Introduction
Pulmonary hypertension (PH) is a heterogeneous disease characterized by increased pulmonary arterial pressure and right ventricular failure. Right ventricular failure is a leading cause of death in patients with PH (1). The right ventricle is functionally and geometrically different from the left ventricle; therefore, the pathophysiology of right ventricular failure should be examined, and the right ventricular function should be evaluated to improve the prognosis of patients with PH (2).
The 14-3-3 protein family has at least seven isoforms (beta, gamma, epsilon, eta, sigma, theta, and zeta), which are ubiquitously expressed in various mammalian tissues and play essential roles in cell regulatory processes (3). Among these isoforms, 14-3-3 epsilon has been implicated in cardiovascular diseases, including cardiac ventricular compaction, aortic aneurysm, and arrhythmia (4-6). As a circulating biomarker, a previous study demonstrated that elevated circulating 14-3-3 epsilon levels were associated with poor prognoses in patients with natural killer/T cell lymphoma (7).
However, its significance in right ventricular failure remains unclear.
14-3-3 epsilon regulates the fibrotic pathway and fibroblast proliferation (8,9). Cardiac fibroblasts play a crucial role in the development of right ventricular failure and fibrosis in the interstitial area of the heart, and are vital for maintaining the extracellular matrix that regulates the cardiomyocyte function (10). Cardiac fibroblasts also secrete matricellular proteins, which can serve as biomarkers of the right ventricular function in patients with PH (11,12).
We hypothesized that right ventricular 14-3-3 epsilon could promote right ventricular fibrosis and be a circulating biomarker reflecting the right ventricular function in patients with PH.
In this study, we showed for the first time that the expression of 14-3-3 epsilon is increased in the interstitial area of the right ventricle in right ventricular failure. Additionally, plasma levels of 14-3-3 epsilon were significantly associated with the right ventricular function in patients with PH.
Materials and Methods
Monocrotaline-induced PH rat model
Male Sprague-Dawley rats (200-250 g body weight; Charles River Laboratories, Wilmington, USA) were used in this study. Four weeks after a single subcutaneous injection of monocrotaline (60 mg/kg, Crotaline, C2401, Sigma-Aldrich, St. Louis, USA) or saline, rats were sacrificed.
The heart and lungs were then harvested. In these rats, the weight ratio of the right ventricle to the left ventricle plus septum and right ventricular systolic pressures were evaluated, and the PH rat model was validated in our previous study (12). The right ventricle and lungs were used for the analysis in this study.
Sugen/hypoxia (SuHx)-induced PH mouse model
Male mice (8-10 weeks old, C57BL/6J) were purchased from Charles River Laboratories. Mice were subcutaneously injected with SU5416 (HY-10374, MedChemExpress, Monmouth Junction, USA) at a dose of 20 mg/kg. The mice were exposed to 10% oxygen in a ventilated chamber for three weeks, after which they were returned to room air for two weeks.
Control mice were maintained in room air for five weeks. The PH mouse model was validated in our previous study (13). The right ventricle was used for this study.
Western blotting
The right ventricles or lungs were washed with phosphate-buffered saline (PBS) on ice. Proteins were extracted from the right ventricles or lungs and homogenized using TissueLyser II (QIAGEN, Venlo, Netherlands) with a protease inhibitor (BD Biosciences, Franklin Lakes, USA). The tissue lysates were then centrifuged for 15 minutes at 16,000×g at 4°C.
A Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, USA) was used to measure protein concentrations. The lysates were diluted with sodium dodecyl sulfate (SDS) sample buffer and heated at 95°C for 5 min. Each protein was then subjected to SDS-polyacrylamide gel electrophoresis at 120 V for 70 min.
Proteins were transferred to a nitrocellulose membrane (Amersham Cytiva, Tokyo, Japan) and blocked for 1 h at room temperature in 5% bovine serum albumin in Tris-buffered saline with Tween 20 (TBS-T). The membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used were 14-3-3 epsilon (1:2,000, sc23957, Santa Cruz Biotechnology, Dallas, USA) and GAPDH (1:10,000, 6004-1-Ig, Proteintech).
