTROLS SIZE OF MNDCM POPULATION AND REGENERATIVE 6
POTENTIAL
CHAPTER 2: SPECIFIC AIMS 9
2.1 PURPOSE AND HYPOTHESIS 9
2.2 STUDY APPROACH 9

CHAPTER 3: MATERIALS AND METHODS 11
3.1 CELL CULTURE AND GROWTH MEDIA 11
3.2 TRANSFECTION AND SELECTION OF ES CELLS 11
3.3 CELL DIFFERENTIATION 12
3.4 IMMUNOFLUORESCENT STAINING 12
3.5 GIEMSA STAINING 13
3.6 ADENOVIRAL INFECTION 13
CHAPTER 4: SKELETAL MYOCYTE MODEL 14
4.1 MYOMIXER IS ESSENTIAL FOR MYOTUBE FORMATION 14
4.1.1 RESULTS 14
4.2 SKELETAL MYOCYTE ADENOVIRUS INFECTION 16
4.2.1 RESULTS 16

iv
CHAPTER 5: ES-DERIVED CARDIOMYOCYTE MODEL 18
5.1 GENETIC ENRICHMENT OF ES-DERIVED CARDIOMYOCYTES 18
5.2 VALIDATION OF CARDIOMYOCYTE ENRICHMENT 18
5.2.1 RESULTS 19
5.3 SINGLE CELL VISUALIZATION OF CARDIOMYOCYTES 21
5.3.1 RESULTS 21
CHAPTER 6: DISCUSSION 22
CHAPTER 7: REFERENCES 25

v
List of Figures

FIGURE 1 TNNI3K KNOCKOUT OR OVEREXPRESSION AFFECTS REGENERATIVE POTENTIAL 8

FIGURE 2 MAP OF MHC-NEO/PGK-HYGRO PLASMID 12

FIGURE 3 KNOCKOUT OF MYOMIXER EXPRESSION REDUCES MYOTUBE FORMATION 15

FIGURE 4 MYOMIXER IS ESSENTIAL FOR MYOBLAST FUSION 17

FIGURE 5 ES-DERIVED CARDIOMYOCYTES AFTER G418 SELECTION 20

FIGURE 6 SINGLE CELL VISUALIZATION OF A CARDIOMYOCYTE 21

vi
Abstract

Although adult mammalian cardiomyocyte regeneration after injury is limited, the
myocardium can be repaired in other contexts such as the hearts in zebrafish, neonatal mice and
many other species in embryonic development. Our lab has shown that the expression of cardiac
troponin I-interacting kinase (TNNI3K) and the population of mononuclear diploid
cardiomyocytes (MNDCM) contribute to varying degrees of heart regeneration, where knockout
of Tnni3k expression in mice raised MNDCM content and increased cardiomyocyte proliferation
after injury while overexpression of Tnni3k in zebrafish promoted cardiomyocyte
polyploidization and compromised heart regeneration. In order to understand the role of
TNNI3K in cardiomyocyte regeneration, I am using two in vitro models, a mouse embryonic
stem cell-cardiomyocyte cell line and a mouse skeletal myoblast cell line. Though the cellular
localization and phosphorylation targets of TNNI3K have not been verified, the kinase has been
shown to play a role in cardiac remodeling after injury and promote progression of
cardiomyopathy in mice. Despite not being expressed in skeletal muscle, TNNI3K could
potentially interact with skeletal sarcomere proteins and could reveal the mechanistic role of
TNNI3K in cell polyploidization. I therefore aim to investigate whether TNNI3K causes
binucleation within the two cell lines. I have demonstrated that cardiomyocytes can be derived
from mouse embryonic stem cells using plasmid transfection. I have also shown that
differentiating mouse skeletal myoblasts can be prevented from forming myotubes when the
Myomixer gene is knocked out. Ongoing work includes virally infecting the two cell lines to
overexpress TNNI3K and observing cell ploidy.

1
Chapter 1: Introduction

1.1 Heart development and disease
Cardiomyocytes are contractile cells that comprise the muscle walls of the heart; to pump
blood to all organs of the body, cardiomyocyte protein structures called sarcomeres respond to
changes in calcium concentration and cause a synchronized contraction of the myocardial tissue.
Cardiomyocyte growth consists two phases: proliferative growth during fetal development and
hypertrophic growth during neonatal development and beyond (Soonpa, et al., 1996).
Cardiomyocytes in proliferative growth are mononuclear and diploid in regards to nuclear
content; the cells undergo DNA replication and cell division to create mononuclear diploid
daughter cells and increase the total cardiomyocyte population. Cardiomyocytes in hypertrophic
growth typically contain 2 or more nuclei and increase in cell size rather than in cell number. The
hypertrophic cells undergo DNA replication but do not proceed through cell division and are
suspended in cycle arrest. Binucleation has been thought to enhance cell survival when coping
with stress or to meet the metabolic demand of cardiomyocytes by enabling the cell to generate
twice the amount of RNA to synthesize proteins. Animal studies have provided evidence of
several factors involved in cardiomyocyte transition from proliferative growth to hypertrophic
growth, such as oxidative stress (Puente, et al., 2014; Bae, et al., 2003), glucocorticoids (Giraud,
et al., 2006) or maternal malnutrition (Corstius, et al., 2005; Bubb, et al., 2007).
Because of this transition into cell cycle arrest, we rarely see primary tumors in the heart nor
an increase in DNA synthesis in normal or injured hearts (Kellerman et al. 1992; Soonpa et
al.,1994). During a myocardial infarction (MI), a blocked blood vessel, unable to supply
sufficient oxygen to the myocardium, causes irreparable damage to cardiomyocytes. Suspended

2
in in cell cycle arrest, cardiomyocytes are incapable of dividing and renewing into healthy tissue
to replace the damaged and dead infarct tissue, leading to scar formation and cardiac remodeling.
Although the surrounding undamaged cardiomyocytes can undergo further hypertrophic growth
to compensate for the loss of contractile force from missing cardiomyocytes, severe damage can
worsen cardiac output and ultimately result in heart failure.

