Compensatory activation of PI3K/AKT signaling and apoptosis-associated protein expression in pulmonary hypertension‑induced right ventricular dysfunction

Introduction

Pulmonary hypertension (PH) is a fatal cardiopulmonary vascular disorder characterized by progressive elevation of pulmonary arterial pressure, driven by a combination of pulmonary vasoconstriction, vascular remodeling and inflammation (1). These pathological changes collectively increase pulmonary vascular resistance, leading to progressive right ventricular (RV) pressure overload and eventual heart failure. PH is estimated to affect ≥1% of the global population, with its prevalence approaching 10% in individuals aged >65 years (2-4). Despite advances in vasodilator therapies, including prostacyclin analogues, endothelin receptor antagonists and phosphodiesterase-5 inhibitors, the long-term outcomes of PH remain poor, with 5-year survival rates of 50-60% (5).

RV dysfunction is increasingly recognized as the primary determinant of survival in patients with PH, surpassing the prognostic importance of pulmonary vascular pathology alone (6-9). The right ventricle is anatomically and functionally distinct from the left ventricle; it is thinner-walled, has a more complex fiber orientation, and possesses limited coronary perfusion reserve and metabolic capacity, rendering it particularly vulnerable to sustained pressure overload (10). Chronic pressure overload triggers a cascade of RV changes, including concentric hypertrophy, metabolic reprogramming toward glycolysis, increased expression of apoptosis-related proteins, such as Bax, interstitial fibrosis and capillary rarefaction, ultimately progressing to chamber dilation, systolic dysfunction and clinical RV failure (11,12). These findings highlight the urgent need for therapeutic strategies that directly target RV protection, rather than focusing exclusively on pulmonary vasodilation.

The PI3K/AKT signaling pathway is a central regulator of cell survival, proliferation, metabolism and apoptosis in the cardiovascular system (13,14). Upon activation by growth factors, cytokines or mechanical stimuli, PI3K phosphorylates its downstream effector AKT, which in turn modulates the expression and activity of numerous target proteins involved in mitochondrial integrity, protein synthesis and redox balance. In cardiomyocytes, PI3K/AKT signaling promotes survival by upregulating anti-apoptotic proteins, such as Bcl-2, and suppressing pro-apoptotic mediators, such as Bax (15-17). However, the role of this pathway in PH-induced RV remodeling remains poorly characterized. While previous studies have focused on the pulmonary vasculature (18,19), whether PI3K/AKT signaling serves as a compensatory protective mechanism in the pressure-overloaded RV, and whether its modulation could attenuate RV dysfunction, remains largely unexplored.

The present study aimed to characterize molecular changes associated with RV remodeling in a murine model of hypobaric hypoxia-induced PH. Following transcriptomic identification of PI3K/AKT pathway enrichment, its potential involvement in apoptosis-associated changes was further evaluated using in vitro inhibition experiments.

Materials and methods

Animals and PH model

Male C57BL/6J mice (age, 8 weeks; weight, 20-25 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. [license no. SCXK (Beijing) 2024-0003]. A total of 16 mice were used in the present study. After 1 week of acclimation, the mice were randomly assigned to the normoxic control (n=8, housed under normal atmospheric conditions with 21% O2) or hypobaric hypoxia-induced PH (n=8) groups. PH was induced by continuous exposure to a hypobaric chamber simulating 5,500 m altitude (10% O2) for 28 days (19). Soda lime and anhydrous calcium chloride were used to absorb CO2 and moisture in the enclosures of PH-group mice. All animal procedures were performed in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines (20) and approved by the Animal Care and Welfare Committee of Beijing Institute of Heart, Lung and Blood Vessel Diseases and Beijing Anzhen Hospital, Capital Medical University (Beijing, China; approval no. 2025205x).

