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Unraveling the Mfn2-Warburg effect nexus: a therapeutic strategy to combat pulmonary arterial hypertension arising from catch-up growth after IUGR

Respiratory Research volume 25, Article number: 328 (2024) Cite this article

Abstract

Background

The interplay between intrauterine and early postnatal environments has been associated with an increased risk of cardiovascular diseases in adulthood, including pulmonary arterial hypertension (PAH). While emerging evidence highlights the crucial role of mitochondrial pathology in PAH, the specific mechanisms driving fetal-originated PAH remain elusive.

Methods and results

To elucidate the role of mitochondrial dynamics in the pathogenesis of fetal-originated PAH, we established a rat model of postnatal catch-up growth following intrauterine growth restriction (IUGR) to induce pulmonary arterial hypertension (PAH). RNA-seq analysis of pulmonary artery samples from the rats revealed dysregulated mitochondrial metabolic genes and pathways associated with increased pulmonary arterial pressure and pulmonary arterial remodeling in the RC group (postnatal catch-up growth following IUGR). In vitro experiments using pulmonary arterial smooth muscle cells (PASMCs) from the RC group demonstrated elevated proliferation, migration, and impaired mitochondrial functions. Notably, reduced expression of Mitofusion 2 (Mfn2), a mitochondrial outer membrane protein involved in mitochondrial fusion, was observed in the RC group. Reconstitution of Mfn2 resulted in enhanced mitochondrial fusion and improved mitochondrial functions in PASMCs of RC group, effectively reversing the Warburg effect. Importantly, Mfn2 reconstitution alleviated the PAH phenotype in the RC group rats.

Conclusions

Imbalanced mitochondrial dynamics, characterized by reduced Mfn2 expression, plays a critical role in the development of fetal-originated PAH following postnatal catch-up growth after IUGR. Mfn2 emerges as a promising therapeutic strategy for managing IUGR-catch-up growth induced PAH.

Introduction

Intrauterine growth restriction (IUGR) is clinically defined as the birth weight lower than the 10th percentile of the average gestational age weight and affects approximately 10–15% of pregnancies worldwide [1]. The advent of advanced nutritional support has led to a significant increase in postnatal weight gain among IUGR infants in recent years [2, 3]. However, emerging evidence from the developmental origins of health and disease suggests that mismatches between intrauterine and early postnatal environments, especially during nutritional transition between generations, may increase the risk of cardiovascular diseases, including pulmonary arterial hypertension (PAH) in adulthood [4,5,6]. Remarkably, one integrated research has revealed dysregulated transcriptomic profiles related to cardiometabolic risk in teenagers who experienced IUGR with postnatal catch-up growth [7]. Moreover, in our previous investigations, we observed that IUGR rats subjected to rapid catch-up growth exhibited elevated pulmonary arterial pressure and pulmonary vascular remodeling in adulthood [5]; however, the underlying mechanism remains to be elucidated.

Pulmonary arterial hypertension (PAH) is primarily characterized by vascular lumen obstruction and elevated mean pulmonary arterial pressure (mPAP), resulting from the increased hypertrophy, proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) [8]. In recent years, the consensus has been growing around the notion that PAH exhibits a Warburg effect akin to tumor cells. Specifically, despite the presence of sufficient oxygen, the cells favor glycolysis over mitochondrial glucose oxidation [9]. The Warburg effect, beyond its association with the tumor environment, plays a pivotal role in immunity, angiogenesis, pathogen infection, and can serve as a marker of cellular proliferation [10]. Notably, proteomic analysis has confirmed the augmented expression of proteins linked to glycolysis and mitochondrial dysfunction in PASMCs from PAH patients, compared to normal controls [11]. This metabolic shift might lead to the hyperpolarization of the mitochondrial membrane through the recruitment of immune or inflammatory cells, increased cytokines or chemokines expression, and ultimately, the increased proliferation of PASMCs [12]. However, the precise mechanisms contributing to the Warburg effect in fetal-originated PAH necessitate further investigation.

Accumulating evidences suggest that imbalanced mitochondrial dynamics significantly contributes to the progression of pulmonary vascular remodeling [13,14,15]. Mitochondria, as dynamic organelles, continuously undergo fusion and division transformations. Maintaining a delicate balance in mitochondrial dynamics is crucial for preserving mitochondrial morphology and vascular function [16]. Among the key players in this process, Mitofusion 2 (Mfn2), a mitochondrial outer membrane protein, plays a pivotal role in regulating mitochondrial fusion and function. Interestingly, Mfn2 was initially identified as a “hyperplasia suppressor gene” due to its anti-proliferative function in vascular smooth muscle cells [17]. Notably, prior investigations have revealed a significant reduced expression of Mfn2 in both PAH patients and animal models, while the restoration of Mfn2 expression in PAH animal models has shown promising outcomes, improving pulmonary vascular remodeling and reducing pulmonary vascular resistance [18, 19]. These findings hold the potential for Mfn2-based interventions to be explored as a viable treatment avenue for PAH.

In this current study, we established a postnatal catch-up growth following IUGR model of PAH to investigate the consequences of adverse mismatched perinatal environments later in life. Our research identifies Mfn2 as a key player in disrupting the metabolic shift of PASMCs via the Warburg effect, thus contributing to the development of fetal-originated PAH. Notably, overexpression of Mfn2 effectively reverses the Warburg effect in PASMCs associated with PAH. Through in vitro and in vivo experiments, we demonstrate the implication of Mfn2 in PAH induced by postnatal catch-up growth following IUGR. This study sheds light on a previously unrecognized mitochondrial-dependent mechanism underlying fetal-originated PAH and provides valuable insights into the role of Mfn2 in this context.