1.2 Heart Regeneration
Although heart regeneration appears to be minimal, there has been evidence of
cardiomyocyte renewal in several contexts.
One of the first examples of heart regeneration came from a study that dated
14
C within the
hearts of humans exposed to radioactivity during the Cold War. The study found that
cardiomyocyte renewal occurred at a yearly rate of 1% and decreased with age (Bergmann, et al.,
2009). An average of about 45% of cardiomyocytes were renewed over an individual’s lifetime.
However, this rate of regeneration does not compensate for the loss of cardiomyocytes in a single
MI event.
Unlike humans, zebrafish hearts have a robust capacity to regenerate. Studies have shown
that zebrafish hearts can undergo complete regeneration after resecting 20% of the ventricular
myocardium after 1-2 months (Poss, et al., 2002) and can retain the ability to regenerate in both
fetal and adult stages after injury (Jopling, et al., 2010). Similar to zebrafish, neonatal mice can
also undergo regeneration after apex resection. However, they are unable to retain the ability to
regenerate 7 days after birth and into adulthood (Ali, et al., 2014, Porrello, et al., 2011; Soonpa,
et al., 1996).

3
In mice, the transition from the mononuclear diploid condition that typifies embryonic
cardiomyocytes to the binucleated condition of most adult cardiomyocytes occurs during the first
postnatal week (Soonpa, et al., 1996). Contrastingly, cardiomyocytes in adult zebrafish remain
mononuclear and diploid throughout their lifespan (Kikuchi, et al., 2010). However, there is now
evidence that the adult mammalian heart is able to retain some ability to regenerate. Adult hearts
from humans and mice have been shown to contain a population of cells that appear
mononuclear and diploid and may be the proliferative population of cardiomyocytes (Bergmann,
et al., 2009; Senyo, et al., 2013).

1.3 MNDCM population varies across inbred mouse strains and predicts regenerative
capacity
Our lab aimed to confirm if mononuclear diploid cardiomyocytes (MNDCM) are the
population of cells that are proliferative within the heart (Patterson, et al., 2017). To verify this,
we surveyed 120 inbred mouse strains for MNDCM content and found that the frequency of
MNDCM varied from strain to strain, ranging from 2.3% to 17% of total ventricular cells. The
continuous distribution of frequency across strains indicated that multiple genes with
polymorphic alleles contributed to the variation in frequency.
Four mouse strains were chosen for further studies: strains A and SWR with high
mononuclear content and C57Bl/6 and SJL with moderately low to very low mononuclear
content. To study the effect of mononuclear content on heart function after injury, the four
strains were subjected to permanent coronary artery ligation and then were evaluated for heart
function using echocardiography. Before injury and 3 days after injury, heart function did not
differ between the four strains. 1 month after injury, ejection fraction worsened or stabilized

4
within the two strains with low mononuclear content, strain C57Bl/6 and SJL, while the two
strains with high mononuclear content, strains A and SWR, showed improved ejection fraction.
There was also less scarring within strains A and SWR compared to the C57Bl/6 and SJL strains.
We next wanted to identify genes that play a role in controlling the size of MNDCM
population and regenerative capacity. We performed genome-wide association for the trait of
mononuclear cardiomyocyte frequency to find single nucleotide polymorphisms that can be
associated with the trait. Chromosome 3 contained several peaks for different protein-coding
genes, one of which is called Tnni3k. The Tnni3k gene contains a T/C polymorphism in a splice
junction in which the T allele causes normal expression of Tnni3k, such as in C57Bl/6 mice,
whereas the C allele causes improper splicing and eliminates protein expression, such as in strain
A. Our analysis showed that expression of the C allele is associated with a higher level of
mononuclear cardiomyocytes, indicating a recessive phenotype, and heterozygous expression of
the T allele in a cross of C57Bl/6 and A strains showed a similar phenotype to homozygous
expressing C57Bl/6 mice.

1.4 Cardiac Troponin I-Interacting Kinase
Cardiac troponin I (cTnI) interacting kinase or TNNI3K is a cardiomyocyte-specific kinase
that is highly expressed in both adult and fetal hearts (Zhao, et al., 2003). TNNI3K contains three
domains: N-terminal ankyrin repeats, a protein kinase domain and a C-terminal serine rich
domain. The protein is highly conserved with little differences between humans and mice.
Although TNNI3K was named after the putative interaction between the kinase and cTnI from
yeast two-hybrid screening (Zhao, et al., 2003), the interaction has not been validated in vivo.

