Targeting REV-ERBα/BNIP3 axis attenuates pulmonary arterial hypertension by repressing mitophagy in mice

Domain: biology
Published: 2026-04-02
Editorial outcome: minor_revision

Abstract
Pulmonary arterial hypertension (PAH) is a life-threatening metabolic disorder. Nuclear receptors REV-ERBα and REV-ERBβ are established regulators of circadian rhythm and metabolic homeostasis, however their roles in PAH remain unclear. Using Rev-erbα+/-, VSMC-specific Rev-erbα-/-, and Rev-erbβ-/- mice (only male mice were used in the study), along with pharmacological activation and AAV-mediated overexpression, we found that Rev-erbα deficiency, particularly in vascular smooth muscle cells (VSMCs), exacerbates Su5416+hypoxia (SuHx)-induced PAH, whereas Rev-erbα activation or overexpression alleviates disease. In contrast, Rev-erbβ loss does not affect PAH. Notably, late-stage administration of REV-ERBα agonist significantly improves established PAH. Mechanistically, REV-ERBα directly represses Bnip3 transcription, thereby inhibiting BNIP3-driven mitophagy and improving mitochondrial function in hypoxic pulmonary artery smooth muscle cells (PASMCs). Bnip3 knockdown phenocopies REV-ERBα activation, while Bnip3 overexpression abrogates REV-ERBα’s anti-proliferative effects and accelerates PAH. Collectively, REV-ERBα protects against PAH by inhibiting BNIP3-driven mitophagy and preserving mitochondrial homeostasis in PASMCs. Targeting the REV-ERBα-BNIP3 axis holds promise as a circadian-based therapeutic strategy for PAH.

Introduction
Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease characterized by the remodeling of pulmonary vasculature, primarily affecting the small pulmonary arteries. This remodeling increases pulmonary vascular resistance, eventually leading to right ventricular (RV) failure1,2. Key underlying mechanisms include hyper-constriction of the hypoxic pulmonary vessels and abnormal proliferation of vascular cells, particularly pulmonary artery smooth muscle cells (PASMCs) in the middle layer of the vessel wall1. Current therapeutic strategies primarily focus on prostacyclin, endothelin, and nitric oxide pathways, offering symptomatic relief by promoting vasodilation3. Despite these advancements, PAH continues to be a life-threatening condition, underscoring the urgent need for innovative therapies that can inhibit PASMCs proliferation, reverse pulmonary vascular remodeling, and improve patient outcomes.

REV-ERBs, including REV-ERBα (also known as NR1D1) and REV-ERBβ (also known as NR1D2), are members of the nuclear receptor (NR) superfamily. They function as transcriptional repressors by binding to receptor-associated orphan receptor elements in target genes, resulting in chromatin condensation and transcriptional silencing4. While primarily recognized for their role in circadian rhythm regulation, REV-ERBα has recently been identified as a key regulator of metabolism and cell survival. Most recently, REV-ERBα has been implicated in the pathogenesis of various metabolic and cardiovascular diseases5-8.

Mitochondrial homeostasis, encompassing mitochondrial biogenesis, dynamic changes, and the process of mitochondrial autophagy, is associated with various cardiovascular diseases, including PAH9,10. Targeting mitochondrial homeostasis has emerged as a promising strategy to mitigate pulmonary vascular remodeling and retard PAH. Notably, REV-ERBs have been extensively studied for their roles in maintaining mitochondrial homeostasis in tissues such as aortic smooth muscle, skeletal muscle and adipose tissues11-14. However, the role of REV-ERBs in PAH progression, particularly their function within pulmonary vascular smooth muscle cells and their relationship with mitochondrial homeostasis, remains poorly explored.

Here we aimed to examine the role of REV-ERBs in the pathogenesis of PAH and to explore how they regulate mitochondrial homeostasis in pulmonary vascular smooth muscle cells.

Results
1. REV-ERBα, but not REV-ERBβ, is downregulated in SPAH models
The protein level of REV-ERBα was decreased in lung tissues of SuHx-induced PAH mice (Figure 1A). Immunofluorescence co-staining of α-smooth muscle actin (α-SMA) and REV-ERBα further confirmed the specific decrease of REV-ERBα in primary cultured PASMCs (Figure 1B). Quantitative PCR (qPCR) also showed a significant down-regulation of Rev-erbα mRNA in pulmonary arteries of the SuHx mice (Figure 1C). Moreover, to investigate whether this downregulation was a direct response to hypoxia, we subjected isolated PASMCs to either physical hypoxia or treatment with cobalt chloride (CoCl₂), a well-established hypoxia mimetic agent15,16. REV-ERBα expression was significantly decreased in CoCl₂-treated (Figure 1D-F) or hypoxia-exposed (Figure 1G-I) PASMCs as well. In contrast, REV-ERBβ expression remained unchanged in either lung tissues, pulmonary arteries or PASMCs (Figure S1A-I). Additionally, considering the crosstalk effects between hypoxia and the circadian clock, we characterized the expression of other core circadian genes in lung tissue from the SuHx mice and hypoxia-exposed PASMCs. While we did observe modest fluctuations in the protein expression of CRY1 and PER1 and the transcriptional amplitude of Cry1 and Per1 in PASMCs, the most consistent, profound, and statistically significant change was the downregulation of REV-ERBα (Figure S2A-I). These findings suggest REV-ERBα may participate in pulmonary vascular remodeling and PAH development.

2. SR9009 attenuates hypoxia-induced PASMC proliferation in a REV-ERBα-dependent manner
Proliferation of PASMCs is a key event in the pathogenesis of pulmonary vascular remodeling and PAH1. To further investigate the role of REV-ERBs in PASMC proliferation, we treated PASMCs with SR9009, a small-molecule agonist targeting REV-ERBs17. The data showed that SR9009 significantly suppressed hypoxia-induced PASMC proliferation, as demonstrated by reduced EdU incorporation, fewer Ki67-positive cells, and lower Cell Counting Kit-8 (CCK-8) assay results (Figure 2A-B, Figure S3A). Considering that SR9009 may exhibit off-target pharmacological effects on REV-ERBs18,19, we knocked down Rev-erbs with siRNA in hypoxic PASMCs. Notably, knockdown of Rev-erbα not only significantly enhanced hypoxia-induced proliferation, but also abrogated the anti-proliferative effect of SR9009 (Figure 2C-D, Figure S3B). In contrast, knockdown of Rev-erbβ neither affected hypoxia-induced proliferation nor blocked SR9009's protective effects (Figure S3C-D). To further confirm the role of REV-ERBα in PASMC proliferation, we transfected a Rev-erbα overexpression plasmid into PASMCs and found that overexpression of Rev-erbα significantly inhibited hypoxia-induced cell proliferation (Figure 2E-F, Figure S3E). These findings collectively demonstrate that REV-ERBα, but not REV-ERBβ, plays a critical role in inhibiting hypoxia-induced PASMC proliferation, and that SR9009 exerts its effects specifically through activation of REV-ERBα.

3. SR9009 mitigates SuHx-induced PAH both during disease development and after its establishment
We next explored the effect of SR9009 on the development of PAH. Mice were given a daily intraperitoneal injection of SR9009 starting at the initiation of SuHx induction (Figure 3A). The results showed that while SR9009 did not change the basal levels of PAH-related parameters in normoxic mice, it significantly attenuated the increased right ventricular systolic pressure (RVSP) and expanded pulmonary artery (PA) wall thickness in the SuHx-induced mice (Figure 3B-C). Echocardiography confirmed that pulmonary acceleration time/pulmonary ejection time (PAT/PET) ratio, a measure of RV afterload correlated with RVSP, was significantly reduced in SuHx mice, and SR9009 effectively restored this decrease (Figure 3D). Pulmonary angiography assessed structural remodeling in small pulmonary vessels, revealing that SuHx treatment reduced total branch length, branch number, and junctions, and SR9009 rescued these changes (Figure 3E). Additionally, SuHx-induced RV remodeling assessed by Fulton index and wheat germ agglutinin (WGA) staining were also significantly mitigated by SR9009 (Figure 3F-G). Fractional area change (FAC), a parameter for evaluating right ventricular systolic function, was decreased in SuHx mice, and this decline was reversed by SR9009 treatment (Figure S4A).
We also evaluated the therapeutic effect of SR9009 on the established PAH by initiating the once-daily treatment of SR9009 at the beginning of the third week after SuHx induction (Figure S5A). As expected, SR9009 demonstrated clear therapeutic potential on PAH, reversing the pulmonary vascular remodeling and mitigating RV remodeling in SuHx-induced PAH mice (Figure S5B-F). These results indicated that SR9009 treatment can alleviate the progression of PAH and limit pulmonary vascular remodeling, both during the development and after the establishment of PAH.

4. Deficiency of Rev-erbα aggravates, while Rev-erbα overexpression alleviates, SuHx-induced PAH
Next, we sought to investigate the effect of genetic disruption of REV-ERBs on SuHx-induced PAH progression. To circumvent the developmental abnormalities and reproductive deficiencies associated with Rev-erbα global knockout mice20, heterozygous Rev-erbα knockout (Rev-erbα+/-) mice were utilized and subjected to SuHx-induced PAH with or without SR9009 treatment (Figure 4A, Figure S6A-D). As shown, compared to the Rev-erbα+/+ control mice, Rev-erbα+/- mice displayed exacerbated SuHx-induced PAH, characterized by increased RVSP and PA wall thickness. Notably, these pathological changes were not mitigated by SR9009 treatment (Figure 4B-C). PAT/PET ratio, pulmonary angiography, Fulton index, and WGA staining further verified that Rev-erbα haploinsufficiency significantly augmented the SuHx-induced arterial remodeling and RV dysfunction, and SR9009 failed to confer protection under this genetic background (Figure 4D-G).
To further explore isoform-specific roles, we employed Rev-erbβ-/- mice to assess the impact of Rev-erbβ depletion on PAH. In contrast to the Rev-erbα deficiency, Rev-erbβ knockout did not alter PAH phenotypes following SuHx induction, as PAH-related parameters in Rev-erbβ-/- mice after SuHx induction were comparable to those in their wild-type littermates (Figure S6E-G, S7A-F). Collectively, these findings strongly indicate that REV-ERBα, but not REV-ERBβ, plays a pivotal role in the pathogenesis of experimental PAH.
Furthermore, we validated the protective function of REV-ERBα in PAH by establishing a Rev-erbα overexpression mouse model through tail vein injection of an adeno-associated virus (AAV) encoding mouse Rev-erbα, followed by exposure of these mice to the SuHx-induced PAH (Figure 5A, Figure S8). As expected, Rev-erbα overexpression significantly alleviated SuHx-induced PAH, pulmonary vascular and RV remodeling, as evidenced by a reduction in the RVSP (Figure 5B) and PA wall thickness (Figure 5C), alongside an increase in the PAT/PET ratio (Figure 5D), and a lower Fulton index (Figure S9A) and smaller cross-sectional area of RV cardiomyocytes (Figure 5E). These findings further highlight the therapeutic potential of targeting Rev-erbα in PAH.

5. VSMC-specific deletion of Rev-erbα aggravates SuHx-induced PAH
Building on the findings that partial loss of Rev-erbα exacerbates PAH and that REV-ERBα activation inhibits PASMC proliferation under hypoxic conditions, we then aimed to determine whether the primary role of REV-ERBα in PAH and pulmonary vascular remodeling is mediated through vascular smooth muscle cells. By generating the vascular smooth muscle cell-specific Rev-erbα knockout mice (VSMC- Rev-erbα-/-) and subjected them to SuHx-induced PAH (Figure 5F, Figure S10A-D), we found that VSMC-specific deletion of Rev-erbα did not affect the baseline pulmonary vascular phenotype (Figure S11); however, similar to the Rev-erbα+/- mice, it drastically exacerbated SuHx-induced PAH and pulmonary vascular remodeling (Figure 5G-K, Figure S9B). These findings collectively provide compelling evidence for a previously unrecognized role for REV-ERBα, particularly VSMC-derived REV-ERBα, as a key mediator in mitigating PAH.