Materials and methods

Establishment of animal model

Sprague-Dawley rats were kept in a constant temperature of 22–24 °C and provided with free access to drinking water. The IUGR-catch up growth rat model was set up as described in our previous study [5]. In brief, virgin Sprague-Dawley female rats weighing 250–300 g were mated overnight. Once pregnant, they were randomly assigned to either the normal diet or restricted diet groups, receiving either regular food or 50% amount of the regular food throughout gestation. Offspring from dams on the normal diet were designated as control rats, while those from the restricted diet dams were labeled as IUGR rats. After birth, the litter size of both control and IUGR offspring was standardized to 8 pups per litter, and they were all fostered by moms from control dams (Fig. 1a). As a result, two experimental groups were formed based on dam/offspring diet: Control/Control (CC) and intrauterine growth restriction with postnatal catch-up growth (RC). To minimize variability associated with the hormonal cycle in female rats, exclusively male rats were chosen for this study.

Fig. 1
figure 1

RC rats developed increased pulmonary arterial pressure and pulmonary arterial remodeling. a, Postnatal catch-up growth rat model design. b, Birth weights of offspring, n = 13–25 rats per group. c, Growth tendency in CC and RC rats before weaning, n = 13–25 rats per group. d, Representative pulmonary arterial pressure measurement of CC and RC group rats at 14 weeks of age. e, Representative images of α-smooth muscle actin staining. Scale bar = 20 μm. f, Mean pulmonary arterial pressure of the rats, n = 8 per group. g, Right ventricular hypertrophy index (RV/ (LV + S)) of the rats, n = 8 per group. h, Medial wall thickness of pulmonary arterioles (20–100 μm in diameter), n = 8 per group. CC, Control/Control; RC, intrauterine growth restriction with postnatal catch-up growth; mPAP, mean pulmonary arterial pressure; PA, pulmonary artery; RV, right ventricular hypertrophy index; LV, left ventricular; S, septum. All data throughout the figures represent the mean ± SEM and analyzed by Student’s two-tailed t-test. *p < 0.05, ***p < 0.001, **** p < 0.0001 compared to CC group

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Hemodynamic and right ventricular hypertrophy measurements

At 14 weeks of age, the rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg, i.p.) and placed on a surgical board. To measure hemodynamic values, a pulmonary artery catheter PE50 was introduced through the right external jugular vein. The catheter was gently rotated leftward and advanced until it traversed the right heart, ultimately reaching the main pulmonary artery. The physiological data acquisition system used for recording these hemodynamic values was the BIOPAC MP150 (Biopac System Inc., Goleta, California, USA).

To assess the degree of right ventricular hypertrophy, the right ventricle was carefully dissected from the left ventricle and septum and weighed individually. The right ventricular hypertrophy index (RVHI) was then determined using the following formula: RVHI = right ventricular weight / (left ventricular weight + septum weight).

Assessment of vascular remodeling in vivo

Immunohistochemistry was conducted on rat lung tissues following previously established protocols [20]. In brief, the paraffin-embedded tissues were sectioned at a thickness of 4–5 μm. The sections were then subjected to de-paraffinization using xylene and rehydration through a series of graded ethanol concentrations until reaching distilled water, followed by antigen retrieval. Subsequently, the samples were incubated in methanol containing 3% hydrogen peroxide for 25 min to quench endogenous peroxidase activity and washed with PBS. Next, the sections were incubated with the primary antibody anti-α smooth muscle actin (anti-α-SMA, GB13044, Servicebio, China).

Isolation and culture of rat PASMCs

Primary pulmonary arterial smooth muscle cells were isolated and cultured from both the RC and CC groups of 14-week-old rats using previously established methods [20]. Briefly, the peripheral pulmonary artery was exposed under a stereoscope, the endothelial and adventitial layers were removed after gentle scraping. The media layer was then sectioned into 1–2 mm [2] fragments, which were subjected to digestion with papain and collagenase (C0130, Sigma Aldrich, USA) at 37 °C for 20 min. The resulting PASMCs were collected via centrifugation at 2000 g for 10 min and cultured in DMEM supplemented with 20% fetal bovine serum (FBS). For the subsequent experiments, PASMCs from passages 3 or 4 were utilized.

PASMCs proliferation assay

Proliferative ability was assessed following the instructions of the EdU kit (C10310-1, Ribobio, China). Briefly, PASMCs were seeded at a density of 5000 cells per well in 96-well microplates and then incubated in EdU for 2 h. Subsequently, cells were fixed, washed, and incubated in glycine, followed by permeabilization using 0.5% Triton X-100 for 10 min. Apollo staining buffer was applied for 30 min at room temperature in the dark. Finally, Hoechst 33,342 was utilized to stain the cell nuclei. The fluorescence microscope used for imaging was Zeiss Observer Z1 (Zeiss, Germany).

Migration assay

To assess the migration ability of PASMCs, we employed the transwell chamber migration assay (3422, Corning, USA). A total of 1 × 105 cells was resuspended in serum-free medium and added to the upper chamber, while the lower chamber was supplemented with 10% FBS. Following 24 h of incubation, non-invaded cells were carefully removed from the upper surface of the filter. The invaded cells were subsequently fixed and stained. To quantify cell migration, the average number of cells in five visual fields was determined.