5
Current studies investigating the molecular function of TNNI3K is limited. To date, no
downstream targets of TNNI3K have been identified and no connection between TNNI3K and
other signaling pathways has been established. However, TNNI3K has been shown to be a
functional kinase through autophosphorylation studies. Confirmed through in vitro and in vivo
studies, the structure of the kinase domain indicates that it is a dual-function kinase and can
phosphorylate both tyrosine and serine/threonine sites, which are mainly located in the ANK-
repeat and serine-rich domains of the TNNI3K protein (Tang, et al., 2013; Feng, et al., 2007).
Also, the cellular location of TNNI3K has yet to be conclusively demonstrated, possibly due
to different anti-TNNI3K antibodies and differences in tissue sample processing.
Immunohistochemical studies in adult mice overexpressing human TNNI3K demonstrated
localization of the kinase to the sarcomeric Z-disk (Tang, et al., 2013). The Z-disk provides
structural support for the sarcomere and is involved in mechanosensation and
mechanotransduction (Frank, et al., 2011). The study also confirmed Z-disk localization within
mice expressing a kinase-dead version of TNNI3K, suggesting that kinase activity is not required
for Z-disk localization (Tang, et al., 2013). However, another investigator reported the
enrichment of TNNI3K in the nuclear fraction in subcellular fractionation studies of neonatal rat
ventricular myocytes (NRVM) and localization around the nuclei in immunofluorescence studies
in neonatal and adult rat cardiomyocytes and did not detect co-localization with alpha-actinin, a
component of the sarcomere (Vagnozzi, et al., 2013). They concluded that TNNI3K did not
interact with cTnI in the sarcomere. Taking all these findings, the cellular localization remains to
be determined.
Although not much is known about the direct role of TNNI3K in heart biology, TNNI3K
kinase activity has been shown to induce cardiac remodeling and promote progression of

6
cardiomyopathy within mice. Overexpression studies of human TNNI3K within DBA/2J mice
sensitized to cardiomyopathy, via overexpression of calquestrin, led to acceleration of heart
failure development, with severe systolic dysfunction, chamber dilation and a profound reduction
in survival in double transgenic mice overexpressing both hTNNI3K and Csq compared to single
transgenic mice (Wheeler, et al., 2009). Other studies investigating cardiac remodeling,
specifically in the development of hypertrophy, have presented conflicting results. One
hTNNI3K overexpression study utilized DBA/2J mice without Csq sensitization, showing an
increase in heart weight to body weight ratio and enlarged cardiomyocytes compared to wildtype
mice (Tang, et al., 2009). However, phosphorylation of several cardiac remodeling signals such
as p38, ERK1/2 and AKT were unchanged, suggesting that TNNI3K may not regulate these
effectors in this setting. The study also showed an increase in cardiac mass and cardiomyocyte
size in mice expressing a kinase-dead version of TNNI3K, implying that cardiac remodeling may
be independent of TNNI3K kinase activity. A different overexpression study utilized a different
mouse strain C57BL/6J and found that hypertrophy was present in mice with a high level of
TNNI3K overexpression and not in mice with intermediate and low expression. (Wang, et al.,
2013). A third study utilizing FVB/N strain mice did not reveal hypertrophy at 3 months of age
in echocardiographic analysis (Vagnozzi, et al., 2013) but did not include histological analysis.

1.5 TNNI3K controls size of MNDCM population and regenerative potential
To study the effects of TNNI3K on MNDCM population and regenerative potential, we
created a conditional knockout mouse for Tnni3k by crossing a conditional loss-of-function
Tnni3k allele in a C57Bl/6 mouse with Cre recombinase mice of similar strain to knockout the
gene globally or specifically in cardiomyocytes (Patterson, et al., 2017). We found a 2.5-fold

7
increase in mononuclear cardiomyocyte frequency and a 3-fold increase in MNDCM population
compared to mice expression wildtype Tnni3k. In response to injury, we also observed a 3-fold
increase in EdU-labeled cardiomyocytes as well as MNDCMs, thus demonstrating that that
injury activates MNDCMs and initiates a proliferative response. Finally, heart function was
evaluated after injury by echocardiography. Compared to control, Tnni3k-null mice did not show
a significant improvement in heart function as well as scar area 1 month after injury. However,
this analysis contained an abundant amount of binucleated cardiomyocytes and may not reflect
changes in proliferative cardiomyocytes.
We also performed overexpression experiments in zebrafish using mouse Tnni3k. Although
the transgenic expression of mouse Tnni3k did not affect the number of binucleated
cardiomyocytes compared to transgenic control fish, polyploidy was found to increase similarly
to mouse cardiomyocytes. To evaluate heart regeneration, scar severity was measured 30 days
after apex resection. While control fish most regenerated efficiently and did not leave prominent
scars, transgenic fish showed a greater degree of scarring. PCNA staining of cardiomyocytes
confirmed that there was a decrease in cardiomyocyte proliferation and inhibited the ability to
regenerate after injury.

8

Figure 1: TNNI3K Knockout or Overexpression affects regenerative potential. TNNI3K
knockout in mice increases cardiomyocyte proliferation in mice while TNNI3K
overexpression decreases cardiomyocyte proliferation in zebrafish. Red nuclei represent
cardiomyocytes with regenerative potential. Image: Vujic, Bassaneze, and Lee, 2017.

9
Chapter 2: Specific Aims
2.1 Purpose and Hypothesis
Taken together, these knockout and overexpression results show that MNDCM content is
associated with cardiomyocyte proliferation and that TNNI3K plays a role in regulating
MNDCM content. However, the mechanism by which TNNI3K controls this remains unclear. It
is also unknown if TNNI3K expression can also affect nucleation or cell cycle regulation within
other cell types. I aim to investigate the role of TNNI3K in cell binucleation in myocytes in vitro.
Based on preliminary results, I hypothesize that TNNI3K expression increases binucleation
in skeletal myocytes and in cardiomyocytes.

