SOX17-mediated LPAR4 expression plays a pivotal role in cardiac development and regeneration after myocardial infarction

doi:10.21203/rs.3.rs-2224276/v1

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SOX17-mediated LPAR4 expression plays a pivotal role in cardiac development and regeneration after myocardial infarction

https://doi.org/10.21203/rs.3.rs-2224276/v1

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published 03 Jul, 2023

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Lysophosphatidic acid receptor 4 (LPAR4) exhibits transient expression at the cardiac progenitor stage during pluripotent stem cell (PSC)-derived cardiac differentiation. Using RNA-sequencing, promoter analyses, and a loss-of-function study in human PSCs, we discovered that the SRY-box transcription factor 17 (SOX17) is an essential upstream factor of LPAR4 during cardiac differentiation. We conducted mouse embryo analyses to further verify our human PSC in vitro findings. We confirmed the transient and sequential expression of SOX17 and LPAR4 during in vivo cardiac development. In the adult bone marrow transplantation model using LPAR4 promoter-driven GFP cells, we observed two LPAR4⁺ cell types in the heart following myocardial infarction (MI). Heart-resident LPAR4⁺ cells, which are SOX17⁺, but not bone marrow-derived infiltrated LPAR4⁺ cells, showed cardiac differentiation potential. Furthermore, we tested various strategies to enhance cardiac repair through the regulation of downstream signals of LPAR4. During the early stage after MI, the downstream inhibition of LPAR4 by a p38 mitogen-activated protein kinase (p38MAPK) blocker improved cardiac function and reduced fibrotic scar, compared with that observed following LPAR4 stimulation. These findings improve our understanding of heart development and suggest novel therapeutic strategies meant to enhance repair and regeneration after injury by modulating the LPAR4 signaling.

Lysophosphatidic acid receptor 4 (LPAR4)

SRY-box transcription factor 17 (SOX17)

p38 mitogen-activated protein kinase (p38 MAPK)

development

regeneration

A precise understanding of pluripotent stem cells (PSCs) and cardiac progenitor cells (CPCs) is required for clinical application of cell therapy targeting the repair and regeneration of the damaged heart.^1–3 In our previous study, we discovered the lysophosphatidic acid receptor 4 (LPAR4), a novel cardiac progenitor-specific marker.⁴ Particularly, LPAR4 is a G-protein coupled receptor (GPCR)⁵ transiently expressed in the cardiac progenitor stage during mouse PSC-derived cardiac differentiation and fades out in the mature stage. In an in vivo mouse study, LPAR4⁺ cells were rarely observed in the uninjured adult heart, whereas their number was significantly increased in the infarcted heart. During mouse PSC-derived cardiac differentiation, sequential LPAR4 stimulation using an LPAR4 agonist followed by blockade of downstream signaling molecules significantly increased the efficiency of cardiac differentiation. We also showed that the cardiac function was improved in a mouse myocardial infarction (MI) model by sequentially stimulating LPAR4 with an agonist and then blocking the downstream signal using a downstream signaling molecule blocker.⁴

Both mouse and human LPAR4 genes and proteins show a homology of over 90%, and transient expression of human LPAR4 was observed during human-induced pluripotent stem cell (iPSC)-derived cardiac differentiation, similar to that of mouse PSC-derived cardiac differentiation.

In this study, we aimed to investigate the regulatory mechanism of LPAR4 expression during cardiac development and the role of LPAR4 in cardiac repair and regeneration. First, we attempted to identify a specific upstream transcription factor that regulates the transient expression of LPAR4 during early stages of cardiac differentiation in human PSCs. Second, we generated LPAR4 promoter-driven GFP transgenic mice to trace and characterize LPAR4⁺ cells during in vivo development. Third, we discovered two types of LPAR4⁺ cells in the adult heart after bone marrow transplantation and MI and examined the role of heart-resident LPAR4⁺ cells. Lastly, we established the most effective LPAR4 modulation strategy for clinical application to enhance cardiac repair and regeneration after injury in adults.

RNA isolation

The total RNA was isolated using the TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA quality was assessed using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands) and an Agilent 2100 bioanalyzer, while RNA quantification was performed using an ND-2000 spectrophotometer (Thermo Fisher Scientific Inc.).

