Association of FOXO3A with right ventricular myocardial fibrosis and its detection by speckle‐tracking echocardiography in pulmonary hypertension

Myocardial fibrosis can result in right ventricular (RV) dysfunction, a critical factor in poor clinical outcomes and high mortality rates among patients with pulmonary hypertension (PH). Decreased RV myocardial strain rates have been reported in PH patients. The expression of FOXO3A may play a crucial role in myocardial fibrosis; however, the relationship between myocardial fibrosis, speckle‐tracking echocardiography (STE), and the transcription factor FOXO3A remains unclear. This study aimed to explore the relationship between the molecular mechanisms of myocardial fibrosis and noninvasive ultrasound evaluation indices to provide a reliable molecular foundation for the early diagnosis of right heart dysfunction in clinical settings.

global, RVLS FW, and collagen deposition. FOXO3A expression gradually decreased with disease progression, while BNP, collagen I, and collagen III expressions gradually increased.

Conclusions: Decreases in RVLS global and RVLS FW in RHF rats occurred earlier than
RVFAC and were associated with RV myocardial fibrosis. Furthermore, FOXO3A may have a protective role in the process of RV myocardial fibrosis.

K E Y W O R D S
FOXO3A transcription factor, myocardial fibrosis, myocardial strain, pulmonary hypertension, right ventricular dysfunction, speckle-tracking echocardiography INTRODUCTION Pulmonary hypertension (PH) is a clinical syndrome that poses a significant threat to human life and health worldwide. 1 Right ventricular (RV) remodeling, fibrosis, and dysfunction caused by pressure overload are associated with adverse outcomes. [2][3][4] Therefore, accurate assessment of RV structure and function is a crucial aspect of PH management. 5 Understanding the mechanisms leading to RV dysfunction and identifying effective imaging techniques for early detection of RV dysfunction may lead to more targeted therapies.
Echocardiography is a widely used noninvasive cardiac imaging technique for assessing the structure and function of the heart. In recent years, advancements in echocardiography have enabled more accurate evaluation of cardiac function. The speckle-tracking echocardiography (STE) technique has proven to be an effective method for assessing RV function. 6 Reduced longitudinal strain of the RV free wall (RVLS FW) is a predictor of poor prognosis in patients with PH. Furthermore, RVLS FW has been shown to correlate with the degree of RV myocardial fibrosis. 7 Professor Xie's team 8 concluded that 3D-RVLS FW can be considered a valuable non-invasive indicator for identifying severe myocardial fibrosis in patients with end-stage heart failure (HF) compared with pathological results. However, RV remodeling is a complex process; although RV fibrosis reduces ventricular wall compliance, leading to RV dysfunction, 9 a certain degree of fibrosis can provide mechanical support to the myocardium and prevent overdistension and deformation of the right ventricle due to pressure overload. 10 Identifying the factors that regulate the transition from protection to dysfunction, which remain largely unknown, is crucial to guiding effective imaging tests for RV dysfunction. Currently, clinical research is limited by the acquisition of myocardial tissue, making it impossible to compare imaging data with pathological results during RV failure. Preclinical studies tend to treat RV dysfunction in animals as a pathological condition, regardless of its stage. Therefore, we aimed to compare echocardiography findings with histological collagen deposition in a progressive RV failure animal model to assess whether STE can be used to evaluate the course of change of RV free wall myocardial fibrosis in PH. Additionally, we will conduct a preliminary study on the possible mechanisms of RV failure.
The FOXO family is a highly evolutionarily conserved group of transcription factors, including FOXO1, FOXO3a, FOXO4, and FOXO6 in mammals. 11,12 Previous studies have shown that FOXO3 can reduce fibrosis in various organs, including the heart, liver, lung, and kidney, by inhibiting fibroblast activation and reducing extracellular matrix deposition. 11 Recent research on cardiac remodeling after myocardial infarction confirmed that activated FOXO3a can inhibit cardiac hypertrophy and fibrosis formation following myocardial infarction. 13 However, the role of FOXO3A in RV myocardial fibrosis has not been investigated. In the final part of the study, we will initially investigate whether FOXO3A is involved in the PH-related RV wall fibrosis process.
The current study, therefore, aims to explore the relationship between the molecular mechanism of myocardial fibrosis and the noninvasive ultrasound evaluation index to provide a reliable molecular basis for the early diagnosis of right heart dysfunction in clinical settings.