1.6 Study Approach
I utilized two cell types cultured in vitro: mouse skeletal myoblasts and mouse embryonic
stem (ES) cell-derived cardiomyocytes.
The skeletal myoblast cell line utilized in these studies is the immortal mouse C2C12 cell
line derived from satellite cells, a mononuclear precursor to skeletal myoblasts that undergo
differentiation when activated by muscle injury or mechanical strain to form new muscle fibers.
The cell line is typically used as a model system for understanding muscle growth and
development. Although skeletal muscle development is profoundly different from cardiac
development, the use of skeletal myocytes has several advantages for studying TNNI3K. For
example, TNNI3K is not expressed in skeletal muscle whereas cardiomyocytes of different
mouse strains express a variation of TNNI3K depending on the polymorphic phenotype. The cell
line can be ectopically induced to express the kinase. Skeletal myocytes are also contractile like
cardiomyocytes and have similar sarcomeric proteins that could possibly interact with TNNI3K.

10
Finally, the cell line is easy to manipulate and culture compared to the arduous task of isolating
cardiomyocytes from primary tissue.
However, skeletal myocytes undergo membrane fusion during myogenesis and muscle
repair, unlike cardiomyocytes. Myoblasts undergo differentiation, recognition of surrounding
fusion-competent cells, and adhesion to surface receptors to allow myoblasts to create a pore
within a neighboring cell and transfer the cytoplasm and nucleus into the founder cell (Kim, et al.
2015). Recently, the micropeptide Myomixer was discovered and was found to control fusion
between myocytes (Bi, et al., 2017). When Myomixer expression was knocked out,
differentiated C2C12 cells showed reduced presence of multinucleated myotubes compared to
wild-type expressing cells without affecting expression of differentiation makers such as myosin
heavy chain. Thus, I can utilize the KO Myomixer C2C12 cell line to investigate whether
TNNI3K would affect nucleation of differentiating myoblasts.
The second in vitro model I aim to use is cardiomyocytes derived from ES cells. Rather
than enzymatically isolating small primary cultures of cardiomyocytes that may be contaminated
with noncardiomyocytes. To create the cell line, I used a genetic enrichment approach to create
pure populations of cardiomyocytes (Pasumarthi & Field, 2002). In this method, long-term
cultures of cardiomyocytes are generated while noncardiomyocytes are removed. With this, I can
investigate the role of TNNI3K in cardiomyocyte nucleation.

11
Chapter 3: Materials and Methods

3.1 Cell Culture and Growth Medium
Undifferentiated ES cells were cultured in ES growth medium: Dulbecco’s Modified
Eagle’s Medium (DMEM) containing 15% heat-inactivated FBS, 0.1 mM nonessential amino
acids, 2mM glutamine, 50U/mL penicillin, 50µg/mL streptomycin, 0.1mM 2-mercaptoethanol,
and 10
3
U/mL LIF. ES cells were fed with growth medium not containing hygromycin or G418
2-3 hours before passaging or treatment.
Undifferentiated C2C12 cell lines were cultured in DMEM containing 10% FBS and
penicillin and streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing
5% CO
2
.

3.2 Transfection and Selection of ES Cells
Before transfection, MHC-neo/pGK-hygro was digested with XhoI at 37°C for 3 hours
and heat inactivated at 65°C for 20 min. ES cells were dissociated with trypsin, resuspended
4x10
6
in 0.8mL of electroporation buffer and transported into a Bio-rad electroporation cuvette
with 15µg of DNA. Cells were electroporated at 400V, 25µF for single copy input into cells.
Cuvette was left to rest in the hood for 15 min. at room temperature. Cells were plated onto 10cm
cell culture dishes in ES growth media for 24 hours before medium was switched to ES growth
medium containing 200µg/mL hygromycin B. Medium was changed daily for seven days to
allow for selection of transfected cells. Cells were passaged and replated when plates became
confluent.

12

3.3 Cell Differentiation
After 7 days of hygromycin selection, ES cells were passaged onto 10cm bacterial Petri
dishes in ES growth media excluding LIF. The next day, fresh medium was added to the dish. On
the third day, the embryoid bodies (EBs) were transferred to a new Petri dish with fresh medium.
On the fourth day, fresh medium was again added to the Petri dish. EBs were then transferred to
cell culture dishes. Noncardiomyocytes were eliminated using ES growth medium excluding LIF
and containing 200µg/mL G418.
At 70-80% confluency, C2C12 cells were treated with DMEM containing 2% horse
serum and penicillin/streptomycin for 7 days to allow for differentiation. Medium was changed
every 3
rd
and 6
th
day.

3.4 Immunofluorescent Staining
Cardiomyocytes fixed in 4% paraformaldehyde for 15 min. and blocked and
permeabilized in goat serum with 0.1% Triton-X 100 for 30 min. Cells were incubated with
Figure 2: Map of MHC-neo/pGK-hygro plasmid.

13
primary antibody in goat serum 4°C overnight. Cells were then washed twice with PBS and
incubated with secondary antibody conjugated with either Alexa Fluorophore 488 or 596. Cells
were then washed in PBS and remained in PBS while imaging.

3.5 Giemsa Staining
C2C12 cells were grown in 4 chamber well slides. Cells were fixed with 4% PFA for 15
min. and washed twice with PBS. Cells were stained with Giemsa diluted (1:20) in dH
2
O for an
hour at room temperature and washed 5 times with dH
2
O. Cells were allowed to air dry and
imaged.

3.6 Adenovirus Infection
C2C12 cells were grown in 4 chamber well slides. One day before imaging, cells were
infected overnight. Cells were then fixed with 4% PFA, washed with PBS twice, and was stained
for DAPI (1:1000) for one hour at room temperature. Coverslips were added with Pro-Long Gold
mounting solution, sealed, and then observed under fluorescence microscope.