Library preparation and Whole mRNA-sequencing

For control and test RNAs, library construction was performed using the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria) according to the manufacturer’s instructions. In brief, 500 ng of total RNA was prepared and an oligo-dT primer containing an Illumina-compatible sequence at its 5′ end was hybridized to the RNA to initiate reverse transcription. After degradation of the RNA template, second strand synthesis was initiated using a random primer containing an Illumina-compatible linker sequence at its 5′ end. The double-stranded library was purified using magnetic beads. Subsequently, the library was amplified to allow the addition of complete adapter sequences required for cluster generation, followed by purification. High-throughput (single-end 75) sequencing was performed using NextSeq 500 (Illumina, Inc., San Diego, CA, USA).

Data analysis

QuantSeq 3′ mRNA-Seq reads were aligned using Bowtie2 (Langmead and Salzberg, 2012). Bowtie2 indices were either generated from the genome assembly sequence or the representative transcript sequences for aligning to the genome and transcriptome, respectively. The alignment file was used for assembling transcripts, estimating their abundances, and detecting the differential expression of genes. Differentially expressed genes were determined based on counts from unique and multiple alignments using coverage in Bedtools (Quinlan AR, 2010). Read count (RT) data were processed based on the global normalization method using the Genowiz ™ version 4.0.5.6 (Ocimum Biosolutions, Telangana, India). Gene classification was based on searches performed using DAVID (http://david.abcc.ncifcrf.gov/) and Medline databases (http://www.ncbi.nlm.nih.gov/).

Human iPS cell (hiPSC) line

We constructed a hiPSC line by reprogramming newborn foreskin fibroblast cells (NuFF, GSC-3006G; AMS Biotechnology (GlobalStem), Abingdon, UK) through transduction with the four Yamanaka factors. The prepared hiPSC line was cultured on a STO feeder cell (SIM, ATCC® number: CRL-1503™, Manassas, VA, USA).

Real-time PCR

RNA was isolated and purified from cells harvested at the representative time points using the RNeasy® mini kit (74104, Qiagen, Hilden, Germany) and QIAshredder. A qPCR RT master mix (FSQ-201, TOYOBO, Osaka, Japan) was used to synthesize cDNA from RNA. Primer sequences are shown in Supplementary Material Online Table S1.

Western blotting

Western blotting was performed to confirm the protein expression patterns of MESP1 and SOX17 during hiPSC-derived cardiac differentiation: anti-MESP1 (sc-130461, Santa Cruz Biotechnology, TX, USA), anti-SOX17 (AF1924, R&D systems, Minneapolis, MN, USA), and anti-GAPDH (sc-47724, Santa Cruz Biotechnology, TX, USA). 30µg of protein was transferred onto the nitrocellulose membrane (IB23001, Thermo Fisher Scientific Inc.) using iBlot 2 Gel Transfer Device (IB21001, Thermo Fisher Scientific Inc.). HRP-donkey anti-mouse IgG (H + L) (715-035-151, Jackson ImmunoResearch, Philadelphia, PA, USA) and HRP-donkey anti-goat IgG (H + L) (705-035-147, Jackson ImmunoResearch) were used as secondary antibodies.

Fluorescence-Activated Cell Sorting (FACS)

We harvested BM cells from BM, tibias, as well as femurs and passed them through a 40 µm cell strainer (352340, Corning, New York, USA) to obtain single cells. We also dissected hearts into the smallest size possible using dissection scissors, incubated them with heart digestion enzymes at 37°C for 1 h, and passed them through a 70 µm cell strainer (352350, Corning, New York, USA) to obtain single cells. Isolated single cells from the BM and heart were incubated on ice for 30 minutes with the following antibodies for flow cytometry analysis: LPAR4 (PA549727, Thermo Fisher Scientific Inc.), GFP (ab13970, Abcam, Cambridge, UK), CD45 (sc-53665, Santa Cruz Biotechnology), and Nkx2.5 (ab91196, Abcam).

