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IMPACT 201
Innovative Minds Partnering to Advance Curative Therapies
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Contributors
Student Name
Major
Academic Year
Email Address
Kwame Opoku
Bio-Medical Sciences Senior
KwameAkyeampong
opoku.akyeampong@m
nsu.edu
Okhumhekho A. Kassim
Bio-Medical Sciences Junior
Okhumhekho.kassim@
mnsu.edu
Ali Oku
Bio-Medical Sciences Junior
[email protected]
Uyi Imasuen
Bio-Medical Sciences Junior
[email protected]
u
Faculty Sponsor
Department
Email Address
Mailing Address
Dr. Penny Knoblich
Biology
penny.knoblich@
Minnesota State
mnsu.edu
University
Department of
Biological Sciences S242 Trafton Science
Center South
Mankato ,MN 56001
Submission Information
IMPACT Question: What is the underlying cause of hypoplastic left heart syndrome
(HLHS)?Hypothesis: During critical periods of heart development, abnormal activity of
the myocyte enhancer factor 2C (MEF2C) as a result of irregular Notch signaling , folic
acid levels, and/or genetic mutations is the underlying cause of HLHS
Background Information:
In trying to uncover the underlying cause of hypoplastic left heart syndrome (HLHS), we
found that a great deal of information from research has become available ever since the
discovery of the disease in 1952 [5]. Despite the amount of research that has been done on
HLHS, the cause still remains unknown. This is a concern because HLHS is a complex heart
disease that affects 1 out of every 4,344 babies in the US every year [4]. There are many surgical
procedures and treatments that have improved the prognosis of HLHS, but HLHS patients still
undergo later complications in life. HLHS has been linked to various chromosomal disorders
including Turner syndrome, Jacobsen syndrome, and Trisomies 10, 11, & 13 [2]. There have also
been links between maternal levels of folic acid during pregnancy and congenital heart disease
[6].However, understanding the origin of the disease could result in disease prevention.
The large array of phenotypes that can be seen in individuals with HLHS complicates the
identification of a cause. In an article by Cambridge Press, the histopathology and pathogenesis
of HLHS was extensively reviewed. The review examined the wide array of phenotypic
expression associated with the disease. Phenotypes ranged from isolated to multiple
combinations of cardiovascular malformations including ventricular, vascular, septal, valvular,
and other defects. It was concluded that because there were many different phenotypic
expressions of HLHS, that there must be a concert of factors working together to cause HLHS as
opposed to just a single factor [2]. The vast array of genetic mutations and copy number variants
linked to this disease, in addition to the differing number of phenotypes observed, has led us to
believe the variants converge on a general signaling/regulatory pathway. There is a plethora of
transcription factors, signaling pathways, and genes that appear a necessity in left and right heart
formation. These include; HAND1, HAND2, NKX2.5, MEF2C, and Notch 1. HAND 1 and
HAND 2 are two basic helix loop transcription factors that are especially important in heart
looping, creating the left and right axis of the heart.
One regulatory pathway that is crucial in cardiac development is the Notch signaling
cascade. Notch signaling is crucial for early cell differentiation in various organ systems. The
Notch receptors and ligands can be regulated by a multitude of different factors. One such factor
that will be discussed is folic acid levels [6]. Folic acid has also been shown to significantly
reduce congenital defects in children. In zebrafish, it has been shown that a competitor molecule,
methotrexate (MTX), diminishes production of tetrahydrofolic acid. This results in decreased
transcription levels of HAND 2 and MEF2C, and abnormal heart formation. These findings led
us to our hypothesis.
Hypothesis and Rationale:
The hypothesis we reached was: “During critical periods of heart development, abnormal
activity of the myocyte enhancer factor 2C (MEF2C) as a result of irregular Notch signaling ,
folic acid levels, and/or genetic mutations is the underlying cause of HLHS.” Knowing that a
plethora of syndromes and diseases can be seen in some individuals with HLHS we concluded
that one of the more general transcription factors must be the causative agent of this disease. In a
study using disease specific induced pluripotent stem cells (HLHS-IPS) and normal bi-ventricle
stem cells, gene expression and cardiomyogenic differentiation potential was compared. In
HLHS-IPS there was a decrease in differentiation potential and gene expression of Hand1,
Hand2, Tbx2, NKX2-5 and a decrease in Notch/Hey1 signaling. [9]. Our research team decided
to look for a possible factor that could affect these different genes/TF. MEF2C is part of the
MEF2 family of transcription factors that has a vital role in regulating gene expression in tissues
such as the heart [1]. We believe that the malfunctioning of this regulator is responsible for the
decreased expression levels and epigenetic modifications shown in HLHS-IPS. We proceeded to
predict what factors could result in the improper functioning of MEF2C activity. In the diagram
below, one of the important receptors that bind to MEF2C is Notch-1. It has been shown that
Notch1 signaling affects the myogenic activity of MEF2C and the activity of other Basic HelixLoop-Helix (BHLH) transcription factors. Wilson-Rawls and colleagues [14] reported that Notch
inhibits myogenesis by inactivating MEF2C. Notch is a regulatory signaling pathway with many
receptors [14]. Mutations in the Notch1 receptor has been strongly linked to the onset of HLHS
[13]. In various articles, it has been reported that HLHS was more prevalent in males than
females [10,2,14]. This is relevant because recent studies have demonstrated a paternally
inherited mutation in the gene for Notch signaling [13]. One study suggested that this mutation,
in addition to diminished myogenic capability, could possibly explain the onset of HLHS. The
diminished myogenic activity may be the result of notch1 effect on MEF2C [14]. MEF2C
binding activity also interacts strongly with an AT-rich sequence of muscle promoters, which
plays specific roles in cardiac muscle structure and enables maximum activity interaction [1].