14
Chapter 4: Skeletal Myocyte Model

4.1 Myomixer is essential for myotube formation
The Myomixer knockout (KO) cell line was created by disrupting the open reading frame
(ORF) of the gene with lentiviruses that expressed Cas9 and two small guide RNAs that targeted
two sections of the Myomixer ORF 122 base pairs away from one another (Bi, et al. 2017). PCR
analysis confirmed the presence of numerous indel mutations in the gene and western blot
analysis confirmed the absence of Myomixer protein expression.
To verify that KO expression of Myomixer reduces the presence of myotubes in
differentiated myoblasts, I grew Myomixer KO and Myomixer wildtype (WT) C2C12 cells in
differentiation media for a week and compared nuclear content between the two cell lines.
Differentiation started when cells were at 60-70% confluency to allow for clear and discernable
staining of individual cells without losing the confluency needed for neighboring cells to initiate
recognition and activation of the fusion process.

4.1.1 Results
After Giemsa staining, C2C12 cells expressing Myomixer KO showed a reduced amount
of large myotubes compared to Myomixer WT cells, confirming that Myomixer is an essential
protein for myocyte fusion (Fig. 3). However, quantification of nuclear content was not possible
with this method of visualization: membrane borders between cells in close proximity were not
discernable and thus individual cells could not be counted as well as the number of nuclei
contain within the cell.

15

Figure 3: Knockout of Myomixer expression reduces myotube formation. WT and KO
Myomixer myoblasts were differentiated for a week and stained with Giemsa to show the
requirement of Myomixer for fusion. Cells stained dark purple represent myotubes.

16
4.2 Skeletal myocyte adenovirus infection
In order to visualize individual myoblasts and quantify nuclear content, I infected
differentiating C2C12 with adenovirus expressing GFP. Using an adenovirus allowed me to
visualize a random portion of the cell population while keeping the needed confluency to form
myotubes, allowing for single cell visualization. Cells were fixed and stained for DAPI on the 7
th

day of differentiation. The number of nuclei per cell were counted against total number of
myonuclei that were GFP positive.

4.2.1 Results
KO Myomixer cells had a decreased amount of myotube formation compared to WT
Myomixer cells (8.25% versus 39.67%) (Fig. 4). Decreased fusion is also reflected within the
increased amount of mononuclear (70.33% versus 47.65%) and binuclear cells (21.41% versus
12.67%) within KO Myomixer cultures compared to WT Myomixer. Because I cannot visually
distinguish between binuclear cells that have fused or have undergone karyokinesis, the increase
in binucleated cells may indicate that there may be a low level of fusion occurring within the KO
Myomixer culture. It could also be possible that the knockout of Myomixer expression promotes
karyokinesis. The experiment must be repeated to obtain more accurate results.

17

0
10
20
30
40
50
60
70
80
1 nuclei/cell 2 nuclei/cell ≥3 nuclei/cell
% total myonuclei
Myomixer Control
Myomixer KO
Figure 4: Myomixer is essential for myoblast fusion. (A) WT and KO Myomixer myoblasts
were differentiated, infected with adenovirus expressing GFP and stained with DAPI. White
arrows indicate mononuclear cells. (B) Quantification of fusion in WT and Myomixer KO
myoblasts cultures (10 images per sample, average of 20 cells counted per image, n=1).

18
Chapter 5: ES-derived Cardiomyocyte Model

5.1 Genetic enrichment of ES-derived cardiomyocytes
To study cardiomyocytes in vitro, investigators developed a genetic enrichment approach
that creates highly enriched cultures of cardiomyocytes from differentiated ES cells (Pasumarthi
& Field, 2002). The method relies on the use of two transcriptional units transfected into
undifferentiated ES cells on a common vector background. The first transcriptional unit is
comprised of a phosphoglycerate kinase (pGK) promoter linked to an hygromycin resistance
gene to enrich for cells carrying the DNA, designated as pGK-hygro. The second transcriptional
unit is comprised of a cardiac specific gene promoter to α-cardiac myosin heavy chain that is
linked to another enrichment gene conferring resistance to aminoglycoside phosphotransferase,
designated as MHC-neo. Undifferentiated ES cells are transfected with the MHC-neo/pGK-
hygro construct and treated with hygromycin to enrich for cells carrying the DNA. The ES cells
are then differentiated and treated with G418 once cardiomyogenesis is observed. Using this
approach, I can generate highly enriched cardiomyocyte cultures as well as investigate the role of
TNNI3K in cardiomyocyte cell cycle arrest.

5.2 Validation of Cardiomyocyte Enrichment from Differentiated ESC
In order to validate the presence of cardiomyocytes from differentiated ES cells, I looked
for spontaneous beating within the culture and I stained for a cardiac-specific protein with
immunofluorescence. Spontaneous beating was observed 4 to 7 days within EB attachment, after
which G418 antibiotic was added to select for MHC expressing cells. Cells were stained for

19
cardiac troponin I (cTnI) 2 weeks after initiating G418 selection. CTnI is the inhibitory subunit
of troponin, the calcium-sensing protein of the thin filament of the sarcomere.

5.2.1 Results
cTnI-positive cells were present within ESC-differentiation cultures. The staining pattern
resembles the sarcomeric structures of contractile cardiomyocytes (Fig. 5). Several cells may
have shown progression of karyokinesis into binucleated cardiomyocytes as seen in the zoomed
in images (Fig. 5). Cultures that were sustained in selection media for 2 months longer displayed
similar staining patterns. It is important to note that the cultures would not expand in
proliferation and would halt migration from the initial EB 2 weeks after attachment. These
observations could suggest that the cardiomyocytes are at terminal differentiation. Interestingly,
several cells showed speckled disorganized staining of cTnI. This could suggest that the
cardiomyocyte could be undergoing sarcomere disassembly to allow for karyokinesis and
cytokinesis. More than likely, the cell could be unhealthy.