Mice were housed in standard polycarbonate cages (maximum, 4 mice/cage) under a 12-h light/dark cycle, at 22±2˚C and 55±5% humidity, with ad libitum access to filtered water and a standard commercial rodent maintenance diet (Beijing Huafukang Biotechnology Co., Ltd.). Animal bedding was enriched with nesting material. For echocardiography, mice were anesthetized with isoflurane (2% induction; 1-1.5% maintenance). For terminal tissue collection, mice were deeply anesthetized with tribromoethanol (250 mg/kg; intraperitoneal) and euthanized by cervical dislocation. Humane endpoints were predefined (≥20% weight loss, severe respiratory distress or immobility), but no early euthanasia was required. All animals were euthanized at the end of the experiment.

Cell culture and treatments

H9C2 cells (cat. no. CRL-1446; American Type Culture Collection) were cultured in DMEM/F-12 (cat. no. D6572; Beijing Solarbio Science & Technology Co., Ltd.) with 10% fetal bovine serum (cat. no. 10099141C; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. P1400; Beijing Solarbio Science & Technology Co., Ltd.) at 37˚C in 5% CO2. Prior to stimulation, cells were serum-starved for 12 h. Cells were subsequently incubated with 1 µM Ang II (cat. no. P1085; Selleck Chemicals) for 24 h at 37˚C to induce apoptosis. Ang II was selected as it is a well-established pro-apoptotic stimulus in cardiomyocytes and has been shown to regulate PI3K/AKT signaling (21). In selected experiments, cells were pretreated with 10 µM LY294002 (cat. no. S1105; Selleck Chemicals) 30 min before Ang II exposure.

Echocardiography

Transthoracic echocardiography was performed using a VINNO 6 system [VINNO Technology (Suzhou) Co., Ltd.] with a 40 MHz transducer at baseline and after 28 days of exposure to either normoxic (control) or hypobaric hypoxic conditions. For this procedure, mice were anesthetized with 2% isoflurane, which was maintained at a concentration of 1-1.5%. Left ventricular dimensions, volumes (including end-diastolic and end-systolic volumes), stroke volume, ejection fraction and fractional shortening were measured and averaged over three cycles.

Tissue collection

On the final day of the experimental protocol, mice were fasted overnight (~12 h) with free access to water prior to tissue collection. After echocardiography, mice were deeply anesthetized with tribromoethanol (250 mg/kg; intraperitoneal) and euthanized by cervical dislocation. Following euthanasia, the hearts and lungs of mice were excised following intratracheal perfusion with cold saline. Tissues were fixed in 4% paraformaldehyde at 4˚C for 24 h or snap-frozen in liquid nitrogen (-196˚C) for subsequent molecular analysis.

RNA sequencing and analysis

Total RNA was extracted from RV tissue using TRIzol® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.), and RNA integrity was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) with an RNA integrity number ≥7.0. Strand-specific libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (cat. no. E7760; New England Biolabs, Inc.) and sequenced on an Illumina NovaSeq 6000 platform (Illumina, Inc.; paired-end; 150 bp). The final library loading concentration was 1.8 pM, as measured by quantitative PCR (qPCR) using the KAPA Library Quantification Kit (cat. no. KK4824; Roche Diagnostics). Raw sequencing reads were quality-controlled with FastQC (v0.11.9; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), aligned to the mouse reference genome GRCm38 (https://www.ensembl.org/Mus_musculus/Info/Index) using HISAT2 (v2.2.1; https://daehwankimlab.github.io/hisat2/), and quantified with featureCounts (v2.0.1; https://subread.sourceforge.net/), which is an R package included in the Subread software suite. Differentially expressed genes were identified using DESeq2 (v1.38.3; https://bioconductor.org/packages/DESeq2/) with thresholds of |log2 fold change (FC)| ≥1 and false discovery rate (FDR) <0.05. Further analyses using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed using the clusterProfiler R package (v4.6.0; https://bioconductor.org/packages/clusterProfiler/). All R packages were run on R (version 4.3.0; R Foundation for Statistical Computing).

