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Identification of SLC44A3 as a novel susceptibility gene for myocardial infarction
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Identification of SLC44A3 as a novel susceptibility gene for myocardial infarction
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Content
Identification of SLC44A3 as a Novel Susceptibility Gene for
Myocardial Infarction
By
Zhiheng Cai
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)
December 2021
Copyright 2021 Zhiheng Cai
ii
Acknowledgements
I would like to express my heartfelt gratitude to my mentor, Dr. Hooman Allayee, for allowing
me to be a member of his lab and the support he provided which allowed me to learn and
complete this thesis. His continuous support and guidance toward the project and thesis writing
are much appreciated.
Also, I want to express thanks to my committee members, Dr. Pragna Patel and Dr. Jian Xu, for
their advice and patience on my project and their guidance on future life directions. I also wish to
thank my lab members, including Dr. Jaana Hartiala and Dr. Yi Han for their bioinformatics
suggestions, as well as Janet Guksyan and James Hilser for their help in wet laboratory methods.
At last, I want to thank to my family for supporting me during study with financial help and in
spirit. Also, thanks to everyone who made the effort to overcome the past pandemic.
iii
Table of Contents
Acknowledgements ......................................................................................................................... ii
List of Tables ................................................................................................................................. iv
List of Figures ................................................................................................................................. v
Abstract .......................................................................................................................................... vi
Introduction ..................................................................................................................................... 1
Coronary Artery Disease and Myocardial Infarction ................................................................................ 1
Genome-wide Association Study and Identified Target SNP ................................................................... 2
Prioritization of Positional Candidate Genes: ........................................................................................... 5
Functional Study of SLC44A3 ................................................................................................................... 6
Summary of Preliminary Results .............................................................................................................. 8
Methods and Material ................................................................................................................... 10
Genetically Modified Mice and Tissue Harvest ..................................................................................... 10
DNA Extraction & PCR .......................................................................................................................... 10
Reverse Transcriptase PCR & Sanger Sequencing ................................................................................. 11
Pathway Analysis .................................................................................................................................... 11
Quantitative Real-time PCR on Mitochondrial DNA ............................................................................. 11
Results ........................................................................................................................................... 13
The SNP rs12743267 Identified is Associated with the Enhancer Region of SLC44A3 in Human
Endothelial cells. ..................................................................................................................................... 13
The Gene SLC44A2 Regulates Thrombosis by Regulating Mitochondrial Function ............................. 14
Pathway Analysis Reveals High Correlation between SLC44A3 Expression and Mitochondrial Function
................................................................................................................................................................. 15
Characterization of Target Allele in Slc44a3 Knockout Mice ................................................................ 17
TS model to determine effect on plaque rupture ..................................................................................... 20
Discussion ..................................................................................................................................... 23
Future Directions .......................................................................................................................... 26
References ..................................................................................................................................... 28
iv
List of Tables
Table 1. Association Signals for MI and CAD at SLC44A3 Locus. .............................................. 3
v
List of Figures
Figure 1. Overall idea of genetic risk factors in atherosclerotic generation and plaque rupture. ... 1
Figure 2. Overview of the design of genetic and functional analyses. ........................................... 2
Figure 3. Manhattan plot showing eight novel loci. ....................................................................... 3
Figure 4. Regional plot of the leading SNP rs12743267 in chromosome 1p.21. ........................... 6
Figure 5. Functional analysis for SLC44A3 in MI-related tissues. ................................................. 8
Figure 6. Standard plot of qPCR and amplification plot of the standards. ................................... 12
Figure 7. Lead SNP at SLC44A3 Locus is Located in Active Enhancer Region. ......................... 14
Figure 8. Proposed model for choline transport and Slc44a2 regulation of platelet activation. ... 15
Figure 9. Slc44a3 Expression is Associated with Mitochondrial Function. ................................. 16
Figure 10. Characterization of Targeted Allele in Slc44a3 knockout mice. ................................. 18
Figure 11. mtDNA Content in Adipose Tissue of Slc44a3
-/-
vs. Wildtype Mice. ........................ 19
Figure 12. Atherosclerosis in the tandem stenosis mouse model. ................................................ 20
Figure 13. Features of the Tandem Stenosis (TS) Model. ............................................................ 21
vi
Abstract
Myocardial infarction (MI) and coronary artery disease (CAD) are leading causes of death.
However, even though most patients with MI have CAD, not all patients with CAD necessarily
develop MI. This suggests that some of the mechanisms that promote atherosclerosis are distinct
from those that predispose to MI. In support of this concept, we recently identified the SLC44A3
locus as being specifically associated with MI but not CAD. Follow up analyses suggested that
SLC44A3 influences MI by modulating mitochondrial function at the vessel wall. To explore this
hypothesis, we have begun characterizing a publicly available knockout mouse in which exon 4
of Slc44a3 has been replaced by the En2 gene, leading to a frameshift and nonsense mutation at
codon 106. Currently ongoing studies are to further validate the Slc44a3 knockout mouse and
characterize it for differences in mitochondrial content and function in various MI-relevant
tissues. In the in vivo mice model, we have validated the pathway analysis result that Slc44a3 is
correlated with mitochondrial function in adipose tissue and have also tested the use of tandem
stenosis surgery to create a thrombosis in carotid artery. Present findings characterized the allele
of the knockout mouse model and validated the previous analyses results. They also as well
provide a strong tool for future experiments.