6. REV-ERBα improves mitochondrial homeostasis in PAH by mitigating mitophagy in PASMCs
Emerging evidence suggests that mitochondrial homeostasis plays a crucial role in pulmonary vascular remodeling and PAH9,21. REV-ERBα has been implicated in regulating mitochondrial function across various metabolic disorders11,22. Next, we sought to establish whether activation of REV-ERBα may improve mitochondrial homeostasis in PAH rodent models. By using Transmission Electron Microscope to study the ultra-structure of mitochondria, we observed significant mitochondrial damage and an increased number of autophagic vesicles in SuHx mice compared to normoxic controls. Notably, SR9009 treatment effectively restored mitochondrial integrity and reduced autophagic vesicles (Figure 6A). Furthermore, pulmonary vascular mitochondrial superoxide levels were assessed using mitoSOX, a marker of reactive oxygen species (ROS) generated during oxidative phosphorylation in the electron transport chain23, and the mitoSOX level was significantly reduced after intervention of SR9009 (Figure 6B). We next examined the expression of key proteins associated with mitochondrial homeostasis and autophagy. Consistent with TEM findings, SRC9009 notably reduced the elevated LC3 II/I ratio, an important indicator of autophagosome activation, observed in SuHx lungs, indicating a mitigation of excessive autophagy (Figure 6C). Supporting these findings, immunofluorescence analysis revealed a significant increase in LC3-positive smooth muscle layers within the pulmonary arteries of SuHx mice compared to normoxic controls, which was significantly reversed by SR9009 treatment (Figure 6D), implying SR9009 modulates mitochondrial homeostasis in vascular smooth muscle cells. Consistent with the in vivo findings, hypoxic PASMCs showed increased total ROS, decreased mitochondrial membrane potential, elevated mitoSOX, and increased mitophagy, as shown by a higher mt-Keima fluorescence intensity ratio, while SR9009 markedly rescued these changes, restored mitochondrial function and reduced mitophagy (Figure 6E-H). Additionally, SR9009 normalized the LC3 II/I ratio, TOMM20 and P62 protein levels without affecting mitochondrial dynamics or biosynthesis (Figure 6I, Figure S12A-B); notably, the hypoxia-induced conversion of LC3B-I to LC3B-II and the decrease in P62 were reversed by SR9009 in mitochondrial fractions (Figure S12C), further supporting its role in maintaining mitochondrial homeostasis through mitophagy.
In contrast to the effects of REV-ERBα activation by SR9009, Rev-erbα+/- mice demonstrated worsening SuHx-induced autophagy and mitochondrial homeostasis, characterized by elevated mitoSOX levels, increased LC3 II/I ratio, and expanded LC3-positive smooth muscle layer areas compared to the control mice (Figure S13A-C). As expected, SR9009 treatment failed to alter these parameters in Rev-erbα+/- mice (Figure S13A-C), further emphasizing the REV-ERBα-dependent mechanism underlying SR9009's effects. Together, these findings suggest that REV-ERBα activation improves mitochondrial homeostasis in PAH by mitigating mitophagy in PASMCs.

7. REV-ERBα regulates mitophagy through transcriptional repression of Bnip3
Mitophagy involves two main mechanisms: (A) PINK1-PARKIN mediated ubiquitination pathway, and (B) receptor-mediated processes24. To further explore the mechanisms by which REV-ERBα regulates mitophagy, we examined the protein expression of the key mitophagy-related proteins in hypoxia-exposed PASMCs with or without SR9009 treatment. While the non-classical mitophagy receptors BNIP3 and BNIP3L were significantly upregulated in hypoxic PASMCs and notably downregulated by SR9009 treatment, SR9009 did not affect the expression of HIF1A, FUNDC1, PINK1 or PARKIN (Figure 7A, Figure S14A). Importantly, co-immunoprecipitation assays further revealed enhanced interaction between BNIP3/BNIP3L and LC3B under hypoxic conditions, which was attenuated by SR9009 treatment (Figure 7C). Simultaneously, mitochondrial isolation revealed that hypoxia-induced accumulation of BNIP3 in the mitochondrial fraction was also reduced by SR9009 (Figure S12C). Moreover, we found that hypoxia significantly upregulated the anti-apoptotic protein BCL2 without altering the levels of its pro-apoptotic counterpart, BAX, whereas SR9009 had no effect on either protein (Figure S14B). Overall, these findings imply that Rev-erbα regulates BNIP3 and BNIP3L-mediated mitophagy rather than apoptosis.
Intriguingly, SR9009 was observed to significantly mitigate the increase in Bnip3 mRNA levels, while having no significant effect on Bnip3l mRNA levels (Figure 7B), implying that Bnip3, but not Bnip3l, might be under direct transcriptional regulation by the transcriptional repressor REV-ERBα. Using the JASPAR database, we identified two potential REV-ERBα binding sites on the Bnip3 promoter (Figure 7D). Chromatin immunoprecipitation (ChIP) assays performed on cultured PASMCs confirmed that REV-ERBα directly binds to the two regions of the Bnip3 promoter (Figure 7E). Additionally, dual-luciferase reporter assay showed that overexpression of Rev-erbα weakened the luciferase activity in cells transfected with a wild-type Bnip3 luciferase plasmids (Figure 7F). Strikingly, when Bnip3 was overexpressed, REV-ERBα lost its inhibitory effect on cell proliferation (Figure 7G-I). These findings strongly suggest that REV-ERBα regulates mitophagy and cell proliferation by transcriptionally repression of Bnip3 (Figure 7J).
To further establish the role of mitophagy in hypoxia-induced PASMC proliferation and mitochondrial dysfunction, we treated the hypoxia-exposed PASMCs with or without mdivi-1, a widely used mitophagy inhibitor25-27, and found that mdivi-1 not only significantly attenuated hypoxia-induced proliferation in PASMCs but also blunted the enhanced proliferation observed in Bnip3-overexpressing cells (Figure S15-S17). Moreover, we found that overexpression of Bnip3 in hypoxic PASMCs did not alter BAX expression (Figure S14C). These results collectively indicate that BNIP3 promotes hypoxia-induced PASMC proliferation through a mitophagy-dependent pathway.
Interestingly, an alternatively spliced variant lacking exon 3, termed Bnip3Δexon3, has been identified as a pro-survival isoform of Bnip3 in cardiomyocytes28,29. Consequently, the ratio of Bnip3Δexon3 to full-length Bnip3 (Bnip3-FL) is considered a critical determinant of whether BNIP3 signaling culminates in cell survival or death30,31. Here we confirmed the expression of both Bnip3-FL and the shorter Bnip3Δexon3 splice variant in PASMCs (Figure S18A-C). Then we found that activation of REV-ERBα by SR9009 not only suppressed the expression of both Bnip3Δexon3 and Bnip3-FL in hypoxia-exposed PASMCs (Figure S18D-E), but it also significantly decreased the ratio of Bnip3Δexon3 to Bnip3-FL (Figure S18F).

8. Suppression of Bnip3 mitigates hypoxic PASMC proliferation, while VSMC-specific Bnip3 overexpression worsens PAH
As demonstrated above, overexpression of Bnip3 alone can enhance hypoxia-induced proliferation of PASMCs (Figure 7G-I), indicating its potential role as an independent driver for pulmonary vascular remodeling. To further validate this, we performed Bnip3 knockdown using siRNA in hypoxic PASMCs. As shown, like the effects of pharmacological activation by SR9009, knockdown of Bnip3 significantly inhibited hypoxia-induced proliferation of PASMCs and suppressed mitophagy (Figure 8A-C, Figure S19A-E), suggesting that BNIP3 plays a critical role in promoting PASMC proliferation under hypoxic conditions. Finally, we generated a VSMC-specific Bnip3 overexpression model by tail vein injecting of an AAV carrying Bnip3 under the control of SM22α promoter, and then exposed them to SuHx-induced PAH (Figure 8D, Figure S20A). As expected, AAV-mediated VSMC-specific Bnip3 overexpression significantly exacerbated both pulmonary vascular remodeling and RV hypertrophy in the SuHx-PAH model, as evidenced by a higher Fulton index (Figure 8E), a significant increase in RVSP (Figure 8F), pronounced thickening of the PA wall (Figure 8G), a notable decrease in the PAT/PET ratio (Figure 8H), and enlarged cross-sectional area of RV cardiomyocytes (Figure 8I). Taken together, these findings establish vascular smooth muscle-derived BNIP3 as a critical mediator of PAH pathogenesis in murine models.

Methods
Animals
Male mice on a C57BL/6N background, aged (8-10 weeks old), were used for all studies. Heterozygous Rev-erbα knockout (Rev-erbα+/-) mice, Rev-erbβ global knockout (Rev-erbβ-/-) mice and their littermate control (Rev-erbα+/+, Rev-erbβ+/+) mice were obtained from the Cyagen Company (Suzhou, China). Vascular smooth muscle cell-specific Rev-erbα knockout (VSMC-Rev-erbα-/-) mice were generated by crossing Rev-erbαflox/flox mice with SM22α-Cre+ transgenic mice, Rev-erbαflox/flox mice that do not express SM22α Cre were used as controls (VSMC-Rev-erbαflox/flox, abbreviated as VSMC-Rev-erbαfl/fl). Male Sprague-Dawley (SD) rats (8-10 weeks old, weighing 200-250 g) were used for primary cell isolation. All mice were exposed to normal light (lights on at 8 a.m., ZT0 [Zeitgeber Time 0])/dark (lights off at 8 p.m., ZT12 [Zeitgeber Time 12]) cycle with free access to food and water, maintained at an ambient temperature of 22 ± 2 °C and a relative humidity of 50 ± 10%. All animal sacrifices and key physiological measurements were performed between ZT6 and ZT8. All animal experiments were performed conforming to the Institutional Animal Care and Use Committee (IACUC) at East China Normal University (permission number: m+RR20211103). All the animal experiments were designed to generate groups using randomization.

PAH model
A hypoxia+Su5416 (SuHx)-induced PAH model in mice were established as follows61. In brief, 8-10-week-old male C57BL/6N mice were subcutaneously injected with Su5416 (20 mg/kg, dissolved in a vehicle consisting of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline) once a week during the 3-week exposure to a hypoxic chamber. Mice underwent cardiac color Doppler ultrasound examination one day before sacrifice. On the final day of the experiment, animals were fully anesthetized via 2% isoflurane inhalation, and the right ventricular pressure (RVP) was immediately measured using a PT-102N pressure transducer (TECHMAN), carefully inserted into the right ventricle (RV). Hemodynamic values were recorded using a physiological data acquisition system (BL-420N, TECHMAN), and right ventricular systolic pressure (RVSP) was subsequently calculated. After the measurement of hemodynamic parameters, animals were euthanized via an overdose of isoflurane anesthesia. For the assessment of RV hypertrophy, after euthanasia, the RV was dissected from the left ventricle and interventricular septum and weighed separately. The weight ratio was then calculated as the ratio of weight of the RV to that of the left ventricle plus the inter-ventricular septum. Data acquisition and analysis were performed by an investigator blinded to the experimental groups. Variations in sample sizes among different assessments within the same animal cohort are due to technical failures during specific procedures (e.g., failure in RVSP measurement) or unexpected mortality during the PAH induction period.

Cell culture
Primary male rat PASMCs were isolated as we previously described61. In brief, pulmonary arteries were isolated under anatomic microscope and dissected out in dissociation medium from adult Sprague-Dawley (SD) rat (Beijing Vital River Laboratory Animal Technology Co., Ltd.). The tissues were digested by 8.5 mg/mL type I collagenase and 2.0 mg/mL BSA dissolved in Ca2+/Mg2+-free Hank's Balanced Salt Solution supplemented with 1% Penicillin-Streptomycin (P/S) incubated for 1 h at 37 °C. Then, the cells were cultured in DMEM supplemented with 20% fetal bovine serum, 1% P/S and in a humidified incubator with 95% air and 5% CO2. Cells between passages 3 and 6 were used for further experiments. PASMCs in hypoxic conditions were incubated for 24 h with a gas mixture containing 3% O2, 5% CO2, and 92% N2, or treated with Cobalt Chloride (CoCl2) (100µM) for 24 h. Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (ATCC). HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% P/S and maintained in a humidified incubator with 95% air and 5% CO2 at 37 °C. Both cell types were seeded in standard cell culture dishes or plates (Jet Biofil).