Western blotting

Proteins were extracted from RV tissue samples and H9C2 cells using radio-immunoprecipitation assay buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) and quantified by bicinchoninic acid assay (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.). Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% gels and transferred to PVDF membranes (cat. no. HVLP09050; MilliporeSigma). After blocking with 5% bovine serum albumin (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) in Tris-buffered saline-0.1% Tween-20 at room temperature for 1 h, the membranes were incubated overnight at 4˚C with primary antibodies against Bcl-2 (1:1,000; cat. no. 68103-1-Ig; Proteintech Group, Inc.), Bax (1:1,000; cat. no. 60267-1-Ig; Proteintech Group, Inc.) and GAPDH (1:10,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.). The membranes were subsequently incubated with HRP-conjugated secondary antibodies (1:5,000; cat. no. ZB-2305; OriGene Technologies, Inc.) at room temperature for 1 h. Bands were visualized using an enhanced chemiluminescence kit (cat. no. G2020; Wuhan Servicebio Technology Co., Ltd.) and semi-quantified using ImageJ software (version 1.53; National Institutes of Health).

Reverse transcription-qPCR (RT-qPCR)

Total RNA was extracted from RV tissues using TRIzol reagent and was reverse-transcribed into cDNA using the PrimeScript™ RT Reagent Kit (cat. no. RR037A; Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol, with the following thermal profile: 37˚C for 15 min, followed by 85˚C for 5 sec. The resulting cDNA was amplified using SYBR Green Master Mix (cat. no. 9109-036-820; Takara Biotechnology Co., Ltd.) on a LightCycler® 96 System (Roche Diagnostics). The thermocycling protocol was as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles at 95˚C for 5 sec and 60˚C for 30 sec, with a final melt-curve analysis to verify amplification specificity. The following primer sequences were used: Bax, forward 5'-AAACTGGTGCTCAAGGCCC-3', reverse 5'-GGTCCCGAAGTAGGAGAGGA-3'; Bcl-2, forward 5'-GACTGAGTACCTGAACCGGC-3', reverse 5'-AGTTCCACAAAGGCATCCCAG-3'; and Gapdh, forward 5'-ACCCTTAAGAGGGATGCTGC-3', reverse 5'-CCCAATACGGCCAAATCCGT-3'. Gapdh served as the internal control. Relative gene expression was calculated using the 2-ΔΔCq method (22). All reactions were performed in triplicate.

Histology and fibrosis quantification

Paraffin-embedded myocardial sections (4 µm) were fixed with 4% paraformaldehyde at 4˚C for 24 h prior to embedding. Sections were then stained with Masson's trichrome at room temperature for 60 min according to the manufacturer's protocol (cat. no. G1340; Solarbio Science & Technology Co., Ltd.). Collagen-positive areas were quantified in five random fields per section using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.) and expressed as a percentage of the total area. Images were captured using a light microscope (BX53; Olympus Corporation).

Immunofluorescence

The aforementioned paraffin-embedded myocardial sections underwent immunofluorescence staining. After dewaxing in xylene I, II and III (10 min each) and rehydration through a graded alcohol series (100, 95, 90, 80 and 70% ethanol; 5 min each), antigen retrieval was performed by heating the sections in citrate buffer (10 mM, pH 6.0) at 95˚C for 20 min, followed by cooling to room temperature. After washing with PBS, the sections were permeabilized with 0.25% Triton X-100 in PBS for 10 min at room temperature and then blocked with 5% normal goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.) in PBS at room temperature for 1 h. Subsequently, the myocardial sections were incubated with primary antibodies against Ki-67 (cat. no. 66555-6-Ig; Proteintech Group, Inc.), Bax (cat. no. 60267-1-Ig; Proteintech Group, Inc.) and Bcl-2 (cat. no. 68103-1-Ig; Proteintech Group, Inc.) overnight at 4˚C. After washing with PBS, the sections were incubated with Multi-rAb® CoraLite® Plus 488-conjugated secondary antibody (cat. no. RGAM002; Proteintech Group, Inc.) and Multi-rAb CoraLite Plus 594-conjugated secondary antibody (cat. no. RGAM004; Proteintech Group, Inc.) at room temperature for 1 h. Nuclei were counterstained with DAPI (cat. no. ab104139; Abcam) at room temperature for 5 min. Images were captured under a fluorescence microscope (BX53; Olympus Corporation), and positively stained cell ratios were quantified using ImageJ software (version 1.53).