1
Introduction
Coronary Artery Disease and Myocardial Infarction
Myocardial infarction (MI) and coronary artery disease (CAD) are leading causes of death in
western societies
1
, where CAD is usually asymptomatic but could have manifestations of MI and
stroke that are caused by atherosclerotic plaque rupture
2
. As both MI and CAD have significant
genetic predisposition, previous large-scale genome-wide association studies have revealed more
than 200 loci that influence risk of CAD and MI
3-9
. However, these genetic determinants still
only explain a small proportion of the heritability of these atherosclerotic cardiovascular
phenotypes. Meanwhile, despite the vast majority of patients with MI having underlying
coronary atherosclerosis, not all patients with CAD develop MI. This phenomenon suggests the
predisposition mechanisms of plaque rupture may be distinct from the ones that promote
atherosclerosis formation
10
.
Figure 1. Overall idea of genetic risk factors in atherosclerotic generation and plaque rupture.
Genetic analyses have revealed that some heritable determinants of plaque rupture and thrombus
formation are distinct from those that contribute to development of coronary atherosclerosis.
ABO was discovered to be correlated only with MI but not coronary artery disease. Identification
of genetic factors with similar properties as ABO would provide new pathways for exploring the
pathophysiology of vulnerable lesions
10, 11
.
2
Genome-wide Association Study and Identified Target SNP
Our lab recently explored the genetic architecture of MI and addressed the hypothesis that
distinct genetic risk factors may underlie susceptibility to MI vs. CAD. The overall study design
involved a large-scale meta-analysis of GWAS data using several datasets, followed by
functional studies (Figure 2). A GWAS was first carried out on the UK Biobank population with
approximately 11 million SNPs and identified 31 loci for MI. Using publicly available GWAS
data from cardiogram+C4D consortium, a meta-analysis was carried out with UK Biobank
results and identified 80 loci
4
. Among these 80 loci, eight loci were novel and not previously
known (Figure 3)
10
. This raises the question of whether the eight novel loci are specific to MI.
Figure 2. Overview of the design of genetic and functional analyses.
3
Figure 3. Manhattan plot showing eight novel loci
10
.
On chromosomes 1p36.11, 1p21.3, 2q13, 2q32.1, 4q22.3, 6q16.1, 9q34.3, and 15q24.2 (orange
dots), eight novel loci were identified to be significantly associated with MI. The horizontal red
and blue lines represent the genome-wide thresholds for significant (P=5.0x10
-8
) and suggestive
(P=5.0x10
-6
) association respectively. P-values are truncated at -log10(P)=40.
Table 1. Association Signals for MI and CAD at SLC44A3 Locus.
4
Association of SLC44A3 Locus with MI: To determine the phenotypic specificity of the 8 new
loci for MI, we used the UK Biobank to compare association of the loci (and used ABO as a
positive control) with MI or CAD only vs. controls, as well as among subjects with both CAD
and MI (CAD
+
/MI
+
) vs. CAD only subjects (CAD
+
/MI
-
)
10
. In both analyses for MI, ABO yielded
associations that were consistent with previous studies but did not exhibit association with CAD
only (Table 1). Similar association patterns with MI were observed with 6 of the 8 novel loci,
including a chromosome 1p21.3 locus harboring SLC44A3
10
. These data suggest that some of
the 8 loci predispose specifically or more robustly to MI than CAD itself. We next used the
Biobank Japan cohort to validate association of ABO with MI but not CAD only as well as carry
out replication for the 6 MI loci. Only the lead SNP (rs12743267) at the SLC44A3 locus was
associated with MI in comparisons to controls or CAD only cases (Table 1). Association of
rs12743267 with MI among CAD patients was further replicated in two independent sets of
angiography-based cohorts (Table 1), which increased in significance when all 16 studies were
meta-analyzed together (P=5.6x10
-8
)
10
. Notably, an all-inclusive meta-analysis with the UK
Biobank, Biobank Japan, and the angiography-based cohorts strengthened the association at the
SLC44A3 locus beyond the threshold for genome-wide significance (Table 1).
Association with Other MI-related Phenotypes: We next explored whether the SLC44A3 locus
was associated with other MI-related thrombotic phenotypes. Based on data from the
MEGASTROKE Consortium
12
, there was no evidence for association of rs12743267 with stroke.
Furthermore, variants at the chromosome 1p21.3 locus had been previously associated with
circulating levels of D-dimer, which is produced when crosslinked fibrin is degraded by plasmin
and the most widely used clinical marker of activated blood coagulation. However, rs12743267
5
was not associated with D-dimer levels (p=0.12) in the GWAS carried out by the CHARGE
Consortium
13
. The lead SNP for D-dimer (rs12029080) was also not associated with MI in the
UKBB, BBJ, or CARDIoGRAM+C4D. Interestingly, SLC44A2, another member of the family
of solute carriers that includes SLC44A3, has been associated with venous thromboembolism
(VTE)
15
. However, we did not obtain evidence for association of our lead SNP with VTE based
on data from the INVENT Consortium.
