THE REGULATION OF CARDIAC SUBSTRATE UTILIZATION BY KRUPPELLIKE FACTOR 15
Abstract
The heart utilizes both fat and glucose to meet its energy demands. In the healthy
myocardium, fatty acids (FA) are the predominant fuel source with glucose serving as the
chief energy substrate under pathological states. This metabolic switch has been linked at
the gene regulatory level wherein a family of transcription factors termed peroxisome
proliferator-activated receptors (PPARs) regulates the expression of FA oxidation (FAO)
gene products as well as glucose oxidation (GOX) gene products. Our laboratory recently
described the zinc finger transcription factor Kruppel–Like Factor 15 (KLF15) as a
critical regulator of glucose metabolism in the liver. This study tested the hypothesis that
KLF15 is a novel molecular regulator of cardiac substrate metabolic gene expression.
Gene expression analysis revealed a perturbation in genes which regulate substrate
oxidation in murine hearts with KLF15 germ-line deletion, as well as in cultured cardiac
myocytes with both adenovirus overexpression (5,000 fold induction) and short-hairpin
knockdown (95% reduction) of KLF15. Interestingly, a significant (p < 0.05, student’s ttest) 40% reduction in PPARα, a PPAR subtype, expression was observed with KLF15
loss of function in vitro, whereas the selective PPARα agonist WY-14643 does not alter
KLF15 levels. Additionally, KLF15 knockdown in cardiac myocytes prevented the
PPARα-dependent induction in FAO gene expression. Similarly, the KLF15 knockdown
also inhibited the PPARα-dependent induction of PDK4, a key intermediate in GOX.
Taken together, these data suggest KLF15 regulates cardiac substrate oxidation through a
mechanism that involves PPARα dependent regulation of FA and glucose oxidation gene
products, and this work has the potential to aid in the discovery of novel drug targets to
improve substrate oxidation and cardiac function in patients with heart failure.
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Executive Summary
THE REGULATION OF CARDIAC SUBSTRATE UTILIZATION BY KRUPPELLIKE FACTOR 15
Cardiomyopathy is a serious affliction that affects the muscular tissue in the heart.
The muscle becomes increasingly rigid and in extreme situations, scar tissue replaces the
muscular tissue. The progression of this disease causes the heart to further weaken in its
ability to maintain a normal heartbeat leading to arrhythmias or heart
failure.
Additionally, this diseased state leads to a weakening in the heart’s ability to generate a
usable form of energy (ATP) for the cells. The heart has the unique capability to utilize
both glucose and fatty acids to meet its energy demands. In a healthy heart, fatty acid
oxidation (FAO) is predominant. However, in a diseased heart, glucose oxidation (GOX)
becomes dominant. While both are valid processes, glucose oxidation is much less
efficient than fatty acid oxidation. When glucose is metabolized, fewer molecules of ATP
are produced per gram of substrate when compared to FAO. This metabolic switch has
been linked to the gene regulatory level wherein a family of transcriptional factors termed
as Peroxisome Proliferator Activated Receptors (PPARS) regulates both FAO and GOX.
This study aims to examine the role of the transcriptional factor Kruppel- Like
Factor 15 (KLF15) in cardiac metabolism. Prior to this study, other studies in the lab
have shown that KLF15 is a novel molecular regulator of glucose metabolism in the liver
and more recent studies have shown that it is also a regulator of lipid metabolism in the
skeletal muscle. With this background, the goal of this study was to test the hypothesis
that KLF 15 is a critical molecular regulator of cardiac metabolism through a mechanism
involving either the direct regulation of or cooperation with the PPAR subtype, PPARa.
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THE REGULATION OF CARDIAC SUBSTRATE UTILIZATION BY KRUPPELLIKE FACTOR 15
Introduction
Adenosine triphosphate (ATP) is the energy currency of all cells in the body. The
importance of ATP generation and utilization is most notable in the heart, in which, on a
beat-to-beat basis, nearly 95% of the myocardial ATP is used and replenished. The heart
is therefore considered the ultimate endurance machine. The heart must maintain a
continuous supply of energy to meets its metabolic demand throughout life. In order to
meet this energy demand, the heart utilizes both glucose and fatty acids. In a healthy
heart, fatty acid oxidation is predominant. However, in the diseased heart, a metabolic
switch occurs which results in an increase in glucose oxidation relative to fatty acid
oxidation. This metabolic switch has been linked to the gene regulatory level wherein a
transcriptional factor member termed peroxisome proliferator activated receptors alpha
(PPARa) regulates fatty acid oxidation and glucose oxidation gene products. Although
fatty acid oxidation and glucose oxidation are both valued processes, fatty acid oxidation
is a much more efficient process because it produces approximately 2 more ATP
molecules per gram of substrate than glucose oxidation.