After rinsing with TBS-T buffer, the membranes were incubated with the appropriate IRDye secondary antibody (1:20,000, 926-32210, LI-COR Biosciences, Lincoln, USA) for 1 h at room temperature. After rinsing with TBS-T buffer again, the Odyssey CLX imaging system (LI-COR Biosciences) was used to detect the fluorescent immunoreactive bands. Empiria Studio (LI-COR Biosciences) was used to evaluate the optical densities of the individual bands (12,14).
Histological evaluation
The harvested right ventricles were fixed using 4% paraformaldehyde (PFA) and embedded in paraffin blocks. Paraffin sections were cut and adhered to microscope slides. For fluorescent immunohistochemical staining, after deparaffinization and rehydration, antigen retrieval was performed using proteinase K (S3004, Agilent, Santa Clara, USA) at 37°C for 10 min.
Slides were then blocked in PBS plus 5% goat serum and incubated with the primary antibody against 14-3-3 epsilon (1:100, sc23957, Santa Cruz Biotechnology) or vimentin (1:100, GT7812, GeneTex Irvine, USA) at 4°C overnight in a moist chamber. The next day, the slides were washed with PBS and incubated with the appropriate secondary antibody, Alexa Fluor 647 (1:500, 4410, Cell Signaling Technology, Danvers, USA) for 1 h at room temperature. Slides were mounted using DAPI (17985-51, Electron Microscopy Sciences, Hatfield, USA).
Masson's Trichrome staining was performed as previously described (15,16). All the slides were scanned using an all-in-one fluorescence microscope (BZ-X700, Keyence, Osaka, Japan) (17).
Human study population
We enrolled 15 patients with pulmonary arterial hypertension and 68 with chronic thromboembolic pulmonary hypertension (CTEPH) at our institution between January 2010 and December 2023. Pulmonary arterial hypertension, including idiopathic pulmonary arterial hypertension, pulmonary arterial hypertension associated with connective tissue disease, and CTEPH, was diagnosed through physical examination, blood tests, right heart catheterization, echocardiography, pulmonary function tests, ventilation-perfusion scintigraphy, computed tomography, and pulmonary angiography (18-20). Clinical information was collected, including age, sex, body mass index, World Health Organization (WHO) classification, history of cancer, natural anticoagulant deficiency (such as protein C and S), blood tests, echocardiographic data, hemodynamic data, and medications.
Control subjects without a history of cardiac or respiratory diseases were also included. Balloon pulmonary angioplasty was performed in patients with CTEPH (21). The interval between balloon pulmonary angioplasties was ＞2 weeks.
Enzyme-linked immunosorbent assay (ELISA)
Venous blood was obtained from all patients upon admission to the hospital and from rats. The blood was centrifuged at 3,000 revolutions per minute (rpm) for 10 min to obtain plasma, which was then stored at -80ºC. We then measured the plasma levels of 14-3-3 epsilon using an ELISA (CSB-EL026287HU, CUSABIO, Wuhan, China) according to the standard curve method based on the manufacturer's protocol.
Optical density was determined using a microplate reader (SpectraMax i3, Molecular Devices, Tokyo, Japan) at 450 nm. We also collected plasma samples from 25 patients with CTEPH before and after balloon pulmonary angioplasty.
Statistics
Normality was analyzed using the Shapiro-Wilk test. Continuous data are shown as the mean±standard deviation, and skewed data are expressed as medians and interquartile ranges. Categorical variables are presented as numbers and percentages.
The unpaired Student's t-test (two-sided) or Mann-Whitney U test was performed for the comparison of two groups. For more than two groups, the Kruskal-Wallis test was followed by Dunn's multiple comparisons test. Spearman or Pearson correlation analyses were performed to investigate the correlations between the two variables.