Prioritization of Positional Candidate Genes:
The lead SNP (rs12743267) on chromosome 1p21.3 is located ~36kb upstream of the
transcriptional start site for SLC44A3 and ~250kb away from the gene encoding tissue factor
(F3) (Fig. 4)
10
. Given the known role of tissue factor in blood coagulation and association of
variants at this locus with circulating D-dimer levels
14
, F3 would be considered a more
biologically plausible candidate gene for a thrombosis-related phenotype such as MI. As noted
above, we did not obtain any genetic evidence prioritizing F3 as a candidate causal gene.
6
Figure 4. Regional plot of the leading SNP rs12743267 in chromosome 1p.21.
The chromosome band and nearest gene (in parentheses) is indicated for each locus. The region
is centered on the lead SNP (purple diamond) and the genes in the interval are indicated in the
bottom panel. The degree of linkage disequilibrium (LD) between the lead SNP and other
variants is shown as r2 values according to the color-coded legend in the box.
Functional Study of SLC44A3
The next question raised is what is the biological role of SLC44A3? Based on the literature, very
little is known about this gene regarding its function or properties. Although SLC44A3 is
categorized as a putative member of the choline transporter family, the function of it as a solute
carrier is not entirely validated. Therefore, we carried out functional analyses using data from
several different cohorts. We first examined the tissue distribution of SLC44A3 in different
7
tissues, from the STARNET study, which is a cohort of CAD patients with genome-wide
genotyping and tissue-wide transcriptomic data
16
. SLC44A3 is expressed at a high level in MI-
relevant tissue, including aorta, visceral adipose, and liver (Fig. 5A). In addition, in
atherosclerotic aorta and mammary artery, the cis eQTLs generated showed that the risk allele
for MI (C) for the SNP rs12743267 was associated with increased expression of SLC44A3 (Fig.
5B). In the GTex project
22
, the same association was observed in aorta and coronary arteries (Fig.
5C). These associations were also consistent with the results found in another independent
human dataset, where mRNA expression of SLC44A3 is significantly higher in ischemic arteries
than normal artery (Fig. 5D). To explore the specific cell type that SLC44A3 affects, we also
observed that SLC44A3 mRNA levels are significantly upregulated in human artery endothelial
cells that were treated with IL-1β than the control group treated with vehicle (Fig. 5E), and
modestly correlated with in vitro migration rate of smooth muscle cells (SMCs) (Fig. 5F). Taken
together, these data provided functional evidence for SLC44A3 as a candidate causal factor for
MI at chromosome 1p21.3 locus and its association with increased plaque rupture risk and
thrombosis.
8
Figure 5. Functional analysis for SLC44A3 in MI-related tissues.
Summary of Preliminary Results
Our data support the concept that some of the heritable factors that contribute to plaque rupture
are distinct from those that promote plaque development. Most importantly, a large body of
evidence from multiple independent approaches converges on SLC44A3 as one such genetic
susceptibility factor. For example, the reproducible association of rs12743267 with MI in
multiple, independent cohorts but the lack of an association with a CAD-only phenotype is
consistent with the concept that the SLC44A3 locus preferentially associates with plaque rupture
in the presence of coronary atherosclerosis. The functional studies also provide supportive
9
evidence that SLC44A3 is at least one candidate causal gene and suggest that this putative solute
carrier could promote risk of plaque rupture through mechanisms at the level of the endothelium.
Thus, our results highlight the potential clinical relevance of SLC44A3 in humans and provide
new avenues for exploring the pathophysiology of vulnerable atherosclerotic plaques. However,
these findings also raise several critical questions: 1) What tissue or cell type(s) does SLC44A3
influence risk of MI through and what is the molecular basis for association of rs12743267 with
MI? 2) Does Slc44a3 deficiency in mice influence plaque instability and rupture and therefore
validate association of SLC44A3 with risk of MI in humans? and 3) What are the biological
mechanisms through which SLC44A3 destabilizes atherosclerotic plaques and can this
information help develop novel therapeutic strategies? We sought to answer these questions
from multiple, complementary directions.
10
Methods and Material
Genetically Modified Mice and Tissue Harvest
The Slc44a3/knockout mice were obtained from the International Mouse Phenotype Consortium
(IMPC). The knockout mice were bred with wild type C57BL/6 mice to generate heterozygous
offspring. The heterozygous mice were bred then to have knockout offspring and wildtype
littermates. The murine tissue was obtained at 8 weeks, immediately after sacrificing and snap
freezing in liquid nitrogen. The tissues harvested include liver, colon, adipose, kidney, and aorta.
The mouse tails were also harvested for genotyping purposes.