Immunofluorescence (IF)

Mouse heart and liver tissues were harvested at representative time points relative to the MI after BMT, and enclosed in paraffin blocks. Following sectioning (4 µm), IF was performed. Sectioned tissues were incubated with the following antibodies: GFP (ab13970, Abcam), αSA (A2172, Sigma-Aldrich, St. Louis, MO, USA), LPAR4 (PA549727, Thermo Fisher Scientific Inc.), SOX17 (AF1924, R&D systems), Sca1 (13-5981-28, Thermo Fisher Scientific Inc.), and Nkx2.5 (ab91196, Abcam).

ChIP assay

To identify the MESP1 and SOX17 binding sites, the consensus sequences of MESP1 and SOX17 were compared with the sequence located 3017 bp upstream from the LPAR4 transcription start site. For cell crosslinking, 1% formaldehyde (F8775, Sigma-Aldrich) was added to a cell culture dish, fixed at 25°C for 10 min, and collected in a tube. ChIP RIPA buffer (50 mM Tris-Cl, 1 mM EDTA, 0.5% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate (SDC), 140 mM NaCl, 1× protease inhibitor cocktail (PIC)) was added to the cell pellet. For DNA shearing, both fractions were sonicated 15 times using a Bioruptor (30 s on/30 s off per cycle). Sonicated DNA was treated with 2 µg each of MESP1 (sc-130461, Santa Cruz Biotechnology, TX, USA) and SOX17 (AF1924, R&D systems) antibodies overnight. Protein A/G sepharose (ab193262, Abcam) was added to pull down the antibody-bound DNA, which was then washed thrice with a wash buffer. DNA was incubated at 65°C for at least 2 h for decrosslinking. Finally, DNA was recovered using a PCR purification kit (28104, Qiagen) and analyzed using semi-quantitative PCR.

Luciferase assay

HiPSC-derived cells on day 4 of cardiac differentiation were simultaneously transfected with the firefly pGL3-control luciferase reporter vector (E1751, Promega, Madison, WI, USA) and the Renilla luciferase reporter plasmid pRL-TK (E2241, Promega) using the FuGENE® HD transfection reagent (E2311, Promega).

The Dual-Glo® Luciferase Assay system (E2920, Promega) was used to detect LPAR4 promoter-driven luciferase activity according to the manufacturer’s protocol.

Animals

For animal experiments, 60 C57BL/6 wild-type mice were purchased from Orient Bio (Seongnam-si, Seoul, Korea) and used for generating the murine MI model. Mice were allowed to adapt to the animal facility for 7 days before the experiments. All mice were free of murine viruses, pathogenic bacteria, and endo- as well as ectoparasites. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC, IACUC no.: SNU-201028-3-1) of the Seoul National University Hospital.

Lineage tracing mouse model and TG DNA sample preparation

The DNA prep for the microinjection of the LPAR4_CreERT2_IRES_EGFP TG vector was as follows:

The LPAR4_CreERT2_IRES_EGFP plasmid DNA for the microinjection comprised a fragment (~ 6.6 kb) containing the LPAR4 promoter. AfeI and FspI restriction enzymes, each located at the start and outside part of the promoter were used to secure the fragment. Consecutively, the ~ 1.9 kb backbone fragment was excised through gel-extraction, whereas the ~ 6.6 kb fragment was introduced.

Generation of TG Mice

LPAR4_CreERT2_IRES_EGFP_TG mice were generated by Macrogen Inc. (Seoul, Korea) as well as inbred and maintained under pathogen-free. All manipulations were conducted with the approval of the Macrogen Institutional Animal Care and Use Committee (IACUC no.: 2018-01). Briefly C57BL/6N female mice were treated with PMSG and hCG for superovulation. More specifically, PMSG (7.5 IU) and hCG were intraperitoneally (i.p.) injected at 48 h intervals (5 IU) into 5- to 8-week-old female mice. After the hCG injection, female mice were mated with C57BL/6N stud male mice. The next day, following an impregnation check using a vaginal plug, female mice were sacrificed (as a method of euthanasia, 5–6 mg of phenobarbital sodium was i.p. injected) and the fertilized embryos were harvested. LPAR4_CreERT2_IRES_EGFP DNA was co-microinjected into single-cell embryos. Standard microinjection procedures were used for the production of transgenic mice (Macrogen Inc.). Briefly, 4 ng/µL DNA was directly injected into the male pronucleus of the zygote using a micromanipulator, and microinjected embryos were incubated at 37°C for 1–2 h. In total, 14 to 16 injected single-cell staged embryos were transplanted by surgical methods into the oviducts of pseudopregnant recipient mice. After F0 was born, genotyping was performed using tail cut samples and the presence of the transgene was confirmed using PCR analyses of their genomic DNA (forward: 5′-CTGCAAAGACAGGCAGACAAG-3′, reverse: 5′-CATTGCTGTCACTTGGTCGTG-3′).