Drug treatment
For the in vivo experiment, SR9009 was prepared in 5% DMSO, 10% Cremophor EL, 85% Saline, and administered intraperitoneally at 100 mg/kg once daily between ZT6-ZT8 for a total duration of 21 days of hypoxia or only for the last week of the experiment. The vehicle group received an equal volume of the identical solvent mixture.
For cell culture experiment, SR9009 (10 μM) was added to the medium for 24 h. Equal concentrations of the solvent DMSO were used as controls. Mitophagy inhibitor mdivi-1 (5 μM) was incubated with cells in the medium for 24 h. Equal concentrations of the solvent DMSO were used as controls.

Echocardiographic measurements
Pulmonary arterial hemodynamics and RV function in the rodent models were assessed by echocardiography. Briefly, the mice were anesthetized with 2% isoflurane and kept warm with a heating plate and then subjected to transthoracic echo using the Vevo 3100 system (VisualSonics) with MX400 (20-46 MHz). The pulmonary artery blood outflow was recorded by Pulsed Wave (PW) Doppler echo in the parasternal short axis view of aortic root and used to measure pulmonary acceleration time (PAT) and pulmonary ejection time (PET). All variables were analyzed from 3 through 5 consecutive peaks or images using Vevo LAB 3.1.1 software (VisualSonics). RV fractional area change was measured in B-mode echocardiograms from the apical 4-chamber view. RV fractional area change (FAC) = (RV end-diastolic chamber area- RV end-systolic chamber area)/RV end-diastolic chamber area. Data acquisition and analysis were performed by an investigator blinded to the experimental groups.

Wheat germ agglutinin
To analyze the cross-sectional area of cardiomyocytes, right heart tissues were stained with wheat germ agglutinin (WGA, Sigma Aldrich, L4895) according to the manufacturer’s instructions. Briefly, mice hearts were dehydrated, embedded in paraffin, cut into 5-μm-thick sections. Paraffin sections were heated in a pressure cooker with Tris-EDTA buffer for 3 min. The sections were stained with WGA working solution (Dissolved in water, 1mg/mL) for 30 min at 37 ℃. Stained sections were scanned under an upright fluorescence microscope (Olympus, BX53). ImageJ software (NIH) was used to estimate the cross-sectional area of the cardiomyocytes.

Histology analysis
The right lungs and pulmonary arteries were collected and placed in liquid nitrogen immediately for preparation of protein and RNA, and the left lungs and part of heart were fixed with 4% paraformaldehyde. Formalin-fixed paraffin-embedded lung tissue was dehydrated with ethanol gradient, embedded in paraffin, and subsequently cut into 5-μm-thick sections. For histological analysis, the sections were stained with hematoxylin and eosin (HE). Images of the HE-stained sections were captured using a digital slide scanning system (SQS-12P, Shengqiang Technology Co.). Or, sections were subjected to immunofluorescence targeting α-smooth muscle actin (α-SMA). To assess medial wall thickness, 15-20 pulmonary arteries categorized by external diameter (<50 μm and >50 μm) were randomly and blindly selected from each lung. The medial area was determined by subtracting the internal luminal area from the total vessel cross-sectional area. The extent of pulmonary vascular remodeling was then evaluated by calculating the ratio of the medial area to the total cross-sectional area [(Total Area − Luminal Area) / Total Area] using ImageJ software (NIH).

Pulmonary angiography
In a separate cohort of mice dedicated to pulmonary angiography, at the termination of the experiments. Briefly, at the termination of the experiments, the animals were anesthetized and then a thoracotomy was carried out to expose their hearts and lungs. The pulmonary artery was subsequently flushed through the right ventricle with PBS containing sodium heparin. Right lung was perfused through the pulmonary artery with 0.1 mL of Microfil, a liquid silicone-based polymer (MV-122, Flow Tech), which was injected slowly and uniformly using a catheter. The lungs were immersed in PBS to cleanse the tissue surface of stains. Subsequently, they were dehydrated and ultimately placed in methyl salicylate for photographic documentation. Total length of arterial branch, number of arterial branch and number of junctions were calculated using ImageJ software (NIH).

Cell proliferation analysis
PASMCs proliferation was quantified using the Cell Counting Kit (CCK-8, MCE), Ki67 immunofluorescence staining, and the EdU Cell Proliferation Image Kit (Abbkine). For the CCK-8 assay, the culture medium of the PASMCs was removed and replaced with 90 μL DMEM and 10μL CCK8, followed by 1 h incubation. The absorbance values were detected at 450 nm wavelength by a microplate reader (Synergy H1, BioTek Instruments). For the EdU assay, living PASMCs were first incubated with 10 μM EdU for 2 h in the cell culture incubator. Following the incorporation, PASMCs were fixed with 4% paraformaldehyde for 20 min, and then incubated with 0.5% Triton X-100 for 20 min, followed by incubation with the click reaction cocktail (provided in the kit) for 30 min in the dark to detect incorporated EdU. Nuclei were then counterstained with DAPI. Images were collected using an upright fluorescence microscope (Olympus, BX53), and the rate of EdU incorporation in the treatment group was normalized with those of the control group. For the Ki67 Immunofluorescence, Cells were fixed with paraformaldehyde, treated with 0.1% Triton X-100 for 30 min, blocked with 5% goat serum and incubated with Ki67 antibody overnight, followed by incubation with anti-rabbit fluorescently conjugated antibody, photographed using confocal and analyzed for fluorescence data using ImageJ software (NIH).

Immunofluorescence
For tissue immunofluorescence, lung tissues were heated in a pressure cooker with Tris-EDTA buffer for 3 min, and then incubated with the blocking buffer 5% goat serum dissolved in PBS. Tissue sections were then incubated with primary antibody over night at 4°C and followed by the incubation of fluorescently conjugated secondary antibody for 1 h at room temperature. Finally, nuclei were counterstained with DAPI. For the quantitative analysis of fluorescence intensity, at least 20 pulmonary vessels per sample were selected, and the images were captured using a confocal microscope (DF505, Andor Technology) for subsequent analysis. For cell immunofluorescence analysis, PASMCs were fixed with 4% paraformaldehyde followed by permeabilized using 0.3% Triton X-100 in PBS and incubated with the 5% goat serum dissolved in PBS. Cells were then incubated with primary antibody overnight at 4°C and followed by the incubation of fluorescently conjugated secondary antibody for 1 h at room temperature. Finally, nuclei were counterstained with DAPI. Antibodies used for immunofluorescence are in Table S2.

Assessment of ROS production
For total ROS, cells were distributed into 96-well black plates and incubated with oxygen radical sensitive probe 5 μM DCFH-DA (S0033S, Beyotime) for 30 min in a 37 °C, 5% CO2 incubator in the dark, and subsequently measure ROS probe fluorescence intensity with a microplate reader (Synergy H1, BioTek Instruments). For the mitoSOX assay of pulmonary vasculature, fresh lung tissues were embedded in Optimal Cutting Temperature compound, and snap-frozen. Unfixed cryosections were then prepared and topically stained with a 5 μM MitoSOX Red superoxide indicator (Yeason, 40778ES50) in a light-protected, humidified chamber. For each group of mice, at least 20 pulmonary vessels were selected from each mouse’s lungs for statistical analysis, with fluorescence images captured using a DF505 fluorescence microscope (Andor Technology) and intensity analyzed via ImageJ software (NIH). Mitochondrial superoxide in PASMCs was detected by staining with 1 µM MitoSOX Red (Invitrogen, M36008) in phenol red-free media for 30 min at 37 °C, followed by two PBS washes. Cells were imaged on an upright fluorescence microscope (Olympus, BX53) . For quantification, ten fields of view per sample were acquired with fixed acquisition settings. Fluorescence intensity was quantified for ≥100 cells per condition using ImageJ (NIH). The mean fluorescence intensity was measured within ROIs manually drawn around whole cells.

Measurement of mitochondrial membrane potential (MMP)
The MMP of PASMCs was monitored using JC-1 (J8030, Solarbio). Briefly, PASMCs were incubated in the dark with JC-1 for 30 min at 37 °C and washed with PBS for three times. When the mitochondrial membrane potential is elevated, JC-1 aggregates within the mitochondrial matrix, exhibiting a maximum excitation wavelength of 585 nm and resulting in enhanced red fluorescence. Conversely, when the mitochondrial membrane potential is diminished, JC-1 remains in its monomeric form with a maximum excitation wavelength of 514 nm leading to an increase in green fluorescence. The alteration in mitochondrial membrane potential is assessed by the shift in fluorescence color. The fluorescence images were captured using an upright fluorescence microscope (Olympus, BX53) and the mitochondrial membrane potential was quantified by calculating the ratio of red (aggregates) to green (monomers) fluorescence intensity using Image J software (NIH).

Chromatin immunoprecipitation (ChIP)
ChIP was performed using anti-REV-ERBα and anti-rabbit IgG. PASMCs were fixed with 1% formaldehyde, and cross-linked protein-DNA complexes were prepared using a ChIP kit. Complexes were incubated with a primary antibody against REV-ERBα or rabbit IgG for 18h at 4°C. Magnetic beads were added with gentle agitation for 2h. DNA was eluted from the beads. Primers flanking the REV-ERBα binding sites on the rat Bnip3 gene promoter were used in PCR assays, and PCR products were visualized using agarose gel electrophoresis with a chemiluminescence imaging system (Bio-Rad, ChemiDoc XSRS+). Primer sequences used for ChIP-PCR are in Table S3.

Co-Immunoprecipitation (Co-IP)
For immunoprecipitation in the endogenous system, PASMCs were seeded 24 h prior to stimulations. After stimulation, PASMCs were lysed in NP-40 lysis buffer. Protein concentrations were determined, and 500 μg of total protein lysate was used for each IP. An aliquot of 10% from each whole-cell lysate was saved as the Input control. Cell lysates were pre-cleared using 20 µl of protein A/G plus-agarose beads (sc-2003, Santa Cruz Biotechnologies) for 2h at 4°C. Primary anti-LC3B antibody was added at a concentration of 1:100 and incubated overnight at 4°C (Normal rabbit IgG was used as a negative control for the immunoprecipitation). The immune complexes were then precipitated by adding 30 μL of Protein A/G plus-agarose beads and incubating for an additional 2 h at 4°C. The beads were subsequently washed three times with lysis buffer, and the immunoprecipitated proteins were eluted by boiling in SDS sample buffer prior to immunoblotting analysis. Antibodies used for immunoprecipitation are in Table S2.

Mitochondrial isolation
Mitochondrial and cytosolic fractions were prepared using a Cell Mitochondria Isolation Kit (C3601, Beyotime Biotechnology). Briefly, cell pellets were lysed via Dounce homogenization. The homogenate was centrifuged at 600 × g for 10 minutes at 4 °C to remove nuclei and cell debris. The resulting supernatant was then centrifuged at 11,000 × g for 10 minutes at 4 °C to pellet mitochondria. The final supernatant was collected as the cytosolic fraction. Subsequently, both components underwent Western blot analysis, with GAPDH serving as the cytoplasmic protein reference and COX IV as the mitochondrial protein reference. Antibodies used for western blot are listed in Table S2.

Western blot, qPCR
For western blot analysis, protein samples were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated overnight with indicated primary antibodies, followed by the appropriate HRP-conjugated secondary antibodies. Finally, immunoblots were visualized with a gel image analysis system (Tanon 5200, Tanon Science & Technology), and the densitometric quantification of protein bands was performed using ImageJ software (NIH). Antibodies used for western blot are in Table S2.
For qPCR analysis, total RNA was extracted by using TRIzol reagent (Invitrogen). The cDNA was obtained using the TIANGEN Reverse Transcription Kit (KR107, TIANGEN) and subsequently the relative mRNA expression levels were quantified using SYBR Green (AQ631, TransGen Biotech) on a Real-Time PCR System (QuantStudio 5, Thermo Scientific), with 18S as an internal control. The relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method. Primer sequences used for qPCR are in Table S3.