DNA Extraction & PCR
The DNA for genotyping purpose was extracted from tails using KAPA Express Extracting Kit
(Cat#07961596001, Roche, Indianapolis, IN). Genotyping for Slc44a3 knockout status was
carried out by multiplex PCR, with one forward primer 5’-GGCTACTCCAGCATTCACAA-3’
and two reverse primers 5’-CTTATCTGAAGCCCTGGCT-3’ and 5’-
CCAACAGCTTCCCCACAACGG-3’. The PCR mixture consisted of PCR buffer (Tris-HCl
1mM, KCl 5mM), 2.5 mM MgCl
2
, 0.2 mM each dNTP, 0.5 mM each primer, 20 ng genomic
DNA, and 1.25 U Taq polymerase in a total volume of 25 µL. The denaturation was performed
at 95°C for 2 mins. Then 10 cycles of touch down process were performed with 94°C
denaturation for 1 minute and 68°C annealing for 2 minutes followed by 25 cycles of
amplification which consisted of denaturation at 94°C for 30 seconds, annealing at 60°C for 30
seconds, and synthesis at 72°C for 45 seconds. Final synthesis was carried out under 72°C for 5
minutes. PCR was carried out in a GeneAmp™ PCR System 9700 instrument (Applied
11
Biosystems, Foster City, CA). Gel electrophoresis was performed at 200V in a 1% agarose gel
stained with ethidium bromide and products visualized under UV light.
Reverse Transcriptase PCR & Sanger Sequencing
Total RNA was isolated using an RNeasy kit following the manufacturer's protocol (Qiagen Inc.,
Valencia, CA). The A260/A280 ratio of all samples was between 1.9 and 2.1 as measured by
spectrophotometry (NanoDrop; Thermo Scientific). cDNA was synthesized by High-Capacity
cDNA Reverse Transcription Kit (Applied Biosystems). The cDNA was amplified with the same
primers for genotyping and subjected to Sanger sequencing through Genewiz, San Diego, CA.
Pathway Analysis
The experiment is based on gene expression data on the Hybrid Mouse Diversity Panel (HMDP),
a collective resource that includes more than 100 mouse strains
19
. Ingenuity Pathway Analysis
(IPA) was performed by characterizing the altered genetic pathway that associated with our
target gene, Slc44a3
20
.
Quantitative Real-time PCR on Mitochondrial DNA
Genomic DNA was extracted from colon, adipose, kidney and aorta using the DNEasy kit
following the manufacturer's protocol (Qiagen Inc, Valencia, CA). Quantitative real-time PCR
was performed by Power SYBR Green Master Mix (Applied Biosystems) for 40 cycles on a
7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The qPCR primers
for mitochondrial DNA were as follows: (16S rRNA: Forward 5’-
CCGCAAGGGAAAGATGAAAGAC-3’. Reverse 5’-TCGTTTGGTTTCGGGGTTTC-3’) and
12
nuclear DNA (HK2: Forward 5’-GCCAGCCTCTCCTGATTTTAGTGT-3’. Reverse 5’-
GGGAACACAAAAGACCTCTTCTGG-3’) as control, respectively
17
.
Quantification was performed in triplicate for each sample for both mtDNA and nuclear DNA,
and tissues were harvested from 10 knockout mice and 7 WT mice. mtDNA content was
calculated by the ΔΔC
T
method with normalization to a DNA pool, which was a mixture of the
DNA extracted from each tissue. A standard curve of C
T
values was generated using undiluted
and 1:10, 1:100, and 1:1000 dilutions of the pooled DNA. This standard curve was then used to
quantitate the amplified mtDNA content from the unknown samples. The ΔΔC
T
was calculated
first with ΔΔC
T
=C
T
(mtDNA gene)-C
T
(nDNA gene). Then, ΔΔC
T
was calculated by using mean
of wildtype mice as control group: ΔΔC
T
= ΔC
T
(Sample of interest)- ΔC
T
(Control sample).
Finally, the relative expression level of each sample was calculated as 2
-ΔΔCT
.
Figure 6. Standard plot of qPCR
and amplification plot of the
standards.
The upper panel was the standard
curve generated with diluted DNA
pool (R
2
=0.998). The lower panel
was the amplification curve of
different dilutions of the DNA
pool. All the following
quantification of unknown DNA
were based on the standard curve.
13
Results
The SNP rs12743267 Identified is Associated with the Enhancer Region of SLC44A3 in Human
Endothelial cells.
As a first step, we determined that rs12743267 maps directly to a genomic region with the
hallmark features of an active enhancer (Fig. 7). These epigenetic marks span a core ~2 kb
window and include histone H3 lysine 4 monomethylation (H3K4Me1), histone H3 lysine 27
acetylation (H3K27Ac), and DNAse I hypersensitive sites. Furthermore, ENCODE designates
this region as a candidate cis-regulatory element (cCRE) and it is predicted by GENEHANCER
to have long-range physical interactions with the proximal promoter of SLC44A3 but no other
genes in the region, including F3 (Fig. 7). Notably, ENCODE shows these epigenetic marks to
only occur in human umbilical vein endothelial cells (HUVECs) (Fig. 7). Taken together, these
observations provide compelling evidence that SLC44A3, but not F3 or other positional
candidates at this locus, is the causal MI gene. These findings also implicate endothelial cells as
the primary cell type through which rs12743267 and SLC44A3 influence risk of MI.
14
Fig. 7. Lead SNP at SLC44A3 Locus is Located in Active Enhancer Region.