Statistical analysis

Data are presented as the mean ± SD. Comparisons between two groups were made using unpaired Student's t-tests and comparisons among multiple groups were made using one-way ANOVA with Tukey's post hoc test. Analyses were performed with GraphPad Prism 9.0 (Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Results

Echocardiography reveals impaired cardiac function in PH model mice

After 28 days of hypoxia, echocardiography (Fig. 1A) demonstrated preserved interventricular septal thickness at diastole (Fig. 1B), left ventricular posterior wall thickness at diastole (Fig. 1D), left ventricular internal diameter at diastole (Fig. 1F), left ventricular end-diastolic volume (Fig. 1H) and left ventricular diastolic mass (Fig. 1M) in PH mice compared with in the control group. However, PH model mice showed significant reductions in interventricular septal thickness at systole (Fig. 1C), left ventricular posterior wall thickness at systole (Fig. 1E), fractional shortening (Fig. 1J), stroke volume (Fig. 1K) and left ventricular ejection fraction (Fig. 1L) compared with those in the control group. These changes were accompanied by significant increases in left ventricular internal diameter at systole (Fig. 1G) and left ventricular end-systolic volume (Fig. 1I) in PH model mice compared with control mice. These findings indicated that PH-induced RV failure was associated with secondary left ventricular systolic dysfunction.

Figure 1 Echocardiographic assessment of cardiac function in PH model mice. (A) Representative M-mode echocardiographic images of the left ventricle from normoxic C mice and PH model mice after 28 days of hypobaric hypoxia exposure. Parameters were measured by echocardiography, including (B) IVSd, (C) IVSs, (D) LVPWd, (E) LVPWs, (F) LVIDd, (G) LVIDs, (H) LVEDV, (I) LVESV, (J) FS, (K) SV, (L) LVEF and (M) LVd mass. Data are presented as mean ± SD (n=8 per group). *P<0.05, **P<0.01 and ***P<0.001 vs. C group. C, control; PH, pulmonary hypertension; IVSd, interventricular septal thickness at diastole; IVSs, interventricular septal thickness at systole; LVPWd, left ventricular posterior wall thickness at diastole; LVPWs, left ventricular posterior wall thickness at systole; LVIDd, left ventricular internal diameter at diastole; LVIDs, left ventricular internal diameter at systole; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; FS, fractional shortening; SV, stroke volume; LVEF, left ventricular ejection fraction; LVd, left ventricular diastolic.

Transcriptomic profiling identifies enrichment of PI3K/AKT signaling-related genes in PH model mice

RNA sequencing revealed 1,101 significantly upregulated genes and 577 significantly downregulated genes in PH model mice compared with in the control group (Fig. 2A). KEGG enrichment analysis identified significant enrichment of the ‘PI3K-AKT signaling pathway’ (FDR<0.01; Fig. 2C). GO enrichment analysis identified significantly enriched terms, including ‘extracellular matrix structural constituent’, ‘DNA-binding transcription factor activity’, ‘response to oxidative stress’ and ‘regulation of cell cycle’ (Fig. 2B), indicating that PH-induced RV remodeling involves alterations in extracellular matrix organization, transcriptional regulation, oxidative stress responses and cell cycle control. KEGG enrichment analysis identified significant enrichment of the ‘PI3K-Akt signaling pathway’ (FDR<0.01; Fig. 2C). GSEA further supported enrichment of the PI3K/AKT signaling pathway in PH model mice (normalized enrichment score, 2.14; P<0.001; Fig. 2D).