Assessment of mitochondrial membrane potential

Mitochondrial Membrane Potential (MMP) was assessed following the instructions provided by the kit (C2006, Beyotime Biotechnology, China). Cells were seeded in 6-well microplates until reaching 60–70% confluence. Next, 1 ml of JC-1 staining solution was added to each well, and the cells were incubated at 37 °C for 20 min after thorough mixing. Subsequently, the cells were washed twice with JC-1 staining buffer, and the fluorescence intensity was measured using a fluorescence microscope (Tecan, Switzerland).

Mitochondrial ROS staining

Mitochondrial ROS levels were examined following the manufacturer’s instructions of the kit (40778ES50, Yeasen, China). Cells were seeded in 24-well plates until reaching a confluence of 60–70%. Subsequently, 500 µl of probe working solution was added to each well, and the cells were incubated at 37 °C for 10 min. Images were captured using a fluorescent microscope (Tecan, Switzerland) with a 567 nm wavelength.

Assessment the opening of MPTP

Mitochondrial permeability transition pore (MPTP) assessment was performed according to the manufacturer’s recommendation (GMS10095.1, Gemmed, China). Cells were seeded in 96-well microplates until reaching a confluence of 60–70%. Following gentle washing, 100 µL of staining solution was added to each well, and the cells were incubated at 37 °C for 20 min. Subsequently, the wells were washed twice with prewarmed washing buffer. The staining product was quantified by measuring the absorbance using a fluorescence microplate reader (Tecan, Switzerland) with a 488 nm excitation wavelength and 505 nm emission wavelength.

RNA-seq and data analysis

RNA was extracted from peripheral pulmonary arteriole of rats and paired-end libraries were sequenced by an Illumina HiSeq 2500 (pair-end 150-nucleotide read length). Sequencing reads were aligned using HISAT2 (version 2.1.0) to rat reference sequence (RGSC 6.0/rn6.0.98)21. Biological coefficient of variation (BCV) is used to exclude differences caused by different biological samples other than technical errors. For quality control, plotBCV was used to perform ANOVA for different expression level values of different samples within the same group. After quality control, FeatureCounts (version 1.6.3) was performed for those qualified gene count from trimmed reads against the rattus norvegicus annotation file [21]. Gene expression levels were quantitated by edgeR [22]. Differential gene was defined as adjusted p value < 0.05 and |log2FC| >0.8.

Quantitative RT-PCR

Total mRNA was extracted from lung tissues and PASMCs according to the instruction of Total RNA kit (Axygen, USA). Subsequently, the RNA was transcribed into cDNA using the reverse transcriptase kit (Takara, Japan). The synthesized cDNA was analyzed using the StepOnePlus Real-Time PCR system, following the SYBR-Green protocol (Takara, Japan). The PCR reaction conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The relative expression of target genes to B2M was quantified using the 2−ΔΔCt method. Primers were listed in Supplement Table 1.

Western blot and quantification

Rat lung tissues and PASMCs were lysed using RIPA buffer (P0013B, Beyotime Biotechnology, China) supplemented with protease and phosphatase inhibitor cocktails. The protein lysates were separated on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride membranes (Merk Millipore, Darmstadt, Germany). The following primary antibodies were used: AMPK (5832T, CST), pAMPK (2535 S, CST), PGC1α (ab54481, Abcam), VDAC (4661 S, CST), Mfn2 (ab124773, Abcam), and β-actin (8457 S, CST).

Assessment of mitochondrial morphology in PASMCs

PASMCs were incubated with 500 nM Mitotracker Red CMXRos probe (M7512, Thermo Fisher Scientific, USA) at 37 °C for 15 min. Subsequently, images were acquired using a confocal laser-scanning microscope (Lecia, Germany).

Transmission electron microscopy (TEM)

A total of 2 × 106 freshly collected primary pulmonary arterial smooth muscle cells (PASMCs) underwent fixation with 2.5% glutaraldehyde in PBS at 4 °C overnight. Subsequently, the cells were fixed with 1% osmium tetroxide for 1 h and stained with a 2% uranium acetate solution for 30 min. The samples underwent dehydration using a density gradient of alcohol and acetone. Finally, the specimens were sectioned into ultrathin slices measuring 60–80 nm (Leica UC7), followed by staining and overnight drying at room temperature. Imaging and analysis of the sections were performed using a TecNAI 10 transmission electron microscope (Philips, FEI, USA).

Up-regulation of Mfn2

Recombinant adenoviral vectors expressing Mfn2 (Ad-Mfn2) and negative control adenoviral vectors (Ad-EV) were constructed by Genechem Co. Ltd (Shanghai, China), with a titre of 4 × 1010 PFU/ml. To overexpress Mfn2 in PASMCs, Ad-Mfn2 was transfected into the cells. Cells were grown for an additional 48 h and were used for western blot to confirm overexpression (Supplement Fig. 1). Mfn2 overexpressing rats were established by intravenously injecting Ad-Mfn2 at a diluted dose of 1 × 1010 PFU/ml via the tail vein when the rats reached 14 weeks of age. The animals were sacrificed based on the expression of GFP-fused Ad-Mfn2 in the lung, as determined from preliminary time-dependent experiments (Supplement Fig. 2).