Animal model and experimental groups
All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 14

Right heart catheterization
Right heart catheterization tests were performed at baseline and 2 weeks after MCT injection. Rats were anesthetized by intraperitoneal injection with 35 mg/kg sodium pentobarbital and secured in a supine position on a heated table. Following routine disinfection, the right internal jugular vein was exposed and cannulated with a sheath connected to a 1.4F pressure catheter (Transonic Scisense Inc) to measure RV pressure. A multi-channel electrophysiology recorder (MP150, BIOPAC system) continuously recorded ventricular pressure and heart rate, and data were analyzed offline using AcqKnowledge 5.0 software.

Echocardiographic evaluation
Transthoracic echocardiography was performed at baseline and

Tissue preparation
At the end of the experiment, hearts were quickly excised under deep anesthesia. Heart tissues were sliced into sections approximately 2 mm thick at the middle of the ventricle and fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin for histology. The remaining heart tissues were separated into right ventricle (RV), interventricular septum (IVS), and left ventricle (LV), snap-frozen with liquid nitrogen, and stored at −80 • C for protein analysis. Total protein of heart tissues was extracted using the tissue protein extraction kit (Solarbio R0010, Beijing, China) according to the manufacturer's instructions. The protein concentration of each group was quantified using the bicinchoninic acid assay kit (Beyotime P0009, Shanghai, China).

Hematoxylin-eosin (HE) and picrosirius red (PSR) staining
Paraffin-embedded tissue was cut into 5 µm sections and stained separately with HE and Picrosirius red (PSR) (BP-DL028, Haeckel Biotechnology, Nanjing, China). HE-stained sections were used for histopathological examination, while PSR-stained sections were used for collagen deposition assessment. For each sample, five visual fields were randomly captured. Image J 1.53 software (National Institutes of Health) was used to calculate cardiomyocyte cross-sectional areas and the percentage of collagen area in each field. The percentages of all these random areas from the same sample were averaged and presented as the result for that sample.

Western blotting
Equal amounts of total heart protein from RV, IVS, and LV (30 µg) were loaded onto a 10% SDS-PAGE gel and transferred onto a PVDF values for each rat in subsequent analyses. The experimental data are expressed as mean ± standard deviations (SD) and were found to meet the normal distribution. One-way analysis of variance (ANOVA) with post hoc Tukey's multiple comparisons test or Sidak's multiple comparisons test was used for multiple comparisons to analyze differences between the baseline and each disease course group, as well as between two adjacent disease course groups. Pearson correlation was used to test the correlation between parameters. The agreement between right heart catheterization and echocardiography was analyzed using the Bland-Altman method. A p-value <.05 was considered statistically significant, with significances indicated as p < .05 (*), p < .01 (**), p < .001 (***) and p < .0001 (****).

General characteristics
General characteristics are summarized in Table 1. The mean baseline body weight of rats was 242 ± 5.6 g, and each group gained weight with increasing age and disease duration (p < .0001). Heart rates were not significantly different among the experimental groups (p = .947).

Hemodynamics
The results of hemodynamics are shown in  (Figure 1), the mean value of the difference between the two methods is 11.06, and the 95% confidence interval of the difference is 5.91 ∼ 16.22. All measured data are within the 95% confidence inter-F I G U R E 1 Agreement of the mPAP measured by right heart catheterization and echocardiography was good.
val, indicating good agreement between the mPAP measured by right heart catheterization and echocardiography.

Alterations in right heart structure and function
The results of conventional echocardiography for each group are presented in Table 2

Myocardial speckle tracking
The results of RVLS global and RVLS FW are shown in Table 2 and the baseline (p < .01), with a sustained reduction at 6 weeks. For RVLS FW, the mean value at baseline was −27.11 ± 3.82% and a decrease was observed at 2 weeks compared to the baseline (p < .05), with no significant differences between 2 and 4 weeks (p = .0526); however, a decrease was observed between 4 and 6 weeks (p < .05).

Histopathological changes of RV myocardial tissue
The results of HE staining of rat myocardial tissue slices are shown in Figure 4. With disease progression, the RV wall gradually thickened and the RV chamber enlarged. RV cardiomyocytes in the baseline were arranged regularly, while after MCT injection, RV cardiomyocytes were gradually abnormal myocardial architecture, as evidenced by an increase in cardiomyocyte diameter, cardiomyocyte disarray and structural disorganization. RV cardiomyocyte cross sectional area increased at 2 weeks compared with baseline (p < .05), and significant increased further at 4-week (p < .0001), 6-week (p < .0001).

