Myocardial fibrosis (MF) is a key stage of heart failure that can exacerbate the associated symptoms and lead to severe outcomes, such as cardiac arrest or sudden death (López et al., 2021). MF is typically caused by prolonged pressure on or damage to the myocardial cells and often causes cardiac hypertrophy. MF is a complex pathological process that is closely related to the extracellular matrix (ECM), immune responses, signaling pathways, and various cardiac cells (Li et al., 2022). Damaged myocardial cells can activate local inflammatory reactions and release pro-inflammatory cytokines, such as interleukin (IL) 1, IL6, IL11, IL17, and tumor necrosis factor alpha (TNFα). Fibroblasts are activated and transformed into myofibroblasts in the heart tissues, which then synthesize and secrete large amounts of collagen and ECM components (Liu et al., 2021). Collectively, these risk factors contribute to MF.

Vasorin (VASN), also known as slit-like 2 (slitl2), contains two exons, of which exon 2 is the main coding region (Pintus et al., 2018). VASN is a transmembrane glycoprotein composed of 673 amino acids and is located on the cell surface (Bonnet et al., 2018). VASN is highly expressed in the cardiovascular system, including the heart, vascular smooth muscles, and umbilical vein endothelial cells (Bonnet et al., 2018; Pintus et al., 2018). Upregulation of VASN expression prevents smooth muscle cell calcification through specific binding to the transforming growth factor (Luong et al., 2019). Downregulation of VASN expression can alleviate adverse reactions to vascular wall injury (Li et al., 2015). However, overexpression or knockout of VASN has been found to cause developmental abnormalities in the heart and blood vessels of zebrafish (Chen et al., 2005). VASN-knockout (VASN^−/−) mice have been reported to die suddenly 3 weeks after birth (Ikeda et al., 2004). Our previous study showed that a VASN-knockout mouse model exhibited pathological cardiac hypertrophy symptoms (Sun et al., 2022).

In the present study, VASN-knockout mice showed the pathological, molecular, and protein features of MF. RNA from the mouse heart tissue was extracted, identified, and sequenced in vitro. Bioinformatic analysis then showed significantly decreased expressions of key ECM genes (COL6A1, COL9A1, and FRAS1); however, the expressions of inflammatory factors IL1β and IL6 were significantly upregulated in the heart tissues of VASN-knockout mice. Our results thus reveal that VASN knockout induces MF by affecting the ECM and inflammation.

MF is a complex pathological process that plays a crucial role in the occurrence and development of cardiovascular disease. MF and cardiac hypertrophy often coexist and interact with each other (Pagourelias et al., 2021), and cardiac hypertrophy could lead to MF. Under long-term pressure or increased volume load, the cardiac cells undergo pathological hypertrophy (Figure 5). However, cardiac hypertrophy is often accompanied by remodeling of the myocardial ECM, including collagen deposition and fibrosis (Detterich, 2017). MF caused by cardiac hypertrophy may be closely related to multiple mechanisms, such as activation of the neuroendocrine system (Qiu et al., 2019), inflammatory responses (Bacmeister et al., 2019), and oxidative stress (Wang et al., 2017). MF exacerbates the progression of cardiac hypertrophy as the stiffening and reduced compliance of the fibrotic myocardial tissue impair both diastolic and systolic heart functions (Lafuse et al., 2020). To maintain the pumping function, the cardiac cells are further enlarged, thereby exacerbating the degree of cardiac hypertrophy. MF can also affect the electrophysiological properties of the myocardial cells, thereby increasing the risk of arrhythmias (Baggett et al., 2023).

One of the typical features of MF is the adverse repair response of the cardiac tissue to various damaging factors. HE staining showed that the gaps between the myocardial cells widened during MF and that there was proliferation of pale pink fibrous tissue in the interstitium (Karur et al., 2024). As the degree of fibrosis worsened, the fibrous tissue increased gradually, and focal or diffuse fibrous cord-like structures became more pronounced. Masson staining showed significantly larger blue areas in the MF heart tissue, indicating greater deposition of collagen fibers. The myocardial interstitium in the fibrotic area was stained dark blue, forming a sharp contrast with the red color (muscle fibers) of normal myocardial tissue (Flori et al., 2024). Sirius staining of MF tissue showed large numbers of type I collagen fibers that appeared strongly positive in red or yellow color, whereas type III collagen fibers were relatively fewer and showed lighter staining (Qi et al., 2022). HYP was increasingly expressed in the fibrotic cardiac tissues (Yang et al., 2021). Collagen fiber types I and III are shown to be significantly increased in MF tissues (Xing et al., 2024). The expression level of α-SMA is reported to be low in normal myocardial cells but high in fibrotic cardiac tissues (Hsieh et al., 2022). Our experimental results are consistent with the findings of the above literature, indicating that VASN-knockout mice exhibit typical symptoms of MF.