Fatty acid oxidation (FAO) and glucose oxidation (GOX) have convergent roles
in that oxidation of their respective substrates generates acetyl CoA in the mitochondria
(Figure 1). However, the intracellular pathways by which FAO and GOX generate acetyl
CoA vary. With regard to FAO, fatty acids (FA) enter the cellular membrane through the
use of a fatty acid transport protein (FATP). Fatty acyl CoA synthase (FACS) then
converts the free fatty acid molecule into fatty acyl CoA (FAC). Through the use of a
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mitochondrial fatty acid translocase, termed Slc25a20, fatty acyl CoA is able to enter the
mitochondrion and generates acetyl CoA through β-oxidation. Similarly to a fatty acid,
glucose also enters the cell through a transport protein termed Glut4. Intracellular glucose
is converted into pyruvate by the activity of pyruvate dehydrogenase (PDH). PDH
activity levels are regulated by its phosphorylation status; phosphorylation of PDH
renders the enzyme inactive.
PDH phosphorylation is mediated by pyruvate
dehydrogenase kinase 4 (PDK4). Active, de-phosphorylated, PDH generates pyruvate,
and this pyruvate enters the mitochondrion and is converted to acetyl CoA. These acetyl
CoA molecules feed into the TCA cycle which generates reducing equivalents NADH
and FADH2; these reducing equivalents pass through the electron transport chain and
donate the electrons necessary to create water and to phosphorylate adenosine
diphosphate (ADP) into (ATP).
A major focus of our lab is on a family of transcriptional factors known as
Kruppel-like factors (KLFs). Prior to this study, other studies in the lab have shown that
KLF15 is a novel molecular regulator of glucose metabolism in the liver and more recent
studies have shown that it is also a regulator of lipid metabolism in the skeletal muscle.
With this background, the goal of this study was to test the hypothesis that KLF 15 is a
critical molecular regulator of cardiac metabolism through a mechanism involving either
the direct regulation of or cooperation with the PPAR subtype, PPARa.
Methods
When working in the lab, no animals were used in this student’s research, although
mouse tissue and rat cells were utilized. Certified adults in the laboratory sacrificed all
animals, and the tissue and cells ere then used.
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Isolation of NRVMs
Certified laboratory members sacrificed neonatal rats in order to isolate Neonatal
Rat Ventricular Myocytes (NRVMs). The purpose of isolating these myocytes is to
extract the RNA contained by these cells. Following RNA isolation, a reverse
transcriptase reaction can be completed and a complimentary strand of DNA (cDNA) can
be produced. This cDNA strand is what is ultimately used for subsequent mRNA analysis
(see details below). The first procedure however is the isolation of NRVMs. A six well
plate and a bottle of Hank’s buffered saline solution (HBSS) were kept on ice. Prior to
the actual isolation, a trypsin solution was made, consisting of 50 mg of Trypsin
dissolved in 25 ml of HBSS. The chest of each neonatal heart was then opened and the
heart was removed. The atrium of each heart was removed and the rest of the heart was
placed in a well with HBSS after being cut into four pieces. The hearts were then placed
in the trypsin solution and were continuously shaken at 4 degrees Celsius over night. The
following day, a collagenase solution was prepared consisting of 50mg of collagenase
dissolved in 50 ml of HBSS. Feeding medium DMEM +5% FBS +1%PSG was then
warmed prior to usage in a 37 degrees Celsius water bath. After the medium was
warmed, 10 ml of DMEM were added to the trypsin solution to stop the reaction. A pipet
was then used to gently remove the trypsin/DMEM solution in order to avoid aspirating
the tissue. 10 ml of the previously prepared collagenase solution was added to the tissue,
which was then placed in a 37 degrees Celsius water bath and shaken at 75 rpm for 2
minutes. The collagenase solution was then removed with a pipette and placed in a sterile