Oxygen consumption rate and glycolytic capacity

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFe96 Extracellular Flux Analyser (Agilent SeaHorse Bioscience, USA). Primary PASMCs were seeded at a density of 40,000 cells per well for measurement. To achieve Mfn2 overexpression, cells were treated with Ad-Mfn2 48 h prior to seeding. The OCR was monitored upon serial injections of oligomycin (1.5µM, ATP-synthase inhibitor), FCCP (2µM, electron transfer chain accelerator), and a combination of rotenone/antimycin A (0.5µM, Complex I inhibitor). The ECAR was monitored upon sequential additions of glucose (10mM, glycolytic substrate), oligomycin (1mM, ATP-synthase inhibitor), and 2-deoxy-glucose (50 Mm, glycolysis inhibitor) as the manufacturer’s instructions. Data analysis was performed using the Wave software and the report generator.

Statistical analysis

Statistical analysis was conducted using the Student’s two-tailed t-test for comparisons between two groups and one-way analysis of variance (ANOVA) and Newman-keuls post-hoc test for multiple groups. Details of statistical tests used are specified in figure legends. All statistical analyses were performed using Prism9 (La Jolla, CA) and SPSS 22.0 (SPSS, Inc). The data were presented as mean ± SEM (standard error of the mean). A P-value < 0.05 was considered statistically significant.

Results

Postnatal catch-up growth following IUGR (RC group) developed increased PAP and pulmonary arterial remodeling

To estimate the effect of postnatal catch-up growth after IUGR, we established a rat model as shown in Fig. 1a. Compared to the control group, the IUGR rats exhibited significantly lower birth weights (Fig. 1b). During the lactation period, the RC offspring showed evident catch-up growth, resulting in no significant difference in body weights between RC and CC offspring at weaning (Fig. 1c). To explore whether the postnatal catch-up growth following IUGR leads to pulmonary vascular dysfunction, we measured the mean pulmonary arterial pressure (mPAP) and assessed the degree of pulmonary vascular remodeling. At 14 weeks of age, the RC offspring demonstrated elevated mPAP levels compared to CC offspring (Fig. 1d, f). Additionally, the RC offspring showed a significant increase in the index of right ventricular hypertrophy, calculated as the ratio of right ventricular weight to the combined weight of the left ventricle and septum (Fig. 1g). Immunostaining revealed a substantial increase in the medial wall area of the pulmonary arteriole in RC offspring compared to CC offspring, indicating a thicker smooth muscle layer in the RC group (Fig. 1e, h). These observations collectively indicate that the RC group offspring exhibited a pulmonary arterial hypertension phenotype in adulthood.

Pulmonary vascular in RC group had dysregulated mitochondrial metabolic genes and pathways

Since the pulmonary arteriole is considered as a proliferative and anti-apoptotic microenvironment in PAH patients, we hypothesized that transcriptomic alterations might occur in the adulthood of the RC group. To investigate this, we conducted a transcriptome analysis of pulmonary arteriole using an NGS RNA-seq approach. For the red line given by plotBCV, its range between 0.2 and 0.4 indicates small heterogeneity among experimental samples (Supplement Fig. 3). Our analysis, based on our defined criteria, revealed a total of 1391 differentially expressed genes using edgeR (Fig. 2a), in which 11 differentially expressed mitochondrial-associated genes were visualized. Further pathway analysis highlighted that these genes were primarily involved in the regulation of cell proliferation, response to hypoxia, response to oxidative stress (Fig. 2b). Given existing researches establishing a significant connection between mitochondrial function and pulmonary hypertension, we subsequently focused on analyzing the pertinent differential genes within the mitochondrial pathway, including mitochondrial fusion and fission (Fig. 2c). For a comprehensive understanding, we have provided a detailed list of these genes in the supplementary materials (Fig. 2d, Supplement Table 2). Collectively, our findings suggest dysregulated mitochondrial metabolism in the pulmonary arteriole of the RC offspring. Such insights contribute to a better understanding of the potential molecular mechanisms underlying PAH pathogenesis in this specific context.

Fig. 2
figure 2

Functional annotations and pathway analysis of differential expression genes. a, A volcano plot visualization of the differentially expressed mitochondrial-associated genes. b, The pathway analysis of DEGs based on DAVID. c, The GO enrichment analysis of mitochondrial-associated genes based on Metascape. d, Representatives differential expression of the energy metabolism associated genes from pulmonary arterioles of CC and RC groups. DEGs represents differential expression genes, n = 3 rats per group

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PASMCs exhibited elevated proliferation, migration and dysregulated mitochondrial functions in RC group

The GO pathway analysis indicated a positive regulation of cell proliferation in the RC group, and PASMC proliferation is recognized as one of the underlying mechanisms of vascular remodeling in PAH, we further investigated the migration and proliferation ability of PASMCs. To assess cell migration, we performed transwell assays after incubating the cells in serum-free medium for 24 h. The results revealed increased cell migration in the RC group compared to CC group (Fig. 3a, b). Additionally, we examined whether catch-up growth following IUGR affected PASMC proliferation. Our findings demonstrated hyperproliferation of PASMCs in the RC group when compared to CC group (Fig. 3c). In our pursuit to explore potential changes in cellular mitochondrial function, we conducted assessments of mitochondrial oxidative stress and mitochondrial membrane channel openness (Fig. 3d-f). The analysis revealed an increase in mitochondrial oxidative stress in the RC group. Previous studies have revealed a decrease in AMPK phosphorylation levels in animals with idiopathic pulmonary arterial hypertension (IPAH) [23]. Moreover, the augmentation of the phosphorylation of AMPK effectively hinders the proliferation of PASMCs in IPAH [23]. We also find that there was no difference in the expression level of total AMPK protein in the lung tissues, while the expression of phosphorylated APMK decreased significantly in the RC group compared to the CC group (Fig. 3g, h). Furthermore, we investigated the expression levels of some mitochondrial proteins: voltage-dependent anion selective channel (VDAC) and PPARγ coactivator 1 alpha (PGC1α), key regulators of mitochondrial quality (Fig. 3i, j). In the RC group lung tissues, we observed a decrease in the expression of VDAC and PGC1α when compared to CC group, indicating a decline in mitochondrial quality. These findings shed light on the potential implications of dysregulated mitochondrial function in the context of catch-up growth following IUGR and its association with PASMC proliferation and migration, contributing to a deeper understanding of the underlying mechanisms of fetal originated PAH pathogenesis.