A receiver operating characteristic analysis was performed to discriminate right ventricular dysfunction (tricuspid lateral annular peak systolic velocity ＜9.5 cm/s). The combined score was calculated using a logistic regression analysis. Paired data were analyzed using paired t-test or Wilcoxon test.
Statistical significance was set at p＜0.05. Data were analyzed using GraphPad Prism 10 (GraphPad Software, San Diego, USA).
Alignment analysis
EMBOSS Needle (European Molecular Biology Laboratory, Cambridge, UK) was used to analyze alignments of the same sequence in rats and human 14-3-3 epsilon (22).
Ethics statement
All animal experiments were conducted with the approval of the Fukushima Medical University Ethics Committee for the Care and Use of Laboratory Animals (2024084). This human study was approved by the Ethics Committee of Fukushima Medical University (No. 823) and was conducted in accordance with the principles outlined in the Declaration of Helsinki.
Written informed consent was obtained from all patients.
Results
To investigate the 14-3-3 epsilon expression in right ventricular failure, we used right ventricular tissue specimens of a monocrotaline-induced PH rat model, validated by increased right ventricular pressure (Fig. 1a) (12). The right ventricular 14-3-3 epsilon expression was higher in the monocrotaline group than in the control group (Fig.
1b). The expression of 14-3-3 epsilon in the lung did not differ between the monocrotaline and control groups (Fig. 1b).
Masson's trichrome staining showed that the right ventricle in the monocrotaline group had increased myocardial fibrosis (Fig. 1c). Immunofluorescence also showed the increased expression of 14-3-3 epsilon and vimentin in the interstitial area of the right ventricle in the monocrotaline group (Fig.
1c). The median plasma level (interquartile range) of 14-3-3 epsilon was lower in the monocrotaline group than that in the control group [monocrotaline, 34 (32-36) ng/mL; control, 48 (46-59) ng/mL; p=0.003]. Plasma levels of 14-3-3 epsilon were significantly correlated with the tricuspid lateral annular peak systolic velocity in rats (r=0.849, p=0.008).
Additionally, the expression of 14-3-3 epsilon in the right ventricle was increased in the SuHx-induced PH mouse model in comparison to that in the control group (Fig. 1d). The ratio of the right ventricle to left ventricle plus septal weight [RV/(LV+IVS)] was increased in the SuHx-induced PH mouse model in comparison to the control group (0.30±0.03 vs.
0.20±0.02; p=0.008).
Next, we validated the amino acid sequence of 14-3-3 epsilon across rats and humans. EMBOSS Needle was used to analyze rat and human 14-3-3 epsilon alignments of the same sequence, which showed an identity of 254/255 (99.6%), similarity of 254/255 (99.6%), and gaps of 0/255 (0.0%), demonstrating high sequence identity. To clarify the significance of 14-3-3 epsilon in human right ventricular dysfunction, we measured the plasma levels of 14-3-3 epsilon in control subjects and patients with pulmonary arterial hypertension and CTEPH.
The clinical characteristics of patients are presented in Table 1. The median plasma levels (interquartile range) of 14-3-3 epsilon were 175 (145-246), 90 (54-113), and 97 (59-169) ng/mL in control subjects and patients with pulmonary arterial hypertension and CTEPH, respectively (Fig. 2a).
In patients with PH, including pulmonary arterial hypertension and CTEPH, plasma levels of 14-3-3 epsilon were higher in females. There were no significant differences in the plasma levels of 14-3-3 epsilon between individuals with and without a history of cancer, natural anticoagulant deficiency, pulmonary vasodilators, or anticoagulants (Fig. 2b).