conical tube on ice. Another 10 ml of collagenase solution were added by pipet to mince
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the tissue that was then shaken in a 37 degrees Celsius water bath at 125 rpm for 10
minutes. The collagenase solution was removed and placed in another sterile conical tube
on ice. The preceding steps were repeated several times until hearts were dissolved;
following dissolution the suspension was saved on ice. The cells were then filtered using
a BD Falcon 70 um nylon filter into 50 ml conical tube starting with most recent
suspension; collagenase was kept on ice. The solution in the conical tubes was then spun
at 2000 rpm for 5 minutes at 4 degrees Celsius. After the spin, the supernatant was
aspirated. Following aspiration, cells were re-suspended in 25 ml of DMEM by
disrupting the pellet. The subsequent pre-plating step was completed in order to ensure no
fibroblasts remained in the solution. Feeding media was mixed with the cells in a filter
flask and were plated in 4 15cm tissue culture plates with 20 ml of cells each. The plates
were then incubated for one hour at 37 degrees Celsius. After incubation, the supernatant
was collected (Myocytes in supernatant) and cells were counted. Cells were then diluted
to 1 x 10^6 ml. Myocytes were then plated on 10 cm plates with 10 ml per plate. 1:100
BrdU was added to cultures to kill the remaining fibroblasts. The media was then
changed after 2 days with fresh feeding media. Following the isolation of NRVMs, the
cells were then quantified for mRNA expression or for Luciferase assays and Western
Blots.
RNA Isolation
Once Myocytes have been harvested, the RNA isolation can be performed. First,
growth medium is aspirated and 1 ml of Trizol is added to each 10cm plate. Samples are
then incubated at room temperature for 5 minutes. Following incubation, per each ml of
Trizol used, 200 ul of chloroform was added. Samples were then mixed vigorously and a
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2-minute room temperature incubation period followed. Samples were then centrifuged at
12000 rcf for 15 minutes at 4 degrees Celsius. The clear aqueous phase was then
transferred to a new tube. 500 ul of iso- propanol was added to the aqueous phase per ml
of Trizol used. Samples were then incubated at room temperature for 10 minutes. After
incubation, samples were centrifuged at 12000 rcf for 10 minutes at 4 degrees Celsius.
Supernatant was then removed and the pellets were washed with 75% ethyl alcohol per 1
ml of Trizol used. Samples were then vortexed to disrupt the pellet for about 10 seconds
each. Samples were centrifuged again at 8000 rcf for 5 minutes at 4 degrees Celsius.
Once centrifugation was finished, supernatant was carefully removed. Additional spin
may be necessary to remove as much supernatant as possible. Remaining pellets were
then air-dried and 20 ul of water was added to each tube. Pellets were dissolved on
heating block for 5 minutes at 60 degrees Celsius. Once pellets were dissolved
completely, a Nano drop machine was used to measure RNA concentrations in each
sample.
DNase I Treatment
DNase I treatment was performed in order to remove genomic DNA from the
isolated RNA samples. In brief, in a 200 ul PCR tube, 1 ug of each RNA sample was
added to a 10 ul solution consisting of 8 ul of water, 1 ul of 10X DNase I buffer and 1 ul
of DNase I. The solution was centrifuged for 5 minutes at 4 degrees Celsius at 1400 rpm.
Following centrifugation, an incubation period of 15 minutes at room temperature was
required. Once the incubation had been completed, 1 ul of EDTA (25mM) was added to
each sample, which was then centrifuged, and another incubation period at 70 degrees
Celsius was required on a Polymerase Chain Reaction (PCR) machine.
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RT using MMLV (NEB)
1 ul of Oligo-dT was added to the tube containing the DNase treated RNA and
centrifuged again. Next the samples were incubated for 2 minutes at 75 degrees Celsius.