ENCODE Project data from HUVECs shows rs12743267 is in the region upstream of SLC44A3
(red rectangle) that is characterized by epigenetic marks for histone H3 lysine 4
monomethylation (K4Me1), H3 lysine 27 acetylation (K27Ac), and DNAse I hypersensitivity
sites. The core 2 kb region is also a candidate cis-regulatory element (cCRE) and predicted to
have long-range physical interactions with the proximal promoter of SLC44A3. These genomic
features are all indicative of open chromatin and active enhancer elements.
The Gene SLC44A2 Regulates Thrombosis by Regulating Mitochondrial Function
One reason for prioritizing SLC44A3 as an MI gene was the previously reported association of
SLC44A2 with VTE
15
. This family of solute carriers (SLC44A1-5) were suggested to function as
choline transporters, but this had only been shown for SLC44A1
23
. Based on these observations,
we tested but did not obtain evidence for association of rs12743267 with plasma levels of
choline and related amines that have been linked to MI-related outcomes
10
. A recent mouse
15
study, however, provided direct evidence that Slc44a2 does in fact function as a choline
transporter
18
. Specifically, the inability of Slc44a2
-/-
mice to transport choline into mitochondria
of platelets led to decreased ATP production, reduced thrombotic potential, and increased
bleeding times. These studies thus provide a plausible biological mechanism for association of
SLC44A2 with VTE in humans.
Figure 8. Proposed model for choline transport and Slc44a2 regulation of platelet activation
18
.
Pathway Analysis Reveals High Correlation between SLC44A3 Expression and Mitochondrial
Function
Based on the intriguing observations with Slc44a2, we investigated whether a similar
relationship existed between Slc44a3 and mitochondria using data from the Hybrid Mouse
Diversity Panel (HMDP)
19
. We first correlated aortic Slc44a3 mRNA levels with all other genes
expressed in aorta and carried out Ingenuity Pathway Analysis (IPA)
20
. Of the top 5 pathways
identified, the 2 most significantly enriched pathways were related to mitochondria (P<10
-14
; Fig.
9A). We carried out the same analysis on data from adipose mRNA levels since mitochondrial
DNA content was also available for this tissue. Genes correlated with Slc44a3 expression levels
16
in adipose tissue were also enriched for mitochondrial pathways, although not as significantly as
in aorta (Fig. 9B). Most importantly, Slc44a3 expression in adipose tissue was positively
correlated with mitochondrial DNA content (Fig. 9C). Since these data were not available in
aorta from the HMDP, we could not determine the same correlation with Slc44a3 mRNA levels.
Taken together with the phenotype of Slc44a2
-/-
mice, these novel findings from our study
suggest that SLC44A3 could also mediate risk of MI in humans through choline-mediated
mitochondrial function. However, we hypothesize that the effect of Slc44a3 on mitochondrial
function would be at the level of the vessel wall as opposed to platelets. This notion is based on
our data in Fig. 5, as well as RNAseq data showing that Slc44a3 is not expressed in mouse and
human platelets.
Fig. 9. Slc44a3 Expression is Associated with Mitochondrial Function.
The top 800 genes correlated with Slc44a3 expression in aorta (A) or adipose tissue (B) are
enriched for oxidative phosphorylation and mitochondrial dysfunction pathways. Compared to
adipose tissue, enrichment in aorta is more significant by several orders of magnitude. The
number (and %) of genes in each pathway that are correlated with Slc44a3 are indicated in or
next to the orange bars. Enrichment was carried out by IPA and only the top 5 pathways are
17
shown. (C) Slc44a3 expression in adipose tissue is strongly correlated with mitochondrial DNA
content in adipose tissue.
Characterization of Target Allele in Slc44a3 Knockout Mice
To further explore the function of SLC44A3, we have obtained a knockout mouse generated by
the IMPC
24
. The exact nature of the genetic modification made to ablate Slc44a3 was not specific
and was only described by IMPC as part of their overall strategy to generate knockout mice on a
large scale for the research community. Therefore, we first characterized the genomic sequence
at the beginning of Slc44a3 in the mice we obtained since the first few exons are typically the
regions of genes that are targeted for genetic modification by the vectors that IMPC uses. PCR
amplification of various exons showed that exon 4 was absent in the knockout mice while all the
other exons examined at the beginning of Slc44a3 were present in the genome (Fig. 10). To
validate this result further, we used reverse transcription to generate cDNA from total RNA.
Sequencing of the cDNA showed that exon 4 in the knockout mice was replaced by a same-
length sequence which was from exon 2 of the En2 gene, a component of the targeting vector
used by the IMPC to genetically modify genes. Furthermore, replacement of exon 4 led to a
frameshift and creation of a nonsense mutation causing early termination of protein translation at
codon 106 (Fig. 10).
18
Fig. 10. Characterization of Targeted Allele in Slc44a3 knockout mice.
PCR amplification of exons 2-5 revealed the expected products in wildtype mice but the absence
of an exon 4 product in Slc44a3
-/-
mice (pink arrows in upper panel), suggesting this region was
replaced by the targeting vector used by IMPC. We next sequenced the cDNA obtained from
adipose tissue of wildtype and Slc44a3
-/-
mice, which confirmed the lack of exon 4 through
19
replacement of the genomic sequence with a portion of the En2 gene (lower panel). As a result
of the frameshift created, a premature stop codon mutation is introduced at codon 106 which
would truncate >80% of Slc44a3 since the protein is comprised of 656 amino acids.
