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The Regulation of PCSK9 and Wnt/β-catenin Signaling
Pathway in Myocardial Ischemic/Reperfusion Injury

Yun-Ji Cha¹ , Hyoeun Kim¹ , Sae-Bom Jeon¹, Sun-Ho Lee¹², Seok-Min Kang³, Ho-Geun Yoon¹², Chan Joo Lee³,
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,
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, ,
Seung-Hyun Lee¹, Sahng Wook Park¹²*
Presenting Author : Yun-Ji Cha¹ ,# #First Author, *Corresponding Author
1 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul 03722, Korea
2 Graduate School of Medical Science, Brain Korea 21 PLUS Project, Yonsei University College of Medicine, Seoul 03722, Korea
3 Division of Cardiology, Severance Cardiovascular Hospital, Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul 03722, Korea
BACKGROUND Figure 3. Overexpression of Pcsk9 downregulated at the mRNA expression levels
of Wnt-related genes.
Real-time PCR data demonstrated the hypoxia-induced downregulated expression of the
Low-density lipoprotein receptors (LDLR) play a critical role in regulating blood cholesterol levels by Wnt/β-catenin signaling-related genes Axin2 (A) and Cyclin D1 (B). And similar to the
binding to and clearing LDLs from circulation. The disruption of the interaction between the proprotein previous results (Fig 1, Fig 2), the expression of Wnt/β-catenin signaling-related genes in the
convertase subtilisin/kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. Circulating PCSK9 control group showed a tendency to recover after H/R, but did not in Pcsk9 overexpressed
binds to the EGF domain of LDLRs causing the co-internalization of both PCSK9 and LDLR and directing HL-1 cells. The data were normalized using Gapdh and presented as the means ± SD, by
the LDLR to degradation in the lysosomes, rather than its recycling to the plasma membrane. Indeed, ANOVA; n=3, *P<0.05.
PCSK9 exerts various effects in the heart independently of LDL-cholesterol regulation. Acute myocardial
infarction (AMI) induces local and systemic inflammation and reactive oxygen species generation, A
resulting in increased PCSK9 expression in cardiomyocytes. Previous study showed that PCSK9 is
upregulated in ischemic heart tissue and determined the development of infarct size and cardiac function. Permanent Ligation Ischemia-reperfusion
The Wnt/β-catenin signaling pathway plays a crucial role during vertebrate heart development, and it is
re-activated in response to cardiac injury. In vertebrates, low-density lipoprotein receptor-related proteins
5 (LRP5), the single-pass transmembrane protein, act as the co-receptors of Wnt ligands and is LAD LAD
indispensable for Wnt signal transduction. However, It is not well known whether other members of the
LDLR superfamily may be targets of PCSK9. The aims of this study were to determine if LDLR-related Occlusion Occlusion
protein 5(LRP5) is a PCSK9 target and to study the association between Wnt/β-catenin signaling, PCSK9
and aiming to identify possible underlying mechanisms.
Infarct Infarct Boarder Zone
RESULTS
B
A C
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* *
* *
C
B
*
*
*
* *
Figure 4. Regulation of Wnt/β-catenin signaling pathway in the PCSK9 cardiac-
specific overexpressed mice after ischemia-reperfusion.
Figure 1. Hypoxia induces Pcsk9 gene expression and suppresses canonical Wnt/β- To examine whether PCSK9 regulates injury of cardiomyocytes in vivo, we subjected transgenic
catenin signaling in cardiomyocytes. mice with cardiac-specific overexpression of PCSK9 (PCSK9 Tg) and age-matched wild-type
(A) Representative western blots of Pcsk9 under hypoxic conditions. Hypoxia induced Pcsk9 mice (WT) to ischemia-reperfusion (IR) injury induced by 1 hour of ligation of the left anterior
expression at the protein level in HL-1 cells (B) Western blot detected the expression of Wnt descending artery and 1 hour of reperfusion. (A),(B) Schematic illustration for the mouse
signaling pathway key molecules. Statistically significant differences were shown that decreased myocardial ischemia‐reperfusion model. (C) Western blot analysis of Wnt signaling pathway key
the values of Lrp5 (0.4-fold) and phospho-β-catenin (0.5-fold) compared with baseline under molecules in WT and hPCSK9-Tg Myh6-cre mouse hearts after 60 min of IR. α-tubulin was internal
hypoxic conditions. After hypoxia and re-oxygenation (H/R), protein levels of Lrp5 and phospho-β- loading control for protein levels. The results showed that 60 min of ischemia was downregulated
catenin recovered to baseline in the control group. However, Pcsk9 overexpressed HL-1 cells, the the Lrp5 and phospho-β-catenin compared with WT sham, similar features were observed that
expression of phospho-β-catenin did not recover compared to control group after H/R. (C) they are recovered after IR with in vitro results. However, sham PCSK9 Tg showed moderately
Quantitative expression levels of proteins were shown. The data were quantified by densitometry reduced expression levels of Lrp 5 and phospho-β-catenin compared with sham WT but did not
and normalized to β-actin and presented as the means ± SD, by ANOVA; n=3, *P<0.05. recover the expression of those genes after IR PCSK9 Tg compared to sham PCSK9 Tg. I,
ischemia; IR, ischemia-reperfusion.
A
CONCLUSION
B C In this study, we investigated that LDLR-related
protein 5(LRP5) is a PCSK9 target, and correlation
with Wnt/β-catenin signaling pathway using Western
blot, Luciferase assay and qPCR in vitro and in vivo.
These results showed that PCSK9 increased under
hypoxic conditions and the Wnt/β-catenin signaling
pathway-related genes were downregulated in
cardiomyocytes, as demonstrated by decreased Lrp5
and active β-catenin protein expression levels and
mRNA expression levels of Axin2, Cyclin D1.
Interestingly, the downregulated Wnt/β-catenin-related
genes were recovered after H/R in the control group
but did not recover in the PCSK9 overexpressed
Figure 2. Effects of Pcsk9 overexpressed HL-1 cells on TOP-/FOP-FLASH analysis group. These results indicated that PCSK9 and Wnt/β-
catenin signaling were correlated at H/R conditions.
in normoxia, hypoxia and hypoxia/re-oxygenation. Additionally, we found that the same results were
(A) Schematic representation of the assay system involving TOP-Flash and FOP-Flash obtained in the PCSK9 Tg mice. Our findings suggest
reporters. (B) TOP-Flash and (C) FOP-Flash reporter activity determined the transcriptional that PCSK9 and Wnt/β-catenin signaling pathway
could be used as a novel drug target in patients with
activity of the β-catenin-TCF complex. (B) Reporter assays showed that hypoxia ischemic cardiomyopathy. We need further studies to
downregulated Wnt reporter (TOP-Flash) luciferase activity in both control and Pcsk9 evaluate the direct mechanisms of PCSK9 to the
overexpressed HL-1 cells. The β-catenin activity was reduced under hypoxic conditions and Wnt/β-catenin signaling pathway and obtain
was rescued after H/R in the control group but not in Pcsk9 overexpressed cells. The values phenotypes in human iPSC-derived cardiomyocytes.
represent the mean ± SD, by ANOVA; n=3, *p<0.05.
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