A master mix consisting of: 3.5 ul of water, 2 ul 10X NEB RT buffer, 1 ul of dNTP mix,
0.5 ul of NEB RNase inhibitor and 1 ul of NEB MMLV was added per sample of RNA
and then centrifuged. All samples were then incubated at 42 degrees Celsius for 1 hour,
Another incubation period of 5 minutes at 94 degrees Celsius was required to inactivate
the MMLV. cDNA was then diluted at a 1:10 ration for 200 ul.
qPCR
Quantitative Polymerase Chain Reaction (qPCR) is done in duplicates. 7.5 ul of
master mix, 5.15 ul of water, 0.6 ul of forward primers, 0.6 ul of reverse primers, and
0.15 ul of the respective probe were added to each well. After the master mix was added
1 ul of cDNA was also added to the respective well. qPCR was done in duplicates in
order to ensure no flaws occurred in the experiment. The plate was then tightly sealed
with film and centrifuged at 4 degrees Celsius for 5 minutes at 1400 rpm.
Luciferase Assay
Following the isolation of NRVMs, the cells were transfected. The media from
the plates was aspirated and the cells were washed with 5 mL of Phosphate Buffered
Saline (PBS). The PBS was then aspirated and 500 ul of lysis buffer was added to each
plate. Next the cells were incubated for 10 minutes on ice. In order to fully lyse the cells,
the cells were scraped and put through a needle. The subsequent lysate was added to an
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eppendorf tube and centrifuged at 10 minutes to pellet the debris. 10 ul of the supernatant
was used for a Bradford Assay and 190 ul was used for a colorimetric assay.
Isolated working heart model. These experiments were performed in collaboration with
the Washington University metabolic core lab.
Results and Discussion
In Figure 2, the objective was to test the in vivo metabolic role of the
transcriptional factor Klf15 in the metabolic switch from Palmitate oxidation dependence
to Glucose oxidation dependence, which often occurs in diseased hearts. An ex vivo
experiment was conducted in which palmitate (fatty acid) and glucose were given to the
removed but beating heart in an isolated working heart model. In Figures 2A and 2B,
mouse hearts deficient in Klf15 were compared against control mouse hearts. In the
control hearts, the rate of Palmitate oxidation was greater than the rate of glucose
oxidation. However in mice that were deficient in Klf15, the rate of glucose oxidation
was higher than the rate of palmitate oxidation. These data suggest that Klf15 is a
regulator of the metabolic switch from palmitate oxidation to glucose oxidation.
Additional hemodynamic and contractile parameters were measured as shown in Figure
2C. Despite the variations in substrate utilization, important cardiac indices were
maintained between the control mice and the Klf15 deficient mice.
In the absence of Klf15, fatty acid oxidation (FAO) is deficient. In order to
discern where this problem occurs, the expression levels of mRNA encoding various
gene products responsible for FAO were observed by qPCR analysis. In figure 3A, the
expression levels of various fatty acid transporters were reduced in mice lacking KLF 15.
The mRNA expression of genes, which coordinate cytoplasmic fatty acid uptake, was
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also measured in both types of mice and these data show the inhibition of the mRNA
expression levels in various FAO and GOX gene products such as Fatp1. This inhibition
occurred in both the cytoplasm and the mitochondria. Additionally in figure 3a, gene
products which regulate glucose oxidation, such as Pdk4, was measured by qPCR. The
expression level of PDK4 also attenuated in the absence of Klf15. These data suggest that
Klf15 is an upstream factor that may regulate the expression of downstream FAO and
GOX gene products. In figure 3b, to ensure that the defects previously shown were
myocyte intrinsic, cultured Neonatal Rat Ventricular Myocytes (NRVMs) were used to
measure the expression of various fatty acid transporters / regulators at multiple levels of
the process, particularly Slc25a20 and Fatp1. In the absence of Klf15, by adenoviral
mediated knockdown, there was a significant reduction seen in each transport molecule’s
expression levels in both the mitochondria and the cytoplasm respectively. Taken
together, these data show further evidence that Klf15 is a regulator of FAO and GOX
gene products.