20

Figure 5: ES-derived cardiomyocytes after G418 selection. Differentiated ESC were
stained for cTnI and DAPI after G418 treatment.

21
5.3 Single cell visualization of cardiomyocytes
En mass differentiation of ES cells within EBs allows for generation of large
cardiomyocyte cultures, but it is difficult to resolve between individual cells that have not
migrated away from the attached EB. Thus, clumping and overlapping of cardiomyocytes makes
it difficult to quantify mono- or binucleation with cardiomyocytes. To address this, I passaged
the cardiomyocytes into less dense cultures and visualized with immunofluorescence.

5.3.1 Results
After growing cardiomyocytes in selection media for 2 weeks, I was able to passage
cardiomyocytes onto glass coverslips at a lower density. Spontaneous beating was confirmed the
day after initial seeding. Staining pattern resembled sarcomeric structures and many of the
cardiomyocytes that had adhered to the plastic appeared mononuclear (Fig. 6). Although the
presence of cTnI staining was present, the number of cardiomyocytes was markedly reduced
compared to the original culture. It could be possible that the trypsinization process rendered the
cells unviable or that the cells may not be able to adhere to the plastic and would need a protein
matrix for future passaging.

Figure 6: Single cell visualization of a
cardiomyocyte. Differentiated ESC
were passaged to a lower density and
stained for cTnI and DAPI after G418
selection.

22
Chapter 6: Discussion
Our results demonstrate that skeletal myocytes and ES-derived cardiomyocytes may be
useful tools for exploring the molecular mechanism for TNNI3K. Although skeletal myocytes do
not have the same function and behavior as cardiomyocytes, both share similar sarcomeric
protein structures that could be involved in TNNI3K function. Our findings have shown that
skeletal myocytes lack the ability to fuse when the open reading frame of Myomixer is disrupted
by Cas9-expressing lentivirus and small guide RNAs targeting the gene's ORF. Membrane fusion
is not a behavior exhibited by cardiomyocytes; thus, KO Myomixer C2C12 cells present a
possible cellular model for studying TNNI3K. However, our results show that there is a low
level of fusion of myocytes after 7 days in low-serum differentiation media, notably with a slight
increase in binucleated cells. This could be due to incomplete selection of pure KO Myomixer
C2C12, allowing cells expressing the WT form of Myomixer to propagate after several rounds of
expansion. Another possible explanation for fusion within the KO Myomixer culture is that there
may be another protein responsible for fusion that does not require Myomixer. It is important to
note that the cells were not stained for differentiation markers such as myosin heavy chain. The
study that discovered Myomixer performed a similar experiment studying the nuclear content of
myotubes and myocytes in WT and KO Myomixer primary mouse myoblasts and C2C12 cells
(Bi, et al., 2017). However, rather than using a general cytoplasmic stain or adenovirus infection
of GFP, the group stained for myosin heavy chain and quantified the nuclei. Not all myocytes
within the culture were stained positive for MHC Although our study is not focused on the
differentiation of myocytes, it is important to take into account that the culture does not
uniformly differentiate and could potentially affect how TNNI3K expression affects the behavior
of cells at different stages of differentiation. To remedy this, I could perform stain the cells for

23
MHC, similar to Bi’s study, and only account for myocytes that have reached a specific stage of
myocyte differentiation. Moreover, this experiment must be repeated in order to accurately
conclude that the amount of multinucleated cells decreases within Myomixer KO cultures
compared to Myomixer control cells.
The cultures of ES-derived cardiomyocytes may also have a similar problem with non-
homogeneity in terms of differentiation. While most of the cardiomyocytes displayed organized
linear structures when stained for cTnI, some of the cells had a speckled pattern of cTnI staining.
Although these cells may just be unhealthy, it could be possible that the disorganized pattern of
cTnI is from disassembly of the sarcomere. The sarcomere occupies a large volume of the cell
and could thus obstruct karyokinesis and cytokinesis. In order to proliferate, cardiomyocytes
must disassemble their sarcomeres during mitosis and reassemble the structure when cell
division is complete and resume contractions (Ahuja, et al., 2004). A study investigating
sarcomere assembly and disassembly in proliferative cardiomyocytes found that alpha-actinin
and titin disassembled as early as prometaphase and that sarcomere disassembly and break down
of the nuclear envelope occurred simultaneously (Fan, et al. 2015). However, it is unlikely that
our ES-derived cardiomyocytes are in proliferative growth since the culture never reaches
confluency and show signs of binucleation. Nonetheless, it would be interesting to further study
the dynamics of sarcomere assembly and disassembly within ES-derived cardiomyocytes before
they reach terminal differentiation.
To quantify the nuclear content of our ES-derived cardiomyocytes, I attempted to
infected the cells with adenovirus carrying the GFP gene, similar to adenoviral infection of
C2C12 cells. Infection did not seem to negatively affect cardiomyocyte health as spontaneous
beating continued up to 2 weeks after infection. GFP signal was not lost over time most likely