The VASN gene is important for the occurrence and development of cardiovascular diseases. In atherosclerosis, abnormal expression of the VASN gene can cause endothelial dysfunction, reduce the resistance of the vascular endothelium to lipid deposition, and promote the formation of atherosclerotic plaques (Louvet et al., 2022). VASN may regulate the expression of adhesion molecules on the surfaces of endothelial cells, increase the adhesion of leukocytes to the vascular walls, and trigger inflammatory reactions to accelerate atherosclerosis (Huang et al., 2015). After myocardial infarction, local tissue ischemia and hypoxia can trigger a series of pathophysiological changes, and VASN is known to be involved in regulating the balance between apoptosis and regeneration of the myocardial cells (Pintus et al., 2018). Abnormal VASN expression leads to increased apoptosis of the myocardial cells, hindered myocardial repair and regeneration capabilities, exacerbated myocardial injury, and pump dysfunction (Shamhart and Meszaros, 2010). Under hypertension, the pressure on the vascular wall increases, and VASN affects the tension and compliance of blood vessels by regulating the contraction and relaxation of the vascular smooth muscle cells (Qin et al., 2024). Owing to dysregulation of VASN expression, the vascular smooth muscles contract excessively, further increasing the blood pressure and exacerbating the burden on the cardiovascular system (Wang et al., 2024).

MF is closely related to the occurrence and development of non-collagenous fibers, which play important roles in normal cardiac tissues. Non-collagen fibers together with collagen fibers form the ECM of the myocardial cells, providing structural support and mechanical stability to the cells (Shamhart and Meszaros, 2010). Non-collagen fibers include various components, such as elastic fibers, fibronectin, and laminin. In MF, changes to the non-collagen fibers often occur before significant changes to the collagen fibers. In the early stages of MF, fibronectin may respond to myocardial injury (Ning et al., 2017). Non-collagen fibers are shown to promote the adhesion, migration, and activation of cardiac fibroblasts, laying the foundation for excessive deposition of collagen fibers (Luther et al., 2012). Elastic fibers endow the myocardium with a certain degree of elasticity in a normal heart, which is beneficial for relaxation and contraction of the heart (Hiesinger et al., 2012). However, these elastic fibers are damaged or replaced by collagen fibers in MF, leading to decreased elasticity and compliance of the heart (Lin et al., 2022). In addition, non-collagen fibers can participate in the regulation of the MF process by interacting with cytokines and growth factors.

Inflammatory factors are another trigger of MF, and there are numerous inflammatory factors that can directly induce MF. Both IL1β and IL6 were observed to play important roles in MF; IL1β activates the nuclear factor kappa B (NF-κB) signaling pathway to promote the production of more profibrotic factors by the cardiac fibroblasts, thereby accelerating MF (Sun et al., 2023); IL6 promotes the activation of cardiac fibroblasts and collagen synthesis through various pathways, such as the downstream signal transduction and transcription activating protein 3 (STAT3) (Rao et al., 2024). MF also triggers inflammatory reactions, resulting in a vicious cycle. As MF progresses, the structure and functions of the myocardial tissue induce local tissue hypoxia, metabolic disorders, and other conditions. Inflammatory cells such as macrophages then aggregate in the fibrotic myocardial tissues, releasing more inflammatory factors like IL1β and IL6 to exacerbate the severity of MF (Fu et al., 2024). The inflammatory factors interact with other signaling pathways to promote MF; they also affect the survival and functions of the myocardial cells, thereby promoting the development of MF (Liu et al., 2020).

VASN deletion leads to MF in mice with cardiac hypertrophy. VASN-knockout mice exhibit typical pathological, imaging, and molecular features of MF. Differential analysis of the various genes involved, especially the ECM genes (COL6A1, COL9A1, and FRAS1), showed strong correlations even as their expression levels decreased significantly in the heart tissues of VASN-knockout mice. The expression levels of inflammatory factors IL1β and IL6 were significantly upregulated in the heart tissues of VASN-knockout mice. These preliminary results reveal that VASN knockout leads to MF by downregulating the non-collagen fibers and promoting inflammation.