As mentioned before, an important regulator of substrate oxidation is the PPAR
subtype PPARa. It was hypothesized that Klf15 was able to regulate FAO and GOX gene
products through the regulation of, or cooperation with PPARa. To further elucidate this
mechanism, PPARa expression levels were observed in KLF15 deficient mice in
comparison to control mice. In figure 4A, PPARa expression was clearly reduced in the
absence of KLF15 suggesting that KLF15 is a regulator of PPARa expression. To
ascertain that PPARa does not regulate KLF15, a similar experiment was conducted in
figure 4B in which KLF15 expression levels were measured in PPARa deficient mice in
comparison to control mice. When PPARa was absent in the mice, it had no significant
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effect on KLF15 expression levels further suggesting that KLF15 is an upstream
regulator of PPARa. In figure 4c, to ensure that the defect again was myocyte intrinsic,
KLF15 knockdown viruses were used to eliminate the expression levels of KLF 15 in the
NRVMs and subsequent PPARa expression was measured. Similar results were seen in
that the absence of KLF15 in NRVM results in a reduction in PPARa expression. To
further test if Klf15 is an upstream regulator of PPARa, experiments were performed in
which Klf15 was overexpressed in cells and the promoter activity of PPARa was
measured. As seen in figure 4d, overexpression of Klf15 results in augmented activity of
the PPARa promoter reporter. In figure 4e, PPARa expression was observed in myocytes
with the addition of virus that solely overexpressed Klf15. An empty vector, a vector in
which there is an expected absence of Klf15 expression, was used as a basis of
comparison and its PPARa expression was compared to that of the myocytes containing
the Klf15 over expression virus. Taken together these data suggest that Klf15 is an
upstream regulator of PPARa, since the over expression and knockdown of Klf15 also led
to the overexpression and knockdown of PPARa expression respectively.
As suggested by the data in the previous figures, Klf15 and PPARa seem to
regulate FAO and GOX gene products. In order to discern whether the two transcriptional
factors interact with one another (protein-protein interaction), a co- immunoprecipitation
(co-IP) test and a Western Blot were done. The IP was done with flag and the Western
Blot was done with KLF 15. As evidenced by the light band in figure 5 top panel, both
Klf15-Myc and PPARa-flag were present suggesting a protein interaction exists between
the two transcription factors.
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In Figure 6, additional experiments were conducted to examine how PPARa and
KLF 15 cooperate to regulate FAO and GOX gene products. Luciferase assays were used
to observe the promoter activity of certain gene products, FATP1 and PDK4. When only
KLF15 was added there was a small induction in the promoter activity of both FATP1
and PDK4. A similar situation occurred when PPARa was added independently to the
promoters, only a slight induction was observed. However when both PPARa and KLF15
were added to the promoters of FATP1 and PDK4, huge inductions in promoter activities
were observed. These data further suggest that KLF15 and PPARa have the ability to
bind to each other and activate the promoters of various gene products. A similar
experiment with the Slc25a20 promoter is currently being done.
The purpose of figure 7 was to determine if there were targets further downstream
of PPARa whose expression were dependent on Klf15. In figure 7, the expression levels
of various metabolic target in the myocytes were measured by qPCR analysis with and
without the Klf15 knockdown virus and/or the PPARa agonist (WY-14-643). In figure
7A, the Klf15 expression levels were recorded and the previous data was reiterated; the
Klf15 virus effectively knocks down Klf15 levels in the cells and the PPARa agonist has
no significant effect on Klf15 expression levels in the cells. In figure 7B, the mRNA
levels of the transporter FATP1 was observed. In the absence of Klf15, there was a slight
reduction in FATP1 expression. As expected, there was an induction in this transporter’s
expression with the PPARa agonist. However, more importantly, when Klf15 expression
is knocked down in the presence of the PPARa agonist, the PPARa dependent induction
of Fapt1 is lost. Again the same observations were made with the fatty acid transporter
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Slc25a20 and the glucose oxidation gene product PDK4, in figures 7C and 7D
respectively. In the absence of Klf15 there was a slight reduction in each gene product’s
expression in addition to an expected induction in each gene product’s activity with the
PPARa agonist. The most significant part of the data is the loss of the PPARa dependent
induction in this transporter’s activity in the absence of Klf15, further suggesting that
there is a correlation between Klf15 expression and the expression and certain metabolic
genes.
Taken together, this study identifies a previously unrecognized role of Klf15 in
regulating cardiac metabolism through a mechanism involving the regulation of PPARa
expression and cooperation with PPARa in regulating metabolic gene products.