Fig. 3
figure 3

RC group rats exhibited dysregulated PASMC functions and mitochondrial protein expressions. a, Representative images of migration of pulmonary arterial smooth muscle cells, scale bar = 50 μm. b, Statistics of the PASMCs migration (n = 6–8 per group). c, Statistics of the PASMCs proliferation (n = 6–7 per group). d, Representative images of PASMC mitochondrial oxidative stress staining, scale bar = 20 μm. e, Statistics of fluorescence intensity (n = 5 per group). f, Statistics of relative fluorescence unit of MPTP (n = 7 per group). g, Representative blot images of AMPK and pAMPK isolated from lung tissues (n = 6 per group). h, analysis of expression of AMPK and pAMPK in lung tissues. i, Representative blot images of mitochondrial specific proteins VDAC and PGC1α isolated from lung tissues (n = 6 per group). j, analysis of expression of VDAC and PGC1α in lung tissues. MPTP, mitochondrial permeability open pores. All data throughout the figures represent the mean ± SEM and analyzed by Student’s two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CC group

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Reconstitution of Mfn2 increased the fusion and mitochondrial functions of PASMCs in the RC group

To elucidate the key gene underlying the dysregulated mitochondrial functions in the RC group, we investigated the mRNA expression levels of main mitochondrial dynamics-related molecules, including Drp1, Fis1, Mfn1, Mfn2, and Opa1. Comparative analysis revealed a significant down-regulation of Mfn2 expression in the lung tissues and particularly in PASMCs of the RC group, when compared to the CC group (Fig. 4a-c). However, the expression of other dynamic-related genes, Fis1 and Opa1, remained unchanged (Fig. 4d). Analysis of mRNA expression in lung tissues and PASMCs (Fig. 4e) indicated that the down-regulation of Mfn2 in the RC group primarily occurred at the transcriptional level. These findings collectively suggest a decrease in mitochondrial fusion during the development of IUGR-catch-up-PAH, with evident changes in Mfn2 expression associated with this process.

Fig. 4
figure 4

Reconstitution of Mfn2 increased the fusion and mitochondrial functions of PASMCs in the RC group. a, Representative blot images of Mfn2 in rat lung tissues. b, Representative blot images of Mfn2 in rat PASMCs. c, Quantitative analysis of Mfn2 protein expression (n = 6–10 per group). d, Quantitative analysis of mitochondrial dynamics gene expression in lung tissues (n = 7 per group). e, Quantitative analysis of Mfn2 expression in rat PASMCs (n = 6 per group). f, Representative confocal microscope images showing mitochondrial morphology stained by MitoTracker Red. g, Representative images of mitochondrial membrane potential, scale bar = 50 μm. h, Quantitative analysis of mitochondrial membrane potential (n = 6 per group). i, Quantitative analysis of mitochondrial permeability transition pore in PASMCs (n = 4–7 per group). Ad-EV, control adenovirus; Ad-Mfn2, recombinant adenovirus encoding Mfn2, MPTP, mitochondrial permeability transition pore. All data throughout the figures represent the mean ± SEM. Figures c-e were analyzed by Student’s two-tailed t-test and Figures h-i were analyzed by one-way analysis of variance (ANOVA) and Newman-keuls post-hoc test. * p < 0.05, ** p < 0.01 compared to CC group, $ p < 0.05 vs. Con + Ad-EV, $$ p < 0.01 vs. Con + Ad-EV, # p < 0.05 vs. RC + Ad-EV. ### p < 0.001 vs. RC + Ad-EV

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To further evaluate mitochondrial dynamics, we utilized MitoTracker Red probe and transmission electron microscopy to track the mitochondrial morphology. MitoTracker staining of the mitochondrial shape indicated elongated and interconnected mitochondrial networks in CC PASMCs (Fig. 4f), the fragmented mitochondrial morphology observed in RC PASMCs (Fig. 4f) indicated a decrease in long rod-shaped elongated interconnected mitochondrial networks and a significant increase in spheroid-shaped fragmented mitochondrial morphology. Further, RC group had longer and more number of mitochondria than CC group, which indicated decreased mitochondrial fusion and increased mitochondrial fission, that is, impaired mitochondrial dynamics (Supplement Fig. 4). To examine the impact of Mfn2 manipulation on primary PASMCs, the gain-of-function approach involving Mfn2 was explored (Supplement Fig. 2), and we observed significant morphological changes in primary PASMCs following transfection with the adenovirus encoding Mfn2 (Ad-Mfn2). This transfection mitigated mitochondrial fission in RC-PASMCs (Fig. 4f and Supplement Fig. 4).