Decreased myogenic capability can explain the underdevelopment of tissue on the left side of the
heart. MEF2C also controls) metalloproteinase -2 (mmp2), which is important in cytoskeleton
remodeling and insuring the left dorsal side of the heart grows faster than the right. In our
explanation, if MEF2C is significantly affected by any of the aforementioned pathways, then the
proliferation of the left side might not occur properly. Another phenotype which is the
overgrowing of endocardial cushions and improper valve formation may also be a result of this
improper mmp2 activity. [6].
Early on in fetal development, folic acid is a key player in ensuring proper cell
differentiation and proliferation. There have been numerous studies linking folic acid
deficiencies during fetal development to various heart malformations. Additionally, Folic acid
has also been shown to increase notch activity [11]. Folic acid deficiency negatively influences
notch signaling by decreasing the production of Notch 1 and Hes 1. Notch 1, a surface cell
protein, together with Hes 1, an effector cell, are critical in proper notch signaling [11].
Therefore, irregular activity of the notch signaling system as a result of insufficient levels of folic
acids during fetal development could lead to HLHS by affecting the production of MEF2C as
discussed above. Additionally it is important to note that folic acid deficiencies have also been
directly connected to the activity of Hand 1, Flk 1 and VEGF which are important parts of the
development of a fetal heart [6]. This hypothesis is further supported with the recorded large
incident of HLHS in Maryland associated with solvents and polychlorinated biphenyls released
from industrial factories into the air. [10]. In the study, trichloroethylene was among the major
compounds found in most of the implicated solvents. Studies have also shown that
trichloroethylene could cause folate deficiencies in mammals [3]. This relationship between
trichloroethylene and folate deficiency together with the observed incident of HLHS in
Baltimore strongly suggests a connection between folate deficiencies and HLHS.
Significance and Innovation:
The significance and innovation of out proposed hypothesis comes from our focus on
genetics. The expression of MEF2C and Notch 1 receptors for Notch signaling depends heavily
on the unaltered presence of the genes coding for them. We believe that there could be mutations
such as; deletions, mutations, or translocations that could be the culprit for the mutations to these
genes. The mutations on the MEF2C and Notch 1 receptor gene would have to be mutations that
negatively alter them; either decreasing the concentration or functionality.
In the medical field, our hypothesis can primarily change the diagnosis of
HLHS. Because we have linked genetic disorders specifically (errors in nucleotide sequences) to
the MEF2C transcription factor and the Notch 1 receptors, it is possible to begin using
embryonic genetic testing to uncover mutations that can lead to HLHS. The concentrations of
mediators necessary for cardiac development could be closely and continuously monitored
through amniotic fluid analysis and fetal biopsy. By noting changes in the concentrations of
these substances that are similar to HLHS patients, healthcare workers can have more assurance
when making their diagnosis.
This hypothesis is suitable for testing with regenerative medical techniques and tools
surrounding genetics. Testing this hypothesis would require the development of knockouts of the
MEF2C and/or Notch 1 receptor gene, which could be done using siRNA in different embryonic
hearts cells at different times. Researchers at the University of California have been able to grow
tiny human beating hearts from reprogrammed skin derived stem cells. These hearts could be
used to test the effects of our knockout or knockdown on heart development [12]. In addition,
another study could be conducting in which folic acid administered to each heart is varied during
the course of development. Through doing this, it can be observed as to whether or not
ventricular malformations or other HLHS associated malformations occur in relationship to the
timing of the MEF2C and/or Notch 1 receptor genes knockdown or varied folic acid levels.
Based on our results we would be able to support or reject our hypothesis that specifically
implicates the MEF2C gene as the main factor of the occurrence of HLHS.