Cardiac fibrosis and its relation to myocardial speckle tracking
The representative myocardial histological sections from different disease duration groups are shown in Figures 5 and 6. With the progression of the disease, PSR staining showed increased collagen volume fractions in the RV free wall, especially at 6 weeks

FOXO3A expression decreased in RV free wall myocardial tissue of RHF rats
As shown in Figure 7, with the progression of the disease, the expression of FOXO3A decreased, while the expression of BNP increased in RV free wall myocardial tissue according to disease duration. However, no significant change was observed in the expression of FOXO3A and BNP among left ventricular and septum myocardial tissue.

DISCUSSION
The principal finding of our study was the gradual decrease of RVLS prognosis of PH patients. 3,18 However, the onset of right heart dys-function in PH is insidious; some PH patients already have changes in the structure and function of the right heart system before the appearance of clinical symptoms. Right heart dysfunction has often progressed into refractory heart failure when RHF is clinically diagnosed, at which point patients may miss the opportunities for early clinical intervention. 19,20 Moreover, the chronic course of PH requires routine monitoring of cardiac function, and noninvasive ultrasound test is the best option. In the present study, we established a rat model that simulates the process of RV hypertrophy and failure in PH, and gathered echocardiography data at different stages of disease. We verified that Speckle tracking echocardiography (STE) could detect myocardial fibrosis early, and it could identify right heart dysfunction earlier than traditional ultrasound index. This provides reliable experimental evidence for routine management of PH patients using STE technology.

F I G U R E 6
As the course of the disease progressed, gradually increase of collagen I and collagen III protein expression in RV free wall. But not observed at left ventricular and interventricular septum. Protein expression levels of collagen I and collagen III were evaluated by western blotting (A, B). Quantitative analysis of collagen I and collagen III protein expression levels (C-F). *p < .05; **p < .01; ***p < .001; ****p < .0001.
In PH, as the pressure in the pulmonary vasculature increases, adaptations in the right ventricle are necessary to manage the augmented afterload. Our study observed that myocardial tissue thickened in response to elevated pressure, but no apparent abnormalities in RA and RV structural parameters, as well as RVOT and MPA, were detected during the early stage of PH (2-week). Significant changes in these parameters were observed later in the disease progression (6week In order to explore the molecular mechanism of RV myocardial fibrosis, we focused on the transcription factors FOXO3A. FOXO3A is a core regulator involved in the modulation of various physiological and pathological processes, including oxidative stress, proliferation, differentiation, apoptosis, and metabolism. 24 It has been reported that decreased FOXO3A expression was associated with enhanced cardiac fibrosis in peripartum cardiomyopathy, which is closely related to the promoting antioxidative defense function of FOXO3A. 25 Additionally, FOXO3A also has a role in inhibiting cardiac fibroblast hyperplasia by promoting p27 expression. 26 In this study, we observed that the expression trend of FOXO3A, Collagen I, Collagen III, and BNP in RV free wall myocardial tissue showed regularity, and the expression of Collagen I, Collagen III, and BNP showed the same trend, all of them increasing during the disease. Conversely, the expression of FOXO3A showed the opposite trend with Collagen I, Collagen III, and BNP, which gradually decreased during the disease. Additionally, this phenomenon was not observed in the interventricular septum and left ventricular myocardial tissues, and the expression of all four proteins remained normal. The results showed that FOXO3A expression gradually decreased, suggesting that FOXO3A may be an intervention site for right heart dysfunction. However, the specific mechanism of how it loses its protective effect is still unclear, which will be further explored in future studies.

CONCLUSIONS
Our study suggested that STE can detect RV myocardial fibrosis dur-

Limitations
Our study has limitations. Firstly, PH is a disease of varying etiology, in our study, we only used one disease model to observe PH-induced RHF.
This is because the pulmonary valve regurgitation model has high surgical mortality, and it is difficult for the two groups to reach a uniform study period. Secondly, Due to technical limitations of our equipment, we were unable to obtain 3D images of the rat's right ventricle for 3DSTE analysis. Consequently, the current 2DSTE RV strain assessment only represents the strain conditions of the RV free wall and interventricular septum, rather than providing a comprehensive analysis. Thirdly, we only observed the changes in FOXO3A expression and did not conduct in-depth research on its mechanism, we will continue this part of the work in future research.

ACKNOWLEDGMENTS
The study was completed with the support of Ms. Rui Bu (Depart-

CONFLICTS OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
All available data can be obtained by contacting the corresponding author on reasonable request.

CONSENT FOR PUBLICATION
Not applicable.