The findings from Jc-1 staining results demonstrated that the RC group exhibited a higher mitochondrial membrane potential compared to the CC group. Furthermore, the overexpression of Mfn2 was observed to significantly reduce the mitochondrial membrane potential (Fig. 4g, h). We also delved into the role in the mitochondrial membrane channel pore (MPTP), a calcium ion-dependent channel comprising components of the mitochondrial inner and outer membrane. During cell apoptosis, mitochondrial content may be released into the cytoplasm through the MPTP. In our study, the mitochondrial membrane channel RFU value of the RC group was found to be higher than that in the CC group, which suggests a reduction in the opening of membrane channel pores in the RC group. Intriguingly, the overexpression of Mfn2 was associated with an increase in the opening of mitochondrial membrane channels in RC PASMCs (Fig. 4i). Furthermore, we found that overexpression of Mfn2 also decreased proliferation ability in RC-PASMC (Supplement Fig. 5). These results collectively provide valuable insights into the role of Mfn2 in mitigating mitochondrial dysfunction and apoptosis, highlighting its potential as a therapeutic target in IUGR-PAH.

Mfn2 overexpression reversed the Warburg effect of PASMCs in RC group

Further investigation of mitochondrial function (respiratory capacity) was carried out in PASMCs. As shown in Fig. 5, the RC group exhibited suppressed mitochondrial respiratory capacity, including basal respiration, ATP-linked respiration, maximal respiration, and spare respiration, all of which were significantly improved after Mfn2 overexpression (Fig. 5a, b). Glycolytic stress testing demonstrated that the RC group displayed a substantially higher level of glycolysis compared to CC group (Fig. 5c, d). However, the glycolysis level was significantly inhibited with overexpression of Mfn2. These findings suggest that Mfn2-mediated mitochondrial fusion plays a crucial role in enhancing mitochondrial respiratory function and influencing energy metabolism.

Fig. 5
figure 5

Mfn2 overexpression reversed the Warburg effect in PASMC of RC groups. a, Oxygen consumption rate of PASMCs. b, Quantitative statistical analysis of OCR. c, extracellular acidification rate of PASMCs. d, Quantitative statistical analysis of ECAR. OCR, oxygen consumption rate; ECAR, extracellular acidification rate; Ad-EV, control adenovirus; Ad-Mfn2, recombinant adenovirus encoding Mfn2, RC + Ad-Ev, RC group PASMCs transfected with control adenovirus; RC + Ad-Mfn2, RC group PASMCs transfected with recombinant adenovirus encoding Mfn2. All data throughout the figures represent the mean ± SEM and analyzed by one-way analysis of variance (ANOVA) and Newman-keuls post-hoc test. * p < 0.05 vs. Con + Ad-EV, ** p < 0.01 vs. Con + Ad-EV, ## p < 0.01 vs. RC + Ad-EV. ### p < 0.001 vs. RC + Ad-EV

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Reconstitution of Mfn2 alleviated the PAH phenotype in RC group

To ascertain whether the reconstitution of Mfn2 offers protection against pulmonary vascular remodeling, we administered adenoviral vectors encoding Mfn2 into 14-week-old RC rats (Fig. 6a). Immunohistochemical staining confirmed that Mfn2 expression in the RC group increased at 1 week and disappeared at 2 weeks following the injection of Ad-Mfn2 (Supplement Fig. 2). Reconstitution of Mfn2 significantly ameliorated pulmonary artery remodeling in the RC group rats, as evidenced by decreased mean pulmonary arterial pressure (mPAP) and right ventricular hypertrophy index (RVHI) (Fig. 6b-e). These results collectively indicate that the reconstitution of Mfn2 prevents and alleviates vascular remodeling in the lungs of RC rats during adulthood. These findings hold significant implications for understanding the potential therapeutic role of Mfn2 in mitigating pulmonary vascular abnormalities associated with catch-up growth following intrauterine growth restriction.

Fig. 6
figure 6

Reconstitution of Mfn2 alleviated the PAH phenotype in RC group. a, Schematic representation of the experimental procedures. b, Representative images of α-smooth muscle actin staining. Scale bar = 20 μm. c, Mean pulmonary arterial pressure of the rats (n = 8 or 4 per group). d, Medial wall thickness of pulmonary arteries in small (20–100 μm in diameter) pulmonary vessels. e, Right ventricular hypertrophy index (RV/ (LV + S)) of the rats (n = 8 or 4 per group). RC + Ad-Ev, RC group rats injected with control adenovirus; RC + Ad-Mfn2, RC group rats injected with recombinant adenovirus encoding Mfn2; mPAP, mean pulmonary arterial pressure; PA, pulmonary artery; RV, right ventricular hypertrophy index; LV, left ventricular; S, septum. All data throughout the figures represent the mean ± SEM and analyzed by one-way analysis of variance (ANOVA) and Newman-keuls post-hoc test. * p < 0.05 vs. Con + Ad-EV, ** p < 0.01 vs. Con + Ad-EV, # p < 0.05 vs. RC + Ad-EV. ## p < 0.01 vs. RC + Ad-EV

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Fig. 7
figure 7

Schematic figure illustrating that imbalanced mitochondrial dynamics induced by decreased Mfn2 promotes PASMC proliferation. Intrauterine growth restricted rats following catch-up growth after birth have decreased expression of Mfn2 in adulthood, resulting in decreased mitochondrial fusion, which affects mitochondrial respiration patterns and mitochondrial oxidative stress, and ultimately leads to the proliferation of PASMCs and the remodeling of pulmonary vessels. PAH, pulmonary arterial hypertension; PVR, pulmonary vascular remodeling; RVHI, right ventricular hypertrophy index; ROS, reactive oxygen species; OCR, Oxygen consumption rate; ECAR, extracellular acidification rate