Bone Marrow Transplantation

C57BL/6 wild-type mice were sub-lethally irradiated (7.5 Gray) using a Cesium-137 Irradiator (IBL437C, CIS Bio Inc., Codolet, France). Donor mice (LPAR4_CreERT2_IRES_EGFP_TG mice) were euthanized and GFP⁺ BM cells were collected from the tibia and femur. Next, 5 × 10⁷ GFP⁺ BM cells were systemically transplanted via the jugular vein into the sub-lethally irradiated recipient mice (C57BL/6 wild-type mice). We used a mouse model and performed a ligation of the left anterior descending artery after BMT. After four weeks of BMT, FACS analysis was performed against GFP⁺CD45⁺ cells for measuring the donor engraftment rate.

Statistical analysis

All data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) using Newman-Keuls multiple comparison tests was used to each compare groups. GraphPad Prism v8 software was used (GraphPad Software Inc, San Diego, CA, USA). A P value < 0.01 was considered statistically significant.

Upstream regulator of LPAR4 expression during cardiac differentiation in human PSCs

To identify the LPAR4 upstream transcription factor that regulates the expression of LPAR4, we performed a whole mRNA-sequencing (RNA-sequencing) analysis using samples from three different differentiation points during human-induced pluripotent stem cell (hiPSC)-derived cardiac differentiation. Figure 1A shows (Test 1) hiPSC-derived cardiac differentiation day 4, mesodermal differentiation state; (Test 2) day 7, cardiac progenitor state; and (Test 3) beating cardiomyocytes (CMC). We used a previously described optimized hiPSC-induced cardiac differentiation protocol (Figure S1A),⁴ and obtained hiPSC-derived CMCs on cardiac differentiation days 14–16. To identify the LPAR4 upstream transcription factor, we selected three clusters from the RNA-sequencing results, and analyzed 25,737 genes (Figs. 1B and 1C). Based on the RNA-sequencing, we clustered 1) genes that were upregulated more than 2-fold on day 4 of hiPSC-derived cardiac differentiation compared with those at day 7 (1492 genes); 2) nuclear transcription factor complex genes (214 genes); and 3) cardiomyocyte differentiation genes (149 genes) (Fig. 1D). Finally, we selected two candidate transcription factors, the mesoderm posterior bHLH transcription factor 1 (MESP1) and the SRY-box transcription factor 17 (SOX17), as LPAR4 upstream regulatory factors (Fig. 1E).

To confirm the RNA sequencing results, we then analyzed the RNA and protein expression levels of MESP1 and SOX17 during cardiac differentiation, using real-time PCR and western blotting, respectively (Fig. 1F). We observed that the MESP1 and SOX17 expression levels were the highest on day 4 of human cardiac differentiation (Figs. 1G and 1H). As their expression was highest at a time point earlier than the cardiac differentiation day 7, at which the LPAR4 expression level was the highest, both could be considered candidate LPAR4 upstream transcription factor genes. We verified the expression of LPAR4 and cardiac lineage markers of hiPSC-derived cells during cardiac differentiation using real-time PCR (Figures S1B, S1C).

SOX17, but not MESP1, regulates transient LPAR4 expression during cardiac differentiation in human PSCs

Next, we aimed to determine which of the two candidate transcription factors (MESP1 and SOX17) binds to the human LPAR4 promoter. We thus examined each consensus sequence capable of binding MESP1 and SOX17 in the LPAR4 promoter region up to 3017 base pairs (bp) upstream of the transcription start site. We discovered that the LPAR4 promoter region had two (E-box, 5′-CACGTG-3′) and four (5′-AACAAT-3′ or 5′-AACAAAG-3′) binding sites for MESP1 and SOX17, respectively (Fig. 2A). To determine whether MESP1 or SOX17 binds to the LPAR4 promoter region, we performed a chromatin immunoprecipitation (ChIP) assay. We observed that MESP1 did not bind to the LPAR4 promoter, whereas SOX17 bound to two of the four sites (Fig. 2B).