Mitochondrial DNA Content of Slc44a3 Knockout Mice
In order to determine the mitochondrial content in tissues, we conducted e real-time quantitative
PCR on purified DNA template using two different sets of primers that amplify mitochondrial
DNA and nuclear DNA, respectively. A standard curve was generated as described in Methods
and used to determine the amount of mtDNA in the tissues. A ΔΔC
T
method was used to
calculate the relative expression level of mtDNA compared with nDNA.
We next sought to carry out a preliminary
characterization of wildtype and Slc44a3
-/-
mice
with respect to mitochondrial DNA (mtDNA)
content. Based on the strong positive
correlation between mtDNA content and
Slc44a3 expression we observed in adipose
tissue from ~100 inbred strains (Fig. 9C), we
focused on this tissue first. Quantitation of
mtDNA content by qPCR in white adipose
tissue revealed decreased mtDNA content in Slc44a3
-/-
mice compared to wildtype animals,
which was highly suggestive and borderline significant (P=0.054; Fig. 11).
WT
KO
0.0
0.2
0.4
0.6
0.8
1.0
Adipose (mtDNA/nDNA)
P=0.054
Fig. 11. mtDNA Content in Adipose Tissue
of Slc44a3
-/-
vs. Wildtype Mice.
20
TS model to determine effect on plaque rupture
Commonly used atherosclerosis models, such as LDLR
-/-
or ApoE
-/-
deficient mice, have
limitations for understanding MI since they do not exhibit spontaneous plaque rupture. Thus,
various surgical and genetic experimental models have been developed to promote plaque
instability and rupture in atherosclerosis-prone mice. One such model, which replicates key
features of human ruptured plaques at high enough frequency and in a reasonable time frame, is
the tandem stenosis (TS) model developed by Dr. Karlheinz Peter at the Baker Institute in
Melbourne, Australia
21
. Therefore, we plan to use the TS model to characterize Slc44a3
-/-
mice
for plaque rupture phenotypes and investigate the underlying mechanisms.
Figure 12. Atherosclerosis in the tandem stenosis mouse model.
The surgery applies two ligations on the carotid artery of mice and creates a thrombosis-like
plaque. (A) shows the main artery is still healthy, whereas (B) and (C) show the carotid artery
with atherosclerosis in different stages. In (B) and (D), the lumen of the vessel is narrowed, and
in (C) and (E) a plaque rupture is formed
21
.
21
The TS model involves two tandem ligations of
the right common carotid artery that reduces wall
shear stress and increases tensile stress, which
both promote plaque instability
21
. Details of the
procedure have been described
21
and used by
several groups to model plaque rupture in mice.
When carried out on an LDLR
-/-
or ApoE
-/-
background, complex atherosclerotic lesions with
large necrotic cores, thin fibrous caps, and
infiltrating leukocytes are evident a few weeks
after surgery and by 7 weeks, 50% of the mice
develop evidence of plaque rupture in the vessel
segment proximal to the first stenosis
21
. Through
Dr. Subramanyan’s expertise, we have
established the TS model at USC, with mice
exhibiting some of the same vascular
abnormalities associated with this surgery (Fig.
13). Through our collaboration, Dr. Peter will
also provide his expertise using the TS model to
study plaque rupture. Therefore, we will use ApoE
-/-
mice (Jackson Labs stock # 002052) on a
C57Bl/6 background (same as Slc44a3
-/-
mice) as the atherosclerosis-prone strain in which to test
Slc44a3 deficiency. ApoE
-/-
mice will be bred with our recently established Slc44a3
-/-
mice
colony to generate Slc44a3
-/-
/ApoE
-/-
mice and Slc44a3
+/+
/ApoE
-/-
WT littermates. At 6 weeks of
Fig. 13. Features of the Tandem Stenosis
(TS) Model.
Color ultrasound shows equivalent flow in
right (red outline) and left (green outline)
carotids of Sham (A) but reduced flow in
right side of TS compared to left (B). Pulse
flow doppler shows comparable flow in
carotid segment proximal to stenosis in
Sham (C) and TS (D). Flow in distal
carotid segment not affected in Sham (E)
but reduced in TS (F). H/E staining of
right carotid shows normal lumen diameter
in Sham (G) but thickened wall (arrow in
H) with atherosclerotic plaque in intima
(arrowhead in H) of TS.
22
age, Slc44a3
-/-
/ApoE
-/-
mice (n=30, 15 of each sex) and Slc44a3
+/+
/ApoE
-/-
littermates (n=30, 15 of
each sex) will be placed on a high fat/high cholesterol diet (Research Diets D12108C), followed
by TS surgery at 12 weeks of age. 7 weeks after TS surgery (~20 weeks of age), fasted mice will
be bled for lipid and metabolic biomarker measurements. Plasma will also be stored at -80°C for
future analyses, such as circulating inflammatory biomarkers or untargeted metabolomics
studies. After euthanization, the entire aortic arch with the brachiocephalic artery and the right
and left carotid artery will be embedded in OCT compound (Fisher Scientific: 23-730-571) and
6μm thick cryosections will be used to determine the degree of plaque hemorrhage, thrombosis,
necrotic cores, thin/ruptured fibrous caps, and immune cell accumulation using H/E, oil red O,
and Masson’s Trichrome staining, as well as immunohistochemistry markers (CD31, CD41,
fibrin, MOMA-2, Mcp1, SMA). Quantification for each carotid artery segment will be
performed on sequential 6μm sections obtained at 120μm intervals.