Transmission electron microscopy (TEM)
Lung tissues were harvested and dissected, rinsed in PBS and then fixed in 2.5% glutaraldehyde in PBS. After being washed with PBS three times, the samples were fixed in PBS-buffered 1% OsO4 for 1 h, washed in ddH2O, stained in uranyl acetate for 30 min, dehydrated in a gradient of ethanol and acetone, permeated by embedding medium containing acetone (1:1), and finally embedded in epoxy resin. The ultrathin electron-stained sections were observed under an electron microscope (Tecnai T10, Philips-FEI) with voltage setting at 100 kV. Micrographs of randomly selected mitochondria were obtained at a final magnification of ×20000.

Plasmids, RNA interference
Recombinant plasmids encoding rat Bnip3 and rat Rev-erbα were purchased from MiaoLing Plasmid for achieving overexpression of Bnip3 and Rev-erbα in PASMCs. For individual transient transfection, the plasmid was transfected into PASMCs at a final concentration of 0.5 μg/ml with the assistance of Lipofectamine 3000 (Invitrogen) for 48 h. For co-transfection, the plasmids were co-transfected into the cells at half of the aforementioned concentration for 48 h.
Rev-erbα/β siRNA, Bnip3 siRNA, and negative control siRNA were sourced from Genepharma. In accordance with the experimental requirements, these siRNAs were individually transfected into PASMCs at a final concentration of 25 nM with the aid of Lipofectamine 3000 (Invitrogen). Following transfection, the cells were incubated for 48 h to facilitate effective gene silencing. All siRNA sequences are shown in Table S3.

Adeno-Associated Virus, Adenovirus
For Rev-erbα overexpression, adeno-associated virus 9 (AAV9) carrying a CMV-promoter-driven FLAG- Rev-erbα (AAV-OE-Rev-erbα) or negative control was administered intravenously in a single dose of 5 × 1011 v.g./mouse. Mice were subjected to SuHx-induced PAH one week after AAV9 injection. For VSMC-specific Bnip3 overexpression, AAV9 carrying a SM22α-promoter-driven FLAG-Bnip3 (AAV-OE-Bnip3) or negative control was administered intravenously in a single dose of 1 × 1012 v.g./mouse. Mice were subjected to normoxia or SuHx treatment one week after AAV9 injection. All viruses were constructed and purified by Obio Technology (Obio Technology Corp).
A mitochondria-targeted Keima adenovirus (Adv-COX8-mt-Keima) (Obio Technology Corp) was employed to evaluate mitophagy in PASMCs, which were infected with the recombinant virus at a multiplicity of infection (MOI) of 100 for 48 h. Mitochondria-targeted Keima (Mt-Keima) is a pH-sensitive fluorescent protein that shifts its excitation peak to 561 nm in lysosomal acidic conditions, compared to 445 nm in other environments. The quantification of mitophagy can be achieved through the detection of mt-Keima fluorescence shift62.

Dual-Luciferase Reporter assay
The dual-luciferase reporter assay was conducted according to the technical manual of Promega Dual-Luciferase Reporter Assay System (E1910, Promega). Firefly luciferase assays were carried out using the luciferase reporter vector pGL4.11-Basic, engineered to contain the wild-type promoter region of Bnip3 (rat), resulting in vectors termed Bnip3-promoter-luc. The firefly reporter plasmid, Renilla luciferase internal control plasmid, and the Rev-erbα overexpression plasmid (or empty vector control) were co-transfected into HEK293T cells (ATCC) by Lipofectamine 3000. After 24 h, cells were homogenized by the passive lysate buffer provided by the manufacturer. Luciferase Assay Reagent II and Stop and Glo Reagent were added to the lysates sequentially. Luminous intensity was detected by the microplate reader using the luminescence model.

Reagents
The reagents utilized in the current study have been outlined in Table S1.

Statistical analysis
Statistical analyses were performed using Prism 8 software (GraphPad). All results are reported as the mean ± SEM. No statistical methods were used to predetermine sample sizes. Sample sizes were chosen based on standard protocols in the field and our previous similar studies. For all animal experiments, n represents the number of individual animals. For cell culture experiments, n represents the number of independent biological replicates. For groups with n > 3, the normality of data distribution was evaluated using the Shapiro-Wilk test. For groups with n = 3, a normal distribution was assumed based on prior literature and historical data. Statistical differences between two groups were analyzed using two-tailed Student's t-tests, whereas differences among multiple groups were analyzed using one-way or two-way ANOVA followed by Tukey’s post hoc test. Exact P values are annotated directly within the figures, and the specific statistical methods applied to each experiment are detailed in the corresponding figure legends.

Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information. The source data underlying all main and supplementary figures are provided as a publicly available Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "An elegant manuscript detailing exhaustive experiments showing that protein Rev-erb-alpha (a canonical molecular core clock associated protein) mitigates development of severe PAH (using the Su/Hx model) through inhibition of BNIP3 and down-stream mitophagy-mediated vascular smooth muscle cell (VSMC) proliferation. The group's work is to be commended, as all transgenic animal models are thoroughly characterized with epistasis demonstrated across all derived-conclusions, and the assessment of end outcomes (invasive RVSP, non-invasive echocardiogram and angiograms) are all thoroughly complete. The use of hypoxia-mimetic CoCl2 in phenotypic display of models is also admirable as a highlight to the multiple in vivo models described. Likewise, the focus on therapeutic interventions (both prevention and treatment regimens) with the SR9009 drug is laudable, and increases the impact of the findings in the opinion of this reviewer. As such, I have only a few modest requests for the author's to help me contextualize their findings, and provide translational applications to patients with disease.\nMajor Comments:\n1. The studies are all well done, but there is no human data provided. I would suggest the following: query one of the many bulk or cell-specific databases for RNASeq of PAH patients, and look for supporting evidence of Rev-erb-alpha or BNIP3 differential regulation. This would greatly add to the translational impact of the findings, as alluded to above.\n2. While the authors are careful to state that they are studying the non-circadian application of Rev-erb-alpha, I do not believe this is adequate. I would suggest that they at the very least characterize BMAL1/CLOCK expression in the Su/Hx baseline model (Figure 1 essentially), to demonstrate variation in the \"brake\" and \"gas\" side of the circadian core clock related to their findings, and as a future area of research. The author's should also specify roughly when harvests and measurements were assess with respect to the murine circadian cycle (awake, asleep, dawn or dusk?); a time-stamp. This is very important given the gross overlap of binding between BMAL1 and nearly all responsible hypoxic-signaling transcriptional components.", "reviewer_3": "This is an interesting study that adds to the growing evidence of a role for Rev-erb(alpha) in pulmonary arterial hypertension and right ventricular hypertrophy. The authors have used SuHx mice as a model of pulmonary arterial hypertension and responses of pulmonary arterial smooth muscle cells in culture to hypoxia as model systems. They show a loss of pulmonary arterial Rev-erb(alpha) in the SuHx mice (relative to normoxic mice) and a down regulation of Rev-erb(alpha) expression in cultured PASMC subject to hypoxia. In cultured PASMC, hypoxia was shown to increase cell proliferation, an effect that could be abrogated by the Rev-erb agonist, SR9009. Small interference RNAs were used to show that the effect of SR9009 was reduced by knockdown of Rev-erb(alpha) but not Rev-erb(beta). Conversely, the effect of SR9009 could be mimicked by overexpression of Rev-erb(alpha). Right ventricular systolic pressure and pulmonary arterial medial wall thickness were increased in SuHx mice, effects that were reduced by treatment with SR9009. Right ventricular systolic pressure and pulmonary artery medial wall thickness were also increased in mice heterozygous for the Rev-erb(alpha) allele. The effect of the Rev-erb(alpha) deficiency could not be reversed by SR9009 treatment. Conversely, overexpressing Rev-erb(alpha) in SuHx mice reduced the pulmonary hypertension and right ventricular hypertrophy. The vascular smooth muscle specific knockout of Rev-erb(alpha) produced a pulmonary hypertension and right ventricular hypertrophy. The effects hypoxia in the SuHx mice and PASMC were associated with increased markers of mitophagy, effects which were inhibited by SR9009. Further, the authors show that the effects of Rev-erb(alpha) on mitophagy are achieved through transcriptional repression of BNIP3, key to the receptor-mediated pathway of mitophagy and that repression of BNIP3 transcription ameliorates the hypoxic PASMC proliferation while VSMC-specific BNIP3 overexpression worsens pulmonary arterial hypertension. Thus, the study presents compelling evidence for the involvement of Rev-erb(alpha) and mitophagy in pulmonary arterial hypertension and suggests that Rev-erb(alpha) may be a suitable therapeutic target. The data are clearly presented and the interpretations are largely justified by the data presented.\nI have the following specific comments\n1) Figure 1, Panels D – F. In the PASMCs, treatment with CoCl2 is shown to reduce Rev-erb(alpha) expression. The target and mechanism of CoCl2 action have not been explained. Text should be added to the Results explaining these data, and to the Discussion on the interpretation of these data. The existing text is wholly inadequate in this regard. Correction is required in panel F to the use of upper case C for Cl and to the use of subscript for Cl2.\n2) Figure 3, panel B. This panel is highly misleading as the Vehicle and SR9009 plots appear to be continuous (presumably there were from different animals?. Similarly, the ‘normoxia’ section appears continuous with the ‘SuHx’ section. Again, presumably there were from different animals. The black horizontal bars beneath the words ‘normoxia’ and ‘SuHx’ could be interpreted as indicating that the duration of the bar represents the period of ‘normoxia’ or ‘SuHx’. The bars should be removed. This figure must be amended so as not to mislead the reader. Also, the label for the y-axis should be ‘RVP’, not ‘RVSP’. The four traces shown are each continuous measures of right ventricular pressure – only the peak values represent right ventricular systolic pressure. The abbreviations need to be clearly defined in the text and the appropriate abbreviations used in the text, on the figures and in the figure legends.\n3) Figure 4. Evidence should be presented that there was a deficiency in Rev-erb(alpha) protein expression in Rev-erb(alpha)+/- mice relative to Rev-erb(alpha)+/+ mice.\n4) Figure 4, panel B. In the trace show for Rev-erb(alpha)+/-/SR9009, the heart rate appears lower than for the corresponding trace in the absence of SR9009. What were the heart rates in Rev-erb(alpha)+/+, Rev-erb(alpha)+/- and Rev-erb(alpha)+/- treated with SR9009? Was heart rate different between Rev-erb(alpha)+/+ and Rev-erb(alpha)+/- mice? Was heart rate changed by SR9009 treatment?\n5) Results sub-section 6, line 249: “… meant to know …’’ does not make sense. Perhaps you meant “… sought to establish …”?\n6) Figure 6, panel H: An explanation of the inset cartoon is required in the Figure Legend. Also, the wavelengths on the y-axis do not correspond to the wavelengths in the cartoon? Why is this? If appropriate, amend (or remove) the cartoon.\n7) Figure 7, panel J. The resolution of this figure panel is very poor. It was effectively illegible on my laptop (even using a larger screen).\n8) Methods, line 536. As explained above, RVSP is an appropriate abbreviation for right ventricular systolic pressure, but the data shown in the traces should be described as right ventricular pressure and abbreviated to RVP. It is necessary to use both terms, and to use them accurately.\n9) Methods, line 543. Define the abbreviation, IVS (inter-ventricular septum?).", "reviewer_4": "Qui et al present a potentially interesting data set describing an Erb-alpha-dependent pathway in the pathogenesis of pulmonary hypertension (PAH). Erb-alpha, but not beta, is down regulated in PAH and hypoxia exposure in culture PASMCs, while inhibition of Erb-alpha with SR9009 prevents and treats manifestations of PAH, but not when Erb-alpha is knocked down. Heterozygous deletion Erb-alpha exaggerates the disease phenotype, similar with VSMC-specific KO, while Erb-alpha overexpression is protective. Finally, the authors identify increased autophagy and mitophagy in the disease phenotype, and identify Erb-alpha regulation of BNIP3 as a necessary and sufficient target to modify the PAH manifestations. Although the data set is compelling, the authors conclusions are not supported by the data. Specifically, the causative role of mitophagy in the pathogenesis of PAH and that Erb-alpha is acting specifically through BNIP3-induced mitophagy to attenuate the disease progression is not supported. BNIP3 is pleiotropic with numerous functions beyond mitophagy and the authors have not established that other BNIP3 functions are not responsible for the PAH phenotype. Although it is an attractive hypothesis that enhanced mitophagy enables cell proliferation, it is also likely that BNIP3 depolarizes/inhibits mitochondrial function, enhancing glycolysis which promotes proliferation. Additional experimentation is needed to support the authors’ conclusion, or the conclusion need to be reworked to match the available data. Finally, BNIP3 splicing needs to be considered. Previous work has shown that alternative splicing of BNIP3 yields smaller variants that both inhibit cell death and proliferation, which should be addressed in the context of the current work. Specific comments are below:\n1) Proof that repressing BNIP3 and mitophagy improves mitochondrial function in vivo in PASMCs is lacking. In most in vivo situations, inhibiting mitophagy worsens mitochondrial function. The authors should address their conclusions or provide more substantial data that inhibiting mitophagy improves mitochondrial function.\n2) BNIP3 small splice variants have been shown inhibit proliferation, along with cell death. In some model systems, it is the ratio of full length to alternatively spliced variants that determine the overall cell phenotype. Do the authors have evidence that its full length BNIP3 vs alternatively spliced BNIP3 vs the ratio is being repressed by erb-alpha? N-terminal antibodies to BNIP3 can detect all spliced variants, while PCR primers can also do this.\n3) As BNIP3 was originally identified as a death gene capable of depolarizing mitochondria and opening the permeability transition pore, I was surprised this wasn’t assessed as a potential mechanism. The authors need additional data to support their conclusions that BNIP3 induced mitophagy is the sole mechanism here, and not other BNIP3 induced mitochondrial perturbations.\n4) In Figure 1 D-I, it should be made clear that these are PASMC in culture? This is a general comment as well for all figures, as the author don’t clearly specify culture based experiments vs in vivo data.\n5) In figure 2 C and D, is the statistical interaction between si-erb-alpha and SR9009 significant (ie. Has a two-way AVOVA been performed?)\n6) Figure 5 relies on controls from previous figures. I find this approach less convincing than if all normoxia and drug controls are included in all experiments. This provides confidence regard the robustness of the data.\n7) The TEM in figure 6A is too small and inconclusive. Improved data quality is needed to support the authors conclusion of mitochondrial perturbations, autophagy/mitophagy.\n8) In figure 6, western blotting isolated mitochondria for P62 and BNIP3 would strengthen the mitophagy argument.\n9) Describe how mito-Sox was performed in vivo\n10) The conclusion that autophagy is ‘excessive’ in SuHx mice is not supported by the data. In addition, the reduction in autophagy by SR9009 does not necessarily mean an improvement in mitochondrial function/homeostasis. The authors should be more careful with the wording of their concluding statements."}, "author_response": "Response to Reviewers for Manuscript # NCOMMS-25-34581\nTitle: Targeting Rev-erbα/BNIP3 axis attenuates pulmonary arterial hypertension by repressing\nmitophagy\nPoint-by-Point Responses\nWe thank the reviewers for their time and constructive comments, which have helped us to\nsignificantly improve our manuscript. Below are our detailed responses to each point.\nReviewer #2\nReviewer #2, Major Comment 1: The studies are all well done, but there is no human data provided.\nI would suggest the following: query one of the many bulk or cell-specific databases for RNASeq of\nPAH patients, and look for supporting evidence of Rev-erb-alpha or BNIP3 differential regulation.\nThis would greatly add to the translational impact of the findings, as alluded to above.\nResponse: We sincerely thank the reviewer for this excellent and constructive suggestion to enhance\nthe translational relevance of our findings. Following the reviewer's advice, we performed a\nbioinformatic analysis using two independent, publicly available human PASMC datasets from the\nGene Expression Omnibus (GEO), GSE113061 (5 PAH vs. 3 controls) and GSE221511 (6 PAH vs.\n3 controls) and specifically examined the expression of Rev-erbα and BNIP3 in PASMCs from\npatients with PAH.\nAs shown in the following heatmap (R-Figure 1), our analysis revealed a consistent and robust\nincrease in BNIP3 mRNA expression in PASMCs from PAH patients compared to non-PAH controls\nacross both datasets, which greatly supports the clinical relevance of our findings and further\nimplicates BNIP3 in the pathophysiology of human PAH. However, the analysis of the expression\nof Rev-erbα yielded variable results: it was downregulated in the GSE113061 dataset but showed\nno consistent alteration in GSE221511. Because the public datasets do not provide the time of day\nat which samples were collected, we assume this discrepancy may stem from the high-amplitude\ncircadian oscillation of Rev-erbα which may introduce substantial uncontrolled variability and\nobscure true disease-related differences.\nGiven the relatively small sample size of these datasets and the potential variability associated with\ncircadian sampling, we hope to not include these exploratory results in the revised manuscript.\nInstead, we have expanded and strengthened the discussion section to fully acknowledge this\nlimitation and further emphasize the need for future clinical investigations (page 28, para 1):\n“Nonetheless, an important limitation of our work is the lack of direct validation in human tissues\nor clinical cohorts. Although our experimental models provide strong mechanistic support, the\nclinical relevance of circadian regulators—including Rev-erbα—remains to be fully defined, as\nhuman studies often face challenges such as limited sample availability, disease heterogeneity, and\nthe influence of circadian timing on gene expression. Future investigations integrating well-\ncharacterized patient samples, ideally with controlled or documented timing of collection, will be\nessential for determining whether dysregulation of the Rev-erbα–BNIP3 axis occurs in human PAH.