24
due to no cardiomyocyte proliferation. Viral titration is needed to control the amount of cells
receiving the virus.
To find if the nuclear content of C2C12 cells expressing KO Myomixer would change
when expressing TNNI3K, we plan to infect the cells with adenovirus expressing GFP and
GFP/TNNI3K in the future. We would expect for an increase in binucleated cells compared to
mononuclear cells within the KO Myomixer culture. Further studies could confirm if TNNI3K
can cause changes in cell morphology, such as hypertrophy or changes in sarcomeric length, as
well as any potential increases in phosphorylation. The same experiments may be performed on
the ES-derived cardiomyocytes in addition to confirming the cellular location of TNNI3K. The
expression profile of the culture would also need to be identified in order to determine if the
cardiomyocytes are naive or highly differentiated cells, which could be change the way TNNI3K
operates within the cardiomyocyte. Future work could also include refinement of the ES-cell
selection process for atrial versus ventricular cardiomyocytes or conduction system versus
working cardiomyocytes can be done with changes in the cell type-restricted promoter to target
aminoglycoside phosphotransferase expression.
Although there are many studies investigating the cardio-protective potential of TNNI3K
inhibition, the downstream targets and binding partners of the protein are still undetermined.
Understanding the molecular mechanism and signaling pathway of TNNI3K in heart
development and pathology is important for developing therapies for myocardial infarct patients
and progression of heart disease.

25
Chapter 7: References
Ahuja, P., Perriard, E., Perriard, J.C., Ehler, E. (2004). Sequential myofibrillar breakdown
accompanies mitotic division of mammalian cardiomyocytes. J Cell Sci. 117: 3295-3306.

Ali, S.R. Hippenmeyer, S., Saadat, L.V., Luo, L., Weissman, I.L., Ardehali, R. (2014). Existing
cardiomyocytes generate cardiomyocytes at a low rate after birth in mice. Proc Natl Acad Sci
USA. 111: 8850–8855.

Bae, S., Xiao, Y., Li, G., Casiano, C.A., Zhang, L. (2003). Effect of maternal chronic hypoxic
exposure during gestation on apoptosis in fetal rat heart. Am J Physiol Heart Circ Physiol. 285:
H983-H990.

Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabe-Heider, F., Walsh, S.,
Zupicich, J., Alkass, K., Buckholz, B.A., Druid, H., Jovinge, S., Frisen, J. (2009). Evidence for
cardiomyocyte renewal in humans. Science. 324: 98–102.

Bi, P., Ramirez-Martinez, A., Li, H., Cannavino, J., McAnally, J.R., Shelton, J.M., Sanchez-
Ortiz, E., Bassel-Duby, R., Olson, E.N. (2017). Control of muscle formation by the fusogenic
micropeptide. Science. 356(63350): 323-327.

Bubb, K.J., Cock, M.L., Black, M.J., Dodic, M., Boon, W.M., Parkington, H.C., Harding, R.,
Tare, M. (2007). Intrauterine growth restriction delays cardiomyocyte maturation and alters
coronary artery function in the fetal sheep. J Physiol. 578: 871-881.

Corstius, H.B., Zimanyi, M.A., Maka, N., Herath, T., Thomas, W., van der Laarse, A., Wreford,
N.G., Black, M.J. (2005). Effect of intraeuterine growth restriction on the number of
cardiomyocytes in rat hearts. Pediatr Res. 57: 796-800.

Feng, Y., Cao, H.Q., Liu, Z., Ding, J.F., Meng, X.M. (2007). Identification of the dial specificity
and the functional domains of the cardiac-specific protein kinase TNNI3K. Gen Physiol Biophys.
26: 104-109.

Giraud, G.D., Louey, S., Jonker, S., Schultz, J., Thornburg, K.L. (2006). Cortisol stimulates cell
cycle activity in the cardiomyocyte of the sheep fetus. Endocrinology. 147: 3643-3649.

Jopling, C., Sleep, E., Raya, M., Martı´, M., Raya, A., Izpisu´ a Belmonte, J.C. (2010). Zebrafish
heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature. 464:
606–609.

26
Kellerman, S., J.A. Moore, W. Zierhut, H.G. Zimmer, J. Campbell, and A.M. Gerdes. (1992).
Nuclear DNA content and nucleation patterns in rat cardiac myocytes from different models of
cardiac hypertrophy. J Mol Cell Cardiol. 24: 497-505.

Kikuchi, K., Holdway, J.E., Werdich, A.A., Anderson, R.M., Fang, Y., Egnaczyk, G.F., Evans,
T., Macrae, C.A., Stainier, D.Y., and Poss, K.D. (2010). Primary contribution to zebrafish heart
regeneration by gata4(+) cardiomyocytes. Nature. 464: 601–605.

Kim, J.H., Ren, Y., Ng, W.P., Li, S., Son, S., Kee, Y.S., Zhang, S., Zhang, G., Fletcher, D.A.,
Robinson, D.N., Chen, E.H. (2015). Mechanical tension drives cell membrane fusion. Dev Cell.
32: 561-573.

Pasumarthi K.B.S., & Field, L.J. (2002). Cardiomyocyte enrichment in differentiating ES cell
cultures: Strategies and applications. Methods Mol Bio. 184: 157-168.

Patterson, M., Barske, L., Van Handel, B., Rau, C.D., Gan, P., Sharma, A., Parikh, S., Denholtz,
M., Huang, Y., Yamaguchi, Y., Shen, H., Allayee, H., Crump, J.G., Force, T.I., Lien, C.L.,
Makita, T., Lusis, A.J., Kumar, S.R., Sucov, H.M. (2017). Frequency of mononuclear diploid
cardiomyocytes underlies natural variation in heart regeneration. Nat Genet. 49(9): 1346-1353.

Porrello, E.R., Mahmoud, A.I., Simpson, E., Hill, J.A., Richardson, J.A., Olson, E.N., and Sadek,
H.A. (2011). Transient regenerative potential of the neonatal mouse heart. Science. 331: 1078–
1080.