23
Discussion
In a recently published study, we used a GWAS approach to identify eight novel loci that
were associated with MI. Of these, the SLC44A3 locus was associated with MI in the presence of
CAD. The comparison analysis in CAD vs. Control was not significant, further validating
SLC44A3 as being a specific MI-susceptibility gene that predisposes to plaque rupture and
thrombosis formation. Based on these observations and the results of more recent unpublished
studies, we hypothesize that SLC44A3 mediates risk of MI through mitochondrial dysfunction.
The lead SNP (rs12743267) at the SLC44A3 locus is located 36 kb upstream of the
transcriptional start site of SLC44A3, and ENCODE Project data suggests that the SNP is in the
enhancer region in endothelial cells. This observation prompted us to carry out a functional
analysis of SLC44A3 in several different datasets. First, in the STARNET cohort
16
, we found
SLC44A3 was expressed in a variety of MI-relevant tissues, including aorta and liver. Second,
the bioinformatics analysis in the same cohort suggested the nucleotide change from T to C at
rs12743267 was only correlated with the elevation of RNA levels of SLC44A3 in atherosclerotic
aorta, but not of any other genes at this locus. These data suggest that SLC44A3 is the causal
gene at chromosome locus 1p21.3. In GTex project data, the same allelic elevation in SLC44A3
expression was observed in the aorta and other coronary arteries, which further validated the
association between the chromosome locus 1p21.3 and SLC44A3
22
. Finally, in two independent
heart donor human-datasets, SLC44A3 was upregulated in ischemic coronary arteries by ~50%
compared to normal arteries and by ~3-fold in HAECs cultured with IL-1β, a pro-inflammatory
cytokine. In summary, these functional data collectively implicate SLC44A3 as the causal gene
on chromosome 1p21.3, where the upregulated expression is positively correlated with plaque
rupture and thrombosis formation in a variety of cell types and tissues.
24
Based on prior observations that Slc44a2 functions as a choline transporter in
mitochondria to modulate platelet activation and thrombosis formation
18
, we hypothesized that
SLC44A3 could also control mitochondrial metabolism to affect risk of MI. These observations
were also supported by a highly significant positive relationship between mitochondrial DNA
content and Slc44a3 expression in adipose tissue. However, due to the lack of expression of
SLC44A3 in platelets, and also the strong correlation of SLC44A3 with endothelial cells, we
postulate that SLC44A3 affects mitochondrial function at the level of the vessel wall. Ingenuity
Pathway Analysis performed on gene expression data on the Hybrid Mouse Diversity Panel
demonstrated that genes highly correlated with Slc44a3 were strongly enriched for mitochondrial
pathways in both aorta and adipose tissue. A strong association between mitochondrial DNA
content and Slc44a3 expression level in adipose tissue was also demonstrated. However, we
could not address whether the same relationship exited in aorta since mitochondrial DNA content
was not quantitated in this tissue.
In order to establish a biological connection between Slc44a3 expression and
mitochondrial DNA content in aorta tissue, we established an Slc44a3 knockout mouse strain as
an in vivo tool. First, we validated the knockout allele using different approaches, including
sequencing the DNA from the genome and cDNA from reverse transcribed RNA. These analyses
validated the targeted genomic structure of Slc44a3 and suggested the absence of Slc44a3
protein in the mice. Based on these observations and the phenotypic data provided by IMPC
(https://www.mousephenotype.org/data/genes/MGI:2384860), these additional preliminary data
validate our mouse model as being a true knockout for Slc44a3 and demonstrate a highly
suggestive difference in mtDNA content between wildtype and Slc44a3
-/-
mice that is
directionally consistent with our observations across a panel of inbred mouse strains. Thus, these
25
data further support our hypothesis that one putative biological mechanism for the association of
SLC44A3 with risk of MI could be mediated through mitochondrial function. Confirmation and
expansion of these observations, including specific evaluation of differences in mitochondrial
function and mtDNA content in vascular tissue, will be pursued through a comprehensive series
of experiments that are currently planned over the next 6 months. We believe these efforts will
lead to a better understanding of the role of SLC44A3 in plaque rupture with potentially
important clinical implications. However, decreased protein levels, which could further support
the characterization of knockout allele, could not be validated directly through Western blot
experiments (data not shown) due to the absence of a suitable commercially available antibody.
However, our analyses at the level of DNA and RNA still point to validation of the knockout of
the gene in the knockout mouse strain.
We then carried out a preliminary analysis of mtDNA content in both knockout and
wildtype mice. Since a high correlation in mtDNA content and Slc44a3 expression was observed
in adipose tissue from our analysis of the HMDP, we first carried out these experiments in
adipose tissue, which revealed a suggestive but non-significant difference. While these results
appeared to validate our analysis in the HMDP, the distribution of the data was scattered and had
a high standard deviation. The inclusion of Slc44a3
-/-
and wildtype mice that were not similar in
age and sex in the experiments could be one factor for the large variations observed in the data.