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Figures
Figure 1. Fatty acid (FA) and glucose metabolism overview. Extracellular FA are taken
up by the FAP transport protein (FATP) and converted to fatty acyl CoA by FAC
synthase (FACS). The FAC transporter (Slc25a20) transports fatty acyl CoA into the
mitochondria where it is used as substrate for b-oxidation and subsequent ATP
production. Extracelllar glucose is taken up by Glut4 and is converted to pyruvate by
pyruvate dehydrogenase (PDH). PDH activity is regulated by its phosphorylation status;
phosphorylation of PDH by pyruvate dehydrogenase kinase-4 (PDK4) results in PDH
inactivation. Pyruvate is converted to acetyl CoA which enters the mitochondria for
subsequent ATP production.
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Figure 2. Klf15 regulates fatty acid and glucose metabolism in the ex-vivo isolated
working heart. Palmitate / fatty acid (A) and glucose (B) metabolism in wild-type (Klf15
+/+, black bars) vs. knockout (Klf15 -/-, white bars) in the ex vivo isolated working heart
model. (C), Cardiac performance indicies measured in the isolated working heart
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Figure 3. Klf15 regulates the expression of metabolic gene targets. (A), qPCR analysis
of metabolic targets involved in cytosolic and mitochondria FA uptake, mitochondria and
peroxisomal b-oxidation, FA cycling, and known transcriptional regulators in Klf15 +/+
(black bars) vs Klf15 -/- (white bars) hearts. (B), qPCR analysis of metabolic targets in
cultured NRVM in the absence (black bars) or presence (white bars) of adenoviral
mediated knockdown of endogenous Klf15.
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Figure 4. Klf15 regulates the expression of PPARa, a known regulator of substrate
utilization in the heart. qPCR analysis of (A) PPARa and PPARd in Klf15 +/+ vs Klf15
-/- hearts, (B) Klf15 in PPARa +/+ vs PPARa -/- hearts, (C) PAPRa in cultured NRVM in
the absence and presence of Klf15 knockdown. (D), PPARa promoter analysis in the
absence and presence of KLF15 overexpression. (E), qPCR analysis of PPARa in
NVRM in the absence and presence of adenoviral mediated overexpression of Klf15.
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Figure 5. Klf15 and PPARa protein-protein interaction. co-immunoprecipitation and
western blot analysis with in vitro overexpression of tagged expression plasmids for
Klf15 and PPARa in cell culture.
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Figure 6. Klf15 and PPARa cooperate on target gene promoters. In vitro
overexpression of Klf15, PPARa, and RXR in the absence and presence of promoter
reporters Fatp1 (A) and Pdk4 (B).
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Figure 7. Klf15 is required for PPARa mediated upregulation of target genes. qPCR
analysis of Klf15 (A), Fatp1 (B), Slc25a20 (C), and Pdk4 (D), in NRVM in the absence
and presence of the PPARa agonist WY-14643 with and without Klf15 adenoviral
mediated knockdown of Klf15.
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Conclusions and Future Work
In conclusion, this research suggests that Klf15 is a novel molecular regulator of
cardiac metabolism. The results give evidence to the possibility that Klf15 is a regulator
of the metabolic switch from Palmitate oxidation to glucose oxidation. In vitro and ex
vivo experiments show that Klf15 is also a regulator of FAO and GOX gene products.
Hypotheses of Klf15 being an upstream regulator of PPARa were also supported when
the over expression and knockdown of Klf15 also led to the overexpression and
knockdown of PPARa expression respectively. Further inquiries about the cooperation
between the two transcription factors were answered when co-IP tests and Western Blot
analysis suggested that a protein interaction exists between the two transcription factors.
Additional evidence of the Klf15 – PPARa cooperation was shown with the luciferase
assay, which provided the additional information that the transcription factors have the
ability to bind to each other and activate the promoters of various gene products. Taken
together, these data suggest that Klf15 regulates cardiac metabolism through a
mechanism that involves either the direct regulation of or cooperation with the PPAR
subtype, PPARa.
Current Research is focused on the cooperation between KLF 15 and PPARa to
gain an understanding of healthy heart metabolism that could be applied to therapeutics
in the unhealthy heart.
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Lopaschuk GD, Ussher JR, Folmes CDL, Jaswal JS & Stanley WC (2010). Myocardial
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Haldar SM et al., (2010). Klf15 deficiency is a molecular link between heart failure and
aortic aneurysm formation. Science Translational Medicine 26, 1-8.
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