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Discussion

In this study, we unveil the crucial role of Mfn2 in the development of pulmonary arterial hypertension (PAH) induced by intrauterine growth restriction (IUGR) and subsequent catch-up growth, as shown in Fig. 7. Firstly, we confirmed increased proliferation and migration in the RC group, accompanied by dysregulated gene expression and pathway related to proliferation. Furthermore, RC group had mitochondrial dysfunction manifested as reduced Mfn2 expression and increased Warburg effect. Intriguingly, overexpressing Mfn2 had a positive impact on ameliorating the mitochondrial dysfunction and reversing the Warburg effect in RC group. Finally, the reconstitution of Mfn2 effectively inhibited pulmonary arterial remodeling and improved pulmonary hemodynamics. These encouraging outcomes suggest that Mfn2 expression plays a pivotal role in the pathogenesis of PAH, and manipulating it could serve as a potential therapeutic strategy for managing this condition in individuals who have experienced catch-up growth following intrauterine growth restriction.

The majority of neonates born with intrauterine growth restriction exhibit a compensatory response by undergoing early catch-up growth, contributing to enhanced adult height and improved cognitive capacity [24]. However, an expanding body of evidence from epidemiological and animal studies underscores the potential association between rapid early growth in IUGR and subsequent cardiovascular ailments [4, 25, 26]. Our findings suggest the pivotal role of IUGR subsequent postnatal catch-up growth, highlighting its potential to instigate pulmonary arterial hypertension (PAH) in adulthood. This phenomenon manifests as elevated mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy, and remodeling of pulmonary arteriole, as previously documented in our research [5]. While sustained postnatal growth restriction can impede the remodeling of pulmonary arteries, it can also have adverse effects on cognitive development [5]. Therefore, a crucial endeavor lies in elucidating mechanisms that facilitate the prevention of arterial remodeling without compromising neurodevelopment.

Our investigation revealed elevated mitochondrial reactive oxygen species (ROS) levels in RC PASMC, concomitant with reduced phosphorylation at threonine 172 (p-T172) of AMPK. In parallel, PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) exhibited a significant decrease in p-T172-AMPK, aligning with our findings and suggesting down-regulation of AMPK signaling in IPAH PASMC [23]. Awad et al. demonstrated that in a PASMC culture under hypoxia (10% O2) for 72 h, elevated ROS levels triggered AMPK activation as a protective response against oxidative stress. Conversely, H2O2 treatment significantly diminished the activation of the AMPK/FoxO1/CAT pathway [27], suggesting that excessive ROS production, surpassing cellular antioxidant defenses, contributes to severe damage and the progression of pulmonary vascular remodeling in persistent pulmonary hypertension [28, 29]. Therefore, we postulate that an unfavorable cellular environment in RC PASMC leads to heightened ROS production, influencing AMPK activation and diminishing its protective effect.

Mfn2, a member of the mitochondrial transmembrane GTP-binding protein family, exhibits highly conserved structural variations across different species [30]. In our study focusing on IUGR postnatal catch-up growth in rat lung tissues, we observed decreased levels of Mfn2 at both transcription and translation stages compared to the CC group. Notably, Mfn2 downregulation has been observed in genetically normal pulmonary hypertension patients and rat models of pulmonary arterial hypertension [19], possibly linked to the expression of PGC-1α, a known transcriptional coactivator of Mfn219, which aligns with our findings. It has also been reported that PGC1 α can perturb mitochondrial structure and function to stimulate PASMC proliferation [31]. To investigate the consequences of Mfn2 deficiency, we conducted experiments using recombinant adenovirus to restore its expression. The results revealed that mitochondrial disruption was rectified, leading to enhanced mitochondrial fusion and a reduction in hyperpolarization changes. Notably, the role of Mfn2 in pleiotropic fusion also facilitates the connection between mitochondria and the endoplasmic reticulum, allowing for Ca2+ transfer from the endoplasmic reticulum to the mitochondria [32]. Furthermore, in vitro down-regulation of Mfn2 resulted in the disruption of the mitochondrial-endoplasmic reticulum unit, leading to reduced calcium influx into the mitochondria and subsequent hyperpolarization [33].

Furthermore, we investigated the impact of Mfn2 overexpression on mitochondrial function. Compared to the control group, we observed an increase in mitochondrial membrane potential in the RC group. Interestingly, subsequent analysis demonstrated a significant reduction in mitochondrial membrane potential after Mfn2 overexpression. Additionally, the overexpression of Mfn2 in the RC group led to an increase in the opening of mitochondrial membrane channels. Notably, both the decrease in membrane potential and the heightened mitochondrial membrane channel opening are hallmark events associated with the early stages of apoptosis. These findings align with previous reports suggesting a potential pro-apoptotic role of Mfn2 mediated by the mitochondrial apoptotic pathway [34,35,36]. Nevertheless, further investigations are required to fully elucidate whether the expression of Mfn2 indeed promotes apoptosis through the mitochondrial apoptotic pathway.