Figure 2 Transcriptomic profiling of RV tissue from PH mode mice. (A) Volcano plot showing differentially expressed genes between control and PH RV tissues. Red dots indicate significantly upregulated genes, green dots indicate significantly downregulated genes (|log2FC|≥1; FDR<0.05) and blue dots represent genes with no significant changes. The dashed vertical line indicates log2FC=0, whereas differentially expressed genes were defined using |log2FC|≥1 and FDR<0.05; the dashed horizontal line indicates -log10(FDR)=1.3. (B) Gene Ontology enrichment analysis of differentially expressed genes. (C) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. (D) Gene Set Enrichment Analysis supporting increased activation of the PI3K/AKT signaling pathway in PH model mice. RV, right ventricular; PH, pulmonary hypertension; FC, fold change; FDR, false discovery rate.

PH induces changes in apoptosis-associated protein expression and RV fibrosis

Western blotting and RT-qPCR showed significantly increased Bax expression and significantly decreased Bcl-2 expression at the mRNA and protein levels in PH model mice compared with those in the control mice, indicating a pro-apoptotic shift in apoptosis-associated signaling (Fig. 3A-E). Immunofluorescence staining supported significantly enhanced Bax and reduced Bcl-2 levels in PH model mice compared with in control mice (Fig. 3H-K). Furthermore, Masson's trichrome staining revealed significantly increased collagen deposition in PH model mice compared with in control mice, which is consistent with RV fibrosis (Fig. 3F and G).

Figure 3 Cardiomyocyte apoptosis and RV fibrosis in PH model mice. (A) Representative western blotting images showing the protein expression of Bax (21 kDa) and Bcl-2 (26 kDa) in RV tissues from C and PH model mice; GAPDH (36 kDa) was used as a reference protein. The protein expression levels of (B) Bax and (C) Bcl-2 were semi-quantified. The relative mRNA expression levels of (D) Bax and (E) Bcl-2 were determined by RT-qPCR and normalized to GAPDH. C-1/C-2 and PH-1/PH-2 denote individual biological replicates from C and PH groups. The bands shown are representative images from 6 biological replicates per group; the samples were divided across three separate gels due to loading capacity. (F) Representative Masson's trichrome-stained myocardial sections and (G) quantitative analysis of collagen deposition (blue staining) in RV tissues. Arrows indicate collagen-positive areas (blue staining). Representative immunofluorescence images of (H) Bax and (I) Bcl-2 protein levels in mouse myocardial tissues. Quantitative analysis of (J) Bax-positive and (K) Bcl-2-positive cell ratios. Data are presented as the mean ± SD (n=6 per group for western blotting, RT-qPCR and histology). *P<0.05 vs. C group. RV, right ventricular; C, control; PH, pulmonary hypertension; RT-qPCR, reverse transcription-quantitative PCR.

PH is associated with a small increase in Ki-67-positive cardiomyocytes

Immunofluorescence analysis showed a small but statistically significant increase in Ki-67-positive cardiomyocytes in RV tissues from PH model mice compared with controls (Fig. 4A and B). However, the proportion of Ki-67-positive cardiomyocytes remained very low (~0.4%). As Ki-67 positivity indicates cell-cycle activity but does not demonstrate completion of cell division (18,19), these findings indicate only limited cardiomyocyte cell-cycle activity in the PH right ventricle, and the biological significance of this low-frequency response remains uncertain.

Figure 4 Ki-67-positive cardiomyocytes in the RV tissues of PH model mice. (A) Representative images of immunofluorescence staining for Ki-67 (green) in RV myocardial tissues from C and PH model mice. Cardiomyocytes were identified based on their characteristic morphology and their nuclear morphology using DAPI (blue) counterstaining. White arrows indicate Ki-67-positive cardiomyocytes (Ki-67+/DAPI+ cells with cardiomyocyte morphology). (B) Quantitative analysis of Ki-67-positive cardiomyocytes, expressed as the percentage of Ki-67-positive cells relative to total DAPI-positive nuclei. Data are presented as the mean ± SD of all biological replicates per group (C and PH), with individual data points overlaid (n=3 biological replicates per group). C-1 to C-3 and PH-1 to PH-3 denote individual biological replicates from C and PH groups. *P<0.05. RV, right ventricular; C, control; PH, pulmonary hypertension.