Poss, K.D., Wilson, L.G., Keating, M.T. Heart regeneration in zebrafish. Science. 298: 2188–
2190 (2002).

Puente, B.N., Kimura, W., Muralidhar, S.A., Moon, J., Amatruda, J.F., Phelps, K.L., Grinsfelder,
D., Rothermel, B.A., Chen, R., Garcia, J.A., et al. (2014). The oxygen-rich postnatal
environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell. 157,
565–579.

Senyo, S.E., Steinhauser, M.L., Pizzimenti, C.L., Yang, V.K., Cai, L., Wang, M., Wu, T.D.,
Guerquin-Kern, J.L., Lechene, C.P., Lee, R.T. (2013). Mammalian heart renewal by pre-existing
cardiomyocytes. Nature. 493: 433-6.

Soonpaa M.H., and Field, L.J. (1994). Assessment of cardiomyocyte DNA synthesis during
hypertrophy in adult mice. Am J Physiol. 266: H1439-445.

27
Soonpaa, M.H., Kim, K.K., Pajak, L., Franklin, M., Field, L.J. (1996) Cardiomyocyte DNA
synthesis and binucleation during murine development. Am J Physiol. 271: H2183–H2189.

Tang, H., Xiao, K., Mao, L., Rockman, H.A., Marchuk, D.A. (2013). Overexpression of
TNNI3K, a cardiac specific MAPKKK, promotes cardiac dysfunction. J Mol Cell Cardiol. 54:
101-111.

Vagnozzi, R.J., Gatto Jr., G.J., Kallander, L.S., Hoffman, N.E., Mallilankaraman, K., Ballard,
V.L.T., Lawhorn, B.G., Stoy, P., Philp, J., Graves, A.P., Naito, Y., Lepore, J.J., Gao, E., Madesh,
M., Force, T. (2013). Inhibition of the cardiomyocyte-specific kinase TNNI3K limits oxidative
stress, injury, and adverse remodeling in the ischemic heart. Sci. Transl. Med. 5(207): 207ra141.

Vujic, A., Bassaneze, V., Lee, R.T. (2017) Genetic insights into mammalian heart regeneration.
Nat Genet. 49(9): 1292-1293.

Wang, L., Wang, H., Ye, J., Xia, R., Song, L., Shi, N., Zhang, Y.W., Chen, X., Meng, X.M.
(2011). Adenovirus-mediated overexpression of cardiac troponin I-interacting kinase promotes
cardiomyocyte hypertrophy. Clin Exp Pharmacol Physiol. 38: 278-84.

Wang, X., Wang, J., Su, M., Wang, C., Chen, J., Wang, H., Song, L., Zou, Y., Zhang, L., Zhang,
Y., Hui, R. (2013). TNNI3K, a cardiac-specific kinase, promotes physiological cardiac
hypertrophy in transgenic mice. PLoS ONE. 8(3): e58570.

Wheeler, F.C., Tang, H., Marks, O.A., Hadnott, T.N., Chu, P.L., Mao, L., Rockman, H.A.,
Marchuk, D.A. (2009) Tnni3k modifies disease progression in murine models of
cardiomyopathy. PLoS Genet. 5(9): e1000647.

Zhao, Y., Meg, X.M., Wei, Y.J., Zhao, X.W., Liu, D.Q., Cao, H.Q., Liew, C.C., Ding, J.F.
(2003). Cloning and characterization of a novel cardiac-specific kinase that interacts specifically
with cardiac troponin I. J Mol Med. 81: 297-304.

Abstract (if available)

Abstract Although adult mammalian cardiomyocyte regeneration after injury is limited, the myocardium can be repaired in other contexts such as the hearts in zebrafish, neonatal mice and many other species in embryonic development. Our lab has shown that the expression of cardiac troponin I-interacting kinase (TNNI3K) and the population of mononuclear diploid cardiomyocytes (MNDCM) contribute to varying degrees of heart regeneration, where knockout of Tnni3k expression in mice raised MNDCM content and increased cardiomyocyte proliferation after injury while overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. In order to understand the role of TNNI3K in cardiomyocyte regeneration, I am using two in vitro models, a mouse embryonic stem cell-cardiomyocyte cell line and a mouse skeletal myoblast cell line. Though the cellular localization and phosphorylation targets of TNNI3K have not been verified, the kinase has been shown to play a role in cardiac remodeling after injury and promote progression of cardiomyopathy in mice. Despite not being expressed in skeletal muscle, TNNI3K could potentially interact with skeletal sarcomere proteins and could reveal the mechanistic role of TNNI3K in cell polyploidization. I therefore aim to investigate whether TNNI3K causes binucleation within the two cell lines. I have demonstrated that cardiomyocytes can be derived from mouse embryonic stem cells using plasmid transfection. I have also shown that differentiating mouse skeletal myoblasts can be prevented from forming myotubes when the Myomixer gene is knocked out. Ongoing work includes virally infecting the two cell lines to overexpress TNNI3K and observing cell ploidy.

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Asset Metadata

Creator Lim, Kimberly (author)

Core Title TNNI3K expression affects nucleation of cardiomyocytes and skeletal myoblasts

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 08/02/2020

Defense Date 08/02/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag binucleation,cardiomyocyte,diploid,heart regeneration,mononuclear,myoblasts,Myocardium,Myomixer,myotubes,OAI-PMH Harvest,skeletal,stem cell,TNNI3K

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Advisor Sucov, Henry (committee chair), Ching-Ling, Lien (committee member), Xu, Jian (committee member)

Creator Email kimlim1992@gmail.com,limka@usc.edu

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