To further clarify the binding of SOX17 to the LPAR4 promoter region, we performed mutagenesis in the SOX17 binding consensus sequence of the LPAR4 promoter region, which was then inserted into a luciferase vector (Fig. 3A) that was used to transfect hiPSC-derived cells. We then performed a luciferase assay on hiPSC-derived cells at day 4 of cardiac differentiation. Consistent with the ChIP assay results, we noticed that the luciferase activity was maintained at a level similar to that of the wild-type (WT) when SOX17 binding sites 1 and 3 were mutated. In contrast, when SOX17 binding site 2 was mutated, the luciferase activity was reduced to approximately half the intensity of that observed in the WT condition. Furthermore, when the SOX17 binding site 4 was mutated, the luciferase activity was reduced to levels similar to that observed in cells transfected with the MOCK vector (Fig. 3B). Thus, we confirmed that SOX17 is a LPAR4 upstream transcription factor and that it binds to the LPAR4 promoter at least at two sites.

Next, we performed loss-of-function (LOF) and gain-of-function (GOF) studies to verify whether the SOX17 expression affects the cardiac differentiation of hiPSCs. We discovered that siRNA-induced SOX17 knockdown inhibited LPAR4 expression as well as cardiac differentiation by reducing the expression of early cardiac lineage markers (Fig. 3C). Interestingly, SOX17 overexpression increased the LPAR4 expression while inhibiting cardiac differentiation; thus, suggesting that transient upregulation of SOX17 and LPAR4 in the early stage of cardiac development and subsequent fading-out in the later stage is necessary to complete cardiac differentiation (Fig. 3D).

LPAR4 lineage tracing in vivo during embryonic and post-natal development

Next, we performed in vivo mouse embryo analyses to confirm the hiPSC in vitro cardiac differentiation findings. We constructed an LPAR4 promoter-driven GFP transgenic mouse line to trace the LPAR4 lineage and verify the expression of LPAR4 during in vivo embryonic development (Figure S2).⁴ We harvested embryos on embryonic days 9.5 (E9.5), 10.5 (E10.5), 12.5 (E12.5), and 16.5 (E16.5) (Fig. 4A).

To confirm the expression of GFP under the LPAR4 promoter activation in mice, we performed a double-stained IF analysis of GFP and the heart-specific marker α-sarcomeric actinin (α-SA) on the harvested embryos (Fig. 4B). Following comparison of the hearts of embryos from different developmental stages, we noticed GFP expression differences between E10.5 and E12.5. As the expression of LPAR4-derived GFP was the strongest in the E12.5 hearts, we expected that the expression of the upstream regulator SOX17 to be mainly detected at E10.5. Expectedly, we observed the presence of SOX17 and GFP double-positive cells at E10.5, but not at E12.5 (Fig. 4C). Interestingly, we observed LPAR4 expression at the E10.5 and E12.5 in the liver and peri-vertebrae (spine); however, no SOX17 and LPAR4 double-positive cells were detected (Fig. 4C). These data indicate that two types of LPAR4⁺ cells are present in vivo and SOX17-mediated LPAR4 expression is specific for cardiac development.

We also assessed the presence of stem cell antigen-1 (Sca1)^6,7 in mouse embryos using fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) analyses, which is both a hematopoietic and cardiac progenitor cell marker. We discovered that both cardiac and hematopoietic lineage cells are LPAR4 and Sca1 double-positive during embryonic (Fig. 4C) and neonatal stage (Fig. 4D). Hematopoietic cells were observed in the embryonic liver and peri-vertebrae (Fig. 4C). Sca1 and LPAR4 double-positive cells were almost non-existent in the neonatal liver, while being common in the neonatal bone marrow (BM); thus, suggesting the migration of hematopoietic cells from embryonic liver to neonatal BM (Fig. 4D).