PI3K inhibition alters Ang II-induced apoptosis-associated protein expression in H9C2 cells)

In H9C2 cells, treatment with Ang II resulted in significant Bax upregulation and Bcl-2 downregulation at both the protein and mRNA levels (Fig. 5A-E), indicating a pro-apoptotic shift in apoptosis-associated protein expression. LY294002, a selective PI3K inhibitor that blocks PI3K/AKT signaling, further upregulated Bax and downregulated Bcl-2 expression in Ang II-treated cells compared with Ang II alone (Fig. 5A-E). These findings support a protective role for endogenous PI3K/AKT signaling against pro-apoptotic protein changes in cardiomyocytes.

Figure 5 Effects of PI3K inhibition on Ang II-induced apoptosis in H9C2 cells. (A) Representative western blotting images and semi-quantitative densitometric analysis of (B) Bax (21 kDa) and (C) Bcl-2 (26 kDa) protein expression in H9C2 cells treated with Ang II (1 µM; 24 h) in the presence or absence of the PI3K inhibitor LY294002 (10 µM; 30 min pretreatment). GAPDH (36 kDa) was used as a reference protein. The relative mRNA expression levels of (D) Bax and (E) Bcl-2 were determined by reverse transcription-quantitative PCR and normalized to GAPDH. Data are presented as the mean ± SD (n=6 biological replicates per group). The western blot bands shown are representative images from the 6 biological replicates; C-1 and C-2 denote representative untreated C replicates; Ang II-1 and Ang II-2 denote representative Ang II-treated replicates; Ang II + LY294002-1 and Ang II + LY294002-2 denote representative replicates treated with both Ang II and LY294002. *P<0.05. Ang II, angiotensin II; C, control.

Discussion

The present study investigated the role of PI3K/AKT signaling in RV remodeling induced by PH. The findings demonstrated that PH model mice developed RV dysfunction with changes in apoptosis-associated protein expression (increased Bax and decreased Bcl-2) and fibrosis, which was accompanied by transcriptomic enrichment of the PI3K/AKT pathway. Inhibition of PI3K/AKT signaling with LY294002 further altered apoptosis-associated protein expression (further upregulated Bax and downregulated Bcl-2) in Ang II-treated H9C2 cells. In addition, Ki-67 staining in PH model mouse RV tissues indicated a limited proliferative response, which was considered insufficient to counterbalance the pro-apoptotic changes observed. Together, these findings suggested that PI3K/AKT pathway enrichment may represent a compensatory response, but this response was insufficient to prevent RV dysfunction.

The PI3K/AKT pathway is a central regulator of cell survival, apoptosis and metabolism in the cardiovascular system (20,21). Previous studies have demonstrated that activation of this pathway promotes the expression of anti-apoptotic proteins, such as Bcl-2, and inhibits pro-apoptotic mediators, such as Bax, thereby enhancing cardiomyocyte survival under stress (22-24). Consistent with these reports, the present study observed that the inhibition of PI3K/AKT signaling with LY294002 further altered apoptosis-associated protein expression (further upregulated Bax and downregulated Bcl-2) in Ang II-treated H9C2 cells. Notably, despite transcriptomic enrichment of the PI3K/AKT pathway in PH model mice, pro-apoptotic Bax expression remained elevated and anti-apoptotic Bcl-2 expression was reduced compared with controls, indicating that compensatory PI3K/AKT signaling was unable to fully counteract the pro-apoptotic shift under sustained pathological stress. This paradox highlights the complexity of signaling regulation in RV remodeling.