In PAH, the energy source of PASMCs undergoes a shift from primarily relying on mitochondrial oxidative phosphorylation to predominantly relying on cytoplasmic glycolysis, akin to the Warburg effect observed in cancer cells [37, 38]. A glycolytic shift is observed in both right and left ventricular cardiomyocytes, promoting cell proliferation and consequent ventricular hypertrophy [39]. Our findings revealed increased glycolysis and reduced oxidative phosphorylation in PASMCs of RC group, indicating metabolic reprogramming in smooth muscle cells. Given that Mfn2 is known to influence mitochondrial function, we examined its effect on the cellular metabolic phenotype using recombinant Mfn2. Remarkably, the administration of Mfn2 overexpression resulted in up-regulated mitochondrial respiratory function and suppressed glycolysis levels in PASMCs of RC group. However, CC group had higher OCR after Ad-Mfn2, suggesting that Mfn2 could indeed increase the OCR level in both CC and RC group PASMC. This highlights the significant impact of Mfn2 on cellular energy metabolism in the context of PAH. Besides, it is worth noting that mitochondrial fusion dynamically regulates oxidative phosphorylation levels in various cellular contexts [40, 41]. Although the reduction in ATP production in the Warburg effect does not fully explain the proliferative advantage derived by PASMCs from this metabolic shift, it is worth noting that glycolysis also supports other metabolic pathways crucial for proliferation, such as the pentose phosphate pathway [42]. Additionally, the lactic acid produced through hyperglycolysis in proliferating cells can serve as a source of nutrients and oxygen, stimulating angiogenesis [43, 44].

Preclinical investigations have demonstrated that upregulating Mfn2 expression in rat PASMCs results in reduced proliferation and increased apoptosis, leading to partial alleviation of monocrotaline-induced pulmonary hypertension [19]. In our study, rats receiving Ad-Mfn2 exhibited significantly lower mean pulmonary artery pressure in the RC group. Although the therapeutic benefit seemed to be associated with restored fusion, the precise link between fusion and the inhibition of proliferation remains a subject of debate. We put forward the hypothesis that Mfn2 might improve the metabolic phenotype of smooth muscle cells and reduce glycolysis. Notably, recent studies have shown that mirabegron, a β3 adrenoceptor agonist, protects human PAECs from hypoxia-induced ROS production and mitochondrial fragmentation by restoring mitochondrial fission/fusion kinetics. Moreover, it demonstrates improvement in monocrotaline and hypoxia-induced pulmonary hypertension in mice [18]. These findings collectively suggest that restoring disrupted mitochondrial dynamics contributes to the relief of the disease phenotype.

However, there are some limitations of this experiment. Unlike Mdivi-1, a drug that specifically targets and inhibits mitochondrial fission [45], the therapeutic effects of Mfn2 currently rely mainly on gene therapy, with no targeted drug available to promote mitochondrial fusion. Although leflunomide, an FDA-approved drug for rheumatoid arthritis, has been found to promote a 2-fold increase in Mfn2 expression in pancreatic cancer cells in other tumor diseases [46], its long-term administration may potentially contribute to pulmonary hypertension [47, 48]. Therefore, further research is required to explore and address potential side effects and limitations associated with Mfn2-based therapies for pulmonary hypertension.

In conclusion, our study provides valuable insights into the significance of down-regulating Mfn2 expression, which leads to impaired mitochondrial fusion, altered mitochondrial respiration patterns, and increased mitochondrial oxidative stress. Consequently, these cellular changes drive the proliferation of PASMC and contribute to pulmonary vascular remodeling. By targeting the Mfn2-Warburg effect axis, there is a promising opportunity to develop novel therapeutic interventions for pulmonary hypertension induced by catch-up growth following intrauterine growth restriction.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors thank PhD Hechen Huang (The First Affiliated Hospital, Zhejiang University School of Medicine) for analyzing the RNA-seq data. The authors thank Professor ZhiChen’s lab for their kindly assistance during the experiments. The authors also thank Beibei Wang in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for her technical assistance on Transmission Electron Microscopy.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 82201889, 82241017, 82001587, 82201890, 82203223) and the Fundamental Research Funds for the Central Universities.

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Authors and Affiliations

  1. Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, No. 3333 Binsheng Road, Hangzhou, Zhejiang Province, People’s Republic of China

    Lingling Yan, Xiaofei Luo, Chengcheng Hang,  YuWang, Ziming Zhang, Shanshan Xu & Lizhong Du

  2. Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China

    Lingling Yan & Xiaofei Luo

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Contributions

LD and LY conceived and designed the study. LY, XL, CH and ZZ performed the study. LY and YW analyzed the data. YL was a major contributor in writing the manuscript. All authors read and approved the final manuscript.

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Correspondence to Lizhong Du.

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In accordance with ethical standards and guidelines, all animal experimental procedures were conducted following the approved Care and Use of Laboratory Animal protocol by the Institutional Animal Care and Use Committee. The Animal Care and Use Committee of Zhejiang University granted approval for these procedures (Approval No. ZJU20160215). During the experimental surgeries, all animals were administered anesthesia to ensure their comfort and minimize any potential discomfort or pain.

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Supplementary Material 1

: Fig. 1. The expression level of Mfn2 in PASCMs after transfection with Ad-Mfn2 for 48 h. Fig. 2. The expression of Mfn2 in pulmonary vascular after injecting Ad-mfn2 for 3, 5, 7 and 14 days. Fig. 3. Biological coefficient of variation of the two experimental NGS samples. Fig. 4. Representative transmission electron microscopic images of the PASMC. Fig. 5. Statistics of the PASMCs proliferation with overexpression of Mfn2. Table 1. Primers for RT-qPCR. Table 2. The expression levels of differential expressed genes

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Yan, L., Luo, X., Hang, C. et al. Unraveling the Mfn2-Warburg effect nexus: a therapeutic strategy to combat pulmonary arterial hypertension arising from catch-up growth after IUGR. Respir Res 25, 328 (2024). https://doi.org/10.1186/s12931-024-02957-1

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