Overall, we confirmed the transient and sequential expression of SOX17 and LPAR4 during in vivo cardiac development and identified two types of LPAR4⁺ cells. Furthermore, we showed that SOX17 is an important indicator that distinguishes the two types of LPAR4⁺ cells. Cardiac lineage LPAR4⁺ cells express SOX17 transiently during the early embryologic stage, while hematopoietic LPAR4⁺ cells do not express SOX17 and migrate from the liver to BM.

Heart-resident and BM-derived LPAR4 cells in adult mouse heart after injury

Next, we designed a bone marrow transplantation (BMT)^8,9 and myocardial injury experiment to examine the dynamics and characteristics of heart-resident LPAR4⁺ cells compared with those of BM-derived LPAR4⁺ cells. We harvested BM mononuclear cells from LPAR4 promoter-driven GFP transgenic mice. Donor BM cells were inoculated into wild-type recipient mice through the jugular vein after ablating BM cells through 7.5 Gray irradiation. Four weeks later, we evaluated the donor cell engraftment using flow cytometry analysis of the recipient BM repopulated cells (Figure S3). We induced MI by ligating the left anterior descending artery (Fig. 5A, experimental scheme).

We first analyzed non-MI sham hearts and 4, 7, as well as 14 days post-MI hearts using flow cytometry.¹⁰ To identify the number of BM-derived cells that infiltrated into the heart after MI, we counted CD45⁺ cells, a pan leukocyte marker expressing cells.¹¹ CD45⁺ cells rapidly increased at day 4 post-MI and decreased as MI progressed (Figure S4A and S4B). Second, we measured the number of heart-resident and BM-derived LPAR4⁺ cells, using LPAR4 and GFP double staining, respectively. We observed that the total number of LPAR4 expressing cells was progressively increased at day 4 post-MI, reached a peak during day 7, and then gradually decreased (Fig. 5B). We observed the different dynamics of two types of LPAR4⁺ cells. The BM-derived LPAR4⁺ cells were the most prevalent on day 4 after MI. Heart-resident LPAR4⁺ cells showed the highest number on day 7 post-MI. Thereafter, the number of both types of cells declined (Fig. 5C).

To further characterize BM-originating LPAR4⁺ cells, we analyzed the GFP⁺ population in the heart 7 days after MI (Fig. 5D). Most cells expressed CD11b and CD115, indicating that they were neither T nor B cells. In this population, we rarely observed neutrophils (CD11b⁺CD115⁻Ly6G⁺F4/80⁻). Taken together, these results suggest that most BM-derived LPAR4⁺ cells that infiltrated the heart following MI were inflammatory cells with monocytes or macrophages characteristics.

Next, we examined the post-MI heart tissue using triple-color immunofluorescent staining to confirm the cardiac differentiation potentials of heart-resident versus BM-derived LPAR4⁺ cells (Fig. 5E). We did not detect the expression of Nkx2.5, a cardiac transcription factor and progenitor marker, in BM-derived GFP⁺LPAR4⁺ cells; however, we observed nuclear Nkx2.5 expression in heart-resident GFP⁻LPAR4⁺ cells from post-MI hearts. Furthermore, flow cytometry analysis provided consistent results indicating that Nkx2.5 was not expressed in BM-derived, heart-infiltrating GFP⁺ cells (Figure S4C).

Modulation of LPAR4 downstream signaling is critical for cardiac differentiation and the repair process after myocardial infarction

As we discovered that heart-resident LPAR4⁺ cells undergo cardiac differentiation, we designed an experiment to increase the cardiac differentiation and repair efficiency of heart-resident LPAR4⁺ cells after MI. In a previous study, we identified the specific protocol to enhance cardiac differentiation efficiency in vitro and improve cardiac function in vivo through the sequential stimulation and inhibition of LPAR4, respectively.

In this study, we aimed to develop a more effective and efficient protocol. We compared the cardiac function of three experimental groups using an in vivo mouse MI model: LPAR4 stimulation for three days immediately after MI, delayed LPAR4 inhibition from post-MI day 3–6 using a LPAR4 downstream signaling molecule, p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, and LPAR4 stimulation for three days immediately after MI followed by LPAR4 inhibition for three days. Echocardiographic examination indicated that the sequential LPAR4 inhibition after stimulation or the delayed LPAR4 inhibition significantly improved the cardiac function compared to that observed following the early LPAR4 stimulation strategy (Fig. 6A). When we analyzed the scar in each group using Masson's trichrome (MT) staining, we observed a reduction in the fibrotic area and alleviated heart wall thinning following sequential LPAR4 stimulation/inhibition or delayed LPAR4 inhibition groups, consistent with the echocardiography results (Fig. 6B). Based on these findings, we concluded that the delayed inhibition of LPAR4, but not LPAR4 stimulations, using a specific downstream signaling blocker improved cardiac repair and restored cardiac function during the early stages after MI.