\nMoreover, studies using patient-derived PASMCs and prospective clinical cohorts will help clarify\nthe translational potential of targeting this pathway. Such efforts will be critical to bridge the gap\nbetween the current basic mechanistic findings and their application in developing effective\ncircadian-based therapeutic strategies for PAH”. We again thank the reviewer for this thoughtful\nsuggestion, which has significantly strengthened the rigor and translational context of our work.\nR-Figure 1. Expression of Rev-erbα and BNIP3 in PASMCs of patients with PAH. Heatmap displaying the\nrelative mRNA expression levels of Rev-erbα and BNIP3 in PASMCs from normal controls and patients with\npulmonary arterial hypertension (PAH). Data were obtained from the GEO dataset GSE113061 and GSE221511.\nExpression values are represented as Z-scores.\nReviewer #2, Major Comment 2: While the authors are careful to state that they are studying the\nnon-circadian application of Rev-erb-alpha, I do not believe this is adequate. I would suggest that\nthey at the very least characterize BMAL1/CLOCK expression in the Su/Hx baseline model (Figure\n1 essentially), to demonstrate variation in the \"brake\" and \"gas\" side of the circadian core clock\nrelated to their findings, and as a future area of research. The author's should also specify roughly\nwhen harvests and measurements were assess with respect to the murine circadian cycle (awake,\nasleep, dawn or dusk?); a time-stamp. This is very important given the gross overlap of binding\nbetween BMAL1 and nearly all responsible hypoxic-signaling transcriptional components.\nResponse: We thank the reviewer for this insightful comment and agree that incorporating context\non the core circadian machinery is critically important. As suggested, we have now performed\nadditional experiments to characterize the expression of other core clock genes in both the SuHx\nmouse lung tissue and hypoxia-exposed PASMCs. While we did observe some modest fluctuations\nin the expression of few clock genes (Cry1 for example), the most consistent, profound, and\nstatistically significant change was the downregulation of Rev-erbα (R-figure 2). This new data\nreinforces our focus on Rev-erbα as the key clock-related mediator in this specific pathological\ncontext. We have now amended this data as Supplemental Figure S2 in the revised manuscript and\nincluded more discussion (page 27, para 1) as the reviewer suggested to suggest future research\ndirections: “While our data point to a primary role for the Rev-erbα in PAH pathogenesis, given\nthat hypoxia may cause multiple core circadian rhythm disturbances [PMID: 33599610, 27773695,\n31848250, 27773697], future studies exploring whether chronic hypoxia in PAH disrupts the entire\ncircadian network to exacerbate the disease might be required”.\nWe also recognize the critical importance of aligning our experiments with the murine circadian\ncycle and apologize for the omission in the original manuscript. We have now added a detailed\ndescription to the Methods section (page 29, para 1), specifying that all animal sacrifices and the\nkey physiological measurements (e.g., right ventricular systolic pressure) were performed between\nZeitgeber Time 6 (ZT6) and ZT8, corresponding to the peak expression period for Rev-erbα1,2. This\nstatement ensures consistency across all experimental groups and enhances the reproducibility of\nour work.\nR-Figure 2. Expression and rhythmic changes of core circadian genes in SuHx mice and hypoxic PASMCs (A)\nSchematic illustration of the experimental protocol for establishing the SuHx-induced PAH mouse model. Western\nblots (B) and corresponding densitometric quantification (C) of core clock proteins (BMAL1, PER1, CRY1, Rev-\nerbα, Rev-erbβ) in lung tissue from normoxia and SuHx groups. GAPDH was used as a loading control. (n=6-10).\n(D) Schematic representation of the in vitro experimental design to assess the effect of hypoxia on circadian rhythms\nin PASMCs. Cultured PASMCs were synchronized by a 2-hour treatment with 100 nM dexamethasone (defined as\nCircadian Time 0, CT0). (E-I) Hypoxia alters the rhythmic mRNA expression of core clock genes in PASMCs.\nSamples were collected every 6 hours over a 48-hour period, and mRNA levels of Rev-erbα (E), Rev-erbβ (F),\nBmal1 (G), Cry1 (H), and Per1 (I) were determined by qPCR. (n=3). Data are presented as mean±SEM. *p<0.05，\n**p<0.01，***p<0.001.\nReviewer #3\nReviewer #3, Comment 1: Figure 1, Panels D – F. In the PASMCs, treatment with CoCl2 is shown\nto reduce Rev-erb(alpha) expression. The target and mechanism of CoCl2 action have not been\nexplained. Text should be added to the Results explaining these data, and to the Discussion on the\ninterpretation of these data. The existing text is wholly inadequate in this regard. Correction is\nrequired in panel F to the use of upper case C for Cl and to the use of subscript for Cl2.\nResponse: We appreciate the reviewer for pointing out these omissions and errors. We have\ncorrected the chemical formula for cobalt chloride to CoCl₂ in Figure 1F as requested. Furthermore,\nas suggested, we have added text to the Results section (page 3, para 3) to clarify that cobalt chloride\n(CoCl₂) is a well-established hypoxia-mimetic agent and expanded more discussion on the\ninterpretation of the CoCl₂ data (page 22, para 1).\nReviewer #3, Comment 2: Figure 3, panel B. This panel is highly misleading as the Vehicle and\nSR9009 plots appear to be continuous (presumably there were from different animals?). Similarly,\nthe ‘normoxia’ section appears continuous with the ‘SuHx’ section. Again, presumably there were\nfrom different animals. The black horizontal bars beneath the words ‘normoxia’ and ‘SuHx’ could\nbe interpreted as indicating that the duration of the bar represents the period of ‘normoxia’ or\n‘SuHx’. The bars should be removed. This figure must be amended so as not to mislead the reader.\nAlso, the label for the y-axis should be ‘RVP’, not ‘RVSP’. The four traces shown are each\ncontinuous measures of right ventricular pressure – only the peak values represent right ventricular\nsystolic pressure. The abbreviations need to be clearly defined in the text and the appropriate\nabbreviations used in the text, on the figures and in the figure legends.\nResponse: We sincerely apologize for the misleading presentation in the original Figure 3B and the\nincorrect terminology. The reviewer is entirely correct. We have now completely revised this panel\nin the revised manuscript (as shown below in R-figure 3) to clearly separate the representative traces\nfrom different animal groups, removing any suggestion of continuity. The horizontal bars have been\nremoved. We have also corrected the y-axis label on all pressure waveform traces to 'Right\nVentricular Pressure (RVP)' and have ensured that only the peak systolic values, as quantified in the\ncorresponding bar graphs, are referred to as 'RVSP'. Furthermore, we have meticulously checked all\nfigures, legends, and the main text for consistent and accurate use of these abbreviations.\nR-Figure 3. SR9009 attenuates right ventricular pressure elevation in the Sugen/hypoxia (SuHx) model of\nPAH. Representative traces of right ventricular pressure (RVP) recordings. The x-axis scale bar indicates 400 ms.\nReviewer #3, Comment 3: Figure 4. Evidence should be presented that there was a deficiency in\nRev-erb(alpha) protein expression in Rev-erb(alpha)+/- mice relative to Rev-erb(alpha)+/+ mice.\nResponse: This is an excellent point, and we apologize for not including this validation data\ninitially. We have now performed Western blot on whole lung tissue lysates from Rev-erbα+/+ and\nRev-erbα+/- mice. As shown, Rev-erbα+/- mice exhibit a significantly reduction in Rev-erbα protein\nexpression compared to their wild-type littermates (R-figure 4). This new data has been added as\nSupplemental Figure S6 in the revised manuscript to molecularly validate our heterozygous mouse\nmodel.\nR-Figure 4. Rev-erbα protein expression is significantly diminished in the lungs of the Rev-erbα heterozygous\nmice. Western blot analysis of Rev-erbα in lung tissue from wild-type (Rev-erbα+/+) and heterozygous (Rev-erbα+/-)\nmice. GAPDH was used as a loading control.\nReviewer #3, Comment 4: Figure 4, panel B. In the trace show for Rev-erb(alpha)+/-/SR9009, the\nheart rate appears lower than for the corresponding trace in the absence of SR9009. What were the\nheart rates in Rev-erb(alpha)+/+, Rev-erb(alpha)+/- and Rev-erb(alpha)+/- treated with SR9009?\nWas heart rate different between Rev-erb(alpha)+/+ and Rev-erb(alpha)+/- mice? Was heart rate\nchanged by SR9009 treatment?\nResponse: We thank the reviewer for his/her careful observation and apologize for the lack of clarity\nin the original trace. We have now systematically analyzed the heart rates of all animals within these\nexperimental groups. As shown in R-figure 5, there was no significant difference in heart rate\nbetween Rev-erbα(+/-) and Rev-erbα(+/+) mice at baseline, and SR9009 administration did not\naffect heart rate in either group. To make it clear, we have replaced the original trace with more\nrepresentative images (R-figure 6) and added timelines to all traces in the revised manuscript.\nR-Figure 5. Genetic deletion or pharmacological activation of Rev-erbα does not alter the heart rate in mice.\n(A) Heart rate in SuHx-induced wild-type (Rev-erba+/+), heterozygous (Rev-erba+/-), and SR9009-treated Rev-erba+/-\nmice (n = 4). (B) Heart rate in mice under normoxia or SuHx-induced pulmonary hypertension, treated with either\nvehicle or SR9009 (n = 4). Data are presented as mean ± SEM.\nR-Figure 6. Deficiency of Rev-erbα exacerbates SuHx-induced PAH, and the protective effect of SR9009 is\ndependent on Rev-erbα. Representative traces of right ventricular pressure (RVP) recordings. The x-axis scale bar\nindicates 400 ms.\nReviewer #3, Comment 5: Results sub-section 6, line 249: “… meant to know …’’ does not make\nsense. Perhaps you meant “… sought to establish …”?\nResponse: Thanks for this professional suggestion. We have now revised the sentence to \"...we\nsought to establish...\" as suggested. We have also carefully proofread the manuscript for any other\nsuch errors.\nReviewer #3, Comment 6: Figure 6, panel H: An explanation of the inset cartoon is required in\nthe Figure Legend. Also, the wavelengths on the y-axis do not correspond to the wavelengths in the\ncartoon? Why is this? If appropriate, amend (or remove) the cartoon.\nResponse: We apologize for the confusion caused by this cartoon. The mt-Keima reporter is a pH-\nsensitive fluorescent protein that is excited at different wavelengths in the neutral environment of\nthe mitochondrial matrix versus the acidic environment of the lysosome3. The discrepancy arose\nbecause the available laser lines on our confocal microscope are slightly different from the protein's\nabsolute peak excitation wavelengths, which were depicted in the cartoon. Nevertheless, as the\nreviewer suggested, to avoid any further confusion for the readers, we have removed the cartoon\nentirely. We have also revised the Methods section (page 36, para 4) and the figure legend to clarify\nthat the key metric for quantifying mitophagy is the ratio of fluorescence emission when excited at\nthese two wavelengths, which reflects the delivery of mitochondria to lysosomes.\nReviewer #3, Comment 7: Figure 7, panel J. The resolution of this figure panel is very poor. It was\neffectively illegible on my laptop (even using a larger screen).\nResponse: We apologize for this oversight. We have re-uploaded a revised graphical abstract in the\nrevised manuscript. It has also been uploaded as a separate file to the submission system for the\nreviewers and editors to review.\nReviewer #3, Comment 8: Methods, line 536. As explained above, RVSP is an appropriate\nabbreviation for right ventricular systolic pressure, but the data shown in the traces should be\ndescribed as right ventricular pressure and abbreviated to RVP. It is necessary to use both terms,\nand to use them accurately.\nResponse: Thanks again for this crucial correction. As noted in the above response to comment #2,\nwe have now systematically reviewed the entire manuscript and corrected all instances, ensuring\nthat 'RVP' is used for pressure traces and 'RVSP' is used only when referring to the quantified peak\nsystolic value.\nReviewer #3, Comment 9: Methods, line 543. Define the abbreviation, IVS (inter-ventricular\nseptum?).\nResponse: Thanks. We have now defined IVS as 'interventricular septum' upon its first use in the\nrevised manuscript.\nReviewer #4\nReviewer #4, Comment 1: Proof that repressing BNIP3 and mitophagy improves mitochondrial\nfunction in vivo in PASMCs is lacking. In most in vivo situations, inhibiting mitophagy worsens\nmitochondrial function. The authors should address their conclusions or provide more substantial\ndata that inhibiting mitophagy improves mitochondrial function.\nResponse: We would like to deeply thank the reviewer for his/her wonderful comments and careful\nconcerns throughout the review process. We fully agree that the role of mitophagy in maintaining\nmitochondrial function is context-dependent and that our initial conclusions would benefit from\nmore direct experimental support. Following this suggestion, we have now performed additional\nexperiments to evaluate the effect of repressing mitophagy and BNIP3 on mitochondrial function\nand cell proliferation, specifically under hypoxic conditions in PASMCs.\nAs shown in the new data, treatment of hypoxia-exposed PASMCs with or without Mdivi-1, a\nwidely used inhibitor of mitophagy4-6, revealed that Mdivi-1 significantly reduced mitochondrial\nROS production, restored mitochondrial membrane potential, and prevented cell proliferation (R-\nfigure 7). This finding suggests that inhibiting mitophagy may preserve mitochondrial function\nunder hypoxia stress in PASMCs. Notably, although our conclusion that mitophagy can become\nmaladaptive under certain cellular challenges is consistent with several recent reports7-9, we\nacknowledge, as the reviewer pointed out, that inhibition of mitophagy is generally detrimental in\nmany contexts. We have now expanded the Discussion section to address this important issue (page\n26, para 1): “Intriguingly, while mitophagy inhibition usually worsens mitochondrial function, our\ncurrent data and findings from other studies suggest that, under certain specific stress conditions,\nsuppressing mitophagy may help preserve mitochondrial integrity [PMID: 40264356, 38916953,\n32351292]. This observation underscores the complex and context-dependent role of autophagy in\nmaintaining cellular homeostasis. However, we must admit that the mitophagy inhibitor used in this\nstudy is not highly specific, and that direct in vivo evidence remains lacking, further investigations\nwill be required to strengthen and refine these findings in the future”.