Vasorin deficiency leads to cardiac hypertrophy by targeting MYL7 in young mice

Abstract Vasorin (VASN) is an important transmembrane protein associated with development and disease. However, it is not clear whether the death of mice with VASN deficiency (VASN −/−) is related to cardiac dysfunction. The aim of this research was to ascertain whether VASN induces pathological cardiac hypertrophy by targeting myosin light chain 7 (MYL7). VASN −/− mice were produced by CRISPR/Cas9 technology and inbreeding. PCR amplification, electrophoresis, real‐time PCR and Western blotting were used to confirm VASN deficiency. Cardiac hypertrophy was examined by blood tests, histological analysis and real‐time PCR, and key downstream factors were identified by RNA sequencing and real‐time PCR. Western blotting, immunohistochemistry and electron microscopy analysis were used to confirm the downregulation of MYL7 production and cardiac structural changes. Our results showed that sudden death of VASN −/− mice occurred 21–28 days after birth. The obvious increases in cardiovascular risk, heart weight and myocardial volume and the upregulation of hypertrophy marker gene expression indicated that cardiac hypertrophy may be the cause of death in young VASN −/− mice. Transcriptome analysis revealed that VASN deficiency led to MYL7 downregulation, which induced myocardial structure abnormalities and disorders. Our results revealed a pathological phenomenon in which VASN deficiency may lead to cardiac hypertrophy by downregulating MYL7 production. However, more research is necessary to elucidate the underlying mechanism.


| INTRODUC TI ON
Vasorin (VASN), also known as SLIT-like 2 (slitl2), is a 673-amino acid transmembrane glycoprotein localized on the cell surface 1 that is cleaved by a disintegrin and metalloproteinase. VASN is considered a potential biomarker and therapeutic target for cancer. 2,3 Notably, VASN is highly conserved in many organs from embryonic development to adulthood. 4,5 There is limited literature on VASN function.
In embryonic development, VASN is primarily expressed in the heart and lungs. 1 VASN is expressed in vascular smooth muscle cells 1 and umbilical vein endothelial cells. 6 Soluble VASN protein can bind to TGFβ, which can inhibit epithelial-to-mesenchymal transition. 7 A previous study showed high VASN expression in human glioblastoma in the context of TNF α-induced apoptosis. 7 CRISPR/Cas9 technology could be used to generate a knockout mouse model to further study the function of VASN and its associated signalling pathway.
However, unexplained death occurs in VASN −/− mice shortly after birth, 7 and it was unclear whether this phenomenon was related to heart damage.
Cardiac hypertrophy may lead to heart damage and sudden death 8-10 but is not always pathological (ie associated with cardiac dysfunction). Cardiac hypertrophy is not present under conditions of increased chamber volume but normal cardiac mass. Pathological cardiac hypertrophy is a compensatory change in cardiac overload induced by pathological stimulation 11,12 that is characterized by increased heart weight or volume, myocardial cell volume and extracellular matrix, 13,14 properties of the inevitable process by which heart disease progresses to heart failure. 15,16 The pathogenesis of cardiac hypertrophy is not clear. In a recent study, monogenic mutation of cardiac sarcomeres was shown to cause pathological cardiac hypertrophy. 17,18 Cardiac myosin is an important structure in cardiac sarcomeres and includes two myosin regulatory light chains (MLC2v and MLC2a). 19 Myosin light chain 7 (MYL7), also known as MLC2a, is expressed in heart ventricles and atria. 20,21 MYL7 inactivation leads to embryonic lethality and abnormal cardiac morphogenesis. 22,23 Based on these studies, a novel pathological phenomenon was hypothesized in which VASN deletion leads to cardiac hypertrophy by affecting MYL7 expression.
In this study, we used CRISPR/Cas9 technology to produce VASNknockout mice and investigated the changes in cardiac function and cardiac hypertrophy index values. Our results indicated that cardiac hypertrophy may be the cause of death in young VASN-knockout mice. We found that VASN deficiency downregulated MYL7 expression, resulting in myocardial structure abnormalities and disorders.
Our results reveal a pathological phenomenon in which VASN deficiency may lead to cardiac hypertrophy by downregulating MYL7 production.

| Mouse lines and embryo manipulation
All mouse experiments were approved by the ethics committee of

| PCR amplification and electrophoresis
Each mouse genotype was identified by PCR amplification and electrophoresis. 27
Approximately 400 μL of blood was collected from each mouse and placed at room temperature for 30 min. The resulting supernatant was stored at −20°C. Serum homocysteine (HCY) levels were measured by an AEROSET-2 000 automatic analyser (Abbott, USA). The levels of serum cardiac enzymes (lactate dehydrogenase (LDH) and creatine kinase (CK)) and myocardial enzymes (creatine kinase isoenzyme (CK-MB)) were measured by an AU5800 automatic analyser (Beckman Coulter, USA).

| Pathological analysis
Haematoxylin and eosin (HE) staining was performed as previously described. 28 Pathological changes in the heart were detected in mice from each group (3 mice, 5 biological replicates per group). The percentage of mice with cardiac hypertrophy was calculated as the number of mice with cardiac hypertrophy divided by the total number of mice in each group.