The correlation analysis revealed that plasma levels of 14-3-3 epsilon were significantly associated with tricuspid lateral annular peak systolic velocity and right ventricular fractional area changes in patients with PH (Table 2). Plasma levels of 14-3-3 epsilon were not correlated with plasma levels of brain natriuretic peptide (BNP, Table 2). Right ventricular dysfunction was defined as a tricuspid lateral annular peak systolic velocity of ＜9.5 cm/s and a receiver operating characteristic analysis was performed to discriminate right ventricular dysfunction.
The combination of BNP and plasma 14-3-3 epsilon showed a higher area under the curve than BNP alone to discriminate right ventricular dysfunction in patients with PH (Fig. 3).
Finally, we evaluated the plasma levels of 14-3-3 epsilon in 25 patients with CTEPH before and after balloon pulmonary angioplasty. The mean number of balloon pulmonary angioplasty sessions was 4.0±1.7. As shown in Table 3, the plasma levels of 14-3-3 epsilon increased from 68 (34-167) ng/mL to 128 (69-179) ng/mL after balloon pulmonary angioplasty (p=0.008), along with improvement of the right ventricular function.
Discussion
This study demonstrated that 14-3-3 epsilon was highly expressed in the right ventricle of animal models of right ventricular failure. Plasma levels of 14-3-3 epsilon are significantly associated with the right ventricular function in patients with PH.
14-3-3 epsilon is a highly conserved member of the 14-3-3 family in mammalian species, and its physiological role varies depending on tissue and cellular type (6,23). 14-3-3 epsilon inhibits autophagy in hepatocytes (24). In addition, 14-3-3 epsilon protects against cerebral infarction in vivo (25).
In cardiac fibroblasts, 14-3-3 epsilon inhibits proliferation (9). Although mechanistic analyses were not performed in this study, the increased expression of 14-3-3 epsilon in the interstitial area of the right ventricle suggests that 14-3-3 epsilon may play a role in promoting right ventricular fibrosis and failure in PH.
Circulating biomarkers are clinically important in the diagnosis, evaluation of severity, prognostication, and monitoring of responses to treatment in patients with PH and right ventricular failure (26-28). BNP, N-terminal pro-BNP, and troponin have been established as biomarkers of right ventricular failure derived from cardiomyocytes (29). In contrast, circulating 14-3-3 epsilon was thought to be derived from right ventricular fibroblasts in the interstitial area of the heart, as suggested by immunostaining of the right ventricle.
Previous research has shown that some cardiac fibroblast-associated circulating biomarkers exhibit a paradox, where decreasing circulating protein levels are accompanied by increased protein levels in the heart of patients with heart failure, such as cartilage intermediate layer protein 1 (30). Similarly, in the present study, plasma 14-3-3 epsilon levels decreased in association with a decline in the right ventricular function. However, myocardial 14-3-3 epsilon levels were increased in patients with right ventricular failure.
This paradox could be explained by the hypothesis that 14-3-3 epsilon is sequestered in the cardiac interstitial area, decreasing plasma levels.
The present study was associated with some limitations. First, the right ventricles of animal models were used to investigate the expression of 14-3-3 epsilon in right ventricular failure. Although the amino acid sequences of 14-3-3 epsilon are conserved among humans and rats, the expression of 14-3-3 epsilon in the human right ventricle in right ventricular failure should be analyzed.
Second, in the present study, the correlation between plasma 14-3-3 epsilon levels and the right ventricular function was weak, and there were no correlations with hemodynamic parameters derived from right heart catheterization. Therefore, further studies with a larger sample size are needed to establish the usefulness of circulating 14-3-3 epsilon levels in evaluating the right ventricular function. Because plasma 14-3-3 epsilon could originate from the right ventricle, plasma 14-3-3 epsilon might better reflect the right ventricular function than the mean pulmonary artery pressure.
In conclusion, this is the first study to demonstrate an association between 14-3-3 epsilon and right ventricular failure. The plasma 14-3-3 epsilon level may be a useful circulating marker for evaluating right ventricular dysfunction in patients with PH.
The authors state that they have no Conflict of Interest (COI).