\nRegarding BNIP3, we found that consistent with evidence from hepatocellular carcinoma cells and\nosteoclasts10,11, siRNA-mediated BNIP3 knockdown phenocopied the effects of Mdivi-1, preserving\nmitochondrial membrane potential, reducing mitochondrial ROS (R-figure 8). Conversely,\noverexpression of BNIP3 decreased mitochondrial membrane potential, increased mitochondrial\nROS levels (R-figure 9). Importantly, BNIP3 overexpression markedly exacerbated PASMC\nproliferation under hypoxia, while these adverse effects were blunted by Mdivi-1 (R-figure 10).\nTogether, these new findings indicate that the pro-proliferative effects of BNIP3 are, at least in part,\nmediated through mitophagy. However, we fully agree that direct in vivo validation of this\nmechanism remains essential. Unfortunately, due to time limitations, we are now having difficulty\nin getting enough VSMC-specific BNIP3 knockout or overexpression mice, which renders the in\nvivo study unfeasible. We hope to pursue this issue in the future.\nThe above new datasets have been added to the revised manuscript as supplementary Figures S15-\nS17 and S19 and the relevant sections have been rewritten to reflect this more nuanced and accurate\ninterpretation, framing our findings as correlational and highlighting the need for future genetic\nmodels to establish causality.\nR-figure 7. Effects of mitophagy inhibitor mdivi-1 on mitochondrial homeostasis in hypoxic PASMCs. (A-E)\nHypoxic PASMCs were treated with to DMSO or mdivi-1. (A) ROS levels in PASMCs measured by fluorescence\nmicroplate reader (excitation: 488 nm, emission: 525 nm) (n=3). (B) JC-1 staining, indicating mitochondrial\nmembrane potential, and the quantification in PASMCs (n=3). (C) MitoSOX Red staining (mitochondrial\nsuperoxide, red) and the quantification in PASMCs. (n=3) (D) Western blot analysis of BNIP3, LC3B, P62 and\nTOMM20 protein expression in PASMCs; GAPDH served as an internal reference (n=3). (E) PASMCs are infected\nwith Adv-COX8-mt-Keima and imaged by confocal microscopy to monitor mitophagy (greenex:445 nm, redex:561\nnm) (n=5). All data are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.\nR-Figure 8. Effects of BNIP3 knockdown on mitochondrial homeostasis in PASMCs. (A-E) PASMCs were\ntransfected with BNIP3-targeting siRNA (BNIP3 siRNA) or negative control siRNA (NC siRNA), followed by\nexposure to normoxic or hypoxic conditions. (A) ROS levels in PASMCs measured by fluorescence microplate\nreader (excitation: 488 nm, emission: 525 nm) (n=3). (B) JC-1 staining, indicating mitochondrial membrane\npotential, and the quantification in PASMCs (n=3). (C) MitoSOX Red staining (mitochondrial superoxide, red) and\nthe quantification in PASMCs. (n=3) (D) Western blot analysis of BNIP3, LC3B, P62 and TOMM20 protein\nexpression in PASMCs; GAPDH served as an internal reference (n=3). (E) PASMCs are infected with Adv-COX8-\nmt-Keima and imaged by confocal microscopy to monitor mitophagy (greenex:445 nm, redex:561 nm) (n=3). All data\nare presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.\nR-Figure 9. Effects of BNIP3 overexpression on mitochondrial homeostasis in hypoxic PASMCs. (A-E)\nHypoxic PASMCs were transfected with BNIP3 overexpression plasmic (OE-BNIP3) or negative control plasmic\n(NC). (A) ROS levels in PASMCs measured by fluorescence microplate reader (excitation: 488 nm, emission: 525\nnm) (n=3). (B) JC-1 staining, indicating mitochondrial membrane potential, and the quantification in PASMCs (n=3).\n(C) MitoSOX Red staining (mitochondrial superoxide, red) and the quantification in PASMCs. (n=3) (D) Western\nblot analysis of BNIP3, LC3B, P62 and TOMM20 protein expression in PASMCs; GAPDH served as an internal\nreference (n=3). (E) PASMCs are infected with Adv-COX8-mt-Keima and imaged by confocal microscopy to\nmonitor mitophagy (greenex:445 nm, redex:561 nm) (n=5). All data are presented as means ± SEM. *p < 0.05, **p <\n0.01, ***p < 0.001.\nR-Figure 10. BNIP3-mediated proliferation was blunted by Mdivi-1. (A-B) Hypoxic PASMCs were transfected\nwith BNIP3 overexpression plasmic (OE-BNIP3) or negative control plasmic (NC), followed by treated with to\nDMSO or mdivi-1. (A) Representative images and quantification of EdU incorporation of proliferative PASMCs\n(n=3). (B) CCK-8 assay to determine the PASMCs viability (n=12). All data are presented as means ± SEM. ***p <\n0.001.\nReviewer #4, Comment 2: BNIP3 small splice variants have been shown inhibit proliferation,\nalong with cell death. In some model systems, it is the ratio of full length to alternatively spliced\nvariants that determine the overall cell phenotype. Do the authors have evidence that its full length\nBNIP3 vs alternatively spliced BNIP3 vs the ratio is being repressed by erb-alpha? N-terminal\nantibodies to BNIP3 can detect all spliced variants, while PCR primers can also do this.\nResponse: We sincerely thank the reviewer for this professional and insightful comment. As noted,\na shorter splice variant of BNIP3 lacking exon 3 (BNIP3Δexon3) has been reported in\ncardiomyocytes as a pro-survival isoform12,13. The absence of exon 3 results in loss of the C-terminal\ntransmembrane domain which is required for proper mitochondrial targeting by full-length BNIP3\n(BNIP3-FL). Consequently, BNIP3Δexon3 is thought to antagonize the cell-death function of\nBNIP3-FL, possibly by forming inactive heterodimers14,15. However, the presence, regulation, and\nfunctional relevance of BNIP3 alternative splicing in PASMCs and pulmonary hypertension have\nnever been investigated and thus warrant attention.\nTo address this important issue, as suggested by the reviewer, we designed specific primers for RT-\nPCR to detect both the BNIP3-FL and BNIP3Δexon3 in PASMCs, with or without hypoxia and\nSR9009 treatments. As shown in R-Figure 11, hypoxia significantly increased the mRNA levels of\nboth BNIP3-FL and BNIP3Δexon3. However, the magnitude of induction was greater for\nBNIP3Δexon3, as reflected by a marked increase in the BNIP3Δexon3/BNIP3-FL ratio under\nhypoxic conditions. These findings suggest that hypoxia preferentially promotes the expression of\nthe anti-apoptotic BNIP3 splice variant, thereby shifting the overall BNIP3 functional output toward\na proliferative phenotype. Moreover, pharmacological activation of Rev-erbα with SR9009\nsuppressed the hypoxia-induced upregulation of both BNIP3-FL and BNIP3Δexon3 and\nsignificantly reduced the BNIP3Δexon3/BNIP3-FL ratio. We have now incorporated these new data\ninto the revised manuscript as Supplementary Figure S18, amended the corresponding Methods and\nResults sections (page 18, para 3), and expanded the Discussion section (page 27, para 2) to include\na more detailed interpretation of these findings and their potential implications: “Another interesting\nfinding of this study is the demonstration of a hypoxia-induced alternative splicing program for\nBNIP3 in PASMCs, resulting in the concurrent expression of both the full-length transcript (BNIP3-\nFL) and a splice variant lacking exon 3 (BNIP3Δexon3). This observation is particularly noteworthy\nwhen viewed in the context of prior research in cardiomyocytes, where BNIP3Δexon3 functions as\na pro-survival isoform that antagonizes its pro-death counterpart, establishing the\nBNIP3Δexon3/BNIP3-FL ratio as a critical rheostat for cell fate decisions [PMID: 21415393,\n40675219, 30275982, 26416963]. Building upon this, we demonstrated that activation of the\nnuclear receptor Rev-erbα not only suppressed the expression of both BNIP3 isoforms but, more\ncritically, induced a significant reduction in the BNIP3Δexon3/BNIP3-FL ratio, suggesting that\nRev-erbα may shift the cellular balance away from a pro-survival state in hypoxic PASMCs.\nAlthough the precise functional consequences of these two variants and the modulation of their ratio\nwithin PASMCs have yet to be elucidated, these results identify a potentially crucial mechanism in\npulmonary vascular pathobiology that warrants in-depth investigation.”\nR-Figure 11. Effect of SR9009 treatment on the expression of BNIP3 transcripts and its splice variants in\nPASMCs. (A) Schematic diagram illustrating the isoform-specific primer design strategy. Primers were designed to\nspecifically amplify total BNIP3 transcripts (targeting exon 6), the full-length isoform (BNIP3-FL, with primers in\nexon 3 and exon 4), and the exon 3-skipped splice variant (Bnip3Δexon3, with a forward primer spanning the unique\nexon 2-4 junction). (B) Representative agarose gel electrophoresis of RT-PCR products from PASMCs cultured\nunder normoxia or hypoxia and treated with or without SR9009. (C) Sanger sequencing validation of the PCR\namplicons. (D) Relative mRNA expression of the BNIP3Δexon3 isoform (n=3). (E) Relative mRNA expression of\nthe BNIP3-FL isoform(n=3). (F) The calculated ratio of BNIP3Δexon3 to BNIP3-FL mRNA(n=3). (G) Relative\nexpression of total BNIP3 mRNA(n=3). Data in bar graphs are presented as mean ± SEM. *p < 0.05, **p < 0.01,\n***p < 0.001.\nReviewer #4, Comment 3: As BNIP3 was originally identified as a death gene capable of\ndepolarizing mitochondria and opening the permeability transition pore, I was surprised this wasn’t\nassessed as a potential mechanism. The authors need additional data to support their conclusions\nthat BNIP3 induced mitophagy is the sole mechanism here, and not other BNIP3 induced\nmitochondrial perturbations.\nResponse: We sincerely thank the reviewer for this insightful and important comment. We fully\nagree that, given the well-established pro-apoptotic role of BNIP3, it is essential to examine whether\nthis canonical apoptotic function also contributes to PASMC proliferation and pulmonary\nhypertension phenotype induced by hypoxia and decreased Rev-erbα.\nTo address this, we performed an additional experiment to examine the expression of key apoptotic\nsignaling molecules in hypoxia-exposed PASMCs. As shown in R-Figure 12, hypoxia robustly\ninduced BNIP3 expression but did not alter the expression of the pro-apoptotic protein BAX, while\nsignificantly upregulating the anti-apoptotic protein BCL2. Similarly, forced overexpression of\nBNIP3 markedly increased BCL2 levels without affecting BAX expression. Importantly,\npharmacological activation of Rev-erbα with SR9009 suppressed BNIP3 expression but did not\naffect either BAX or BCL2. Together, these data suggest that the hypoxia-induced increase and\nSR9009-mediated decrease in BNIP3 expression occur independently of changes in BNIP3-\nassociated apoptotic signaling.\nAs is well known, the canonical mechanism for BNIP3-induced apoptosis involves its\nheterodimerization with anti-apoptotic proteins such as BCL2, thereby liberating the pro-apoptotic\nproteins like BAX to oligomerize and permeabilize the mitochondrial outer membrane16. However,\nsince the upregulation of BNIP3 in our study was not accompanied by an increase in BAX, and\nactivation of Rev-erbα also failed to affect apoptotic signaling molecules, we speculate that under\nhypoxic conditions, BNIP3 may not primarily exert a pro-apoptotic role. The concurrent elevation\nof BCL2 expression may sequester available BAX, thereby raising the threshold for apoptosis.\nTherefore, we propose that in this specific cellular context, a high anti-apoptotic tone likely\n\"channels\" the function of BNIP3 away from apoptosis and towards its alternative role in promoting\nmitophagy. However, we acknowledge that our data is still preliminary and does not directly exclude\nthe potential contribution of BNIP3-induced apoptotic or other mitochondrial perturbations to PAH\npathogenesis. To be more precise and cautious, we have now revised the manuscript to temper our\nconclusions and state the limitations in the Discussion section (page 26, para 1): “Although our\ncurrent finding mainly points to the major role of BNIP3-mediated mitophagy in PASMC\nproliferation and PAH, other BNIP3-induced mitochondrial perturbations may also participate in\nRev-erbα associated PASMC proliferation and PAH development. Future studies will be necessary\nto further delineate the relative contributions of BNIP3-induced mitophagy and apoptosis, as well\nas to elucidate how BNIP3 interacts with other mitochondrial quality control pathways under\nhypoxic conditions”. These new datasets have been added to the revised manuscript as\nSupplemental Figure S14 and the relevant interpretations have also been included in the Results\nsection (page 17, para 1 and page 18, para 2).\nR-Figure 12. Effects of Rev-erbα activation and BNIP3 overexpression on BAX and BCL2 protein levels under\nhypoxia. (A) Representative Western blots and corresponding quantification showing the effects of SR9009 on\nBNIP3, BAX and BCL2 protein levels in PASMCs treated with DMSO or SR9009 for under normoxia or hypoxia.\n(B) Representative Western blots and corresponding quantification showing the effects of BNIP3 overexpression on\nBNIP3, BAX, and BCL2 protein levels in PASMCs transfected with a negative control (NC) or BNIP3-\noverexpressing (OE-BNIP3) vector, followed by incubation under hypoxia. GAPDH was used as a loading control.\nData in bar graphs are presented as mean ± SEM. **p < 0.01, ***p < 0.001.\nReviewer #4, Comment 4: In Figure 1 D-I, it should be made clear that these are PASMC in\nculture? This is a general comment as well for all figures, as the author don’t clearly specify culture\nbased experiments vs in vivo data.\nResponse: Thanks for this careful criticism. We have now revised all figure legends and the\nMethods and Results section to explicitly state at the beginning of each description whether the\nexperiments were performed in vitro using cultured primary PASMCs or in vivo using mouse\nmodels.\nReviewer #4, Comment 5: In figure 2 C and D, is the statistical interaction between si-erb-alpha\nand SR9009 significant (ie. Has a two-way AVOVA been performed?)