In addition to changes in apoptosis-associated protein expression, the present study observed a small but significant increase in Ki-67-positive cardiomyocytes in PH model mice compared with in the controls.Ki-67 is a marker associated with active phases of the cell cycle and is generally absent in quiescent cells (25). Therefore, the presence of Ki-67-positive cardiomyocytes suggests cell-cycle activity in a small subset of cardiomyocytes; however, Ki-67 staining alone cannot determine whether these cells progressed through the cell cycle or completed cytokinesis. Moreover, Ki-67-positive cardiomyocytes accounted for only ~0.4% of the cells examined. Accordingly, these findings should be interpreted as evidence of limited cell-cycle activity rather than definitive cardiomyocyte proliferation or regeneration. Previous studies have reported that cardiomyocytes under pathological conditions can re-express cell cycle markers (26-28). However, such activity rarely translates into effective regeneration (26). The findings of the present study supported this notion, indicating that proliferative attempts in RV tissues were insufficient to compensate for ongoing pro-apoptotic changes, ultimately leading to cardiac functional decline.

Clinically, RV failure remains the leading cause of mortality in patients with PH (29), and current therapies predominantly target pulmonary vasculature (30,31). The data of the present study suggested that modulation of PI3K/AKT signaling could help to preserve cardiomyocyte survival and delay RV dysfunction, as supported by the following observations: i) Transcriptomic enrichment of the PI3K/AKT pathway in PH model mice; ii) the pro-apoptotic shift indicated by increased Bax and decreased Bcl-2 expression in PH model mice; and iii) further exacerbation of these apoptosis-associated protein changes by LY294002 in Ang II-treated H9C2 cells. However, excessive activation of this pathway may promote maladaptive remodeling through crosstalk with mTOR, autophagy- or fibrosis-related pathways (32). Thus, therapeutic strategies for PH using the PI3K/AKT pathway must emphasize precise signaling regulation rather than indiscriminate pathway activation.

From a translational perspective, the findings of the present study suggested that targeting the PI3K/AKT signaling pathway may offer a promising adjunctive strategy for preventing RV dysfunction in PH. Currently, approved PH therapies primarily target pulmonary vasodilation and exhibit limited direct effects on myocardial survival pathways (33,34). The observation that transcriptomic enrichment of the PI3K/AKT pathway was associated with a pro-apoptotic shift in PH model mice (increased Bax and decreased Bcl-2 expression), and that inhibition of this pathway with LY294002 further exacerbated these changes in Ang II-treated H9C2 cells, implies that endogenous PI3K/AKT signaling may provide partial protection against pro-apoptotic protein changes. Therefore, augmenting this pathway, either through pharmacological activators or gene therapy approaches, could potentially delay or attenuate RV failure in patients with PH, particularly in those at high risk for RV decompensation (16,35,36). However, given the pleiotropic nature of PI3K/AKT signaling and its involvement in cell proliferation, fibrosis and metabolic regulation (17,27,37), indiscriminate activation carries the risk of promoting maladaptive remodeling or oncogenic effects. Therefore, future clinical strategies should prioritize precise, context-dependent modulation of this pathway, possibly through isoform-specific inhibitors or activators, or via combination with existing PH therapies to achieve synergistic benefits. Additionally, the development of biomarkers reflecting PI3K/AKT pathway activity in RV tissues could facilitate patient stratification and personalized therapeutic approaches. Although these possibilities are encouraging, notable challenges remain, including the requirement for cardiac-specific delivery systems and rigorous preclinical safety evaluation. Therefore, the findings of the present study provide a rationale for further translational studies to explore these opportunities.