| Transcriptome sequencing
Transcriptome sequencing of heart tissue from the three groups was completed at the Wuhan Genome Institute (BGI-Shenzhen).
Twelve total RNA samples (4 mice per group) were sequenced.
Whole transcriptome data were collected and compared with the ribosome database to identify known transcripts (mRNA), perform quantitative analysis of known and new mRNAs, analyse differences between samples (at least 2 samples) and analyse differences between groups (at least 2 samples and at least 3 biological replicates per group). Differentially expressed genes were analysed using the DAVID database by identifying Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps.

| Real-time quantitative PCR analysis
RNA was extracted from heart tissue from each group (3 samples, 3 biological replicates per group) as previously described. 29 Reverse ance with a previous article. 30 Primers (Table 1) were produced by Sangon Biotech (Shanghai). Each gene was subjected to 40 cycles of PCR, and this process was repeated three times. Expression of the endogenous gene 18S was used for comparison. The relative expression levels of target genes were calculated by using the 2 -△△CT method.

| Western blotting analysis
Hearts from mice in each group (2 samples, 4 biological replicates per group) were prepared as previously described. 31  were used, and the secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (AS014, ABclonal, 1:1000). Protein expression was calculated by using an automatic analysis system (Image Lab 6.0). The target protein data were normalized to GAPDH data.

| Electron microscopy analysis
The isolated hearts from mice in each group (2 samples, 2 biological replicates per group) were prepared as previously described. 33

| Statistical analysis
All results are shown as the mean and standard deviation. Data were analysed by Duncan's multiple comparison. p < 0.05 was considered indicative of a significant difference. p < 0.01 was considered highly significant.

| VASN-knockout results in premature death and cardiovascular risk in young mice
To observe the effect of VASN knockout on mice, we analysed

| VASN deficiency leads to cardiac hypertrophy in young mice
To explore whether the cause of death of VASN −/− mice is related to the heart, we detected pathological changes in this organ.

| VASN deficiency reduces MYL7 expression in young mice
To investigate the changes in potential downstream genes caused by VASN deficiency, we performed transcriptome analysis of hearts from VASN −/− , VASN −/+ and VASN +/+ mice using RNA sequencing.
According to standard expression value ≥100 and log absolute value ≥2, we identified 61 upregulated genes and 82 downregulated genes ( Figure 4A-4B). VASN was among the most drastically downregulated genes. KEGG analysis revealed that VASN deficiency affected signalling pathways ( Figure 4C). We selected genes for qRT-PCR verification and validated MYL7 among the genes following the selected trend with a high correlation ( Figure 4D). Realtime PCR analysis further confirmed that MYL7 expression in the heart was significantly downregulated in VASN −/− mice compared to VASN −/+ and VASN +/+ mice ( Figure 4E). These data suggest that VASN deficiency regulates MYL7 expression.

| VASN deficiency induces cardiac hypertrophy by downregulating MYL7 expression
To further study the potential molecular mechanism by which VASN deficiency leads to cardiac hypertrophy, we studied whether Western blotting analysis showed that MYL7 protein expression was significantly lower in VASN −/− mice hearts than in VASN −/+ and VASN +/+ mice hearts ( Figure 5A1-A2). The relative density of MYL7 in VASN −/− mice hearts was significantly lower than that in VASN −/+ and VASN +/+ mice hearts ( Figure 5B1-B4). . This result implies that the downregulation of MYL7 production causes abnormal myocardial structure. Taken together, these findings show that VASN deficiency reduces MYL7 expression, which leads to cardiac hypertrophy (Figure 7, mechanism diagram).

| DISCUSS ION
In zebrafish gastrula, VASN is diffusely expressed in the brain and central nervous system. 34 In the early stage of mouse embryonic development, VASN is strongly expressed in the hindbrain and neural tube midline. 4 In adult mice, VASN is expressed in the heart, liver, kidney and inner follicle, 1,5  The third generation of artificial endonucleases for use in the CRISP/Cas9 system has been successfully developed. Due to its high mutation efficiency, simple production and low cost, this system is considered a molecular tool for genome site-specific modification with broad application prospects. At present, this technology has been successfully applied to the precise genome modification of human cells, 26 zebrafish 37 and mice. 38 In this study, we used CRISP/Cas9 technology to study VASN function. We generated a 2384-nucleobase deletion affecting the transcription region that could be inherited stably ( Figure 1). Our CRISPR/Cas9 system achieved rapid, efficient and ac-  21 and MYL7-deficient hearts showed abnormal myocardial organization and embryonic atrial function. 40 Our results showed that MYL7 downregulation caused abnormal myocardial structure, as reported in previous studies ( Figure 6).
Therefore, these studies suggest that the downregulation of MYL7 expression may lead to impaired cardiac development and damaged myocardial structure. However, the mechanism by which VASN regulates MYL7 expression must be further explored.
These results will contribute to understanding the pathogenesis of hypertrophic cardiomyopathy and identifying treatment strategies ( Figure 7).

ACK N OWLED G EM ENT
This study was supported by grants from the Central Government Guides the Development of Local Science and Technology (GUIKEZY1949025 and GUIKEZY20198024).

CO N FLI C T O F I NTE R E S T
All authors have no conflicts of interest.