\nResponse: Thanks for raising this important statistical point. We confirmed that a two-way ANOVA\nwas indeed used to analyze the data in Figures 2C and 2D. While our initial intention was to\nstreamline the figure by highlighting only the most critical pairwise comparisons related to our\ncentral hypothesis: whether the effect of SR9009 is dependent on the presence of Rev-erbα.\nHowever, we agree that this approach lacked the necessary statistical transparency and rigor. To\naddress this, we have now revised Figure 2 to display the complete statistical results of the analysis\nand included the p-value for the statistical interaction between si-Rev-erbα and SR9009 treatment\ngroups. The corresponding figure legend has also been updated to reflect this more complete\nstatistical description.\nReviewer #4, Comment 6: Figure 5 relies on controls from previous figures. I find this approach\nless convincing than if all normoxia and drug controls are included in all experiments. This provides\nconfidence regard the robustness of the data.\nResponse: We appreciate this valuable suggestion. In response, we have now conducted additional\nexperiments to incorporate the necessary normoxia controls, as recommended. As the new data\npresented in R-figure 13&14 shown, under normoxic conditions, neither systemic AAV-mediated\noverexpression of Rev-erbα nor VSMC-specific knockout of Rev-erbα has a significant effect on\nRVSP, right ventricular hypertrophy, or pulmonary vascular remodeling. This confirms that the\nphenotypes we observed are specific to the hypoxia context and not confounded by the genetic\nmanipulations themselves. These new data have now been added to the revised manuscript as\nFigures S8 and S11 for clarity and transparency.\nR-Figure 13. Overexpression of Rev-erbα does not impact pulmonary hemodynamics or induce right\nventricular and vascular remodeling in normoxic mice. WT mice were treated with AAV-NC or AAV-OE-Rev-\nerbα for 30 days. (A) Representative RVP traces and quantification of RVSP (n=4). The x-axis scale bar indicates\n400 ms. (B) Fulton index assessing RV hypertrophy (n=4). (C) Echocardiography measurement of PA function and\nthe ratio of pulmonary artery blood flow acceleration time (PAT) to pulmonary artery ejection time (PET) (n=4). (D)\nRepresentative images of HE staining and the quantification of the vascular medial thickness to total vessel size\nratios of PAs (n=4). Data in bar graphs are presented as mean ± SEM.\nR-Figure 14. VSMC-specific Rev-erbα knockout does not impact pulmonary hemodynamics or induce right\nventricular and vascular remodeling in normoxic mice. WT mice were treated with VSMC-Rev-erbαflox/flox or\nVSMC-Rev-erbα-/- for 30 days. (A) Representative RVP traces and quantification of RVSP (n=4). The x-axis scale\nbar indicates 400 ms. (B) Fulton index assessing RV hypertrophy (n=4). (C) Echocardiography measurement of PA\nfunction and the ratio of pulmonary artery blood flow acceleration time (PAT) to pulmonary artery ejection time\n(PET) (n=4). (D) Representative images of HE staining and the quantification of the vascular medial thickness to\ntotal vessel size ratios of PAs (n=4). Data in bar graphs are presented as mean ± SEM.\nReviewer #4, Comment 7: The TEM in figure 6A is too small and inconclusive. Improved data\nquality is needed to support the authors conclusion of mitochondrial perturbations,\nautophagy/mitophagy.\nResponse: Sorry for the confusion. We have now replaced the original images with new, higher-\nmagnification and higher-quality micrographs which clearly show the changes in mitochondrial\nnumber and the state of autophagy (R-figure 15).\nR-figure 15. SR9009 improves mitochondrial homeostasis and inhibition of mitophagy in SuHx mice.\nTransmission electron microscopy (TEM) shows subcellular structures; arrows indicate autophagosomes.\nRepresentative TEM images of pulmonary from normoxia-vehicle, SuHx-vehicle, and SuHx-SR9009 treated mice.\nScale bars: 2 µm (main images), 500 nm (insets).\nReviewer #4, Comment 8: In figure 6, western blotting isolated mitochondria for P62 and BNIP3\nwould strengthen the mitophagy argument.\nResponse: Thanks for this excellent suggestion. In response, we have performed additional\nexperiments in which we separated the cytosolic and mitochondrial components of PASMCs and\nassessed the expression of LC3B, P62, and BNIP3 via Western blot. The new data (R-Figure 16)\nshowed under hypoxic conditions, the conversion of LC3B-I to LC3B-II, the decrease in P62, and\nthe accumulation of BNIP3 specifically occur within the mitochondrial fraction. Furthermore, these\nchanges were reversed following SR9009 treatment. As presented in Supplementary Figure S12C\nof the revised manuscript and described in the Results section (page 15, para 1), these findings\nprovide direct biochemical evidence for the recruitment of the autophagic machinery to the\nmitochondria, strongly supporting our conclusion that Rev-erbα inhibits mitophagy.\nR-Figure 16. SR9009 inhibits the hypoxia-induced recruitment of autophagic machinery to mitochondria in\nPASMCs. Western blot analysis of the expression of LC3B, P62, and BNIP3 in cytosolic (Cyto) and mitochondrial\n(Mito) fractions isolated from PASMCs. Cells were subjected to normoxia or hypoxia in the absence or presence of\nthe Rev-erbα agonist SR9009. The purity of the subcellular fractionation was confirmed by the localization of the\nmitochondrial marker COX IV and the cytosolic marker GAPDH.\nReviewer #4, Comment 9: Describe how mito-Sox was performed in vivo.\nResponse: We apologize for this omission. We have now clarified the in vivo MitoSOX Red staining\nprocedure in the Manuscript Methods section (page 33, para 2). Briefly, immediately following\neuthanasia, fresh lung tissues were excised, embedded in Optimal Cutting Temperature (OCT)\ncompound, and snap-frozen. Unfixed cryosections were then prepared and topically stained with a\nMitoSOX Red working solution in a light-protected, humidified chamber. Following incubation and\nwashing, the sections were immediately analyzed by fluorescence microscopy to assess\nmitochondrial superoxide production.\nReviewer #4, Comment 10: The conclusion that autophagy is ‘excessive’ in SuHx mice is not\nsupported by the data. In addition, the reduction in autophagy by SR9009 does not necessarily mean\nan improvement in mitochondrial function/homeostasis. The authors should be more careful with\nthe wording of their concluding statements.\nResponse: We thank the reviewer for his/her wonderful comments and apologize that our original\nconcluding statements were not framed with sufficient caution. Specifically, we acknowledge that\nthe term 'excessive' was not directly supported by a quantitative baseline, and have now revised the\nmanuscript throughout to use more precise and cautious wording. Additionally, as per the reviewer’s\nsuggestion, we have toned down our original conclusion and now state that the Rev-erbα-mediated\ninhibition of pathologically elevated, BNIP3-driven mitophagy is a key, but probably not the sole,\ncontributor to the maintenance of mitochondrial function and the attenuation of PAH in our model.\nReferences\n1. Preitner, N., et al. The orphan nuclear receptor REV-ERBalpha controls circadian transcription\nwithin the positive limb of the mammalian circadian oscillator. Cell 110, 251-260 (2002).\n2. Wang, S., Li, F., Lin, Y. & Wu, B. Targeting REV-ERBα for therapeutic purposes: promises and\nchallenges. Theranostics 10, 4168-4182 (2020).\n3. Sun, N., et al. Measuring In Vivo Mitophagy. Molecular cell 60, 685-696 (2015).\n4. Jiang, X.L., et al. PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via\ninhibiting FUNDC1-mediated mitophagy. Acta pharmacologica Sinica 45, 1809-1820 (2024).\n5. Li, X.T., et al. The UCP2/PINK1/LC3b-mediated mitophagy is involved in the protection of\nNRG1 against myocardial ischemia/reperfusion injury. Redox biology 80, 103511 (2025).\n6. Li, J., et al. Restricting intracellular Salmonella proliferation by coordinating p-TBK1 mediated\nmitophagy and xenophagy. Autophagy, 1-23 (2025).\n7. Qasim, W., et al. PTEN-induced kinase 1-induced dynamin-related protein 1 Ser637\nphosphorylation reduces mitochondrial fission and protects against intestinal ischemia\nreperfusion injury. World journal of gastroenterology 26, 1758-1774 (2020).\n8. Lin, Y., et al. Targeting DRP1 with Mdivi-1 to correct mitochondrial abnormalities in ADOA+\nsyndrome. JCI insight 9(2024).\n9. Zhang, C., et al. Gestational Exposure to Black Phosphorus Nanoparticles Induces Placental\nTrophoblast Dysfunction by Triggering Reactive Oxygen Species-Regulated Mitophagy. ACS\nnano 19, 16517-16533 (2025).\n10. Zhou, R., et al. Brevilin A, a novel BNIP3 inhibitor suppresses osteoclastogenesis and prevents\novariectomy-induced bone loss via impairing mitophagy and mitochondrial metabolism.\nPhytomedicine : international journal of phytotherapy and phytopharmacology 143, 156774\n(2025).\n11. Sun, Y., et al. L-Phenylalanine promotes liver steatosis by inhibiting BNIP3-mediated\nmitophagy. Molecular medicine (Cambridge, Mass.) 31, 250 (2025).\n12. Gang, H., et al. A novel hypoxia-inducible spliced variant of mitochondrial death gene Bnip3\npromotes survival of ventricular myocytes. Circulation research 108, 1084-1092 (2011).\n13. Pandey, A., Jithin, B., Mutnuru, S.A., Samaiya, A. & Shukla, S. Identification of SRSF9 through\npooled shRNA screening links BNIP3 splicing to autophagy and metabolic reprogramming in\nbreast cancer. The Journal of biological chemistry 301, 110482 (2025).\n14. Gang, H., et al. PDK2-mediated alternative splicing switches Bnip3 from cell death to cell\nsurvival. The Journal of cell biology 210, 1101-1115 (2015).\n15. Field, J.T., et al. Misoprostol regulates Bnip3 repression and alternative splicing to control\ncellular calcium homeostasis during hypoxic stress. Cell death discovery 4, 37 (2018).\n16. Kubli, D.A., Ycaza, J.E. & Gustafsson, A.B. Bnip3 mediates mitochondrial dysfunction and cell\ndeath through Bax and Bak. The Biochemical journal 405, 407-415 (2007)."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have adequately addressed all of reviewer comments.", "reviewer_3": "My comments have largely been addressed by the authors. The following points must be addressed before publication:\n1) Line 84: Cite a reference to support CoCl2 as a hypoxia mimetic agent.\n2) Line 345: The scientific method proceeds by refutation; consequently, it is not possible to prove a hypothesis. This sentence should read, “To further establish the role of mitophagy …”\n3) Figure 7J: the resolution of this panel in the pdf download for review was inadequate for publication. The editors should be aware of this limitation and the authors should check proofs carefully before publication.", "reviewer_4": "Thank you for addressing my previous concerns with additional experimentation. Although mdivi-1 inhibits mitochondrial fission, the combination with targeted mKeima provides more convincing evidence of mitophagy. The addition of isolated mitochondria probed for autophagy components is also more convincing. Finally, thank you for the additional discussion and more carefully worded and thoughtful conclusions that are more closely aligned with your data."}, "author_response": "Response to Reviewers for Manuscript # NCOMMS-25-34581A\nTitle: Targeting REV-ERBα/BNIP3 axis attenuates pulmonary arterial hypertension by repressing\nmitophagy\nPoint-by-Point Responses\nWe would like to express our sincere gratitude to the Editor and all Reviewers for their positive\nevaluation of our revised manuscript. We are thrilled that the reviewers found our revisions and\nadditional experiments satisfactory.\nIn accordance with the minor requests from Reviewer #3 and the editorial formatting guidelines,\nwe have made the final adjustments to the manuscript. Below is our point-by-point response.\nReviewer #2 (Remarks to the Author):\nThe authors have adequately addressed all of reviewer comments.\nResponse: We sincerely thank the reviewer for his/her rigorous evaluation and positive feedback\non our revised manuscript.\nReviewer #3 (Remarks to the Author):\nMy comments have largely been addressed by the authors. The following points must be\naddressed before publication:\n1) Line 84: Cite a reference to support CoCl2 as a hypoxia mimetic agent.\nResponse: We thank the reviewer for pointing this out. We have now added two references\n[Marsboom et al., 2012; PMID: 22511751; and Ryan et al., 2013; PMID: 23449689] to support\nthe use of CoCl as a hypoxia-mimetic agent in the revised manuscript.\n2\n2) Line 345: The scientific method proceeds by refutation; consequently, it is not possible to prove\na hypothesis. This sentence should read, “To further establish the role of mitophagy …”\nResponse: We completely agree with the reviewer's rigorous perspective on scientific\nepistemology. As requested, we have corrected the sentence to: \"To further establish the role of\nmitophagy...\" to ensure objective and precise scientific phrasing.\n3) Figure 7J: the resolution of this panel in the pdf download for review was inadequate for\npublication. The editors should be aware of this limitation and the authors should check proofs\ncarefully before publication.\nResponse: We appreciate the reviewer's attention to image quality. The low resolution in the peer-\nreview PDF was likely an artifact of the system's auto-compression during file generation. For this\nfinal submission, we have uploaded all figures, including Figure 7, as separate, high-resolution\nvector/PPT files as required by the journal. We will absolutely ensure its clarity and fidelity\nduring the final proofreading stage.\nReviewer #4 (Remarks to the Author):\nThank you for addressing my previous concerns with additional experimentation. Although mdivi-\n1 inhibits mitochondrial fission, the combination with targeted mKeima provides more convincing\nevidence of mitophagy. The addition of isolated mitochondria probed for autophagy components is\nalso more convincing. Finally, thank you for the additional discussion and more carefully worded\nand thoughtful conclusions that are more closely aligned with your data.\nResponse: We sincerely thank the reviewer for his/her rigorous evaluation and profound insights.\nWe are particularly grateful for your exceptional expertise in mitochondrial biology. Your highly\nprofessional critiques regarding the context-dependent roles of mitophagy, the dual functions of\nBNIP3, and its alternative splicing have been truly invaluable. Addressing these expert comments\nhas fundamentally elevated the mechanistic depth and overall rigor of our manuscript."}]

Decision letter