In this study, we observed several novel findings related to the regulatory mechanism of LPAR4 in vitro and in vivo and its potential clinical implication. First, we identified SOX17, an upstream transcription factor that induces the expression of LPAR4 during early cardiac differentiation. Second, we identified two types of LPAR4⁺ cells in the infarcted heart and delineated the fate of heart-resident LPAR4⁺ cells using a LPAR4 lineage tracing mouse model. Third, we suggested an efficient cardiac repair protocol using the molecular blockage of LPAR4 downstream signaling.

In a previous study,⁴ we identified a transient expression pattern of LPAR4, in which its expression was increased until the cardiac progenitor stage was reached during the cardiac differentiation and then rapidly decreased at the cardiac maturation stage. The expression of LPAR4 is essential for cardiac differentiation to proceed, and its suppression during the early differentiation stages inhibits cardiac maturation. Therefore, we investigated the transcription factors that regulate the expression of LPAR4 during cardiac differentiation. Using RNA sequencing, ChIP-assays, and mutagenesis analyses we identified SOX17 as the main LPAR4 upstream transcription factor and assessed the importance of the SOX17-induced LPAR4 activation during the early stages of cardiac differentiation using an LPAR4 lineage tracing mouse model. We further found that SOX17 exhibits a transient expression pattern similar to that of LPAR4 during cardiac differentiation.

SOX17 is a member of the SOXF subgroup, which is involved in vascular development, and as such, SOX17^−/− mice show embryonic lethality.¹² Moreover, SOX17 is a representative endoderm marker¹³ and a well-known transcription factor essential for endothelial differentiation.^12,14 Moreover, SOX17 is also expressed in the early stages of cardiac lineage differentiation.^15,16 Our study revealed that SOX17 induces the expression of LPAR4, which is turned off when differentiation into the cardiac lineage proceeds. In addition, we confirmed through IF analysis that cells expressing SOX17 will subsequently differentiate into the cardiac lineage, not the cells surrounding cardiac lineage cells. We further confirmed through SOX17 overexpression experiments that cardiac differentiation does not proceed further unless the SOX17 expression is halted. A decrease in SOX17 expression is expected to suppress the expression of LPAR4; however, the detailed mechanism of action remains to be elucidated.

Next, we investigated the identity of LPAR4⁺ cells by creating an in vivo LPAR4 lineage tracing mouse model. Using SOX17, GFP, and LPAR4 triple staining, we detected the presence of SOX17⁺LPAR4⁺ cells, which were predicted to be heart-resident cells, in E10.5 hearts. However, we did not detect any SOX17⁺ cells in E12.5 hearts. These in vivo findings confirm that the LPAR4 upstream transcription factor (SOX17) was initially expressed and then suppressed at an earlier time point compared to the LPAR4 expression patterns observed during mouse embryonic development. In contrast to our previous studies, LPAR4 was strongly expressed in both the heart and liver during embryonic development in the LPAR4 lineage tracing mouse model.

We newly discovered that two types of LPAR4⁺ cells exist in mouse embryos. The main difference between the two types of LPAR4⁺ cells is the transient expression of SOX17. We demonstrated that LPAR4⁺ cells that transiently expressed SOX17 were subsequently becoming cardiac cells that were continuously present in the heart, while the LPAR4⁺ cells that do not express SOX17 were hematopoietic lineage cells that migrated from the liver to the BM. LPAR4⁺ cells that migrated from the liver to BM were identified using Sca1 expression tracing.