Therefore, we will repeat these experiments as well as analyze mtDNA content in aorta which is
our primary tissue of interest. We may also examine other tissues, such as kidney and colon
since Slc44a3 is expressed at the highest level in colon (GTEx Project), and kidney has the
highest mitochondria content in mice
25
.
26
Finally, even though the relationship between Slc44a3 and mitochondrial function will be
tested, it will still be necessary to directly determine whether Slc44a3 deficiency influences
plaque rupture in the presence of atherosclerosis in vivo. The TS model we have elected to use
will provide us with an established protocol to prove this hypothesis. Preliminary experiments
show our ability to establish the TS model at USC and we plan to perform further experiments
on Slc44a3
-/-
mice after sufficient numbers of mice have been bred.
Future Directions
As described above, we will need to expand the mitochondrial DNA content experiments
with more mice and in other tissues (aorta), as well as in a cell-specific manner. More
importantly, in order to reduce the variation in data, our studies should use mice of the same age
and equivalent numbers of both sexes. Also, we will use a microscope to dissect the descending
aorta and remove the adventitia to make the fraction of tissue we get more enriched for vessel
wall cells, including endothelial cells. Subsequently, mtDNA content will be determined by
qPCR, as in our preliminary analyses.
Concurrently, we will perform gene expression analysis to determine whether
mitochondrial pathways are perturbed in Slc44a3
-/-
mice. Since we are interested in
understanding the pathways that are perturbed as a result of Slc44a3 deficiency in the context of
atherosclerotic lesions and unstable/ruptured plaques, we will use vascular tissues from mice that
have undergone TS surgery for these experiments. One set of single cell (sc)RNA-seq
experiments will be performed with aortas from these mice, and due to the small size and low
RNA yields of carotid artery segments with unstable/ruptured plaques, scRNA-seq experiments
27
will be carried out on pools of mice. These analyses will focus on the endothelium since we
hypothesize that SLC44A3 affects risk of MI by perturbing mitochondrial function in these cells.
Finally, the gold standard to demonstrate the lack of protein in knockout mice is by
Western blot analysis. However, the only commercial antibody that we tested which yielded a
band of the correct size for Slc44a3 did not show a difference in the amount of protein in the
knockout mice compared with wildtypes. Based on epitope analysis and sequence alignments
between members of the Slc44 family of choline transporters, it is possible that the antibody we
used cross reacted with Slc44a1. For example, these two proteins have a similar size and epitope
sequence recognized by the antibody. Thus, at present, we cannot definitively demonstrate the
absence of protein in Slc44a3
-/-
mice, but this can potentially be circumvented by establishing
other phenotype changes in knockout and wildtype mice.
In summary, our experiments strongly suggest that SLC44A3 could be correlated with
mitochondrial function in endothelial cells, which is the biological basis for the association of
this gene with risk of MI in humans. The mouse model we have established will be a valuable
tool for our planned experiments as well future studies regarding the relationship between
SLC44A3 and atherosclerotic plaque stability.
28
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Abstract (if available)
Abstract
Myocardial infarction (MI) and coronary artery disease (CAD) are leading causes of death. However, even though most patients with MI have CAD, not all patients with CAD necessarily develop MI. This suggests that some of the mechanisms that promote atherosclerosis are distinct from those that predispose to MI. In support of this concept, we recently identified the SLC44A3 locus as being specifically associated with MI but not CAD. Follow up analyses suggested that SLC44A3 influences MI by modulating mitochondrial function at the vessel wall. To explore this hypothesis, we have begun characterizing a publicly available knockout mouse in which exon 4 of Slc44a3 has been replaced by the En2 gene, leading to a frameshift and nonsense mutation at codon 106. Currently ongoing studies are to further validate the Slc44a3 knockout mouse and characterize it for differences in mitochondrial content and function in various MI-relevant tissues. In the in vivo mice model, we have validated the pathway analysis result that Slc44a3 is correlated with mitochondrial function in adipose tissue and have also tested the use of tandem stenosis surgery to create a thrombosis in carotid artery. Present findings characterized the allele of the knockout mouse model and validated the previous analyses results. They also as well provide a strong tool for future experiments.
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Asset Metadata
Creator
Cai, Zhiheng
(author)
Core Title
Identification of SLC44A3 as a novel susceptibility gene for myocardial infarction
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Publication Date
11/15/2021
Defense Date
06/24/2021
Publisher
University of Southern California
(original),
University of Southern California. Libraries
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Tag
genome-wide association study,meta-analysis,mitochondrial DNA content,mouse model,myocardial infarction,OAI-PMH Harvest,SLC44A3,tandem stenosis
Language
English
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Allayee, Hooman (
committee chair
), Patel, Pragna (
committee member
), Xu, Jian (
committee member
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caizhihe@usc.edu,zhcai2018@gmail.com
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Tags
genome-wide association study
meta-analysis
mitochondrial DNA content
mouse model
myocardial infarction
SLC44A3
tandem stenosis