It is important to acknowledge that the experiments in the present study were conducted exclusively in male mice, which may limit the generalizability of findings to female populations. Notably, PH exhibits well-recognized sex disparities, with higher prevalence but potentially improved survival outcomes in female patients compared with male patients (28,38). The underlying mechanisms of these differences are complex and have been shown to involve sex hormones, particularly estrogen, sex chromosomes and differential RV adaptation (39-42). In the context of PI3K/AKT signaling, estrogen has been shown to activate this pathway in various cell types, raising the possibility that the compensatory activation observed in male mice in the present study might differ in magnitude or functional consequence to PI3K/AKT activation in females (43-45). Given that RV failure remains the leading cause of mortality in patients with PH regardless of sex (46,47), the findings of the present study derived from male PH model mice still provide valuable insights into RV dysfunction. Future studies incorporating both male and female animals, as well as investigations into the effects of estrogen and other sex-specific factors on PI3K/AKT signaling in the right ventricle, will be important for determining whether the observations of the present study are universally applicable or sex-dependent. Furthermore, although this limitation warrants consideration, the use of male mice in the present study aligns with common practice in numerous preclinical cardiovascular studies, in which male animals are used to minimize variability associated with the estrous cycle. Notably, the results of the present study established a clear mechanistic foundation that may be extended to female models in subsequent investigations. Several additional experimental limitations should be acknowledged. Primarily, the present study focused on apoptosis and did not explore other forms of programmed cell death, such as pyroptosis or ferroptosis, which may also contribute to RV remodeling. Furthermore, downstream effectors and crosstalk with other signaling cascades were not investigated in detail. Additionally, the conclusions of the present study were drawn from animal and cell models only, and validation in human PH samples is required to establish clinical relevance. Furthermore, although Ki-67 staining indicated limited cell-cycle activity in a small subset of cardiomyocytes, Ki-67 positivity alone cannot determine whether these cells completed the cell cycle or cytokinesis; therefore, definitive conclusions regarding cardiomyocyte proliferation or regeneration cannot be drawn from the present data.

In summary, the present study demonstrated that transcriptomic enrichment of the PI3K/AKT pathway was observed in the right ventricle during PH. Although this pathway enrichment was accompanied by a pro-apoptotic shift (increased Bax and decreased Bcl-2 expression) and inhibition of PI3K/AKT signaling with LY294002 further exacerbated these changes in vitro, this compensatory response appeared insufficient to prevent progressive cell loss and functional deterioration. Targeted and precise modulation of PI3K/AKT signaling may therefore represent a promising therapeutic strategy for preventing RV failure in patients with PH.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the Capital's Funds for Health Improvement and Research (grant no. CFH-2024-1-2062) and the Beijing Natural Science Foundation - Beijing Economic-Technological Development Area Innovation Joint Fund (grant no. L258027).

Availability of data and materials

The RNA sequencing data generated in the present study may be found in the Genome Sequence Archive repository under accession number CRA033550 or at the following URL: https://ngdc.cncb.ac.cn/gsa/browse/CRA033550. The other data generated in the present study may be requested from the corresponding author.

Authors' contributions

QW and GL were responsible for the conceptualization and design of the study. QZ wrote the original draft of the manuscript. HZ was responsible for reviewing, editing and critically revising the manuscript for important intellectual content. JS was responsible for data curation, including the collection, organization and verification of experimental datasets derived from echocardiography, molecular assays and histological analyses; checking the completeness and consistency of data records; and preparing the curated datasets for subsequent statistical analysis and figure preparation. YD contributed to the acquisition of resources and materials essential for the study, and participated in the experimental design discussions. GX performed the bioinformatics analysis and software implementation for the RNA-seq data processing and analysis. WD contributed to the visualization and presentation of the results, including the preparation of figures. All authors participated in the interpretation of the data, reviewed the manuscript, and read and approved the final manuscript. All authors agree to be accountable for all aspects of the work and to ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. QW and GL confirm the authenticity of all the raw data.

Ethics approval and consent to participate

All animal experiments were authorized by the Animal Care and Welfare Committee of Beijing Institute of Heart, Lung and Blood Vessel Diseases and Beijing Anzhen Hospital, Capital Medical University (approval no. 2025205x).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References