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The effects of abnormal MEF2C activity induced by Irregular Folic acid levels and Notch Signaling on Early Heart Development
Kwame Opoku Akyeampnog, Okhumhekho Kassim, Uyi Jefferson Imasuen, Ali Eastman Oku
Faculty Mentor: Dr. Penny Knoblich
Minnesota State University, Mankato, MN, Department of Biological Sciences.
Abstract
HLHS is a complex heart disease that affects 1 out of every 4,344
babies in the US every year (Facts about Hypoplastic Left Heart
Syndrome, 2015). Myocyte enhancing factor (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 (Black, et.
al, 2010).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 It is therefore plausible that a malfunction of this
regulator (MEF2C) during critical periods od heart development,
would lead to HLHS. The notch signaling pathway is one of the
major regulators of the expression of MEF2C (Lui1, 2010). Folic
acid has strongly been linked to different congenital heart
diseases and plays a key role in function of the notch signaling
system. We, therefore, plan to knockout the MEF2C gene using
CRISPR-Cas 9 (or siRNA) under varying concentrations of folic
acid. The cells with the knocked-out mef2c gene will then be
used to develop miniature hearts (using stem cells ) to
determine if any abnormal heart phenotypes develops.
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.
Figure 1
Figure 3
MEF2C has both trans-activating and DNA binding activities that is
significant for cardiac myogenesis. The transcription factor is
controls morphogenesis and myogenesis, and can be inhibited by
different regulating activity pathway. The team believed the major
activities that affects MEF2C regulation of gene expression in
cardiac tissues are:
Significance
•
The significance and innovation of our proposed hypothesis
comes from our focus on genetics and/or gene expression. The
expression of MExF2C and Notch 1 signaling depends heavily on
the presence of the genes coding for them as well as
transcription factors. We believe that either genetic mutations,
or maternal conditions altering gene expression, could underlie
the development of HLHS
•
Our hypothesis can primarily change the timing of the diagnosis
of HLHS. Because we have linked genetic disorders specifically
(errors in nucleotide sequences) to the MEF2C transcription
factor and the Notch signaling cascade, it is possible to begin
using embryonic genetic testing to uncover mutations that can
lead to HLHS.
•
This hypothesis is suitable for testing with tools surrounding
genetics. Testing this hypothesis would require the development
of knockouts of the MEF2C gene, which could be done using
siRNA in different embryonic hearts cells at different times.
•
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
• Notch signaling: Notch is a regulatory signaling pathway with
many receptors. Mutations in the Notch1 receptor has been
strongly linked to the onset of HLHS. Several studies have
demonstrated a paternally inherited mutation in the gene for
Notch signaling and more prevalent in males than females. The
down regulation of notch2 receptor affecting MEF2c could
result the increase of HLHS in males
• Decreased Folic Acid Levels: Folic acid is a key player in
ensuring proper cell differentiation and cell proliferation, and
reducing heart malformations during fetal development. Folic
acid has also been shown to increase notch signaling including
notch1 activity. 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
The 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. Decreased
myogenic capability can explain the underdevelopment of tissue on
the left side of the heart.
Background
Figure 3. A picture depicting the function of the CRSPR Cas 9.
The CRISPR/Cas9 system for targeted genome editing. (n.d.). Retrieved
February 1, 2016, from
http://www.clontech.com/US/Products/Genome_Editing/CRISPR_Cas
9/Resources/About_CRISPR_Cas9
Figure 2
Innovation
Axis label here
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 9, 11, & 13 (Cole & Eghtesady 2016).
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 (Cole &
Eghtesady, 2016). 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.
One regulatory pathway that is crucial in cardiac development is
the Notch signaling cascade. 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 (Gilbert, 2014).
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.
Rationale
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.
Figure 1. Visual representation of transcription factors that
interact and are crucial in cardiac development.
Heart Development Pathway in Heart Development SuperPath.
(n.d.). Retrieved December 20, 2015, from
http://pathcards.genecards.org/pathway/74
References
Figure 2. A picture outlining early heart development of an
embryo. (a)3D cardiac microchamber generated from WTC
hiPSCs on a 400-mm pattern, where cardiomyocytes only
appeared in the centre and myofibroblasts on the perimeter.
Panels above and to the right of the main panel represent the
z axis projection images at their respective x and y crosssections
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.
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 ( Ma et. al, 2015). In addition,
another study could be conducted in which the amount of folic acid
administered to each heart is varied during the course of
development. These manipulations can determine whether or not
ventricular malformations or other HLHS associated
malformations occur in relationship to the timing of the MEF2C
knockdown or varied folic acid levels
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. Facts about Hypoplastic Left Heart Syndrome. (2015). Retrieved January 15,
2016, from http://www.cdc.gov/ncbddd/heartdefects/hlhs.html
4. Gilbert, S. F. (2014). Developmental Biology. Sunderland, MA: Sinauer
Associates, Inc
5. 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