After detecting two types of LPAR4⁺ cells in mouse embryos, we designed experiments to determine whether both types of LPAR4⁺ cells were also present in adult mice. Because liver-to-BM migration of Sca1-expressing LPAR4⁺ cells was confirmed through mouse embryo experiments, we hypothesized that Sca1-expressing LPAR4⁺ cells would be present in the adult mouse BM. As few LPAR4⁺ cells were observed in the normal adult mouse hearts, we analyzed MI adult mouse hearts after BMT and detected both types of LPAR4⁺ cells immediately after MI, similar to that in embryonic mouse hearts. Infiltration of BM cells into the heart following MI has been well-documented.^17–19 Although there were few LPAR4⁺ cells in normal hearts (non-MI hearts), the number of LPAR4⁺ cells was significantly increased immediately after MI, with FACS analysis showing a more than two-fold increase. Immediately after MI, the number of BM-derived LPAR4⁺ cells in the heart was significantly increased, confirming that the number of BM-derived LPAR4⁺ cells infiltrating in the heart was more than two-fold higher than the number of heart-resident LPAR4⁺ cells. Additionally, the number of CD45⁺ BM-derived cells infiltrating the heart was more than four-fold that of the total number of heart-resident LPAR4⁺ cells. Heart-resident LPAR4⁺ cells and BM-derived LPAR4⁺ cells showed a decreasing trend as MI progressed. We performed an IF assay to investigate the expression of Nkx2.5²⁰ and determine the cardiac lineage differentiation capacity of both types of LPAR4⁺ cells and discovered that only heart-resident LPAR4⁺ cells expressed Nkx2.5.

We showed that SOX17 is an upstream regulator of LPAR4. Moreover, by identifying the role of LPAR4⁺ cells cardiac lineage residing in the heart, we established an efficient cardiac regeneration method applicable to MI hearts cases in clinical settings. Although a method for inhibiting LPAR4 after stimulation was established previously, our method is more efficient for improving cardiac function by eliminating the LPAR4 stimulation step and only blocking the LPAR4 downstream signaling molecules. Using this method, we confirmed that LPAR4 inhibition during the late cardiac differentiation phase is more critical for cardiac maturation than LPAR4 stimulation during the early cardiac differentiation phase. The p38 MAPK²¹ signaling pathway in cardiomyocytes has been described in previous studies²² and the enhancement of MI cardiac function through p38 MAPK inhibition was also established.²³ Here, we showed that p38 MAPK blockers promote cardiac regeneration by inhibiting the LPAR4 downstream signal of heart-resident LPAR4⁺ cells, but not in BM-derived LPAR4⁺ cells.

In summary, our findings improve the general understanding of heart development and suggest a new therapeutic strategy to enhance repair and regeneration after injury through LPAR4 signaling modulation.

Supplemental information

Supplementary information is available at Experimental & Molecular Medicine’s website.

Acknowledgments

This research was supported by the “Korea Research-Driven Hospital” (grant number: HI14C1277) through the Korea Health Industry Development Institute, funded by the Korean Ministry of Health & Welfare, and the National Research Foundation (NRF), funded by the Ministry of Science and Information and Communication Technologies (ICT) (grant number: 2022M3A9E4017151), the Republic of Korea.

Author contributions

J.-W.L., C.-S.L., H.-J.C., and H.-S.K. designed the study; J.-W.L. and C.-S.L. performed the experiments; J.-W.L., J.L., and J.C. performed the animal experiments; J.-W.L., C.-S.L., and H.-J.C. wrote the manuscript; HJ.S. and M.K. assisted in performing the experiments; H.-J.C. and H.-S.K. acquired the funding.

Conflicts of interest

The authors declare no competing interests.

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SOX17-mediated LPAR4 expression plays a pivotal role in cardiac development and regeneration after myocardial infarction

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

RNA isolation

Library preparation and Whole mRNA-sequencing

Data analysis

Human iPS cell (hiPSC) line

Real-time PCR

Western blotting

Fluorescence-Activated Cell Sorting (FACS)

Immunofluorescence (IF)

ChIP assay

Luciferase assay

Animals

Lineage tracing mouse model and TG DNA sample preparation

Generation of TG Mice

Bone Marrow Transplantation

Statistical analysis

Results

Upstream regulator of LPAR4 expression during cardiac differentiation in human PSCs

SOX17, but not MESP1, regulates transient LPAR4 expression during cardiac differentiation in human PSCs

Heart-resident and BM-derived LPAR4 cells in adult mouse heart after injury

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1