The CRISPR-Cas 9, a fairly new and recent genomic engineering tool, would be
invaluable in this experiment. Cas9 is a protein that comes from a single RNA strand that is then
cleaved into a smaller RNA strands known as crRNA which are found in many organisms. The
crRNA is then translated into nucleolytic enzymes [7]. This tool would allow alteration of
MEF2C gene, which is crucial to this experiment. If MEF2c is implicated as the main factor
interfering with successful heart formation. This tool could be used to amend a germline cells
with mutations in MEF2C or Notch-1.
References:
1. Black, B. L., & Cripps M. R. (2010). Heart development and regeneration: Myocyte
enhancer factor 2c transciption actors in heart development and disease. Oxford:
Academic Press.
2. Cole, C. R., & Eghtesady, P. (2016). The myocardial and coronary histopathology and
pathogenesis of hypoplastic left heart syndrome. Cardiology in the Young, 26(01), 19-29.
doi:10.1017/S1047951115001171
3. Dow, J. L., & Green, T. (2000). Trichloroethylene induced vitamin B12 and folate
deficiency leads to increased formic acid excretion in the rat.Toxicology, 146(2-3), 123136.
4. Facts about Hypoplastic Left Heart Syndrome. (2015). Retrieved January 15, 2016, from
http://www.cdc.gov/ncbddd/heartdefects/hlhs.html
5. Fruitman, D. S. 2000. Hypoplastic left heart syndrome: prognosis and management
options. Paediatr Child Health.5(4),219-225
6. Gilbert, S. F. (2014). Developmental Biology. Sunderland, MA: Sinauer Associates, Inc
7. Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and Applications of
CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262–1278.
http://doi.org/10.1016/j.cell.2014.05.010
8. Jeffrey A. Feinstein, D. Woodrow Benson, Anne M. Dubin, Meryl S. Cohen, Dawn M.
Maxey, William T. Mahle, Elfriede Pahl, Juan Villafañe, Ami B. Bhatt, Lynn F. Peng,
Beth Ann Johnson, Alison L. Marsden, Curt J. Daniels, Nancy A. Rudd, Christopher A.
Caldarone, Kathleen A. Mussatto, David L. Morales, D. Dunbar Ivy, J. William Gaynor,
James S. Tweddell, Barbara J. Deal, Anke K. Furck, Geoffrey L. Rosenthal, Richard G.
Ohye, Nancy S. Ghanayem, John P. Cheatham, Wayne Tworetzky, Gerard R. Martin,
Hypoplastic Left Heart Syndrome: Current Considerations and Expectations, Journal of
the American College of Cardiology, Volume 59, Issue 1, Supplement, 3 January 2012,
Pages S1-S42, ISSN 0735-1097, http://dx.doi.org/10.1016/j.jacc.2011.09.022.
9. Kobayashi, J., Yoshida, M., Tarui, S., Hirata, M., Nagai, Y., Kasahara, S., Naruse, K.,
Ito, H., Sano, S., & Oh, Hidemasa. (2014). Directed Differentiation of Patient-Specific
Induced Pluripotent Stem Cells Identifies the Transcriptional Repression and Epigeneteic
Modification of NKX2-5, HAND1, and NOTCH1 in Hypoplastic Left Heart Syndrome.
PLoS ONE. 9(7): e102796. doi: 10.13/1/o102796
10. Kuehl, K. S., & Loffredo, C. A. (2006). A Cluster of Hypoplastic Left Heart
Malformation in Baltimore, Maryland. Pediatric Cardiology, 27:25–31. DOI:
10.1007/s00246-005-0859-x
11. Lui1, H., Huang1, G., Zhang1, X., Ren1, D., & Wilson2, J. X. 2010. Folic Acid Supplementation
Stimulates Notch Signaling and Cell Proliferation in Embryonic Neutral Stem Cells.174-180
12. Ma, Z., Wang, J., Loskill, P., Huebsch, N., Koo, S., Svedlund, F. L., . . . Healy, K. E.
(2015). Self-organizing human cardiac microchambers mediated by geometric
confinement. Nature Communications Nat Comms, 6, 7413.
13. Theis, J.L., Hirstka, S.C.L., Evans, J.M., O’Byrne, M.M., Andrade, M., O’Leary, P.W., &
Nelson, T.J. (2015). Compound heterozygous NOTCH1 mutations underlie impaired
cardiogenesis in a patient with hypoplastic left heart syndrome. CrossMark. Hum Genet
134:1003–1011. doi:10.1007/s00439-015-1582-1
14. Wilson-Rawls, J., Molkentin, J. D., Black, B,L,. & Olson, N. E. (1998). Activated notch
inhibits myogenic activity of the MADS-box transcription factor myocyte enhancer factor
2c. Molecular and Cellular Biology, 19 (4). Retrieved from
http://mcb.asm.org/content/19/4/2853.short.